Comprehensive accounts of pharmaceutical research and development : from discovery to late-stage process development v2 9780841231887, 0841231885, 9780841231894, 9780841231917, 9780841231900

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Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.fw001

Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage Process Development Volume 2

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.fw001 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

ACS SYMPOSIUM SERIES 1240

Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.fw001

to Late-Stage Process Development Volume 2 Ahmed F. Abdel-Magid, Editor Therachem Research Medilab Jaipur, India

Jaan Pesti, Editor Gelest Inc., Morrisville Pennsylvania

Rajappa Vaidyanathan, Editor Bristol-Myers Squibb Bangalore, India

Sponsored by the ACS Division of Organic Chemistry

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.fw001

Library of Congress Cataloging-in-Publication Data Names: Abdel-Magid, Ahmed F., 1947- editor. | Pesti, Jaan A., 1954- editor. | Vaidyanathan, Rajappa, editor. | American Chemical Society. Division of Organic Chemistry, sponsoring body. | American Chemical Society. Title: Comprehensive accounts of pharmaceutical research and development : from discovery to late-stage process development / Ahmed F. Abdel-Magid, editor, Jaan A. Pesti, editor, Rajappa Vaidyanathan, editor ; sponsored by the ACS Division of Organic Chemistry. Other titles: ACS symposium series ; 1239-1240. Description: Washington, DC : American Chemical Society, [2016] | Series: ACS symposium series ; 1239-1240 | Includes bibliographical references and index. Identifiers: LCCN 2016052701 (print) | LCCN 2016053570 (ebook) | ISBN 9780841231894 (v. 1 : alk. paper) | ISBN 9780841231917 (v. 2 : alk. paper) | ISBN 9780841231900 (ebook) Subjects: | MESH: Chemistry, Pharmaceutical--methods | Drug Discovery | Technology, Pharmaceutical | Pharmaceutical Preparations--chemistry Classification: LCC RS403 (print) | LCC RS403 (ebook) | NLM QV 745 | DDC 615.1/9--dc23 LC record available at https://lccn.loc.gov/2016052701

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2016 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

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Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Preface Until the latter part of the 19th century, the majority of known drugs were either herbs or extracts of active ingredients from botanical sources. At this point, the world witnessed two major cornerstone achievements that laid the foundation of modern drug discovery and development. The first was the emergence of pharmacology as a contemporary science through the work of Schmiedeberg (considered by many as the father of modern pharmacology) at the University of Strasbourg. He studied the correlation between the chemical structure of substances and their therapeutic effectiveness, and opened the door to understanding drug actions and effects. The second was Wöhler’s landmark synthesis of urea from ammonium cyanate (the first synthesis of an ‘organic’ molecule from an ‘inorganic’ source) that signaled the end of the old belief in the ‘vital force’ theory and its limitations, and heralded the birth of modern organic chemistry. Shortly thereafter, researchers identified salicylic acid as a natural constituent and active ingredient in the willow tree bark extract, which was used for centuries as an analgesic. In 1897, scientists at Bayer performed one of the earliest known developments of a small molecule single-component drug when they modified the structure of salicylic acid to form acetyl salicylic acid. This modified structure exhibited improved efficacy and fewer side effects compared to salicylic acid and was commercialized as aspirin. It is still today one of the most used over-thecounter drugs. Further advancements were introduced in the first half of the 20th century with the identification and manufacturing of products such as penicillin and insulin. Major advancements in drug discovery and development occurred during the Second World War to meet the large demands for analgesics, antibiotics and many other vital drugs. Thus, pharmaceutical research and development was expanded to identify new drugs for the treatment of a wide range of conditions. While many of the earlier drugs were used only for alleviating symptoms rather than for completely curing diseases, starting in the 1950s, pharmaceutical research and development soared to new heights and introduced many novel drugs capable of treating a plethora of ailments including infections, pain, CNS disorders, heart diseases and cancer, as well as “non-traditional” drugs such as oral contraceptives. During this evolution of pharmaceutical research, the role of synthetic organic chemistry has manifested in two distinct functions—Discovery or Medicinal Chemistry, and Process Chemistry (also known as Chemical Process Research). Drug discovery has evolved from relying on luck, accident and serendipity to a complex endeavor that is at the interface of several disciplines (e.g. pharmacology, biology, chemistry), and is built on the understanding of mechanisms and causes of diseases. Modern approaches involve identifying ix Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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therapeutic targets such as enzymes and receptors that are implicated in the pathogenesis of diseases. Medicinal chemists will then design and test molecules that can modulate (inhibit or enhance) the activities of these enzymes or receptors to find new potential drugs that can treat or prevent a disease. The deciphering of the human genome has added new opportunities for the development of novel drugs capable of specifically targeting the sites where diseases are caused. Process chemistry’s role is to produce high quality drug substances on large scales. Once the discovery chemists identify a drug candidate and advance it into development, process chemists start designing a practical and safe synthesis, referred to as a process, capable of producing large quantities of this drug candidate to support all the needs of drug development, such as toxicology, preclinical and clinical studies. Process chemists must design routes that are scalable, can routinely produce drug substance that meets its pre-determined acceptance criteria (purity, physical form, polymorph etc.), are economically viable and environmentally responsible, while meeting a myriad of growing regulatory and legal requirements, as well as internal deadlines. The increasing structural complexity of drug molecules has challenged process chemists to be more creative and innovative by discovering and adopting new methodologies and technologies for the synthesis of these molecules. The implementation of new methodology may demand the use of highly complex and potentially expensive reagents, catalysts and/or equipment and that may add new safety and logistical concerns. This book is produced to celebrate the evolution of drug discovery and development. We put this book together hoping it will be helpful to synthetic organic chemists in both pharmaceutical industry and in academia. We think it can serve as a teaching tool to students who want to learn and understand the processes and challenges of drug discovery and development with real examples from top pharmaceutical companies. The chapters contain citations of a large number of valuable selected references to the primary literature. The book highlights the tireless efforts of discovery and process chemists, and their roles in the advancement of drug discovery and development. We were motivated to create this book by our appreciation of the value of chemical research by both discovery and process chemists in producing new pharmaceutical entities. Their combined efforts make it possible to introduce novel and effective drugs into the market to treat millions of patients and alleviate their suffering, improve their quality of life and possibly save their lives from killer diseases and disorders. The chapters presented in this book are written by a selected group of outstanding, very accomplished medicinal and process chemists with noted experiences and diverse backgrounds representing some of the top pharmaceutical companies. The chapters highlight examples of emerging concepts, new developments and challenges arising in the discovery of new drug candidates and the development of new practical synthetic chemistry processes to produce these drug candidates on large scale. The discovery of each drug or drug candidate is presented by the discovery chemist(s), and then the process chemist(s) who developed this same drug candidate describe its development to give the reader a complete story of drug discovery and development. Some groups have separate discovery and development chapters while others selected to include both in the same chapter. x

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.pr001

Either way, the reader will experience a rare and unique opportunity to get the complete perspectives of Discovery and Process chemistry in a single book. While most of these topics have appeared in the primary literature where space is critical and brevity valued, the book setting permits us to tell the complete tales as stories, from start to finish or to the current state of the drug development. We aimed to increase the value of this book by imposing few limits on relevant details. Our particular thanks to all the authors and the coauthors who are acknowledged in the individual chapters listed below. Their outstanding contributions to this book made it a worthy project and a valuable contribution to the literature and their hard work and courtesy made it an enjoyable experience: Seb Caille and John G. Allen of Process Development – Drug Substance Technologies, and Therapeutic Discovery of Amgen, respectively, report on the discovery of protein fucosylation inhibitors and development of a manufacturing process to prepare the inhibitor 6,6,6-trifluorofucose. Inhibitors of fucosylation enable the preparation of monoclonal antibodies displaying improved antibody-dependent cell-mediated cytotoxicity (ADCC) and in vivo efficacy. Jason S. Tedrow and Wenge Zhong of Process Development and Discovery Research Amgen Asia R&D Center, respectively, report the hydroxyethylamine (HEA)-derived potent and orally efficacious BACE1 inhibitors as potential treatments for Alzheimer’s disease. They eliminated hazardous chemistry, shortened of the overall route from 29 to 19 steps, and increased the yield from 4% to 19%. Xianglin Shi, William F. Kiesman and Donald G. Walker of Chemical Process R&D at Biogen discuss Hsp90 inhibitors for cancer treatment. The initial compound discovered through SAR studies was efficacious for the treatment of HER-2 positive solid cancers in clinical trials. As the trials progressed, a follow-up compound was discovered to meet intravenous formulation requirements. Processes were developed to prepare multi-kilogram quantities of these inhibitors. Hidenori Takahashi, Alessandra Bartolozzi and Thomas Simpson of Small Molecule Discovery Research, and Keith Fandrick, Jason Mulder, Jean-Nicolas Desrosiers, Nitin Patel, Xingzhong Zeng, Daniel Fandrick, Carl A. Busacca, Jinhua J. Song, and Chris H. Senanayake of Chemical Development, Boehringer Ingelheim discuss the discovery and synthesis of a novel oxadiazole-containing 5-lipoxygenase activating protein inhibitor. The key transformation was a dual boronate rearrangement process. Christian Harcken and Hossein Razavi of the Department of Small Molecule Discovery Research and Jonathan T. Reeves, Daniel R. Fandrick, Jinhua J. Song, Zhulin Tan, Soojin Kim, Bing-Shiou Yang, Nathan K. Yee and Chris H. Senanayake of the Department of Chemical Development, at Boehringer Ingelheim discuss the discovery and synthesis of a series of novel, non-steroidal, trifluoromethylcarbinol glucocorticoid receptor agonists. Multi-parameter optimization led to the identification of a clinical candidate. To support both pre-clinical and clinical studies, the team has developed an asymmetric process for the large scale and economical synthesis of the drug candidate and increased the overall yield by 88-fold. xi

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Remy Angelaud, Mark Reynolds, Scott Savage and Andreas Stumpf of Small Molecule Process Chemistry, and Daniel P. Sutherlin of Discovery Chemistry, all of Genentech, describe the discovery and development of the hedgehog inhibitor, vismodegib. This is the first inhibitor of the hedgehog pathway to be approved for treating metastatic and locally advanced basal cell carcinoma, and represents an option for patients where surgery is not recommended. Timothy P. Heffron of Discovery Chemistry, and Andrew McClory and Andreas Stumpf of Small Molecule Process Chemistry, all again of Genentech, detail the discovery and process chemistry of GDC-0084, a blood-brain-barrier penetrating inhibitor or PI3K and mTOR, a treatment for brain tumors. Daniel Sutherlin and Alan Olivero of Genentech Discovery Chemistry, and Srinivasan Babu, Francis Gosselin, Theresa Humphries, and Qingping Tian of Small Molecule Process Chemistry discuss the discovery and process research to make multi-kilogram quantities of apitolisib and pictilisib—therapies to influence the PI3K signaling pathway. This pathway has received a great deal of interest as a potential target for cancer treatment. Ian M. Bell of the Department of Discovery Chemistry, and Paul G. Bulger and Mark McLaughlin of the Department of Process Research and Development at Merck & Co., Inc., detail the discovery and preparation of the CGRP receptor antagonist MK-3207. The compound was developed for the treatment of migraine, a common, highly disabling neurovascular disorder that can significantly impact quality of life. This chapter describes how MK-3207 was discovered, and the development of an efficient synthetic route that enables large scale production of this complex molecule. Jason D. Burch of Inception Sciences Canada, and Benjamin D. Sherry, Donald R. Gauthier, Jr. and Louis-Charles Campeau of Process Research & Development of Merck Research Laboratories present the discovery and development of doravirine—a drug currently in phase III clinical trials for the treatment of HIV infection. The future looks encouraging as the drug showed similar efficacy to efavirenz but with fewer side effects. Debra J. Wallace and Ian Mangion (Department of Process Chemistry) and Paul Coleman (Department of Medicinal Chemistry) at Merck & Co., Inc. outline the discovery and subsequent synthetic development of suvorexant, a dual orexin antagonist for sleep disorder. The discussion follows progress from the medicinal chemistry approach through an initial fit-for-purpose (FFP) process synthesis to finally an optimized asymmetric route. The value of a complete mechanistic understanding of the chemistry underscores the research undertaken. Jessica Reed formerly of Pfizer Global Research & Development and Jeff Smaill of the University of Auckland write about the discovery of dacomitinib, an irreversible small molecule epidermal growth factor receptor (EGFR) inhibitor. In a separate chapter, Shu Yu and Olivier Dirat of Pfizer Chemical Research and Development, discuss the process work that has produced over 800 kg of drug. Dacomitinib is currently in phase III clinical development for the treatment of non-small cell lung cancer (NSCLC) and other cancers. Jed L. Hubbs, Ruichao Shen, Nathan O. Fuller, Wesley F. Austin, and Brian. S. Bronk of Satori Pharmaceuticals Inc. describe the discovery and process development of the natural product derivative SPI-1865, a gamma-secretase modulator. As such, the drug is a potential therapy for Alzheimer’s disease. xii

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The drug preparation is initiated by fractionation of black cohosh extract. Julian P. Henschke and Erick W. Co of ScinoPharm Taiwan Ltd. discuss the discovery and process development of the 5-azacytosine-based nucleosides azacitidine and decitabine as an introduction to the main topic, cladribine, a chlorinated, deaminase-resistant derivative of the naturally occurring nucleoside, 2′-deoxyadenosine. Cladribine possesses both antineoplastic and immunosuppressive activity. Hirotaki Mizutani, Steven Langston, and Stepan Vyskocil of the Medicinal Chemistry Pharmaceutical Research Division and Ian Armitage, Ashley McCarron, and Lei Zhu of the Process Chemistry Department of the Chemical Development Laboratories, Takeda Pharmaceuticals, describe the discovery and the process development of the NEDD8 activating enzyme inhibitor pevonedistat. A novel Burgess-type reagent was developed in the final sulfamoylation step. Gerald J. Tanoury, Stephen Eastham, Cristian L. Harrison, Benjamin J. Littler, Piero L. Ruggiero, and Zhifeng Ye, of the Process Chemistry Department, and Anne-Laure Grillot of the Chemistry Department, of Vertex Pharmaceuticals Inc. discuss the drug discovery, process development, and commercial synthesis of telaprevir, an HCV NS3•4A protease inhibitor. The manufacturing process has delivered >60 metric tons of drug substance. We gratefully acknowledge the many people whose hard work, dedication and expertise made this book possible. We appreciate the work of our referees who made constructive suggestions for improvement. Many thanks to our colleagues at ACS Books who encouraged and facilitated the compilation of this book: Bob Hauserman, Elizabeth Hernandez, and Arlene Furman. We acknowledge Syngene International Ltd. for allowing us to use a photograph of their facilities on the cover of this book. A special thanks to Trevor Laird whose foreword elegantly states the importance of pharmaceutical research, and whose work at Org. Process Res. Dev. and Scientific Update has contributed significantly to the advancement of process chemistry. Finally we thank the Organic Chemistry and Medicinal Chemistry Divisions of the American Chemical Society for their joint sponsorship of our biennial symposium.

Ahmed F. Abdel-Magid Therachem Research Medilab Jaipur, India

Jaan A. Pesti Gelest Inc. Morrisville, Pennsylvania

Rajappa Vaidyanathan Bristol-Myers Squibb Bangalore, India xiii Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Chapter 1

Synthesis of a Potent NAE Inhibitor: Pevonedistat Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch001

Hirotaki Mizutani, Steven Langston,* and Stepan Vyskocil Medicinal Chemistry Pharmaceutical Research Division, Takeda Pharmaceuticals International Company, 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States *E-mail: [email protected].

Pevonedistat (MLN-4924, TAK-924) is a first-in-class inhibitor of Nedd-8 activating enzyme (NAE) currently undergoing clinical evaluation in oncology. This chapter will discuss the identification of pevonedistat and describe the synthetic routes undertaken by discovery chemists to provide material for pre-clinical studies.

Introduction Within the cellular environment, the levels of particular proteins at any given time are highly controlled by regulation of protein synthesis and also by regulation of protein degradation. The organelle responsible for the regulated decomposition of proteins within the cell is the proteasome, which acts as a multi-subunit protease with broad substrate specificity. Proteins tagged for proteolysis by the proteasome are first labeled with a poly-ubiquitin chain by way of a cascade of enzymes within the cell, a process known as ubiquitination. Ubiquitin (Ub) is a highly conserved, low molecular weight protein that is attached to the protein to be degraded through its carboxylic acid terminus and a side chain amine of a lysine residue of the protein. The enzyme responsible for initial activation of ubiquitin is known as ubiquitin activating enzyme (UAE), and is generically termed an E1. UAE utilizes ATP to activate the terminal carboxylic acid of ubiquitin to facilitate formation of a thioester bond through a © 2016 American Chemical Society

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch001

cysteine residue of UAE and release both adenosine 5′-O- monophosphate(AMP) and pyrophosphoric acid (PPi). Ubiquitin thus activated is transferred to an intermediary protein, known as a conjugating enzyme or E2, through thiol exchange with a cysteine residue on the E2 and then is finally attached, or ligated, to the protein substrate via an E3 ligase. This general pathway for regulated degradation of proteins is known as the ubiquitin-proteasome system or UPS (Figure 1) (1–3).

Figure 1. The Ubiquitin Proteasome System (UPS).

The E3 ligases are a broad family of enzymes which play a critical role in imparting substrate specificity to which proteins are ubiquitinated and ultimately degraded by the proteasome. Their activity is thus closely regulated. A subclass of E3 ligases are known as the Cullin-Ring-Ligases (CRLs) based on the make-up of subunits that compose the whole enzyme. For this class of ligases to be fully enzymatically active they are required to be modified (on the Cullin subunit) by a protein—Nedd-8. This is a protein closely related to ubiquitin and the process of attachment of Nedd-8 to its protein substrate is known as neddylation. The process of neddylation is analogous to ubiquitination with Nedd-8 activating enzyme (NAE) being the E1, and a protein known as UBC12 serving as the E2 before transferring to the CRL family of E3 ligases, (Figure 2) (1–3). Because neddylation is necessary for the activity of the CRLs it plays an important role in regulating which proteins are ubiquitinated and degraded by the proteasome. These proteins, such as phosphorylated IκB, Cdt-1 etc., often play critical roles in cellular processes such as DNA replication, cell division and cell signaling (1–3). 2

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 2. The ubiquitination and neddylation processes.

Identification of Pevonedistat (TAK-924, MLN4924) At the beginning of this program, the clinical success of the first proteasome inhibitor bortezomib (Velcade®) from our company gave us encouragement to investigate additional targets within the ubiquitin proteasome pathway. The inhibition of the proteasome essentially prevents the degradation of all protein substrates within the UPS. However, only a subset of those proteins would be expected to be stabilized by inhibition of NAE, i.e. those proteins targeted for proteasome degradation through ubiqutination via CRL E3 ligases, and thus dependent upon neddylation (and NAE activity) for activity, (Figure 2). It is known that AMP is a product of the NAE-catalyzed reaction and is a weak inhibitor of NAE (4, 5). The knowledge that ATP and AMP are recognized by E1 enzymes, including NAE, indicated that this class of enzymes may be druggable 3 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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targets within the UPS. Indeed, a high throughput screen followed up by array synthesis identified N-6 substituted adenosine derivatives as μM inhibitors. Incorporating the 5’-O-phosphate with one of the preferred N-6 substituents, 1-(+)-(S)-aminoindane, gave a sub-μM inhibitor, compound 2. A key finding for us was that replacing the phosphate with the neutral sulfamate group (3, X=O, Figure 3 and Table 1) substantially boosted potency to low nM and exhibited good activity in cellular assays. However, the sulfamate group can act as a weak leaving group and analogs were prone to degradation upon storage through an internal cyclization reaction, (Scheme 1).

Figure 3. Compounds on route to the discovery of pevonedistat.

Scheme 1. Degradation of Adenosine Sulfamate Analogs through Internal Cyclization 4 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The Medicinal Chemistry effort was thus focused on stabilizing the compounds to intrinsic degradation and exploring the SAR (structure activity relationship) to identify more selective compounds for NAE over related E1 enzymes such as ubiquitin activating enzyme (UAE). Replacing the sulfamate group with the more stable sulfonamide (X = CH2, 5) or sulfamide (X = NH, 4) rendered reduced potency. A broad diversity of substituents at the 6-position of the adenine ring was also investigated with 1-(+)-(S)-aminoindane remaining one of the preferred groups. Replacing the ribose for a cyclopentane, and removal of the 2' OH group and N-7 of the adenine ring (adenosine numbering) were all well-tolerated. Combining these modifications gave compounds that were selective for NAE over UAE, for example 6 (Figure 4 and Table 1). However, the compounds remained somewhat prone to degradation through intramolecular cyclization. A breakthrough came with the surprising finding that inversion of the configuration at the methylene sulfamate position was tolerated (TAK-924, Figure 4, and Table 1). This places the sulfamate on the opposite face to the adenine type ring and thus immune to intramolecular attack by the adenine ring due to lack of proximity between the groups. These changes ultimately led to the identification of the candidate molecule pevonedistat (MLN4924, TAK-924) (Figure 4 and Table 1) (6).

Table 1. Inhibitory Activity against the Target Nedd-8 Activating Enzyme (NAE) and the Related Enzyme Ubiquitin Activating Enzyme (UAE) NAE *IC50 (nM)

UAE *IC50 (nM)

1

1600

>10000

2

120

>1000

3 X=O

0.5

2

4 X=NH

2

450

5 X=CH2

12

>1000

6

8

>1000

pevonedistat

3

>1000

Compound

*

HTRF assay. (8)

During the course of this Medicinal Chemistry effort, it was discovered that such adenosine sulfamate analogs, including pevonedistat, act as a unique class of mechanism based inhibitors, termed substrate assisted inhibition (7). The biological consequences of NAE inhibition through pevonedistat, including efficacy of cellular outcomes, pharmacodynamics markers and efficacy in in vivo xenograft models are described in detail elsewhere. (8, 9). 5 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 4. Structures of adenosine sulfamate analogs, including pevonedistat, in relation to adenosine mono-phosphate (AMP).

Scheme 2. Retrosynthesis of Pevonedistat

Synthesis of Pevonedistat Retrosynthesis of Pevonedistat The initial route for the synthesis for pevonedistat was designed to allow flexibility in the choice of a purine-like base and enantiomerically pure carbocycle, and utilized building blocks that could be prepared using chemistry precedented in the literature. Pyrrolopyrimidine 7 and cyclopentane building block 8 with an epoxide function were chosen as synthons (Scheme 2). While 7 could be easily accessed from commercially available materials, the construction of 8 with all cis-tetrasubstituted cyclopentane stereochemistry was significantly more challenging. We envisioned that it could be accessed 6 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

from the alkene 9, which in turn could be derived from the precursor 10 via a metathesis reaction (10). Enantiomerically pure diene 10 could be then constructed using an Evans aldol reaction utilizing an oxazolidinone as the chiral auxiliary. Subsequently, a more direct route towards intermediate 9 was designed, utilizing a hetero-Diels-Alder reaction of cyclopentadiene with glyoxalic acid via hydroxylactone 11 (11), followed by enzymatic resolution of rac-9.

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First Synthesis of Pevonedistat The first synthesis of pevonedistat used an Evans asymmetric aldol reaction as the key step for building the appropriate cyclopentane stereochemistry (Scheme 3) (12). (S)-Benzyloxazolidinone 12 was deprotonated using n-BuLi and treated with 4-pentenoyl chloride to form the oxazolidinone intermediate 13 in almost quantitative yield. Compound 13 underwent a highly stereoselective enolization using dibutylboron triflate in the presence of a base, and the corresponding boron enolate was then allowed to react with acrolein. Following oxidative workup, 14 was isolated in 61% yield in enantiomerically pure form (>99% ee) (12). Diene 14 was then cyclized to cyclopentene 15 using ring closing metathesis with Grubbs second generation catalyst (10) in 79% yield. The chiral auxiliary was then removed by reduction with lithium borohydride (13), to furnish the enantiomerically pure diol 9 in 61% yield. Diol 9 was protected and then subjected to diastereoselective epoxidation to cis-epoxydiol 16. High (>9:1) cis/trans selectivity could be rationalized by syn-stereodirecting effect of the allylic hydroxyl group on one of the oxygens of the peroxy group (14). The PMP-acetal protecting group with good basic pH stability was introduced using condensation with PMP-dimethylacetal and a catalytic amount of TsOH. This group was used in anticipation that the epoxide-opening step may require forcing basic conditions. The key coupling to produce the skeleton of pevonedistat was effected by treating the protected epoxide-diol 16 with pyrrolopyrimidine 7 under basic conditions with heating. The epoxide was opened regioselectively to give nucleoside precursor 17. The regiochemistry can be explained by steric factors associated with the acetal. The hydroxyl group at the 2-position was removed using a two-step sequence. This reaction proceeded through a thiocarbamoyl intermediate which underwent Barton-McCombie (15) radical deoxygenation to 18. Deprotection of the acetal-protecting group PMP was achieved using AcOH (16) to furnish diol 19. Initial attempts to introduce the sulfamoyl group with sulfamoyl chloride were unsuccessful as they led to mixtures of mono- and bis-sulfamoylated products. This final step required a sequence of protection/deprotection steps to selectively sulfamoylate the primary hydroxyl group, starting with TBS protection, followed by acetate protection of the secondary alcohol to yield 20. The primary TBS group was removed using HF•TEA, and the resulting alcohol was sulfamoylated using sulfamoyl chloride. Sulfamate 21 was then deacetylated using ammonia to furnish pevonedistat. 7 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

While this route enabled the synthesis of the initial quantities of pevonedistat, it had some drawbacks: • • •

long synthesis (15 synthetic steps). it required multiple chromatographic purifications. provided a low overall yield of product (5.5 %).

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch001

Thus, we needed an alternative route for the preparation of multigram quantities of pevonedistat required for in vivo testing.

Scheme 3. The First Synthesis of Pevonedistat

Gram Scale Synthesis of Pevonedistat The second synthetic iteration (Scheme 4) started with a hetero-Diels-Alder reaction of cyclopentadiene with glyoxalic acid (11). This [4+2] addition followed by rearrangement (17) produced hydroxylactone 11 which was converted to racemic cis-diol 9 via global reduction with LAH followed by periodate cleavage of the triol 22 (18). This sequence required only one chromatographic purification 8 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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and was performed successfully on 250 g of hydroxylactone 11. Following selective TBS protection, racemic 23 was isolated by column chromatography in 75% overall yield for the 3 steps. This material was obtained as a single diastereosisomer with cis-orientation of cyclopentene substituents set by syn-addition in the first step.

Scheme 4. Gram Scale Synthesis of Pevonedistat Enantiomeric resolution of racemic 23 was achieved using enzyme-catalyzed acetylation with Candida antarctica lipase B on acrylic beads (19). The desired enantiomer 23 was separated from acetate 24, which possessed the opposite stereochemistry, in high yield (88% of theory) and high enantiomeric purity (>98% ee). Subsequent deprotection of 23 with TBAF yielded 9, which was then converted to 19 using the same chemistry as described in Scheme 3. It was desired to avoid the protection/deprotection protocol used in the first synthesis to differentiate the hydroxyls of 19. Since it would be difficult to functionalize the secondary in the presence of the primary alcohol, we sought to identify conditions for selective sulfamoylation at the desired primary position. Considering the steric hindrance of the secondary versus the primary alcohol, we studied bulky reagents to differentiate the two positions. We ultimately discovered that reagent 25 selectively sulfamoylated the primary hydroxyl group, and provided pevonedistat in 81% yield after hydrolysis. 9

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

This new process possessed a number of advantages: • • • •

fewer steps than the first synthesis (13 vs 15). higher overall yield (6.8 % vs 5.5%). this route provided robust access of to up to 50 g of key cyclopentene diol 9 in one synthetic sequence. it enabled the synthesis of multigram quantities of pevonedistat.

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These two synthetic routes completed the Medicinal Chemistry synthesis of pevonedistat.

Conclusion The modular character of the synthetic routes described in Schemes 3-4 enabled modifications on both carbocyclic and purine-like portions of the molecule, helping to explore SAR of the series, and select pevonedistat for IND-enabling studies. The second generation route described in Scheme 4 provided a ~ 20 gram supply of pevonedistat for pharmacological and early toxicological evaluation of NAE inhibition. The main improvement over the initial approach (described in Scheme 3) was a robust and scalable supply of rac-23 cyclopentene. This second generation synthesis however had several segments with poor atom economy. While resolution of rac-23 provided the desired enantiomer in excellent enantiomeric purity, inability to utilize the undesired cyclopentene enantiomer 24 significantly reduced the overall yield. Also, the melding of pyrrolopyrimidine 7 with epoxide 16 required protecting group manipulations, and the removal of the undesired 2-hydroxyl group in PMP-acetal 17 which added two synthetic steps requiring chromatography purifications. While this was a suitable synthesis for Medicinal Chemistry purposes, the low yield, number of synthetic steps and need for multiple chromatography purifications prevented further scaling to provide larger quantities of pevonedistat. Therefore, a search for new, GLP/GMP-compatible routes was warranted to provide supply of pevonedistat for clinical development and is discussed in the next chapter.

Acknowledgments If it takes a village to raise a child it most certainly takes a team to discover and develop a drug candidate. We would like to express our thanks all those involved in the NAE program.

10 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

References 1. 2. 3.

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4. 5. 6.

7.

8.

9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Bedford, L.; Lowe, J.; Dick, L. R.; Mayer, R. J.; Brownell, J. E. Nat. Rev. Drug Discovery 2011, 10, 29–46. Nalepa, G.; Rolfe, M.; Harper, J. W. Nat. Rev. Drug Discovery 2006, 5, 596–613. Soucy, T. A.; Dick, L. R.; Smith, P. G.; Milhollen, M. A.; Brownell, J. E. Genes Cancer 2010, 1, 708–716. Haas, A. L.; Rose, I. A. J. Biol. Chem. 1982, 257, 10329–10337. Bohnsack, R. N.; Haas, A. L. J. Biol. Chem. 2003, 278, 26823–26830. Ciavarri, J.; Dick, Langston, S. P., The Discovery of First-in-Class Inhibitors of the Nedd8 Activating Enzyme (pevonedistat, TAK-924, MLN4924) and the Ubiquitin Activating Enzyme (TAK-243) Compr. Med. Chem. III Submitted for publication. Brownell, J. E.; Sintchak, M. D; Gavin, J. M.; Liao, H.; Bruzzese, F. J.; Bump, N. J.; Soucy, T. A.; Milhollen, M. A.; Yang, X.; Burkhardt, A. L.; Ma, J.; Loke, H. K.; Lingaraj, T.; Wu, D.; Hamman, K. B.; Spelman, J. J.; Cullis, C. A.; Langston, S. P.; Vyskocil, S.; Sells, T. B.; Mallender, W. D.; Visiers, I.; Li, P.; Claiborne, C. F.; Rolfe, M.; Bolen, J. B.; Dick, L. R. Mol. Cell 2010, 37, 102–111. Soucy, T. A.; Smith, P. G.; Milhollen, M. A.; Berger, A. J.; Gavin, J. M.; Adhikari, S.; Brownell, J. E.; Burke, K. E.; Cardin, D. P.; Critchley, S.; Cullis, C. A.; Doucette, A.; Garnsey, J. J.; Gaulin, J. L.; Gershman, R. E.; Lublinsky, A. R.; McDonald, A.; Mizutani, H.; Narayanan, U.; Olhava, E. J.; Peluso, S.; Rezaei, M.; Sintchak, M. D.; Talreja, T.; Thomas, M. P.; Traore, T.; Vyskocil, S.; Weatherhead, G. S.; Yu, J.; Zhang, J.; Dick, L. R.; Claiborne, C. F.; Rolfe, M.; Bolen, J. B.; Langston, S. P. Nature 2009, 458, 732–736. Milhollen, M. A.; Traore, T.; Adams-Duffy, J.; Thomas, M. P.; Berger, A. J.; Dang, L.; Dick, L. R.; Garnsey, J. J.; Koenig, E.; Langston, S. P.; Manfredi, M.; Narayanan, U.; Rolfe, M.; Staudt, L. M.; Soucy, T. A.; Yu, J.; Zhang, J.; Bolen, J. B.; Smith, P. G. Blood 2010, 116, 1515–1523. Monfette, S.; Fogg, D. E. Chem. Rev. 2009, 109, 3783–3816. Auge, J.; Gil, R.; Kalsey, S.; Lubin-Germain, N. Synlett 2000, 6, 877–879. Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem. Soc. 1981, 103, 2127–2109. Williams, D. R.; Nold, A. L.; Mullins, R. J. J. Org. Chem 2004, 69, 5374–5382. Ye, D.; Fringuelli, F.; Piermatti, O.; Pizzo, F. J. Org. Chem 1997, 62, 3748–3750. Barton, D. H. R.; McCombie, S. W. J. Chem. Soc., Perkin Trans. 1. 1975, 16, 1574–1585. Toshima, K.; Jyojima, T.; Miyamoto, N.; Katohno, M.; Nakata, M.; Matsumura, S. J. Org. Chem 2001, 66, 1708–1715. Lubineau, A.; Auge, J.; Lubin, N. Tetrahedron Lett. 1991, 32, 7529–7530. An, G-I.; Rhee, H. Nucleosides, Nucleotides Nucleic Acids 2002, 21, 65–72. Johnson, C. R.; Bis, S. J. Tetrahedron Lett. 1992, 33, 7287–7290. 11

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Chapter 2

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Process Development and GMP Production of a Potent NEDD8-Activating Enzyme (NAE) Inhibitor: Pevonedistat Ian Armitage, Ashley McCarron, and Lei Zhu* Chemical Development Laboratories, Millennium Pharmaceuticals, Inc., a subsidiary of Takeda Pharmaceutical Company Limited, 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States *E-mail: [email protected].

Development efforts for a manufacturing process of a novel NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924) are described. Highlights include an enantioselective synthesis of an aminodiol cyclopentane intermediate containing three chiral centers and a novel, regioselective sulfamoylation using N-(tert-butoxycarbonyl)N-[(triethylenediammonium)sulfonyl]azanide. The linear process, involving six isolations, has been carried out in multiple cGMP productions on 15 to 30 kg scale to produce pevonedistat in 98% purity and 25% overall yield.

Section 1: Process Development for GLP and First GMP Production of Pevonedistat Introduction (((1S,2S,4R)-4-{4-[(S)-2,3-dihydro-1H-inden-1-ylamino]-7H-pyrrolo[2,3d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methyl sulfamate hydrochloride) (pevonedistat, Figure 1), also known as MLN4924 and TAK-924, a novel NEDD8-activating enzyme (NAE) inhibitor, has demonstrated in vitro cytotoxic activity against a variety of human malignancies. It is currently being developed © 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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by Takeda Pharmaceuticals Company Limited in phase I/II clinical trials for the treatment of hematological and solid tumor cancers (1–17). The five-membered ring, carrying three chiral centers and an acid/base sensitive terminal sulfamoyl group, presented considerable challenges for the development of a practical and scalable synthesis of pevonedistat. This report details our successful efforts in developing a laboratory-scale synthesis to a multikilogram, reproducible preparation.

Figure 1. Chemical Structure of Pevonedistat.

General Strategy The original medicinal chemistry synthetic route to produce pevonedistat involved 13 linear steps and multiple chromatographic purifications (Scheme 1). More than half of the chemical transformations in the synthesis were carried out to construct the chiral five-membered ring. The poor overall efficiency of this route resulted from: • • •

a low yielding resolution (step 4). removal of an extra chiral hydroxyl group (step 10). installation of an acetal protecting group (step 7).

Furthermore, the late stage terminal sulfamoylation on the primary hydroxyl group was poorly selective and low yielding. Although the original route was reproducible on gram scale and able to supply material needs for early drug metabolism and pharmacokinetics (DMPK), toxicological, and pharmacology studies, the process chemistry group assessed that it would not be viable for producing kilogram quantities to support further preclinical and clinical studies. It would have been a considerable challenge to complete the first GMP API campaign without incorporating significant process improvements. However, due to the time constrains from candidate selection to IND, we set our short-term goal to develop a synthetic route capable of delivering up to 200 g of API to supply IND enabling toxicology studies. Subsequently, we needed to prepare 1–2 kg of clinical material for clinical studies. This section describes our activities towards achieving these goals. 14

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Scheme 1. Original Medicinal Chemistry Synthesis of Pevonedistat

Research Towards a Scalable Route Several alternative syntheses were considered on a theoretical basis in the development of a scalable route. The selected bond disconnection strategy that enabled the cGMP production of multiple hundred gram and ultimately multi kilogram quantities of pevonedistat is displayed in Scheme 2.

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Scheme 2. Retrosynthetic Analysis of Pevonedistat

The basis of this strategy was supported by literature precedent of the synthesis of the acetal derivative of aldehyde 5 (18–20). Coupling of substrates similar to 4 and 5 was also demonstrated in the same reference, although it was not clear whether aldehyde and acetal would work equally in our case. Coupling of 6 with 3 and subsequent sulfamoylation were carried out previously by medicinal chemistry. The key development focuses envisioned were: • • •

establishment of a scalable route towards enantiomerically pure cyclopentane aminodiol intermediate 4. successful installation of the labile sulfamate group. optimization of the chemically inefficient process.

Research Strategies To Enable a Scalable Route To Produce up to 200 g for IND Enabling Toxicology Studies Focusing on the desired enantiomerically pure building block aminodiol 4, two routes were examined thoroughly based on retrosynthetic analysis (Scheme 3). The first one centered on a Sharpless epoxidation of an allyl alcohol intermediate and the other on a chiral lactone intermediate which would be then reduced to result in the diol.

Scheme 3. Retrosynthetic Analysis of Aminodiol 4 16 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Sharpless Epoxidation Route to Intermediate 4

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The preparation of the allylic alcohol 11 necessary to test the Sharpless epoxidation route was inspired by the work from Bray et al. (Scheme 4) (21). A bulkier protecting group, trityl, was installed to aid steric control of the epoxidation.

Scheme 4. Sharpless Epoxidation Process to Intermediate 4 Thionyl chloride (2.05 equivalents) in methanol resulted in ring opening of commercially available lactam 7. Isolation of the desired product 8 was initially achieved by complete removal of solvent, but this method afforded a gum or a sticky solid and was not scalable. Subsequently, it was found that distillation of methanol to approximately half of the original volume followed by the addition of an antisolvent induced successful precipitation of aminoester 8. Toluene was initially used as the antisolvent, resulting in > 85% yields, but on a multi hundred gram scale, the use of toluene resulted in the isolation of unacceptably sticky solid. Thus, upon further investigation, we identified methyl t-butyl ether (MTBE) to be a more effective antisolvent. The product precipitated as a free-flowing white solid that was isolated easily by filtration on up to 500 g scale in quantitative yield. Trityl protection of 8 provided Tr-protected aminoester 9, which was isolated via solvent removal and afforded an oil that could be taken directly into the double bond migration step as crude material. On gram scale, once double bond migration 17

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was complete by HPLC, the solvent was removed. The crude material was purified via a silica gel plug filtration followed by solvent removal to provide the α,βunsaturated ester 10. Subsequent process development led to an aqueous work-up whereby DBU was simply removed by washing the crude reaction mixture with water and telescoping the DCM extract into the next step of the synthesis. For the ester reduction step (Scheme 4, 10 to allylic alcohol 11), the amount of DIBAL-H (1.0 M solution in toluene) was optimized to 2.2 equivalents for a cleaner reaction profile. Isolation of this intermediate was accomplished by solvent removal resulting in a viscous oil. We later switched the reaction solvent from DCM to toluene to avoid this clumsy oil isolation and to allow telescoping into the key transformation of this strategy: the Sharpless epoxidation. The oxidation of allylic alcohol 11 to epoxide 12 was examined under the following conditions: 1) the standard Sharpless epoxidation conditions using (+)-diethyl L-tartrate. 2) the standard Sharpless epoxidation conditions using (–)-diethyl D-tartrate (22–25). 3) with m-chloroperbenzoic acid in the presence of triethylamine with DCM used as solvent. 4) with methyl trioxorhenium and t-butyl hydroperoxide. 5) with vanadyl acetylacetonate. The NMR studies indicated that the Sharpless conditions using (+)-diethyl Ltartrate (condition 1) produced the desired epoxide 12 whereas the corresponding diastereomer 13 (Figure 2) was the major product obtained from conditions 2–4. The epoxidation reaction in the presence of vanadyl acetylacetonate (condition 5) resulted in a mixture of 12 and 13.

Figure 2. Isomers from Epoxidation.

While the stereoselective Sharpless chemistry (condition 1) was effective, it involved extensive work-up, and the product required column purification. With the desired diastereomer epoxide 12 in hand, we were in a position to test the key transformation of the route: the regioselective ring opening of 12 to form 1,3-diol 14. We hypothesized that the use of Red-Al would provide the desired regioselectivity (26–28), but unfortunately little reaction occurred under these conditions. Thus, screening was initiated to find an alternative reducing agent (Table 1). 18 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Table 1. Ring-Opening Conditions for Epoxide 12 to Diol 14

a

Conditionsa

Solvent

Results

Red-Al, 20 °C, 2.5 equivalents

THF

No reaction

Red-Al, 1.0 equivalent

DME

Loss of the protecting group

BF3•Et2O, NaBH3•CN, 1.0 equivalent

THF

Decomposition

B2H6, 1.0 equivalent

HMPA

Multiple nonpolar products

NaBH4, 1.0 equivalent

THF

No reaction

Super-Hydride, 1.0 equivalent

THF

Formation of a less-polar product

LAH, 1.0 equivalent

THF

Formation of 15

BH3•THF, NaBH4, 1.0 equivalent

THF

1:2 ratio of 14/15

LiBH4, 2.0 equivalents

THF

Formation of 15

Na(OAc)3BH, 2.0 equivalents, 16 h

THF

Unknown compound

BH3•THF, 2.0 equivalents, 16 h

THF

1:1.3 ratio of 14/15

BH3•THF, 2.0 equivalents, 4 h

THF

1:1.4 ratio of 14/15

BH3•THF, 2.0 equivalents, 4 h

DCM

1:1.5 ratio of 14/15

BH3•DMS, 2.0 equivalents, 16 h

THF

1:1.5 ratio of 14/15

Reactions were carried out at room temperature overnight if not specified.

The use of BH3•THF appeared to be the most useful for maximizing the conversion to diol 14. After further investigation, it was determined that the simultaneous presence of two reductants (BH3•THF and NaBH4), was efficacious. The reaction was performed in the presence of sodium borohydride (1.0 equivalent) and BH3•THF (2.0 equivalents) in DCM at 35 °C, which provided the best balance of conversion to the required 1,3-diol regioisomer and isolated yield on small scale. After aqueous work-up, separation of regioisomers was achieved on small scale via preparative column chromatography; however, no crystallization conditions could be developed to separate the two isomers. Therefore, a different approach was needed to separate the isomers. Another method explored to affect this separation was to utilize protecting groups and exploit the potential difference in reactivity between isomeric diols 14 and 15. Two options were investigated: •

•

attempted acetonide formation with 10-camphorsulfonic acid/2,2dimethoxypropane did not reach completion and resulted in loss of the trityl protecting group (Scheme 5). selective reaction of the 1,3-diol 14 in the product mixture with 1,3-dichloro-1,1,3,3-tetra-isopropyldisiloxane, followed by a chromatographic separation of derivative 16 from the unreacted diol 15. The deprotection of compound 16 regenerated the desired 1,3-diol, 14. Removal of Tr group afforded the desired aminodiol 4 (Scheme 5). 19

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Scheme 5. Purification and Separation of 14

On treating the diol mixture (1:2 ratio of 14/15) with 1,3-dichloro-1,1,3,3tetraisopropyldisiloxane in the presence of triethylamine (Et3N) and DCM, there was complete consumption of 14 to protected product while the isomeric 15 remained largely unreacted. The desired acyclic product 16 was purified by column chromatography but still retained 2–5% of the silylated undesired isomer 17. Reaction optimization focused on temperature, reaction time, and equivalents of silylating agent to further minimize the reaction of compound 15 and to reduce formation of cyclic compounds 18 and 19, which formed over extended reaction times. Reducing the charge of silylating agent in accordance with the mole ratio of desired diol in the mixture, halting the reaction once complete consumption of 14 was achieved, and performing the reaction at ambient temperature proved successful in reducing the amount of undesired compounds 17, 18, and 19. Ultimately, the product was purified by column chromatography. Simple deprotection conditions were developed using TBAF to afford the desired amine-protected diol, 14, in excellent yield (~90% based on moles available in starting mixture). Deprotection was also readily achieved in similar yields and purity using a solution of hydrofluoric acid in triethylamine but ultimately this method was not selected for scale-up due to concerns over the use of HF. Final trityl deprotection of 14 was effected by catalytic hydrogenation in methanol. Complete conversion was achieved, but column chromatography was required to obtain aminodiol 4 in desired quality. No further optimization was carried out for this step. 20

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The Sharpless epoxidation route was successful in providing 550 g of material in a multibatch strategy to support development and scale-up work for later steps. However, due to several intermediates being isolated as oils and frequent column chromatography being required, the route was deemed unsuitable for the kilogram scale production of material. Instead, the significantly shorter bromolactonization route (discussed in the following section) was considered and proved to be ultimately the answer.

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Bromolactonization Process to Aminodiol 4 The starting material, trans amino acid 20 for this route is a known compound, but not widely available commercially (Scheme 6) (29). We initiated our own development work to produce this desired starting material based on ring opening of Vince lactam and epimerization (30), while sourcing strategies for 20 occurred in parallel. While internal chemistry efforts were showing progress, a commercial supplier capable of producing 20 on multikilogram scale and delivering required amounts was identified to meet project timelines.

Scheme 6. Bromolactonization Process to Intermediate 4

Successful lactonization directed by the existing stereochemistry in Boc-amino acid 20 was considered the key transformation in this route (31–34). Similar to the Sharpless route, internal work began with the trityl protecting group. However, since the reaction outcomes between 20 and its trityl protected analog were identical, the Boc-protected variant was selected on the basis of better atom efficiency and lower cost. Only research on the Boc-protected synthesis is described below.

Scheme 7. Bromolactonization of Compound 20 21 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Bromolactonization to produce β-lactone 21 (Scheme 7) was first attempted on a small scale, utilizing bromine and tetrabutylammonium hydroxide in DCM under cryogenic conditions and varying the equivalents of bromine. This method proved successful on a laboratory scale with 2.0 equivalents of bromine showing optimal performance at 65–70% yield. The solid product was isolated by removal of solvent and this method was deemed suitable for the production of material necessary for toxicological studies. A further-developed, process-friendly isolation would be addressed at a later time. Screening efforts were undertaken to investigate alternatives to bromine for lactonization, e.g. iodine, NBS, silver triflate, and copper iodide. However, only bromine led to the desired product.

Scheme 8. Reduction of β-Lactone 21 We investigated the use of both NaBH4 and LiBH4 for the reduction of βlactone 21 (Scheme 8). LiBH4 in solution proved superior with respect to reaction rate and purity of product for the formation of Boc-bromo diol 22 as well as ease of use and the ability to add in a controlled manner. Reaction at 0–5 °C was necessary to control purity. The reaction was rapid and complete immediately after the addition of LiBH4 was finished. A simple NH4Cl aqueous quench followed by extractive work-up with MTBE and removal of solvent, yielded product in almost quantitative yield as a thick oil or glassy solid.

Scheme 9. Reductive Debromination of β-Lactone 21 Several attempts at this stage were also made for simultaneous reductive dehalogenation (Scheme 9) by modification of the reaction conditions, either through the addition of Lewis acids to the LiBH4, alternative reducing agents, or via elevating the temperature. If successful, this approach would shorten the 22 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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synthesis to aminodiol 4 by one step. Unfortunately, most conditions yielded Boc-bromo diol 22, or instigated degradation of 22, with no desired des-bromo product seen. Nevertheless, the LiBH4 conditions were deemed suitable for the scale required of the chemistry to form the bromo-diol 23 and we considered telescoping this step into the following step for the first GMP production. A set of standard conditions for removal of the Boc group utilizing HCl in dioxane and IPA was employed to obtain bromo-diol 23, an HCl salt, and removal of Boc was typically complete within 3–5 h (Scheme 9). Isolation of intermediate 23 was not attempted at that time, and the resultant mixture was concentrated and hydrogenated under typical conditions with Pd/C as the catalyst (35). The debromination of 23 was clean, and the desired aminodiol 4 was formed exclusively. We initially pursued a combination of methanol and IPA as solvent in conjunction with sodium bicarbonate to quench any residual HCl from the deprotection reaction. Methanol provided high solubility for both the starting material as well as the product. Simple filtration to remove catalyst and inorganic salts followed by removal of solvent yielded the desired product, the HBr salt of 4 (36).

Coupling Steps Preparation of Dichloropyrimidine Acetal

Scheme 10. Preparation of Dichloropyrimidine Acetal

The literature synthesis of 27 is provided in Scheme 10. From a process chemistry perspective, the areas of concern were: • • •

the use of sodium methoxide solid or solution in step 1. the use of neat POCl3 in step 2. the availability of ozonolysis capabilities for step 3. 23

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Synthetic procedures available from the literature (18–20) were utilized on small scale to produce both sufficient quantities of 25, ultimately from guanidine, and diester 24 for gram scale development work. For the production of compound 25, the use of either sodium methoxide solution or solids resulted in a similar reaction profile. Eventually, solid NaOMe was used in production. For step 2, the literature procedure called for reaction in neat POCl3 with diethylaniline. The transformation of 25 to dichloride 26 could be achieved using toluene/acetonitrile as a solvent system and DIPEA as the base. Yields after work-up using these cosolvents were generally ~15% lower than when neat POCl3 was utilized. The yield became much lower as the chemistry was scaled to > 100 g. Reaction in neat POCl3 was reassessed and successful conditions were developed using five volumes of neat POCl3. The work-up for this step was modified by introducing toluene at the end of reaction in order to facilitate azeotropic removal of residual POCl3. This azeotropic removal of POCl3 was followed by an extractive aqueous work-up. The product was isolated as an oil by concentration. Ozonolysis utilizing the literature conditions proved effective for the conversion of alkene 26 to aldehyde 5 (18). The work-up was modified from the ether extractions described in the literature to isolation by trituration with EtOAc/heptanes in order to obtain better product quality. Once product was formed, ethyl acetate was added to achieve phase separation, a series of aqueous washes was performed, and then the organic phase was concentrated to a low volume. Crystalline product was obtained by the addition of heptanes as antisolvent. The conversion of 26 to aldehyde 5 was also investigated using catalytic osmium tetroxide with NaIO4 or NMO. Although use of osmium tetroxide was successful in obtaining product in similar yield and quality to the ozonolysis process, the toxicity of osmium and potential for residual osmium in intermediates was a concern for large scale reactions. Ultimately, we identified vendors with the required large scale capabilities for ozonolysis to produce aldehyde 5. The osmium method henceforth was no longer considered. No development work was performed on the acetal formation step to produce 27. The chemistry described in Scheme 10 using solid sodium methoxide in step 1, five volumes of neat POCl3 in step 2, ozonolysis for step 3, and literature conditions for step 4 was successfully implemented at two different Contract Manufacturing Organizations (CMOs) to produce 1 kg of 5, 200 g of 27 for non-GMP work, and 6 kg of 27 for the first GMP campaign. Later, commercial suppliers were identified for 5 during the first GMP production campaign and no further development work to prepare 5 was performed by Takeda.

Preparation of Diol Intermediate 2 The conditions for the coupling of aminodiol 4 and acetal 27 are based on literature (18–20). The reference highlights the importance of using an acetal derivative to avoid unwanted imine formation. For this reason, acetal 27 was used in this reaction. A screen of alternative solvents and bases was performed. 24 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Triethylamine in IPA showed the cleanest reaction profile. After extractive workup with EtOAc, chloropyrimidine 3 was obtained by removal of solvent under reduced pressure. Crystallization conditions were not fully developed prior to production of material for toxicological studies, due to time constraints.

Scheme 11. Chemistry To Prepare Diol Intermediate 2

Coupling of the aminoindane 6 to chloropyrimidine 3 had been previously performed on other scaffolds by medicinal chemists utilizing microwave chemistry on small scale. However, during early process research on the coupling step, it proved challenging to drive the transformation fully to diol 2. The reaction was originally carried out in high boiling solvents such as DMAc and NMP in the presence of inorganic bases. The desired organic bases were prone to vaporization due to operating temperatures above the boiling point of the base. However, in order to drive this transformation to completion in a reasonable timeframe, a temperature of greater than 125 °C was necessary. After multiple failed attempts to perform the reaction at atmospheric pressure, a screen in a pressurized system was conducted. This revealed that 2-butanol with DIPEA provided a clean reaction profile with a completion time of ~70 h at 135 °C under 80 psig. While this appears extreme, all solvent and base combinations investigated required longer reaction time and higher temperatures in order to drive the reaction to completion. 25

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

With reaction completion achieved, extractive work-up was employed to remove excess aminoindane and the DIPEA•HCl salts. The organic solvent (EtOAc) was subsequently concentrated to a low volume and crystallization was initiated by the addition of DCM to yield crystalline diol 2 in high yield (> 70%) and purity (Scheme 11).

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Final Steps

Scheme 12. Final Steps for the Synthesis of Pevonedistat The final steps include the sulfamoylation of diol 2 and salt formation of free base 1 to produce pevonedistat (Scheme 12). Sulfamoylation chemistry presented another significant challenge for the synthesis of pevonedistat. Both the primary and secondary hydroxyl groups of diol 2 had similar reactivities toward the two sulfamoylation reagents 29 and sulfamoyl chloride 30 (37–39) (Scheme 13) used in the original synthesis. A mixture of the desired monosulfamoylated product 1 with its regioisomer 31 and the bis-sulfamoylated byproduct 32 were produced, requiring purification of free base 1 by chromatography. Both approaches with the two different sulfamoylation reagents posed challenges to overcome with regards to efficiency (37, 40, 41) and safety (gas evolution and exothermicity) (40) in order to achieve successful scale-up.

Scheme 13. Sulfamoylation Chemistry with Reagents 29 and 30 26 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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We adopted two main approaches to differentiate the two hydroxyl groups in the conversion of 2 to 1. The first is to use a protecting group strategy and the second is to modulate the reactivity of sulfamoyl chloride. The protecting group approach explored TBSCl and TBSOTf in conjunction with a variety of bases (Scheme 14). Poor mono- versus di-protection and modest regioselection necessitating chromatographic purification rendered this approach unattractive. Instead, we chose to pursue the alternative approach of modulating the reactivity of the sulfamoyl chloride.

Scheme 14. Sulfamoylation via Protecting Group

Sulfamoylation Using Sulfamoyl Chloride or Derivatives Thereof Early sulfamoylation work was based on the literature conditions for accomplishing this transformation (i.e. NH2SO2Cl/DMAc, with or without base) (40, 41). Use of sulfamoyl chloride alone with no base gave minimal to no selectivity between the desired free base 1, the regioisomer 31, and the undesired bis-sulfamated product 32. We subsequently screened the use of sulfamoyl chloride 30 in DMAc and THF (Scheme 13) as possible reaction solvents with DIPEA, Et3N, DBU, DABCO, or t-BuOLi as base. The reactions were carried out at both 0 °C and room temperature with sulfamoyl chloride and base combined together in either DCM or MeCN before addition to diol 2 in the main reaction. Many of these conditions yielded the desired free base 1, however in low purity (Table 2). Clearly a different approach was needed. To better control the formation of the desired free base 1 in the large scale sulfamoylation reaction, a protected variant of sulfamoyl chloride was investigated. This strategy was a focus for process development after the first GMP API campaign was complete. For this early investigation, Boc was selected as the protecting group of choice due to its simplicity (42) and the bases Et3N, 2,6-lutidine, DBU, DABCO, and DIPEA were examined (Scheme 15). We hypothesized that the bulkiness of a protecting group (e.g. Boc) would sterically block access of Boc-sulfamoyl chloride 33 to the secondary alcohol, improving the relative reactivity of the primary position. Although there was some evidence that the rate of addition of Boc-sulfamoyl chloride 33 to diol 2 would help to reduce the overall amount of bis-sulfamoyl impurity formation, poor stability of 33 in solution made this option less feasible. Along this line of thought, multiple 27

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

charges of the sulfamoylating agent were often required to consume all the starting material, thus necessitating further exploration.

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Table 2. Sulfamoylation of 2 with Different Bases at 0 °C Solvent

Base

1:31

(1+31):32

DMA

none

6.5:1

5.9:1

DMA

DIPEA

HPLC not resolved

1:2.2

THF

tBuOLi

6.0:1

5.4:1

THF

DBU

8.7:1

3.9:1

THF

DABCO

7.1:1

9.7:1

Scheme 15. Sulfamoylation Chemistry with Boc-Sulfamoyl Chloride 33

Further studies of the bases utilized in this reaction revealed that DABCO worked best to modulate the reactivity of 33. This approach involved precomplexation of 2 equivalents of Boc-sulfamoyl chloride with 2 equivalents of DABCO, followed by the addition of 1 equivalent of diol 2. Under these conditions, the reaction with the diol was relatively slow, and thus allowed facile control of the reaction endpoint. Also, the need for adding multiple charges of sulfamoylating reagent via slow addition was eliminated. Instead, a relatively fast addition was possible, resulting in reduced overall reaction time. Once sulfamoylation was complete, the Boc group could be removed in situ by quenching the reaction mixture with an equal volume of 9 M HCl. Upon complete deprotection and extractive work-up, the solution was neutralized. Efforts were made to crystallize the desired product and remove the impurities. All crystallization attempts proved unsuccessful due to the similar physical properties of the compounds. Silica gel column purification was still required, but due to the minimal separation seen in a multitude of eluents investigated, the isolated product unfortunately still contained 1–2% of 32 and/or 31. 28

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

This procedure proved consistent up to 300 gram scale. While the ultimate goal in preparation for GMP production by using the same process to produce pure free base 1 was achieved, the challenges still remained to develop a manufacturing process with the ability to control the amounts of undesired regioisomer 31 or bis-sulfamate 32 in API.

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Salt Formation To Provide the Final API The final step to produce pevonedistat was a salt formation (Scheme 12). From salt screening and polymorph studies, the HCl salt, an anhydrous form (Form 1), was selected. Form 1 was not readily crystallized directly, but rather was produced via drying of several intermediate solvated forms. Of the isostructural solvates identified, the ethanolate (Form 3) most readily converted to Form 1 upon drying. A crystallization method was developed by dissolving the free base of pevonedistat (1) in ethanol followed by the addition of one equivalent of ethereal HCl to provide pevonedistat in high yield. Unfortunately, the undesired regioisomer 31 or bissulfamate 32 impurities were not readily rejected in the salt formation, which highlighted the need to consider these impurities in further work on pevonedistat. During salt formation, one of the main impurities in the drug substance, a chloro derivative (Figure 3), was formed during the reaction. While this impurity was rejected by crystallization, it was unfortunately regenerated upon drying of the final API. Salt formation and drying conditions needed to be carefully studied later in order to minimize the formation of this impurity.

Figure 3. Chloro Impurity. The chemistry described thus far starting from the bromolactonization transformation to the protecting group mediated sulfamoylation outlined in Schemes 6–13 was successfully employed to produce ~200 g of material to support IND enabling toxicological studies and to provide material for the development of a phase 1 formulation. However, further scale-up required modified conditions to overcome residual impurities and other scalability concerns. Process Development for the First cGMP API Production The first cGMP production targeted a delivery of ~1.5 kg and was initiated almost immediately after the internal GLP campaign leaving minimal time for additional process development (Scheme 16). However, development work focusing on peripheral aspects (work-up, isolation, etc.) was performed to enable 29

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further scale-up. There was also a continued effort to improve the selectivity and efficiency of the sulfamoylation chemistry on large scale. In order to meet the delivery timeline for the first cGMP API production, the team agreed to limit the scope of its development activities.

Scheme 16. GLP/First GMP Productions of Pevonedistat In the first GMP production, the bromolactonization (production of β-lactone 21) reaction proved robust; it was decided that only isolation of the product needed to be addressed for this stage of development. After reaction to form the bromolactone, excess bromine was quenched and aqueous washes were carried out to remove tetrabutylammonium bromide. The final ethyl acetate extracts were reduced to a low volume to initiate precipitation. After screening a number of potential antisolvents, 10% MTBE in heptanes afforded the product in desirable yield and purity in the form of a free-flowing solid. For the reductive opening of the β-lactone, we needed to address improving the work-up and isolation of the Boc-bromodiol 22 as a high priority to start the GMP campaign. Despite screening a multitude of solvents for crystallization, we could not identify any solvent system that precipitated the product as a filterable 30

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free-flowing solid. In a few cases, we obtained solid products, however these solids had poor isolatable properties (e.g. a gum or sticky solid). Because of these difficulties, for this first GMP campaign, isolation of product was achieved by concentration of the final organic extracts to dryness. For the deprotection and subsequent dehalogenation steps there were concerns about telescoping the reaction. It was believed that isolation of the HCl salt of bromodiol 23 might be advantageous to aid in improving the purity of this intermediate. Precipitation of product occurred in the reaction media, but in suspension and upon isolation, the solid had a sticky morphology. Switching the main reaction solvent to MTBE and adding HCl/dioxane was successful in generating a free-flowing slurry. Unfortunately, upon isolation, the solid exhibited hygroscopic properties and melted together. This issue of hygroscopic solid was addressed through isolation of the solid under a positive nitrogen pressure and readily enabled air-free manipulation for short periods of time, sufficient to isolate the salt. The HCl salt of compound 23 was then subjected to hydrogenation to effect the debromination. A 1:1 ratio of MeOH and IPA was selected to afford a balance between the solubility of starting material and product, and carry-through of inorganic salts. Removal of inorganic impurities and the catalyst was initially performed by passing the reaction mixture through a pad of filter aid. However, during scale-up studies performed in preparation for GMP manufacture, several washes of the filter aid bed were required to fully elute the product which appeared to be stuck to the filter pad. Filtration through glass fiber paper readily solved this issue and afforded complete recovery of product. After filtration, the organic filtrate was concentrated to near dryness before adding a mixture of 1:1 IPA:ethyl acetate to resuspend the solid before the final isolation by filtration. The product, a mixture of 4•HBr and 4•HCl, from these development experiments contained higher amounts of residual inorganic impurities than desired. Fortunately, additional studies performed indicated that these impurities would not affect the subsequent coupling reaction. However, further investigation into the optimal base and isolation of product from the hydrogenation step was deemed to be necessary prior to subsequent API production campaigns. A single salt of aminodiol 4 was also preferred for compound characterization and pursued later. Coupling of aminodiol 4•HBr/4•HCl and acetal 27 to produce compound 3 was carried out under the same reaction conditions as described for the 200 g GLP run. However, one disadvantage was that the product was isolated by evaporating the solvent to dryness, a long procedure, which would require modification in the future. For the coupling of the aminoindane to chlorodiol 3, the only modification made from the GLP run (Scheme 16) was to concentrate the ethyl acetate extract, after the aqueous work-up, to a thick slurry/paste. Adding DCM to the mixture further reduced the solubility of 2. Compound 2 was readily isolable as a freeflowing solid with this improved work-up and isolation method. Sulfamoylation of diol 2, particularly with respect to selectivity of the sulfamoylation reagent and the ability to eliminate 31 and bis-sulfamate 32 impurities (Scheme 15) from the desired product, remained as a top concern 31

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

and significant challenge for the first GMP campaign. Poor selectivity resulted in mixtures of product and undesired byproducts, and required column chromatography for the isolation of free base 1. Yields for this step were inevitably low. Moreover, the inability to eliminate these impurities from the desired product in the final salt formation limited our options to purge the impurities. Thus, we focused on investigating alternative reagents to improve selectivity and minimize impurities. Factors examined were:

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• • •

Bases: DABCO, imidazole, sparteine, 2,6-lutidine, DMAP, or DBU. Solvents: DMAc, THF, 2-Me THF or EtOAc. Ratio of base to Boc-sulfamoyl chloride 33.

THF proved to be the best solvent and DABCO remained the best base to use, with sparteine giving similar results. Excess base (beyond 3 equivalents) extended the reaction time and only provided a negligible improvement to the ratio of product and undesired byproducts. We decided to utilize these conditions for the internal 200 g preparation for the GMP production. Continued efforts to crystallize or recrystallize free base 1 after the initial column chromatography proved unsuccessful for removing the isomer 31 or bis-sulfamate 32 (Scheme 15). We did change the solvent elution mixture for column chromatography from MeOH/DCM to MeOH/EtOAc, and this eluent change reduced the overall amount of solvent required. We also noted that the MeOH/EtOAc eluent made it easier to separate bis-sulfamate impurity 32 from the desired product than the regioisomeric sulfamate impurity 31. To take advantage of this physiochemical property of the bis-sulfamate, the reaction time in the GMP production was lengthened to convert the regioisomeric impurity 31 to bis-sulfamate 32 prior to work-up, in order to increase the efficiency of the column chromatography. While this change did aid in purification, it reduced the yield by about 10–15% for the step compared to the prior internal results. This was considered a fair trade-off for the present maturation of the project. An atypical result was observed when isolated parent free base 1 was taken into the salt formation step. In the demonstration batch prior to the GMP production, the final HCl salt crystallized much slower and the yield was significantly lower than that obtained from previous development lots. After carefully examining the isolated free base 1, we discovered that there was some contamination with polybutylene glycol. This contamination was attributed to polymerization of THF during the Boc deprotection with HCl. Even after work-up of post-sulfamoylation and column chromatography, high amounts (visible by NMR) of polybutylene glycol were still present. This polymer contamination was the root cause for inhibiting crystallization and led to the low yields. A solution to the problem was urgently needed to prevent delays to manufacture and release of API. A study on recrystallization of free base 1 identified DCM as a suitable solvent to remove the polymeric impurity at this stage, and allowed production to continue on schedule. The following salting step provided API in acceptable quality with no extra development work. 32

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Conclusions for First GMP Preparation

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The chemistry and route utilized for the internal 200 g production with the modifications as described vide supra were successfully employed to produce 1.5 kg of cGMP material to support phase I clinical trials (Scheme 16). However, almost every step required concentration of the worked-up solutions for isolation of product, and the penultimate step required chromatographic purification. It was widely recognized within our group that further work was needed to improve the chemistry from an operational efficiency standpoint for future GMP productions on larger scales.

Section 2: Process Development for the Current GMP Preparation of Pevonedistat Overall Development Strategy It was rewarding to the process chemistry team that many aspects of the chemistry developed for the GLP API production were readily transferable to the first GMP campaign. This enabled us to produce enough API to initiate clinical trials. However, it was also clear that many parts of the process needed further development and optimization for robustness and efficiency if the process was to be successful on multikilogram scale. The yields of a number of steps needed to be improved. Concentration of reaction mixture to dryness needed to be replaced by larger scale/manufacturing process friendly isolation methods (e.g. crystallization). Column chromatography needed to be avoided (Scheme 16 GLP/first GMP). Post first GMP production, we focused our development efforts on the challenging areas discussed vide supra, as well as directed at improving process efficiency and lowering the production costs. Reaction conditions, choice of reagents, and isolation procedures were carefully examined for each step. This section details our progression from a process used to produce kilogram quantities of API to a potential commercial-scale process to support multikilogram preparation of pevonedistat. Process Development for the Current GMP Synthesis of β-Lactone 21 Main Issues with the First GMP Process The original laboratory scale procedure for the preparation of β-lactone 21 was unsuitable for a large scale production due to the cryogenic requirements (i.e. –25 °C) for the bromolactonization and the subsequent necessary multibatching due to size restrictions of the cryogenic plant equipment. In the first GMP production, the bromolactonization conditions were modified for larger scale manufacture and utilized NaHCO3 as base with bromine in an aqueous system at 0–5 °C (Scheme 17). These conditions caused significant foaming in the reaction mixture on the manufacturing scale during the addition of bromine and the aqueous sodium ascorbate quench. Another drawback of these conditions was 33

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impurity formation. For this reason, the development efforts for this step focused on identifying alternative conditions that could eliminate these issues and provide the product in high yield and purity.

Scheme 17. Modified GMP Conditions To Prepare 21

Initial Attempts to Address Undesired Foaming Issues We investigated several approaches to suppress or eliminate the foaming without significantly changing the reaction conditions (e.g. replacing NaHCO3). Simple techniques were attempted such as the application of vacuum, nitrogen, and the introduction of antifoaming agents, all of which were ineffective. Additional studies were then carried out on the remaining parameters to see what effect each played on the foaming and if refining these parameters would offer a solution to the troublesome issue. Base amount, bromine addition rate, reaction temperature and time, quench solution amount, and rate of addition were each assessed. Several factors were found to offer improvement toward reducing foaming: • • •

an increase in base from 2 to 4 equivalents. a slow controlled addition of bromine at 5 °C over ≥ 2.5 h. a controlled addition of the sodium ascorbate quench over a minimum of 1.5 h.

A clean reaction profile was found to be heavily dependent on the reaction temperature and time. A reaction temperature of 0 ± 5 °C was determined to be critical and an increase in temperature above this range significantly impacted the purity profile. A prolonged reaction time, e.g. > 6 h, also led to notable impurity formation. Fortunately, it was discovered that water and heptanes reslurries were effective at reducing the levels of residual sodium bromide and impurities, respectively, in the product. Although these efforts helped to minimize the foaming and related processing issues, they did not completely eliminate the problems. For this reason, further development was deemed necessary. 34 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Further Process Development for the Conversion of Acid 20 to β-Lactone 21

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Screening Organic Bases Attempts at redesigning the process sought to replace NaHCO3 with an organic base to eliminate the foaming. In purely aqueous conditions, pyridine and DIPEA proved to be acceptable, and produced the bromolactone in high purity but in low yield. In comparison, the use of Et3N led to an incomplete reaction. The introduction of an organic base helped to avoid the foaming issue, but unfortunately, the organic base also promoted the formation of solid agglomerates in the reaction mixture, which were identified as residual bromine and product. This agglomeration suppressed the reactivity of the bromine, which led to low yields and incomplete reaction. However, this effect was minimal with the use of pyridine which was chosen as base for further development efforts.

Organic Cosolvent The switch to an organic base prompted us to investigate the addition of an organic cosolvent as a way of improving the homogeneity of the reaction mixture and suppressing the agglomeration, which was still a problem with pyridine. 1,2Dimethoxyethane (DME) was studied at varying ratios with results showing only a small amount was necessary to enhance solubility, suppress agglomeration, and improve the isolated yield. In early work, 30% DME in water by volume gave the best results. This ratio of DME in water provided an appropriate solubility of starting acid 20, while precipitating the product, β-lactone 21. Under these conditions, the precipitation of 21 from the reaction mixture limited its interaction with other reagents in situ and increased the product purity. This change also offered the potential of isolation via filtration without a quench. Lower amounts of DME (10–20% v/v, DME/water) promoted the precipitation of pyridinium hydrobromide, while higher concentrations (> 30% DME) enhanced the solubility of product in the reaction mixture. Both of these events contributed to a decrease in product purity, so a more thorough investigation to define the optimal amount of DME was carried out. Ultimately, the yield and purity achieved from these solvent experiments did not offer an improvement over the 30% DME/water system. There was also a difference observed in the ease and manageability of the reaction as the amount of DME was increased. Greater DME content hindered bromine solubility causing it to oil out and settle at the bottom of the vessel. This immiscibility not only made it difficult for the reaction to reach completion, but also resulted in a higher level of residual bromine and an orange hue to the final solid. As was mentioned, limiting the solvent ratio to 30% DME/water allowed for β-lactone 21 to precipitate from the reaction mixture. Further study proved this solvent ratio did in fact allow for the successful isolation of the bromolactone solid without a sodium ascorbate quench. This process proceeded well and eliminated the second foaming event observed with the quench. Additionally, the earlier implementation of the water and heptanes reslurry/wash protocol was sufficient 35

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

for upgrading the product purity. This permitted scale-up of the lactonization step to multikilogram scale.

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Bromine Charge The addition of DME to the reaction system suppressed the observed agglomeration of bromine and product, which occurred as a result of the use of pyridine. This improvement allowed for a decrease in the bromine charge from the standard 2.0 to 1.25 equivalents. A range of 1.1–1.3 equivalents was investigated to determine the potential point of failure around this parameter, yet comparable yields and purity were achieved across this span. An increase in the amount of residual bromine in β-lactone 21 was noted when this range was exceeded, so it was deemed beneficial to minimize the bromine content to avoid potential problems associated with carry-through in the downstream chemistry.

Outcome of Process Optimization Process redevelopment led to new conditions (Scheme 18) which utilized a 30% DME in water (v/v) solvent system, pyridine (2.5 equivalents) as base, and bromine (1.25 equivalents) at 0–5 °C, with isolation at –10 °C followed by water and heptanes washes. The new reaction rate was slightly slower than with the previous process, but the trade-off was a more process-friendly reaction and workup. This process has been successfully proven eight times to date, on scales up to 75 kg. Yields resulting from this process are typically in the 60–70% range with product purity ≥ 95%.

Scheme 18. Step 1 Current GMP Process To Prepare 21

Process Development for β-Lactone 21 to Boc-bromodiol 22 Main Issues with the First GMP Process By the time that this step reached GMP manufacture, the reduction of βlactone 21 to bromodiol 22 using lithium borohydride was a fairly well-established process (Scheme 19). Efforts for improvement of this step focused on increasing 36 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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product yield and purity, as well as improving the cumbersome isolation of a sticky, glass-like solid which was undesirable from a process handling perspective. First to be examined was the choice of reducing agent.

Scheme 19. Modified GMP Conditions for 21 to 22

Screen of Reducing Agents A screen was carried out comparing our initial reducing agent, LiBH4/THF, with various other reducing agents including NaBH4, NaBH(OAc)3, BH3·THF, L-Selectride™, and Super Hydride™, in several different solvents: MeOH, IPA, MeCN, THF, and toluene. Of the alternative systems investigated, only NaBH4 in IPA at 25 °C showed formation of the desired Boc-bromodiol 22 while all others resulted in either no reaction or a complex mixture. However, upon scaling up the reduction with NaBH4 in IPA, it resulted in about 20–30% decrease in yield and up to a 20% decrease in purity. Based on this result, we returned our focus to improving the LiBH4 reduction conditions.

Water as Cosolvent Subsequently, we investigated whether the presence of a small amount of water would enhance the reaction rate and improve the purity profile (43). Our initially developed LiBH4 process in THF at 0 °C was attempted in the presence of 0 to 30% v/v water. Experimental results indicated that the presence of 5–15% water improved this reaction. The modified conditions provided a cleaner purity profile and a faster reaction rate than the standard anhydrous conditions. Increasing the water content above 15% resulted in the formation of a significant amount of an unknown impurity. Conditions utilizing 1.0 equivalent of LiBH4 in 10% water in THF proved optimal and were successfully scaled to produce multigram quantities of 22, giving excellent yields (> 99%) and high purity (> 96%). This change to the solvent system mandated a reassessment of the reaction parameters in order to understand their edges of failure. Using the 10% water in THF solvent system, LiBH4 was varied from 0.50 to 1.5 equivalents (compared to reactant) of 2.0 M LiBH4/THF to find the optimal operating range. This work 37

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demonstrated that quantities at < 0.9 equivalent LiBH4 resulted in incomplete reaction, while 0.9 to 1.50 equivalents gave normal conversion. Longer reaction times were required at the lower end of the range of equivalents, while the upper end of the range, (1.25 –1.50 equivalents), displayed a decrease in purity. The yield in the experiment utilizing 1.50 equivalents was compromised, due to the formation of the same unknown impurity observed in the initial screen when using 20–30% water/THF. This impurity was isolated and identified to be the triol structure (Figure 4) using 1H NMR, 13C NMR, COSY, and HMQC analyses.

Figure 4. Impurity in Boc-Bromodiol 22. An investigation linked the triol formation to elevated temperatures (> 10 °C) experienced during an exotherm caused by the presence of excess LiBH4 and its reaction with water. Based on this information, the optimal amount of LiBH4 was further evaluated employing 5% water in THF as a solvent system in order to reduce the exotherm that occurred when 10% water was used (Table 3). These studies revealed that 1.05 equivalents of LiBH4 in 5% water/THF led to complete conversion, while suppressing the exotherm and minimizing triol formation, even allowing that a small portion of the reagent is quenched during addition on larger scale.

Table 3. Optimization of LiBH4 Content Using 5% Water in THF as Solvent

a

LiBH4 (equiv)

Conversion (%, a/a)

Triol (%, a/a)

Comments

0.50

95

0

Reaction stalled at 78% conversion after 2 h, reached 95% conversion after stirring at ambient temperature.

0.75

100

0

Completion in 2 h at 0–5 °C.

1.00

100

0

Completion in 2 h at 0–5 °C.

1.25

100

1.9

Completion in 2 h at 0–5 °C, 23% triola observed after 16 hours at ambient temp.

HPLC area %

This process was scaled multiple times to 100 g quantities, which provided yield and purity comparable to the 10% water/THF process. Good control of the exotherm was achieved through the appropriate amount of LiBH4, water, and a slow, controlled addition rate, resulting in low levels of the triol impurity. The Bocbromodiol product 22 resulting from these conditions was then carried through 38 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

the subsequent two steps to ensure this material performed appropriately in the downstream processes. Quality was deemed sufficient by NMR and yields were essentially quantitative.

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Work-up Modifications With the reduction process under control, work focused on improving the work-up. The initial work-up protocol utilized EtOAc extraction. Following a concentration to dryness, 22 was isolated as a sticky foam that settled into a glassy solid. This solid was difficult to handle at any scale, so efforts were made to improve the work-up through either modification of the product’s physical form, or elimination of the isolation along with an implementation of a telescoped process. Once attempts to attain crystallization conditions to avoid the sticky solids failed, several alternative extraction solvents were investigated. We sought to replace EtOAc in attempts to identify conditions that might improve the purity profile and allow a simplified isolation. MTBE, isopropyl acetate, and n-propyl acetate were screened with MTBE providing the highest yield based on NMR assay and a purity profile most similar to EtOAc. The decision was made to replace EtOAc with MTBE based on these results. Since the subsequent Boc deprotection is carried out in IPA, a solvent swap was implemented to substitute MTBE with IPA. We took into consideration the solubility of Boc-bromodiol 22 and investigated the distillation process to determine the amount of solvent required to keep 22 in solution while achieving the most effective removal of MTBE via the solvent swap. It was determined that 22 remained in solution in as little as 2 volumes of MTBE. Analysis proved concentrating to 2 volumes MTBE, implementing two IPA additions and subsequent concentrations to 2 volumes, sufficiently removed the residual THF and MTBE to a suitable extent that they would not have any impact on the downstream process.

Outcome of Process Scale-Up Improvements Process redevelopment efforts led to the following improvements: • • • • •

utilized 5% water in THF as the solvent system. charged 2.0 M LiBH4 in THF as the reducing agent (1.05 equivalents) slowly at –7.5 ± 2.5 °C. stirred at 0 ± 5 °C for 2 h. performed the work-up with MTBE. solvent exchanged to IPA in preparation for the next step.

This process (Scheme 20) was successfully carried out nine times to date, on scales up to 56 kg. Yields resulting from this process were typically ≥ 90% with purity ≥ 85%. 39 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 20. Current GMP Process To Prepare 22

Process Development for the Current GMP Process of des-Boc Bromo-Aminodiol 23 Main Issues with the First GMP Process The main concern for this transformation on large scale was the use of HCl as the acid source to affect carbamate deprotection, which could potentially produce mixed salt forms of bromo-aminodiol 23 (HCl and HBr salts) after the subsequent debromination (Scheme 21). While the formation of a mixed salt would create a problem in characterization and reproducibility, we believed that the formation of a homogeneous salt could be achieved by replacing HCl with HBr. A single HBr salt of aminodiol 4 would be formed.

Scheme 21. Initial GMP Conditions for 22 to 23•HCl

Replacement of HCl with HBr to Influence the Boc Deprotection of 22 Various hydrobromic acid reagents were studied for the carbamate deprotection including 48% aqueous HBr, 33% HBr in AcOH, and 20–30% HBr in EtOH. The reagents were furthermore investigated in IPA, EtOH, and MTBE from ambient to 50 °C. The 48% aqueous HBr and 20–30% ethanolic HBr in IPA and EtOH proved successful at elevated temperatures, while all others resulted in 40 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

incomplete reaction and/or a poor purity profile. Further work revealed that the use of aqueous HBr resulted in slightly lower yields of aminodiol salt 4•HBr than the ethanolic HBr reagent. This was due to the presence of water, which resulted in higher solubility of 4•HBr. Although ethanolic HBr provided a higher yield, 48% aqueous HBr was still chosen for this reaction due to its wider commercial availability in bulk quantities.

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Outcome of Process Optimization We determined that adding 48% aqueous HBr (1.3 equivalents) slowly to Boc-bromodiol 22 in IPA and heating the reaction mixture to 50 ± 5 °C were the optimal conditions for the deprotection. Once reaction completion was achieved, the reaction mixture containing 23•HBr was transferred directly into the debromination step as a solution in IPA. This process (Scheme 22) has been successfully carried out eight times to date, on large scales up to 127 kg. Because of the telescoped nature of this process, the yield is assumed to be quantitative and purity is in the range of 85–93%.

Scheme 22. Step 3 Current GMP Process To Prepare 23•HBr

Process Development for Bromo-Aminodiol 23•HBr to Aminodiol 4•HBr Main Issues with the First GMP Process During our initial GLP development work, we discovered that free base 4 darkened in color over six months, which raised concern for long term storage. Isolation of a salt was much more desirable from a stability standpoint (Scheme 23). Further process challenges with this debromination were: • •

NaHCO3 utilized as base caused CO2 evolution. crystallization was carried out by evaporating to dryness then adding a mixture of IPA and EtOAc (1:1). Significant amounts of inorganic salts precipitated and were isolated together with 4. 41

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Scheme 23. Initial GMP Conditions for 23•HBr/HCl to 4•HBr/HCl

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Use of Organic Base Initial Work to Replace Sodium Bicarbonate with N,N-Diisopropylethylamine The original method for the debromination utilized NaHCO3 as base and was performed as an isolated step (i.e. no telescoping). Due to the problematic issue of inorganic residue carry-through to downstream chemistry using NaHCO3, we decided that the use of an organic amine base was preferable. DIPEA was selected as the best from those screened. The optimal DIPEA stoichiometry (from 1.0 to 3.5 equivalents) for the debromination was investigated. Early work using isolated 23•HCl indicated that 1.0 equivalent of DIPEA was insufficient and led to poor conversion due to reaction with the acid equivalent. A slight excess of DIPEA (1.2 equivalents), however, drove the reaction to completion in good yield in only 8 h. Additional amounts of DIPEA (≥ 1.6 equivalents) resulted in no further benefits to the reaction rate and isolated yield of 4•HBr/HCl, and thus 1.2 equivalents of DIPEA was adopted as the new stoichiometry.

Telescoped One-Pot Process from 22 Through to 4 The telescoped process from 22 through to 4 was also being considered at this time. Thus, 23•HBr, obtained from the newly developed HBr deprotection conditions containing residual solvent and acid, was also investigated to determine how it would behave under the organic base debromination conditions. This work indicated that a minimum of 2.0 equivalents of DIPEA were required to drive the reaction to completion. If the reaction stalled and hydrogen uptake ceased, charging additional DIPEA was effective at driving the reaction to completion with no detriment to the yield or purity of 4•HBr. This data supported the feasibility of a telescoped process, but highlighted the need for further investigation. In order to determine whether the two-step deprotection/debromination of 22 to 4•HBr could be carried out in a one-pot process, several experiments were carried out. Keeping all other factors constant, the amounts of HBr (1.2–2.0 equivalents) for the deprotection and DIPEA (1.4–3.0 equivalents) in the dehalogenation were varied. This work indicated that the use of a 1:1 ratio of HBr and DIPEA led to poor conversion and a slight excess of DIPEA was necessary to drive the reaction to completion. Reducing the amount of 48% aqueous HBr in 42

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the previous deprotection step significantly improved the isolated yield of 4•HBr. This is a result of minimizing the residual water content, which in turn, also minimized the solubility of 4•HBr in the reaction mixture. Based on these results, the optimal conditions for the two-step, one-pot process were 1.2 equivalents of HBr for the deprotection and 1.8 equivalents of DIPEA for the debromination, which led to an overall yield of ~60%. Unfortunately, on larger scale, this one-pot process of converting 22 to 4 resulted in a lower yield. Investigations demonstrated that this was a result of 4•HBr loss to the filtrate. Lost 4•HBr was not recoverable in acceptable purity due to the excess of DIPEA salts present. Previous work indicated that 1.8 equivalents of DIPEA were sufficient for the process, but later development work revealed that actually 3.0 equivalents were necessary for the optimal yield. Catalyst Issues Catalyst Removal from the Debromination Process A factor leading to the large amount of residual inorganic impurities and loss of product was the process for catalyst removal. At this time, palladium removal was achieved by filtration of the reaction mixture through Celite®, but this filtration required excessive rinsing of the filter cake with MeOH to fully remove the product from the filter medium. Catalyst removal by filtration through glass fiber filter paper with a 1:1 methanol/IPA rinse resulted in no loss of product when the mixed solvent wash was used. The combined mass balance of inorganic solids and isolated product was consistent with complete recovery. This filtration protocol provided a simple and high recovery method for catalyst removal.

Effect of Catalyst Loading, Pressure, and Base on the Debromination of 23 In efforts to improve both the yield and purity, other reaction parameters were investigated. An assessment of catalyst loading using 10% Pd/C indicated 2 wt% was optimal, with lower catalyst loadings resulting in minimal conversion to the desired aminodiol and 18% of a new impurity. ES-MS analysis supported an epoxide structure for this impurity (Figure 5). Further investigation of the process and impurity formation on larger scale highlighted inconsistencies in the application of hydrogen pressure. It was determined that the resulting low hydrogen pressure in combination with low catalyst loading and a side reaction between 23•HBr and DIPEA over extended periods of time contributed to the formation of this impurity and had a significant impact on the overall yield and purity.

Figure 5. Epoxide Impurity Observed During Debromination Optimization. 43 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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In order to determine what catalyst loading and pressure were sufficient to avoid impurity formation, several experiments were carried out varying catalyst loading over 2 to 3 wt% and hydrogen pressure up to 55 psig. Although all reactions reached completion, longer reaction times were required and as much as 10% of the undesired epoxide was observed with 2 wt% catalyst loading at lower pressures, e.g. < 30 psig. This result was deemed unacceptable for large scale development. This study revealed that 3 wt% catalyst loading and 50 psig resulted in a faster reaction rate with no impurity formation. This process was found to be suitable for large scale and was therefore utilized in the future process.

Crystallization: Identification of an Antisolvent for the Isolation of Aminodiol 4•HBr Knowing that the inorganic content (from the previous steps) of the reaction mixture had such a significant impact on the isolation, yield, and purity of aminodiol 4•HBr, solubility studies were performed on both 4•HBr and the reaction byproduct DIPEA•HBr. We hoped to identify conditions that would provide the lowest 4•HBr solubility and best rejection of the base salt. DCM, MeCN, acetone, MTBE, and THF were all investigated but only DCM and MeCN were viable antisolvent options. Studies with DCM resulted in a slow, controlled crystallization of 4•HBr with better recovery and purity over any process using MeCN. It was decided to therefore utilize DCM as antisolvent in the isolation of 4•HBr.

Optimization of the DCM Crystallization Process The other crystallization parameters were investigated in efforts to optimize the new DCM process. This work looked into the concentrated volume of the crude reaction mixture prior to DCM addition; the rate, volume, and temperature of antisolvent addition; and finally the stir time after antisolvent addition. Once the debromination was complete, the reaction mixture was concentrated to remove the MeOH and most of the IPA to allow for a less hindered crystallization of 4•HBr. Based on this process, we first investigated the optimal volume to which the reaction mixture should be reduced before the DCM addition. When less than 3 volumes were utilized, an uncontrolled and spontaneous crystallization occurred and a thick slurry was formed. For this reason, 3 to 4 volumes were deemed optimal for the process. Additionally, the ratio of IPA/DCM added during the isolation of 4•HBr was investigated by slurrying mixtures of 4•HBr in solutions of IPA/DCM at 30 °C. The data indicated that when 2 volumes of IPA were used, there was a difference observed on recovery when varying the amount of DCM from 4 to 10 volumes. However, when 3 volumes of IPA were used, at least 8 volumes of DCM were necessary to achieve 90% recovery. Based on these results, it was decided to use 3:10 IPA/DCM as the optimal ratio for the isolation of 4•HBr. 44

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The DCM addition rate to the IPA reaction mixture was also investigated. A comparison of a fast, one portion addition to a dropwise addition over 4 h showed no significant impact on yield. The purity, however, was greater when a longer addition time was applied. We decided to add the DCM over a minimum of 60 minutes. The temperature of aging was investigated from –5 to 20 °C and lower temperatures gave a higher yield due to the precipitation of DIPEA salts from solution. For this reason, the slurry was stirred for a period of time at 20 °C and cooled further to 5 °C just prior to filtration.

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Outcome of Process Optimization Redevelopment of the debromination conditions resulted in a process utilizing DIPEA (3.0 equivalents) as base with Pd/C (3 wt%) under 50 psig hydrogen pressure in a solution of IPA and MeOH at 25 ± 5 °C. Once completion of the reaction was achieved, a solvent swap to IPA was performed to drive off MeOH (≤ 0.2%), followed by addition of DCM antisolvent, allowed for the isolation of 4•HBr as an off-white solid. This improvement provided yields in the range of 61–77% of aminodiol 4•HBr in ≥ 96% purity. The process (Scheme 24) has been successfully carried out ten times on scales up to 40 kg.

Scheme 24. Current GMP Process To Prepare 4•HBr

Process Development for Pyrimidine-Aldehyde 5 to Cyclopentane-diol 3 Main Issues with the First GMP Process Due to the time constraint for delivery of material, the first GMP process (Scheme 25) to prepare chloropyrimidine 3 was carried out based on a two-step procedure using literature conditions (18–20). The aldehyde 5 was converted to acetal 27, followed by reaction with aminodiol salt 4 and cyclization to give chloropyrimidine 3. Due to the switch from a mixture of HCl/HBr salts of 4 to 4•HBr as the other reactant, development efforts for this step (27 to 3) in post-first GMP production were focused on base, solvent, and reaction temperature. 45 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 25. Initial GLP/GMP Process To Convert Aldehyde 5 to Chloropyrimidine 3

Starting Material Switch from Acetal 27 to Aldehyde 5 We hypothesized if aldehyde 5 could react directly with aminodiol 4 and thus, the need for the acetal starting material 27 would be eliminated. However, unsuccessful reactions and undesired imine formation had been reported to occur with the aldehyde. (18, 20). The result was surprisingly good for the reaction of 5 with 4 using the Et3N, IPA conditions, and a similar reaction profile was obtained. This new one-step process eliminated the need for the additional step of converting aldehyde 5 to the acetal 27. Use of solid 5 versus the oily acetal 27 also made it easier to charge the starting material into the reactor.

Solvent and Base Screening Optimization work for this step included a comprehensive solvent and base screen in efforts to achieve a better reaction profile. For the solvent, toluene, THF, DMF, NMP, IPA, MeOH, EtOH, and 2-butanol were explored. Both organic and inorganic bases, such as Et3N, DIPEA, K2CO3, DABCO, DMAP, pyridine, and Cs2CO3 were examined. The best conversion and purity resulted from conditions using IPA or DMF as the solvent and Et3N or DIPEA as the base. Further study led to the findings that 3 could not be isolated by crystallization readily when DMF was used as the solvent, while IPA provided better results. It was determined that 8 to 12 volumes of IPA could be utilized with no significant impact on the extent of reaction, yield, or product purity. Several reactions were then carried out in IPA, investigating both the base options and the type and quality of 4, i.e. if 4 was 46 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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more effective and stable as free base than its HBr salt and if additional purification was necessary before each use. The use of an alternative base (DIPEA, DABCO, DMAP, or pyridine) in IPA with 4•HBr did not offer any improvement to yield over use of Et3N and was therefore not investigated further. The reaction with 4•HBr and aldehyde 5 was also examined under acidic conditions (without addition of base). This resulted in the disappearance of starting material and formation of another compound, identified as the imine intermediate by MS (M+ 304) (Figure 6). However, further cyclization of the imine to the desired product 3 did not occur, indicating that base was required for this condensation.

Figure 6. Imine Intermediate When Base Was Not Charged.

Equivalents of Aldehyde 5 and Reaction Temperature Aldehyde 5 stoichiometry was initially reviewed between 0.8 and 1.1 equivalents versus the diol 4, with 0.9 equivalent being selected to ensure a complete reaction of aldehyde. Later, the ratio of starting materials was reinvestigated keeping other parameters constant as originally developed (2 equivalents of Et3N, 75–80 °C) to identify the optimal range. From this study, it was discovered that the isolated yield of product increased with the use of a slight excess of aldehyde. However, the benefit from increasing 5 was compromised by the decreased purity of the product, especially when more than 1.15 equivalents of aldehyde 5 was used (Table 4). The improved reaction profile using more aldehyde can be explained as related to stability concerns with the aldehyde. We hypothesized that thermal decomposition of the aldehyde competes with the desired reaction pathway, thus an excess of the aldehyde is beneficial to the overall reaction yield. However, more than 1.15 equivalents of aldehyde 5 used in the reaction led to lower product yields as more impurities were produced in the reaction (Table 4). Evaluation of the reaction temperature revealed 75 ± 5 °C as the optimal reaction temperature range for achieving a high isolated yield. Although high reaction temperatures are required for an acceptable reaction rate, thermal decomposition of 5 became significant when the reaction temperature exceeded 80 °C, thus defining the range. 47

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Table 4. Screen for Optimal Amount of 5 4HBr (equiv)

5 (equiv)

Isolated Yield of 3 (%)

Purity (% a/a)

1.0

0.9

67

98

1.0

1.0

66

98

1.0

1.05

76

99

1.0

1.10

78

96

1.0

1.15

74

99

1.0

1.25

79

89

1.0

1.35

76

87

Isolation Water was used as the antisolvent to induce product crystallization from the reaction mixture in IPA. Slow addition of water after the reaction completion at 45–50 °C provided a cleaner product and more filterable crystals than addition at lower temperatures. The amount of water used for isolation was also investigated. Due to the minimal solubility of the product in water, the addition of 4–12 volumes had little impact on yield or product purity. Larger volumes of water, however, were found to be beneficial to remove the triethylamine salts and produce an overall better purity profile. The benefits of using larger volumes of water led to our final process.

Outcome of Process Optimization Process redevelopment led to the currently executed step 5 conditions (Scheme 26), which utilized IPA (20 volumes) as solvent and triethylamine (2.2 equivalents) as base to affect the reaction of 4•HBr (1.0 equivalent) and aldehyde 5 (1.1 equivalents) at 75 ± 5 °C for 18 ± 2 h. Isolation was carried out via crystallization at 45 ± 5 °C using water (15.5 volumes) as antisolvent, followed by filtration and a water wash. This process has been successfully proven six times to date, on scales up to 59 kg. Yields resulting from this process are typically in the 75–83% range with purity ≥ 98%.

Process Development for the Current GMP Process for Conversion of Chloropyrimidine 3 to Indane-Diol 2 The main issue with the first GMP process for conversion of chloropyrimidine 3 to indane-diol 2 was the long reaction time and low yield. As the high temperatures and high pressures required for the conversion were untenable in 48 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the plant, milder conditions were preferred. In the search for an alternative to this SNAr reaction, a cross-coupling strategy was explored. A number of literature reported catalysts with various ligands and bases were screened (Table 5) (44–46). However, none of the experiments provided results worthy of further effort. The initial GMP process was maintained (Scheme 27).

Scheme 26. Current GMP Process To Prepare Chloropyrimidine 3

Process Development for the Preparation of Free Base 1 Main Issues with the First GMP Process Burgess Reagent Development This sulfamoylation step represents the culmination of the addition of the final piece to produce pevonedistat. In the first GMP campaign, the Burgess-type reagent 33 was generated in situ in a separate vessel (Scheme 28). A large excess (4.0 equivalents) was prepared and used in order to ensure complete conversion of diol intermediate 2. Use of THF as solvent led to polymerization of the solvent during acidic deprotection, which produced an impurity that proved difficult to remove. Most problematic of all, the reaction was not clean and chromatographic purification was required to obtain pure free base 1. These problems were successfully addressed as detailed below.

Optimization of the First GMP Process An early strategy to improve the first GMP process was considered by retaining the use of in situ generated Boc-sulfamoyl chloride/DABCO reagent. These studies examined various solvents to replace THF and increase solubility of diol 2. DMAc was optimal over DMF, NMP, and DMSO. 49 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Table 5. Cross-Coupling Strategy To Prepare Diol 2

Most of our work on this transformation involved changes to the preparation and the manipulation of the Burgess-type reagent in order to improve the selectivity and efficiency of this reaction. Preliminary results indicated that having a small amount of acid present helped to speed up the reaction and improve selectivity. We hypothesized that the protonated form 38 is more electrophilic than 37 (Scheme 29). A series of reactions was also carried out in DMAc incorporating a group of selected acids (2 M HCl/THF, TFA, acetic acid and isobutyric acid) to determine their effect on the process. All acids increased 50 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the reaction rate; however, faster decomposition of the Burgess-type reagent also occurred simultaneously and a large excess of the Burgess-type reagent (5 equivalents) was required to drive the reaction to completion. Use of HCl was the most effective, as acetic acid and TFA resulted in the formation of a new impurity and isobutyric acid showed only a small increase in the reaction rate.

Scheme 27. Current GMP Process To Prepare Diol 2

Scheme 28. Initial GLP/GMP Process To Convert diol 2 to Free Base 1

Scheme 29. Acid Effect on Burgess-type Reagent Based on these results, development reactions were carried out in DMAc exploring: 51 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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• • • • •

various reaction temperatures. order of addition. volume of solvent. amount of Burgess-type reagent. amount of HCl.

One possibility was that a low temperature might help with the selectivity, but it was found impractical due to the poor low temperature solubility of both diol 2 and the Boc-sulfamoyl chloride/DABCO reagent in DMAc. Better selectivity was seen with dilution of the reaction mixture and a higher ratio of Burgesstype reagent/HCl to 2. The Burgess-type reagent (or the activated form) was not stable under the reaction conditions and therefore an excess was required to reach completion. Portionwise addition of the Burgess-type reagent was initially considered to circumvent the degradation and need for excess reagent, but this strategy would add operational inconvenience and thus, was not pursued.

Development of a Solid Burgess-type Reagent Although the above improvements had addressed some of the problematic issues of the early process, they failed to provide a fundamental improvement in the process, which would allow the sulfamoylation to be carried out on a manufacturing scale. The ideal reaction conditions for this step would be to use a reagent that is solid and stable for the purposes of utility and storage. It would then be possible to charge the reaction mixture easily with small quantities of Burgess-type reagent when required. (47, 48). A second highly desirable property of the reagent would be for the reagent to selectively react with the primary hydroxyl group over the secondary hydroxyl group in diol intermediate 2. A reagent with these superior properties was in fact developed by Takeda’s process team as described below.

Preparation and Isolation of a t-Bu/DABCO Burgess-type Reagent Early in the process development phase, the unexpected discovery was made that the addition of DABCO to Boc sulfonyl chloride, resulted in a precipitation. This was later determined to be a mixture of 37 and DABCO•HCl salt, where the ratio depended on the type of reaction solvent (Scheme 30). Efforts were spent to isolate this solid in order to further develop a sulfamoylation reaction that would use a solid t-Bu/DABCO Burgess-type reagent. The first process to form solid t-Bu/DABCO Burgess-type reagent using acetone as solvent produced a mixture with 70% Burgess-type reagent and 30% DABCO•HCl salt byproduct by NMR. Apparently, DABCO•HCl salt has better solubility than t-Bu/DABCO Burgess-type reagent in acetone. Attempts to purify the Burgess-type reagent via an aqueous work-up to remove the DABCO•HCl salt via extraction failed since the reagent decomposed rather quickly in water (< 10 min). Due to the reactive nature of the reagent, isolation under nonaqueous 52

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conditions with a minimum number of operational steps was targeted. The crude Burgess-type reagent reaction mixture obtained after solvent removal was slurried in various solvents (49) in the hope that a suitable recrystallization solvent could be identified. Unfortunately, none of the solvents worked; the reagent salt complex either decomposed slowly (> 18 h) in some solvents or did not result in clean separation of the t-Bu/DABCO Burgess-type reagent and DABCO•HCl. These many unsuccessful attempts forced us to consider a different approach. Isolation of the reagent as a fixed ratio with the byproduct, DABCO•HCl salt, was then pursued. Toluene was ultimately chosen as the reaction solvent since both the Burgess-type reagent and DABCO•HCl salt had very low solubility in toluene. The strategy resulted in collection of a 1:1 solid mixture of 37 and DABCO•HCl salt (39). The mixture was isolated from toluene in > 98% yield and > 98% purity by quantitative NMR analysis (Scheme 30) (48). The mixture was as effective in the sulfamoylation reaction as the original isolated reagent mixture that contained only 30% DABCO•HCl salt. It has been stored at ambient temperature for 12 months without a detectable decrease of activity. This synthesis proved to be robust and the desired solid Burgess-type reagent 39 has been prepared on 100–150 kg scale.

Scheme 30. Synthesis and Isolation of t-Bu/DABCO Burgess-type Reagent This optimized Burgess-type reagent, a solid, allowed a better control of the reaction with precise addition of reactants. We determined that the Burgess-type reagent/DABCO•HCl only needed to be used in slight excess. A solvent and temperature screen was carried out using the complex (2.0 equivalents) in MeCN. The key findings from this study were: •

•

Use of DMF or DMAc as a cosolvent in acetonitrile was examined for better solubility but neither was found beneficial to the rate of reaction or improvement of the product purity. The reaction rate at 30 °C was slow, while complete reaction was observed in 2 h at 60–70 °C. 53

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•

The effect of solvent volume was studied from 5–10 volumes of solvent. When 5 volumes of acetonitrile were used, the reaction went to completion in 2 h. However, under these conditions, the reaction mixture was too thick to stir efficiently. An increase to 7.5 volumes of acetonitrile at 50 °C was optimal to achieve the desired rate of reaction and purity of product 34. Reaction at 10 volumes showed a similar reaction rate and product profile.

This work helped to define the extent and conditions for use of our Burgesstype reagent. While development of the t-Bu/DABCO Burgess-type reagent/DABCO•HCl mixture was ongoing, investigation into sulfamating reagents bearing other carbamate protecting groups (e.g. Boc, tertiary, or Bn-like) were considered. Unfortunately, none of the other Burgess-type reagent analogs demonstrated better results than the original reagent 39. The decision was made to remain with the current sulfamating agent.

Optimization of the Deprotection with the Newly Developed Burgess-Type Reagent Elimination of Impurities After effectively driving the reaction of diol 2 with 39 to a mixture of 34 and 36, the remaining challenge for a chromatography-free process would be the removal of the bis-sulfamate 36 side product (Scheme 31). Based on our previous experience from the first GMP production, separation of the free base 1 from the bis-sulfamate side product would not be an easy task due to the similarity of their structures. In early development work, use of an acid (HCl, TFA, or isobutyric acid) in the sulfamoylation was investigated as a way to improve the reaction rate and selectivity. In the course of these studies, we discovered that the Boc-protected bis-sulfamate impurity underwent elimination to a cyclopentene intermediate under aqueous acidic conditions at ambient temperature. This observation was not surprising as the Burgess-type reagent is known to be a mild reagent for alkene formation. (50, 51). Further investigation to find a better acid to perform this conversion identified 0.5 N aqueous HCl (6 volumes) to be optimal for this elimination reaction to produce the cyclopentene. We hoped that the structurally different cycloalkene impurities 40 or 41 compared to free base 1 would allow facile separation. Full conversion of the bis-sulfamate 36 to 40 was achieved in 1 h after adding 0.5 N aqueous HCl (6 volumes). No deprotection of 34 or 40 was observed during this transformation. Work-up procedures were investigated in attempts to identify conditions that would isolate Boc product 34 from the cyclopentene analog 40. Studies were carried out to determine if and how the cyclopentene impurity could be removed from the reaction mixture. 54

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Scheme 31. Bis-Sulfamate Elimination to Cyclopentene Impurity After full conversion of 40 from 36, MTBE and water were introduced to the reaction system to allow separation of organics (mainly the product) from the inorganic salts (e.g. DABCO•HCl). We noticed that the cyclopentene impurity 40 was less soluble in MTBE; however, the amount of MTBE required to force precipitation of 40 from a MTBE/acetonitrile solution would be too high for large scale manufacture. We hypothesized that if a realistic amount of MTBE was added to the acetonitrile layer and the combined organic layer was then extracted portionwise with water, the cyclopentene impurity 40 could be separated from the organic layer when acetonitrile was washed away with water. This extractive process was tested and the results indicated that indeed the cyclopentene impurity 40 appeared as a solid at the interphase after water washes (2 × 10 volumes), and was filtered off from the organic layer. This process produced product 34 that retained a low level of impurities (5–10%). However, the aqueous washes also had a tendency to result in a gummy material coming out of the organic layer as acetonitrile was removed by water washes. The gummy material, likely being 34, made the work-up difficult, unless the washes were conducted at slightly elevated temperature (40 °C). Unfortunately, low levels of Boc deprotection of 34 now occurred at the higher temperatures. To circumvent this issue, we decided to telescope the MTBE solution without extensive water washes into the deprotection step, and utilize the crystallization operations in the downstream steps to reduce the levels of cyclopentene 40. This is described in the following section.

Deprotection Conditions To the solution of 34/40 in MTBE was added more acid for the deprotection. In the absence of THF in the system—and thus, no concern for polymerization—stronger acids were screened for this transformation. We also noted the cleavage of the indane moiety as a side reaction in the presence of aqueous solutions of strong acids. Thus, a balance of speedy Boc deprotection 55 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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and minimum indane hydrolysis was required. To fine-tune the conditions for this step, the intermediate Boc-sulfamate 34 was isolated and a number of alternative deprotection conditions were investigated focusing on HCl of varying concentrations in EtOH, IPA, dioxane/MeCN, dioxane/EtOH, MeCN, and water. We determined that the use of 3.0 M HCl/MeCN system gave the cleanest reaction of > 99% deprotection, while retaining > 99% of the indane. It should be noted that higher concentrations of HCl also led to an increase in the formation of the chloride impurity (Figure 3), and therefore higher concentrations should be avoided. In order to further simplify the process and avoid MTBE extraction, a small amount of concentrated HCl was used to convert cyclopentane analog 36 to cyclopentene 40, followed by another addition of small amount of concentrated HCl to adjust the reaction pH to ~3 for deprotection of the Boc. Unfortunately, the bis-sulfamated byproduct underwent elimination easily, but the subsequent deprotection was extremely slow, taking multiple days and further acid additions to reach completion. Apparently, when DABCO salts and other inorganic impurities were not removed by aqueous work-up, deprotection was difficult. The reaction also resulted in only 35% yield, lower than the typical 45–55% achieved under the process with the MTBE/aqueous extractive work-up. The MTBE extraction of 34 was beneficial to the Boc deprotection and therefore was retained. After complete conversion of 34/40 to 1/41, the reaction mixture was diluted with water/EtOAc. The organic layer was separated and then washed with NaHCO3 solution. The level of cyclopentene impurity 41 was reduced to < 1% in the EtOAc layer, leaving free base 1 as the main component (> 90%).

Free Base 1 Crystallization The initial free base 1 purification/isolation process under the sulfamoyl chloride reaction conditions required the use of column chromatography for acceptable purification. In order to remove this column chromatography, efficient crystallization of free base 1 was required. An antisolvent screen identified EtOAc/DCM system to be optimal for the crystallization. As the recrystallization did not remove several polar impurities at borderline high levels, we sought to add an adsorbent treatment as an orthogonal purification step. The use of silica gel (100 wt%) and charcoal (100 and 50 wt%) showed comparable purity improvements, but the material loss at these loadings was slightly greater with charcoal. Thus, our recrystallization protocol included a silica gel filtration to remove impurities prior to the recrystallization.

Outcome of Process Optimization Using t-Bu/DABCO Burgess-type reagent/DABCO•HCl (1:1 mixture, 39), the current process for sulfamoylation of diol 2 was established (Scheme 32). Diol 2 was consumed with 2.0 equivalents of reagent 39 in 4–5 h at 50 °C. The end reaction mixture contained ~80% 34 and ~15% bis-byproduct 36. Byproduct 36 56 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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was first hydrolyzed to mono-Boc-sulfamate olefin 40 under mild acidic conditions (44–46). Upon full conversion of 36, an increase in HCl concentration successfully removed the Boc groups to afford 1 and sulfamate olefin 41. Compound 41 was purged completely to the aqueous washes during work-up. Charcoal plug filtration followed by crystallization of the crude product from DCM afforded free base 1 in > 96% purity and > 50% yield. This procedure has been successfully carried out in multiple 30 kg API manufacturing campaigns.

Scheme 32. Current GMP Process To Prepare Free Base 1

Process Development for the Salt Formation of the API An early polymorph screening on pevonedistat identified eight forms, as follows: • • •

Among them, Form 1, being the most stable and with acceptable bulk drug physical properties, is the desired form for development. Form 2 is obtainable through interconversion of Form 1 under high moisture conditions. The other six forms were identified as solvates, many of which transform to Form 1 after desolvation.

In any event, we required a convenient process to isolate the desired Form 1. Initially, the salt formation was carried out in pure ethanol using ethereal HCl. However, this process produced a mixture of polymorphic forms. For one of them, comparison of the XRPD trace from the prequalification batch with known crystal forms, along with KF analysis, identified this mixture as consisting of Form 3, 57 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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the ethanol solvate, and the hydrated Form 2. Unfortunately, Form 2 does not convert to Form 1 on drying, so a rework procedure and redevelopment of the crystallization process was necessary. It was known that a hot slurry in ethanol would force the conversion of the hydrated Form 2 to the EtOH solvate, Form 3. Form 3 converts to the desired crystal form (Form 1) under drying. Therefore, ethanolic HCl was chosen to replace ethereal HCl to allow a higher reaction temperature (Figure 7). Also, the use of pure Form 1 seed crystals was determined to be critical to prevent the formation of polymorphic mixtures.

Figure 7. Three Major Crystalline Forms of Pevonedistat. In summary, the desired polymorph was obtained from the following procedure making use of the crude pevonedistat obtained from our multikilogram procedure. Heating to reflux to achieve dissolution was implemented as higher purity free base 1 had lower solubility in EtOH at 50 °C. Addition of 1.25 M HCl in ethanol at 50 °C was carried out in two portions. Approximately half of the HCl was added rapidly and the remaining portion was added post seeding, over the course of an hour. After addition of HCl, the reaction mixture was then cooled to ambient over several hours. This procedure: • • •

aided in better control of the crystallization. provided the desired form. provided improved purity of the API.

A final issue after the first GMP campaign was the presence of ethyl chloride, a genotoxic impurity (GTI) in the final API (52). An investigation was carried out to determine the origin of ethyl chloride and effectiveness of drying to remove ethyl chloride in the API. Analytical methods could not detect ethyl chloride in ethanolic HCl. This suggested that ethyl chloride was generated during the HCl salt formation under heat. Drying at 35 °C for only one day resulted in an EtCl level comparable to drying at 25 °C for ten days. However, in both cases, the data showed that although there was EtOH and HCl trapped within the crystal structure, the close proximity and heating did not appear to generate any additional EtCl. While confusing, the levels of ethyl chloride were acceptable. All lots of API have met the acceptance criterion for ethyl chloride levels (NMT 4630 ppm) which was calculated based on the highest clinical daily dose. This procedure has been successfully carried out to afford multiple cGMP lots of pevonedistat. 58 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Summary A manufacturing process was developed for the synthesis of pevonedistat, a novel and potent NAE inhibitor. The chromatography free, six isolation process has been demonstrated on a 50 kg scale for multiple cGMP productions to afford drug substance with greater than 98% (a/a) chemical purity and 25% overall yield (Scheme 33). A high yielding chiral aminodiol 4·HBr synthesis was developed through bromolactonization of 20, followed by reductive lactone opening, deprotection, and removal of bromide. This work also showcased the development and use of a novel Burgess-type reagent 39 in the final selective sulfamoylation step. The increase in yield and removal of chromatography for this step also contributed to the overall improvement of the synthesis of pevonedistat. The total amount of API prepared by this process is > 100 kg.

Scheme 33. Current GMP Pevonedistat Manufacturing Process

Acknowledgments The authors thank the entire Takeda NAE team for helpful discussions. Special thanks go to Eric L. Elliott, Frederick Hicks, and Steve Langston for assistance in preparing the manuscript. 59 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

References 1.

2.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch002

3.

4.

5.

6.

7.

8.

9. 10.

11. 12.

13. 14. 15.

16.

WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells. MackIntosh, C.; Garcia-Dominguez, D. J.; Ordonez, J. L.; Ginel-Picardo, A.; Smith, P. G.; Sacristan, M. P.; de Alava, E. Oncogene 2013 Mar14, 32 (11), 1441–51. DOI: Epub 2012 May 2810.1038/onc. 2012. 153. Dezube, B. J.; Petruzzelli, L. M. Administration of Nedd8-activating enzyme inhibitor. PCT Int. Appl. (2012), WO 2012061551 A1. Genome-wide screen for modulators of cell death induced by the NEDD8activating enzyme inhibitor MLN4924. Blank, J. L.; Liu, J. X.; Bouck, D.; Garcia, K.; Hugues, B.; Liu, R.; Liao, H.; Lightcap, E. S. Pacifichem 2010, International Chemical Congress of Pacific Basin Societies, Honolulu, HI, December 15–20, 2010, BIOL-1050. Soucy, T. A.; Smith, P. G.; Milhollen, M. A.; Berger, A. J.; Gavin, J. M.; Adhikari, S.; Brownell, J. E.; Burke, K. E.; Cardin, D. P.; Critchley, S. Nature 2009, 458, 732–736. Brownell, J. E.; Sintchak, M. D.; Gavin, J. M.; Liao, H.; Bruzzese, F. J.; Bump, N. J.; Soucy, T. A.; Milhollen, M. A.; Yang, X.; Burkhardt, A. L. Mol. Cell 2010, 37, 102–111. Swords, R. T.; Kelly, K. R.; Smith, P. G.; Garnsey, J. J.; Mahalingam, D.; Medina, E.; Oberheu, K.; Padmanabhan, S.; O’Dwyer, M.; Nawrocki, S. T. Blood 2010, 115, 3796–3800. Milhollen, M. A.; Traore, T.; Adams-Duffy, J.; Thomas, M. P.; Berger, A. J.; Dang, L.; Dick, L. R.; Gamsey, J. J.; Koenig, E.; Langston, S. P. Blood 2010, 116, 1515–1523. Gavin, J. M.; Chen, J. J.; Liao, H.; Rollins, N.; Yang, X.; Xu, Q.; Ma, J.; Loke, H.; Lingaraj, T.; Brownell, J. E. J. Biol. Chem. 2012, 287, 15512–15522. Toth, J. I.; Yang, L.; Dahl, R.; Petroski, M. D. Cell Rep. 2012, 1, 309–316. Milhollen, M. A.; Thomas, M. P.; Narayanan, U.; Traore, T.; Riceberg, J.; Amidon, B. S.; Bence, N. F.; Bolen, J. B.; Brownell, J.; Dick, L. R. Cancer Cell 2012, 21, 388–401. Kee, Y.; Huang, M.; Chang, S.; Moreau, L. A.; Park, E.; Smith, P. G.; D’Andrea, A. D. Mol. Cancer Res. 2012, 10, 369–377. Heiser, L. M.; Sadanandam, A.; Kuo, W.; Benz, S. C.; Goldstein, T. C.; Ng, S.; Gib, W. J.; Wang, N. J.; Ziyad, S.; Tong, F. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 2724–2729. Wei, D.; Li, H.; Yu, J.; Sebolt, J. T.; Zhao, L.; Lawrence, T. S.; Smith, P. G.; Morgan, M. A.; Sun, Y. Cancer Res. 2012, 72, 282–293. Zhao, L.; Yue, P.; Lonial, S.; Khuri, F. R.; Sun, S. Mol. Cancer Ther. 2011, 10, 2415–2425. Chen, J. J.; Tsu, C. A.; Gavin, J. M.; Milhollen, M. A.; Bruzzese, F. J.; Mallender, W. D.; Sintchak, M. D.; Bump, N. J.; Yang, X.; Ma, J. J. Biol. Chem. 2011, 286, 40867–40877. Chou, T.; Deshaies, R. J. J. Biol. Chem. 2011, 286, 16546–16554. 60

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17. Lin, J. J.; Milhollen, M. A.; Smith, P. G.; Narayanan, U.; Dutta, A. Cancer Res. 2010, 70, 10310–10320. 18. Montgomery, A. J.; Hewson, K. J. Med. Chem. 1967, 10, 665–667. 19. Secrist, J. A., III; Clayton, S. J.; Montgomery, A. J.; Hewson, K. J. Med. Chem. 1984, 27, 534–536. 20. Legraverend, M.; Ngongo-Tekam, R. N.; Bisagni, E.; Zerial, A. J. Med. Chem. 1985, 28, 1477–1480. 21. Bray, B.; Dolan, C. S.; Halter, B.; Lackey, J. W.; Schilling, B. M.; Tapolczay, D. J. Tetrahedron Lett. 1995, 36, 4483–4486. 22. Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974–5976. 23. Hill, J. G.; Sharpless, K. B.; Exon, C. M.; Regenye, R. Org. Syn. 1990, 7, 461–467. 24. Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune, H.; Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765–5780. 25. Johnson, R. A.; Sharpless, K. B. Comp. Org. Syn. 1991, 7, 389–436. 26. Finn, J.; Yoshito, K. Tetrahedron Lett. 1982, 23, 2719–2722. 27. Viti, S. Tetrahedron Lett. 1982, 23, 4541–4544. 28. Dominguez, B.; Cullis, M. Tetrahedron Lett. 1999, 40, 5783–5786. 29. Allan, R.; Fong, J. Aust. J. Chem. 1986, 39, 855–864. 30. Smith, M.; Lloyd, M.; Derrien, N.; Lloyd, R.; Taylor, S.; Chaplin, D.; Casy, G.; McCague, R. Tetrahedron Asymm. 2001, 12, 703–705. 31. Knapp, S.; Zhao, D. Org. Lett. 2000, 2, 4037–4040. 32. Banwell, M. G.; Edwards, A. J.; Lupton, D. W.; Whited, G. Aust. J. Chem. 2005, 58, 14–17. 33. Bodkin, J. A.; Humphries, E. J.; McLeod, M. D. Tetrahedron Lett. 2003, 44, 2869–2872. 34. Mead, K. T.; Park, M. Tetrahedron Lett. 1995, 36, 1205–1208. 35. Rylander, P. N. Hydrogenation Methods; Academic Press: New York, 1985. 36. Although both HCl and HBr salts were possible, elemental analysis confirmed this solid was mainly a HBr salt. 37. Claiborne, C. F.; Critchley, S.; Langston, S. P.; Olhava, E. J.; Peluso, S.; Weatherhead, G. S.; Vyskocil, S.; Visiers, I.; Mizutani, H.; Cullis, C. Preparation of carbocyclic purine nucleoside analogs as antitumor agents and inhibitors of E1 activating enzymes. Application: PCT Int. Appl. (2008) WO2008019124. 38. Langston, S. P.; Olhava, E. J.; Vyskocil, S. Preparation of purine nucleoside derivatives as antitumor agents and inhibitors of E1 activating enzymes. Application: PCT Int. Appl. (2007) WO 2007092213. 39. Lukkarila, J. L.; da Silva, S. R.; Ali, M.; Shahani, V. M.; Xu, G. W.; Berman, J.; Roughton, A.; Dhe-Paganon, S.; Schimmer, A. D.; Gunning, P. T. ACS Med. Chem. Lett. 2011, 2, 577–582. 40. Geisler, J.; Schneider, F.; Lovis, K.; Lopez, H. F. Industrially applicable process for the sulfamoylation of alcohols and phenols. (Schering AG, Berlin, Germany). WO Pat. 2003/053992 A2. 41. Arvai, G.; Garaczi, S.; Mate, A. G.; Lukacs, F.; Viski, Z.; Schneider, G. Process for the preparation of Topiramate. US Pat. Appl. Publ. (2006), US2006040874 A1. 61

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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42. Nicolaou, K. C.; Snyder, S. A.; Huang, X. Synthesis of sulfamidates. WO Pat. 2003/066549 A2. 43. Morales-Serna, J. A.; García-Ríos, E.; Bernal, J.; Paleo, E.; Gavino, R.; Cárdenas, J. Synthesis 2011, 1375–1382. 44. Navarro, O.; Kaur, H.; Mahjoor, P.; Nolan, S. P. J. Org. Chem. 2004, 69, 3173–3180. 45. Zim, D.; Buchwald, S. L.; Song, W. Org. Lett. 2003, 5, 2413–2415. 46. Marion, N.; Ecarnot, E. C.; Navarro, O.; Amoroso, D.; Bell, A.; Nolan, S. P. J. Org. Chem. 2006, 71, 3816–3821. 47. Armitage, I.; Elliott, E. L.; Langston, M.; Langston, S. P.; McCubbin, Q. J.; Mizutani, H.; Stirling, M.; Zhu, L. Process for the synthesis of 4-(7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-(hydroxymethyl)cyclopentanol derivatives as E1 activating enzyme inhibitors. U.S. Pat. Appl. Publ. (2009) 20090036678. 48. Armitage, I.; Berne, A. M.; Elliott, E. L.; Fu, M.; Hicks, F.; McCubbin, Q. J.; Zhu, L. Org. Lett. 2012, 14, 2626–2629. 49. Other solvents investigated: MeCN, acetone, THF, dioxane, DCM, MEK, EtOAc and iPrOAc. 50. Atkins, G. M.; Burgess, E. M. J. Am. Chem. Soc. 1968, 90, 4744–4745. 51. Burgess, E. M.; Penton, H. R.; Taylor, E. A. J. Org. Chem. 1973, 38, 26–31. 52. Yang, Q.; Haney, B. P.; Vaux, A.; Riley, D. A.; Heidrich, L.; He, P.; Mason, P.; Tehim, A.; Fisher, L. E.; Maag, H.; Anderson, N. G. Org. Process Res. Dev. 2009, 13, 786–791.

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Chapter 3

The Discovery and Synthesis of the CGRP Receptor Antagonist MK-3207 Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

Ian M. Bell,1 Paul G. Bulger,2 and Mark McLaughlin*,2 1Department

of Discovery Chemistry, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States 2Department of Process Research & Development, Merck & Co., Inc., 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States *E-mail: [email protected].

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and neuromodulator. Multiple lines of evidence demonstrate that CGRP plays a key role in the pathogenesis of migraine and it has become a major target for migraine drug discovery efforts. This chapter reviews the discovery of MK-3207, a novel, potent, orally acting CGRP receptor antagonist and the development of a highly efficient synthetic route that allows for large scale production of the compound.

Introduction Migraine is a common, highly disabling, neurovascular disorder that affects about 11% of adults worldwide and results in a significant burden to society in terms of lost productivity and diminished quality of life (1, 2). Migraine attacks are characterized by moderate to severe headache accompanied by other symptoms, including photophobia, phonophobia, allodynia, nausea, and vomiting (1). The duration of these attacks can be from a few hours to several days and the frequency is typically around one or two attacks per month (3). The “gold standard” agents for the acute treatment of migraine are the triptans, which are selective 5-HT1B and 5-HT1D receptor agonists. Triptans are believed to act via vasoconstriction of cranial blood vessels and by inhibition of the release of neuropeptides, including calcitonin gene-related peptide (CGRP) (4). Although triptans are effective antimigraine agents, they also cause constriction of coronary arteries and are consequently contraindicated in patients © 2016 American Chemical Society

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with cardiovascular disease and uncontrolled hypertension (5). There continues to be significant interest in the development of new and effective antimigraine drugs that are safe and that lack the cardiovascular liabilities of the triptans. Migraine is a complex disorder and the precise details of its pathogenesis are still being studied. There is, however, general agreement that a key player is CGRP, a 37-amino acid neuropeptide that is a member of the calcitonin family of peptides (6). CGRP, which is a potent vasodilator and neuromodulator, is found throughout the peripheral and central nervous systems and appears to play a role in a number of biological functions (7). Among these, its apparent involvement in cerebrovascular regulation led to the hypothesis that it could be a key player in the pathophysiology of migraine and subsequent studies have confirmed this (8). For example, it was shown that the craniovascular levels of CGRP increased significantly during migraine attacks (9). Additionally, these migraine-associated increases in CGRP concentrations appeared to return to basal levels following successful treatment of the migraine headache with sumatriptan (10). In another compelling study, it was found that intravenous infusion of CGRP induced a migraine-like headache in migraineurs (11). These lines of evidence demonstrating that CGRP was playing an important role in migraine led to speculation that a “CGRP blocker” could represent a new therapeutic approach with potential advantages over the triptans (12). The present review will focus on the discovery of one such small molecule antagonist of the CGRP receptor and on the development of novel synthetic routes that allow for large scale production of this clinical candidate, MK-3207.

CGRP and the CGRP Receptor The calcitonin family of peptides consists of calcitonin (CT), CGRP, amylin (AMY) and adrenomedullin (AM) (13, 14). The peptides share a number of features, including a cyclic structure at the N-terminus formed by a Cys-Cys disulfide bond, and a C-terminal amide group (13). There are two forms of CGRP: α-CGRP, which is produced by alternate splicing of the CT gene, and β-CGRP, which is encoded by a separate gene and differs from α-CGRP by three amino acids in humans (14). The receptors for these peptides are members of the secretin family (also known as family B or family 2) of G-protein-coupled receptors (GPCRs) (15). These family B GPCRs contain large N-terminal extracellular domains (ECDs) that are involved in binding of their peptide ligands (15). The CGRP receptor is a heterodimeric receptor, composed of the calcitonin receptor-like receptor (CLR) in association with receptor activity-modifying protein 1 (RAMP1) (16, 17). CLR can also associate with RAMP2 or RAMP3 to produce high affinity AM receptors that are designated AM1 and AM2, respectively (17). In a similar way, the calcitonin receptor (CTR) can partner with RAMP1 to produce a receptor for AMY that is designated AMY1 (17). Because CTR and CLR have significant homology, the AMY1 receptor (CTR/RAMP1) has a similar binding site to the CGRP receptor (CLR/RAMP1). 64

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The interaction of CGRP with the CGRP receptor has been studied by a number of approaches, including mutational studies and cross-linking experiments (18–20). These studies demonstrated that the ECDs of both CLR and RAMP1 are important for ligand binding and receptor activation (17). Residues in the transmembrane domains of CLR and the loops in between these transmembrane domains have also been found to play key roles in agonist binding and receptor function (21). The available data are consistent with the two-domain model for family B GPCRs described by Hoare (22). According to this model, the C-terminal region of the peptide CGRP first binds to ECDs of CLR and RAMP1. This binding event brings the N-terminal portion of CGRP into close proximity with the juxtamembrane region of CLR, allowing them to interact and produce receptor activation (22). This model neatly explains why the truncated peptide CGRP8-37, which lacks the N-terminal cyclic structure of the first seven amino acids, binds potently to the CGRP receptor but cannot activate it and acts as an antagonist (14).

Figure 1. The CGRP receptor antagonists olcegepant (1) and telcagepant (2) showing the privileged structures.

Small molecule CGRP receptor antagonists, such as olcegepant (23) (1, Figure 1) and telcagepant (24) (2), are also thought to bind to the ECDs of CLR and RAMP1 and thereby prevent binding of CGRP and receptor activation. A number of residues in the ECDs have been shown to be important for binding of small molecule receptor antagonists, including CLR Met42, RAMP1 Trp74, and RAMP1 Trp84 in the human CGRP receptor (25–28). One known exception to this antagonist binding mode is the hydroxypyridine class of antagonists, which do not seem to bind to the ECDs but appear to interact with transmembrane domain 7 in CLR (26, 29). The understanding of how small molecules antagonize the CGRP receptor was greatly enhanced by a group of researchers at Vertex, who expressed and purified a stable complex of the ECDs of CLR and RAMP1 (30). This purified ECD complex bound to small molecule CGRP receptor antagonists with high affinity but exhibited relatively weak binding to CGRP itself, consistent with the two-domain model for binding of the peptide agonist to its receptor (22). The Vertex group 65

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was able to crystallize the CLR:RAMP1 complex and solve the structures of the unliganded ectodomain as well as tertiary complexes with the small molecule antagonists olcegepant and telcagepant (31). A schematic representation of the binding of telcagepant to the CGRP ECD, as described by ter Haar et al. is shown in Figure 2, with an emphasis on the key interactions that are thought to contribute to potency (31). Telcagepant, like many small molecule CGRP receptor antagonists, has a “privileged structure” (32) that contains a terminal cyclic amide (CONH) moiety – in the case of telcagepant an azabenzimidazolone ring system that is known to be very important for CGRP receptor affinity. In the published crystal structure, this moiety makes hydrogen binding interactions with the backbone elements of CLR Thr122 (Figure 2) (31). Another residue that makes key interactions with telcagepant is CLR Trp72, which stacks against the piperidine ring of the “privileged structure” and also engages in a hydrogen bond between the tryptophan indole NH and the carbonyl oxygen of the caprolactam ring. The difluorophenyl ring in telcagepant, a group known to be crucial for binding affinity, occupies a hydrophobic pocket formed, in part, of CLR Met42, RAMP1 Trp 74, and RAMP1 Trp 84 (31). Consistent with the importance of these hydrophobic contacts, all three of these residues have been shown to be key contributors to the potency of small molecule receptor antagonists (27, 28). Overall, the complex of the CGRP receptor ECD with olcegepant is similar to that described for telcagepant and these same key residues interact with both small molecule antagonists (31).

Figure 2. Telcagepant bound to the CGRP receptor ECD. Telcagepant is shown in dark gray and the CGRP receptor ECD is shown in light gray. Key residues involved in antagonist binding are labeled. 66 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Both telcagepant and olcegepant bind in an extended conformation, bridging a distance of about 18 Å between the critical hydrogen bonding interactions at CLR Thr122 and the important hydrophobic pocket formed at the CLR:RAMP1 interface (31). The crystal structures of these small molecule antagonists bound to the CGRP receptor ECD afford insight into the challenges of designing orally acting antagonists for this family B GPCR. In order to bind to this site on the protein with high affinity and block the binding of the peptide agonist, a molecule must apparently be relatively large and have multiple hydrogen bond donors and acceptors. This profile tends to be at odds with guidelines for orally bioavailable drugs, such as the Lipinski “Rule of Five” (33).

Discovery of MK-3207 Based on the significant evidence of a key role for CGRP in migraine headache, Merck initiated a program to develop orally bioavailable CGRP receptor antagonists in the early 2000s. High-throughput screening (HTS) identified the micromolar benzodiazepinone-based lead 3 (Figure 3), which was originally synthesized as a cholecystokinin receptor antagonist (34). In many ways, 3 was not an optimal lead structure for an oral drug discovery program. It only had modest binding affinity (Ki = 4.8 µM) and similar potency in a functional assay based on CGRP-stimulated production of cyclic adenosine monophosphate (cAMP) in cells (cAMP IC50 = 6 µM) (35). The combination of modest potency and large molecular size meant that it had relatively poor ligand efficiency (LE = 0.18 kcal/mol) (36). Compound 3 also possessed a significant number of hydrogen bond donors (four) and acceptors (five), which correlated with a calculated polar surface area (PSA) that predicted poor passive permeability (PSA = 147 Å2) (37). However, the structure of 3 was novel when compared with other known CGRP receptor antagonists, many of which were based on a peptidic backbone. Moreover, it was the only novel and tractable lead identified from the high-throughput screen and the team investigated the optimization of this benzodiazepinone lead. One approach to optimizing the HTS lead was based on the structural analogy between the spirohydantoin moiety in 3 and the piperidinyldihydroquinazolinone in olcegepant (1). Both contained a secondary amide hydrogen bond donor-acceptor pair embedded in a heterocycle and it was hypothesized that these rings could be key components in related “privileged structures” (32) that played a key role in binding to the GPCR. This hypothesis prompted the team to evaluate a number of piperidinyl privileged structures and led to the identification of a novel piperidinylazabenzimidazolone that provided the optimal balance of potency and chemical stability (38). Reengineering of the benzodiazepinone portion of the molecule, with the goal of improving physicochemical properties, led to caprolactam-based antagonists and ultimately to the discovery of telcagepant (2, MK-0974), the first orally bioavailable CGRP receptor antagonist to advance to the clinic (24). Thus, the initial approach to optimization of HTS lead 3 was to replace the tetralin-spirohydantoin part of the structure with a piperidinyl privileged 67

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structure. In a complementary approach, the benzodiazepinone was removed and the spirohydantoin portion of compound 3 was used as the basis of a rapid analogue screen for benzodiazepinone replacements with lower molecular weight (39). Early results indicated that the tetralin ring could be effectively replaced by an indane and led to the identification of a number of submicromolar, racemic lead structures (39). Resolution of the indanylspirohydantoin and further optimization afforded 4 (Figure 3, Ki = 21 nM; cAMP IC50 = 78 nM), in which a substituted benzimidazolone replaced the benzodiazepinone. In the simplified lead 4, the (R)-enantiomer of the spirohydantoin was preferred in terms of CGRP-R affinity and it exhibited about 200-fold higher affinity for the CGRP-R than the higher molecular weight HTS lead 3. Compound 4 exhibited a good pharmacokinetic profile in rat, dog and monkey, with low plasma clearance and good oral bioavailability (F = 29–83%) (39).

Figure 3. Spirohydantoin lead compounds and tricyclic CGRP receptor antagonists.

Although 4 was an attractive lead structure, its potency was suboptimal and incompatible with a low projected clinical dose. It was found that the 2-pyridyl ring could be replaced by a glycine substituent to provide an analogue with similar affinity for the CGRP-R. An interesting approach to potency enhancement was realized when this glycine substituent was constrained to give the tricyclic moiety found in 5 (Figure 3, Ki = 0.51 nM; cAMP IC50 = 2.4 nM) (40). This tricyclic benzimidazolone represented a 40-fold increase in CGRP-R affinity relative to the pyridyl-substituted 4. 68 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Additional improvements in potency were achieved by rational modification of the spirohydantoin moiety based on its analogy to the azabenzimidazolone privileged structure in compounds like telcagepant. Specifically, replacement of the spirohydantoin in 5 with a spiroazaoxindole led to the highly potent antagonist 6 (Figure 3, Ki = 0.04 nM; cAMP IC50 = 0.28 nM), which possessed approximately 500-fold enhanced CGRP-R affinity compared with 4 (41). Unfortunately, spiroazaoxindole 6 was not orally bioavailable in preclinical species and it appeared that low passive permeability was at least partly responsible. It was known that low permeability could limit the oral absorption of related compounds and the calculated PSA of the compounds proved to be a useful guide: when PSA was greater than 130 Å2 the oral bioavailability was usually very low (39). For 6, the PSA was 149 Å2 and the aqueous solubility was poor (0.24 µg/mL at pH 7.4 for amorphous material) (42). In order to reduce the PSA, the benzimidazolone was replaced with an indoline and it was hoped that the weakly basic indoline nitrogen would afford improved solubility at acidic pH (42). These design considerations led to 7 (Figure 3, Ki = 0.35 nM; cAMP IC50 = 2.4 nM), which was orally bioavailable in rat, dog, and monkey (F = 25–49%), and possessed similar potency to telcagepant (42). Before the publication of the crystal structures determined by the Vertex team (31), conformationally constrained analogues were studied as one way to provide information on the bioactive conformation of CGRP receptor antagonists. One such example was the use of a quinoline-based central constraint as a replacement for the central amide bond in compounds such as spirohydantoin 4 (43). This modification had the added advantage that it reduced PSA and therefore might be expected to improve membrane permeability. This strategy led to quinoline 8 (Figure 3, Ki = 0.52 nM; cAMP IC50 = 2.2 nM), in which the spirohydantoin was reintroduced, in an effort to improve solubility (43). This quinoline central constraint appeared to be a good mimic of the bioactive conformation of the central amide and it also helped to impart good oral bioavailability for compound 8 in rat, dog, and monkey (F = 38–59%) (43). Not only did quinoline analogues like 8 shed light on the bioactive conformation of spiroindane-based CGRP receptor antagonists, but they also provided inspiration for the design of 9 (Figure 4, Ki = 1.9 nM), which was part of an effort to simplify the highly constrained nature of compounds like 8 to facilitate rapid exploration of SAR (44). Somewhat surprisingly, the preferred stereochemistry of the spirohydantoin 9 had switched to (S) from (R) in earlier compounds like 4. This stereochemical inversion effectively resulted from a change in the position of attachment between the amide nitrogen and the indane and this led to very different SAR for the new series. Compound 9 represented an attractive lead compound, in part because of its impressive ligand efficiency (LE = 0.34 kcal/mol), which is excellent for a CGRP receptor antagonist and compares favorably with olcegepant (1) (LE = 0.27 kcal/mol) and telcagepant (2) (LE = 0.31 kcal/mol). One area for improvement was the 370-fold selectivity of 9 for the CGRP-R over the AM2 receptor (CLR/RAMP3), which was deemed to be suboptimal (44). Initial exploration of SAR in this new series focused on improving potency and selectivity vs. the AM2 receptor. It was quickly established that fluoro 69

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substitution of the terminal benzyl group helped to address both concerns. Additionally, cyclization of the N-benzylpivalamide end group was well tolerated, affording the substituted piperidinone analogue 10 (Figure 4, Ki = 0.23 nM; cAMP IC50 = 0.91 nM) (44). Further potency enhancement was achieved by replacing the spirohydantoin with the corresponding spiroazaoxindole, in analogy with 5 and 6 (Figure 3). In the context of these piperidinones, incorporation of the spiroazaoxindole privileged structure for spirohydantoin provided a 6-fold increase in CGRP receptor affinity to give the picomolar antagonist 11 (Figure 4, Ki = 0.039 nM; cAMP IC50 = 0.16 nM) (44, 45).

Figure 4. Spiroindane CGRP receptor antagonists. Piperidinone 11 exhibited improved selectivity versus the AM2 receptor (AM2/CGRP selectivity = 4100-fold) compared with earlier analogues like 9 (44). In the cell-based functional assay, 11 had subnanomolar potency in the presence of 50% human serum (cAMP + HS IC50 = 0.35 nM). Compound 11 was found to be orally bioavailable in rat (F = 12%) and dog (F = 44%) but not in monkey (F = 0%), and this deficit was significant because it was important to fully evaluate the in vivo pharmacology of such small molecule CGRP receptor antagonists in monkeys because they exhibit significantly reduced affinity for non-primate CGRP receptors (12). In analogy with data for telcagepant, the low monkey oral bioavailability of 11 was thought to be due in part to intestinal first-pass metabolism (45). It was known that the piperidinone ring of 11 was subject to 70 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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significant metabolism in vitro, so the team sought to both reduce metabolism and increase aqueous solubility by incorporating polar functionality into this ring. This strategy led to morpholinones such as 12 and piperazinones such 13 (Figure 4) (45). While morpholinone analogue 12 (Ki = 0.018 nM; cAMP IC50 = 0.32 nM) was highly potent and had good oral bioavailability in rat (F = 59%) and dog (F = 72%), it was not orally bioavailable in monkey (F = 0%) (45). Piperazinone 13 was not only potent (Ki = 0.034 nM; cAMP IC50 = 0.17 nM) but was also more soluble than the corresponding morpholinones, especially at acidic pH. The improved aqueous solubility correlated with modest oral bioavailability in monkey (F = 7%) and this observation led to a significant effort to optimize the potency, selectivity, and pharmacokinetic properties of such piperazinone analogues (45). This led to the discovery that replacement of the gem-dimethyl substituents on the piperazinone ring of 13 with a spirocyclopentyl ring provided MK-3207 (14) (Figure 4, Ki = 0.021 nM), a picomolar CGRP receptor antagonist with an excellent overall profile (45).

Preclinical Profile of MK-3207 MK-3207 was a picomolar CGRP receptor antagonist with a binding affinity (Ki = 0.021 nM) only slightly lower than the much larger olcegepant (Ki = 0.014 nM), leading to a significantly higher ligand efficiency for MK-3207 (0.35 kcal/mol) compared with olcegepant (0.27 kcal/mol). MK-3207 was also highly potent in a cell-based functional assay (cAMP IC50 = 0.12 nM) and this cell-based potency was slightly shifted in the presence of 50% human serum (cAMP + HS IC50 = 0.17 nM), suggesting that the compound was relatively free in human serum (45). The radiotracer [3H]MK-3207 was used in detailed binding studies and it was determined that the KD of MK-3207 was 60 pM and that it had a reduced off-rate (0.012 min-1) and longer dissociation half-life (59 min) compared with telcagepant (KD = 1.9 nM) (46, 47). The high in vitro potency of MK-3207 translated to an in vivo monkey pharmacodynamic model, based on capsaicin-induced dermal vasodilation (CIDV) (48). In this CIDV model, MK-3207 was found to block 90% of the capsaicin-induced increase in dermal blood flow at a plasma concentration of 7 nM (EC90 = 7 nM) (46). For telcagepant, the corresponding EC90 value in the rhesus monkey CIDV model was found to be 994 nM (49). Thus, compared to telcagepant, MK-3207 was about 40-fold more potent than in vitro and approximately 100-fold more potent in vivo. Small molecule CGRP receptor antagonists typically display pronounced species selectivity, with significantly higher affinity for the CGRP receptors of primates than for those of non-primates, and MK-3207 was no exception. Thus, while it had high affinity for rhesus monkey CGRP-R (Ki = 0.024 nM) it exhibited markedly reduced affinity for the CGRP receptors of rat (Ki = 10 nM) and dog (Ki = 10 nM) (46). MK-3207 had excellent selectivity for the human CGRP-R against the related AM1 (> 600,000-fold), AM2 (> 6,500-fold) and AMY3 (> 5,000-fold) receptors but only modest selectivity against the AMY1 receptor 71

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(30-fold). MK-3207 was screened against a panel of 169 receptors, enzymes, and transporters and was found to be greater than 50,000-fold selective for human CGRP-R versus any of these other targets (46). The preclinical pharmacokinetic profile of MK-3207 is summarized in Table 1. In rat and dog, the compound exhibited low plasma clearance and good oral bioavailability (F = 67–74%). In contrast, the plasma clearance in rhesus monkey was moderate and the oral bioavailability was relatively low at lower doses (F = 9% at 2 mg dose/kg bodyweight/day (mpk)) but improved at higher doses (F = 41% at 20 mpk) (45). These results suggest that saturable first-pass metabolism played a role in limiting the oral bioavailability in monkeys, similar to observations with the earlier compound telcagepant (50).

Table 1. Preclinical Pharmacokinetic Properties of MK-3207 Species

Dose (mpk)

Fa (%)

Clb (mL/min/kg)

Vdssb (L/kg)

IV t1/2b (h)

Rat

10 (PO); 2 (IV)

74

11

0.3

0.6

Dog

2 (PO); 0.5 (IV)

67

8.0

0.6

1.0

Monkey

2 (PO); 0.5 (IV)

9

15

1.7

1.5

Determined after dosing in 0.5% or 1% methocel vehicle. DMSO vehicle.

a

b

Determined after dosing in

As detailed in Table 1, the plasma half-life was short in preclinical species but a short half-life is quite compatible with acute treatment of migraine in the clinic. Overall, MK-3207 had an excellent preclinical profile in terms of its potency, selectivity and pharmacokinetics and it was advanced for clinical evaluation as a novel treatment for migraine with the potential for a low human dose based upon its significantly improved preclinical potency relative to telcagepant (51).

Clinical Profile of MK-3207 Consistent with results in preclinical species, MK-3207 was orally bioavailable in humans. Following oral dosing, the compound exhibited a Tmax of ~ 1–2 h and a terminal plasma half-life of ~ 9–18 h (52). The CIDV pharmacodynamic assay that was developed in rhesus monkeys could be readily translated to the clinical setting by virtue of its non-invasive nature and provided results in good agreement with the preclinical observations, with a human CIDV EC90 ≈ 14 nM (52). A detailed PK/PD analysis indicated that a 20 mg oral dose of MK-3207 should produce effective blockade of the CGRP receptor in the periphery, similar to clinically efficacious doses of telcagepant (52). 72 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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A placebo-controlled Phase II study evaluated seven doses of MK-3207 (from 2.5 to 200 mg) with a primary endpoint of pain freedom at 2 h (53). In this study, doses of 10 mg, 100 mg, and 200 mg were found to be superior to placebo at the primary endpoint. Based on the available data, the 200 mg dose did not appear to be more effective than any dose at or above 10 mg, and the 10 mg dose may have represented a plateau in terms of efficacy (53). This possibility is consistent with the PK/PD modeling (52). Unfortunately, although MK-3207 was generally well tolerated in this clinical trial, in extended Phase I studies there were a number of observations of delayed liver test abnormalities, typically after cessation of dosing. As a result of these findings, the development of MK-3207 was discontinued (53). The liver transaminase elevations seen for MK-3207 raised the possibility of a mechanism-related effect, since liver enzyme elevations were also observed for telcagepant. However, the pattern of clinical findings with telcagepant was quite different from that observed with MK-3207, suggesting that these effects may be compound-related rather than mechanism-based (54).

Medicinal Chemistry Synthesis of MK-3207 The synthesis of piperazinones such as MK-3207 was accomplished by amide coupling of aniline and carboxylic acid fragments described in the following two schemes. The route to aniline 25 was essentially the same as the original published synthesis (Scheme 1) (41). The 2-(trimethylsilyl)ethoxymethyl (SEM) protecting group was selected for protection of 7-azaindole (15) based on its stability under a range of conditions. This protected azaindole 16 was converted to the corresponding azaoxindole 18 using methodology described by Marfat and Carta (55). 4-Nitrophthalic acid (19) underwent standard reduction and bromination to afford 1,2-bis(bromomethyl)-4-nitrobenzene (21) in essentially quantitative yield. The key step was bis-alkylation of oxindole 18 with dibromide 21 to provide spirocycle 22 in 75% yield. This bis-alkylation employed Cs2CO3 as base, and was run at relatively low concentration in DMF to reduce the formation of oligomeric material. The nitro group in 22 was reduced under catalytic hydrogenation conditions to give the corresponding racemic aniline (±)-23, which could be separated into its individual enantiomers using chiral column chromatography. Alternatively, racemic aniline (±)-23 could be protected with a Boc group and the chiral separation conducted on carbamate (±)-24 (as shown in Scheme 1), which had improved solubility relative to the unprotected aniline. Finally, the desired enantiomer was deprotected to afford aniline (R)-25. To facilitate exploration of the piperazinone series, a practical, two-step synthesis of 6-phenylpiperazin-2-ones was developed (56). This route allowed rapid access to piperazinones such as (±)-30 (Scheme 2), albeit in moderate yield. Thus, alkylation of methyl 1-aminocyclopentanecarboxylate (27) with 3,5-difluorophenacyl bromide (26) using trisodium phosphate as base proceeded in 55% yield. The resulting aminoketone 28 was found to have poor stability 73

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and underwent oxidation at the aminoketone α-carbon to produce a symmetrical dimer. This oxidative process could be minimized by storing aminoketone 28 as the mesylate salt and by protecting it from air (45). Reductive amination of ketone 28 with glycine ethyl ester (29) and in situ cyclization of the resulting amine afforded racemic piperazinone 30 in 45% yield.

Scheme 1. Medicinal Chemistry Synthetic Route to Aniline (R)-25

To facilitate the subsequent chemistry and chromatography steps, the piperazinone was protected with a Boc group, and separation of the enantiomers provided the desired (R)-enantiomer of ester 31. Saponification of 31 led to the corresponding lithium salt 32, which was coupled with aniline 25 under standard conditions to provide, after deprotection, MK-3207 (14). 74 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 2. Medicinal Chemistry Synthetic Route to MK-3207 (14) The route to aniline (R)-25 consisted of eleven total steps with an overall yield of 15% from 7-azaindole. The route from bromoacetophenone 26 to MK3207 consisted of six total steps with an overall yield of 9%. These synthetic approaches enabled the medicinal chemistry team to rapidly explore the structureactivity relationships of these piperazinone CGRP receptor antagonists. The route also allowed the synthesis of multigram quantities of MK-3207 to support early toxicological and pharmacological characterization of the compound, but it was clear that significant improvements to the chemistry would need to be made as MK-3207 advanced into development.

Process Chemistry Development Within the pharmaceutical industry, organic synthesis encompasses both medicinal chemistry and process chemistry activities. Medicinal chemists focus on the discovery of appropriate small molecules that interact with biological mechanisms in such a way as to provide a beneficial effect on a given disease state. Process chemists aim to design and develop efficient, practical and economical chemical syntheses for the drug candidates identified by medicinal chemists. The differing goals of medicinal and process chemists have a significant bearing on the nature of the synthetic chemistry strategies employed by either group of chemists. Broadly, medicinal chemists typically design flexible synthetic routes that quickly access common building blocks from which many candidate compounds 75 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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can be prepared, allowing structure-activity relationships to be determined and the appropriate molecular entity to be identified. Reaction yield, expense, robustness and green chemistry considerations are of secondary importance relative to speed of compound synthesis and acquisition of data from biological assays. Conversely, the industrial manufacture of drug compounds at commercial scale puts a premium on these other synthesis attributes. Consequently, process chemists seek to devise an ideal route that has maximum synthetic efficiency and is high yielding. Often this necessitates the invention of new synthetic methods, providing an excellent venue for creative chemists to conceive novel applications of existing transformations or even to develop entirely new reactions. The commercial process must be operationally safe, cost-effective and sufficiently robust to deliver active pharmaceutical ingredient (API) in consistently high quality. Furthermore, given the larger scale of operation relative to medicinal chemistry activities, minimizing the environmental impact of industrial drug production is a priority and drives the application of green chemistry principles wherever possible. Lastly, intellectual property (IP) aspects cannot be neglected when conducting commercial operations and therefore manufacturing processes need to have freedom to operate without infringing upon competitor patents. In addition to the myriad technical objectives faced by process chemists, attention must also be given to broader, cross-functional facets of development programs including project drivers and timelines. Typically, in the early stages of pre-clinical and clinical drug development, relatively small quantities of drug substance are required to support initial in vitro and in vivo toxicology studies and the speed of API delivery is normally prioritized over definition of an ideal synthesis. This being the case, it is not unusual to have an interim synthesis (“supply route”) used to support early API deliveries with a parallel (or staggered) effort to develop increasingly refined routes that are aligned with the overall program context, considering factors such as drug demand, synthesis complexity/cost and clinical timelines. In an ideal world, the best, most efficient synthesis is established as early as possible in the overall timeline but in practice it is common to observe an evolution in efficiency over the development cycle, culminating in the manufacturing route being ready as the program moves into Phase III. Excellent reviews are available that discuss chemical process development in the pharmaceutical industry at greater length (57–60). The discussion above highlights several common themes of process chemistry development across the industry. However, the details of route development, timing, and selection criteria vary, based on the unique features of individual projects and also according to the experiences and philosophies of different companies. In particular, decision-making on long-term manufacturing routes can be influenced not only by quantifiable metrics such as cost-of-goods, but also more subjective measures such as scientific elegance, novelty and broader impact in the field. The following sections in this chapter describe the process chemistry development for MK-3207. One objective is to provide instructive case studies on how and why the chemistry evolved with increasing scale and changing priorities as MK-3207 progressed into and through clinical development. Another aim 76

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is to highlight our cornerstone philosophy at Merck of implementing the best possible chemistry for our manufacturing routes and continuing to build upon a tradition of process chemistry innovation. Pursuing this goal resulted not only the identification of a highly efficient long-term route to MK-3207 but also the discovery and development of a number of synthetic methodologies; these transcended the unfortunate demise of this specific compound by subsequently impacting other projects as well as being novel scientific advances in their own right.

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MK-3207 Fragment Coupling Strategy The medicinal chemistry synthesis of MK-3207 featured the use of an amide coupling between aniline 25 and N-Boc-piperazinone 32 to complete the assembly of the molecular framework of the API (Scheme 2). This transformation was also appealing from a process chemistry perspective in that it provided a convergent approach with late-stage coupling of two fragments of approximately equal size and complexity, and also had high likelihood of being able to be developed into a robust, scalable reaction. Therefore at the outset of our work the decision was made to retain this key bond disconnection. In the forward synthetic direction, an opportunity was identified to streamline the endgame by eliminating the use of protecting groups, resulting in development of the coupling strategy illustrated in Scheme 3.

Scheme 3. Modified Fragment Coupling Strategy to Generate MK-3207 The benefit of using of unprotected piperazinone (R)-33 came at the expense of introducing a chemoselectivity challenge, as aniline (R)-25 needed to react in preference to the secondary amine in the piperazinone (R)-33. The sterically hindered nature of the latter meant that the desired coupling mode was 77 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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indeed feasible. However, further coupling of the initially formed MK-3207 product to another molecule of piperazinone (R)-33 resulted in the formation of a troublesome impurity 34 that was difficult to reject, thus the coupling reagents and conditions needed to be optimized. A panoply of methods exists for effecting amide couplings, many of which have been employed on scale in the pharmaceutical industry (61). It was determined that EDC-mediated coupling minimized impurity formation and generated MK-3207 in high yield. After aqueous workup the API was crystallized from EtOH and isolated as a solvated form. This provided good rejection of other impurities (including stereoisomers), and enabled recrystallization in a subsequent purification step to obtain the desired final API form. Initially this was the HCl salt, before a freebase monohydrate form was selected for early clinical development. Subsequently, a more stable anhydrate phase was discovered and was to be developed for use in the commercial tablet formulation prior to discontinuation of MK-3207. The following sections describe development of routes to the key aniline and piperazinone fragments, highlighting new chemistry that was developed along the way.

Piperazinone – Process Chemistry First-Generation Synthesis The original synthesis of the chiral piperazinone fragment of MK-3207 was racemic in nature, and utilized preparative chiral column HPLC purification in order to access enantioenriched material (Scheme 2). This approach was perfectly suited to the goal of rapid discovery of the optimal drug candidate for further development. However, upon transition into the pre-clinical development space, identification of an asymmetric approach became a high priority objective for the process chemistry team. As is typical for early development projects, speed to the clinic was also highly desirable. To this end, the team focused early efforts around modifying the discovery synthesis to introduce control of chirality while maintaining many of the already established bond formations, so as to minimize development time on the path to the first GMP delivery of MK-3207 API.

Scheme 4. First-Generation Retrosynthesis of Piperazinone (R)-33 78 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Retrosynthetic analysis (Scheme 4) suggested that the piperazinone ring could be assembled via cyclization of the chiral benzylic amine 35 that would ultimately be derived from stereoselective displacement of an appropriate leaving group. Assuming the requisite leaving group would take the form of an activated alcohol (e.g., 36), ketone 28 could serve as a viable precursor to introduce the benzylic stereocenter via an asymmetric reduction process. Further disconnections to the simpler materials 26 and 37 would mirror that in the medicinal chemistry route.

Scheme 5. First Generation Synthesis of Piperazinone Acid (R)-33 The first generation process chemistry route to the chiral piperazinone acid (R)-33 began with addition of the Grignard reagent derived from 1-bromo-3,5-difluorobenzene (37, Scheme 5) to acetyl chloride in the presence of CuCl and AlCl3. The resulting 3,5-difluoroacetophenone (38) was then subjected to bromination with NBS under acidic conditions to afford 79 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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3,5-difluoro-α-bromoacetophenone (26). Bromination of 38 with molecular bromine resulted in the formation of around 11% of the dibromo impurity 42 (Scheme 6). This was problematic because the dibromo impurity participated in the subsequent step to form an imine 43 which reacted with another molecule of ketone 28 to generate the highly insoluble dimeric impurity 44. This impurity was first observed in the medicinal chemistry synthesis and it was difficult to remove from intermediate 28. Investigation of alternative reagents established that NBS gave a cleaner reaction profile with an acceptable level of 4% of the dibromo impurity 42.

Scheme 6. Fate of Dibromo Impurity 42: Generation of Dimer Impurity 44

Nucleophilic displacement of bromide 26 by cycloleucine 27 provided the α-aminoacetophenone derivative 28 which, as our medicinal chemistry colleagues had identified earlier, was somewhat unstable and required isolation as the more stable methanesulfonate salt (Scheme 5). Suitable conditions for the key asymmetric reduction of ketone 28 were then quickly identified using high-throughput experimentation (HTE) techniques that have been extensively developed and applied in our laboratories by our Chemocatalysis group (62–69). This transformation was performed under hydrogen in the presence of a chiral non-racemic ruthenium catalyst in MeOH/MsOH solvent mixture to deliver the aminoalcohol 39 in 60–70% enantiomeric excess (ee). In order to activate the benzylic hydroxyl group in 39 as a leaving group it was decided to form a cyclic sulfamate in a two-step process via the reaction with thionyl chloride followed by periodate oxidation. This choice of activation served to simultaneously protect the adjacent nitrogen and prevent any undesired participation from this group. The nucleophile to open cyclic sulfamate 36 was ethyl glycinate freebase (29), which must first be prepared via treatment of the commercially available hydrochloride salt 29•HCl with an appropriate base. Attempts to form the freebase process in situ using Hünig’s base were plagued by unwanted interference from soluble chloride ion, which was sufficiently nucleophilic to open the cyclic sulfamate and generate a benzyl chloride by-product 46, (Scheme 7). 80

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Scheme 7. Generation of Chloro Impurity 46 To avoid this issue, we investigated the option of forming the glycine ethyl ester free-base from the hydrochloride salt in a separate step using a two-phase aqueous Na2CO3/MTBE extraction. Unfortunately, this approach was also problematic because ethyl glycinate free-base is slightly volatile and some material was lost during the extended distillation required for azeotropic drying (water was deleterious to the cyclic sulfamate ring-opening). Further, the free-base is inherently unstable with a tendency to polymerize over time, which causes additional loss of yield. A practical solution to this dilemma that combined the most favorable attributes of aqueous and non-aqueous free-base techniques was ultimately identified. Pre-treatment of the hydrochloride salt with tetramethylguanidine (TMG) in MTBE resulted in the formation of ethyl glycine free-base in solution and concomitant precipitation of the TMG hydrochloride salt. The extremely low solubility of TMG•HCl in MTBE ensured near complete removal of the chloride ions from the solution by filtration, thereby avoiding the formation of the benzyl chloride by-product 46. The anhydrous nature of the process obviated the need for prolonged azeotropic drying. Application of this process allowed displacement of the oxygen leaving group at the benzylic center in a stereospecific fashion (inversion) to yield the intermediate 45 that spontaneously cyclized to the piperazinone ring 40 after hydrolytic cleavage of the N-sulfate residue. At this stage, the moderate enantiomeric excess stemming from the asymmetric ketone reduction was upgraded via diastereomeric salt formation using tartaric acid derivative 41 (Scheme 5). This provided material in greater than 99% ee that could be taken through the remaining chemistry with no loss of stereochemical integrity. To complete the synthesis of the piperazinone fragment, the tartrate salt was first broken and then the ethyl ester was hydrolyzed to reveal the target acid (R)-33.

Piperazinone – Process Chemistry Second-Generation Synthesis Early GMP deliveries of MK-3207 utilized the first generation synthesis of the piperazinone acid (R)-33 described above. Although this route ably supported initial animal toxicology and clinical studies, it was recognized that an alternative approach would be required in the long term. The first-generation route to piperazinone acid 33 comprised ten steps in the longest linear sequence. There were several key issues with respect to process robustness/efficiency and economics. Despite extensive study, the asymmetric hydrogenation to set the benzylic stereocenter remained only moderately effective and typically delivered 81 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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material with enantiomeric excess in the range of 60–70%. This relative lack of stereocontrol necessitated an upgrade via the dibenzoyltartaric acid salt in the downstream chemistry, compromising the overall process efficiency. Additionally, both the aminoketone 28 and ethyl glycinate (29) intermediates had limited chemical stability and created issues for process robustness. Furthermore, cost analysis of this route to piperazinone acid 33 identified the synthetic amino acid "cycloleucine" (27) as a major contributor. Since this reagent constitutes an integral part of the molecular structure of MK-3207, its use is essentially mandatory in any synthesis. Consequently, introduction of this component at such an early stage in a ten-step sequence was not the ideal strategy from an economic standpoint. Taking all of these factors into account, together with the Merck goal to implement the best chemistry for our commercial products, the process chemistry team set out to design a second-generation approach to the synthesis of piperazinone acid 33 that would provide higher synthetic efficiency, better stereocontrol and greater overall economy (70).

Scheme 8. Second-Generation Retrosynthesis of Piperazinone 33 Retrosynthetic analysis for the second-generation route to the piperazinone acid 33 intermediate is shown in Scheme 8. The first disconnection revealed the core piperazinone heterocycle 47 and it was anticipated that selective alkylation of the amide in the presence of the secondary amine would be achievable under appropriate conditions. A benefit of this disconnection was that the source of the acid side-chain was now a readily available α-haloacetate reagent. This would obviate a process robustness issue in the first-generation synthesis, where the unstable glycine ethyl ester freebase was used as the source of this molecular fragment. To access the core piperazinone 47, it was recognized that a cyclic sulfamate intermediate 48 with “inverted” regiochemistry from the first generation synthesis could significantly shorten the synthetic sequence and confer several additional advantages. In addition, cyclic sulfamates participate in ring-opening/ring-closing reaction sequences with bifunctional reagents (e.g., amino acids) to afford piperazinones in a single step (71, 72). Due to steric encumbrance, cycloleucine 82

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appeared to be a challenging reaction partner for this process, but in light of the overall synthetic advantage conferred by this disconnection, we deemed it worthy of investigation. Having identified the key chiral intermediate, a solution to the problem of the benzylic stereocenter was required. Asymmetric hydrogenation of unsaturated compounds represents one of the most attractive and practical options for control of functionalized stereocenters (73, 74). Consequently, cyclic imine 49 was targeted as a likely precursor of the chiral sulfamate and the team sought to derive this cyclic imine from hydroxyacetophenone 50, itself available from 1-bromo-3,5difluorobenzene 37 as the raw material. Hydroxyacetophenone Synthesis α-Hydroxyacetophenones are useful synthetic intermediates amenable to various asymmetric transformations capable of generating valuable chiral compounds (75–77). Although α-hydroxyacetophenones had featured many times in the chemical literature, at the time of this research there were only a limited number of reports directly focused on general procedures for their preparation (78–87). This is perhaps indicative of underlying stability issues associated with these intermediates. Indeed, significant stability problems were encountered during early attempts to work with intermediate 50. However, these difficulties were eventually resolved after we understood the instability of the compound towards both oxygen and neutral-to-basic pH. Thus, a straightforward and general approach to the synthesis of α-hydroxyketones using readily available reagents was developed for the synthesis of the desired compound 50 (Scheme 9) (88).

Scheme 9. Synthesis of Hydroxyacetophenone 50 The arylzinc intermediate derived from 3,5-difluorobromobenzene was acylated with α-acetoxy acetyl chloride in the presence of CuCl to produce α-acetoxy-3,5-difluoroacetophenones (51) (Scheme 9). Treatment of 51 with 5 N aqueous HCl at 40 °C in MeOH led to the formation of α-hydroxy-3,5-difluoroacetophenone (50). We noticed that the hydroxyketone 50 is relatively sensitive to oxygen in solution, presumably via facile oxidation of equilibrium concentration of the enol tautomer. For this reason, we carried out the hydrolysis of 51 with 5 N aqueous HCl under nitrogen in deoxygenated MeOH. Under these conditions the formation of polymeric degradants was minimized and the assay yield of 50 became generally good. The product 50 is isolated by direct crystallization from the reaction mixture after dilution with water. With the ready access to hydroxyacetophenone 50 secured, the team was positioned to develop the subsequent planned synthetic transformations. 83

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Cyclic Sulfamate Synthesis Cyclic sulfamates similar to 49 have been described previously in the literature. Initial laboratory investigations quickly revealed the known methods of preparation to be entirely unsuitable for large scale operation (89). The literature method (Scheme 10) relies on an in situ preparation of sulfamoyl chloride, an unstable compound that is not readily commercially available on scale. Reported procedures involve a neat reaction between N-chlorosulfonylisocyanate (CSI) and 95% aqueous formic acid, which is relatively hazardous as it releases one mole equivalent each of CO and CO2. Small scale laboratory testing revealed this reaction is highly exothermic and the mixture solidified as conversion proceeded, preventing agitation and making control of the exotherm even more problematic. In the next stage, sulfamoyl chloride was combined with the α-hydroxyacetophenone 50 in the presence of pyridine to yield the O-sulfamoyl intermediate 54, which was cyclized and dehydrated to the cyclic sulfamate via thermal treatment during workup. A significant side-reaction was the nucleophilic attack by chloride ion on intermediate 54 to yield the α-chloroacetophenone 55. Pyridine is a poor choice of base in this regard because pyridinium hydrochloride has reasonable solubility in the reaction medium and facilitates side-product formation (vide infra). In addition to having a detrimental effect on yield, chloroketone 55 is a severe lachrymator, creating handling/industrial-hygiene issues during workup.

Scheme 10. Literature Conditions for Preparation of Cyclic Sulfamate 49 This procedure was deemed unsuitable for large-scale synthesis and spurred development of a new process that was both safer and higher yielding (Scheme 11). The reaction of t-BuOH with CSI was essentially quantitative and generated N-Boc-sulfamoyl chloride (56) cleanly. Since this process is a simple addition reaction there are no gaseous by-products. Also, the reaction was conveniently carried out in 2-Me-THF, allowing for good control of the exotherm via rate of addition of reagent. Range-finding experiments indicated that over-reaction of excess t-BuOH with the initially formed N-Boc-sulfamoyl chloride was not an issue under the reaction conditions. The resulting solution of N-Boc-sulfamoyl chloride 56 had good stability, which afforded an acceptable operating window for large scale processing. 84 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Combination of 56 with the hydroxyacetophenone 50 generated negligible exotherm because there was no reaction until the subsequent addition of triethylamine; the O-sulfamoylation exotherm was then controlled via addition rate of the base. O-Sulfamoylation generates intermediates 54 and 57 that are activated towards nucleophilic displacement by chloride ion, generating the undesired α-chloroacetophenone side-product 55. The reaction temperature and age time also had a significant impact on the amount of impurity 55 formed, with greater than 90% conversion to this compound after 24 h age at room temperature using pyridine as the base. To mitigate these issues, we used triethylamine as the base and carried out the reaction at –5 to 0 °C. Under these conditions, triethylamine hydrochloride precipitated from solution, and that reduced the concentration of dissolved chloride ion to suppress the formation of 55. Cooling below –10 °C decreased the solubility of substrate 50, and thus the rate of reaction, significantly. Above 0 °C the solubility of triethylamine hydrochloride increased and the level of α-chloroacetophenone side-product 55 would accumulate over time. Under the optimal conditions, the O-sulfamoylation reaction was typically complete within 30 min and the reaction was then quenched by the addition of 0.5 M NaHSO4, maintaining the batch temperature around 0 °C. The use of 2-Me-THF as solvent allowed direct phase separation and the rejection of triethylamine hydrochloride into the lower aqueous phase. A second wash with additional 0.5 M NaHSO4 ensured negligible concentration of chloride ion in the organic phase and rendered the intermediate O-sulfamoyl compound 57 stable for continued processing at elevated temperatures.

Scheme 11. Improved Synthesis of Cyclic Sulfamate 49 Although it was possible to isolate the N-Boc-protected-O-sulfamoyl intermediate 57 via crystallization, the opportunity to continue in a through process to the desired cyclic sulfamate 49 was attractive. Accordingly, the wet 2-Me-THF solution of uncyclized intermediate 57 was treated with a catalytic quantity of p-TsOH•H2O (1 mol%) and the reaction mixture was heated at reflux to effect sequential N-Boc deprotection and cyclization/dehydration to the cyclic sulfamate 49. The N-Boc group remained largely intact until the batch 85 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

temperature reached at least 60 °C and then cleaved smoothly under the action of catalytic acid. Over the course of several hours the conversion to cyclic sulfamate 49 reached greater than 95%, and then close to complete conversion was attained via azeotropic removal of water by distillation. The final isolation involved cooling and washing with water to remove residual inorganics (NaHSO4) from the organic phase, followed by crystallization of 49 from a mixture of 2-Me-THF and heptanes. The overall assay yield of sulfamate 49 from hydroxyketone 50 was 94% and the isolated yield was 86%.

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Asymmetric Hydrogenation of Cyclic Sulfamate Concurrent with this process development work, a literature report appeared from Zhou and coworkers on the asymmetric hydrogenation of cyclic sulfamates (89). In this paper, the levels of conversion and enantiocontrol were excellent across a variety of substrates, so we were optimistic around the use of 49. However, the optimal conditions described by Zhou had several potential drawbacks with respect to large scale operation. From an economic perspective, the use of 2,2,2-trifluoroethanol (TFE) as solvent, Pd(TFA)2 as a catalyst precursor and (S,S)-f-binaphane as the chiral ligand would all contribute to a high cost for the key asymmetric transformation in the second-generation route. Furthermore, in practical terms, the requirement for relatively high pressures of hydrogen gas (500 psig) could limit options for implementing this chemistry at vendors lacking appropriate plant equipment. To discover an improved process for this key transformation, the project team embarked upon a systematic study of the reaction conditions. Determination of the optimal conditions for this asymmetric hydrogenation was again achieved through HTE techniques. For initial screening of reaction conditions, certain aspects of the published procedure (such as the TFE solvent and the 500 psig pressure of hydrogen) were retained while the catalyst precursor and ligand were varied. A control experiment where the exact literature conditions were used was also conducted. A summary of results is shown in Table 2. The control experiment using Pd(TFA)2 and the (S,S)-f-binaphane ligand 58 gave a similar result to that published. Complete conversion was reached in all cases and excellent enantiomeric excess was also observed using Rh, Ir and Pd catalyst precursors in conjunction with several alternative commercially available phosphine ligands. The Josiphos ligand 60 was selected for further study because this was readily available on production scale as part of the process for another Merck product (Januvia®) (90). By examining the other reaction parameters (Table 3), it was established that Pd(OAc)2 could replace Pd(TFA)2 with similar performance. More significantly, in contrast to the literature report, replacement of TFE with MeOH as the reaction solvent was equally effective and conferred several process advantages such as cost reduction, alleviation of industrial hygiene concerns around TFE and the opportunity for a simple product isolation (vide infra). Also notable was the option to conduct the hydrogenation using significantly lower hydrogen pressures. 86 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Table 2. Conditions for Initial Evaluation of Metal/Ligand for Asymmetric Hydrogenation of Cyclic Sulfamate 49

In the final process (Scheme 12), cyclic sulfamate 49 was dissolved in 5 volumes MeOH and subjected to 0.3 mol% catalyst loading (0.33 mol% ligand 60) under 40 psig of hydrogen at 40 °C. After complete conversion the batch was treated with carbon, filtered and the desired product was isolated via crystallization following addition of water. The isolated yield was 94% and the white solid was typically of greater than 99 wt% purity. The measured enantiomeric excess of the isolated material matched the in-process-control assay, indicating no upgrade was available via this crystallization. Subsequent crystallization of a downstream intermediate afforded the necessary upgrade in stereochemical purity (91). Piperazinone Formation Cyclic sulfamates are known to undergo reactions with amino esters that ultimately cascade to piperazinones (71, 72). For the target piperazinone 33 the amino ester required was the unnatural but commercially available "cycloleucine" methyl ester (27). Due to steric hindrance, cycloleucine is an extremely poor nucleophile. In contrast to many other amino esters, cycloleucine freebase is relatively stable for extended periods and did not polymerize to any significant extent upon storage. This lack of reactivity necessitated significant process development in order to achieve an acceptable rate of reaction with cyclic sulfamate 48. Scheme 13 illustrates the reaction conditions initially used to gain proof-of-concept for this particular piperazinone formation, as well as the subsequently optimized process. 87

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Table 3. Optimization of Catalyst Precursor and Loading, Solvent, Hydrogen Pressure and Reaction Temperature

Scheme 12. Optimized Conditions for Asymmetric Hydrogenation of 49

Cycloleucine methyl ester 27 is available commercially as the hydrochloride salt, which needed to be converted to the corresponding freebase before reaction with cyclic sulfamate 48. In our initial studies, cycloleucine freebase was extracted into toluene from aqueous potassium phosphate tribasic. Concentration of the organic phase allowed for azeotropic drying prior to reaction with the cyclic sulfamate. Heating the dry toluene solution of cycloleucine 27 and cyclic sulfamate 48 at 70 °C led to ring opening to give intermediate 61, and then 5 M aqueous HCl was added to effect the cleavage of the N-sulfate to generate 1,2-diamine 62. However, the two-phase nature of the toluene/water system made the sulfate hydrolysis very slow. Addition of an acid stable co-solvent (1,2-dimethoxyethane) was necessary to make the phases partially miscible and increase the rate of hydrolysis to a practical level. After hydrolysis, the majority of diamine 62 was present in the acidic aqueous phase (as determined by HPLC assay) while the organic phase contained some dark polymerized material, which was separated via a phase cut at this stage. Adjustment of the pH via treatment 88

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with 10 M aqueous NaOH resulted in piperazinone formation and partitioning into the freshly replaced organic phase. After phase cut and solvent switch the desired piperazinone 47 was isolated via crystallization. Although the overall yield was reasonable, a close examination of the various operations revealed several opportunities for streamlining the unit operations. The overall piperazinone process involves four distinct stages: •

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• • •

formation of cycloleucine methyl ester free base from hydrochloride 27•HCl ring opening of cyclic sulfamate (48→61) N-sulfate cleavage (61→62) pH adjustment/ring closure to form the piperazinone (62→47)

Scheme 13. Reaction Sequence for Formation of Piperazinone 47 In developing this overall process, the following aspects received close attention: • • • •

the volatility of cycloleucine methyl ester 27 during free-basing process. the dipolar aprotic solvent used to enhance nucleophilic ring opening. the potentially deleterious presence of extraneous nucleophiles (including water and certain solvents). solvent/water miscibility and pH stability across a wide range for Nsulfate cleavage and lactamization. 89

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To address the sluggish nature of the ring-opening step, we evaluated several solvents with particular focus on those likely to facilitate formation of the highly polar reaction intermediate (N-sulfate 61). Protic solvents such as MeOH and EtOH were excluded after experiments confirmed partial solvolytic opening of the cyclic sulfamate starting material 48. More surprising, but unfortunately also discouraging, were observations that typical dipolar aprotic solvents such as THF, MeCN, DMF, DMAc and NMP did not provide improved results. In fact, in the case of DMF and DMAc, these solvents were sufficiently nucleophilic to be competitive with cycloleucine 27 and consequently led to unwanted side reactions and poor reaction profiles. Following these observations attention was turned to sulfolane, a solvent that appeared a good candidate for this particular process (92). Sulfolane has one of the highest dielectric constants of any solvent and was expected to promote the initial desired ring opening to yield N-sulfate 61. Additionally, and in contrast to other common dipolar aprotic solvents, sulfolane is non-nucleophilic, which virtually eliminated non-productive solvolytic processes such as those encountered with DMF. Sulfolane is also miscible with water and stable at low pH, which helped streamline the overall piperazinone formation process. The finalized process for piperazinone formation was as follows (Scheme 13). Cycloleucine methyl ester freebase 27 was generated via partitioning of the hydrochloride salt 27•HCl between aqueous K3PO4 and MTBE followed by azeotropic drying of the MTBE layer. The relative volatility of MTBE minimized loss of the cycloleucine methyl ester free base 27 during this distillation process. Upon reaching the desired water content specification (below 100 ppm) the MTBE solution was concentrated and transferred into a solution of cyclic sulfamate 48 in sulfolane. Further distillation under reduced pressure removed MTBE from the system and the resulting highly concentrated sulfolane solution of reactants was heated to 70 °C to promote the desired ring opening. Typical conversion to intermediate 61 was greater than 95% after heating for 10 h. Addition of 5 M aqueous HCl and continued heating led to hydrolysis of the N-sulfate; the water-miscibility of sulfolane rendered this organic/aqueous system homogeneous and facilitated hydrolysis. Next, the system was adjusted to pH 10 using aqueous NaOH and heated to ensure complete lactamization. At the end of reaction, the mixture was extracted with MTBE and, after washing with brine, the majority of the sulfolane was rejected to the aqueous phase. The product piperazinone 47 was isolated via crystallization following solvent switch into n-heptane. The typical corrected isolated yield was 70% (96% ee, unchanged from the input stream of starting material 48). Given the lack of stereochemical upgrade achieved via the isolation of piperazinone 47, it appeared more attractive to through-process this intermediate into the final N-alkylation step. The crystallization after the N-alkylation step proved highly robust, consistently affording good overall purity and sufficient stereochemical upgrade, as described in the next section.

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N-Alkylation of Piperazinone Amide Chemoselective N-alkylation of the piperazinone amide was accomplished via deprotonation using NaHMDS in THF followed by treatment with ethyl bromoacetate (Scheme 14). The resulting ester 30 was hydrolyzed in situ during workup with aqueous LiOH, then acidification to pH 1 with aqueous HCl led to precipitation of the crude intermediate hydrochloride salt. Re-slurrying of this salt in water followed by treatment with one equivalent NaOAc and heating to 80 °C allowed neutralization and crystallization of the product 33 in the free form. This crystallization also afforded the necessary upgrade in enantiopurity to deliver material that matched the previously established purity specifications for this regulated API starting material (> 99 LCAP, 99.9 HPLC wt% purity, 99.7% ee). Additionally, using piperazinone acid 33 from this new route in the final amide bond formation with aniline 25 generated MK-3207 API that met the established acceptance criteria.

Scheme 14. Alkylation of Amide 47 and Hydrolysis to Generate Piperazinone Acid 33 The overall second-generation process chemistry route is illustrated in Scheme 15. The synthesis is highly enantioselective, with cascade reactions and through-processing of reaction streams figuring prominently to rapidly build up molecular complexity. The novelty and efficiency of this piperazinone route set a high bar for spirooxindole aniline (R)-25 to match, and the development efforts for this latter intermediate are described in the following section.

Spirooxindole – Process Chemistry First-Generation Synthesis As MK-3207 entered preclinical development, the problem statements for the scale-up of spirooxindole (R)-25 were analogous to those presented by the piperazinone fragment (R)-33. Spirooxindole (R)-25 is a complex synthetic target. The molecule is chiral, in this case bearing an all-carbon quaternary stereocenter that has the unique feature of being rendered stereogenic by the remote aniline substituent four bonds away. To enable initial entry to the clinic, a first GMP delivery of MK-3207 was needed on kilogram scale, representing a quantity of material two orders of magnitude greater than the total amount that had been produced up to that point. The program was on an aggressive timeline, and there 91 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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was only a narrow window to develop chemistry that could be scaled to deliver material that met the appropriate attribute and quality requirements.

Scheme 15. The Second-Generation Synthesis of Piperazinone Acid (R)-33 Such challenges are commonly encountered by process chemists across industry, necessitating early decisions be made on a case-by-case basis as to whether existing chemistry can be developed for initial clinical supply or if a new route should be developed. The former benefits from knowledge and experience with a proven way of making API on small scale but may encounter significant issues that slow or even prevent scale-up; the latter can yield a more efficient synthesis but at the cost of up-front commitment of time and resources at a phase of a program where industry-wide compound attrition rates are above 90% (93, 94). Successfully bridging from a discovery-focused synthesis that prizes rapid generation of diverse analogs for profiling to a scalable route for clinical supply of a specific target is one of the critical ways process chemists use their skillsets to enable programs in early development. For spirooxindole (R)-25, the initial route utilized by our medicinal chemistry colleagues offered an expedient approach to carbon-carbon bond formation with the spirocyclic ring system being generated in a single step by double alkylation of oxindole 18 with dibromide 21 (Scheme 16). This generated spirocycle 22 as a racemate, with the desired (R)-enantiomer being isolated using preparative HPLC on a chiral stationary phase. 92

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Scheme 16. Initial Synthesis of Spirooxindole (R)-25

Attracted by the good yields for the individual chemical steps and the convergent assembly of the spirocycle, the process chemistry team elected to focus on development of this route for the first GMP API delivery. Key primary objectives of this work were to: • • • •

establish robustness of the chemistry and address potential safety issues to enable successful kilogram-scale production. increase volumetric productivity of the reactions. improve product isolations. identify quick wins for yield improvement.

Additional opportunities were also identified to shorten the sequence by reducing the use of protecting groups and functional group interconversions, and potentially substitute the preparative HPLC chromatography with a more productive method for resolution of enantiomers. The modified spirooxindole route utilized for the first GMP API delivery is illustrated in Scheme 17. The commonality with the medicinal chemistry route is readily apparent, but it also features a number of developments and innovations to enable the larger-scale production. Dibromide 21 was prepared from 4-nitrophthalic acid 19 following the initial reduction/bromination sequence, but with some procedural modifications. Reduction was accomplished by addition of a THF solution of diacid 19 to 1 M BH3•THF followed by quenching with MeOH, aging with 2 M aq NaOH to break up boron complexes (95), and extraction with EtOAc. A crystallization of the resulting diol 20 from EtOAc/n-heptane was developed that eliminated the need to concentrate to dryness to obtain a solid product. For the subsequent bromination, Et2O was replaced as the reaction solvent with a safer mixture of MTBE/THF (96), and after aqueous workup the product 21 could be crystallized from EtOH/water in good overall yield. As in the initial synthesis, commercially available 7-azaindole 15 was used as the starting material for the preparation of the azaoxindole coupling partner for the dialkylation reaction. In this case, however, a one-step access to unprotected 93

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oxindole 63 was developed in collaboration with our Biocatalysis group. Mediated by chloroperoxidase in an aqueous-rich solvent system and with H2O2 as the oxidant, this direct transformation proceeded under much more environmentally benign conditions than the previous oxidation/reduction sequence and also reduced processing time, improved overall yield, and eliminated the use of the costly SEM protecting group. Critical to the success of the oxidation of 7-azaindole 15 to oxindole 63 was slow addition of H2O2 to avoid prolonged build-up of reagent in the reaction medium, as the enzyme was deactivated in the presence of excess peroxide.

Scheme 17. First Kilogram-Scale Synthesis of Spirooxindole (R)-25

The use of unprotected oxindole 63 increased the complexity of the spirooxindole formation by introducing the amide nitrogen as a competing reactive site for alkylation with dibromide 21. Use of the original Cs2CO3/DMF conditions gave an assay yield of only 21%, despite an ostensibly clean reaction profile by HPLC analysis. The formation of polymeric impurities was suspected to be the root cause of this discrepancy. Significant screening and optimization was required to ultimately arrive at the conditions shown in Scheme 17, utilizing LiOH in a mixture of THF/water at room temperature. Reaction workup was accomplished by extracting the desired product into a basic aqueous layer, with the majority of the impurities partitioning into the organic phase. Acidification then resulted in crystallization of spirooxindole 64, which was isolated in 60% yield. Conversion of racemic alkylation product 64 to final spirooxindole (R)-25 closely paralleled the initial medicinal chemistry route. Heterogeneous hydrogenation was used to reduce the aromatic nitro group to the corresponding aniline (±)-25, which was then converted to N-Boc-protected derivative 65. Initial attempts at classical resolution of intermediates in this sequence were unsuccessful, thus a chromatographic resolution was retained. Resolution was accomplished by preparative supercritical fluid chromatography (SFC) on a chiral 94

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stationary phase with reasonable recovery of the desired (R)-enantiomer. The free aniline was then liberated by acidic cleavage of the Boc group followed by neutralization to isolate spirooxindole (R)-25 as the free-base. The first-generation process chemistry route to aniline fragment (R)-25 proceeded in eight total steps (removing three steps from the original route), and was used to produce multikilogram quantities of material for the first GMP API delivery, enabling MK-3207 to rapidly advance to into Phase I clinical trials. Behind the headline numbers of step count and material quantity, this work featured a number of examples of chemistry innovation and collaborative problem-solving across the Merck process chemistry network, all conducted against a tight timeline. The successful completion of the first GMP API delivery reflected the collective efforts and accomplishments of all the team members. It also specifically exemplifies the potential for beneficial impact of applying novel enzymatic processes early in compound development. This route would also serve as the foundation for subsequent deliveries of spirooxindole (R)-25 that supported continued progression of MK-3207 through early clinical development. However, there was a critical bottleneck that needed to be addressed to enable production of this intermediate on increasing scale beyond the first GMP delivery.

Spirooxindole - Classical Resolution for Clinical Supply While the chromatographic resolution to obtain enantiomerically pure (R)-25 supported the program for the first GMP API delivery, the limitations of this methodology for larger preparation of MK-3207 were well understood. The chromatography of aniline derivative 65 was sandwiched between installation and subsequent removal of a Boc group on the aniline nitrogen (Scheme 17). This was a necessary maneuver, implemented solely for the purpose of increasing the solubility of the spirocycle enough to enable preparative-scale separation to be feasible at all. Generally poor solubility across a wide pH range was found to be one of the defining physicochemical characteristics of the rigid core spirocyclic ring system. The Boc protection/deprotection was an unfortunate sequence but considered manageable for clinical supply. The more pressing issue was that, even under the optimized conditions, the productivity and material throughput of the chromatography was still very low. The projected long cycle times and high cost (not to mention the environmental impact of large solvent volume usage) meant that it would not be practical for larger deliveries, and developing an alternative resolution protocol was viewed as a priority objective by the team. Efforts were focused on revisiting classical resolution based on diastereoselective salt formation, taking advantage of the (weakly) basic aniline and/or pyridine nitrogen atoms. Extensive salt screening was performed on the nitro compound 64 and free aniline (±)-25 intermediates in the existing synthetic sequence, as well as on a number of aniline amide derivatives (66, Figure 5). 95

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Figure 5. Substrates evaluated for classical resolution.

Following many failed attempts, there was a key breakthrough while investigating the use of di-p-toluoyl-L-tartaric acid (67) as the resolving agent with aniline (±)-25 (Scheme 18). A crucial experimental finding was that resolution was only successful when AcOH was used as the solvent. In this system the desired (R)-aniline salt preferentially formed a crystalline material that was determined to be an AcOH solvate (1:1:1 molar ratio of aniline:tartaric acid:AcOH); the undesired (S)-aniline remained primarily in solution. Other solvent systems did not exhibit this phenomenon and did not lead to efficient diastereoselective salt crystallization. Aniline salt (R)-25•DTTA•AcOH was isolated in high diastereomeric excess and in excellent recovery from the racemate. Subsequent neutralization afforded the free base (R)-25 in 96% ee, an acceptable level of stereochemical purity as the minor (S)-enantiomer could be rejected during the crystallization of the final API following coupling to the piperazinone fragment 33.

Scheme 18. Classical Resolution of Aniline (R)-25

This classical resolution eliminated the chromatography and the Boc protection/deprotection steps, and was implemented in pilot plant campaigns to produce aniline (R)-25 for further clinical supply. Persistence and attention to detail by the team members were critical to the discovery and implementation of this resolution, highlighting these as valuable traits needed for process development. 96 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Spirooxindole - Development of an Asymmetric Route

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Initial Route Scouting As the MK-3207 program continued to advance, our attention turned to developing chemistry to enable the production of spirooxindole (R)-25 on much larger scale to support late-stage clinical development and ultimately commercial manufacture. The route described above is relatively short and supported early clinical supply well. However, viewed through the lens of long-term manufacturing needs, our analysis was that it did not meet key criteria. This was both in direct measures such as cost, productivity, and sustainability but also, as for the piperazinone fragment 33, our high expectations of utilizing the best chemistry for our commercial products. In the route described in Scheme 17, azaindole 15 and the chloroperoxidase enzyme were expensive raw materials that were both used in the first step. The throughput of some steps was quite low—for example, the dialkylation reaction had to be run in ~ 100 volumes of solvent to maximize yield at 60%, contributing to a high overall Process Mass Intensity (PMI) (97) for the route. The overall yield was lower than desired. Ultimately, a key issue was that the synthesis was racemic. The decision was therefore made to focus on development of a new asymmetric route. 3,3′-Disubstituted oxindoles in general, and spirocyclic systems in particular, are prevalent in molecules of biological and therapeutic interest, and work across academia and industry continues to be directed towards developing methodologies for their enantioselective synthesis (98–101). Many elegant approaches have been reported, however we felt that the unique structure of spirooxindole (R)-25 presented us with an opportunity for innovation and the possibility of making our own impact in this field. Our strategy consisted of initial scouting of multiple routes in parallel, so that we may establish proof-of-principle on key transformations and make rapid decisions to focus efforts on the more promising options, and then finally select one route for full process development. Many creative ideas were generated and evaluated by the team, a selection of which is highlighted in Scheme 19. These routes feature a number of different bond disconnections and strategies for assembly of the spirocycle, but each is characterized by a unique proposal for generation of the quaternary stereocenter using asymmetric catalysis. Early no-go decisions were made on three of these routes. The reductive Heck approach was conceptualized as an extension of intramolecular Heck chemistry for the synthesis of 3,3′-disubstituted oxindoles pioneered by the Overman group (102). However, the position of the trisubstituted alkene in indene 68 was found to be challenging to control during its synthesis and prone to subsequent migration, negatively impacting the potential enantioselectivity of any cyclization. This regioisomer issue was negated in the asymmetric arylation approach by the use of saturated indane substrates 69, but in this case efficient, enantioselective Pd-mediated cyclization was thwarted by the low reactivity of this system towards generating the sterically congested quaternary center, and also the lack of discrimination between the two prochiral faces of the indane due to the remote location of the aniline substituent. 97 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 19. Overview of Some Routes Evaluated for Long-Term Manufacture of Spirooxindole (R)-25

The enzymatic desymmetrization route offered a more distinct strategy for sequential assembly of the spirocyclic ring system from an acyclic precursor, but there was a lack of differentiation between the ester groups in preliminary experiments. The two approaches that were most extensively investigated were the tandem asymmetric Heck/arylation route and asymmetric phase-transfer catalysis.

Tandem Heck/Arylation Reaction The premise of this approach is illustrated in Scheme 20. It was proposed that treatment of substrate 72 with an appropriate palladium catalyst precursor and chiral ligand could lead to the formation of two carbon-carbon bonds, both the oxindole and indane rings, and the quaternary stereocenter in a single operation. This was to be achieved by combining sequential asymmetric Heck and C–H functionalization steps. The intramolecular Heck reaction is established as a powerful method for the construction of (poly)cyclic ring systems (102–105), and in recent years direct arylation has become an important part of the blossoming field of C–H activation chemistry (106). However, precedent for combination of these methodologies into a single catalytic cycle was limited (107). 98 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 20. Proposed Catalytic Cycle for Tandem Pd-Mediated Reaction Cyclization precursor 83 was expeditiously prepared as shown in Scheme 21, with a key step being generation of the requisite 1,1-disubstituted alkene in a regiocontrolled manner via a Mannich reaction/decarboxylation/elimination cascade. Gratifyingly, proof-of-concept for efficient bond formation in the cascade was quickly established. Using Pd(OAc)2 and the achiral phosphine ligand P(t-Bu)3 with carbonate base in DMAc, cyclization to the desired racemic spirooxindole 84 could be effected in very high yield.

Scheme 21. Preparation and High-Yielding Cyclization of Substrate 83 Encouraged by this result, we turned our attention to the goal of developing an asymmetric variant of this transformation. Unfortunately, and despite considerable efforts with extensive screening of chiral ligands and reaction conditions, no hits were identified and ultimately it was not possible to perform 99 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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this chemistry in an enantioselective manner. Proposals of mechanistic rationale for this negative outcome were that a fast, but reversible, alkene insertion may be followed by a minimal discrimination between the two diastereomers of alkylpalladium(II) intermediate 76 (Scheme 20) in the subsequent C-H activation, and/or that 1,1-disubstituted alkene substrate 83 lacks the structural elements required for enantiofacial discrimination by the catalyst (1,1,2-trisubstituted alkenes are more commonly employed in asymmetric Heck reactions). The use of alternative protecting groups (or none) on the amide nitrogen did not resolve this issue. Consequently this approach was deprioritized for the long-term manufacture of spirooxindole (R)-25. Although a disappointing outcome in the specific context of the synthesis of MK-3207, this chemistry nevertheless offers a rapid entry into the complex spirooxindole architecture, and the broader scope was successfully explored as follow-up to this work (Scheme 22) (108). More recently, Zhu and co-workers have further developed Heck/C-H functionalization methodology into a cascade for the synthesis of [3,4]-fused oxindoles (109).

Scheme 22. Broader Scope of Tandem Heck/C–H Functionalization for Spirooxindole Synthesis

Phase-Transfer Catalysis The original oxindole dialkylation chemistry, first employed by our Medicinal Chemistry colleagues and subsequently modified for kilogram-scale deliveries, provided a rapid access to the spirocyclic framework in racemic form. The proposal that this chemistry could somehow be performed in an enantioselective manner (Scheme 19) was thus attractive in its simplicity; reducing it to practice was anticipated to be anything but such. We chose to focus on evaluation of asymmetric phase-transfer catalysis (PTC). Our motivation for this was driven partly by practical considerations, such as the potential to employ more environmentally benign, mild reaction conditions inherent to this organocatalytic methodology, but also by Merck’s experience and tradition in this field. It is now more than three decades since a landmark publication from our laboratories describing a synthesis of (+)-indacrinone (90, Scheme 23) employing an enantioselective methylation of indanone 87 catalyzed by the cinchonine-derived quaternary salt 88 (110, 111). This was one of the first reports of a practical method for catalytic enantioselective alkylation and helped open the door for further research in asymmetric PTC across academia 100

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and industry (112), including many additional examples from these laboratories (113–116). Today, asymmetric PTC is a burgeoning area that has seen remarkable advances in reaction scope, catalyst design, and practical application (117–119).

Scheme 23. Enantioselective PTC Alkylation in the Synthesis of (+)-indacrinone 90 The opportunity to make a novel contribution to the field with development of spirooxindole (R)-25 was therefore enticing, but this chemistry was anticipated to be challenging. At the outset of this work, only a single example of asymmetric PTC alkylation of an oxindole substrate (Scheme 24) had been reported (120) (other elegant asymmetric oxindole PTC reactions have been reported more recently) (121–126), and application to generate spirooxindoles was unprecedented. Differentiation between the two leaving groups in a bis(benzylic) electrophile 73 was not expected to be trivial (127, 128). From a practical perspective, challenges are presented by the heterogeneous nature of PTC reactions, as well gaps in detailed understanding of specific phase-transfer mechanistic pathways which often necessitate an empirical approach to reaction development.

Scheme 24. Precedent for Oxindole PTC Alkylation

Initial Results The first lesson learned from our PTC development work was that protection of the oxindole amide nitrogen was going to be required. Attempted asymmetric alkylation of the unsubstituted substrate 63 led to tarry, intractable mixtures in 101 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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which the starting materials were consumed but minimal amounts of the desired product 25 were formed (Scheme 25), with competing oligomerization believed to be a significant issue. That a scalable racemic alkylation of oxindole 63 using LiOH in THF/water had previously been successfully developed for the first GMP delivery underscored both the sensitivity of this chemistry to the reaction conditions and the skill of the team members in developing the earlier clinical supply route.

Scheme 25. Inauspicious First Attempts at PTC Alkylation

Hopes were initially raised with use of PMB-protected oxindole 94 (Table 4). Encouragingly, reaction with nitro-containing dibromide 21 using the original Merck catalyst 88 provided a cleaner reaction profile and measurable enantioselectivity for formation of spirooxindole (S)-64 (Table 4, entry 2). Less encouragingly, the level of enantioselectivity was mediocre. Alkylation was more rapid using the structurally distinct first- and second-generation binaphthyl-derived quaternary ammonium salt catalysts pioneered by the Maruoka group (97 (129) and 98 (130), respectively), but the enantioselectivity was not significantly higher (entries 3 and 4). The low conversion observed in the absence of catalyst (entry 1) indicated that competing background (i.e., uncatalyzed) alkylation was not a major contributing factor to the low enantioselectivities. Switching the aromatic substituent in the electrophile from nitro to methoxy resulted in marginal improvements (entries 5–7), but the enantioselectivity could not be improved further with modification of the base, solvent, or reaction temperature.

Modified PTC Strategies and Unexpected Findings A generous claim could be made that these initial experiments established proof-of-principle for an asymmetric PTC dialkylation approach. However, the results were clearly unsatisfactory. The low enantioselectivity was rationalized as resulting from either, or both, of two factors: poor regioselectivity in the first intermolecular alkylation and/or a low level of stereochemical induction imparted by the chiral catalyst during the subsequent ring-closure. Scheme 26 illustrates the multiple reaction pathways that are possible for the dialkylation. Formation 102 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

of spirooxindole (R)-64 requires selective formation of one of the monoalkylated intermediates followed by high facial selectivity for the cyclization (solid arrow pathway). Low selectivity in the initial coupling of oxindole 94 and dibromide 21 and/or during the ring-closure leads to competing formation of (S)-64 (dotted arrows), reducing the overall enantioselectivity.

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Table 4. First Signs of Enantioselectivity in PTC Alkylation of Oxindole 94

To eliminate regioselectivity as a liability, a modified PTC strategy was proposed (Scheme 27). Alkylation of ester-substituted oxindole 101 with monohalide 102 would, by design, furnish intermediate 103 as a single benzylic regioisomer and establish the quaternary stereocenter with, ideally, high 103 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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selectivity. Subsequent manipulation involving intramolecular Friedel–Crafts chemistry followed by ketone reduction would then complete assembly of the spirocyclic ring system.

Scheme 26. Control of Both Intermolecular Regioselectivity and Intramolecular Facial Selectivity Is Required for High Enantioselectivity

Scheme 27. Modified Monoalkylation/Friedel–Crafts Proposal

Initial results from this approach were equally disappointing (Scheme 28): reaction of acylated oxindole 101 (which existed in the enol tautomeric form shown) with 3-nitrobenzylbromide 102 using various catalysts in the presence of hydroxide base gave the desired monoalkylated product 103 with low yield and enantioselectivity. However, it was observed that one side-product was consistently formed in significant amounts from these reactions; isolation and 104 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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characterization established it to be the unexpected bis-alkylated compound 105. It was subsequently confirmed that this material was formed from monoalkylated oxindole 103, presumably via a sequence of ester hydrolysis, decarboxylation, and alkylation. Evidently, the ester group in monoalkylated intermediate 103 is more prone to hydrolysis than in starting material 101, as the latter was slow to hydrolyze under the reaction conditions. The hydrolysis of intermediate 103 was promoted by the catalyst and did not occur at an appreciable rate in its absence, an observation which was recorded as a footnote at the time but which would become significant later on.

Scheme 28. Unexpected Formation of Bis-Alkylated Compound 105

At this point, we looked to turn this unexpected result to our advantage. Since dialkylation of acylated oxindole 101 was clearly feasible, we proposed that use of a bis-benzylic halide could provide an alternative entry to spirocyclic system (Scheme 29). The hope was that acylated oxindole 101 would be a more discriminating partner than the corresponding unsubstituted compound 94 in the intermolecular alkylation, resulting in improved regioselectivity for this elementary step and consequently higher enantioselectivity for the overall cascade in the presence of the chiral catalyst.

Scheme 29. Second Modified PTC Proposal

Gratifyingly this hypothesis was experimentally validated, at least to a degree, in the first experiments. As shown in Scheme 30, using catalyst 88 on screening scale the enantioselectivity for formation of (S)-spirocycles 64 and 95 was more than doubled compared to using unsubstituted oxindole 93, reaching a level of 45% ee using the methoxy-containing dibromide 94. 105 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 30. Improved Spirocycle Formation Using Acylated Oxindole 101

The next round of experimentation was directed by the observation that the presence of the electron-donating methoxy group in dibromide 94 had consistently resulted in higher enantioselectivities than the corresponding electron-poor nitroanalog 21. To exploit this theme, we proposed to evaluate a stronger electrondonating aniline substituent with the use of electrophile 108.

Scheme 31. Unexpected Result in Bromination of Aniline Diol 107

Bromination of aniline diol 107 using HBr in AcOH did not afford the expected dibromide 108, but instead monobromide hydrobromide salt 109 could be isolated in good yield by crystallization from PhMe/MTBE (Scheme 31). This unexpected result was followed by two positive findings (Scheme 32): not only was monobromide 109 a viable coupling partner for the dialkylation cascade, but also the resulting spirocycle (S)-84 was generated with the highest level of enantioselectivity seen up to that point (65% ee). 106 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 32. Improvement in Stereoselectivity Using Aniline Derivative 109

The former finding was more unexpected than the latter. In studying the reaction sequence further, it was shown to proceed via intermediate 110. Using a weaker base (K2CO3), the reaction stopped at this monoalkylated stage and ester hydrolysis did not occur. Intermediate 110 could be isolated, characterized, and resubjected to the reaction using a stronger, more nucleophilic base (KOH) to generate the spirocycle. The formation of intermediate 110 was rapid, and it was found that the catalyst was not required for this first alkylation step. We rationalized that the formal displacement of a hydroxyl group from compound 109 proceeded via aza-quinone methide 111. This type of reactive intermediate had previously been implicated in nucleophilic substitution of 4-hydroxymethylanilines by activated carbonyl compounds (131). Encouraged by this breakthrough, we conducted an initial screen of a small number of catalysts. The focus was on derivatives of cinchonine, largely because a modestly-sized collection of about a dozen of these had been accumulated from previous projects within the group and was thus immediately on-hand. As the number of commercially available chiral PTCs was quite limited at the time, this in-house collection served as a valuable resource for a rapid first-pass assessment of the impact of catalyst structure in this series. A selection of results is highlighted in Table 5 (132). As far as trends in catalyst structure-activity relationships could be discerned from this small data set, it appeared that an electron-deficient aromatic ring at the quaternized N-benzylic position was preferred (e.g., entries 1 and 3), as was the presence of the hydroxyl group in unprotected form (e.g., entries 4 and 5). The real breakthrough from this screen was the finding that 3,5-bis(trifluoromethyl)benzyl derivative 120 generated the spirocycle in 93% ee (entry 10). 107

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Table 5. Initial Screen Resulted in Identification of Highly Selective Catalyst 120

This result gave us the confidence to continue developing this PTC chemistry for the long-term manufacturing route, focusing on the use of aniline derivatives as the electrophile. By this stage we had also developed a better empirical understanding of the influence of the structure of the electrophilic coupling partner on the outcome of the PTC cascade. As highlighted previously, both the inter- and intramolecular steps of the cascade needed to be controlled for overall high stereoselectivity. Isolation of the monoalkylated intermediates allowed the influence of each of these steps to be characterized. As shown in Table 6, the nature of the C-4 substituent in the electrophile was found to have some influence on the facial selectivity of the intramolecular ring closure, but strongly impacted the regioselectivity of the intermolecular alkylation. Whereas the nitro-substituted compound gave a mixture of regioisomeric intermediates in the first step, formation of the ‘para’ intermediate 122 was highly selective using the electron-rich methoxy and aniline analogues, which largely translated into the overall enantioselectivity of the direct one-pot process. 108

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Table 6. Investigation of Inter- and Intramolecular Alkylation Steps

The astute reader would have noted that the studies described so far have generated the (S)-enantiomer of the spirocyclic products, i.e. the undesired enantiomer of that required for MK-3207. This was driven by our desire to establish rapid proof-of-concept on a PTC approach in our initial route-scouting efforts using the cinchonine-derived catalysts we already had in hand, with the expectation that catalyst optimization would form part of subsequent process development. That we identified 3,5-bis(trifluoromethyl)benzyl catalyst 120 during these initial efforts was a fortuitous discovery, but we were able to capitalize on this serendipity to significantly accelerate the subsequent development. We also knew that access to the desired spirooxindole (R)-84 should be provided by catalysts derived from cinchonidine (126, Figure 6), another Cinchona alkaloid described as a ‘pseudoenatiomer’ of cinchonine 124 (technically, the two are diastereomers). For both alkaloids their low cost, bulk availability, functional handles for derivatization, and generally predictable stereochemical outcomes when used for asymmetric transformations have made them popular choices in organocatalysis (133–136). These facets, combined with our assessment that we would have freedom-to-operate with these catalysts, resulted in us focusing on the cinchonidine-derived series. 109

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Figure 6. Pseudoenantiomeric cinchonine and cinchonidine scaffolds for PTC.

We quickly established that the corresponding 3,5-bis(trifluoromethyl)benzylcinchonidinum catalyst 128 indeed generated spirooxindole (R)-84 (Scheme 33), albeit with slightly diminished enantioselectivity on screening scale relative to the earlier result for (S)-84 using the cinchonine-derived analog 120. However, an issue that emerged when this chemistry was run on larger laboratory scale for the first time was that inconsistent results were observed, with the enantiomeric excess varying from 70–85% between experiments. In addition, the optimum oxindole and aniline coupling partners also needed to be defined and the downstream conversion to final MK-3207 API rigorously established. The assessment was that while the spirocyclization chemistry shown in Scheme 33 would provide a general framework for the manufacturing route, systematic study of substrates and parameters, optimization of all steps in the sequence, and detailed process characterization was going to be required. The following sections describe how each of these was successfully addressed and the manufacturing route for spirooxindole (R)-25 finalized.

Optimized Oxindole Synthesis PMB-protected oxindole 101 had served a valuable purpose to this point in discovery of the PTC cascade, but it was clear that the utility of this intermediate had run its course. Both the synthesis of substrate 101 and later removal of the PMB group from protected spirooxindole product (R)-84 were found to be challenging, and an alternative protecting group that mitigated these issues was desired. 110 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 33. Generation of Spirooxindole (R)-84 Using Cinchonidinium-Derived Catalyst 128 As we had access to a supply of the parent 7-azaoxindole (63) from the early GMP deliveries of MK-3207, our first attempts involved selective protection of the amide nitrogen in this material (Scheme 34). However, this was thwarted by the formation of complex mixtures resulting from competing O- and C-alkylations. Even if it had been successful, the question of how to efficiently access 7-azaoxindole 63 from a long-term manufacturing perspective would have remained.

Scheme 34. Chemoselectivity Issues in the First Attempted Synthesis of PMB-Protected Oxindole 94 As an alternative approach we sought to construct the oxindole ring from a more readily available 2,3-disubstituted pyridine precursor, building upon an example of an anionic cyclization that had been reported by Snieckus and co-workers (137). The route used to synthesize PMB-protected compound 101, together with its unacylated progenitor 94, is illustrated in Scheme 35. Sequential N-acylation and alkylation of commercially available 2-amino3-picoline (129) gave compound 130, which upon treatment with LDA in THF underwent lateral metalation followed by cyclization to afford the desired oxindole 94 as the major product. The subsequent C-acylation could be effected in good yield using methyl cyanoformate (138), with the product being isolated as the aromatic enol tautomer. Although this was a serviceable method for obtaining gram quantities of oxindoles 94 and 101, it suffered from a liability in the formation of a significant amount of rearrangement product 133 during the anionic cyclization step. This side-product presumably resulted from competitive deprotonation at the other benzylic site in substrate 130 followed by acyl migration (formally, a 111

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[1,2]-aza-Wittig rearrangement) (139). Formation of the desired oxindole 94 was favored by higher reaction temperatures and addition of the substrate to the LDA base, but the formation of side-product 133 could not be completely suppressed.

Scheme 35. Different Chemoselectivity Issues in the Gram-Scale Synthesis of PMB-Protected Oxindoles 94 and 101

After considerable further experimentation, we settled on the use of the tertbutyl group for the oxindole protection. Somewhat underutilized as a protecting group for nitrogen in organic synthesis (140), this simple alkyl group was found to meet our criteria of efficient introduction and removal, stability during the PTC cascade, as well as being cost and (relatively) atom economical. Direct tert-butylation of 2-amino-3-picoline 129 could not be achieved due to the low nucleophilicity of the amino group, thus a high-yielding amination reaction was utilized to access compound 135 (Scheme 36). A novel one-pot anionic cascade sequence was then developed to generate the acylated oxindole. Treatment of compound 135 with n-HexLi followed by methyl chloroformate resulted in conversion to carbamate 136; subsequent treatment with n-HexLi followed by further charges of i-Pr2NH and n-HexLi (to generate LDA in situ) resulted in cyclization to Li-enolate 137 which, in turn, was acylated upon the final addition of a further equivalent of methyl chloroformate. The acylated oxindole 139 was formed in 69% solution assay yield for the overall sequence. tert-Butyl intermediate 136 could not undergo competing rearrangement as was observed for PMB-protected analog 130, however control of the reaction conditions was required to minimize formation of dimeric impurity 140 which was difficult to reject. It was found that addition of a substoichiometric amount of n-HexLi to the solution of carbamate 136 and aging for 30 min prior to addition of the subsequent reagents suppressed the formation of dimer 140 by consuming any excess methyl chloroformate still present before the cyclization. 112

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Scheme 36. Synthesis of Tert-Butyl-Protected Oxindole 139

While evaluating acid/base aqueous workup procedures for the isolation of product 139, the serendipitous finding was made that the corresponding potassium salt 138 could be efficiently partitioned into the organic phase with rejection of impurities to aqueous layers, and then crystallized from THF/n-heptane with good recovery and purity. A subsequent salt break (dissolution of potassium salt 138 in MeOH, acidification with AcOH, addition of water antisolvent, and filtration) enabled isolation of 139 in the free form.

Optimized Chloride Synthesis The first synthesis of aniline bromide electrophile 109 is illustrated in Scheme 37. Beginning with 4-bromophthalic acid (141), esterification followed by palladium-catalyzed amination gave aniline 143. The implementation of the esterification step was primarily to improve substrate solubility; attempted amination of the diacid starting material resulted in poor conversion even at high catalyst/ligand loadings. Reduction of diester 143 to diol 107 was accomplished using LiAlH4. Conversion to monobromide 109 then proceeded as described previously. Chloride electrophile 144 could be formed from diol 107 by treatment with SOCl2. Analogously to the corresponding bromide 109, electrophile 144 was isolated as the monobenzylic halide. A small, but nonetheless significant, improvement in enantioselectivity (typically, 2–3% ee) was observed when chloride 144 was used in place of bromide 109 for the PTC spirocyclization cascade, therefore it was selected for further development. 113

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Scheme 37. First Route to Aniline Electrophiles 109 and 144

Scheme 38. Improved Route to Chloride Electrophile 144

An issue with the first synthesis of chloride 144 was that 4-bromophthalic acid (141) was relatively expensive, and a more cost-effective alternative was needed for long-term manufacture. The route that was subsequently developed is shown in Scheme 38. We returned to 4-nitrophthalic acid (19) as a cheap, commercially 114 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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available starting material. Conversion to aniline diester 145 was achieved via a one-pot protocol. Fischer esterification using a small excess of methanesulfonic acid in MeOH (141, 142) at 80 °C for 16 h was followed by cooling, addition of 10% Pd/C (3 wt% relative to diacid 19) and hydrogenation below 40 °C at 60 psig of hydrogen. At the end of reaction, the solution was filtered to remove the catalyst, the bulk of the MeOH was removed and the solvent switched to EtOAc for the basic aqueous workup. Finally, crystallization from PhMe/n-heptane gave the product 145 in 84% overall yield. Dibenzylation was accomplished using BnCl, K2CO3, and a catalytic amount of KI as promoter in DMAc at 90 °C (143). This was a heterogeneous reaction and the particle size of the K2CO3 base was important. Competitive acylation of the aniline by the DMAc solvent would result in significant levels of side-products 147 and 148 when granular K2CO3 was used; this liability was avoided using a powdered form of the base. After an organic/aqueous workup using MTBE, the product stream was switched into THF for use directly in the next reduction step. We used the commercially available solution of LiAlH4(1 M in THF) for the reduction of diol 110 to avoid challenges of handling the solid reducing agent on scale. Thus, LiAlH4 solution was added slowly to the diester solution while maintaining the temperature below 5 °C. Reduction was complete within a few hours, and then the reaction was worked up using a modified Fieser protocol. Excess hydride reagent was first quenched by the slow addition of solution of 20% v/v water in THF below 10–15 °C (144), followed by the addition of 15% aq NaOH and then finally water. The resulting slurry was then filtered through a bed of cellulose as a filter aid, and the filtrate was concentrated and the product was crystallized from toluene/n-heptane. Diol 107 was isolated in 87% overall yield for the two steps and in greater than 97% purity. Chlorination was accomplished by charging diol 110 in portions to a solution of SOCl2 in MeCN below 20 °C. Once the starting material had been fully consumed, the reaction mixture was diluted with MTBE. Seeding of the solution resulted in crystallization of the monochloride HCl salt 144. Alternative combinations of solvents for reaction and crystallization were evaluated, but MeCN/MTBE provided the best reaction profile and crystallization attributes. The product was isolated in 90% yield corrected for the 94% weight purity; the mass balance was primarily residual solvents together with small amounts of dimeric impurities.

Optimization of PTC Chemistry and Deprotection We set out on our development of the PTC spirocyclization reaction with a number of goals in mind, namely to: • •

define critical parameters and address reproducibility issues seen in early lab-scale reactions. optimize the yield, enantioselectivity and practicality of the process. · establish a robust product isolation protocol. 115

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The path to establishing reproducibility was guided by piecing together a number of observations from earlier experiments: •

•

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•

a catalyst was not necessary for the formation of the monoalkylated intermediate but was required for the subsequent ester hydrolysis to occur at an appreciable rate under the reaction conditions. the enantioselectivity of the spirocyclization was consistently higher starting from isolated monoalkylated intermediate than in the one-pot double-alkylation process. the enantioselectivity was also highly dependent on the structure of the catalyst.

Thus, based on the these goals and guidelines, we arrived at the protocol shown in Scheme 39. Oxindole 139 and chloride 144 were added to a heterogeneous mixture of KOH, water and toluene at 8–12 °C and allowed to react in the absence of catalyst. Once formation of intermediate 149 was complete (in less than 1 h), catalyst 128 (5 mol%) was charged and agitation continued for a further 12–14 h to generate the spirooxindole. A number of parameters were important for the efficiency and reproducibility of this process, including reagent stoichiometry and order of addition, base and solvent, concentration, temperature, and agitation rate. Each of these was extensively investigated and optimized.

Scheme 39. Optimized PTC Chemistry

To achieve the highest enantioselectivity, it was critical that chloride 144 be almost completely consumed prior to introduction of the catalyst. As an explanation for the variability in performance when the catalyst was present in the mixture from the beginning, we believed that the electrophile would competitively alkylate the catalyst (e.g., on the hydroxy group) to generate a new species that was still catalytically active but afforded much lower enantioselectivity. 116 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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In practice, control of residual chloride 144 was achieved by employing a slight excess of oxindole 139 and establishing an in-process control of less than 1% chloride 144 remaining before charging the catalyst 128. Sampling and analysis of the reaction was non-trivial given the heterogeneous nature of the mixture and the limited stability of chloride 144 in solution, highlighting the skill of our analytical colleagues in establishing a reliable method. We were also able to reduce the loading of catalyst 128 to 5 mol% without impacting the enantioselectivity (145). Evaluation of organic solvent and base systems confirmed that toluene and aqueous KOH were the optimum combination, used in a 2.6:1 (v/v) ratio. The yield and enantioselectivity increased with the concentration of the base solution. For convenience, commercially available 50 wt% KOH in water was initially used for lab-scale development, but it was noted that the overall reaction generated molar equivalents of water, thus the concentration of KOH decreased slightly over the course of the reaction. By using a slurry of 55 wt% KOH in water (prepared by charging solid KOH to the commercial 50 wt% solution prior to addition of the other reagents), the concentration of KOH remained above the saturation limit in water (~51 wt% at the reaction temperature) throughout the sequence, maximizing the enantioselectivity. The reaction mixture was therefore comprised of a triphasic system of KOH(s), water, and organic phases (146). The enantioselectivity was also dependent on the total reaction volume, increasing at greater dilution. On laboratory scale, spirooxindole (R)-84 was formed in 90% ee at a reaction volume of 40 mL/g oxindole starting material 139, but the selectivity dropped to 85% ee at 20 mL/g. Analogous dependence on concentration has previously been reported for asymmetric PTC reactions (114). To balance overall yield against material throughput, a reaction volume of 40 mL/g oxindole 139 was selected for scale-up. Similarly, a reaction temperature of 8–12 °C was selected as the best compromise between two competing trends as the temperature was lowered: for the intrinsic enantioselectivity of the spirocyclization to increase, but at the expense of increasing viscosity of impeding agitation of the thick, heterogeneous mixture. Effective agitation and mixing of the heterogeneous mixture was critical to maximizing reaction performance. On kilo-lab scale using a simple U-shaped paddle stirrer in a 100 L cylindrical vessel, (R)-84 was obtained in 88% ee. But in a pilot plant setting with more efficient agitation (using either a retreat-curve impeller or Rushton disk turbine), higher enantioselectivities of up to 93% ee were achieved. At the end of reaction, an aqueous workup afforded crude spirocycle (R)84 as a solution in toluene in 89–92% assay yield. Product isolation was greatly facilitated by the discovery of a crystalline toluene solvate that afforded upgrade of both chemical and enantiopurity. Concentration of the crude product solution to reach supersaturation and trigger crystallization was followed by addition of MeOH as antisolvent to reduce liquor losses and then filtration. In the first kilo-lab demonstration of this chemistry, spirocycle (R)-84 was obtained in 77% yield and 95% ee. Upgrade of stereochemical purity to the required level could be achieved in crystallizations of subsequent steps to the MK-3207 API. Further development of the isolation of spirocycle (R)-84, in particular definition of the minimum amount of toluene required to maintain the 117

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solvate form during the crystallization, filtration and cake washes (147), enabled the product to be isolated in 83% yield and greater than 99% ee in a later pilot plant campaign. Two impurities observed at low levels during early lab scale experiments were the novel pyran-containing spirocycle 150 (the configuration at the chiral center was not determined) and the ‘aniline dimer’ 151 (Figure 7). Pyran 150 was proposed to arise from enolate oxidation via adventitious intrusion of oxygen into the reaction mixture (148), and its formation was easily eliminated using standard inertion techniques. Aniline dimer 151, shown to arise by alkylation of monoalkylated intermediate 149 with a second molecule of chloride 144, was more problematic as it was only partially rejected during the crystallization of spirocycle (R)-84. The downstream fate of aniline dimer 151 was to react in subsequent steps to generate a new impurity in the API that had not been qualified in toxicological studies. The control strategy implemented was to restrict the level of formation of aniline dimer 151 to no more than 0.6% by the end of the PTC reaction. This was achieved in practice by controlling the stoichiometry of oxindole 139 and chloride 144 starting materials to limit the amount of the latter remaining after formation of intermediate 149.

Figure 7. Impurities 150 and 151 formed during the PTC reaction.

A through-process was developed for the two-stage removal of the protecting groups from spirocycle (R)-84 to give free aniline (R)-25 in excellent yield and purity (Scheme 40). The tert-butyl group was first cleaved from the oxindole nitrogen using excess methanesulfonic acid and a small volume of toluene at 90 °C. The main role of the toluene was to sequester the liberated tert-butyl cation and prevent undesired alkylation of the aniline core. The residual toluene present in the solvate form of starting material (R)-84•PhMe also served this purpose to a degree, but we found that using additional PhMe as co-solvent improved the scavenging efficiency. After cooling to ambient temperature, the solution of oxindole (R)-152 was diluted with MeOH and hydrogenated at 60 psig of H2 in the presence of catalytic Pd/C. Once hydrogenolysis of the benzyl groups was complete, the mixture was filtered and a workup performed in which the product (R)-25 was extracted into the aqueous layer as the corresponding MsOH salt. Adjustment of pH to 6–8 using aq. NaOH resulted in crystallization of free aniline (R)-25. 118

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Scheme 40. Deprotection To Generate Free Aniline (R)-25

Spirooxindole Route Summary The overall route that was developed for the long-term manufacture of (R)-spirooxindole (R)-25 is shown in Scheme 41 (149). The synthesis comprised ten steps, and features at its heart a novel PTC-mediated reaction to generate the spirocyclic ring system and quaternary stereocenter in high yield and enantioselectivity. The overall yield was 40%, representing a four-fold improvement over the earlier classical resolution route. One of the goals of this chapter section on the spirooxindole has been to describe the evolution of the chemistry from the medicinal chemistry synthesis that enabled discovery of MK-3207, through the first generation process chemistry route for speed to the clinic, to the manufacturing route for practical, economical long-term supply. Beyond the specific chemistry details, however, another aim has been to use this story to convey more general lessons learned from our experiences as well as some of our broader thoughts on chemical process development. The first is that effective teamwork and communication are essential to deliver on a project of any complexity. The successes described above reflect the collective efforts of dozens of outstanding colleagues and collaboration between many different groups. A second key theme is the impact of bringing innovation to process development, and being willing to take risks in developing new chemistry to tackle the most challenging synthetic problems. From the enzymatic oxidation used in the first GMP delivery to the Heck/C–H functionalization chemistry and then the PTC spirocyclization, this spirooxindole work exemplified: • • • •

the development of novel reactions. new extensions of established reactions. cascade processes to rapidly assemble molecular complexity. the enabling ability of asymmetric catalysis and HTE.

A third lesson is that persistence, attention to detail, mechanistic understanding, and the ability to take advantage of opportunities presented by serendipity are all important traits in process development. The optimized PTC chemistry to prepare (R)-25 (Scheme 41) ended up being quite different to original 119 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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proposal at the outset of long-term route scouting (Scheme 25). The connection between the two was neither intuitively obvious nor direct, but instead resulted from several rounds of analysis and strategy revision based on unanticipated findings.

Scheme 41. Summary of New Route to Spirooxindole (R)-25 Finally, the development of novel methodologies and capabilities to solve challenges on one project can have broader benefits, as is described below. Development of a PTC Library and Its Application Reflecting back on the development of the PTC spirocyclization chemistry, there was an element of good fortune in 3,5-bis(trifluoromethyl)benzylcinchoninium bromide 120 (Table 5) being present among the small collection of catalysts we had in hand at the time of initial screening, and that we were thus able to identify a high-performing catalyst so quickly. Establishing catalyst structure-activity relationships for any given PTC reaction has been largely empirical and qualitative to date, although more quantitative approaches to catalyst design have begun to be developed (150, 151). In addition, the number of commercially available phase-transfer catalysts is fairly limited. To address these capability gaps and improve the odds of success on future projects, we sought to generate a much larger library of catalysts that would be available for colleagues to use in-house. In its initial form, this library consisted of derivatives of the cinchonine and cinchonidine alkaloids quaternized on the quinuclidine nitrogen with a diverse 120

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set of commercially available benzyl halides. Over 100 of these catalysts were prepared in each pseudoenantiomeric series (Figure 8). The goal was for this library to enable efficient screening and hit generation for various transformations using HTE techniques. The following two examples highlight some of the applications and new discoveries that this library has enabled so far.

Figure 8. A library of cinchonine- and cinchonidine-derived catalysts. One of the early applications was in our studies towards developing a cost-effective synthesis of vinylcyclopropane 156 (Scheme 42), a structural motif present in several NS3/4A protease inhibitors, including grazoprevir (157) (152, 153), for the treatment of hepatitis C virus (154, 155). A number of synthetic routes to this valuable building block had been reported (156), including racemic dialkylation of a glycine imine derivative to generate the cyclopropane followed by enzymatic resolution (157). In our hands, screening of this cyclopropanation reaction with the set of cinchonidinium derivatives under PTC conditions resulted in the rapid identification of catalyst 155 as a promising lead that could serve as a starting point for further development (158, 159).

Scheme 42. Early Demonstration of Utility of the PTC Library Screening for Synthesis of Vinylcyclopropane 156 The second example serves as another case study of the value of capitalizing on serendipity in research. MK-8825 (158, Figure 9) was identified as another CGRP receptor antagonist candidate (160) following MK-3207, and 121 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

process chemistry was engaged in developing a scalable synthesis of the core spirooxindole component 159.

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Figure 9. MK-8825 (158) and spirooxindole 159. The direct one-pot PTC dialkylation cascade developed for the related MK3207 spirooxindole proved less effective when applied to this new system. The team evaluated a number of other routes, including the stepwise approach shown in Scheme 43 in which alternative chemistry was used to access intermediate 160, but asymmetric PTC was still envisaged as being used to effect the critical intramolecular ring closure.

Scheme 43. Initial Screening of Intramolecular Cyclization of Oxindole 160 and Discovery of ‘Bis-Quat’ Catalyst 163 Screening of the cinchonidinium-derived catalysts generated an initial data set comprised largely of incomplete conversions and poor-to-middling enantioselectivities. However, catalyst 162 was a unique outlier in furnishing spirocycle 161 in high yield and 92% ee. Encouraged by this promising result the team began laboratory-scale development, at which point the performance of the cyclization promptly dropped (Scheme 43). The team noted that different batches of catalyst 162 were used between screening and scale-up, and closer inspection indicated that the newer batch of catalyst prepared for the scale-up studies was of 122 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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higher purity than the screening material that had been prepared earlier as part of the library generation. After further excellent detective work, doubly-quaternized species 163 (Scheme 43) was identified as the true actor responsible for the high performance of the screening catalyst batch, despite being present only as a low-level impurity in this material. The remarkable catalytic efficiency of compound 163 was underscored in the optimized cyclization conditions (Scheme 44) which required only 0.3 mol% catalyst loading (161), an unprecedentedly low level for Cinchona alkaloid-based phase-transfer catalysis.

Scheme 44. Optimized Spirocyclization Using Doubly-Quaternized Catalyst 163

Diligent investigation of some initially confounding results therefore led to the serendipitous discovery of a novel class of doubly-quaternized Cinchona alkaloid derivatives that we believe will have broader utility in asymmetric PTC. As an example, development of an efficient intramolecular aza-Michael PTC reaction for the enantioselective synthesis of the clinical drug candidate letermovir (167, Scheme 45) has recently been reported by these laboratories (162).

Scheme 45. Application of ‘Bis-Quat’ PTC in the Asymmetric Synthesis of letermovir 167 123 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

The advances in PTC chemistry described in this chapter, from development of the MK-3207 spirooxindole cascade to the discovery of a new catalyst class, build upon Merck’s legacy in this field and offer exciting new directions for the future.

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Conclusions The discovery of MK-3207, an orally acting, picomolar CGRP receptor antagonist with the potential for a low clinical dose, together with the development of scalable methods for its production, were significant achievements. Unfortunately, there were a number of liver enzyme abnormalities observed in the clinic after dosing with the compound and its development was discontinued after the work described in this chapter had been completed. Nonetheless, the preclinical and clinical studies with MK-3207 demonstrated that it is possible to achieve CGRP receptor blockade with a low oral dose of a suitable antagonist. Moreover, the challenge of synthesizing such a complex molecule on large scale led to novel approaches and scientific discoveries that highlight the impact of process chemistry in a complex multidisciplinary area and the importance of driving innovation in chemistry research.

References 1.

Goadsby, P. J.; Lipton, R. B.; Ferrari, M. D. Migraine – current understanding and treatment. N. Engl. J. Med. 2002, 346, 257–270. 2. Chan, K. Y.; Vermeersch, S.; de Hoon, J.; Villalón, C. M.; MaassenVanDen Brink, A. Potential mechanisms of prospective antimigraine drugs: A focus on vascular (side) effects. Pharmacol. Ther. 2011, 129, 332–351. 3. Silberstein, S. D. Emerging target-based paradigms to prevent and treat migraine. Clin. Pharmacol. Ther. 2013, 93, 78–85. 4. Hargreaves, R. New migraine and pain research. Headache 2007, 47 (Suppl. 1), S26–S43. 5. Silberstein, S. D. Migraine. Lancet 2004, 363, 381–391. 6. Ho, T. W.; Edvinsson, L.; Goadsby, P. J. CGRP and its receptors provide new insights into migraine pathophysiology. Nat. Rev. Neurol. 2010, 6, 573–582. 7. Edvinsson, L.; Ho, T. W. CGRP receptor antagonism and migraine. Neurotherapeutics 2010, 7, 164–175. 8. Edvinsson, L. Functional role of perivascular peptides in the control of cerebral circulation. Trends Neurosci. 1985, 8, 126–131. 9. Goadsby, P. J.; Edvinsson, L.; Ekman, R. Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann. Neurol. 1990, 28, 183–187. 10. Goadsby, P. J.; Edvinsson, L. The trigeminovascular system and migraine: studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats. Ann. Neurol. 1993, 33, 48–56. 11. Lassen, L. H.; Haderslev, P. A.; Jacobsen, V. B.; Iversen, H. K. CGRP may play a causative role in migraine. Cephalalgia 2002, 22, 54–61. 124

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

12. Bell, I. M. Calcitonin gene-related peptide receptor antagonists: New therapeutic agents for migraine. J. Med. Chem. 2014, 57, 7838–7858. 13. Poyner, D. R.; Sexton, P. M.; Marshall, I.; Smith, D. M.; Quirion, R.; Born, W.; Muff, R.; Fischer, J. A.; Foord, S. M. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol. Rev. 2002, 54, 233–246. 14. Woolley, M. J.; Conner, A. C. Comparing the molecular pharmacology of CGRP and adrenomedullin. Curr. Protein Pept. Sci. 2013, 14, 358–374. 15. Barwell, J.; Gingell, J. J.; Watkins, H. A.; Archbold, J. K.; Poyner, D. R.; Hay, D. L. Calcitonin and calcitonin-like receptors: common themes with family B GPCRs? Br. J. Pharmacol. 2012, 166, 51–65. 16. McLatchie, L. M.; Fraser, N. J.; Main, M. J.; Wise, A.; Brown, J.; Thompson, N.; Solari, R.; Lee, M. G.; Foord, S. M. RAMPs regulate the transport and ligand specificity of the calcitonin receptor-like receptor. Nature 1998, 393, 333–339. 17. Moore, E. L.; Salvatore, C. A. Targeting a family B GPCR/RAMP receptor complex: CGRP receptor antagonists and migraine. Br. J. Pharmacol. 2012, 166, 66–78. 18. Hilairet, S.; Foord, S. M.; Marshall, F. H.; Bouvier, M. Protein-protein interaction and not glycosylation determines the binding specificity of heterodimers between the calcitonin receptor-like receptor and the receptor activity-modifying proteins. J. Biol. Chem. 2001, 276, 29575–29581. 19. Koller, D.; Born, W.; Leuthauser, K.; Fluhmann, B.; McKinney, R. A.; Fischer, J. A.; Muff, R. The extreme N-terminus of the calcitonin-like receptor contributes to the selective interaction with adrenomedullin or calcitonin gene-related peptide. FEBS Lett. 2002, 531, 464–468. 20. Kuwasako, K.; Kitamura, K.; Nagoshi, Y.; Cao, Y.-N.; Eto, T. Identification of the human receptor activity-modifying protein 1 domains responsible for agonist binding specificity. J. Biol. Chem. 2003, 278, 22623–22630. 21. Barwell, J.; Conner, A.; Poyner, D. R. Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochim. Biophys. Acta 2011, 1813, 1906–1916. 22. Hoare, S. R. J. Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptors. Drug Discovery Today 2005, 10, 417–427. 23. Rudolf, K.; Eberlein, W.; Engel, W.; Pieper, H.; Entzeroth, M.; Hallermayer, G.; Doods, H. Development of human calcitonin gene-related peptide (CGRP) receptor antagonists. 1. Potent and selective small molecule CGRP antagonists. 1-[N2-[3,5-dibromo-N-[[4-(3,4-dihydro2(1H)-oxoquinazolin-3-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-L-lysyl]-4(4-pyridinyl)piperazine: The first CGRP antagonist for clinical trials in acute migraine. J. Med. Chem. 2005, 48, 5921–5931. 24. Paone, D. V.; Shaw, A. W.; Nguyen, D. N.; Burgey, C. S.; Deng, J. Z.; Kane, S. A.; Koblan, K. S.; Salvatore, C. A.; Mosser, S. D.; Johnston, V. K.; Wong, B. K.; Miller-Stein, C. M.; Hershey, J. C.; Graham, S. L.; Vacca, J. P.; Williams, T. M. Potent, orally bioavailable calcitonin gene-related peptide 125

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

25.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

26.

27.

28.

29.

30.

31.

32.

33.

34.

receptor antagonists for the treatment of migraine: discovery of N-[(3R,6S)6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide (MK-0974). J. Med. Chem. 2007, 50, 5564–5567. Mallee, J. J.; Salvatore, C. A.; LeBourdelles, B.; Oliver, K. R.; Longmore, J.; Koblan, K. S.; Kane, S. A. Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists. J. Biol. Chem. 2002, 277, 14294–14298. Salvatore, C. A.; Mallee, J. J.; Bell, I. M.; Zartman, C. B.; Williams, T. M.; Koblan, K. S.; Kane, S. A. Identification and pharmacological characterization of domains involved in binding of CGRP receptor antagonists to the calcitonin-like receptor. Biochemistry 2006, 45, 1881–1887. Miller, P. S.; Barwell, J.; Poyner, D. R.; Wigglesworth, M. J.; Garland, S. L.; Donnelly, D. Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74. Biochem. Biophys. Res. Commun. 2010, 391, 437–442. Moore, E. L.; Gingell, J. J.; Kane, S. A.; Hay, D. L.; Salvatore, C. A. Mapping the CGRP receptor ligand binding domain: Tryptophan-84 of RAMP1 is critical for agonist and antagonist binding. Biochem. Biophys. Res. Commun. 2010, 394, 141–145. Zartman, C. B.; Bell, I. M.; Gallicchio, S. N.; Graham, S. L.; Kane, S. A.; Mallee, J. J.; Rutledge, R. Z.; Salvatore, C. A.; Vacca, J. P.; Williams, T. M. Identification of a novel RAMP-independent CGRP receptor antagonist. Bioorg. Med. Chem. Lett. 2011, 21, 6705–6708. Koth, C. M.; Abdul-Manan, N.; Lepre, C. A.; Connolly, P. J.; Yoo, S.; Mohanty, A. K.; Lippke, J. A.; Zwahlen, J.; Coll, J. T.; Doran, J. D.; Garcia-Guzman, M.; Moore, J. M. Refolding and characterization of a soluble ectodomain complex of the calcitonin gene-related peptide receptor. Biochemistry 2010, 49, 1862–1872. ter Haar, E.; Koth, C. M.; Abdul-Manan, N.; Swenson, L.; Coll, J. T.; Lippke, J. A.; Lepre, C. A.; Garcia-Guzman, M.; Moore, J. M. Crystal structure of the ectodomain complex of the CGRP receptor, a class-B GPCR, reveals the site of drug antagonism. Structure 2010, 18, 1083–1093. Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.; Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P. A.; Chang, R. S. L.; Lotti, V. J.; Cerino, D. J.; Chen, T. B.; Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J. Methods for drug discovery: development of potent, selective, orally effective cholecystokinin antagonists. J. Med. Chem. 1988, 31, 2235–2246. Lipinksi, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 1997, 23, 3–25. Bock, M. G.; DiPardo, R. M.; Evans, B. E.; Rittle, K. E.; Whitter, W. L.; Garsky, V. M.; Gilbert, K. F.; Leighton, J. L.; Carson, K. L.; 126

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

35.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

36. 37.

38.

39.

40.

41.

42.

43.

Mellin, E. C.; Veber, D. F.; Chang, R. S. L.; Lotti, V. J.; Freedman, S. B.; Smith, A. J.; Patel, S.; Anderson, P. S.; Freidinger, R. M. Development of 1,4-benzodiazepine cholecystokinin type B antagonists. J. Med. Chem. 1993, 36, 4276–4292. Williams, T. M.; Stump, C. A.; Nguyen, D. N.; Quigley, A. G.; Bell, I. M.; Gallicchio, S. N.; Zartman, C. B.; Wan, B.-L.; Della Penna, K.; Kunapuli, P.; Kane, S. A.; Koblan, K. S.; Mosser, S. D.; Rutledge, R. Z.; Salvatore, C.; Fay, J. F.; Vacca, J. P.; Graham, S. L. Non-peptide calcitonin gene-related peptide receptor antagonists from a benzodiazepinone lead. Bioorg. Med. Chem. Lett. 2006, 16, 2595–2598. Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful metric for lead selection. Drug Discovery Today 2004, 9, 430–431. Clark, D. E. Rapid calculation of polar molecular surface area and its application to the prediction of transport phenomena. 1. Prediction of intestinal absorption. J. Pharm. Sci. 1999, 88, 807–814. Burgey, C. S.; Stump, C. A.; Nguyen, D. N.; Deng, J. Z.; Quigley, A. G.; Norton, B. R.; Bell, I. M.; Mosser, S. D.; Salvatore, C. A.; Rutledge, R. Z.; Kane, S. A.; Koblan, K. S.; Vacca, J. P.; Graham, S. L.; Williams, T. M. Benzodiazepine calcitonin gene-related peptide (CGRP) receptor antagonists: Optimization of the 4-substituted piperidine. Bioorg. Med. Chem. Lett. 2006, 16, 5052–5056. Bell, I. M.; Bednar, R. A.; Fay, J. F.; Gallicchio, S. N.; Hochman, J. H.; McMasters, D. R.; Miller-Stein, C.; Moore, E. L.; Mosser, S. D.; Pudvah, N. T.; Quigley, A. G.; Salvatore, C. A.; Stump, C. A.; Theberge, C. R.; Wong, B. K.; Zartman, C. B.; Zhang, X.-F.; Kane, S. A.; Graham, S. L.; Vacca, J. P.; Williams, T. M. Identification of novel, orally bioavailable spirohydantoin CGRP receptor antagonists. Bioorg. Med. Chem. Lett. 2006, 16, 6165–6169. Theberge, C. R.; Bednar, R. A.; Bell, I. M.; Corcoran, H. A.; Fay, J. F.; Hershey, J. C.; Johnston, V. K.; Kane, S. A.; Mosser, S.; Salvatore, C. A.; Williams, T. M.; Zartman, C. B.; Zhang, X.-F.; Graham, S. L.; Vacca, J. P. Potent benzimidazolone-based CGRP receptor antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 6122–6125. Stump, C. A.; Bell, I. M.; Bednar, R. A.; Bruno, J. G.; Fay, J. F.; Gallicchio, S. N.; Johnston, V. K.; Moore, E. L.; Mosser, S. D.; Quigley, A. G.; Salvatore, C. A.; Theberge, C. R.; Zartman, C. B.; Zhang, X.-F.; Kane, S. A.; Graham, S. L.; Vacca, J. P.; Williams, T. M. The discovery of highly potent CGRP receptor antagonists. Bioorg. Med. Chem. Lett. 2009, 19, 214–217. Bell, I. M.; Bednar, R. A.; Corcoran, H. A.; Fay, J. F.; Gallicchio, S. N.; Johnston, V. K.; Hershey, J. C.; Miller-Stein, C. M.; Moore, E. L.; Mosser, S. D.; Roller, S. A.; Salvatore, C. A.; Theberge, C. R.; Wong, B. K.; Zartman, C. B.; Kane, S. A.; Williams, T. M.; Graham, S. L.; Vacca, J. P. The identification of potent, orally bioavailable tricyclic CGRP receptor antagonists. Bioorg. Med. Chem. Lett. 2009, 19, 4740–4742. Stump, C. A.; Bell, I. M.; Bednar, R. A.; Fay, J. F.; Gallicchio, S. N.; Hershey, J. C.; Jelley, R.; Kreatsoulas, C.; Moore, E. L.; Mosser, S. D.; 127

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

44.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

45.

46.

47.

48.

49.

50.

51.

Quigley, A. G.; Roller, S. A.; Salvatore, C. A.; Sharik, S. S.; Theberge, C. R.; Zartman, C. B.; Kane, S. A.; Graham, S. L.; Selnick, H. G.; Vacca, J. P.; Williams, T. M. Identification of potent, highly constrained CGRP receptor antagonists. Bioorg. Med. Chem. Lett. 2010, 20, 2572–2576. Wood, M. R.; Schirripa, K. M.; Kim, J. J.; Quigley, A. G.; Stump, C. A.; Bell, I. M.; Bednar, R. A.; Fay, J. F.; Bruno, J. G.; Moore, E. L.; Mosser, S. D.; Roller, S.; Salvatore, C. A.; Vacca, J. P.; Selnick, H. G. Novel CGRP receptor antagonists through a design strategy of target simplification with additional molecular flexibility. Bioorg. Med. Chem. Lett. 2009, 19, 5787–5790. Bell, I. M.; Gallicchio, S. N.; Wood, M. R.; Quigley, A. G.; Stump, C. A.; Zartman, C. B.; Fay, J. F.; Li, C.-C.; Lynch, J. J.; Moore, E. L.; Mosser, S. D.; Prueksaritanont, T.; Regan, C. R.; Roller, S.; Salvatore, C. A.; Kane, S. A.; Vacca, J. P.; Selnick, H. G. Discovery of MK-3207: A highly potent, orally bioavailable CGRP receptor antagonist. ACS Med. Chem. Lett. 2010, 1, 24–29. Salvatore, C. A.; Moore, E. L.; Calamari, A.; Cook, J. J.; Michener, M. S.; O’Malley, S.; Miller, P. J.; Sur, C.; Williams, D. L.; Zeng, Z.; Danziger, A.; Lynch, J. J.; Regan, C. R.; Fay, J. F.; Tang, Y. S.; Li, C.-C.; Pudvah, N. T.; White, R. B.; Bell, I. M.; Gallicchio, S. N.; Graham, S. L.; Selnick, H. G.; Vacca, J. P.; Kane, S. A. Pharmacological properties of MK-3207, a potent and orally active calcitonin gene-related peptide receptor antagonist. J. Pharmacol. Exp. Ther. 2010, 333, 152–160. Moore, E. L.; Burgey, C. S.; Paone, D. V.; Shaw, A. W.; Tang, Y. S.; Kane, S. A.; Salvatore, C. A. Examining the binding properties of MK-0974: A CGRP receptor antagonist for the acute treatment of migraine. Eur. J. Pharmacol. 2009, 602, 250–254. Hershey, J. C.; Corcoran, H. A.; Baskin, E. P.; Salvatore, C. A.; Mosser, S.; Williams, T. M.; Koblan, K. S.; Hargreaves, R.; Kane, S. A. Investigation of the species selectivity of a nonpeptide CGRP receptor antagonist using a novel pharmacodynamic assay. Regul. Pept. 2005, 127, 71–77. Salvatore, C. A.; Hershey, J. C.; Corcoran, H. A.; Fay, J. F.; Johnston, V. K.; Moore, E. L.; Mosser, S. D.; Burgey, C. S.; Paone, D. V.; Shaw, A. W.; Graham, S. L.; Vacca, J. P.; Williams, T. M.; Koblan, K. S.; Kane, S. A. Pharmacological characterization of MK-0974 [N-[(3R,6S)-6-(2,3difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide], a potent and orally active calcitonin gene-related peptide receptor antagonist for the treatment of migraine. J. Pharmacol. Exp. Ther. 2008, 324, 416–421. Roller, S.; Cui, D.; Laspina, C.; Miller-Stein, C.; Rowe, J.; Wong, B.; Prueksaritanont, T. Preclinical pharmacokinetics of MK-0974, an orally active calcitonin gene-related peptide (CGRP)-receptor antagonist, mechanism of dose dependency and species differences. Xenobiotica 2009, 39, 33–45. Bell, I. M. Abstracts of Papers, 237th ACS National Meeting & Exposition, Salt Lake City, UT, United States, March 22-26, 2009; American Chemical Society: Washington, DC, 2009; MEDI 28. 128

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

52. Li, C.-C.; Vermeersch, S.; Denney, W. S.; Kennedy, W. P.; Palcza, J.; Gipson, A.; Han, T. H.; Blanchard, R.; De Lepeleire, I.; Depré, M.; Murphy, M. G.; Van Dyck, K.; de Hoon, J. N. Characterizing the PK/PD relationship for inhibition of capsaicin-induced dermal vasodilatation by MK-3207, an oral calcitonin gene related peptide receptor antagonist. Br. J. Clin. Pharmacol. 2015, 79, 831–837. 53. Hewitt, D. J.; Aurora, S. K.; Dodick, D. W.; Goadsby, P. J.; Ge, Y. J.; Bachman, R.; Taraborelli, D.; Fan, X.; Assaid, C.; Lines, C.; Ho, T. W. Randomized controlled trial of the CGRP receptor antagonist MK-3207 in the acute treatment of migraine. Cephalalgia 2011, 31, 712–722. 54. Ho, T. W.; Connor, K. M.; Zhang, Y.; Pearlman, E.; Koppenhaver, J.; Fan, X.; Lines, C.; Edvinsson, L.; Goadsby, P. J.; Michelson, D. Randomized controlled trial of the CGRP receptor antagonist telcagepant for migraine prevention. Neurology 2014, 83, 958–966. 55. Marfat, A.; Carta, M. P. Oxidation of indoles with pyridinium bromide perbromide: a simple and efficient synthesis of 7-azaindoles. Tetrahedron Lett. 1987, 28, 4027–4030. 56. Gallicchio, S. N.; Bell, I. M. Convenient synthesis of 1,3-substituted-6phenylpiperazin-2-ones. Tetrahedron Lett. 2009, 50, 3817–3819. 57. Dach, R.; Song, J. J.; Roschangar, F.; Samstag, W.; Senanayake, C. H. The eight criteria defining a good chemical manufacturing process. Org. Process Res. Dev. 2012, 16, 97–1706. 58. Leng, R. B.; Emonds, M. V. M.; Hamilton, C. T.; Ringer, J. W. Holistic route selection. Org. Process Res. Dev. 2012, 16, 415–424. 59. Butters, M.; Catterick, D.; Craig, A.; Curzons, A.; Dale, D.; Gillmore, A.; Green, S. P.; Marziano, I.; Sherlock, J.-P.; White, W. Critical assessment of pharmaceutical processes–a rationale for changing the synthetic route. Chem. Rev. 2006, 106, 3002–3027. 60. Anderson, N. G. Practical Process Research & Development, 2 ed.; Elsevier: Oxford, 2012; p 475. 61. Dunetz, J. R.; Magano, J.; Weisenburger, G. A. Large-scale applications of amide coupling reagents for the synthesis of pharmaceuticals. Org. Process Res. Dev. 2016, 20, 140–177. 62. Buitrago Santanilla, A.; Regalado, E. L.; Pereira, T.; Shevlin, M.; Bateman, K.; Campeau, L.-C.; Schneeweis, J.; Berritt, S.; Shi, Z.-C.; Nantermet, P.; Liu, Y.; Helmy, R.; Welch, C. J.; Vachal, P.; Davies, I. W.; Cernak, T.; Dreher, S. D. Nanomole-scale high-throughput chemistry for the synthesis of complex molecules. Science 2015, 347, 49–53. 63. Friedfeld, M. R.; Shevlin, M.; Hoyt, J. M.; Krska, S. W.; Tudge, M. T.; Chirik, P. J. Cobalt precursors for high-throughput discovery of base metal asymmetric alkene hydrogenation catalysts. Science 2013, 342, 1076–1080. 64. Pei, T.; Tellers, D. M.; Streckfuss, E. C.; Chen, C.; Davies, I. W. [1,2]-Aryl migration in the synthesis of substituted indoles: Scope, mechanism, and high throughput experimentation. Tetrahedron 2009, 65, 3285–3291. 65. Dreher, S. D.; Dormer, P. G.; Sandrock, D. L.; Molander, G. A. Efficient cross-coupling of secondary alkyltrifluoroborates with aryl 129

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

66. 67. 68.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

69.

70.

71.

72.

73. 74.

75.

76.

77.

78.

79.

chlorides–reaction discovery using parallel microscale experimentation. J. Am. Chem. Soc. 2008, 130, 9257–9259. Shultz, C. S.; Krska, S. W. Unlocking the potential of asymmetric hydrogenation at Merck. Acc. Chem. Res. 2007, 40, 1320–1326. Collins, K. D.; Gensch, T.; Glorius, F. Contemporary screening approaches to reaction discovery and development. Nature Chem. 2014, 6, 859–871. Schmink, J. R.; Bellomo, A.; Berritt, S. Scientist-led high-throughput experimentation (HTE) and its utility in academia and industry. Aldrichimica Acta 2013, 46, 71–80. Rubin, E.; Tummala, S.; Both, D. A.; Wang, C.; Delaney, E. J. Emerging technologies supporting chemical process R&D and their increasing impact on productivity in the pharmaceutical industry. Chem. Rev. 2006, 106, 2794–2810. McLaughlin, M.; Belyk, K.; Chen, C.; Linghu, X.; Pan, J.; Qian, G.; Reamer, R. A.; Xu, Y. Practical asymmetric synthesis of a chiral piperazinone derivative. Org. Process Res. Dev. 2013, 17, 1052–1060. Williams, A. J.; Chakthong, S.; Gray, D.; Lawrence, R. M.; Gallagher, T. 1,2-Cyclic sulfamidates as versatile precursors to thiomorpholines and piperazines. Org. Lett. 2003, 5, 811–814. Bower, J. F.; Rujirawanicha, J.; Gallagher, T. N-Heterocycle construction via cyclic sulfamidates. Applications in synthesis. Org. Biomol. Chem. 2010, 8, 1505–1519. Chen, Q.-A.; Ye, Z.-S.; Duan, Y.; Zhou, Y.-G. Homogeneous palladiumcatalyzed asymmetric hydrogenation. Chem. Soc. Rev. 2013, 42, 497–511. Etayo, P.; Vidal-Ferran, A. Rhodium-catalysed asymmetric hydrogenation as a valuable synthetic tool for the preparation of chiral drugs. Chem. Soc. Rev. 2013, 42, 728–754. Asymmetric hydrogenation affording 1,2-diols: Ohkuma, T.; Utsumi, N.; Watanabe, M.; Arai, N.; Murata, K. Asymmetric hydrogenation of α-hydroxy ketones catalyzed by MsDPEN–Cp*Ir(III) complex. Org. Lett. 2007, 9, 2565–2567. Asymmetric Mannich-type reactions affording β-amino-alcohols: Matsunaga, S.; Kumagai, N.; Harada, S.; Shibasaki, M. anti-Selective direct catalytic asymmetric Mannich-type reaction of hydroxyketone providing β-amino alcohols. J. Am. Chem. Soc. 2003, 125, 4712–4713. Asymmetric Mannich-type reactions affording β-amino-alcohols: Trost, B. M.; Jaratjaroonphong, J.; Reutrakul, V. A direct catalytic asymmetric Mannich-type reaction via a dinuclear zinc catalyst: Synthesis of either anti- or syn-α-hydroxy-β-amino ketones. J. Am. Chem. Soc. 2006, 128, 2778–2779. Acetophenone oxidation: Moriarty, R. M.; Berglund, B. A.; Penmasta, R. Direct α-hydroxylation of ketones under acidic conditions using [bis(trifluoroacetoxy)]iodobenzene. Tetrahedron Lett. 1992, 33, 6065–6068. Acetophenone oxidation: Chen, C.; Feng, X.; Zhang, G.; Zhao, Q.; Huang, G. An efficient method for the synthesis of α-hydroxyalkyl aryl ketones. Synthesis 2008, 3205–3208. 130

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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80. Via the dimethyl acetal: Moriarty, R. M.; Hou, K.-C.; Prakash, I.; Arora, S. K. α-Hydroxylation of a ketone using o-iodosylbenzoic acid: α-hydroxyacetophenone via the α-hydroxy dimethylacetal. Org. Synth. 1986, 64, 138. 81. Oxidation of silyl enol ethers: Moriarty, R. M.; Prakash, O.; Duncan, M. P. Hypervalent iodine oxidation of enol silyl ethers using boron trifluoride etherate. A direct route to aryl hydroxymethyl ketones. Synthesis 1985, 943–944. 82. Oxidation of silyl enol ethers: Le, J. C.-D.; Pagenkopf, B. L. Asymmetric hydrogenation of o-alkoxy-substituted arylenamides. J. Org. Chem. 2004, 69, 4177–4180. 83. From α-haloacetophenones: Zhu, G.; Casalnuovo, A. L.; Zhang, X. Practical syntheses of β-amino alcohols via asymmetric catalytic hydrogenation. J. Org. Chem. 1998, 63, 8100–8101. 84. From α-haloacetophenones: Wong, F. F.; Chang, P.-W.; Lin, H.-C.; You, B.J.; Huang, J.-J.; Lin, S.-K. An efficient and convenient transformation of α-haloketones to α-hydroxyketones using cesium formate. J. Organomet. Chem. 2009, 694, 3452–3455. 85. Enzymatic cleavage of α-acetoxyacetophenones: Paizs, C.; Tosa¸, M.; Majdik, C.; Bódai, V.; Novák, L.; Irimie, F. D.; Poppe, L. Chemo-enzymatic preparation of hydroxymethyl ketones. J. Chem. Soc., Perkin Trans. 1 2002, 2400–2402. 86. Hydroxymethylation of benzaldehydes: Demir, A. S.; Ahyan, P.; Igdir, A. C.; Duygu, A. N. Enzyme catalyzed hydroxymethylation of aromatic aldehydes with formaldehyde. Synthesis of hydroxyacetophenones and (S)-benzoins. Tetrahedron 2004, 60, 6509–6512. 87. Hydroxymethylation of benzaldehydes: Kuhl, N.; Glorious, F. Direct and efficient N-heterocyclic carbene-catalyzed hydroxymethylation of aldehydes. Chem. Commun. 2011, 47, 573–575. 88. McLaughlin, M.; Belyk, K. M.; Qian, G.; Reamer, R. A.; Chen, C. Synthesis of α-hydroxyacetophenones. J. Org. Chem. 2012, 77, 5144–5148. 89. Wang, Y.-Q.; Yu, C.-B.; Wang, D.-W.; Wang, X.-B.; Zhou, Y.-G. Enantioselective synthesis of cyclic sulfamidates via Pd-catalyzed hydrogenation. Org. Lett. 2008, 10, 2071–2074. 90. Hansen, K. B.; Hsiao, Y.; Xu, F.; Rivera, N.; Clausen, A.; Kubryk, M.; Krska, S.; Rosner, T.; Simmons, B.; Balsells, J.; Ikemoto, N.; Sun, Y.; Spindler, F.; Malan, C.; Grabowski, E. J. J.; Armstrong, J. D. Highly efficient asymmetric synthesis of sitagliptin. J. Am. Chem. Soc. 2009, 131, 8798–8804. 91. The utility of cyclic unsaturated sulfamates such as 49 has recently been extended, by these laboratories and others, beyond asymmetric hydrogenation to the enantioselective addition of aryl groups to generate tertiary amine stereocenters. See: Kong, J.; McLaughlin, M.; Belyk, K.; Mondschein, R. Enantioselective Rh(I)-catalyzed addition of arylboronic acids to cyclic ketimines. Org. Lett. 2015, 17, 5520–5523 and references therein. 131

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

92. Tilstam, U. Sulfolane: A versatile dipolar aprotic solvent. Org. Process Res. Dev. 2012, 16, 1273–1278. 93. Waring, M. J.; Arrowsmith, J.; Leach, A. R.; Leeson, P. D.; Mandrell, S.; Owen, R. M.; Pairaudeau, G.; Pennie, W. D.; Pickett, S. D.; Wang, J.; Wallace, O.; Weir, A. An analysis of the attrition of drug candidates from four major pharmaceutical companies. Nat. Rev. Drug Discovery 2015, 14, 475–486. 94. Kola, I.; Landis, J. Can the pharmaceutical industry reduce attrition rates? Nat. Rev. Drug Discovery 2004, 3, 711–715. 95. A trial acidic workup gave diol product 21 of low weight% purity and contaminated with boron salts. 96. In MTBE alone, formation of a gummy mixture was observed during the addition of PBr3 to a solution of diol 21. With a small amount of THF cosolvent a homogeneous reaction mixture was maintained throughout. 97. Jimenez-Gonzalez, C.; Ponder, C. S.; Broxterman, Q. B.; Manley, J. B. Using the right green yardstick: Why process mass intensity is used in the pharmaceutical industry to drive more sustainable processes. Org. Process Res. Dev. 2011, 15, 912–917. 98. Hong, L.; Wang, R. Recent advances in asymmetric organocatalytic construction of 3,3′-spirocyclic oxindoles. Adv. Synth. Catal. 2013, 355, 1023–1052. 99. Trost, B. M.; Brennan, M. K. Asymmetric synthesis of oxindole and indole spirocyclic alkaloid natural products. Synthesis 2009, 3003–3025. 100. Galliford, C. V.; Scheidt, K. A. Pyrrolidinyl-spirooxindole natural products as inspirations for the development of potential therapeutic agents. Angew. Chem., Int. Ed. 2007, 46, 2–13. 101. Marti, C.; Carreira, E. M. Construction of spiro[pyrrolidine-3,3′-oxindoles] – recent applications to the synthesis of oxindole alkaloids. Eur. J. Org. Chem. 2003, 2209–2219. 102. Dounay, A. B.; Overman, L. E. The asymmetric intramolecular Heck reaction in natural product total synthesis. Chem. Rev. 2003, 103, 2945–2963. 103. McCartney, D.; Guiry, P. J. The asymmetric Heck and related reactions. Chem. Soc. Rev. 2011, 40, 5122–5150. 104. Link, J. T.; Wada, C. In The Mizoroki-Heck Reaction; Oestreich, M., Ed.; Wiley-VCH: Weinheim, 2009; pp 433–462. 105. Link, T. J. Organic Reactions; Wiley-VCH: Hoboken, 2002; Vol. 60, pp 157–534. 106. Catalytic Transformations via C-H Activation; Yu, J.-Q., Ed.; Science of Synthesis; Thieme: Stuttgart, 2016; p 1044. 107. Grigg, R.; Fretwell, P.; Meerholtz, C.; Sridharan, V. Palladium catalysed synthesis of spiroindolines. Tetrahedron 1994, 50, 359–370. 108. Ruck, R. T.; Huffman, M. A.; Kim, M. M.; Shevlin, M.; Kandur, W. V.; Davies, I. W. Palladium-catalyzed tandem Heck reaction/C–H functionalization—preparation of spiro-indane-oxindoles. Angew. Chem., Int. Ed. 2008, 47, 4711–4714. 109. Piou, T.; Bunescu, A.; Wang, Q.; Neuville, L.; Zhu, J. Palladium-catalyzed through-space C(sp3)–H and C(sp2)–H bond activation by 1,4-palladium 132

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

110.

111.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch003

112. 113.

114.

115.

116.

117. 118. 119. 120.

121.

122.

123.

migration: Efficient synthesis of [3,4]-fused oxindoles. Angew. Chem., Int. Ed. 2013, 52, 12385–12389. Dolling, U.-H.; Davis, P.; Grabowski, E. J. J. Efficient catalytic asymmetric alkylations. 1. Enantioselective synthesis of (+)-indacrinone via chiral phase-transfer catalysis. J. Am. Chem. Soc. 1984, 106, 446–447. Hughes, D. L.; Dolling, U.-H.; Ryan, K. M.; Schoenewaldt, E. F.; Grabowski, E. J. J. Efficient catalytic asymmetric alkylations. 3. A kinetic and mechanistic study of the enantioselective phase-transfer methylation of 6,7-dichloro-5-methoxy-2-phenyl-1-indanone. J. Org. Chem. 1987, 52, 4745–4752. Bulger, P. G. In Comprehensive Chirality; Carreira, E. M., Yamamoto, H., Eds.; Elsevier: Amsterdam, 2012; Vol. 9, pp 228–252. Scott, J. P.; Ashwood, M. S.; Brands, K. M. J.; Brewer, S. E.; Cowden, C. J.; Dolling, U.-H.; Emerson, K. M.; Gibb, A. D.; Goodyear, A.; Oliver, S. F.; Stewart, G. W.; Wallace, D. J. Development of a phase transfer catalyzed asymmetric synthesis for an estrogen receptor beta selective agonist. Org. Process Res. Dev. 2008, 12, 723–730. Huffman, M. A.; Rosen, J. D.; Farr, R. N.; Lynch, J. E. Synthesis of a selective estrogen receptor β-modulator via asymmetric phase-transfer catalysis. Tetrahedron 2007, 63, 4459–4463. Conn, R. S. E.; Lovell, A. V.; Karady, S.; Weinstock, L. M. Chiral Michael addition: Methyl vinyl ketone addition catalyzed by Cinchona alkaloid derivatives. J. Org. Chem. 1986, 51, 4710–4711. Bhattacharya, A.; Dolling, U.-H.; Grabowski, E. J. J.; Karady, S.; Ryan, K. M.; Weinstock, L. M. Enantioselective Robinson annelations via phase-transfer catalysis. Angew. Chem., Int. Ed. 1986, 25, 476–477. Herchl, R.; Waser, M. Stereoselective cyclization reactions under phase-transfer catalysis. Tetrahedron 2014, 70, 1935–1960. Shirakawa, S.; Maruoka, K. Recent developments in asymmetric phase-transfer reactions. Angew. Chem., Int. Ed. 2013, 52, 4312–4348. Hashimoto, T.; Maruoka, K. Recent development and application of chiral phase-tansfer catalysts. Chem. Rev. 2007, 107, 5656–5682. Lee, T. B. K.; Wong, G. S. K. Asymmetric alkylation of oxindoles: An approach to the total synthesis of (–)-physostigmine. J. Org. Chem. 1991, 56, 872–875. Shirakawa, S.; Koga, K.; Tokuda, T.; Yamamoto, K.; Maruoka, K. Catalytic asymmetric synthesis of 3,3′-diaryloxindoles as triarylmethanes with a chiral all-carbon quaternary center: Phase-transfer-catalyzed SNAr reaction. Angew. Chem., Int. Ed. 2014, 53, 6220–6223. Deng, F.; Moteki, S. A.; Maruoka, K. Catalytic asymmetric alkylation of 3-aryl-substituted oxindoles to give 3,3-disubstituted oxindoles under phasetransfer conditions. Asian J. Org. Chem. 2014, 3, 395–398. Chen, W.; Yang, W.; Yan, L.; Tan, C.-H.; Jiang, Z. Bicyclic guanidiniumcatalyzed enantioselective phase-transfer alkylation: direct access to pyrroloindolines and furoindolines. Chem. Commun. 2013, 49, 9854–9856.

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124. Wu, X.; Liu, Q.; Liu, Y.; Wang, Q.; Zhang, Y.; Chen, J.; Cao, W.; Zhao, G. Amino acid-derived phosphonium salts-catalyzed Michael addition of 3-substituted oxindoles. Adv. Synth. Catal. 2013, 355, 2701–2706. 125. He, R.; Ding, C.; Maruoka, K. Phosphonium salts as chiral phase-transfer catalysts: Asymmetric Michael and Mannich reactions of 3-aryloxindoles. Angew. Chem., Int. Ed. 2009, 48, 4559–4561. 126. He, R.; Shirakawa, S.; Maruoka, K. Enantioselective base-free phase-transfer reaction in water-rich solvent. J. Am. Chem. Soc. 2009, 131, 16620–16621. 127. Racemic PTC alkylation of 1,2-bis(benzyl)halides with glycine equivalents have been reported, but these electrophiles have not been used for enantioselective alkylations. See: Kotha, S.; Brahmachary, E. Synthesis of indan-based unusual α-amino acid derivatives under phase-transfer catalysis conditions. J. Org. Chem. 2000, 65, 1359–1365. 128. Ellis, T. K.; Hochla, V. M.; Soloshonok, V. A. Efficient synthesis of 2-aminoindane-2-carboxylic acid via dialkylation of nucleophilic glycine equivalent. J. Org. Chem. 2003, 68, 4973–4976. 129. Ooi, T.; Kameda, M.; Maruoka, K. Molecular design of a C2-symmetric chiral phase-transfer catalyst for practical asymmetric synthesis of α-amino acids. J. Am. Chem. Soc. 1999, 121, 6519–6520. 130. Kitamura, M.; Shirakawa, S.; Maruoka, K. Powerful chiral phase-transfer catalysts for the asymmetric synthesis of α-alkyl- and α,α-dialkyl-α-amino acids. Angew. Chem., Int. Ed. 2005, 44, 1549–1551. 131. Takahashi, H.; Kashiwa, N.; Kobayashi, H.; Hashimoto, Y.; Nagasawa, K. Nucleophilic substitution on 4-hydroxymethylanilines under ‘neutral’ conditions via aza quinone methide intermediate. Tetrahedron Lett. 2002, 43, 5751–5753. 132. The use of binaphthyl-derived catalysts 97 and 98 gave 22% and 33% ee, respectively. 133. Melchiorre, P. Cinchona-based primary amine catalysis in the asymmetric functionalization of carbonyl compounds. Angew. Chem., Int. Ed. 2012, 51, 9748–9770. 134. Marcelli, T.; Hiemstra, H. Cinchona alkaloids in asymmetric organocatalysis. Synthesis 2010, 1229–1279. 135. Cinchona Alkaloids in Synthesis & Catalysis: Ligands, Immobilization and Organocatalysis; Song, C. E., Ed.; Wiley-VCH: Weinheim, 2010; p 546. 136. Jew, S.; Park, H. Cinchona-based phase-transfer catalysts for asymmetric synthesis. Chem. Commun. 2009, 7090–7103. 137. MacNeil, S. L.; Gray, M.; Briggs, L. E.; Li, J. J.; Snieckus, V. Directed ortho and remote metalation - cross coupling connections. Buchwald-Hartwig synthesis of 2-carbamoyl diarylamines. Regioselective anionic routes to acridones, oxindoles, dibenzo-[b,f]azepinones, and anthranilate esters. Synlett 1998, 419–421. 138. Mander, L. N.; Sethi, S. P. Regioselective synthesis of β-ketoesters from lithium enolates and methyl cyanoformate. Tetrahedron Lett. 1983, 24, 5425–5428. 139. A related acyl migration has been reported, see: Kise, N.; Ozaki, H.; Terui, H.; Ohya, K.; Ueda, N. A convenient synthesis of N-Boc-protected 134

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140. 141.

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142.

143.

144. 145.

146.

147. 148.

149. 150.

151.

tert-butyl esters of phenylglycines from benzylamines. Tetrahedron Lett. 2001, 42, 7637–7639. Wuts, P. G. M. Greene’s Protective Groups in Organic Synthesis, 5th ed.; Wiley: Hoboken; 2014. A potential concern with the use of MsOH and MeOH was the formation and persistence of mutagenic methyl methanesulfonate, however this was well controlled during the processing. For discussion of alkyl sulfonate impurities in pharmaceutical manufacturing, see: Snodin, D.; Teasdale, A. Mutagenic alkyl-sulfonate impurities in sulfonic acid salts: Reviewing the evidence and challenging regulatory perceptions. Org. Process Res. Dev. 2015, 19, 1465–1485. Elder, D.; Facchine, K. L.; Levy, J. N.; Parsons, R.; Ridge, D.; Semo, L.; Teasdale, A. An approach to control strategies for sulfonate ester formation in pharmaceutical manufacturing based on recent scientific understanding. Org. Process Res. Dev. 2012, 16, 1707–1710. Attempts to perform the PTC spirocyclization cascade using an unprotected aniline electrophile resulted in complex, intractable product mixtures, thus protection of the amino group as the dibenzyl derivative was necessary. The use of this diluted aqueous THF solution enabled better control of exotherm and effervescence compared to the addition of pure water. Catalyst 128 was prepared in 84% yield by refluxing cinchonidine with 3,5-bis(trifluoromethyl)benzyl bromide (1.1 equiv) in 2-propanol, followed by cooling and filtration of the crystalline product. Commercially available cinchonidine typically contain up to 15% dihydrocinchonidine which also forms the corresponding quaternized salt, the level of which did not impact the selectivity of the PTC cascade. Albanese, D.; Landini, D.; Maia, A.; Penso, M. Key role of water for nucleophilic substitution in phase-transfer-catalyzed processes: A mini-review. Ind. Eng. Chem. Res. 2001, 40, 2396–2401. A downgrade of enantiopurity in the product cake was observed when pure MeOH was used as the wash. A synthetically useful oxindole hydroxylation PTC protocol has been developed, see: Sano, D.; Nagata; Itoh, T. Catalytic asymmetric hydroxylation of oxindoles by molecular oxygen using a phase-transfer catalyst. Org. Lett. 2008, 10, 1593–1595. Belyk, K. M.; Bulger, P. G.; Linghu, X.; Maloney, K. M.; McLaughlin, M.; Pan, J.; Xiang, B.; Xu, Y.; Yin, J. PCT Int. Appl. WO 201105731, 2011. Denmark, S. E.; Gould, N. D.; Wolf, L. M. A systematic investigation of quaternary ammonium ions as asymmetric phase-transfer catalysts. Application of quantitative structure activity/selectivity relationships. J. Org. Chem. 2011, 76, 4337–4357. Denmark, S. E.; Gould, N. D.; Wolf, L. M. A systematic investigation of quaternary ammonium ions as asymmetric phase-transfer catalysts. Synthesis of catalyst libraries and evaluation of catalyst activity. J. Org. Chem. 2011, 76, 4260–4336.

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152. Williams, M. J.; Kong, J.; Chung, C. K.; Brunskill, A.; Campeau, L.-C.; McLaughlin, M. The discovery of quinoxaline-based metathesis catalysts from synthesis of grazoprevir (MK-5172). Org. Lett. 2016, 18, 1952–1955. 153. Kuethe, J.; Zhong, Y.-L.; Yasuda, N.; Beutner, G.; Linn, K.; Kim, M.; Marcune, B.; Dreher, S. D.; Humphrey, G.; Pei, T. Development of a practical, asymmetric synthesis of the hepatitis C virus protease inhibitor MK-5172. Org. Lett. 2013, 15, 4174–4177. 154. Kazmierski, W. M.; Jarvest, R. L.; Plattner, J. J.; Li, X. In Macrocycles in Drug Discovery; Levin, J., Ed.; RSC Drug Discovery Series 40; Royal Society of Chemistry: London, 2014; pp 235–282. 155. Hui, C.-Y.; Xie, X.-B.; Cao, H.; Huang, S.-H. The development of novel HCV NS3-4A protease inhibitors. Anti-Infect. Agents 2013, 11, 125–135. 156. Sato, T.; Izawa, K.; Aceña, J. L.; Liu, H.; Soloshonok, V. A. Tailor-made α-amino acids in the pharmaceutical industry: Synthetic approaches to (1R,2S)-1-amino-2-vinylcyclopropane-1-carboxylic Acid (vinyl-ACCA). Eur. J. Org. Chem. 2016, 2757–2774. 157. Beaulieu, P. L.; Gillard, J.; Bailey, M. D.; Boucher, C.; Duceppe, J.-S.; Simoneau, B.; Wang, X.-J.; Zhang, L.; Grozinger, K.; Houpis, I.; Farina, V.; Heimroth, H.; Krueger, T.; Schnaubelt, J. Synthesis of (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid (vinyl-ACCA) derivatives: Key intermediates for the preparation of inhibitors of the hepatitis C virus NS3 protease. J. Org. Chem. 2005, 70, 5869–5879. 158. Belyk, K. M.; Xiang, B.; Bulger, P. G.; Leonard, W. R., Jr.; Balsells, J.; Yin, J.; Chen, C. Enantioselective synthesis of (1R,2S)-1-amino2-vinylcyclopropanecarboxylic acid ethyl ester (Vinyl-ACCA-OEt) by asymmetric phase-transfer catalyzed cyclopropanation of (E)-Nphenylmethyleneglycine ethyl ester. Org. Process Res. Dev. 2010, 14, 692–700. 159. Another investigation of the asymmetric PTC approach to ester 156 was subsequently reported from Bristol-Myers Squibb laboratories, see: Lou, S.; Cuniere, N.; Su, B.-N.; Hobson, L. A. Concise asymmetric synthesis of (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid-derived sulfonamide and ethyl ester. Org. Biomol. Chem. 2013, 11, 6796–6805. 160. Bell, I. M.; Stump, C. A.; Gallicchio, S. N.; Staas, D. D.; Zartman, C. B.; Moore, E. L.; Sain, N.; Urban, M.; Bruno, J. G.; Calamari, A.; Kemmerer, A. L.; Mosser, S. D.; Fandozzi, C.; White, R. B.; Zrada, M. M.; Selnick, H. G.; Graham, S. L.; Vacca, J. P.; Kane, S. A.; Salvatore, C. A. MK-8825: A potent and selective CGRP receptor antagonist with good oral activity in rats. Bioorg. Med. Chem. Lett. 2012, 22, 3941–3945. 161. Xiang, B.; Belyk, K. M.; Reamer, R.; Yasuda, N. Discovery and application of double quaternized cinchona-alkaloid-based phase-transfer catalysts. Angew. Chem., Int. Ed. 2014, 53, 8375–8378. 162. Humphrey, G. R.; Dalby, S. M.; Andreani, T.; Xiang, B.; Luzung, M. R.; Song, Z. J.; Shevlin, M.; Christensen, M.; Belyk, K. M.; Tschaen, D. M. Asymmetric synthsesis of letermovir using a novel phase-transfer-catalyzed aza-Michael reaction. Org. Process Res. Dev. 2016, 20, 1097–1103. 136

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Chapter 4

Design and Enabling Development of Hydroxyethylamine-Derived BACE1 Inhibitor Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch004

Jason S. Tedrow*,1 and Wenge Zhong2 1Process

Development, Amgen Inc, One Amgen Center Drive, Thousand Oaks, California 91320, United States 2Discovery Research, Amgen Asia R&D Center, 99 Haike Road, 4th Floor, Building 6, Shanghai 201210, P. R. China *E-mail: [email protected].

Herein we report the hydroxyethylamine (HEA)-derived potent and orally efficacious BACE1 inhibitors as potential treatments for Alzheimer’s disease. These compounds were designed for low efflux and in vivo clearance to effect robust reduction of Aβ levels in the central nervous system (CNS). Key design strategies feature an amide masking approach for mitigating PGP-mediated efflux and the incorporation of CYP 3A4 inhibitory activity for decreased in vitro and in vivo clearance. Lead molecules demonstrated sufficient oral bioavailability and CNS penetration and were shown to be orally efficacious in pre-clinical rodent models. Collaboration between medicinal and process chemistry on the key synthetic challenges is presented including new chemistry towards challenging fragments of the HEA core structure. The new routes were designed for scalability and improved overall safety (elimination of hazardous reagents). Additionally a new, templated assembly route toward the HEA core structures was developed to overcome key challenges using traditional methods for HEA construction.

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Medicinal Chemistry and Discovery of HEA-Derived BACE1 Inhibitors as Potential Disease Modifying Treatments for Alzheimer’s Disease Alzheimer’s Disease (AD) is a debilitating neurodegenerative disease and the most common form of dementia. AD afflicts more than five million people in the United States (1). It is estimated that by the year 2050, the total number of AD patients will reach 16 million in the U.S. and over 50 million worldwide. There is tremendous medical and socioeconomic burden associated with the disease. Currently only symptomatic treatments are available and they provide modest temporary benefits (2). Thus finding a disease modifying treatment to slow, stop or even reverse AD represents a huge unmet medical need. One of the key characteristics of AD is the accumulation of insoluble amyloid plaques in the brain. The principal components of the amyloid plaques are the amyloid β peptides (Aβ) of various lengths, typically 38-43 amino acids (3). A large body of evidence has suggested that increased formation and/or impaired clearance of Aβ peptides are the underlying pathological mechanism for the disease (3, 4). Aβ peptides are produced in the brain via a tandem two-step proteolytic cleavages of the amyloid precursor protein (APP) by BACE1 (β-site APP cleaving enzyme or β-secretase) and γ-secretase. Thus, inhibition of secretase functions could provide a potential disease modifying approach for AD. During the last several decades, substantial effort across the pharmaceutical industry has been dedicated to the finding of viable γ-secretase inhibitors but has met with no clinical success so far (5). In more recent years, BACE1 emerged as the secretase target of focused interest across the industry. Genetic data from familial AD patients indicated that mutations in APP around the BACE1 cleavage site lead to increased processing of APP and accumulation of Aβ peptides (6). Interestingly, it was also found that a low frequency APP mutation, A673T, two amino acids after the BACE1 cleavage site, reduces APP processing by BACE1 and decreased risk of AD and cognitive decline in the aged people (7). On these genetic bases and the additional notion that the cleavage of APP by BACE1 is the rate limiting first step (8, 9), many groups committed substantial amount of efforts to the finding of effective BACE1 inhibitors as possible disease modifying agents (10, 11). It should be noted that many different BACE1 substrates other than APP have been discovered and the implications of overall inhibition of BACE1 are not well understood. It remains to be proven if BACE1 inhibitors can be effective treatments for AD with significant benefits over any potential side effects (12). Currently, two of the most advanced molecules MK-8931 (verubecestat) and AZD3293 (Figure 1) are in phase III clinical trials for mild-to-moderate and early AD, respectively (13). Amgen’s discovery research on BACE1 dates back to the cloning of the enzyme in 1999 (14). Since then, we have embarked on a long and committed journey of finding promising BACE1 inhibitors for AD. With the availability of numerous in-house crystal and co-crystal structures, we initiated a structure-based drug discovery program and started working with a chemical series that was based on the hydroxyethylene (HE) transition state isostere. Despite the fact that 138 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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single digit nM BACE1 inhibitors were quickly identified, they suffered from many drawbacks, among which most noteworthy were poor cellular activity and chemical instability due to lactone formation. For example, a representative compound in the series, 1, (Figure 2A) in the fluorescence resonance energy transfer (FRET) based BACE1 enzyme assay exhibited an IC50 of 6.1±2.3 nM, but in the cellular assay its IC50 was 500±120 nM, indicating an enzyme-to-cell shift of over 80-fold.

Figure 1. Structures of MK-8931 and AZD3293. Additionally, during the synthesis and purification of these compounds, an impurity identified as the lactone (Figure 2B) was often observed in amounts up to 15%. In order to circumvent these issues and to reduce size of the molecules, we moved on to a new series that is based on the hydroxyethylamine (HEA) transition state isostere. In this article, we describe briefly the evolution of the series leading to the highly efficacious BACE1 inhibitors and the chemistry that was developed for the large scale synthesis of these promising molecules.

Figure 2. A. Structure of a Representative HE-derived Inhibitor; B. Formation of the Lactone. Compound 2 was the first promising HEA-derived BACE1 inhibitor in our program that afforded modest enzymatic activity and very small enzyme-to-cell shift (BACE1 IC50: 76±2.3 nM; Cell IC50: 210±160 nM) (Figure 3). Ring formation gave rise to a chroman derivative 3 that displayed potent BACE1 activity with an IC50 of 4.5±2.5 nM and Cell IC50 of 64±18 nM. Introduction of spirocyclobutane ring onto the chroman scaffold further improved enzymatic activities consistently, for example, 4 was identified as a 1.7±0.6 nM BACE1 inhibitor with good cellular activity (IC50: 22±4.9 nM). Based on the available structural information and modeling, the binding mode of 4 with the BACE1 enzyme is illustrated in Figure 3. We reported previously that P2′ (15) interactions with the enzyme were optimized with the introduction of a neopentyl group at this site (16). We found 139

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that the combination of the spirocyclobutane P1′ and neopentyl P2′ groups allowed us to truncate the left hand pyridone-derived acyl group substantially to just a simple acetyl group, rendering analogs such as 5 to retain potent activity (BACE1 IC50: 7.2±2.9 nM; Cell IC50: 81±12 nM). Ultimately, realizing the 8-position of the chroman is solvent exposed when binding to the BACE1 enzyme, we replaced chroman with 8-aza chroman which consistently preserved potent enzyme activities and minimized the enzyme-to-cell shift (6, BACE1 IC50: 5.8±6.3 nM; Cell IC50: 3.1±4.2 nM).

Figure 3. Evolution of the Early HEA-derived BACE1 Inhibitors. With potent cellular activities and chemical stability accomplished in the HEA series, we next turned our attention to achieving sufficient exposure in the central nervous system (CNS). It is well documented in the literature that major impediments for CNS penetration by small molecules are poor passive permeability and P-glycoprotein (PGP)-mediated efflux (17). Our early HEA-derived BACE1 inhibitors displayed good to excellent passive permeability as measured in the LLC-PK1 parental cell line assay, however, they suffered from high PGP-mediated efflux in the LLC-PK1 PGP-transfected cell line assay. Furthermore, these early inhibitors exhibited high clearance in rat pharmacokinetics studies, leading to poor systemic and brain exposure in vivo. In order to understand the importance of PGP-mediated efflux and in vivo clearance for achieving adequate BACE1 target coverage in the CNS for Aβ lowering efficacy, we performed co-dosing studies with a representative inhibitor 7 (Figure 4A; BACE1 IC50: 31±19 nM; Cell IC50: 34±6.8 nM). Compound 7 showed excellent passive permeability (average Papp: 26E-06 cm/s) with very high rat MDR1-mediated efflux ratio of 41. It also had a moderately high intravenous (iv) clearance in Sprague-Dawley rats at 2.6 L/h/kg. In one co-dosing study in rat, 7 was dosed at 30 mg/kg orally (i.e., per os, or p.o.) in combination with the known PGP inhibitor GF-120918 (Figure 4B; 100 mg/kg, p.o.). In another study, 7 was dosed at 30 mg/kg orally in combination with the well-known cytochrome 140

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P450 3A4 CYP 3A4 inhibitor ritonavir (Figure 4C; 10 mg/kg, p.o.). Two h after dosing, cerebrospinal fluid (CSF) samples were taken, Aβ40 levels were determined, and compared to the vehicle-treated group and the 7 alone treated group (30 mg/kg, p.o.). Plasma and CSF drug levels were also measured. We should point out that in all our rodent studies, CSF Aβ lowering effects and drug levels are appropriate surrogates for brain Aβ lowering efficacy and free drug concentrations, respectively.

Figure 4. A. Structure of Compound 7; B. Structure of GF-120918; C. Structure of Ritonavir. Table 1 summarizes the results from the PGP inhibitor GF-120918 co-dosing study. When 7 was dosed alone, the plasma drug concentration ([Plasma]) and the CSF drug concentration ([CSF], an approximate indicator of free drug level in the brain) were 0.609 μM and 0.007 μM, respectively. This gave a very poor [CSF]/[Plasma] ratio of 0.011 which was suggestive of poor brain exposure, and the CSF drug level was only about 20% of the cellular IC50 of compound 7, thus no significant CSF Aβ40 reduction was observed. In contrast, when 7 was co-dosed with GF-120918, [Plasma] was modestly increased to 1.65 μM. More importantly, [CSF] was increased to 0.222 μM by a factor of greater than 30-fold. This represented a roughly ten-fold improvement in [CSF]/[Plasma] ratio to 0.134 and [CSF] was about seven-fold of the cellular IC50 for 7. As a result, a robust CSF Aβ40 reduction of 71% was achieved.

Table 1. Co-dosing Results with GF-120918 Additive

[Plasma] (μM)

[CSF] (μM)

[CSF]/[Plasma]

↓CSF Aβ40

30 mg/kg, p.o.

__

0.61

0.007

0.011

< 10%

30 mg/kg, p.o.

GF120918

1.65

0.222

0.134

71%

Compound 7

Results from the co-dosing study with CYP 3A4 inhibitor ritonavir are provided in Table 2. In this study, dosing 7 alone yielded drug levels in both plasma and CSF that were about twice as high as in the co-dosing study with GF-120918 (Table 1). This difference was presumably due to study-to-study 141 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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variations. Nevertheless, the [CSF]/[Plasma] ratio remained very low at 0.010 and CSF Aβ40 reduction was less than 10%. Co-dosing with ritonavir increased both [Plasma] and [CSF] similarly by about ten-fold with the [CSF]/[Plasma] ratio essentially unchanged. Since [CSF] reached 0.176 μM, which was five-fold of the cellular IC50 for 7, a significant CSF Aβ40 reduction of 55% was observed. Taken together, we concluded from these two co-dosing studies that reducing PGP-mediated efflux and in vivo systemic clearance for improving CNS exposure should be the main optimization strategies for identifying efficacious HEA-derived BACE1 inhibitors.

Table 2. Co-dosing Results with Ritonavir Compound 7

Additive

[Plasma] (μM)

[CSF] (μM)

[CSF]/[Plasma]

↓CSF Aβ40

30 mg/kg, p.o.

__

1.47

0.015

0.010

< 10%

30 mg/kg, p.o.

Ritonavir

14.2

0.176

0.012

55%

Studies have shown that the total number of H-bond donors (HBD’s) in a molecule is a critical physicochemical parameter and typically less than a total of three HBD’s is desirable for achieving good brain exposure (18). Since the HEA core already possesses two HBD’s that are necessary for direct interactions with the BAC1 enzyme catalytic residues at the active site, we sought to reduce the apparent total number of HBD’s by masking the amide N-H via a possible intramolecular H-bond. We contemplated that such a masking strategy could retain all key interactions with the enzyme and good passive permeability but significantly improve efflux. This indeed proved to be one of the most fruitful approaches we employed to mitigate PGP-mediated efflux for the HEA derivatives. For example, we introduced a suitably-positioned methoxy group on the acyl moiety to be capable of forming an interamolecular H-bond with, and mask the amide N-H bond (Figure 5). Similar modification of 8 thus provided analog 9 (Figure 5B). In order to augment the possibility of forming an intramolecular H-bond, we further introduced an additional methyl group to afford analog 10. To our gratification, we found that 8 and 9 displayed essentially identical enzyme and cell activities and 10 was just about two-fold less potent in both the enzyme and cell assays. The average passive permeabilities for the three compounds were comparable. Perhaps most importantly, the efflux ratios were dramatically improved, suggesting that PGP-mediated efflux was significantly reduced. In the rat MDR1- transfected LLC-PK1 cell assay, the efflux ratios for 8, 9 and 10 were 43, 18, and 7.7, respectively. When these compounds were dosed orally at 30 mg/kg in rat, 8 reduced CSF Aβ40 levels by less than 10%. In contrast, 9 and 10 reduced CSF Aβ40 levels by 39% and 50%, respectively. 142

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Figure 5. Masking the Amide N-H via an Intramolecular H-bond.

Having established methods for improving efflux, we then turned our attention to focus on improve in vivo systemic clearance. Towards this end, we designed and prepared a large number of analogs that were very stable in the liver microsomes. However, these analogs continued to suffer from moderate to high in vivo clearance in rodents that led to limited exposures in plasma and in the brain for significant Aβ lowering efficacy (unpublished data). As detailed in our previous report (18), we committed to a strategy to build a CYP 3A4 inhibitory property into the HEA-derived molecules. After surveying the effects of various CYP 3A4 inhibitory functional groups around the molecule, we prioritized our effort on modifying the P1 aryl group. Among these, the benzodioxole P1 derived analogs designated as ‘699 and ‘359 emerged as the leading molecules in our program (Figure 6). While ‘699 appeared to be three-fold more potent than ‘359 in the BACE1 enzyme assay, they exhibited comparable cellular activity in the low double digit nM range (Cell IC50 for ‘699: 17 ± 5.0 nM: for ‘359: 26± 11 nM). Both compounds displayed excellent passive permeability with average Papp > 15E-06 cm/s. The efflux ratios as measured in the human and rat MDR1-transfected LLC-PK1 cell lines for ‘699 were still in the high range (hMDR1 efflux ratio: 16; rMDR1 efflux ratio: 27), however, those in the same assays for ‘359 were in the low range (hMDR1 efflux ratio: 4.0; rMDR1 efflux ratio: 6.0). As expected from the incorporation of benzodioxole group, both ‘699 and ‘359 showed potent 3A4 inhibitory activities (human 3A4 IC50 for ‘699: < 0.1 μM; for ‘359: 0.1 μM), resulting in low to moderate in vitro clearance in the human and rat microsome incubation experiments. We further profiled ‘699 and ‘359 in rat pharmacokinetics studies. When ‘699 was dosed at 2.0 mg/kg intravenously in fed male Sprague-Dawley rats, the observed clearance was 1.3 L/h/kg, volume distribution (Vss) was 5.3 L/kg, and t1/2 was 8.5 h, respectively. Its oral bioavailability was estimated to be greater than 100% from another oral pharmacokinetics study in fasted Sprague-Dawley 143

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rats. We observed a similar rat pharmacokinetics profile for ‘359 and its dog and monkey pharmacokinetics profiles were also very favorable. The results were detailed in our earlier publication (18). Owing to their overall properties of potency, reduced PGP-mediated efflux, and improved in vivo clearance and systemic exposure, ‘699 and ‘359 showed robust in vivo CSF Aβ lowering of 52% and 57%, respectively (dose: 30 mg/kg as a solution in 1% Tween/2%HPMC; sampling at 4 h after dosing). With these data in hand, the team selected both ‘699 and ‘359 for further advancement.

Figure 6. Structures of Lead Molecules ‘699 and ‘359. During the course of the SAR development, the medicinal chemistry team implemented several lines of chemistry to quickly access these P1 analogs. Most noteworthy is the chiral sulfinylimine chemistry that was published by our group (15, 19). With the identification of lead molecules ‘699 and ‘359, Amgen’s medicinal chemistry and process chemistry teams started to work together. In the next section, we describe in detail the medicinal chemistry routes and process development for preparing the advanced HEA-derived BACE 1 inhibitors.

Process Research Toward Synthesis of the Hydroxyethylamine BACE Inhibitor Class: A New Templated Approach to the HEA Core Due to the inherent synthetic complexity and challenges to prepare gram amounts of the lead HEA molecules for the BACE program, the process chemistry team embarked on an early engagement strategy with our discovery group. The goals of the collaboration were to support and/or accelerate lead candidate selection and develop a fit-for-purpose synthetic route to enable early development trials. Process research concentrated on enabling support for the BACE program with focus on elimination of any key technological road block(s) and early process development for kilogram production with built-in flexibility to pivot on emerging SAR. Key to this was to build in technology to intercept intermediates common to the medicinal chemistry synthesis where applicable. 144

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While medicinal chemistry was continuing to optimize the final candidate, the lead series contained most of the key synthetic challenges and thus we focused our enabling route selection efforts toward targeting ‘699 and ‘359 as prototypical of the ultimate compound. The medicinal chemistry route to the final molecule, while ideally positioned for maximal flexibility, presented a number of difficulties from a process chemistry perspective (Schemes 1-3). Outlined from the start, some of the key challenges toward scalability of the route was the long synthetic sequence (29 steps in total), three non-contiguous stereocenters which are set independently from one another and a challenging fragment coupling with an epimerization prone α-alkoxy aldehyde 12. Additionally the use of protecting groups and heavy reliance on chromatographic purification to control quality attributes presented substantial hurdles toward future scale-up.

Scheme 1. Discovery Assembly Route to ‘359 and ‘699

On its surface, the discovery disconnection around the central dialkyl amine (Scheme 1) was attractive and we sought to exploit similar tactics in hopes of maintaining overall convergence of the route. This approach then distilled the process chemistry challenges to definition of the disconnection strategy which in turn defines the structure of the fragments to join. 145 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 2. Discovery Synthesis of P1 Alcohol 11

Two alternate reactivity modes to build the target were considered as alternatives to the previous P1 aldehyde reductive amination (Figure 7). Route 1 relied on a fully elaborated azachromyl amine 14 reacting with an electrophilic P1 epoxide, building upon precedent from the HEA literature surrounding commercially available / late stage clinical candidates (vide infra). Multiple routes were known and likely applicable to our epoxide fragments, so technical feasibility regarding the epoxide aminolysis was the key question. Route 2 had limited precedent in the literature regarding a stereoselective reductive amination of a P1 amine fragment and an azachromyl ketone 13. Each of these pathways require redefinition of both of the key fragments and thus their synthetic routes. As the majority of the SAR optimization was focused in on the P1 fragment of the molecules, initial process development focused in on synthesis of the amine and ketone fragments to enable both synthetic approaches in Figure 7. 146

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Scheme 3. Discovery Synthesis of Azachromyl Amine 14

Process Research Targeting a Scalable Synthesis of Azachromanone 13 and Azachromylamine 14 The discovery synthesis of the azachromylamine fragment (Scheme 3) presented several inherent throughput difficulties which led us toward rethinking the entire assembly strategy for the intermediate. The key transformations were built around an intramolecular SnAr of the tertiary cyclobutyl alcohol 30 into a fluoropyridine to construct the azachroman structure (Scheme 4). Intermediate 30 is built from the pyridyl Grignard addition to a functionalized cyclobutane aldehyde, which is four steps from commercially available cyclobutane. Basic concerns with this sequence surrounded the instability of the pyridyl organometallic, overall length in synthesis of the cyclobutane linker and lack of robust crystalline intermediates to control purity outside of chromatography. In rethinking the overall strategy to synthesize the target azachromylamine, a need for access to both the neopentyl and the bromoazachromyl amine systems was required by medicinal chemistry to further SAR at the 6-position. The target then became developing a synthesis to ketone 37 (Scheme 5). 147

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Figure 7. Retrosynthetic Strategy Targeting Penultimate 15.

Scheme 4. Discovery Approach to Azachromanone 13

A prominent approach to access 2-aryl substituted chromanones involves intramolecular conjugate addition of a phenol to an enone (20, 21). In the case of the 2-aryl chromanones, the precursor enone is easily accessed via aldol condensation of the arylmethylketone and an aryl aldehyde. This type of method was viewed to be a challenge to target the cyclobutyl enone, however similar systems have been generated via olefination reactions of the Wittig (22, 23), Horner-Wadsworth-Emmons (HWE) (24, 25), and Peterson (26) type. Of the aforementioned transformations, the HWE approach was particularly attractive as the requisite ketophosphonates may be accessed from the corresponding esters (27–29). 148 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 5. Alternate Retrosynthesis to Azachroman Structures 38 & 13 Bromination of the readily available 2-methoxynicotinic acid 41 in the biphasic CH2Cl2/water (30) mixture afforded 5-bromo-2-methoxynicotinic acid. The product was precipitated upon antisolvent induced crystallization and was isolated directly by filtration. Esterification (refluxing H2SO4/MeOH) (31) gave the methyl ester 40 in 97% for the combined two steps. Ketophosphonate 42 was prepared by the reaction of bromonicotinic ester 40 and methyl dimethylphosphonate using previously optimized conditions (32) in 82% yield (Scheme 6).

Scheme 6. Synthesis of Ketophosphonate 42 With the ketophosphonate 42 in hand, its HWE reaction with cyclobutanone was addressed. Cyclobutanone is known to undergo HWE reactions with stabilized phosphonates (33, 34), however the range of these substrates is limited and its reaction with more elaborate ketophosphonates is unknown. Screening of various conditions (35), illustrated that polar solvents and/or bases which generated a protic by-product (hexamethyldisilazine (HMDS), alcohols, water) showed diminished yields. Alkyl lithium bases with toluene as solvent showed clean reaction to the corresponding enone. Simple use of LiOMe in toluene, followed by azeotropic removal of methanol and then treatment with cyclobutanone delivered the desired enone 44 on gram scale (Scheme 7). In practice however, we found that we could isolate the lithium enolphosphonate salt and eliminate the need for extensive distillations. Additionally, the salt 42 was found to be bench stable, non-hygroscopic and served as an important crystalline holding point in the process. Formation of the lithioketophosphonate 43 was effected with a solution of LiOMe/MeOH in i-PrOH directly telescoped from the crude reaction mixture of 42. Horner-Wadsworth Emmons reaction of 43 was performed with two equivalents of cyclobutanone relative to 43 in five volumes of toluene at 90 °C. Aqueous workup and distillation 149

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afford the enone 44 as a brown oil in 60% yield. Further attempts to increase the yield beyond 60% were not productive due to competing decomposition of the lithioketophosphonate under the reaction conditions (36). Demethylation and cyclization was easily effected by in situ generated iodotrimethylsilane (TMSCl/NaI in MeCN), and the product ketone could be isolated following aqueous workup in 75% yield when the purified 44 was used. However, when the crude material was subjected to the cyclization conditions, a short plug of silica gel was required for isolation of the crystalline 38. We demonstrated the robustness of the overall sequence (Scheme 7) on a ten kilogram scale and obtained 38 in 45% yield from the lithioketophosphonate 43 in two steps and in 39% yield for the overall sequence starting from 41.

Scheme 7. Optimized HWE approach to chromanone 38

With a scalable synthesis in hand of the chromanone 38, focus shifted on amine installation. From the medicinal chemistry work, the asymmetric ketone reduction / azide displacement (37) sequence constituted a dependable approach toward small-scale synthesis of azachromyl amine 46 (Scheme 8). Corey-Bakshi-Shibata (CBS) reduction of the alcohol proved unreliable and often required a significant excess of borane to achieve full conversion. Replacing the CBS reduction with a ruthenium-based transfer hydrogenation proved more robust (38). Slight modification of the alcohol reduction / azide inversion sequence could be implemented from the ketone 38 to deliver the amine tartrate 46 in 78% yield and 95% ee (Scheme 8). To circumvent the safety concerns around the use of DPPA, we chose to explore if the amine could be prepared via a diastereoselective reduction of a chiral imine which could be selectively deprotected to reveal the parent amine.

Scheme 8. Synthesis of Bromoamine 46

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The chiral imine route commenced from ketone 38 already in hand. Installation of the neopentyl group using a Negishi coupling, similar to the medicinal chemistry route, revealed that the azachromanone structure 13 was prone to retrocyclization. Optimization of the coupling could only deliver a 1:2 ratio of desired cyclized to retrocyclized ketone. Fortunately, with the carbonyl functionality intact, re-cyclization could be easily achieved by treatment of the crude reaction mixture with ethanolic HCl. The neopentyl ketone 13 can be isolated in 75% yield from 38 (Scheme 9) using this recycle procedure.

Scheme 9. Negishi Route Toward Ketone 13

Investigation of imine formation of the chromanone series showed comparable propensity towards retrocyclization as with the palladium-catalyzed alkylzinc coupling (Scheme 9). Treatment of 13 with a model amine (4-fluorobenzylamine, 48) under typical imine formation conditions, revealed significant decomposition (Scheme 10). Methyl ketone 49 could be isolated in 65% yield indicating that not only retrocyclization, but retroaldolization was occurring under the conditions for imine formation. Side-product analysis by NMR and mass spectrometry led to the identification of several products along the retrocyclization/retroaldolization pathway (Figure 8). Similar results were seen with the use of other strong Brønsted acids (i.e. 4-MePhSO3H, etc) and Lewis acids.

Scheme 10. Decomposition of Ketone 13 Under Imine Formation Conditions

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Figure 8. Intermediates Arising from Decomposition of 13 (Scheme 10).

Postulating that the strongly acidic conditions may be leading to the unravelling of the desired product imine / ketone, we chose to focus on imine formation under milder conditions. We obtained better results by lowering the reaction temperature and using ammonium acetate as a milder acid catalyst. Under these conditions, we achieved up to 60% conversion of 13 to the desired N-(4-fluorobenzyl)imine 50. Further analysis revealed that amine 48 was being consumed via a side reaction with acetate to generate the corresponding acetamide. Suppressing this side reaction by switching to ammonium pivolate alleviated this problem. Full conversion of the ketone to imine 50 can be achieved with 7) workup.

Scheme 11. Modified Imine Formation with Ammonium Pivolate

With suitable imine formation conditions now accessible, condensation of the 13 with S-α-methylbenzyl amine and reduction with sodium borohydride in ethanol revealed serviceable chirality transfer toward the desired stereochemistry (95:5 desired: undesired diastereoisomers). The product amine was not amenable to direct crystallization, however the bis-HCl salt is a non-hygroscopic benchstable solid and can be conveniently isolated by direct crystallization from the crude reaction mixture. Upon removal of the ethanol following the reduction, dissolving the crude product in acetone and treatment with two equivalents of HCl, the product 51 crystalized out and was isolated in 92% yield for the two steps. The crystallization served also to improve the diastereomeric purity (99:1 d.r.) of the isolated product.. Selective hydrogenolysis (39, 40) to cleave the α-methylbenzyl 152 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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group was easily accomplished with palladium on charcoal in methanol/water under 45 psig of hydrogen (Scheme 12). While the freebase of 14 product lacked suitable physical properties for a robust direct isolation, the bistosylate salt of 14 was found to be a convenient bench-stable solid and provided an upgrade in overall purity with despite variability in diastereomeric purity of the incoming stream of 51. On >100 g scale, the product could be isolated in >99% ee and 94% yield. In total (Scheme 12), the target amine was generated in 29% yield and >99% ee from 2-methoxynicotinic acid.

Scheme 12. Optimized Chiral Auxiliary Route to 14

Epoxide Aminolysis Investigations Toward Target HEA Structure With a scalable synthesis of the azachromylamine 14 and ketone 13 in hand, we focused our attention on the strategy for the assembly of the hydroxyethylamine (HEA) core. Traditional HEA transition state isosteres are common in commercial and investigatory HIV protease inhibitors (41). Typical HEA-based inhibitors such as saquinavir (42), amprenavir (43) and palanavir (44) contain a common phenyl group in the P1/S1 region of the molecule (45). The diamino alcohol moieties are all synthesized from a key intermediate epoxide 52 via an epoxide aminolysis reaction (Figure 9). 153 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 9. Examples of Commercial / Late-stage Clinical Aspartyl Protease Inhibitors Based Upon the Hydroxyethyl Amine Transition-State Isostere. Commonly seen in a number of syntheses, the epoxide ring opening to form the intermediate for amprenavir/fosamprenavir (eq 1) is accomplished by heating the starting epoxide 52 with several equivalents of isobutyl amine (3–10 are generally used) (41). Lowering the amine stoichiometry resulted in slower reaction rates and may also result in the formation of dialkylated amine. While the complication of the dialkylation was not possible in the reaction to form saquinivir or alanavir (eq 2), the aminolysis was still low yielding (60–70%) in these cases (42, 44). These reactions often consume excess epoxide (~1.2–2 equiv) to reach full conversion, owing to competitive decomposition via anchimeric opening of the epoxide by the pendant carbamate (eq 3) (46, 47).

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Initial work in this arena began with both screening a thermal aminolysis and Lewis acid-mediated epoxide openings with epoxide 52. Lewis acid mediated (48) epoxide openings with amine 14 were screened extensively with little to no avail. Desired product 56 was produced, however this was typically in less than 30% assay yield and was accompanied by several side-products. The uncatalyzed reactions of 14 with 52 in 2-propanol(IPA) (49) at 70 °C, afforded a cleaner reaction profile than the Lewis acid reactions, but could only be classified as marginally successful (assay yields of 30–40%) and useful yields (>60%) could only be obtained from using excess of the amine (50). Portionwise addition of the epoxide to a solution of the amine at elevated temperatures did not offer any improvement over the original approach and the dialkylation of the amine became a significant side-product (usually up to 20% LCAP) as the reaction progressed, resulting in lower solution yield of the desired product 57 (Scheme 13). Alkylated amine derivatives such as allyl or benzyl versions of amine 13 completely suppressed any desired product formation even under forcing conditions. Metallated (nBuLi, iPrMgCl, Et3Al) amine 14 or carbamate 14b resulted in either no reaction or complex product mixtures (Scheme 14) (48). Further attempted optimizations of thermal mediated or metallated amine derivative epoxide 50a ring-opening reactions with 13 were unsuccessful. Based upon the compilation of these results it was felt that the epoxide aminolysis approach was not viable at this juncture and an alternative disconnection needed to be explored.

Scheme 13. Epoxide 52 Aminoloysis with AzachromylAmine 14 155 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Scheme 14. Epoxide 52 Aminoloysis with AzachromylAmine 14 Derivatives

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Reductive Amination Investigations Toward HEA Assembly While the epoxide route initially proved unsuccessful in generating our target HEA inhibitor, the success of the amine installation work with the azachromylamine directed us toward a possible reductive amination as a potential disconnection strategy. In this case a P1 amine moeity with the already available ketone 13. The key question would be that while methylbenzylamine was an appropriate chiral auxillary, could a P1 amine serve to successfully transfer chirality to the azachromyl center. A prototypical P1 amine was quickly generated in three steps from the commercially available epoxide (51). Condensation under the mild conditions developed for the azachromanone (Scheme 12) delivered 89% isolated yield of the target imine 58 (Scheme 15 Excess (>1.5 equiv) of the P1 amine 57 was required to achieve conversions of 12 over 80%.

Scheme 15. Imine Formation Between 59 and 13

Reduction of the imine under a variety of conditions illustrate that borohydride-type reagents showed little to no diastereoselectivity, and assay yields were moderate (53-88%; Table 3, entries 1-4). Hydrogenation with platinum on carbon in IPA showed some modest success with 100% assay yield and a ratio of 6:1 desired : undesired (Table 3, entry 5). Unfortunately, the reaction requried significant pressure of hydrogen (400 psig) to reach full conversion and while initial diastereoselectivity did show promise, further optimization of the reaction conditions did not further increase the selectivity (52). Based upon these results and the success of an alternate approach (vide infra), the imine reduction route was deprioritized. 156 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Table 3. Conditions Screen for Reduction of Imine 60 (eq 4)

Development of a Novel HEA Assembly Method: A Templated Approach While the intermolecular bond assembly strategy toward our target HEA molecules proved challenging, we chose to explore an alternate, but underutilized option which we hoped could provide improved process control. Intramolecular C-N bond formation of two tethered fragments would, in theory, mitigate reactivity issues with the epoxide aminolysis and prevent over reaction. Of additional benefit, the amine stereocenter would be controlled in the fragment amine instead of relying on the coupling chemistry to govern the selectivity. Critical to this approach we would need to select an appropriate tether which could be easily installed, facilitate the desired C-N bond formation and also be seamlessly removed (Figure 10). 157 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Initial work focused in on the use of the urethane as a tether which would cyclize to an oxazolidinone and be cleaved under basic conditions (53). Two possible disconnection strategies were conceived with this templated type of approach. The first disconnection involved acylation of an amine isocyanate with a P1 halohydrin or other such activated species (Figure 10, Path A). A similar strategy has been employed by Das and others via halohydrins (54) or other activated species (55) and simple isocyanates to form vicinal amino alcohols. No report exists of this type of strategy utilized in a complex fragment coupling such as the one we are proposing. Alternatively, an activated carbonate derivative could react with the amine to form the halourethane which upon cyclization would afford the targeted oxazolidinone intermediate (Figure 10, Path B). For purposes of testing the validity of the disconnection, path A was chosen first due to the ready availability of both coupling partners.

Figure 10. Proposed Templated Approach to HEA Core.

With limited amounts of key intermediates available at the start of this exercise, a model study was explored initially with a 4-Cl derived P1 chlorohydrin 63 (56) and commercially available S-α-methylbenzyl isocyanate 64 (Scheme 16). Treatment of chlorohydrin 63 with isocyanate 64 with 30 mol% of DABCO (57) in THF afforded a 67% isolated yield of chlorourethane 65 Further reaction with sodium tert-butoxide (NaOt-Bu) in THF was rapid (99% ee and 95% yield (Scheme 18).

Scheme 18. Enantioselective Hydrogenation Route Towards Aminoester 78 160 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Hydrolysis of ester 78 with lithium hydroxide in THF/H2O revealed the corresponding carboxylate, which upon pH adjustment and Steglich esterification with 4-nitrophenol and DCC delivered the aryl ester 77 which was crystallized from THF/n-heptane. Condensation with trimethylsulfoxionium ylide showed in minimal epimerization and after crystallization from IPA, the ylide 78 was delivered in 86% yield and >99% ee (Scheme 19).

Scheme 19. Generation of Ketoylide 80 Treatment of the ketoylide 80 with LiCl and methanesulfonic acid in THF as a source of anhydrous HCl, afforded the chloroketone 81 in 79% yield and >99% ee following a recrystallization from IPA (Scheme 20). Diastereoselective reduction using aluminum tri-isopropoxide in IPA following the protocol from Yin and coworkers (63) delivered 96:4 d.r. favoring the desired diastereoisomer. Workup with isopropyl acetate, Rochelle’s salt and crystallization from IPA resulted in 85% yield of the desired chlorohydrin 82 as essentially a single stereoisomer (>99% ee and >99:1 diastereomeric ratio). Overall the target chlorohydrin yield from piperonal 74 was 55% with seven steps and five isolations.

Scheme 20. Diastereoselective Synthesis of Chlorohydrin 82

Application and Development of the Templated Assembly Strategy toward ‘699 and ‘359 With the desired chlorohydrin 82 in hand, optimization of the final assembly route commenced by selecting the appropriate coupling mode. While the isocyanate 67 and nitrophenylcarbamate derivatives 70 of the azachromylamine were shown to synthetically deliver the desired HEA array (Schemes 16 & 17), the inherent instability of functionalized amines combined with our reluctance toward further elaboration of the already synthetically intensive azachromylamine, a more convergent mixed carbonate route was pursued. 161 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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To choose the appropriate coupling partner for the amine 14, a screen of a variety of P1 electrophiles with 14 was undertaken (Table 4). While the nitrophenylcarbonate derivative 84 showed smooth conversion to deliver the desired chlorourethane 86 (Table 4, entry 2), the corresponding phenyl carbonate 83 showed no desired product with prolonged heating up to 120 °C (Table 4, entry 1). Acyloxyimidazolide 85 (64) was contemporaneously pursued as cheaper and safer alternative to 4-nitrophenylchloroformate. Similar to 83, little to no reaction was seen with 85 under similar conditions which afforded full conversion of 84 (Table 4, entry 3).

Table 4. Electrophilic P1 Coupling with Amine 14 (eq 5)

Screening a variety of activators (65) showed that while Brønstead and Lewis acids afforded little to no conversion, common peptide coupling additives showed promise. After extensive screening N-hydroxy succinimide (HOSu) and N-hydroxypthalimide afforded >90% assay yields of the desired product 86. The loading of HOSu could be reduced to 5 mol% relative to 85 without any loss in 162 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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yield (Table 4, entry 4). Due to the time constraints and the lack of a sufficient purity control point (66) for the acyl imidazolide coupling partner, the nitrophenyl carbonate 84 was chosen over 85 as a phase-appropriate solution. Initial screen of reaction conditions coupling chlorohydrin 82 and 4-nitrophenylchloroformate in THF (67) showed that pyridine as a mild base formed the desired acylated chlorohydin 84 in nearly quantitative assay yield. While the overall solution yields in the acylation reaction were high, the reactions were contaminated with two significant impurities: bis-4-nitrophenylcarbonate 88 and 4-nitrophenol 89. The origin of both of these impurities is likely due to the hydrolysis of 4-nitrophenylchloroformate by adventitious water either before or during the reaction. Impurities 88 and 89 were generally produced in variable amounts but often >10% LCAP once the reaction has reached completion. Recrystallization of crude 84 from isopropyl acetate (IPAc) could remove the bisnitrophenylcarbonate 88 but only at considerableloss to the mother liquor (17% loss) and the isolated product still containes significant amounts of phenol 89. Screening of different solvent systems showed that a 1:1 mixture of dimethyoxyethane (DME) and water is uniquely competent to remove the two impurities with minimal loss of the desired product (100 g scale resulted in 99% wt% adjusted yield and 99% purity (Scheme 21).

Scheme 21. Synthesis of Activated Carbonate 84 The acylation of amine 14 with carbonate 84 performed well in a number of solvents to generate chlorourethane 90. Reactions in THF, 2-MeTHF, IPAc, and 2-butanol afforded 100% conversion to 90 in 18 h at 65 °C. Cyclization of 90 was easily accomplished by the treatment the crude reaction mixture with NaOt-Bu. Cyclization of crude 90 produced in either THF or 2-MeTHF afforded 88–90% assay yield, where as other solvents were found to be suboptimal due to low reactivity or competing side-reactions. In practice we chose 2-MeTHF for both 163 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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coupling and cyclization. because of the high conversion and the ease of reaction workup. On further optimization, we found potassium tert-amyloxide (1.7 M solution in toluene) to be a convenient replacement for solid sodium tert-butoxide, and could be added directly to the crude acylation reaction mixture. This change afforded the oxazolidinone 91 in 93% assay yield. Due to lack of suitable control points in 90 and 91, the choice was made to pursue a telescopic procedure through to the isolated bis-HCl salt 92. While an extensive screen was not performed, the bis-HCl salt was selected due it’s physical characteristics and ease of deprotection of the Boc group with HCl. Solvent exchange of the crude reaction tream of 91 to n-BuOH followed by treatment of 3 equiv.of anhydrous HCl at 70 °C afforded smooth Boc-deprotection. Addition of n-BuOAc as antisolvent and cooling afforded the desired bis-HCl salt 92 in 90% yield over three steps and >99% purity on >100 g scale (Scheme 22).

Scheme 22. Telescoped Process to Oxazolidinone 92

Cleavage of the oxazolidinone in 92 was most efficiently accomplished with ethanolic potassium hydroxide at elevated temperature. Treatment of an ethanol solution of the 92 with 10 equivalents of aqueous 5N KOH and heating for 18 h at 70 °C afforded full conversion and >95% assay yield of HEA 14. While the deprotection operationally simple to perform, isolation of the product was a challenge. Cooling the reaction mixture to 40 °C and charging 6 equiv of aqueous HCl (6N) led to a phase separation. Addition of toluene facilitated extraction of the diaminoalcohol 14 into the organic layer. Conveniently, the tris-HCl salt could be crystallized from a mixture of toluene and IPA. Overall on the penultimate 85 was isolated in 93% yield and 99.8% purity on >100 g scale (Scheme 23). 164 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 23. Deprotection and Isolation of Penultimate 15

Scheme 24. Summary of Demonstration Run of the Templated Route to Penultimate 15

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Conclusions In summary, a new templated approach to the hydroxyethylamine core has been demonstrated on >100 g scale toward penultimate HEA 15. The new synthesis involves the use of a urethane tether to facilitate intramolecular C-N coupling of azachromylamine 14 and a P1 electrophile, followed by seamless removal and revealing the target diaminoalcohol array. Key highlights to this method include activation of chlorohydrin 82 to a stable crystalline 4-nitrophenyl carbonate 84 which occurs in excellent yield and purity (99 wt% adjusted yield). Activated carbonate 84 is readily coupled with azachromyl amine 14 to form an intermediate chlorourethane 90 which is cyclized and deprotected to afford oxazolidinone 92 in 90 wt% adjusted yield over three steps. Cleavage of the oxazolidinone tether under basic conditions affords the penultimate 15 in high yield and purity (93 wt% adjusted yield, 102 wt%). Overall 82% yield over three isolations from P1 chlorohydrin 82 (Scheme 24). In addition to the new HEA route, the early process development efforts for the BACE program were able to discover and demonstrate a new and scaleable route toward the azachromylamine 14. The route eliminated the hazardous azide chemistry, replacing it with a robust chiral auxillary route and overall shortened the sequence from 16 overall steps to 9 steps from 2-methoxy nicotinic acid. Combining the new amine synthesis with the new templated HEA route resulted in a shortening of the overall synthesis of the lead molecules from 29 steps (4% overall yield) to 19 steps. Additional process improvements resulted in of elimination of all chromatographic purifications and the overall yield was increased by nearly four-fold to 19%. The new technology described above, not only was suited for future larger scale deliveries, but also accelerated and enabled supply of several hundered grams of intermediates for molecule selection and pre-clinical toxicology work.

Acknowledgments The authors would like to acknowledge the following: Thomas A. Dineen, Matthew M. Weiss, Toni Williamson, Paul Acton, Safura Babu-Khan, Michael D. Bartberger, James Brown, Kui Chen, Yuan Chen, Martin Citron, Michael D. Chrogan, Robert T. Dunn, Joel Esmay, Russell F. Graceffa, Scott S. Harried, Dean Hickman, Stephen A. Hitchcock, Daniel B. Horne, Hongbing Huang, Ronke Imbeah-Ampiah, Ted Judd, Matthew R. Kaller, Charles R. Kreiman, Daniel S. La, Vivian Li, Patricia Lopez, Steven Louie, Holger Monenschein, Thomas T. Nguyen, Lewis D. Pennington, Tisha San Miguel, E. Allen Sickmier, Hugo M. Vargas, Robert C. Wahl, Paul H. Wen, Douglas A Whittington, Stephen Wood, Qiufen Xue, Bryant H. Yang, Vinod F. Patel, Eric Bercot, Emilio Bunel, Seb Caille, Johann Chan, Evan DiVirgilio, Jinkun Huang, Liang Huang, Anil Guram, Ken McRae, Rob Milburn, Charles Papageorgiou, Silas Wang, Filisaty Vounatsos, Jamie Zigterman, Jenny Chen, Tiffany Correll, Troy Soukup, J. Preston, Judy Ostovic, Jiemin Bao, Fang Wang, Helming Tan, Susanna Lai, Kelly Nadeau, Kevin Turney, Peter Grandsard, Margaret Faul, Mike Martinelli and Paul Reider. 166

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References 1. 2. 3. 4. 5. 6.

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7.

8. 9. 10. 11.

12. 13.

14.

15.

2014 Alzheimer’s Disease Facts and Figures; http://www.alz.org/downloads/ Facts_Figures_2014.pdf (accessed October 13, 2016). van Marum, R. J. Fundam. Clin. Pharmacol. 2008, 22, 265–274. Vassar, R.; Kuhn, P.; Haass, C.; Kennedy, M. E.; Rajendran, L.; Wong, P. C.; Lichtenthaler, S. F. J. Neurochem. 2014, 130, 4–28. Yan, R.; Vassar, R. Lancet 2014, 13, 319–329. De Strooper, B. Cell 2014, 159, 721–726. Mullan, M.; Crawford, F.; Houlden, H.; Axelman, K.; Lilius, L.; Winblad, B.; Lannfelt, L. Nat. Genet. 1992, 1, 345–347. Johnsson, T.; Atwal, J. K.; Steinberg, S.; Snaedal, J.; Jonsson, P. V.; Bjornsson, S.; Stefansson, H.; Sulem, P.; Gudbjartsson, D.; Maloney, J.; Hoyte, K.; Gustafson, A.; Liu, Y.; Lu, Y.; Bhangale, T.; Graham, R. R.; Huttenlocher, J.; Bjornsdottir, G.; Andreassen, O. A.; Jönsson, E. G.; Palotie, A.; Behrens, T. W.; Magnusson, O. T.; Kong, A.; Thorsteinsdottir, U.; Watts, R. J.; Stefansson, K. Nature 2012, 488, 96–99. Sinha, S.; Lieberberg, I. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11049–11053. Vassar, R. J. Mol. Neurosci. 2004, 23, 105–113. See review and references cited therein: Ghosh, A. K.; Osswald, H. L. Chem. Soc. Rev. 2014, 43, 6765–6813. See review and references cited therein: Wyss, D. F.; Cumming, J. N.; Strickland, C. O.; Stamford, A. W. Fragment Based Drug Discovery. In BACE Inhibitors; Erlanson, D. A., Jahnke, W., Eds.; Wiley-VCH: New York, 2016; pp 329−354. Barão, S.; Moechars, D.; Lichtenthaler, S. F.; De Strooper, B. Trends Neurosci. 2016, 39, 158–169. News for MK-8931: http://www.alzforum.org/therapeutics/verubecestat. For AZD3293: https://www.astrazeneca.com/media-centre/pressreleases/2016/AstraZeneca-and-Eli-Lilly-and-company-announcecontinuation-of-pivotal-clinical-trial-for-people-with-early-alzheimersdisease-08042016.html. Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J. C.; Collins, F.; Treanor, J.; Rogers, G.; Citron, M. Science 1999, 286, 735–741. Standard nomenclature for the proteases and their substrates in terms of the active site binding subpockets. At the protease active site with the catalytic amino acids positioned in the center (and potential substrate binding to the active site in extended form with N- and C-termini on the left and right, respectively), going to the left, the sub-binding pockets are named S1, S2, S3,.. sequentially; going to the right, the subpockets are named S1′, S2′, S3′, … For the substrates, the corresponding amino acid side chains binding to these pockets are called P1, P2, P3, …, and P1′, P2′, P3′… 167

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16. Kaller, M. R.; Harried, S. S.; Albrecht, B.; Amarante, P.; Babu-Khan, S.; Bartberger, M. D.; Brown, J.; Brown, R.; Chen, K.; Cheng, Y.; Citron, M.; Croghan, M. D.; Graceffa, R.; Hickman, D.; Judd, T.; Kriemen, C.; La, D.; Li, V.; Lopez, P.; Luo, Y.; Masse, C.; Monenschein, H.; Nguyen, T.; Pennington, L. D.; Miguel, T. S.; Sickmier, E. A.; Wahl, R. C.; Weiss, M. M.; Wen, P. H.; Williamson, T.; Wood, S.; Xue, M.; Yang, B.; Zhang, J.; Patel, V.; Zhong, W.; Hitchcock, S. A. ACS Med. Chem. Lett. 2012, 3, 886–891. 17. Hitchcock, S. A.; Pennington, L. D. J. Med. Chem. 2006, 49, 7559–7583. 18. Weiss, M. M.; Williamson, T.; Babu-Khan, S.; Bartberger, M. D.; Brown, J.; Chen, K.; Cheng, Y.; Citron, M.; Croghan, M. D.; Dineen, T. A.; Esmay, J.; Graceffa, R.; Harried, S. S.; Hickman, D.; Hitchcock, S. A.; Horne, D. B.; Huang, H.; Imbeah-Ampiah, R.; Judd, T.; Kaller, M. R.; Kreiman, C. R.; La, D. S.; Li, V.; Lopez, P.; Louie, S.; Monenschein, H.; Nguyen, T. T.; Pennington, L. D.; Rattan, C.; San Miguel, T.; Sickmier, E. A.; Wahl, R. C.; Wen, P. H.; Wood, S.; Xue, Q.; Yang, B. H.; Patel, V. F.; Zhong, W. J. Med. Chem. 2012, 55, 9009–9024. 19. Harried, S. S.; Croghan, M. D.; Kaller, M. R.; Lopez, P.; Zhong, W.; Hungate, R.; Reider, P. J. J. Org. Chem. 2009, 74, 5975–5982. 20. Cotterill, W.; Johnson, D. A.; Livingstone, R. J. Chem. Res., Synop. 1995, 12–13. 21. Sarges, R.; Goldstein, S. W.; Welch, W. M.; Swindell, A. C.; Siegel, T. W.; Beyer, T. A. J. Med. Chem. 1990, 33, 1859–1865. 22. Taylor, R. J. K. Synthesis 1977, 564–565. 23. Danappe, S.; Boeda, F.; Alexandre, C.; Aubertin, A.; Bourgougnon, N.; Huet, F. Synth. Commun. 2006, 36, 3225–3239. 24. Samuel, S. P.; Niu, T. Q.; Erickson, K. L. J. Am. Chem. Soc. 1989, 111, 1429–1436. 25. Bernard, A. M.; Frongia, A.; Ollivier, J.; Piras, P. P.; Secci, F.; Spiga, M. Tetrahedron 2007, 63, 4968–4974. 26. Danappe, S.; Pal, A.; Alexandre, C.; Aubertin, A.; Bourgougnon, N.; Huet, F. Tetrahedron 2005, 61, 5782–5787. 27. Paterson, I.; Lyothier, I. Org. Lett. 2004, 6, 4933–4936. 28. Palacios, F.; Ochoa de Retanam, A. M.; Alonso, J. M. J. Org. Chem. 2006, 71, 6141–6148. 29. Westermann, J.; Schneider, M.; Platzek, J.; Petrov, O. Org. Process Res. Dev. 2007, 11, 200–205. 30. Chen, C.; Wilcoxen, K. M.; Huang, C. Q.; Xie, Y.; McCarthy, J. R.; Webb, T. R.; Zhu, Y.; Saunders, J.; Liu, X.; Chen, T.; Bozigian, H.; Grigoriadis, D. E. J. Med. Chem. 2004, 47, 4787–4798. 31. Goldstein, S. W.; Sarges, R. Azolidinedione Derivatives. European Patent 0306251A2, August 30, 1988. 32. Milburn, R. R.; McRae, K.; Chan, J.; Tedrow, J.; Larsen, R.; Faul, M. Tetrahedron Lett. 2009, 50, 870–872. 33. Samuel, S. P.; Niu, T. Q.; Erickson, K. L. J. Am. Chem. Soc. 1989, 111, 1429–1436. 168

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34. Bernard, A. M.; Frongia, A.; Ollivier, J.; Piras, P. P.; Secci, F.; Spiga, M. Tetrahedron 2007, 63, 4968–4974. 35. (Bases: DIPEA, NaOH, NaOEt, LiHMDS, LiOMe, MeLi, n-BuLi, trityllithium, PhLi, K2CO3. Solvents: toluene, THF, EtOH, MeCN). 36. Although the reaction was extremely clean by HPLC, the mass balance was very low. An alternative workup of the reaction mixture revealed a second pyridine-containing product which was tentatively assigned as the demethylated phosphonate 13 by 1H NMR. This assignment was supported by experiments showing the lithioketophosphonate to be unstable to the reaction conditions. 37. Thompson, A. S.; Humphrey, G. R.; DeMarco, A. M.; Mathre, D. J.; Grabowski, E. J. J. J. Org. Chem. 1993, 58, 5886–5888. 38. Wang, F.; Liu, H.; Cun, L.; Zhu, J.; Deng, J.; Jiang, Y. J. Org. Chem. 2005, 70, 9424–9429. 39. Nugent, T. C; Negru, D. E.; El-Shazly, M.; Hu, D.; Sadiq, A.; Bibi, A.; Umar, M. N. Adv. Synth. Catal. 2011, 353, 2085–2092. 40. Kanai, M.; Yasumoto, M.; Kuriyama, Y.; Inomiya, K.; Katsuhara, Y.; Higashiyama, K.; Ishii, A. Chem. Lett. 2004, 33, 1424–1425. 41. Ghosh, A.; Bilcer, G.; Schlitz, B. Synthesis 2001, 15, 2203–2209. 42. Göhring, W.; Gokhale, S.; Hilpert, H.; Roessler, F.; Schlageter, M.; Vogt, P. Chimia 1996, 50, 532–537. 43. Kim, E. E.; Baker, C. T.; Dwyer, M. D.; Murcko, M. A.; Rao, B. G.; Tung, R. D.; Navia, M. A. J. Am. Chem. Soc. 1995, 117, 1181–1182. 44. Beaulieu, P. L.; Lavallée, P.; Abraham, A.; Anderson, P. C.; Boucher, C.; Bousquet, C.; Duceppe, J-S.; Gillar, J.; Gorys, V.; Grand-Maître, C.; Grenier, L.; Guse, I.; Planmondon, L.; Soucy, F.; Valois, S.; Wernic, D.; Yoakim, C. J. Org. Chem. 1997, 62, 3440–3448. 45. Abbenanted, G.; Fairlie, D. P. Med. Chem. 2005, 1, 71–104. 46. Romeo, S.; Rich, D. H. Tetrahedron Lett. 1994, 35, 4939–4942. 47. Agami, C.; Couty, F. Tetrahedron 2002, 58, 2702–2724. 48. Extensive screens of Lewis acids (LiX, MgX2, CaX2, ScX3, TiX4, ZnX2, CuX2, AlX3; X= Cl,OTf)), solvents (THF, DCM, IPAC, MeCN, IPA, DMF, toluene), additives (Et2BOMe; silica, alumina, etc.) led mostly to low assay yields (at most up to 40%) with numerous side products . 49. A relative decrease in epoxide degradation and subsequent increase in conversion of amine 7 was noticed upon increasing the steric bulk of the alcohol (rate of decomposition: MeOH>EtOH>IPA~tBuOH) 50. Amine equiv : epoxide equiv (1:1 = 36% assay yield; 2:1 = 58% assay yield; 3:1 = 67% assay yield; 4:1 = 70% assay yield). 51. Miller, J. F.; Furfine, E. S.; Hanlon, M. H.; Hazen, R. J.; Ray, J. A.; Robinson, L.; Samano, V.; Spaltenstein, A. Bioorg. Med. Chem. Lett. 2004, 14, 959–963. 52. An additional P1 amine with an oxazolidinone linkage between the two stereogenic N and O in the P1 amine was tested with similar diastereoselectivity under same conditions. 169 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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53. Knapp, S.; Sankar Lal, G.; Sahai, D. J. Org. Chem. 1986, 51, 380–383. 54. Das, J. Synth. Commun. 1988, 18, 907–915. 55. Tiecco, M.; Testaferri, L; Temperini, A.; Bagnoili, L.; Marini, F.; Santi, C. Chem.−Eur. J. 2004, 10, 1752–1764. 56. Prepared in analogy: Honda, Y.; Katayama, S.; Kojima, M.; Suzuki, T.; Izawa, K. Org. Lett. 2002, 3, 447–449. 57. Reaction of chlorohydrin with isocyanate requires a nucleophilic catalyst (DABCO, DMAP) to achieve >20% conversion. DABCO = 1,4-diaza-bicyclo[2.2.2]octane. 58. Prepared from amine 11 and bisnitrophenylchloroformate in analogy to: Izdebski, J; Danuta, P. Synthesis 1989, 423–425. 59. Wang, D.; Schwinden, M. D.; Radesca, L.; Patel, B.; Kronenthal, D.; Huang, M.; Nugent, W. A. J. Org. Chem. 2004, 64, 1629–1623. 60. Schmidt, U.; Lieberknect, A.; Wild, J. Synthesis 1984, 53–60. 61. He, Z-T.; Zhao, Y-S.; Tian, P.; Wang, C-C.; Dong, H-Q.; Lin, G-Q. Org. Lett. 2014, 16, 1426–1429. 62. Liu, D.; Zhang, X. Eur. J. Org. Chem. 2005, 646–649. 63. Yin, J.; Huffman, M. A.; Conrad, K. M.; Armstrong, J. D. J. Org. Chem. 2006, 71, 840–843. 64. Bertolini, G.; Pavich, G.; Vergani, B. J. Org. Chem. 1998, 63, 6031–6034. 65. ZnCl2, MgCl2, LiI, PPTS, Pivalic acid, HOAt, HOBt, 5-nitro-2hydroxypyridine, 4-nitrophenol, 4-nitrothiophenol, N-hydroxyphthalimide and N-hydroxy succinimide were screened at 70 °C for 15 h with 1 equivalent of additive. 66. Reactions with both target and parent chlorohydrins were sluggish with CDI and the products proved to be intractable from an isolation standpoint. Alternatively the 4-nitrophenyl chloroformate was a bench stable crystalline solid. 67. Bases evaluated: triethylamine, diisopropylethylamine, 2,6-lutidine and pyridine with and without DMAP. Highest assay yield (100%) achieved with pyridine in absence of a nucleophilic counterion. 68. Typically 2-5% of the aminoalcohol remained in the aqueous layer and additional toluene extractions were implemented to recover all product from the aqueous (up to 3 extractions).

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Chapter 5

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Identification and Optimization of a Series of Non-Steroidal Trifluoromethylcarbinol Glucocorticoid Receptor Agonists Christian Harcken*,1 and Hossein Razavi2 1Department of Immunology and Respiratory Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06778, United States 2Department of Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06778, United States *E-mail: [email protected].

Steroidal glucocorticoids (GCs) are among the most used classes of anti-inflammatory drugs. However, their chronic use is severely limited by deleterious side effects such as GC-induced osteoporosis. A series of non-steroidal trifluoromethylcarbinol glucocorticoid receptor (GR) agonists was identified from literature-known nuclear hormone receptor binder motifs. The series was successively optimized for potency, selectivity, and in vivo activity through rational design. Compounds with reduced GR-related side effects were identified empirically, ultimately the drug-like properties of these compounds were optimized resulting in the identification of the clinical candidate BI 653048.

Introduction Glucocorticoids (GCs) are among the most widely used anti-inflammatory drugs. More than 60 years ago, cortisone (1) was first used for the treatment of rheumatoid arthritis (Figure 1) (1). Soon thereafter, the first synthetic steroidal glucocorticoids were developed: dexamethasone (2) and prednisolone (3) are still widely used in clinical practice today. At the time, these treatments were hailed as the “cure” for inflammatory and auto-immune diseases; however, a © 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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plethora of side effects upon their chronic use were soon discovered. These undesired effects included weight gain, fat redistribution, obesity, GC-induced diabetes, GC-induced osteoporosis, cataracts, hypertension and CNS effects (2). In particular, GC-induced diabetes and osteoporosis severely limited the suitable doses and durations for the chronic administration of glucocorticoids (3). Thus, the discovery of a GC with substantially reduced side effects is highly desirable and has led to numerous drug discovery and development programs. However, a proof of clinical concept for a therapeutically beneficial GC with diminished side effects remains elusive to this day.

Figure 1. Steroidal Glucocorticoids (GCs).

GCs elicit their effects through interaction with the glucocorticoid receptor (GR) — a nuclear hormone receptor (NHR) and transcription factor. GCs function as GR agonists. Two distinct functional mechanisms for GR agonists have been proposed (4). Upon binding of a GC to the cytosolic GR, a receptor-ligand complex forms that can dimerize, translocate to the nucleus and directly bind to glucocorticoid response elements (GREs) on the DNA resulting in the transcriptional up-regulation of selected genes. This direct activation of transcription has been termed “transactivation”. Alternatively, the receptor-ligand complex can translocate to the nucleus as a monomer and bind to transcription factors such as nuclear factor κB (NFκB) and activating protein 1 (AP1), thereby, impeding their activities and leading to the down-regulation of gene expression. This indirect suppression of transcription has been termed “transrepression”. Originally, it was hypothesized that transactivation was responsible for the majority of the undesired GC side effects while transrepression mediated their desired anti-inflammatory effects. The predominant approach for the discovery of a GC with reduced side effects has been to achieve functional selectivity or “dissociation” between these two pathways. However, during the past decade, it has become increasingly clear that the GR’s actions are more complex and that the concept of achieving reduced side effects based on functional dissociation may be too simplistic and does not account for the effects of negative GREs (i.e., DNA-binding-mediated direct transrepression), non-genomic effects through membrane bound GR and post-transcriptional effects, or the existence of GRα/β isoforms (5, 6). Additionally, many steroidal GCs elicit unwanted side effects by modulating other NHRs such as the mineralocorticoid receptor (MR) and progesterone receptor (PR). Hence, sufficient selectivity over these related receptors is a requirement for reducing off-target side effects. 172

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Lead Identification

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Schering AG (now Bayer-Schering) identified a non-steroidal trifluoromethylcarbinol series of GCs that is presumed to be derived from the androgen receptor antagonist bicalutamide (4; Figure 2) (7). Compound 5 is an example of an early Schering GR agonist (GR IC50 8 nM) (8) that has demonstrated in vitro and in vivo anti-inflammatory activity (9). However, this compound suffered from low selectivity and poor pharmacokinetic properties. At Boehringer Ingelheim (BI), we found that replacement of the amide linkage in 5 with alkyl linkers retained GR binding activity and led to the discovery of a novel class of compounds (10). The SAR showed that a methylene linker as in 6 was the optimal linker length for retaining GR binding affinity (GR IC50 610 nM).

Figure 2. Origin of Trifluoromethylcarbinol Glucocorticoids.

In general, compounds in this scaffold were synthesized through Friedel-Crafts alkylation of an appropriately substituted arene 7, followed by reduction and glycol cleavage of the product 8, and addition of the A-ring moiety to the intermediate trifluoromethylketone 9 (Scheme 1). Docking studies using a GR homology model showed that 6 was bound to the GR ligand-binding domain with its left-hand side (i.e., the phenyl ring) occupying the steroid A-ring pocket, while the methoxyfluorophenyl group resided in the steroid D-ring pocket. Increased steric bulk of the A-ring moiety improved GR binding affinity substantially such as in compound 10 (GR IC50 55 nM); however, this compound showed only marginal agonist activity in a cellular assay (IL-6 IC50 280 nM, 60% maximum efficacy vs. prednisolone) (11). Modelling studies suggested that the incorporation of a hydrogen bond acceptor (HBA), which would mimic the steroid A-ring carbonyl group should improve potency. The SAR of various substitution patterns on the left-hand side phenyl ring (i.e., the A-ring mimetic) led to compound 11 that showed potent GR binding activity (GR IC50 17 nM), moderate NHR selectivity (PR IC50 140 nM, MR IC50 320 nM) and partial agonism in a cellular assay (IL-6 IC50 20 nM, 60% maximum efficacy vs. prednisolone). 173

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Scheme 1. First-Generation Synthesis Route

It is interesting to note that only the combination of a HBA (e.g., CN) and a lipophilic substituent (e.g., Cl) on the left-hand side phenyl ring was able to strike the right balance of properties to achieve cellular activity. Compound 11 showed low aqueous solubility and low metabolic stability that precluded its evaluation in vivo; nevertheless, it represented a novel class of non-steroidal GR agonists.

Potency Optimization Due to the apparent dramatic effect of the left-hand side substitution on agonist activity, we screened a variety of heterocyclic ring systems as A-ring mimetics (12). One of the preferred ring systems identified was the quinolone in compounds such as 12a (Figure 3). These compounds were synthesized using a second-generation route: starting from the addition of an appropriately substituted organometallic reagent such as 14 to the trifluoromethylenone 13, a shorter synthesis of the key trifluoromethylketones such as 9 was achieved (Scheme 2). The ketone 9 was transformed to the corresponding epoxide 15 using sulfur ylide chemistry. The final compound 12a was synthesized by nucleophilic opening of the epoxide with quinolone. This route was later modified to allow the preparation of enantiopure compounds by using epoxide (R)-15 that was synthesized in three steps through a chiral sulfoxide addition to provide intermediate (R)-16, separation of diastereomers, reduction to thioether (R)-17, and cyclization (Scheme 3) (13). Most of the GR activity in the trifluoromethylcarbinol series resides in the (R)-enantiomers. Compound 12a retained potent binding affinity (GR IC50 10 nM), moderate selectivity (PR IC50 470 nM, MR IC50 120 nM), and agonist cellular activity (IL-6 IC50 20 nM, 82% maximum efficacy). The right-hand side SAR revealed that a hydroxyl group at the C-2 position of the phenyl ring provided the highest agonist efficacy while a C-5 hydroxy group had a deleterious effect. The phenol analog 12b showed almost full transrepression efficacy (IL-6 IC50 6 nM, 92% maximum efficacy); however, this translated to only partial efficacy in an acute in vivo mouse model of TNF-α production 174

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(55% inhibition of TNF-α at 10 mg/kg) (14). Further optimization to compounds such as 18 did not lead to a significant improvement of the in vivo profile (58% inhibition of TNF-α at 10 mg/kg). Due to these challenges, we turned to a different heterocyclic ring system.

Figure 3. Heteroaryl A-Ring Mimetics.

Scheme 2. Second-Generation Synthesis Route 175 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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We had previously identified the indole ring as a left-hand side replacement in our survey of heterocyclic ring systems. Compounds such as 19 had displayed potent GR binding (GR IC50 22 nM), moderate selectivity (PR IC50 290 nM, MR IC50 380 nM), and agonist cellular activity (IL-6 IC50 7 nM, 87% maximum efficacy) (15). A systematic SAR investigation on the indole ring system revealed a number of trends. In particular, it was discovered that the space available for substitution at the C-4 and C-7 positions was limited. In fact, substitutions at these positions led to lower agonist activity, and upon further increase of the substituents’ size, binding affinity was ultimately lost. Due to the proximity of the C-5 and C-6 positions to the Arg611/Glu570 pair in the GR binding pocket, substitution in these positions with an HBA should be beneficial for activity. Since unsubstituted indoles are electron rich and prone to metabolism, electron-withdrawing groups (EWG) such as CN that satisfied the HBA requirement appeared to be ideal substituents. Indeed, 5-CN and 6-CN substituted indoles showed good agonist activity and NHR selectivity. For instance, 6-cyanoindole 20 retained potent binding (GR IC50 14 nM), moderate selectivity (PR IC50 400 nM, MR IC50 440 nM) and agonist cellular activity (IL-6 IC50 14 nM, 91% maximum efficacy). The SAR of the right-hand side (D-ring mimetic) revealed that the selectivity could be further improved by introducing bulky substitution at the C-5 position as in the sulfonyl dihydrobenzofuran 21. These compounds showed potent GR affinity (GR IC50 6 nM) with >100-fold selectivity over MR and PR while maintaining moderate agonist activity (IL-6 IC50 100 nM). Thus, to impart potent agonist activity and NHR selectivity, a combination of 5-CN-indole A-ring and 5-sulfonyl-substituted dihydrobenzofuran D-ring mimetics was synthesized: Substituted indole analog (R)-22 (GR IC50 2 nM, MR IC50 230 nM, PR IC50 750 nM, IL-6 IC50 28 nM, 88% maximum efficacy) showed potent acute in vivo anti-inflammatory effects (97% inhibition of TNF-α production at 3 mg/kg). However, despite moderate to good mouse PK properties (4 mL/min/kg clearance, 0.9 L/kg VSS, 26% bioavailability; dosed at 1 mg/kg iv and 30 mg/kg po), the efficacy in the acute mouse model only translated to partial inhibitory effects in a chronic mouse model of collagen induced-arthritis (CIA) (45% maximum inhibition of disease score) (16).

Scheme 3. Enantiopure Synthesis Route 176 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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An alternative way of improving upon the profile of indoles like 19 was the incorporation of an additional ring nitrogen atom to form azaindoles (17). The systematic SAR exploration of all possible azaindole isomers revealed that the 5and 6-azaindole moieties not only increased potency, but also imparted further improvement in NHR selectivity. These compounds were synthesized using a third-generation synthesis route (Scheme 4). The key epoxide intermediate (R)15 was converted to the corresponding alkyne (S)-23 which was treated with an appropriately substituted and protected amino pyridine (e.g., iodopyridine 29) in a Sonagashira coupling followed by cyclization to yield the desired compounds such as (R)-24a. The base-mediated cyclization process used in this synthesis was developed in our labs and was a significant improvement over the literature-known processes (18). Compound (R)-24b was the first compound from this scaffold that showed dexamethasone-like potency (GR IC50 2 nM, IL-6 IC50 3 nM, 93% maximum efficacy) with greater than 100-fold selectivity over MR and PR (Figure 4). This compound demonstrated acceptable PK properties in rat (49 mL/min/kg clearance, 7.6 L/kg VSS, 48% bioavailability; dosed at 5 mg/kg iv and 30 mg/kg po) and potent acute anti-inflammatory effects in vivo (ED50 < 0.3 mg/kg for TNFα inhibition in mouse) that translated into dose-responsive efficacy in a chronic CIA mouse model. The compound inhibited disease progression approximately equipotent to prednisolone (daily dosing of 30 mg/kg of (R)-24b resulted in 92% inhibition as assessed by the arthritic score AUC compared to 77% with 30 mg/kg of prednisolone).

Dissociation Optimization Previously discussed compounds 12b, (R)-22 and (R)-24b showed a partial dissociation profile, since they were dissociated in a cellular transactivation assay with a direct read-out such as a MMTV reporter gene assay (19) but not in a functional aromatase transactivation assay (20). For example, (R)-24b was dissociated based on reduced potency and maximum efficacy in the MMTV assay (IC50 80 nM, maximum efficacy 30%), but did not show a dissociated profile in the aromatase assay (IC50 11 nM, 84% maximum efficacy) as compared to the transrepression activity (IL-6 IC50 3 nM, maximum efficacy 93%). To assess how this partially dissociated profile of (R)-24b would translate into in vivo dissociation, the compound and prednisolone were dosed in healthy mice for 5 w, and their metabolic side effect profile (e.g., body fat content, triglyceride, free fatty acid (FFA) and insulin levels) at equi-efficacious doses was analyzed. Body fat increase and increased insulin secretion were significantly reduced for (R)-24b as compared to prednisolone; however, FFA and triglyceride increases were comparable to prednisolone. Thus for the first time in this series, a partially dissociated in vitro profile translated to an in vivo reduction in metabolic side effects. Unfortunately, this in vivo dissociated profile did not extend to bone dissociation since micro-CT analysis showed that (R)-24b did not reduce GCinduced osteoporosis when compared to prednisolone. We were also able to achieve a comparable balance of potency, selectivity and partial dissociation by exploiting an induced D-ring binding pocket with related 177

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compounds such as biaryl (R)-25 (IL-6 IC50 20 nM, 78% maximum efficacy; 85% inhibition in CIA model at 100 mg/kg) (17), or by subtle modifications of the central alkyl carbinol group as in t-butyl analogue (R)-26 (IL-6 IC50 27 nM, 76% maximum efficacy; 82% inhibition in CIA model at 100 mg/kg) (21).

Figure 4. Azaindole Glucocorticoids.

Scheme 4. Third-Generation Synthesis Route 178 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Bone Dissociation To assess the potential of the compounds for reduced bone side effects, we tested their ability to suppress osteocalcin (OC) production (22). A retrospective analysis of historical compounds revealed that partial agonist compounds (with 2000 nM, CYP3A4 IC50 2 µM). Therefore, we used the morpholinyl substituted 6-azaindole A-ring to re-investigate the D-ring SAR. Substitution with EWG at C-2 of the phenyl D-ring mimetic further increased maximum agonist efficacy, but provided reduced hERG and CYP inhibition such as in 32 (IL-6 IC50 4 nM, 95% maximum efficacy, hERG IC50 20 µM, CYP3A4 IC50 3 µM). The combination of these pharmacophores ultimately resulted in a panel of compounds that balanced all the desired properties. (R)-33 (BI 653048) was identified as the preferred combination from this exercise. This compound displayed a favorable selectivity profile, potent transrepression activity with partial agonism (IL-6 IC50 23 nM, 88% maximum efficacy), the desired degree of in vitro dissociation (MMTV maximum efficacy 33%, osteocalcin maximum efficacy 39%), reduced DDI potential (CYP3A4 IC50 8 µM), good metabolic stability (11% Qh HLM), and no risk of potential arrhythmia (hERG IC50 > 30 µM). Solely the aqueous solubility remained low (5 µg/mL at pH 6.8); however, a solid form with excellent dissolution properties was identified. For reasons that were poorly understood, this compound showed a species-difference in GR affinity. For example, the compound was significantly less potent in mouse in vitro assays, which precluded its profiling in our established mouse inflammation models. However, in a rat CIA model, the compound showed good potency (ED50 14 mg/kg). (R)-33 demonstrated acceptable safety margins in rat and dog pre-clinical safety pharmacology and toxicology studies and advanced to clinical trials in humans. Clinical data from a phase I proof-of-concept study will be disclosed in the near future.

Figure 5. Substituted Azaindole Glucocorticoids. 180 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

During the pre-clinical development phase, a more efficient route for the synthesis of (R)-33 was devised by Process Chemistry that improved upon the 17-step Medicinal Chemistry route, which was based on the third-generation synthesis route (Scheme 4). Significant optimization for the development of this scalable route will be discussed in the next chapter.

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Conclusions In conclusion, a series of non-steroidal trifluoromethylcarbinol glucocorticoid receptor (GR) agonists was identified from literature-known nuclear hormone receptor binder motifs. The series was successively optimized for potency, selectivity, and in vivo activity through rational design. Compounds with reduced GR-related side effects were identified empirically, ultimately the drug-like properties of these compounds were optimized resulting in the identification of the clinical candidate BI 653048.

References 1.

2. 3. 4. 5. 6.

7.

8.

9.

Buttgereit, F.; Saag, K. G.; Cutolo, M.; da Silva, J. A. P.; Bijlsma, J. W. J. The molecular basis for the effectiveness, toxicity, and resistance to glucocorticoids: focus on the treatment of rheumatoid arthritis. Scand. J. Rheumatol. 2005, 34, 14–21. Schäcke, H.; Döcke, W.-D.; Asadullah, K. Mechanisms involved in the side effects of glucocorticoids. Pharmacol. Ther. 2002, 96, 23–43. Weinstein, R. S. Glucocorticoid-induced bone disease. N. Engl. J. Med. 2011, 365, 62–70. Adcock, I. M. Molecular mechanisms of glucocorticosteroid actions. Pulm. Pharmacol. Ther. 2000, 13, 115–126. Clark, A. R.; Belvisi, M. G. Maps and legends: the quest for dissociated ligands of the glucocorticoid receptor. Pharmacol. Ther. 2012, 134, 54–67. Newton, R.; Holden, N. S. Separating transrepression and transactivation: a distressing divorce for the glucocorticoid receptor? Mol. Pharmacol. 2007, 72, 799–809. He, Y.; Yin, D.; Perera, M.; Kirkovsky, L.; Stourman, W.; Dalton, J. T.; Miller, D. D. Novel nonsteroidal ligands with high binding affinity and potent functional activity for the androgen receptor. Eur. J. Med. Chem. 2002, 37, 619–634. GR, PR and MR binding assays were performed in a fluorescence polarization format that measures competition for binding to the nuclear receptor between a test compound and a fluorescently labeled receptor ligand. Schäke, H.; Schottelius, A.; Döcke, W. D.; Strehlke, P.; Jaroch, S.; Schmees, N.; Rehwinkel, H.; Hennekes, H.; Asadullah, K. Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 227–232. 181

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10. Kuzmich, D.; Kirrane, T.; Proudfoot, J.; Bekkali, Y.; Zindell, R.; Beck, L.; Nelson, R.; Shih, C.-K.; Kukulka, A. J.; Paw, Z.; Reilly, P.; Deleon, R.; Cardozo, M.; Nabozny, G.; Thomson, D. Identification of dissociated nonsteroidal glucocorticoid receptor agonists. Bioorg. Med. Chem. Lett. 2007, 17, 5025–5031. 11. The IL-6 assay measures the ability of test compounds to inhibit the production of IL-6 by human foreskin fibroblasts following stimulation by IL-1 in vitro. The maximum inhibition by the test compound is compared to prednisolone at 2 µM which is set to 100%. 12. Regan, J.; Lee, T. W.; Zindell, R. M.; Bekkali, Y.; Bentzien, J.; Gilmore, T.; Hammach, A.; Kirrane, T. M.; Kukulka, A. J.; Kuzmich, D.; Nelson, R. M.; Proudfoot, J. R.; Ralph, M.; Pelletier, J.; Souza, D.; Zuvela-Jelaska, L.; Nabozny, G.; Thomson, D. S. Quinol-4-ones as steroid A-Ring mimetics in nonsteroidal dissociated glucocorticoid agonists. J. Med. Chem. 2006, 49, 7887–7896. 13. Lee, T. W.; Proudfoot, J. R.; Thomson, D. S. A concise asymmetric route for the synthesis of a novel class of glucocorticoid mimetics containing a trifluoromethyl-substituted alcohol. Bioorg. Med. Chem. Lett. 2006, 16, 654–657. 14. The model measures the ability of a single dose of test compound to inhibit the production of serum TNF-α after stimulation with LPS in Balb/c mice. 15. Betageri, R.; Gilmore, T.; Kuzmich, D.; Kirrane, T. M.; Bentzien, J.; Wiedenmeyer, D.; Regan, J.; Kukulka, A. J.; Fadra, T. N.; Nelson, R. M.; Zuvela-Jelaska, L.; Souza, D.; Pelletier, J.; Proudfoot, J.; Dinallo, R.; Panzenbeck, M.; Torcellini, C.; Lee, H.; Pack, E.; Harcken, C.; Nabozny, G.; Thomson, D. S. Non-steroidal Dissociated Glucocorticoid Agonists: lndoles as A-Ring Mimetics and Function-regulating Pharmacophores. Bioorg. Med. Chem. Lett. 2011, 21, 6842–6851. 16. In this model B10.RIII mice immunized with type II collagen are scored daily for paw swelling for 5 weeks. 17. Riether, D.; Harcken, C.; Razavi, H.; Kuzmich, D.; Gilmore, T.; Bentzien, J.; Pack, E. J., Jr.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra, T. N.; Zuvela-Jelaska, L.; Pelletier, J.; Dinallo, R.; Panzenbeck, M.; Torcellini, C.; Nabozny, G. H.; Thomson, D. S. Nonsteroidal Dissociated Glucocorticoid Agonists Containing Azaindoles as Steroid A-Ring Mimetics. J. Med. Chem. 2010, 53, 6681–6698. 18. Harcken, C.; Ward, Y.; Thomson, D.; Riether, D. A General and Efficient Synthesis of Azaindoles and Diazaindoles. Synlett 2005, 3121–3124. 19. The MMTV transactivation assay measures the ability of test compounds to activate MMTV promoter in HeLa cells stably transfected with MMTV luciferase construct. The maximum activation by the test compound is compared to prednisolone at 2 µM which is set to 100%. 20. The aromatase assay measures the ability of test compounds to induce aromatase activity in human foreskin fibroblasts. The maximum activation by the test compound is compared to prednisolone at 2 µM which is set to 100%. 182

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21. Razavi, H.; Riether, D.; Harcken, C.; Bentzien, J.; Dinallo, R. M.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra-Khan, T. N.; Pack, E. J., Jr.; ZuvelaJelaska, L.; Pelletier, J.; Panzenbeck, M.; Torcellini, C. A.; Proudfoot, J. R.; Nabozny, G. H.; Thomson, D. S. Discovery of a potent and dissociated non-steroidal glucocorticoid receptor agonist containing an alkyl carbinol pharmacophore. Bioorg. Med. Chem. Lett. 2014, 24, 1934–1940. 22. The osteocalcin assay measures the suppression of the production of osteocalcin upon stimulation with vitamin D in human MG-63 cells, an osteosarcoma cell line of osteoblast lineage. The maximum inhibition by the test compound is compared to prednisolone at 2 µM which is set to 100%. 23. Harcken, C. ; Riether, D.; Kuzmich, D.; Liu, P.; Betageri, R.; Ralph, M.; Emmanuel, M.; Reeves, J. T.; Berry, A.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra, T. N.; Zuvela-Jelaska, L.; Dinallo, R.; Bentzien, J.; Nabozny, G. H.; Thomson, D. S. Identification of highly efficacious glucocorticoid receptor agonists with a potential for reduced clinical bone side effects. J. Med. Chem. 2014, 57, 1583–1598. 24. Harcken, C.; Riether, D.; Liu, P.; Razavi, H.; Patel, U.; Lee, T.; Bosanac, T.; Ward, Y.; Ralph, M.; Chen, Z.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra-Khan, T. N.; Zuvela-Jelaska, L.; Patel, M.; Thomson, D. S.; Nabozny, G. S. Optimization of Drug-Like Properties of Nonsteroidal Glucocorticoid Mimetics and Identification of a Clinical Candidate. ACS Med. Chem. Lett. 2014, 5, 1318–1323.

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Chapter 6

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Development of an Asymmetric Route for Large-Scale Synthesis of a Glucocorticoid Agonist Jonathan T. Reeves, Daniel R. Fandrick, Jinhua J. Song,* Zhulin Tan, Soojin Kim, Bing-Shiou Yang, Nathan K. Yee, and Chris H. Senanayake Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06778, United States *E-mail: [email protected].

Glucocorticoids are one of the most used classes of anti-inflammatory drugs. However, their chronic use causes deleterious side effects such as GC-induced osteoporosis. In an effort to discover non-steroidal anti-inflammatory agents, a trifluoromethylcarbinol and azaindazole containing glucocorticoid receptor agonist was identified from our Discovery Program and advanced as a development candidate. The evolution of the synthesis of this candidate from early discovery to multi-kilogram synthesis is described. The ultimate pilot plant route was based on a highly efficient synthesis of the trifluoromethylketone intermediate via an enolization/bromine–magnesium exchange/electrophile trapping sequence and the discovery of a new asymmetric propargylation reaction of the resultant trifluoromethylketone.

Introduction In the preceding chapter, the discovery of 1 as a non-steroidal glucocorticoid receptor agonist was described (1–3). In order to further profile and investigate this drug candidate in both pre-clinical and clinical studies, large quantities of the drug substance with both high chemical and optical purities were required. The drug substance demand in terms of amounts and timelines are dependent on the stage of the project. © 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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At the very beginning of the preclinical studies, e.g. for a four week toxicological study, speed takes the top priority. Often times Chemical Development only has 4-6 m before the first kilogram batch is due. As the program moves along towards clinical phases, both speed and cost become critical. Accordingly, our chemistry program must be designed and executed in such a way as to provide stage-appropriate support for the project. In the following sections, we describe the evolution of the synthesis of the drug substance. This includes the modification of the discovery route to supply early toxicological studies as well as the development of the ultimate pilot plant route to provide multi-kilograms of the required drug substance in a more cost-effective manner (4). In addition to cutting cost many other factors needed to be taken into account. First of all, the new synthetic route must avoid any limitation derived from external intellectual property (IP) and ideally be patentable. Furthermore, a considerable amount of attention is required to ensure compliance with regulatory requirements for drug development. The FDA mandates in-depth understanding of the process parameters as well as tight controls of process impurities, in particular potential genotoxic impurities, to below pre-defined limits, down to ppms in many cases. During the chemical development phase of the project, all these aspects must be properly addressed.

Modified Discovery Route Compound 1 was originally synthesized using a 16-step sequence depicted in Scheme 1. The stereogenic center was established using a diastereoselective addition of chiral sulfoxide 5 to trifluoromethylketone 4. However, only a 2:1 diastereoselectivity was obtained. Separation of the two diastereomers entailed repeated crystallizations and tedious chromatography. Another issue was the installation of the primary carboxamide functionality. A total of seven linear steps were required to elaborate a tolyl group into the desired primary carboxamide. Besides the length of the route and the low overall yield (0.2%), the most critical concern was the use of a radical bromination reaction (8 → 9, Scheme 1). Due to the unreliability in reaction initiation for radical bromination (0-80% yield), it posed a potential process safety concern. Therefore, our most urgent task was to immediately address this issue in order to meet the drug substance requirements for early toxicological studies.

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Scheme 1. Discovery Route for Synthesis for 1 To avoid the free-radical bromination reaction, several alternative carboxy group precursors or equivalents were evaluated. Eventually a styrene-based route was pursued as shown in Scheme 2. The vinyl group served as a handle for a later conversion to the requisite carboxamide functionality. Under the time pressure, our top priority was to quickly establish the feasibility of this approach by intercepting a known synthetic intermediate as early as possible in the Discovery route. To this end, we converted 2-bromo-5-fluorostryrene to amide 17 in 8 steps as shown in Scheme 2 and our synthetic sample of 17 matched that synthesized by the discovery route, thus establishing the vinyl route for the synthesis of the target molecule. The longest linear sequence was reduced to 11 steps from the original 16 steps. The new route also avoided the need for Dess-Martin oxidation which was one of the high cost steps at that time. 187

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Scheme 2. Modified Discovery Route for 1

Since trifluoromethyl enone 3 is a key intermediate in the synthesis, a significant amount of work was devoted to improving its synthesis. The original protocol had a large Vmax (~70 L/kg) (5), used Et2O as the extraction solvent and required ~12 phase separations in the workup as well as multiple dryings over MgSO4. Despite this elaborate workup, the procedure provided enone 3 of poor quality which was black-colored and contained significant amounts of water (>0.5 molar equiv relative to 3). This poor quality enone gave very low yield (~5%) in the subsequent conjugate addition reaction with either styryl or vinyl substituted aryl Grignard reagents. 188 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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By adding dodecane during the workup, using five water/MeOH washes to remove most of the THF (the reaction solvent), drying over molecular sieves (beads) and finally distilling the enone from dodecane, very pure, anhydrous enone (< 0.1% water by Karl Fisher titration) was obtained which gave a much higher yield in the next step (92%, Scheme 2). The Vmax was reduced to 25 L/kg and the number of phase separations was reduced to 5 from the original 12. The new procedure was amenable to kilogram-scale operations. For the subsequent Michael addition, 2-bromo-5-fluorostyrene was converted into its Grignard derivative. Treatment of 2-bromo-5-fluorostyrene with i-PrMgCl and i-PrMgCl-LiCl led to decomposition. On the other hand the Grignard was readily prepared by treating the aryl bromide with Mg metal (85% yield by titration). By using the CF3 enone 3 prepared as outlined above, excellent yield was obtained for the conjugate addition (92% isolated yield). The original process used 1.2 equiv of CuI for this type of reaction. It was found in our laboratories that using a catalytic amount (10 mol%) of copper (particularly with 10 mol% LiCl) gave much higher yields. The volume efficiency was greatly improved to a Vmax of ~25 L/kg from 70 L/kg). In summary, we have developed a modified Discovery route based on the use of a vinyl group as a latent carboxy equivalent. The new route reduced the count of linear steps from 16 to 11 and increased the overall yield from 0.2% to 5.4%. Most importantly, it avoided the use of the free-radical bromination, was carried out safely in the Kilolab, and enabled us to meet the early stage drug substance need from the project. In the following section, we provide a full account of the development of a concise and asymmetric route to the target molecule for longterm drug substance supplies.

Chiral Amide Route While the styrene route described in the previous section enabled the delivery of a first batch of 200 g of drug substance, this route was still deemed less than optimal for further scale-up. Although it had solved the major safety issue of the free-radical bromination reaction, several key safety and cost issues remained. The expensive chiral sulfoxide 5 was still employed, and the diastereoselectivity of its addition to trifluoromethyl ketone 20 remained low. The elaboration of the sulfoxide moiety in 21 to alkyne 17 necessary for Sonogashira cross coupling required three steps, including the use of the highly reactive and genotoxic Meerwein’s salt (trimethyloxonium tetrafluoroborate). Furthermore, the conversion of the vinyl group to the primary amide required three steps, including two oxidation reactions. These six steps of functional group conversions contributed heavily to the overall step count of the synthesis. A more concise synthesis was envisioned based on a chiral amide. The key concepts for this route (Scheme 3) were: 1) Propargylation of a CF3 ketone with an N-chiral amide (A) may occur with diastereoselectivity; 2) The high crystallinity imparted by the amide may allow efficient crystallization for separation of the diastereomers of B; and 3) The N-chiral amide may be deprotected simply by 189

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treatment with acid, leading to 17, a known intermediate which has been converted to 1 in high yield in two steps.

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Scheme 3. Concept for the Chiral Amide Route

The first challenge for this route was finding a way to synthesize the trifluoromethyl ketone A. Numerous attempts to add Grignard reagents with a pendant amide sidechain, with copper catalysis, to enone 3 were unsuccessful. While the corresponding ester or acid functionalized CF3 ketones could be made in low yield by this approach, conversion of the ester or acid groups to the chiral amide was fruitless. Finally, it was found that the bromo-CF3 ketone 29 could be made by i-PrMgCl-mediated metal-halogen exchange with 2-bromo-4-fluoro-1-iodobenzene 27 and subsequent CuI-catalyzed 1,4-addition to 3 (Scheme 4). This reaction was further optimized to minimize the competing 1,2-addition byproduct. A yield of 75% of 29 was obtained after distillation.

Scheme 4. Synthesis of Trifluoromethyl Ketone 29 by Conjugate Addition

Elaboration of the aryl bromide in ketone 29 to a chiral amide was next required. An initial approach based on Pd-catalyzed aminocarbonylation was unsuccessful (Scheme 5). Aminocarbonylation of 29 resulted in only trace amounts of desired amide 33, with the ketone arylation product 31 and the amination product 32 as the major products. Subsequently, an approach based on metal-halogen exchange of the aryl bromide and addition to isocyanate 34 was explored. In order for this reaction to be possible, the highly reactive trifluoromethyl ketone needed to be protected. It was reasoned that the enolate may be an ideal in situ generated protecting group (Scheme 6). Upon formation of enolate 35, a reagent for bromine/metal exchange could be added, and the resultant dianion 36 could be treated with isocyanate 34. In theory the stronger aryl anion would add to the isocyanate, and after an aqueous quench the trifluoromethyl ketone would be regenerated to give 33. 190 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 5. Attempted Synthesis of 33 by Aminocarbonylation

Scheme 6. Concept for Grignard/Isocyanate Route to 33

The base for enolization needed to be both non-nucleophilic and also have a conjugate acid which would not react with the reagent employed for bromine/metal exchange. These requirements eliminated bases such as organolithiums, lithium amides, amines, and alkoxides. Sodium hydride met these two requirements nicely, and proved effective at generating the enolate 35 in THF at 0-20 °C. The enolization could be conveniently monitored by React-IR™ spectroscopy (Scheme 7). The 60 wt.% dispersion of sodium hydride in mineral oil was employed, as it can safely be handled in dry atmosphere (6). 191 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 7. React-IR™ Monitoring of the Enolization of 29

The enolization required catalytic amounts of water to proceed efficiently. Reactions conducted under anhydrous conditions, with THF containing only ~10 ppm of water, proceeded very slowly. Upon addition of 5 mol % of water, however, the enolization rapidly went to completion (Scheme 8). Thus a water specification of 300-500 ppm was incorporated for the THF to ensure reproducible enolization. It is speculated that the water reacts with sodium hydride to form sodium hydroxide, which is the actual base that enolizes ketone 29.

Scheme 8. Accelerating Effect of Water on the Enolization of 29 192 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The reagent for bromine/metal exchange was next explored. The exchange was extremely fast using n-BuLi or n-Bu3MgLi (< 15 min), but the yields of product 33 after isocyanate quench were low (entries 1 and 2 in Table 1). The use of the milder Grignard reagents i-PrMgCl and i-PrMgCl-LiCl gave only small amounts of exchange after 24 h at 20-25 °C (7). When Knochel’s modification of adding 1,4-dioxane to i-PrMgCl-LiCl to generate in situ the highly reactive “iPr2Mg-LiCl” was tried, the bromine/magnesium exchange was completed within 5 h at 20-25 °C, and the product 33 was formed cleanly and isolated in 85% yield by crystallization (8).

Table 1. Conditions for Bromine/Metal Exchange of 35

The final process for conversion of bromide 29 to amide 33 is shown in Scheme 9. This process was employed for production of 20 kg of ketone 33.

Scheme 9. Grignard/Isocyanate Route to 33 193 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Table 2. Conditions for Propargylation of 33

The propargylation of 33 was investigated next. The Barbier-type reaction of propargyl bromide using Al metal and catalytic HgCl2 cleanly gave the desired propargylation product, but as a 1:1 mixture of diastereomers 37 and 38 (entry 1, Table 2). Although the lack of diastereoselectivity was disappointing, a single recrystallization of the diastereomeric mixture produced the desired diastereomer in 30% isolated yield with a diastereomeric purity of 99:1. Thus the homopropargyl stereocenter could be easily obtained in high chiral purity even from a non-selective propargylation reaction. Due to the toxicity of HgCl2 and the shock-sensitivity of propargyl bromide, we sought safer alternative reagents for the propargylation. 194 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Heating 33 and propargyl bromide with Zn dust in THF also gave a clean conversion to the product, but again with no diastereoselectivity (entry 2). To replace propargyl bromide, 1-trimethylsilylpropyne was lithiated with n-BuLi in the presence of TMEDA at low temperature according to the procedure of Corey and co-workers (9). Addition of the lithiated propyne to 33 resulted in a low conversion (entry 3). When the lithiated propyne was transmetallated to zinc by the addition of ZnBr2 and ketone 33 was subsequently introduced, variable yields of product were obtained with 1:1 diastereoselectivity (entry 4). After significant process research, it was found that the source of variability in conversion was TMEDA. The lithiation proceeded equally well in the absence of TMEDA, and when the transmetallation and propargylation were performed, consistently high yields were obtained, and the desired isomer 37 could be crystallized out in 33% yield and 98:2 diastereomeric purity (entry 5). It was speculated that the TMEDA coordinates to zinc and renders the propargyl zinc species more basic and thus more likely to deprotonate rather than propargylate ketone 33. The final process employed for the non-selective propargylation is shown in Scheme 10. The lithiation and transmetallation steps to give the putative allenylzinc species were performed in THF at -20 °C. Subsequent introduction of the ketone 33 resulted in propargylation to give a 1:1 mixture of the TMS-protected alkynes 39 and 40. The reaction mixture was directly quenched with aqueous NaOMe, which cleaved the TMS groups to give a mixture of 37 and 38. Aqueous workup and crystallization from i-PrOAc and heptane gave 37 in 32% yield and 98:2 dr. i-PrOAc was a preferred solvent for large scale processing compared to EtOAc.

Scheme 10. Process for Non-selective Propargylation of 33 195 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Azaindole Formation and Amide Deprotection The installation of the heterocycle was examined next (Scheme 11). Initially, a two-step procedure as was done in the Discovery route was employed. Sonogashira cross-coupling with aryl iodide 18 using PdCl2(MeCN)2 and CuI gave the alkyne 41 in 74% yield. Cyclization to give azaindole 42 was performed with DBU in MeOH in 77% yield. It was subsequently discovered that the cross-coupling reaction did not require CuI, and could be carried out in MeOH as solvent with Pd(OAc)2 as the catalyst and a loading of just 0.5 mol %. Furthermore, the cyclization reaction could be run in the same pot by simply adding DBU upon completion of the cross-coupling reaction. The product 42 was isolated by crystallization upon addition of MeCN and water to the reaction mixture. Notably, this protocol gave the product in higher overall yield (82%) than the two-step process and with high purity (>98%) and low residual Pd (10-30 ppm).

Scheme 11. Two-step and One-pot Processes for Synthesis of Azaindole 42

The final chemical step in the synthesis was removal of the N-4methoxyphenethyl group from the amide. An initial screen of various acids for this purpose demonstrated neat TFA at 65 °C to be effective, but the workup for this process was tedious and the use of large quantities of TFA was not desirable on large scale. It was subsequently found that 48% aqueous HBr in combination with AcOH gave a relatively clean conversion to product after 8 h at 80 °C (Scheme 12). After addition of toluene and neutralization of the HBr with aq. NaOH, the product crystallized directly as an acetic acid solvate. A recrystallization from toluene/AcOH was necessary to upgrade the purity. This procedure was used for initial scale-up to prepare ~20 kg of 1•AcOH. 196 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Scheme 12. Deprotection of the Chiral Amide to give 1•AcOH

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Summary of the First Generation Propargylation Route The first generation propargylation route to 1•H3PO4 is shown in Scheme 13. This process reduced the step count from 16 (discovery route) and 11 (modified discovery route) to just 6 steps. The route avoided the multiple functional group manipulations required to transform the sulfoxide group into a propargyl group and the vinyl group into the primary amide. This synthesis enabled the first scale-up process to deliver kilogram quantities of API in the kilo-lab. For further scale-up to pilot plant scale, however, further improvements were deemed necessary to increase the scalability, safety and efficiency of the route. Ideally, the enone 3, which required an expensive Grignard reagent and a tedious thin-film distillation, would be replaced with a solid intermediate. The isocyanate 34, which required toxic phosgene or a derivative reagent for its preparation, and which was not an ideal intermediate in terms of stability and safety (liquid, propensity to trimerize on distillation), was also desirable to replace. The non-selective propargylation represented a major loss in yield for the synthesis, and the identification of a selective alternative was a key objective for increased efficiency and decreased cost. Finally, the final amide deprotection step was modest yielding and the need for a recrystallization to upgrade purity was not desirable. A more efficient deprotection was needed.

Second Generation Propargylation Route The second generation synthesis of amide ketone 33 is shown in Schemes 14-16. As a replacement for the liquid enone 3, the alkylidene Meldrum’s acid derivative 44 was prepared by a simple condensation reaction between the inexpensive and widely available Meldrum’s acid 43 and acetone (10). More than 500 kg of the crystalline product was prepared. Next, the aryl Grignard reagent 28, prepared as outlined previously for CuI-catalyzed conjugate addition to enone 3, was added to Meldrum’s acid derivative 44. Notably, this highly activated system required no Cu for 1,4-addition (11). The intermediate adduct 45 was not isolated, but directly hydrolyzed/decarboxylated to give the acid 46, which was directly crystallized from the reaction mixture in 80% yield. Over 420 kg of acid 46 was produced by this process. 197

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Scheme 13. First Generation Propargylation Route to 1•H3PO4

Scheme 14. Synthesis of Acid 46 The conversion of the acid 46 into the requisite trifluoromethyl ketone 29 was achieved using chemistry we had previously applied for structurally related compounds. Zard and co-workers reported the conversion of aliphatic acid chlorides to trifluoromethyl ketones using TFAA and pyridine (12, 13). We subsequently reported the same reaction could be performed directly from the acid 198 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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(14, 15). Thus, treatment of acid 46 with excess TFAA and pyridine in toluene at 65 °C followed by hydrolysis/decarboxylation on addition of water gave 29 in 83% yield (Scheme 15). Notably, ketone 29 produced by this process was free of the impurities formed in the previous Cu-catalyzed 1,4-addition to enone 3, and thus did not require the vacuum distillation necessary for the previous process. Over 330 kg of 29 was produced by this route.

Scheme 15. Conversion of Acid 46 to Trifluoromethyl Ketone 29 To avoid the use of isocyanate 34, a two-step carboxylation/amide formation approach was employed (Scheme 16). In the first step, the same enolization/Grignard formation sequence was applied, but instead of quenching with the isocyanate, a carbon dioxide quench was utilized. This gave the carboxylic acid 47 in 78% yield. The acid chloride was then formed using SOCl2 in toluene, and to the resultant acid chloride solution was added 2,6-lutidine followed by the amine 30. The product ketone 33 was isolated in 75% yield. A total of 318 kg of 33 was produced by this process.

Scheme 16. Carboxylation/Amide Coupling Route to 33 The second generation synthesis of amide ketone 33 avoided the preparation and isolation of liquid enone 3, eliminated the need to distill bromo ketone 29, and avoided the use of isocyanate 34. The second generation process was scaled up successfully at pilot plant scale. 199 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Development of the Asymmetric Propargylation After establishing a suitable process to secure material to support non-clinical toxicological evaluations, process research changed focus to the development of a long term economical and green process. Toward this endeavor, the low yielding non-selective propargylation at an advanced operation became the principal focus. A goal to double the yield in this operation was formulated which would reduce the raw material cost, wastes produced and the number of batches from the beginning of the process through to this advanced operation in half (16). The first principle approach toward a stereoselective propargylation was to modify the allenyl zinc reagent utilized in the non-selective propargylation with a chiral ligand which may differentiate the diastereotopic faces of the trifluoromethyl ketone (Scheme 17).

Scheme 17. General Strategy for Stereoselective Induction

Before venturing into reagent modification in 2007, an assessment of the prior art was conducted revealing poor precedence for asymmetric propargylation reactions (Scheme 18). Although asymmetric additions with catalytic chiral ligands mediated by zinc were precedented, addition with nucleophiles that invert, i.e. allylations, propargylations and allenylations, were sparse. Attempts with zinc mediated asymmetric propargylations by Marino (17) and Kocienski (18) provided poor stereoselective induction with catalytic or stoichiometric amino-alcohol ligand. One of the only examples with asymmetric induction through a zinc mediated process and a nucleophile which inverts in the addition was the Nakamura (19) allylation utilizing the stoichiometrically prepared chiral zinc complex with generality specific to ynones. Although the precedence was poor, examination of chiral ligands to effect stereoselectivity in our propargylation was pursued. 200 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 18. Prior Art in 2007 for Zinc Mediated Asymmetric Additions with Nucleophiles that Invert

The screen and ultimate stereoselective propargylation toward the development candidate retained the phenethyl chiral component as it would allow facile and efficient stereochemical enrichment and ease of deprotection. The first attempted reagent controlled approach, through modification of the alkynyl-Zn-“ate” complex developed by Tan (20) to an allenyl intermediate, was examined. However, no conversion was observed, likely due to enolization of the ketone 33 (Scheme 19).

Scheme 19. Initial Reagent Controlled Approach for Selective Propargylation

The screen of ligands to effect diastereoselectivity were focused on readily available per-N-alkyl amino alcohol and amino acids ligands. The later series were attractive due to the rapid generation of a library through a one step reductive hydrogenation from the corresponding chiral amino-acid (Scheme 20). The screens utilized a pre-complexation to the zinc reagent prior to subjection to the ketone substrate. Amino-alcohol ligands, typical for zinc mediated asymmetric additions with nucleophiles that do not invert, (Table 3) proceeded with no asymmetric induction. However, ligands derived from N-alkyl proline provided the first observation of stereoselective induction (Table 4). 201 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 20. Preparation of Per-N-Alkyl Amino-Acids

Table 3. Amino-Alcohol Effect on the Diastereoselective Propargylation of 33

The main drawback to the above process was the generally poor conversion and yields observed in the propargylation utilizing a transmetallation approach to generate the allenyl zinc reagent. Therefore, a B/Zn exchange process (21–23) was pursued wherein a propargyl borolane (24) reagent was employed. This approach demonstrated a productive improvement in the conversion and yields while also demonstrating a facile B/Zn exchange process (Table 5). More importantly, optimization of the proline scaffold was continued wherein the N-isopropyl-L-proline system provided a modest to high diastereoselectivity. This 202 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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system was optimized in a straightforward manner with a focus on temperature, stoichiometry, concentration and addition. For complete and robust conversion, a stoichiometric process with a slight excess of diethyl zinc to ligand was necessary. However, the most pronounced effect was the requirement of a metered addition of the substrate and borolane to the ligated ethyl-zinc complex (Table 6). Increasing the addition time from 99.5 A% purity by HPLC after crystallization from acetone/water (Scheme 3).

Scheme 3. Oxo-dihydropyrimidinium Route to 18 221 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

The second starting material 2-chloro-4-(methylsulfonyl)benzoic acid (20) was commercially available and was directly purchased in >99.5 A% purity with a reproducible impurity profile. Both starting materials 18 and 20 are well characterized and their respective manufacturing processes routinely produce them in high purity with no single impurities >0.2 A% by HPLC. Now that we had a robust supply chain for both starting materials in hand, we focused our effort towards Stage 1of the manufacturing process to implement a catalytic hydrogenation of 18.

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Stage 1: Hydrogenation of Nitroaromatic 18 to Aniline 19 Although selective catalytic hydrogenation of nitroaromatic compounds to the corresponding aromatic amines is a well-known process in the literature (16–19), our initial attempts evaluating the effect of catalysts such as Pd(OH)2/C, Pd/C, and Pt/C, hydrogen pressure (50-110 psig) and temperature (25-80 °C) on the reduction reaction resulted in the formation of aniline 19 with various impurity profiles. Particularly, hydroxylamine 25 and its associated by-products (20–23), 26, 27 and 28 were generated at room temperature and low pressure from incomplete reduction. On the other hand, when the hydrogenation was performed at high temperature and high pressure, over-reduction led to des-chloro amine 29 and piperidine 30 as indicated in Scheme 4. Plainly, the reaction conditions would need to be balanced.

Scheme 4. Under- and Over-Reduced Impurities Generated during Hydrogenation of 18. From ref (12), copyright American Chemical Society, 2016. This was clearly a problem since most of these impurities gave a structural alert when using predictive quantitative structure-activity relationship (QSAR) (24) methods to predict mutagenicity and therefore require an Ames25 test to assess their mutagenic potential. Hydroxylamine 25 was found to be Ames-positive and is therefore considered a genotoxic impurity (GTI) in the process. Furthermore, impurities 25, 28, 29 and 30 could potentially be acylated during the next stage of manufacturing and lead in turn to even more by-products. 222 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Based on literature precedent (25, 26), combinations of 1% Pt + 2% V/C catalyst in the presence of acid additives (HCl and AcOH) were tested in MeOH (for solubility of 18). By using a combination of 1% Pt + 2% V/C in MeOH/AcOH (9:1) at 60 °C and 88 psig (6 bar) the desired aniline 19 was obtained in ~97 A% by HPLC. More importantly, hydroxylamine amine 25, a GTI in the process, was not detected with this catalyst. However, slight variations in reaction conditions had a tremendous impact on the outcome of the hydrogenation. For example, diazene impurity 27 was produced exclusively in 95 A% when using 1 wt% of catalyst loading in pure AcOH. To better understand the criticality of the process parameters, a statistical DOE was performed (Table 4) under 6 bar of hydrogen for 3 h on 1 g of nitroaromatic 18. An extract of the DOE monitoring aniline 19, hydroxylamine 25 and des-chloro 29 as response factors is reported in Table 4.

Table 4. DOE Process Optimization to 19

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From these experiments it was clear that when using the %Pt + 2%V/C catalyst in MeOH/AcOH within the investigated range: • • • • •

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•

Formation of hydroxylamine 25 was no longer significant. Formation of des-chloro 29 was only seen in the high catalyst loading experiments, even then it is only formed in low levels (NMT 0.35 A%). The impact of acetic acid concentration was small in the range evaluated. Increased reaction temperatures and catalyst loadings led to higher aniline 19 levels but also higher levels of the deschloro impurity, 29. Catalyst loads under 5 wt% resulted in low levels of conversion within the investigated reaction time of 3 h. The ideal temperature ranges between 40 and 60 °C.

Based on these experiments, optimal conditions were arrived at. These were then slightly modified in order to make them more conducive to manufacturing (for instance, the total volume was slightly increased to assure good dissolution, the acetic acid amount was slightly lowered to minimize the need for its neutralization later in the work up), leading to the final ranges of parameters (Table 5). On scale (20 kg), the completion of the reaction was monitored by hydrogen uptake as determined by a flow of 10 and the resulting solid was isolated by centrifugation. On commercial production, this step is routinely run on 18 kg scale of 18 and produces amine 19 in 95-98 % yield (corrected for residue on drying) with >99.9 A% purity by HPLC (residual Pt and V, 99% conversion to acid chloride 21 within the range investigated and demonstrated that the reaction proceeds to completion with as little as 1.2 molar equivalents of SOCl2 and 0.05 wt of DMF in 6 volumes (6 mL/g) of DCM. While the first generation process performed well at the proposed commercial scale (approximately 20 kg of 20), we decided to replace dichloromethane as the Stage 1 solvent with toluene for safety (due to toluene’s higher boiling point) and environmental reasons. As the reagent stoichiometry data generated in dichloromethane should apply regardless of solvent, these data were used as a starting point for further optimization of the process. The low end of the reaction temperature range in toluene was limited to 65 °C for safety reasons, ensuring that the reagents are consumed at a faster rate than they are charged, avoiding a potential adiabatic exothermic runaway reaction if a build-up were to occur. The upper end of the temperature range was limited by the boiling point of the reaction mixture (~107–109 °C) at atmospheric pressure. Thus, the reaction was routinely run at 70 °C. Additionally, a large volume of HCl and SO2 by-product gases (191 L/kg of 20) had to be removed from the production 225

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vessel. Thus, the gas evolution rate must be low enough to ensure that there was no build up of pressure in the vessel, and this was achieved by controlling the addition rate. Complete dissolution signaled the end of reaction. The homogeneous solution was then partially concentrated at atmospheric pressure, which serves to remove excess thionyl chloride, HCl, and SO2 reaction gases, and then cooled to 10 °C while the compound crystallized out (solubility of 21 at isolation temperature was approximately 2%). Isolating the intermediate at higher dilution will not lead to a significant yield loss. On scale, this step was run reproducibly on 23 kg of 20 and provided acid chloride 21 in 92-95% isolated yield (28) (corrected for residue on drying) with >99.8 A% purity by HPLC. The only impurity detected at this Stage was the starting acid 20 in 15 wt% at 20 °C) and because crude vismodegib could be isolated directly from THF/water mixture through crystallization. As it was likely that the base used in Stage 3 would have an impact not only on the reaction, but also on the level of inorganic impurities in vismodegib, the type and amount of base was studied. Due to the higher solubility of potassium salts over sodium salts in water (K2CO3: 111 g/mL; Na2CO3: 21.5 g/mL at 20 °C) a study was designed to compare the use of the two bases and their stoichiometry with respect to reaction completion and inorganic impurities as measured by residue on ignition (ROI) (Table 8). 226

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Table 8. Impact of Type and Stoichiometry of the Base

Use of acid chloride 21 in 1.1 molar equivalent excess ensured that all amine 19 was consumed. The solvent ratio (THF/water) was designed so amine 19 was fully soluble at the beginning of the reaction. Before addition of 21, the THF/water ratio is 60:40 and solubility of 19 is ~7.5 wt% and well in range of the solubility of ~5.3 wt% in 50:50 THF/water at 20 °C. The results from Table 8 screen clearly showed that K2CO3 was a more effective base, giving higher yields and purities. However, the type of base had no impact on the ROI. So, 0.66 molar equivalent of K2CO3 was chosen based on the resulting yield and purity. Using an excess of base also reduced yield loss from the higher solubility of the vismodegib HCl salt in the aqueous layer. To ensure good purging of the potassium salt of 20, a pH check to verify that the aqueous layer was basic (pH > 7) was implemented. For the isolation, the aqueous layer was drained after completion of the reaction and THF was removed by distillation under atmospheric pressure with concomitant addition of water. The batch was then seeded with vismodegib and the remaining THF was distilled off until a boiling point of 82 °C was reached. At this temperature point, the ratio of water/THF was 97.5:2.5 (verified by KF-titration). After cooling to 15 °C (the solubility of vismodegib was 99.9 A% HPLC purity. Table 9 summarizes the operating ranges and criticality assessment for the Stage 3 amide formation. 227

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Table 9. Stage 3 Operating Ranges Comparison and Criticality Assessment. Reproduced from ref. (12). Copyright 2016 American Chemical Society.

Target

Normal Operating Range

Key for Yield or Purity

Critical Process Parameter

Acid chloride 21 (molar equivalents)

1.1

± 1%

Yes

No

THF (wt)

5.3

± 5%

No

No

Potassium Carbonate (molar equivalents)

0.66

± 5%

Yes

No

Water (wt)

4.3

± 5%

No

No

Reaction Temperature

2 °C

0–5 °C

No

No

Final Temperature

20 °C

15–30 °C

No

No

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Parameter

Stage 4: Control of Vismodegib Physical Properties Stage 4 consists of three steps: • • •

crystallization delivered the API with the required polymorphic form and purity drying ensured acceptable residual solvent levels milling the API furnished the specified particle size distribution (PSD)

Two polymorphic forms of the API were identified during development: Form A and Form B. The latter is the thermodynamically more stable polymorph observed to date as shown in a differential scanning calorimetry (DSC) trace (Figure 1) containing a mixture of Form A ( m.p. = 177 °C) and Form B (m.p.= 187 °C). Form B formation in Stage 4 of the manufacturing process is controlled by seeding the crystallization with the appropriate Form B material. Importantly, however, upon seeding with a mixture of Forms A and B, a mixture of the two forms was obtained, so seeding with the correct polymorph is critical. Vismodegib is poorly soluble in most organic solvents like iso-propyl acetate, 1- or 2-propanol, toluene or acetonitrile (4 ppm by LC-MS with selective ion monitoring, giving a purging of >2500X for these 2 nitro GTIs. Therefore 22 and 23 could be specified at 1 ppm, giving a purging factor of >10,000X. This purging factor justified the specification for 25 of 76% over 4 steps) with the required critical quality attributes.

Scheme 5. Vismodegib Manufacturing Process

References 1. 2. 3. 4.

5.

Nüsslein-Volhard, C.; Wieschaus, E. Mutations affecting segment number and polarity in Drosophila. Nature 1980, 287, 795–801. Ingham, P. W.; McMahon, A. P. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001, 15, 3059–3087. Hui, C.-C.; Angers, S. Gli proteins in development and disease. Annu. Rev. Cell Dev. Biol. 2011, 27, 513–537. Teh, M-T; Blaydon, D.; Chaplin, T.; Foot, N. J.; Skoulakis, S.; Raghavan, M.; Harwood, C. A.; Proby, C. M.; Philpott, M. P.; Young, B. D.; Kelsell, D. P. Genomewide single nucleotide polymorphism microarray mapping in basal cell carcinomas unveils uniparental disomy as a key somatic event. Cancer Res. 2005, 65, 8597–8603. Danaee, H.; Karagas, M. R.; Kelsey, K. T.; Perry, A. T.; Nelson, H. H. Allelic loss at Drosophila patched gene is highly prevalent in basal and squamous cell carcinomas of the skin. J. Invest. Dermatol. 2006, 12, 1152–1158. 233

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

6.

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7.

8. 9.

10.

11.

12.

13.

14. 15.

16.

Kool, M.; Korshunov, A.; Remke, M.; Jones, D. T.; Schlanstein, M.; Northcott, P. A.; Cho, Y. J.; Koster, J.; Schouten-van Meeteren, A.; van Vuurden, D.; Clifford, S. C.; Pietsch, T.; von Bueren, A. O.; Rutkowski, S.; McCabe, M.; Collins, V. P.; Bäcklund, M. L.; Haberler, C.; Bourdeaut, F.; Delattre, O.; Doz, F.; Ellison, D. W.; Gilbertson, R. J.; Pomeroy, S. L.; Taylor, M. D.; Lichter, P.; Pfister, S. M. Molecular subgroups of medulloblastoma: an international meta- analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas. Acta Neuropathol. 2012, 123, 473–484. Northcott, P. A.; Jones, D. T.; Kool, M.; Robinson, G. W.; Gilbertson, R. J.; Cho, Y. J.; Pomeroy, S. L.; Korshunov, A.; Lichter, P.; Taylor, M. D.; Pfister, S. M. Medulloblastomics: the end of the beginning. Nat. Rev. Cancer 2012, 12, 818–834. Ingham, P. W.; Nakano, Y.; Seger, C. Mechanisms and functions of Hedgehog signalling across the metazoa. Nat. Rev. Genet. 2011, 12, 393–406. Briscoe, J.; Therond, P. P. The mechanisms of hedgehog signalling and its roles in development and disease. Nat. Rev. Mol. Cell Biol. 2013, 14, 416–429. Robarge, K. D.; Brunton, S. A.; Castanedo, G. M.; Cui, Y.; Dina, M. S.; Goldsmith, R.; Gould, S. E.; Guichert, O.; Gunzner, J. L.; Halladay, J.; Jia, W.; Khojasteh, C.; Koehler, M. F.; Kotkow, K.; La, H.; Lalonde, R. L.; Lau, K.; Lee, L.; Marshall, D.; Marsters, J. C.; Murray, L. J.; Qian, C.; Rubin, L. L.; Salphati, L.; Stanley, M. S.; Stibbard, J. H.; Sutherlin, D. P.; Ubhayaker, S.; Wang, S.; Wong, S.; Xie, M. GDC-0449-a potent inhibitor of the hedgehog pathway. Bioorg. Med. Chem. Lett. 2009, 19, 5576–5581. Gould, S. E.; Low, J. A.; Marsters, J. C.; Robarge, K.; Rubin, L. L.; de Sauvage, F. J.; Sutherlin, D. P.; Wong, H.; Yauch, R. L. Discovery and preclinical development of vismodegib. Expert Opin. Drug Discovery 2014, 9, 969–984. Angelaud, R.; Reynolds, M.; Venkatramani, C.; Savage, S.; Trafelet, H.; Landmesser, T.; Demel, P.; Levis, M.; Ruha, O.; Rueckert, B.; Jaeggi, H. Manufacturing development and genotoxic impurity control strategy of the hedgehog pathway inhibitor vismodegib. Org. Process Res. Dev. 2016, 20, 1509–1519. Gunzner, J. L.; Sutherlin, D. P.; Stanley, M. S.; Bao, L.; Castanedo, G.; Lalonde, R. L.; Wang, S.; Reynolds, M. E.; Savage, S. J.; Malesky, K.; Dina, M. S. Pyridyl inhibitors of hedgehog signaling. WO2009126863, October 15, 2009. Sonoda, T.; Shintou, T.; Onoue, K. Nagayama, T. Process for production of 2,3′-bipyridyl-6′-one. WO2008093392, July 08, 2008. Kuwabara, H.; Zhang, C. S.; Saitoh, H.; Sonoda, T. Preparation of pyridine derivatives using pyrimidium derivatives as intermediates. EP1559711, August 03, 2005. Blaser, H. U.; Siegrist, U.; Steiner, H.; Studer, M. In Fine Chemicals through Heterogeneous Catalysis; Sheldon, R. A., van Bekkum, H., Eds.; WileyVCH: Weinheim, 2001; pp 389–406. 234

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17. Kosak, J. R. Catalysis in Organic Synthesis; Jones, W. H., Ed.; Academic Press: New York, 1980; pp 107–117. 18. Corma, A.; Serna, P. Chemoselective hydrogenation of nitro compounds with supported gold catalysts. Science 2006, 313, 332–334. 19. Hoogenraad, M.; van der Linden, J. B.; Smith, A. A.; Hughes, B.; Derrick, A. M.; Harris, L. J.; Higginson, P. D.; Pettman, A. Accelerated process development of pharmaceuticals: selective catalytic hydrogenations of nitro compounds containing other functionalities. J. Org. Process Res. Dev. 2004, 8, 469–476. 20. Takenaka, Y.; Kiyosu, T.; Choi, J-C.; Sakakura, T.; Yasuda, H. Selective synthesis of N-aryl hydroxylamines by the hydrogenation of nitroaromatics using supported platinum catalysts. Green Chem. 2009, 11, 1385–1390. 21. Becker, A. R.; Sternson, L. A. J. Org. Chem. 1980, 45, 1708–1710. 22. Corma, A.; Concepción, P.; Serna, P. A different reaction pathway for the reduction of aromatic nitro compounds on gold catalysts. Angew. Chem., Int. Ed. 2007, 46, 7266–7269. 23. Siegrist, U.; Baumeister, P.; Blaser, H.-U. The selective hydrogenation of functionalized nitroarenes: new catalytic systems. Chem. Ind. 1998, 75, 207–219. 24. Ames, B. N.; Durston, W. E.; Yamasaki, E.; Lee, F. D. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 2281–2285. 25. Baumeister, P.; Blaser, H-U.; Studer, M. Strong reduction of hydroxylamine accumulation in the catalytic hydrogenation of nitroarenes by vanadium promoters. Catal. Lett. 1997, 49, 219–222. 26. Crump, B. R.; Goss, C.; Lovelace, T.; Lewis, R.; Peterson, J. Influence of reaction parameters on the first principles reaction rate modeling of a platinum and vanadium catalyzed nitro reduction. Org. Process Res. Dev. 2013, 17, 1277–1286. 27. Conversion to 21 is monitored by HPLC through derivatization to the corresponding methyl ester. 28. To reduce the potential for hydrolysis during isolation, drying and handling, it was decided not to incorporate a drying step and to store acid chloride 21 as a toluene wet cake cold (2–8 °C) in sealed polyethylene bags purged with an inert gas (nitrogen or argon) and use it as is in the amide coupling Stage 3. 29. ICH Q3c. Impurities: guideline for residual solvents; Step 4 version, February 11, 2011. http://www.ich.org/fileadmin/Public_Web_Site/ ICH_Products/Guidelines/Quality/Q3C/Step4/Q3C_R5_Step4.pdf 30. ICH Q6A. Specifications: test procedures and acceptance criteria for new drug substances and new drug products: chemical substances; Step 4 version, October 6, 1999. http://www.ich.org/fileadmin/Public_Web_Site/ ICH_Products/Guidelines/Quality/Q6A/Step4/Q6Astep4.pdf 31. The d(v,0.5) is the median for the particle size distribution by volume. d(v,0.9) describes the distribution where ninety percent of the distribution volume has a smaller particle size and ten percent has a larger particle size. The d(v,0.1) distribution has ten percent smaller and ninety percent larger. 235

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32. ICH M7. Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk; Step 4 version, June 23, 2014. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Multidisciplinary/M7/M7_Step_4.pdf 33. Threshold of Toxicological Concern or TTC corresponding to a theoretical 10-5 excess lifetime risk of cancer.

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Chapter 8

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Discovery and Process Development of Class I PI3K and Class I PI3K/mTOR Inhibitors GDC-0941 and GDC-0980 Srinivasan Babu,1 Francis Gosselin,1 Theresa Humphries,1 Alan Olivero,2 Daniel Sutherlin,2 and Qingping Tian*,1 1Small

Molecule Process Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States 2Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States *E-mail: [email protected].

The PI3K signaling pathway has attracted significant attention in drug development due to its role in tumorigenesis. Two orally available clinical compounds which have been developed include GDC-0941 (pictilisib), a selective Class I PI3K inhibitor, and GDC-0980 (apitolisib), a selective Class I PI3K/mTOR inhibitor. Chemical process development has been conducted to support both compounds, which share a common thienopyrimidine core, from the pre-clinical development stage to the clinical study. An enabling process (the first-generation route) was employed to produce multikilogram of API for the GLP and initial GMP production while an improved process (the second-generation route) was developed as the projects were advanced to late stage development. The development of a practical and protecting-group-free synthesis for GDC-0980 and an efficient synthesis of a key intermediate for GDC-0941 through aminoalkylation is discussed in detail in this chapter.

Discovery Chemistry The phosphoinositide-3-kinase ( PI3K) pathway is involved in the transmission of growth and survival signals from the outside of the cell to the nucleus (1). As such, this signaling pathway has received a great © 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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deal of interest as a potential target for cancer therapy where unregulated cell growth and survival plays a large role in the pathogenesis of the disease (2). Functionally, signal transduction occurs when PI3K proteins are recruited to the cell membrane whereupon PI3Ks phosphorylate the membrane-bound lipid phosphatidylinositol-(4,5)-bis-phosphate (PIP2) to phosphatidylinositol-(3,4,5)-tris-phosphate (PIP3) which serves to activate and recruit the oncogene Protein Kinase B (AKT). Subsequently, AKT activates a number of pathways that ultimately result in the transcription of genes that promote cell survival and growth (2). PI3Ks are lipid kinases, distinguishing them from serine and threonine kinases, and exist in four different isoforms, PI3Kα, β, δ, and γ (Class I PI3Ks). While these isoforms have been implicated in cancer to varying degrees, PI3Kα is considered to be influential in tumorigenesis based on the identification of PI3Kα activating mutations and through observation that Phosphatase and Tensen Homolog (PTEN), a negative regulator of PI3K signaling, is deleted in many tumor samples (2). Based on this strong genetic evidence for PI3Ks’ significant role in cancer, we sought to develop inhibitors of this pathway. In our early efforts to identify potent inhibitors of PI3K signaling, we developed two clinical molecules: GDC-0941/pictilisib and GDC-0980/apitolisib (Figure 1) in collaboration with Piramed Pharmaceuticals (later acquired by Roche) (3, 4). While both compounds were potent inhibitors of all four Class I PI3K isoforms, they differed in their selectivity for mTOR, an additional kinase that plays an important role in cell signaling and cancer. We hypothesized that these biological differences could lead to a unique efficacy, and safety profiles that would require further exploration in the clinic to truly understand the therapeutic potential for each compound.

Figure 1. Structures for Class I PI3K inhibitor GDC-0941 and Class I PI3K/mTOR inhibitor GDC-0980. Early discovery efforts initiated by Piramed began with thienopyrimidine 1 (Table 1), a compound that was chosen for its excellent potency for PI3K and good overall properties (3). Analysis of a molecular model and literature data for other morpholine-containing compounds led to speculation that the morpholine oxygen bound to the hinge region of the kinase and would position the phenol in the affinity pocket to make a hydrogen bond contact to the protein via the phenol OH. Both of these interactions were observed to be critical for PI3K potency. 238 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Unfortunately regardless of the R1 substitution, every compound with a phenol at R3 had unacceptable oral bioavailability. A thorough investigation of phenol replacements that might retain the same back-pocket donor interaction led to discovery of indazole 2. Although indazole 2 lost four-fold potency compared to phenol 1, we were gratified to see the oral bioavailability in rats improve from 2% to 37%. Once the imidazole was discovered, R1 substitutions were explored to optimize solubility, potency, and PK properties. Ultimately the sulphonylated piperazine was incorporated to yield GDC-0941 (3). This change improved potency significantly while also improving microsomal stability relative to 2.

Table 1. Summary of Major Structural Changes in the Transformation of Lead Compound 1 to Identify GDC-0941 and GDC-0980

GDC-0941 was shown to be a broad class I PI3K inhibitor (IC50s for PI3Kα, 3 nm; PI3Kβ, 33 nM; PI3Kδ, 3 nM; PI3Kγ, 75 nM). Consistent with results from many lipid kinase inhibitors, this compound was found to be very selective relative to a large number of serine/threonine kinases in the kinome. The potent biochemical activity for both PI3Kα and PI3Kβ isoforms contributed to cellular potency of this compound in cell lines with activating mutations of 239 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

PI3Kα (MCF7.1) and with deleted PTEN (PC3-NCI) (5, 6). This trend was consistent across a larger panel of cell lines. Specifically, GDC-0941 inhibited the downstream phosphorylation of AKT with an IC50 of 28 nM and 37 nM in PC3-NCI and MCF7.1 cells respectively. Based on potent inhibition of PC3 and MCF7 cell proliferation and significant efficacy in their corresponding xenograft models, GDC-0941 was nominated for clinical development. Additional preclinical PK and safety data also supported this nomination (7). The subsequent discovery of GDC-0980 (6) began with the identification of 4 (Table 1), where the indazole of GDC-0941 is replaced with an aminopyrimidine that occupies the affinity pocket (8). This change was significant for two reasons:

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•

•

First the aminopyrimidine lowered the cLogP of otherwise identical indazoles by nearly two log units which contributed to an increase in stability in rat microsomes, and a decrease in plasma protein binding ultimately combining to lower the human dose projection. Secondly, this change brought about an approximate 20-fold increase in mTOR potency, a property that was anticipated to increase efficacy and broaden the scope of activity across cell lines.

Despite these advantages, 4 had high clearance in dogs. This liability was addressed by the addition of a methyl group to the thienopyrimidine scaffold to yield 5, that served to reduce in vivo clearances in multiple species and across multiple scaffolds (9). In an effort to improve the intrinsic solubility of 5, the sulfonamide moiety was replaced with a (S)-lactic amide to yield GDC-0980 (6), which increased the solubility of GDC-0980 by nearly 10-fold relative to 5. Like GDC-0941, GDC-0980 was found to be a broad class I PI3K inhibitor (5, 27, 7, and 14 nM IC50s for PI3Kα, β, δ, γ, respectively), very selective in a large kinase panel, and potent in proliferation assays for cell lines with both PI3K activating mutations and PTEN deletions (MCF7-neo/HER-2, 240 nM and PC3-NCI, 120 nM IC50s). GDC-0980 was active in a larger number of cell lines when compared to GDC-0941 (10, 11) and was attributed to the addition of mTOR inhibition to the scaffold. Based primarily on the improved properties of GDC-0980 relative to GDC-0941 (better overall PK properties and a higher free-drug fraction), GDC-0980 was found to be active at doses as low as 5 mg/kg in xenograft efficacy studies. With this differentiated profile relative to GDC-0941, GDC-0980 was also selected for clinical development (12, 13). The medicinal chemistry route to both GDC compounds and their analogs began with thiophene amino esters 7 and 8, for GDC-0941 and GDC-0980 respectively (Scheme 1). High temperature condensation with molten urea gave the thienopyrimidones 9 and 10. Bis-chlorination was effected by treatment with POCl3 to generate the corresponding dichloro intermediates 11 and 12, followed by a selective displacement of the most electrophilic chlorine by morpholine at room temperature, yielding monochloro-morpholines 13 and 14. Selective deprotonation of the CH adjacent to sulfur followed by addition of DMF gave the late stage aldehydes 15 and 16 which could be used to rapidly generate analogs that had varying functionality in two important areas of the binding pocket, the solvent exposed region through functionalization of the aldehyde, and the affinity 240

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pocket through aromatic substitution and cross coupling reactions with the aryl chloride (indicated by R1 and R3 in Table 1). For GDC-0941, a reductive amination was performed with aldehyde 15 and methylsulphonylpiperazine to yield 17. Next, a Suzuki-Miyaura coupling reaction with indazole boronic ester 19 furnished the final molecule. A similar reductive amination sequence with Boc-piperazine on 16, followed by Suzuki-Miyaura coupling with commercially available aminopyrimidine boronic ester 20 provided 18. The synthesis of GDC-0980 was completed through Boc removal under acidic conditions followed by an amide coupling with (S)-lactic acid. This consisted of the Med Chem route to these PI3K inhibitors.

Scheme 1. Medchem Synthesis of GDC-0941 and GDC-0980 241 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Process Chemistry Development Process chemistry development has been pursued as both compounds (GDC-0941 and GDC-0980) were advanced to the clinic. We employed a similar synthetic strategy for both compounds due to the structural similarity of GDC-0941 and GDC-0980 (14, 15). As such, the process development for GDC-0980 will be discussed in this chapter and some interesting route scouting work on GDC-0941 will be added.

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The Enabling Synthesis As the GDC-0980 project was advanced to the development stage, it was important to produce multikilogram quantities of API to support the GLP tox study and the clinical study. Due to the tight timeline, we elected to modify the medicinal chemistry route (Scheme 1) to enable the synthesis on large scale. Several issues were identified when we were attempting to scale up the medicinal chemistry synthesis. • • •

Step 1 was run neat at 200 °C. Long reaction time (3 days) was required for step 2. Microwave radiation (MW) was used in the Pd-catalyzed SuzukiMiyaura coupling reaction which was not practical on large scale.

Those issues required resolution prior to the scale-up of the synthesis. Synthesis of intermediate 10 relied on condensation of commercially available methyl 3-amino-4-methylthiophene 2-carboxylate 8 with urea at 200 ºC (Scheme 1). Although the reaction afforded a good yield of the desired product, there were concerns about the safety of this reaction on large scale. We noted that urea could solidify in the condenser and block the outlet of the NH3 gas formed in the reaction. To avoid this potential hazard, we sought milder conditions for the condensation reaction. When urea was replaced with potassium cyanate in aqueous AcOH, the reaction proceeded smoothly at room temperature to afford 10 in 84% yield (Scheme 2) (16). Pyrimidinone 10 was subsequently chlorinated with POCl3 to afford the dichloropyrimidine 12. The yield was significantly improved in the presence of N,N-dimethylaniline (0.70 equiv) and the reaction time was reduced from 3 d to 24 h. Subsequent site-selective SNAr reaction (17, 18) with morpholine in MeOH proceeded under mild conditions and gave thienopyrimidine 14 in 96% yield and 98% purity by HPLC. This process was then scaled up to >100 g scale in our lab and subsequently at contract manufacturing organizations (CMO) to >10 kg scale. The synthesis continued with the metalation/formylation of thienopyrimidine 14. Thus, 14 was treated with n-BuLi at –70 °C and the mixture was warmed up to –50 °C to achieve complete deprotonation, as ascertained by 1H NMR spectroscopic analysis of aliquots quenched into D2O. Subsequent formylation of the resulting organolithium compound with DMF at –70 °C, followed by quenching into cold aqueous HCl, afforded aldehyde 16 in 87% yield (Scheme 3). 242

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Scheme 2. Synthesis of the Thienopyrimidine 14

Scheme 3. First-Generation Route to GDC-0980 The reductive amination of aldehyde 16 with Boc-piperazine was performed in the presence of trimethyl orthoformate as the dehydrating agent. The resulting aryl-chloride 21 proceeded in a Suzuki-Miyaura coupling reaction with the boronate 22 to produce coupled-compound 23. A protecting group was needed for the primary amino group of boronate 22 to improve the solubility of the product 23 and avoid interference during the removal of the residual Pd. Since a Boc protecting group was already placed in aryl-chloride 21, it was convenient to use the same protecting group for boronate 22. The main task on the Suzuki-Miyaura coupling reaction was to replace the microwave conditions used in the medicinal chemistry route. We conducted a brief screening of reaction conditions and identified PdCl2(PPh3)2 and Na2CO3 as the suitable catalyst and base for the reaction. It was proved that 1.20 equiv of the boronate 22 and 0.01 equiv of the Pd catalyst were sufficient to drive the reaction to completion. 1,4-Dioxane was initially used as the solvent, however, 243

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1,4-dioxane is not a preferred solvent (19). Therefore, it was desirable to explore other solvents in the reaction. After a survey of several solvents, we identified 2-propanol as a suitable solvent for this reaction. The reaction proceeded faster in 2-propanol/water and was complete in 2–3 h. The use of 2-propanol/water also simplified the workup procedure as the crude product was isolated by simply filtering the slurry after further dilution with more water. When 1,4-dioxane was used as the solvent, a solvent swap from 1,4-dioxane to acetonitrile was needed before the filtration. The crude product typically contained 200–1000 ppm residual Pd and was then treated with Florisil®(2.0 wt) and Thio-Silica® (0.40 wt) in dichloromethane for 16 h to reduce residual Pd to < 20 ppm. The final step of the synthesis incorporated two chemical transformations; the deprotection of the two Boc groups and the amidation with (S)-lactic acid (Scheme 3). Deprotection was readily achieved without any issues by using HCl in ethanol. However, the amidation reaction turned out to be problematic. We explored several coupling agents including 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and an additive such as 1-hydroxy-1,2,3-benzotriazole hydrate (HOBt•H2O) (20). However, no desired product resulted when these coupling reagents were employed. It appeared that the combination of EDC•HCl and HOBt•H2O would work as the reaction proceeded well on lab scale. However, when we performed the reaction at kilogram scale, the reaction did not reach completion after 24 h as 10–20% of intermediate 24 was not consumed. Additional amounts of EDC•HCl and (S)-lactic acid were added to drive the reaction to completion. Another issue was the isolation and purification of the product. HOBt (0.89 wt%) and N,N-diisopropylethylamine (DIPEA) (0.1 wt%) appeared in the crude product, so an acid/base extraction procedure was performed to purge out those two residual reagents. Two major process impurities in the tox lot were identified as the des-lactate 25 at 1.80 % and di-lactate 26 at 3.70 %, (Figure 2). When the crude product was re-slurried in a mixture of methanol (7.5 vol) and THF (2.5 vol), the impurities were reduced to 0.53 % and 1.10 %, respectively, with the overall purity being improved to 97%. However, the overall yield of this step was only 61% due to loss of the product during the above purification process. We anticipated this loss could still be lessened once additional process research was conducted.

Figure 2. Process impurities in final step of first-generation route. 244 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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API Salt Selection, Polymorph Screening, and Particle Size The salt screening of GDC-0980 (measured pKa: 3.1 and 4.7) was conducted and several salts were identified, but all of them including the HCl and phosphate salt were hygroscopic. On the other hand, the free base was not hygroscopic and showed good physical properties. The dog PK data did not show any difference between the free base, the HCl salt, and the phosphate salt. Therefore, the free base was selected for bulk drug preparation. As for the polymorph screening, a total of 15 crystalline forms of GDC0980 have been identified. The initial observed form (anhydrous form A) was the predominant crystalline form identified in the screening and also the most stable one. Thus, anhydrous crystalline form A was selected for development. Investigations have also been made on the particle size and the morphology of GDC-0980. Both factors had significant impact on the flow property of the API, which was a critical parameter for making the powder in capsule (PIC) and the initial Phase I formulation. In the tox lot preparation, the crude product was triturated with 10 volumes of 3:1 methanol:THF. The resulting API had a poor flow property, presumably due to fine particle size (Table 2, entry 1). When the total solvent volume was increased to 25 and the ratio of MeOH/THF was changed from 3:1 to 1:1, the resulting particle size was significantly larger (Table 2, entry 2). Also, SEM confirmed the transformation from small particles to large ones. In Table 2, entry 3, the reference standard lot was prepared through a heating/ cooling crystallization, i.e., the crude GDC-0980 was dissolved in 100 volumes of 1:1 MeOH:THF and the resulting solution was gradually cooled to ambient temperature, followed by filtration to isolate the product with a purity of > 99.0% by HPLC. The reference standard lot showed a good flow property in the formulation process; however, the yield of the crystallization process was low (58%). To improve the yield, a significant amount of solvent (65 vol) was removed and the product was obtained in 86% yield while retaining good flow property (Table 2, entry 4) and acceptable purity. Therefore, this procedure (first-generation crystallization process) was selected for the first GMP production. The SEM results suggested agglomeration of GDC-0980 crystals. Therefore, the API was milled through a Fitzmill to break the agglomerates. While the first-generation synthesis was successfully employed in the preparation of the GLP tox batch and first GMP batch, it suffered from several shortcomings that were not ideal for large-scale implementation:

• • • •

The synthesis was not convergent and also required protection /deprotection steps. One of the starting materials, the boronate 22, was very expensive ($25000/kg). The yield of the final step was low. It was difficult to remove impurities 25 and 26 from the API generated in the final step. 245

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Table 2. Crystallization Process and the API Properties (PSD, SEM, and Flow)

Thus, a more convergent and efficient synthesis was needed to produce the large amount of API required for the advanced clinical studies. Second-Generation Route. Since the reductive amination and SuzukiMiyaura coupling reactions performed well in the first-generation synthesis, we elected to utilize both reactions for the second-generation synthesis. We envisioned that GDC-0980 could be assembled in a highly convergent manner via Suzuki-Miyaura coupling of unprotected boronic acid 27 and 2-chloro-thienopyrimidine 28 (Scheme 4). The thienopyrimidine core 28 would then be assembled through metalation and formylation of 14 followed by reductive amination of the resulting aldehyde 16 with piperazine 29 bearing the lactamide moiety. The key feature of the synthesis is to install the lactamide in the new starting material 29, thus avoiding the problematic late stage lactamide formation in the first-generation synthesis.

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Scheme 4. Retrosynthetic Analysis of the Second-Generation Route

Improved Formylation Process Although metalation of the thiophene ring could be performed with n-BuLi under cryogenic conditions, the instability of the resulting organolithium species precluded its use on large scale. Lithium trialkylmagnesiates have been used successfully in halogen-magnesium exchange (21–23), and for deprotonation of a variety of heterocycles including furans and thiophenes (24–26). Lithium triarylmagnesiates are generally more thermally stable than the corresponding organolithium species, and thus reactions can be performed under non-cryogenic conditions. To our delight, we found that use of n-Bu2i-PrMgLi allowed for deprotonation and formylation under non-cryogenic conditions (–10 °C) and provided aldehyde 16 in 96% yield (Scheme 5, R = CH3). Both the resulting lithium triarylmagnesiate 31 and the components of the reaction mixture (after addition of DMF) were very stable at reaction temperature between −10 °C and –5 °C for an extended time (>15 h). In an optimized procedure, i-PrMgCl and n-BuLi were added sequentially to a solution of 14 in THF at –10 °C. This operationally simple process proved easy to perform on 50 kg scale and obviated the need for a separate vessel to prepare n-Bu3MgLi as reported previously (26). It is noteworthy that the presence of the adjacent methyl group significantly improved the stability of the lithium triarylmagnesiate 31 and thus led to the excellent yield of aldehyde 16. Without the adjacent methyl group (Scheme 5, R = H), the resulting lithium triarylmagnesiate 30 and the components of the reaction mixture (after addition of DMF) were stable at −5 °C for > 6 h, and the desired product 15 was produced in 87% yield. 247

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Scheme 5. Non-Cryogenic Conditions for Metalation and Formylation The aldehyde product 16 was isolated by filtration; however, the filtration of the crude mixture was slow. We found that removal of THF was beneficial to the filtration. The filtration was also significantly improved through an ripening process. After ripening for 1–2 h at 50 °C, the filtration was about 10× faster. This is the result of a crystalline form change confirmed by DSC and XPRD. We also noticed that the ripening process afforded larger crystals as indicated by the microscopy data. Under the optimal conditions, the desired product was reproducibly produced in 94‒98% yield. Reductive Amination As previously mentioned, the lactamide moiety was incorporated into the new starting material 29 (Scheme 4); however, piperazine 29 was an oil, so salt formation was required to obtain the material in a preferable solid state. Initially, the HCl salt of 29 was tested in the reaction, but a significant amount of the starting material 16 was not consumed, presumably due to the fact that the HCl salt was deliquescent under normal lab conditions and water was thus brought into the reaction. After screening a variety of acids, we found that the corresponding oxalic acid salt 32 (Scheme 6), was less hygroscopic and thus easier to handle, and it performed well in the reaction (27). Therefore, piperazine lactamide oxalate 32 was chosen as the starting material for the reductive amination. Initially, Na(OAc)3BH was employed as the reducing reagent (Scheme 6). Several solvents including dichloromethane, acetonitrile, methanol, and tetrahydrofuran were examined in the reaction. We observed the best conversion and the least amount of the alcohol impurity 33 when acetonitrile was used as the solvent. A brief survey of bases, which was needed to free base the oxalate 32 in situ, identified sodium acetate as optimal (28). Addition of acetic acid (0.50 equiv) was critical in order to suppress the formation of alcohol impurity 33. This impurity 33 was further controlled by using 1.50 equiv of 32 and adding the Na(OAc)3BH in multiple portions. A dehydrating reagent was needed for the iminium ion formation in the reaction. When HC(OCH3)3 was employed, the outcome of the reaction was not reproducible. In some runs, we observed significant amounts of the unconsumed starting material 16 and alcohol impurity 33. We eventually resolved this problem by the use of molecular sieves, with the reaction proceeding consistently well when powdered 3Å molecular sieves were employed. Use of 100 wt% of sieves was sufficient to suppress the formation of alcohol impurity 33. 248 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 6. Reductive Amination of the Second-Generation Synthesis

Besides the alcohol impurity 33, other impurities were also observed in the reaction mixture (Figure 3). Considerable efforts were made to control these and the downstream impurities. The alcohol impurity 33 would be purged out in the work-up and its derivative in the downstream process would be purged out in the following step. As for other impurities, it was also critical to control them in this step since the impurities derived from these impurities in the following steps would be difficult to remove.

Figure 3. Impurities of the reductive amination. 249 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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It was anticipated that all three impurities 34-36 would be generated from the corresponding impurities in the starting material 32, such as piperazine 37 and acetyl piperazine 38 (Figure 4). Therefore, we set the specification for the starting material 32 with a limit of ≤ 0.20% for each of these two impurities.

Figure 4. Impurities in starting material 32.

Another source for the impurities was the hydrolysis of the product 28 to afford the piperazine impurity 34, which would be further converted to the acetyl impurity 35 under the reaction conditions in the presence of acetic acid. Therefore, the reaction conditions and the workup process were extensively studied. We found out that the amounts of the piperazine impurity 34 and the acetyl piperazine impurity 35 were significantly increased when the reaction mixture was aged at 55–60 °C. It was particularly striking that the amount of the piperazine impurity 34 was increased to 3.38% after the mixture was aged at 60 °C for 23 h. Therefore, the reaction temperature was lowered from 55–60 °C to 35–45 °C so as to suppress 34. We also discovered that the amount of the piperazine impurity 34 was increased 2x from 1.73% to 3.36% at pH 0.4 after 24 h. As such, the acidic aqueous workup should be operated at a higher pH 1.2 while maintaining the temperature at 0–10 °C. The optimal procedure using Na(OAc)3BH has been successfully scaled up to 50 kg; however, it was not desirable to use molecular sieves and add Na(OAc)3BH in multiple portions as solid via a special solid dosing unit or as a slurry. To address these shortcomings, we explored other reducing agents such as sodium borohydride, pyridine•BH3 and 2-picoline•BH3 (29, 30). A significant amount of the alcohol impurity 33 (7–10%) formed when sodium borohydride was used. On the other hand, the reaction with pyridine•BH3 or 2-picoline•BH3 proceeded smoothly and fewer impurities were observed, although the level of alcohol impurity 33 was slightly higher than that when Na(OAc)3BH was used. This prompted us to further investigate the reductive amination reaction using 2-picoline•BH3 (31). We examined the reaction with 2-picoline•BH3 at different temperatures, and in the presence or absence of the dehydrating reagent HC(OCH3)3 (Scheme 7 and Table 3): 250

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•

• •

•

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•

We were able to achieve the best conversion and the lowest % of the alcohol impurity 33 when the reaction was run in the presence of 10 equiv of HC(OCH3)3 at 50 °C (entry 3). We demonstrated that the addition of 2-picoline•BH3 in 3 portions was effective. We explored other solvents such as ACN, THF, and EtOH in the reaction, but higher amounts of the alcohol impurity 33 were observed (entries 4-6). We determined that 1.20 equiv of 2-picoline•BH3 and 10 equiv of HC(OCH3)3 would be needed to drive the reaction to completion (entries 3, 7, 8 and 10). We were able to charge 2-picoline•BH3 as solutions in either methanol or THF and both reactions performed well (entries 9-10).

Since 2-picoline•BH3 in THF is commercially available, it was selected for large scale production.

Scheme 7. Reductive Amination with 2-Picoline•BH3

2-Picoline•BH3 has several advantages over Na(OAc)3BH: • •

•

The reaction proceeded well in the presence of HC(OCH3)3, so no molecular sieves were needed. 2-Picoline•BH3 could be added as a solution in THF in a continuous mode. As a result, the time required for the addition of the reducing reagent was significantly reduced. The reaction was cleaner and fewer impurities were generated, presumably due to the improved stability of 28 in the presence of 2-picoline.

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Table 3. Reductive Amination of 16 and 32 with 2-Picoline•BH3a)

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Entry

Temp (°C)

2-Picoline•BH3 (equiv)

Solvent

% of the reaction mixture by HPLC 28

33

16

1

rt

1.50 (solid)

MeOH

84.0

9.6

6.4

2

rt

1.50 (solid)

MeOH

93.0

7.0

0.0

3

50

1.50 (solid)

MeOH

95.1

4.9

0.0

4

50

1.50 (solid)

ACN

84.3

13.6

2.1

5

50

1.50 (solid)

THF

18.6

72.7

8.7

6

50

1.50 (solid)

EtOH

63.0

36.8

0.2

7

50

1.25 (solid)

MeOH

94.5

5.0

0.5

8

50

1.00 (solid)

MeOH

93.8

5.6

0.6

9

50

1.25 (13wt% solution in MeOH)

MeOH

94.9

4.4

0.6

10

50

1.20 (30wt% solution in THF)

MeOH

96.0

3.5

0.5

All the reactions were run with compound 16 (10.0 g, 1.00 equiv) and compound 32 (12.5 g, 1.50 equiv) in 190 mL of solvent for 20 h. The reaction of entry 1 was run without HC(OCH3)3. All other reactions were performed in the presence of 10 equiv of HC(OCH3)3. a)

The reductive amination reaction using 2-picoline•BH3 has been successfully scaled up to 8.75 kg with a reproducible yield of 79‒86% and >99.0% purity by HPLC. Synthesis of Oxalate 32. The enabling synthesis of oxalate 32, illustrated in Scheme 8, employed five steps with an overall yield of 40%. Several steps were utilized for the protection and deprotection of both the hydroxyl group and piperazine. As the project was advancing to late stage process development, we developed a more concise and efficient synthesis. N-Benzylpiperazine Route We initially envisioned a one-pot amidation-deprotection process starting from N-benzylpiperazine with (S)-ethyl lactate as solvent at elevated temperatures (70–100 °C) to produce benzylpiperazine lactate 43 (Scheme 9). However, these reactions proved difficult to reach completion with up to 15% by HPLC of N-benzylpiperazine remaining unconsumed. (S)-Methyl lactate was also investigated, as we reasoned the lower boiling point of the methanol by-product and its distillation could aid the reaction conversion. The reaction also failed to progress to completion and therefore offered no advantages over the cheaper (S)-ethyl lactate. A further issue with these reactions was the formation of the ester impurity 44 in ~ 13% where the hydroxyl group of the desired product was esterified in the presence of the excess (S)-ethyl lactate. 252

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Scheme 8. The Enabling Synthesis of Oxalate 32

Scheme 9. Amidation of (S)-ethyl Lactate and N-Benzylpiperazine Next, in an attempt to eliminate the formation of the ester impurity 44, we decided to concentrate our efforts on reaction conditions that could be carried out at ambient temperature. The amidation between amines and (S)-ethyl lactate in the presence of an alkoxide base has previously been reported (32–34). Thus, we explored this method in the synthesis of oxalate 32 (Scheme 10).

Scheme 10. Benzylpiperazine route to oxalate 32 253 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Several alkoxide bases were examined in the amidation reaction (Table 4). We selected NaOMe (25wt% in MeOH) (entry 1) as the base since significant erosion of enantiomeric ratios (er) resulted when other bases were employed (entries 2-4).

Table 4. Effect of Alkoxide Bases on Enantiomeric Ratio of the Amidation Reactiona)

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Entry

Base (0.15 equiv.)

(S)-Ethyl lactate (equiv)

HPLC assay yield (%)

Enantiomeric ratio (er)

1

NaOMe 25wt% solution in MeOH

1.03

34

99.3:0.7

2

t-BuONa (solid)

3.00

90

85.0:15.0

3

t-PentONa 1.4 M solution in THF

1.03

61

96.7:3.3

4

NaOEt 21wt% solution in EtOH

1.03

37

98.9:1.1

The reaction was run with N-benzylpiperazine (10.0 g, 1.00 equiv), (S)-ethyl lactate (20.1 g, 3.00 equiv) and base (0.15 equiv) at ambient temperature for 23 h.

a)

Table 5. Optimization of N-Benzylpiperazine Reactiona) Entry

NaOMe (25wt% in MeOH) (equiv)

(S)-Ethyl lactate (equiv)

Assay yield of 43

Remaining N-benzylpiperazine (wt%)

Enantiomeric ratio

1

0.25

3.00

73

9.0

99.0:1.0

2

0.50

3.00

90

2.0

99.1:0.9

3

0.75

3.00

91

0.9

99.2:0.8

4

0.75

2.00

64

1.4

99.1:0.9

5

0.75

2.50

70

1.5

99.2:0.8

6

0.75

3.50

82

0.9

99.2:0.8

7

0.75

4.00

84

1.0

99.2:0.8

The reaction was run with N-benzylpiperazine (10.0 g, 1.00 equiv), (S)-ethyl lactate (2.50–4.00 equiv) and base (0.25–0.75 equiv) at ambient temperature for 20 h.

a)

254 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Further optimization of the reaction conditions was focused on the amount of base and (S)-ethyl lactate (Table 5). 0.75 Equivalents of the base was required to drive the reaction to completion and control the residual N-benzyl piperazine at < 1.0% (entries 1−3). On the other hand, excess of (S)-ethyl lactate (3.00 equiv) was needed to fully consume the N-benzyl piperazine (entries 3-7). Thus, the reaction conditions selected were N-benzylpiperazine (limiting reagent), (S)-ethyl lactate (3.00 equiv) and sodium methoxide (25 wt% in methanol) (0.75 equiv) at ambient temperature (entry 3). Monitoring the reaction progress hourly by HPLC showed that >7 h of reaction time was required. The reaction was complete at ambient temperature in ~ 20 h, affording less than 1% of residual N-benzylpiperazine and an enantiomeric ratio of 99.2:0.8. The effect of temperature on the reaction was also investigated. At both 40 °C and 70 °C, the reaction did progress at a faster rate, achieving 70% when 3.0 equiv of water was added as the result of the conversion of the bis-lactamide 45 to the desired product 29 presumably by hydrolysis (35). Our next task was to isolate the product from the reaction mixture. We thus needed to purge out the residual sodium salt, present as sodium ethoxide and /or sodium methoxide, residual piperazine and (S)-ethyl lactate, as well as the by-products, the bis-lactamide 45 and lactic acid, the latter presumably being generated from the hydrolysis of (S)-ethyl lactate and the bis-lactamide 45. Since piperazine 29 is soluble in water, an aqueous work up was not an option to remove the residual salts. Our initial attempt was to use an Amberlite IRC-748 resin treatment to remove sodium salts. However, we observed significant loss of product on the resin (~ 20%), even when using the minimum amount of resin (0.80 equiv) that was required. We next investigated formation of a sodium salt as a way to remove residual sodium. We chose oxalic acid since the final product 32 is an oxalate. After exploring different amounts of oxalic acid, we discovered that 0.25 equiv of oxalic acid was able to generate the di-sodium oxalate salt which is insoluble in ethanol and could therefore be readily removed by filtration (36). To the resulting filtrate, additional oxalic acid was then added to adjust the pH to 7–7.5. The residual piperazine was precipitated out as the oxalate salt and subsequently removed through a filtration while the product 29 remained in the mother liquor. Direct treatment of the filtrate with excess oxalic acid (1.12 equiv) afforded the desired oxalate 32 in 59% isolated yield and > 99% purity. The piperazine route to oxalate 32 is significantly more efficient than the enabling synthesis with a 55% reduction in process mass intensity (PMI) (37). We were also able to achieve a 53% reduction in total solvent volume used in the process. More significantly, the undesirable solvent, dichloromethane, heavily used in three of the five steps of the enabling synthesis, was replaced with preferred solvents and a small amount of a usable solvent, THF (4.3%) being used (38). 257

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Another improvement for the piperazine route is a 41% increase in atom economy as the result of the elimination of the non-value adding steps (installation and removal of the protecting group), and thus producing oxalate 32 in a single chemical step and a protecting group-free synthesis (39). Overall, the piperazine route is more concise, cleaner and safer, and ready for scale-up.

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Aminoalkylation Approach The reductive amination reaction performed well; however, there were concerns about the alcohol impurity 33 which was carried in the downstream chemistry resulting in formation of additional impurities that were difficult to remove. We therefore explored an alternative route involving aminoalkylation to avoid the formation of the alcohol impurity 33. As shown in Scheme 13, we envisioned that the aminoalkylation could be performed by direct addition of lithium triarylmagnesiate 31 to iminium salt 46 to produce intermediate 18. The same strategy could potentially be employed for the synthesis of 17, an intermediate in the synthesis of GDC-0941 (Scheme 13).

Scheme 13. Synthesis of 17 and 18 by Aminoalkylation

Our investigation on the aminoalkylation commenced with the preparation of the iminium salt 47. As shown in Scheme 14, the iminium salt 47 was generated from the aminal 49 or aminol ether 50 (40, 41). The resulting iminium salt was then subjected to reaction with lithium triarylmagnesiate 30 to afford the desired product 17. However, a significant amount of the starting material 48 was observed in the crude product, possibly due to the impurities present in the iminium salt (42). 258 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 14. Synthesis of Intermediate 17 via the Iminium Salt In another approach the iminium salt was also generated in situ by treating the aminol ether 50 with a Lewis acid (Scheme 15), followed by addition of the lithium triarylmagnesiate 30 which was generated using non-cryogenic conditions. We identified ZnCl2 as the preferred Lewis acid with the desired product being obtained in ~ 80% yield.

Scheme 15. Synthesis of 17 from Iminium Salt Generated in Situ from Aminol Ether To further improve the aminoalkylation process, our efforts were then turned towards the benzotriazole substrates that have been widely used in the aminoalkylation reactions (43–45). Treatment of 48 with benzotriazole, paraformadehyde and MeOH in the presence of KHCO3 afforded benzotriazolyl-piperazine 51 in 90% yield after isolation by simple filtration 259 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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(Scheme 16). Unlike the aminol ether 50, compound 51 is not hygroscopic and can be isolated as a bench-stable solid. Treatment of compound 51 with ZnCl2 followed by addition to a solution of lithium triarylmagnesiate 30 afforded the desired product 17 in 93% yield.

Scheme 16. Aminoalkylation via Benzotriazolyl-Piperazine 51

This route achieved a slightly higher yield than the reductive amination route (Scheme 17) and did not generate the corresponding alcohol impurity 52. The synthesis has been demonstrated on kilogram scale. Although a large excess of ZnCl2 (4.00 equiv) was needed, the aminoalkylation route offered a complementary process to the reductive amination route which has been scaled up to > 35 kg.

Scheme 17. Synthesis of 17 by the Reductive Amination Route 260 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

As for the synthesis of GDC-0980, we also explored the aminoalkylation strategy for the synthesis of intermediate 28 using the optimal conditions for the synthesis of intermediate 17 shown in Scheme 16; however, the strategy did not work for intermediate 18 as the reaction suffered with low conversion, presumably due to presence of the hydroxyl group of piperazine lactate 32. Therefore, the reductive amination process was selected for the synthesis of intermediate 28 (Scheme 7).

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Suzuki-Miyaura Cross-Coupling With intermediate 28 in hand, our attention was then turned to the final carboncarbon bond-forming step of the synthesis of GDC-0980, the Suzuki-Miyaura cross-coupling reaction (Scheme 18). We used the boronic acid 27 to replace the expensive boronate 22 employed in the first-generation route. Unlike the firstgeneration synthesis, no protecting group was employed for boronic acid 27 which was prepared directly from 2-amino-5-bromopyrimidine in a one-step synthesis (46).

Scheme 18. Suzuki-Miyaura Cross-Coupling Reaction of the Second-Generation Synthesis

After a brief screening of solvents (2-propanol, 2-propanol/water, acetonitrile and 1-propanol), we identified 1-propanol as the reaction solvent due to the better solubility of the product GDC-0980 and the solvent’s higher boiling point which allowed us to run the reaction at a higher temperature. We also evaluated a variety of bases in the reaction when the corresponding boronate ester 22 was used. We found that a significant amount of amide hydrolysis byproduct des-lactate 25 (7–17%, Figure 2) was generated when either Na2CO3 or Cs2CO3 was used. To our delight, the use of K3PO4 as the base has significantly reduced the formation of the des-lactate 25 in the reaction with the boronate ester 22 or when we later made the switch to the corresponding boronic acid 27. Further optimization showed that only 1.18 equiv of boronic acid 27 were sufficient to achieve complete conversion of 28 to GDC-0980. 261 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Expensive scavengers were employed in the first-generation route for the removal of the residual Pd, which would not be practical on large scale. To resolve this issue, we first reduced the Pd catalyst loading from 0.25 mol% to 0.15 mol% as the result of a faster reaction at higher temperature with 1-propanol as the solvent. Further, we eliminated the use of scavengers by filtering the crude GDC-0980 solution in 1-propanol and water through an activated carbon cartridge, and the resulting solution was concentrated followed by filtration to afford the crude product which contained 20-40 ppm of the residual Pd. The crude product contained three major impurities: • • •

des-lactate impurity 25 (Figure 2) in ~ 0.60%. homo-coupling impurity 53 (Figure 5) in ~ 0.65%. alcohol impurity 54 (Figure 5) in ~ 0.25%, stemming from alcohol impurity 33 in the precursor.

The levels of those impurities were reduced to < 0.20% after the crude product went through a recrystallization process from 25:75 w/w mixture of 1-propanol and water, and the product was produced on ~ 10 kg scale reproducibly in 79‒83% yield.

Figure 5. Homo-coupling Impurity 53 and Alcohol Impurity 54.

API Recrystallization The recrystallization process of the first-generation synthesis employed large volumes of methanol and THF (total 100 vol) in order to dissolve the API, and a distillation was subsequently required to remove the excess solvent to improve the yield. To develop a more efficient recrystallization process, we started with the investigation of the solubility of GDC-0980. We found that GDC-0980 has relatively low solubility (< 1 mg/mL) in organic solvents; however, the API did show good solubility in a mixture of water and 1-propanol, with maximum solubility in 40:60 w/w (Figure 6). The solubility and super-saturation curves in this solvent composition are displayed in Figure 7 262 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 6. Solubility of various concentrations of GDC-0980 in water/1-propanol.

Figure 7. Clear and cloud curves of GDC-0980 in water/1-propanol (40:60 w/w). 263 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The boiling point of the water/1-propanol mixture (40:60 w/w) was 87–88 °C. As shown in Figure 7, the maximum achievable solubility is ~14 wt% under atmospheric pressure. For practical purposes and to avoid the possible precipitation of API during the polish filtration, we set the concentration for dissolution at a slightly lower level (11.7wt%), affording a final concentration of 10 wt% after the polish filtration and rinses. The resulting solution was then cooled slowly to ‒10 °C while spontaneous nucleation started at a temperature between 60 °C and 65 °C. The solid product was collected by filtration and dried to afford the API. The implementation of this new API recrystallization process has consistently produced the desired crystalline form and particle size distribution as well as it further depleted the levels of residual Pd and process impurities. The purity of the API was >99.0% by HPLC and all identified impurities were within the specifications and all unidentified impurities < 0.15% (Scheme 19). The residual Pd was < 10 ppm. The recrystallization process has been scaled up reproducibly in multiple batches (13.9 kg to 15.7 kg) in 89 ̶996% yields.

Scheme 19. API Crystallization

Summary Two clinical compounds, GDC-0941(pictilisib) and GDC-0980 (apitolisib), have been discovered. While both compounds inhibit the Class I PI3Ks, GDC-0980 also inhibits the kinase mTOR. Chemical process development has been conducted to support both compounds in the clinical study. For GDC-0980, the first-generation route was employed to produce API for the GLP tox and the initial clinical study while the second-generation route (Scheme 20) was developed as the project advanced to the late stage of the clinical study. The second-generation route was convergent, efficient and robust. The metalation was performed under non-cryogenic conditions via triarylmagnesiate intermediates. 2-Picoline•BH3 was employed to replace Na(OAc)3BH in the reductive amination and to eliminate the use of molecular sieves. A concise one-step synthesis was developed for the piperazine lactamide starting material through the selective mono-amidation of piperazine with (S)-ethyl lactate. The second-generation route has been demonstrated on >10 kg scale affording GDC-0980 in 59% overall yield in four steps and >99.0% purity by HPLC. 264

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Scheme 20. The Second-Generation Route to GDC-0980

Employing a similar synthetic strategy, we have developed a practical synthesis for GDC-0941 (Scheme 21). Non-cryogenic conditions were employed in the formylation of 13 via a triarylmagnesiate intermediate. The synthesis of the key intermediate 17 was also achieved through an aminoalkylation approach, a complementary process to the reductive amination. The THP deprotection and salt formation were combined in one operation in the final step to afford GDC-0941 in >99.0% purity by HPLC and 54% overall yield over four steps.

265 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 21. A Practical Synthesis of GDC-0941

References 1. 2. 3.

4.

Cantley, L. C. The Phosphoinositide 3-kinase Pathway. Science 2002, 296, 1655–1657. Bauer, T. M.; Patel, M. R.; Infante, J. R. Targeting PI3 Kinase in Cancer. Pharmacol. Ther. 2015, 146, 53–60. Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.; Box, G.; Chuckowree, I. S.; Clarke, P. A.; Depledge, P.; Eccles, S. A.; Friedman, L. S.; Hayes, A.; Hancox, T. C.; Kugendradas, A.; Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J.; Patel, S.; Pergl-Wilson, G. H.; Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.; Sohal, S.; Ultsch, M. H.; Valenti, M.; Wallweber, H. J. A.; Wan, N. C.; Wiesmann, C.; Workman, P.; Zhyvoloup, P.; Zvelebil, M. J.; Shuttleworth, S. J. The Identification of 2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a Potent, Selective, Orally Bioavailable Inhibitor of Class I PI3 Kinase for the Treatment of Cancer. J. Med. Chem. 2008, 51, 5522–5532. Sutherlin, D. P.; Bao, L.; Berry, M.; Castanedo, G.; Chuckowree, I.; Dotson, J.; Folks, A.; Friedman, L.; Goldsmith, R.; Gunzner, J.; Heffron, T.; Lesnick, J.; Lewis, C.; Mathieu, S.; Murray, J.; Nonomiya, J.; Pang, J.; 266

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Pegg, N.; Prior, W. W.; Rouge, L.; Salphati, L.; Sampath, D.; Tian, Q.; Tsui, V.; Wan, N. C.; Wang, S.; Wei, B.; Wiesmann, C.; Wu, P.; Zhu, B.-Y.; Olivero, A. Discovery of a Potent, Selective, and Orally Available Class I Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Kinase Inhibitor (GDC-0980) for the Treatment of Cancer. J. Med. Chem. 2011, 54, 7579–7587. 5. Wallin, J. J.; Guan, J.; Prior, W. W.; Edgar, K. A.; Kassees, R.; Sampath, D.; Belvin, M.; Friedman, L. S. Nuclear Phospho-Akt Increase predicts Synergy of PI3K Inhibition and Doxorubicin in Breast and Ovarian Cancer. Sci. Transl. Med. 2010, 2, 48ra66. 6. Salphati, L.; Wong, H.; Belvin, M.; Bradford, D.; Edgar, K. A.; Prior, W. W.; Sampath, D.; Wallin, J. J. Pharmacokinetic-Pharmacodynamic Modeling of Tumor Growth Inhibition and Biomarker Modulation by the Novel Phosphatidylinositol 3-kinase Inhibitor GDC-0941. Drug Metab. Dispos. 2010, 38, 1436–1442. 7. Sarker, D.; Ang, J. E.; Baird, R.; Kristeleit, R.; Shah, K.; Moreno, V.; Clarke, P. A.; Raynaud, F. I.; Levy, G.; Ware, J. A.; Mazina, K.; Lin, R.; Wu, J.; Fredrickson, J.; Spoerke, J. M.; Lackner, M. R.; Yan, Y; Friedman, L. S.; Kaye, S. B.; Derynck, M. K.; Workman, P.; de Bono, J. S. First-in-Human Phase I Study of Pictilisib (GDC-0941), a Potent Pan-class I Phosphatidylinositol-3-kinase (PI3K) Inhibitor, in Patients with Advanced Solid Tumors. Clin. Cancer Res. 2015, 21, 77–86. 8. Sutherlin, D. P.; Sampath, D.; Berry, M.; Castanedo, G.; Chang, Z.; Chuckowree, I.; Dotson, J.; Folkes, A.; Friedman, L.; Goldsmith, R.; Heffron, T.; Lee, L.; Lesnick, J.; Lewis, C.; Mathieu, S.; Nonomiya, J.; Olivero, A.; Pang, J.; Prior, W. W.; Salphati, L.; Sideris, S.; Tian, Q.; Tsui, V.; Wan, N. C.; Wang, S.; Wiesmann, C.; Wong, S.; and Zhu, B.-Y. Discovery of (Thienopyrimidin-2-yl)aminopyrimidines as Potent, Selective, and Orally Available Pan-PI3-Kinase and Dual Pan-PI3-Kinase/mTOR Inhibitors for the Treatment of Cancer. J. Med. Chem. 2010, 53, 1086–1097. 9. Heffron, T. P.; Berry, M.; Castanedo, G.; Chang, C.; Chuckowree, I.; Dotson, J.; Folkes, A.; Gunzner, J.; Lesnick, J. D.; Lewis, C.; Mathieu, S.; Nonomiya, J.; Olivero, A.; Pang, J.; Peterson, D.; Salphati, L.; Sampath, D.; Sideris, S.; Sutherlin, D. P.; Tsui, V.; Wan, N. C.; Wang, S.; Wong, S.; Zhu, B. Y. Identification of GNE-477, a Potent and Efficacious Dual PI3K/ mTOR Inhibitor. Bioorg. Med. Chem. Lett. 2010, 20, 2408–2411. 10. Wallin, J. J.; Edgar, K. A.; Guan, J.; Berry, M.; Prior, W. W.; Lee, L.; Lesnick, J. D.; Lewis, C.; Nonomiya, J.; Pang, J.; Salphati, L.; Olivero, A. G.; Sutherlin, D. P.; O’Brien, C.; Spoerke, J. M.; Patel, S.; Lensun, L.; Kassees, R.; Ross, L.; Lackner, M. R.; Sampath, D.; Belvin, M.; Friedman, L. S. GDC-0980 is a Novel Class I PI3K/mTOR Kinase Inhibitor with Robust Activity in Cancer Models driven by the PI3K Pathway. Mol. Cancer Ther. 2011, 10, 2426–2436. 11. Salphati, L.; Pang, J.; Plise, E. G.; Lee, L. B.; Olivero, A. G.; Prior, W. W.; Sampath, D.; Wong, S.; Zhang, X. Preclinical Assessment of the Absorption and Disposition of the Phosphatidylinositol 3-Kinase/Mammalian Target of 267

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

12.

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch008

13.

14.

15.

16.

17. 18. 19.

20. 21. 22.

23. 24.

Rapamycin Inhibitor GDC-0980 and Prediction of its Pharmacokinetics and Efficacy in Human. Drug Metab. Dispos. 2012, 40, 1785–1796. Dolly, S.; Wagner, A. J.; Bendell, J. C.; Kindler, H. L.; Krug, L. M.; Seiwert, T. Y.; Zauderer, M. G.; Lolkema, M.; Apt, D.; Yeh, R. F.; Fredrickson, J.; Spoerke, J. M.; Koeppen, H.; Ware, J. A.; Lauchle, J. O.; Burris, H. A.; de Bono, J. S. Phase I Study of Apitolisib (GDC-0980), Dual Phosphatidylinositol-3-Kinase and Mammalian Target of Rapamycin Kinase Inhibitor, in Patients with Advanced Solid Tumors. Clin. Cancer Res. 2016 DOI:10.1158/1078-0432.CCR-15-2225. Powles, T.; Lackner, M. R.; Oudard, S.; Escudier, B.; Ralph, C.; Brown, J. E.; Hawkins, R. E.; Castellano, D.; Rini, B. I.; Staehler, M. D.; Ravaud, A.; Lin, W.; O’Keeffe, B.; Wang, Y.; Lu, S.; Spoerke, J. M.; Huw, L. Y.; Byrtek, M.; Zhu, R.; Ware, J. A.; Motzer, R. J. Randomized Open-Label Phase II Trial of Apitolisib (GDC-0980), a Novel Inhibitor of the PI3K/Mammalian Target of Rapamycin Pathway, Versus Everolimus in Patients With Metastatic Renal Cell Carcinoma. J. Clin. Oncol. 2016 DOI:10.1200/JCO.2015.64.8808. Tian, Q.; Cheng, Z.; Yajima, H. M.; Savage, S. J.; Green, K.; Humphries, T.; Reynolds, M. E.; Babu, S.; Gosselin, F.; Askin, D.; Kurimoto, I.; Hirata, N.; Iwasaki, M.; Shimasaki, Y.; Miki, T. Org. Process Res. Dev. 2013, 17, 97–107. Tian, Q.; Hoffmann, U.; Humphries, T.; Cheng, Z.; Hidber, P.; Yajima, H.; Guillemot-Plass, M.; Li, J.; Bromberger, U.; Babu, S.; Askin, D.; Gosselin, F. Org. Process Res. Dev. 2015, 19, 416–426. Bandurco, V. T.; Schwender, C. F.; Bell, S. C.; Combs, D. W.; Kanojia, R. M.; Levine, S. D.; Mulvey, D. M.; Appollina, M. A.; Reed, M. S.; Malloy, E. A.; Falotico, R.; Moore, J. B.; Tobia, A. J. J. Med. Chem. 1987, 30, 1421–1426. Fairlamb, I. J. S. Chem. Soc. Rev. 2007, 36, 1036–1045. Large, J. M.; Clarke, M.; Williamson, D. M.; Collins, I. Synlett 2006, 861–864. Guidance for Industry Q3C-Tables and List , Feb 2012, Revision 2; http://www.fda.gov/downloads/Drugs/ GuidanceComplianceRegulatoryInformation/Guidances/ucm073395.pdf. Valeur, E.; Bradley, M. Chem. Soc. Rev. 2009, 38, 606–631. Gallou, F.; Haenggi, R.; Hirt, H.; Marterer, W.; Schaefer, F.; SeegerWeibel, M. Tetrahedron Lett. 2008, 49, 5024–5027. Mase, T.; Houpis, I.; Akao, A.; Dorziotis, I.; Emerson, K.; Hoang, T.; Iida, T.; Itoh, T.; Kamei, K.; Kato, S.; Kato, Y.; Kawasaki, M.; Lang, F.; Lee, J.; Lynch, J.; Maligres, P.; Molina, A.; Nemoto, T.; Okada, S.; Reamer, R.; Tschaen, D.; Wada, T.; Zewge, D.; Volante, R. P.; Reider, P. J.; Tomimoto, K. J. Org. Chem. 2001, 66, 6775–6786. Dolman, S. J.; Gosselin, F.; O’Shea, P. D.; Davies, I. W. Tetrahedron 2006, 62, 5092–5098. Bayh, O.; Awad, H.; Mongin, F.; Hoarau, C.; Bischoff, L.; Trécourt, F.; Quéguiner, G.; Marsais, F.; Blanco, F.; Abarca, B.; Ballesteros, R. J. Org. Chem. 2005, 70, 5190–5196. 268

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25. Mongin, F.; Bucher, A.; Baureau, J.; Bayh, O.; Awad, H.; Trécourt, F. Tetrahedron Lett. 2005, 46, 7989–7992. 26. Bayh, O.; Awad, H.; Mongin, F.; Hoarau, C.; Trécourt, F.; Quéguiner, G.; Marsais, F.; Blanco, F.; Abarca, B.; Ballesteros, R. Tetrahedron 2005, 61, 4779–4784. 27. The water content remained stable (1–3%) when the oxalic salt 32 was exposed to the atmosphere for a short period of time (2–3 h). 28. When other bases (potassium acetate, potassium carbonate and N,Ndiisopropylethylamine) were employed, a greater amount of alcohol impurity 33 (27–48% by HPLC) was generated. 29. Sato, S.; Sakamoto, T.; Miyazawa, T.; Kikugawa, Y. Tetrahedron 2004, 60, 7899–7906. 30. Wu, P-L.; Chen, H.; Line, M. J. Org. Chem. 1997, 62, 1532–1533. 31. Pyridine•BH3 was not further investigated due to its short, 6-month shelf life. 2-Picoline•BH3 is more stable and was deemed suitable for large-scale production.29 32. Ohshima, T.; Hayashi, Y.; Agura, K.; Fujii, Y.; Yoshiyamab, A.; Mashima, K. Chem. Commun. 2012, 48, 5434–5436. 33. Pesti, J.; Chen, C-K.; Spangler, L.; Delmonte, A. J.; Benoit, S.; Berglund, D.; Bien, J.; Brodfuehrer, P.; Chan, Y.; Corbett, E.; Costello, C.; DeMena, P.; Discordia, R. P.; Doubleday, W.; Gao, Z.; Gingras, S.; Grosso, J.; Haas, O.; Kacsur, D.; Lai, C.; Leung, S.; Miller, M.; Muslehiddinoglu, J.; Nguyen, N.; Qiu, J.; Olzog, M.; Reiff, E.; Thoraval, D.; Totleben, M.; Vanyo, D.; Vemishetti, P.; Wasylak, J.; Wei, C. Org. Process Res. Dev. 2009, 13, 716–728. 34. Tasaka, A.; Tamura, N.; Matsushita, Y.; Teranishi, K.; Hayashi, R.; Okonogi, K.; Itoh, K. Chem. Pharm. Bull. 1993, 41, 1035–1042. 35. A key development was the observation of a significant change in the NMR spectra of the crude mixture in D2O after 18 h at ambient temperature. With careful examination of the NMR spectra, we discovered the conversion of the bis-lactamide 45 to the desired product 29 presumably by hydrolysis. We therefore envisioned that we could improve the yield of 29 through the hydrolysis of the bis-lactamide 45. 36. Patnaik, P. Handbook of Inorganic Chemicals; McGraw-Hill: New York, 2003; p 873. 37. Anastas, P.; Warner, J. Green Chemistry: Theory and Practice; Oxford University Press: New York, 1998. 38. Jimenez-Gonzalez, C.; Poechlauer, P.; Broxterman, Q. B.; Yang, B. S.; am Ende, D.; Baird, J.; Bertsch, C.; Hannah, R. E.; Dell’Orco, P.; Noorman, H.; Yee, S.; Reintjens, R.; Wells, A.; Massonneau, V.; Manley, J. Org. Process Res. Dev. 2011, 15, 900–911. 39. Trost, B. M. Angew. Chem., Int. Ed. Engl. 1995, 34 (3), 259–281. 40. Heaney, H.; Papageorgiou, G.; Wilkins, R. Tetrahedron 1997, 53, 2941–2958. 41. Bryson, T. A.; Bonitz, G. H.; Reichel, C. J.; Dardis, R. E. J. Org. Chem. 1980, 45, 524–525. 269

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42. The assay purity of the isolated iminium iodide salt was only 90% based on quantitative 1H NMR analysis. One of the major impurities in the iminium salt was tentatively assigned as the residual 48 based on the 1H NMR data. In addition, other iminium salts (chloride, trifluoromethanesulfonate and trifluoroacetate) were also investigated, but the iodide salt performed best in the reaction. 43. Katritzky, A. R.; Manju, K.; Singh, S. K.; Meher, N. K. Tetrahedron 2005, 61, 2555–2581. 44. Katritzky, A. R.; Lan, X.; Yang, J. Z.; Denisko, O. V. Chem. Rev. 1998, 98, 409–548. 45. Katritzky, A. R.; Suzuki, K.; He, H. J. Org. Chem. 2002, 67, 3109–3114. 46. Boronic acid 27 was initially produced from 2-amino-5-bromopyrimidine in 38% overall yield through a sequence of Boc protection and metalation/ borylation followed by the Boc deprotection. A concise and protecting group free synthesis was developed to produce boronic acid 27 directly from 2amino-5-bromopyrimidine in one step in 40% yield.

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Chapter 9

The Development and Manufacture of Azacitidine, Decitabine, and Cladribine: Stereoselective Ribonucleoside Drug Synthesis Using the Vorbrüggen Glycosylation Erick W. Co and Julian P. Henschke* ScinoPharm Taiwan, Ltd., No. 1, Nan-Ke 8th Road, Tainan Science Industrial Park, Shan-Hua, Tainan, Taiwan 74144 *E-mail: [email protected].

The process development of 5-azacytosine-based nucleosides azacitidine and decitabine are described as a backdrop to a more detailed account of the discovery and development of cladribine, a chlorinated, deaminase-resistant derivative of the naturally occurring nucleoside, 2′-deoxyadenosine, that possesses both antineoplastic and immunosuppressive activity. To address the distinct challenges in the regio- and stereoselective syntheses of these molecules, and their stability, each process employs unique variations of the Vorbrüggen glycosylation for the key C-N bond-forming step.

The Discovery and Preclinical Development of Cladribine Background As a manufacturer of APIs, ScinoPharm receives a significant number of customer inquiries about the production of many agents, including nucleoside derivatives, both for use in generic and brand drugs. As a consequence of this interest in nucleoside drugs, the process, analytical development, and ultimately cGMP manufacture, of eight nucleoside derivatives and analogs were conducted in our company starting in the mid-2000s. In this chapter we will focus on the discovery and process development of cladribine (β-1). With the development of © 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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azacitidine (2) and decitabine (β-3) occurring prior to cladribine (Figure 1), and with much having been learned about the Vorbrüggen glycosylation from these projects, their development will also be described here.

Figure 1. Ribonucleoside APIs produced using the Vorbrüggen glycosylation.

While the use of these drugs in the treatment of disease is much more recent, a relatively large amount of the academic and patent literature describing their syntheses already existed. These syntheses were often directed towards the preparation of collections of related nucleoside derivatives, rather than being tailored for single molecules. As a result, the synthetic methods were often inefficient and cumbersome, low yielding, and not directly scalable. Around the time that these molecules received FDA approval, [2004 for azacitidine (Vidaza®); 2006 for decitabine (Dacogen®); 1993 and 2010 for cladribine (Leustatin®)], reports of their use in the treatment of disease, as well as process and polymorph patent literature began to emerge. The existence of this literature meant that if we were to manufacture these drugs, we had to develop methods that provided us freedom-to-operate. This necessity spurred the innovation and the development of novel processes and intellectual property that will be discussed later in this chapter. The most significant lesson from the development of these compounds was that seemingly small differences between apparently very similar compounds often lead to different synthetic and process strategies for their manufacture. For example, the absence of the 2′-hydroxyl group of decitabine and cladribine resulted in lower stereoselectivity than azacitidine during the glycosylation step. The substitution of a carbon atom in the cytosine ring for nitrogen made the nucleobase rings of azacitidine and decitabine very susceptible to hydrolysis as compared to their natural counterparts, cytidine and 2′-deoxycytidine. These examples were only a few of the challenges of this set of related compounds. The Advent of Nucleoside Analogs as Viable Therapeutics The study and use of nucleoside analogs as agents for anticancer and antiviral therapeutics has spanned the last fifty years. The advent of the field can be traced back firmly to the emergence of cytarabine (for Acute Myeloid Leukemia (AML)) and edoxudine (for Herpes Simplex Virus), agents both approved by the FDA in 1969 (Figure 2). Though edoxudine since has been superseded by improved treatments and is no longer clinically relevant, more than 25 nucleoside or 272

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nucleotide analogs have been approved for antiviral applications such as hepatitis B and HIV (1). Cytarabine, on the other hand, has withstood the test of time and is listed on the World Health Organization’s Model List of Essential Medicines (2). Cytarabine has paved the way for six additional approved nucleosides in the arena of anticancer treatments (Table 1), including cladribine, the primary focus of this chapter.

Figure 2. Cytarabine and edoxudine signaled the start of the nucleoside era for cancer and viral therapeutics.

Nucleosides consist of a nucleobase, usually from the purine (adenine or guanine) or pyrimidine (cytosine, uracil, thymine) families, attached to a sugar derivative. Nucleotides, which comprise many of the antiviral agents, incorporate an extra diversity point by the installation of a variable number of phosphate or polar groups to the sugar moiety. Not surprisingly, this additional functionality is a primary reason for the greater number of antiviral therapies available on the market today. Figure 3 illustrates the nomenclature basis in this class of therapeutic agents.

Figure 3. Nomenclature of nucleosides and nucleotides. 273 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

agent (trademark name)

current manufacturer or licensor

FDA approval

mechanism of action (target)

indication(s)

cytarabine (Aracytine®)

Pfizer

1969

DNA incorporation

Acute Myeloid Leukemia; Acute Lymphocytic Leukemia Lymphomas

gemcitabine (Gemzar®)

Eli Lilly

1996

DNA incorporation

Non-small cell lung cancer; Pancreatic, bladder, and breast cancers

azacitidine (Vidaza®)

Celgene

2004

DNA methyltransferase inhibitor

Myelodysplastic syndrome

decitabine (Dacogen®)

Eisai and JanssenCilag

2006

DNA methyltransferase inhibitor

Myelodysplastic syndrome; Acute Myeloid Leukemia

capecitabine (Xeloda®)

Hoffmann-La Roche (Genentech)

1998

Irreversible inhibitor of thymidylate synthase

Metastatic breast cancer; Metastatic colon cancer Gastric cancer (off-label) Esophageal cancer (off-label)

entecavir (Baraclude®)

Bristol-Myers Squibb

2004

Reverse Transcriptase Inhibitor

Hepatitis B

cladribine (Leustatin®, Movectro®)

Janssen-Cilag

1993 2010a

Adenosine Deaminase inhibitor

Hairy Cell Leukemia; Relapsing Remitting Multiple Sclerosisa

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Table 1. Prominent Nucleoside Analogs, Approval Dates, and Indications

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current manufacturer or licensor

FDA approval

mechanism of action (target)

indication(s)

clofarabine (Clolar®)

Genzyme

2004

DNA incorporation

Acute Lymphoblastic Leukemia

tegafurb

Merck-Serono

N/A

RNA & DNA incorporation

Advanced Colorectal Cancer

a Cladribine was approved for multiple sclerosis in 2010 in Russia and Australia; Merck consequently withdrew all marketing applications in 2011 voluntarily after failure to obtain additional approvals in Europe and North America. b Tegafur, a prodrug of 5-fluorouracil (5-FU), is a component of the combination drug tegafur/uracil (Uftoral®) approved in many countries, but excluding the United States.

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agent (trademark name)

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ScinoPharm’s Involvement with Cladribine ScinoPharm entered into the nucleoside class of therapeutics in 2005 with gemcitabine which had presented a promising opportunity for generic manufacturing. As a result, we developed a novel, patentable process for the synthesis of gemcitabine. Subsequent customer inquiries turned our focus onto the synthesis of the DNA demethylating agents: azacitidine and decitabine. Capitalizing on our academic, albeit pragmatic approach to development, we established efficient routes to the synthesis of both azacitidine and decitabine. (3, 4). In 2007, we became aware that cladribine, already approved in 1993 for Hairy Cell Leukemia (HCL), was being positioned as an oral multiple sclerosis treatment by Merck-Serono, having obtained fast-track status from the FDA that same year (5). The prospect of drug repositioning into an indication with a large patient population, coupled with the significant relevance of our platform synthesis technology, made the option to pursue cladribine very attractive. In addition to cladribine, ScinoPharm completed the preparation of a number of other nucleoside analogs. (Figure 4).

Figure 4. Various nucleoside analogs manufactured by ScinoPharm.

Rationale Toward the Discovery of Cladribine Cladribine (2-chloro-2′-deoxyadenosine a.k.a. 2-CdA, Leustatin®, ® Movectro ) is a synthetic nucleoside analog composed of a modified purine nucleobase and a deoxyribose component. Biological activity of ribose and various halogenated analogs, including the individual components of cladribine, had been studied as early as the 1950s (6). The first mention of 2-chloro-2′-deoxyadenosine was in the early 1960s as an uncharacterized intermediate (7) and as an isolated compound by Ikehara (8, 9). Ikehara and coworkers prepared the novel derivative 8,2′-cyclonucleoside 4 from chlorosugar 5 and studied its synthetic utility for the synthesis of 2′-deoxyadenosine (6) (Scheme 1). 276 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Scheme 1. Cladribine as a Synthetic Intermediate toward 2′-Deoxyadenosine

The earliest suggestion of 2-chloro-2′-deoxyadenosine as an in vitro anti-proliferative agent, however, was reported in 1972 by Broom et al. (10) In their studies, they described the structure-activity relationship (SAR) of various substituted 2′-deoxyadenosine derivatives at the 2-position of the purine. Table 2 aptly demonstrates that the β-anomer β-1 is equivalent to, or more potent than its corresponding α-anomer α-1. It is important to note that cladribine (first entry) is the most potent analog, with 7 x 10-8 M inhibitory activity against L-1210 cells. Cladribine is also several orders of magnitude more potent than its corresponding ribose derivative, 2-chloroadenosine 7, in the same cell line. The rationale for directed use of 2-chloro-2′-deoxyadenosine in its subsequently approved use in lymphoproliferative diseases, however, was pioneered through the drug design and research of Carson et al. (11) Rather than conducting a protracted high-throughput and medicinal chemistry approach to identifying hits and leads, they elected to approach drug discovery by exploiting the unique mechanism of action of deoxyadenosine analogs. They took note of a rare pediatric immunodeficiency in which lymphocyte levels were depleted by a malfunction of the enzyme adenosine deaminase (ADA) (12). They postulated that lymphospecific toxicity in this particular ailment may arise from the selective buildup of deoxyadenosine nucleotides by lymphocytes, which are known to have higher levels of deoxycytidine kinase, and conversely, low dephosphorylation activity (13). Armed with this hypothesis, Carson et al. sought to design an analog of deoxyadenosine that could induce similar lymphocytic effects as experienced in autoimmune diseases. Their strategy revolved around the chief principles that: • •

The agent should be a close analog of deoxyadenosine to retain activation properties. The agent should exhibit stability against key enzymes such as ADA and nucleotide phosphorylases. 277

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They found that substitution of the hydrogen at the 2-position of adenine afforded resistance to ADA (Table 3). Furthermore, choice of a chlorine substituent effectively retained its substrate specificity for deoxycytidine kinase, allowing for proper phosphorylation inside the cell. Out of a focused set of 25 deoxyadenosine analogs, 2-chloro-2′-deoxyadenosine was determined to have the best potency and selectivity in in vitro experiments and in vivo with L-1210 leukemia in mice (14).

Table 2. Early SAR Studies on Nucleoside Components and Analogs

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Table 3. Toxicity of ADA-Resistant Analogs toward Various Lymphoblasts

Preclinical and Clinical Development of Cladribine Compared to other anticancer therapeutics, cladribine possesses a unique dual-mode of action in being able to act upon lymphocytes in both the dividing and dormant states (15). Figure 5 illustrates the cellular metabolism of cladribine. Upon internalization, cladribine, which is resistant to ADA, is sequentially phosphorylated by deoxycytidine kinase (dCK), which is present in higher levels than its counterpart, 5′-nucleotidase (5′-NT). Once cladribine triphosphate is generated, the mechanisms of both dividing and quiescent lymphocytes are disrupted, leading to apoptosis. 279 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Figure 5. Cellular uptake and activation of cladribine leads to apoptosis of both dividing and quiescent lymphocytes.

Figure 6. Hairy Cell Leukemia cells are characterized by a hair-like layer surrounding the cell surface. (Reproduced with permission from Dr. William Karkow, M.D., F.A.C.S. (16)) 280 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Regarding the selection of an optimal indication, hematological malignancies dependent on lymphocyte proliferation were quickly identified during preclinical evaluation. Though shown to be effective for diseases such as chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma, and acute myelogenous leukemia (AML), cladribine proved superior in a rare form of chronic leukemia, Hairy Cell Leukemia (HCL). HCL, whose cells have clear hair-like protrusions from its surface (Figure 6) (16), accounts for approximately 2% of the leukemic population. In one of the earliest clinical trials for cladribine, Piro et al. followed a 144 patient cohort for a median of 14 months. They reported (17) that 123 (85%) achieved complete responses, 17 (12%) partial responses, and 3 (2%) recorded no response. At 36 months post treatment, only 4 patients had relapsed. Cladribine for early clinical trials was produced via enzymatic methods. As shown in Scheme 2, a Lactobacillus helveticus-derived transdeoxyribosylase, with thymidine as the deoxyribose source, effectively glycosylated 2-chloroadenine (8). Subsequent purification by ion-exchange chromatography afforded pure cladribine in unspecified yields (18).

Scheme 2. Carson’s Enzymatic Synthesis of Cladribine for Clinical Trial Use Despite facing the uncommon patent challenges of a drug that has been described in the literature for decades prior to its approval, the determination and perseverance of Carson safely ushered cladribine through clinical trials (18). Under the Orphan Drug Act, cladribine was approved by the FDA in 1993, licensed as Leustatin®, and became a prominent standard of care for lymphoproliferative diseases. Current and Potential Use of Cladribine for Lymphoproliferative Indications Compared to its analogs, azacitidine and decitabine, cladribine’s current market demand and use is very small (Table 4) (19). Cladribine’s low demand, despite still being considered widely as the first option therapy for HCL (20), may be attributed to several factors: • • • •

HCL accounts for only 2% of total leukemic incidence. HCL symptoms may subside before medical intervention is required. The majority of patients require only a single course of therapy. The treatment cycle for an average 70 kg adult male is 1 equiv TfOH. As with TfOH in MeCN, the β/α ratio increased with decreasing temperature when TMSOTf in DCM was used instead (β/α was 1.3:1 at 20 °C, 2.0:1 at -20 °C and 2.6:1 at -40 °C). In contrast to TfOH, however, as long as the amount of TMSOTf was kept at 2:1). Further, increasing the reaction concentration or the molar equivalents of nucleophile 16 resulted in decreased selectivity for the β-nucleoside.

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Stereoselectivity Explained When the completed glycosylation product mixture comprising 22a was allowed to stand at ambient temperatures for an extended period of time without work-up, the initial >2:1 β/α ratio degraded to a 90% HPLC-purity was conveniently isolated from the deprotection slurry by filtration of the mixture. The impurities were efficiently cleared into the filtrate. In fact, the β/α ratio was typically amplified from 2–3:1 in crude 22b to >60–105:1 of decitabine/α-3 due to the relatively high solubility of the α-anomer in MeOH.

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Purification of Decitabine While MeOH proved to be an excellent solvent to obtain crystalline, high-purity (99.9%) decitabine (72, 73, 83, 84), its poor solubility (91) meant that ≥80 volumes were required for dissolution. Although small amounts of DMSO significantly reduced the amount of MeOH (≤20 volumes) required, the recovery yield was unsatisfactory. With the threat of residual DMSO contaminating the recovered API, anhydrous MeOH was therefore selected. Yields of API-grade decitabine were about 70% and another 15% could be recovered by partial concentration of the mother liquors, isolation and recrystallization.

cGMP Manufacture of Decitabine Prior to the first cGMP manufacture runs, further process development was conducted. The most significant process change was an increase in glycosylation temperature from -40 °C to ~ 0 °C. In addition to the warmer temperature being more suitable for manufacturing, it ensured complete and rapid conversion of 21 without impacting product quality. Despite the β/α ratio inevitably being reduced due to the rate of in situ anomerization being higher at 0 °C, the β-anomer still comprised around 50% of the crude 22b mixture, which was in fact similar if not slightly better than had been achieved in the exploratory phase of the development. Further changes to the process allowed enrichment of the β-anomer by partial precipitation of the less soluble α-anomer α-22b during work-up with β/α ratios of the isolated 22b being about 3:1. In fact, not only was the yield increased from around 50% up to 70%, based on β-22b, but the quality of crude decitabine was increased from around 90% up to ≥97%. The purification step was modified by incorporation of an activated charcoal treatment step, partial concentration, and holding at a cooler temperature, resulting in an improved 75% recovery yield. Together these changes resulted in process and operational simplification and reduced manufacturing times while increasing the overall yield to 15% (based on 21). The first cGMP manufacturing campaign proceeded smoothly. Silyl 5-azacytosine 16 was prepared as for the manufacture of azacitidine (see cGMP Manufacture of Azacitidine). Using 3.6–3.7 kg batches of 2-deoxyribosyl acetate 21, 4-chlorobenzoyl protected decitabine 22b was obtained as a mixture of anomers (β-purity was 48-55%) from the glycosylation of an equimolar amount 299

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of 16 using 1.05 equiv TMSOTf in DCM at 0 °C (5 h). Stabilization of the glycosylation mixture was achieved using about 1 equiv MeNH2 in MeOH, prior to dilution with DCM and treatment with aqueous NaHCO3 at 25 °C. Of the 22b mixture, β-22b was produced in yields of about 40% based on 21. Following its isolation by evaporation, milling (FitzMill®) and drying, deprotection of 22b as a slurry on 5.4–6.2 kg batch scales using 30% NaOMe in MeOH furnished 96.4–97.1% HPLC area %-pure crude decitabine following filtration in yields of 59–66% (based on β-22b). Crystallization of the crude API on 1.1–1.2 kg scales from MeOH provided high purity decitabine meeting the established specification in ~ 66–72% yield. Overall yields of up to almost 20% were achieved reflecting improvements upon the laboratory process.

Conclusion As per azacitidine, decitabine was also produced using a two-step/two-pot silylation/Vorbrüggen glycosylation. Although the process strategy could be used on both nucleosides (4), modifications (TMSOTf in DCM) to the glycosylation step allowed better stereocontrol and complete conversion of the glycosyl donor 21. The lack of high stereoselectivity in the glycosylation step and the unexpected anomerization of protected decitabine 22b, inherently limited the efficiency of the process. Deactivation of the glycosylation catalyst with MeNH2 was helpful in inhibiting anomerization after the reaction. Unlike that for azacitidine, omission of an aqueous work-up after glycosylation was not feasible. The deprotection step proved the most challenging step during process development. Crude protected decitabine 22b had to be sufficiently exposed to aqueous NaHCO3 in the prior step to destroy the silicon residues, had to be devoid of residual DCM and moisture, and had to be ground into a fine powder to ensure reproducibility of the reaction. Heterogeneous conditions were preferred to homogeneous conditions but ultimately crude decitabine of 97% purity was achieved by filtration in up to about 70% yield (based on β-22b) due to excellent clearance of the α-anomer and other impurities into the filtrate. In all, only one dedicated crystallization step was required and chromatography was not necessary. The process was cost-effective and allowed the industrial scale manufacture of decitabine of very high quality. The novelty, non-obviousness and industrial applicability of the process resulted in the granting of our US patent application (90) in 2013 (4). With the next target, cladribine, also being a 2′-deoxyribonucleoside, it was anticipated that the use of low temperatures in the glycosylation step would improve stereoselectivity.

Process Development and Manufacture of Cladribine Reported Methods for the Synthesis of Cladribine Cladribine (Figure 8) has been prepared using a diverse range of approaches over the last fifty years. As compared to azacitidine and decitabine, for which 300 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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much larger numbers of manufacturers exist, cladribine has received less attention. This may be due to the very small annual volume (1:1 (following dissolution with moist DMSO) were only witnessed when using combinations of strong catalysts (TMSOTf, TBSOTf or TfOH) with BSTFA (but not for weaker MsOH, p-TsOH, TFA or Ms2O with BSA or BSTFA). reactions conducted under homogeneous conditions were nonstereoselective.

We realized that selective precipitation of the β-anomer β-30a during glycosylation combined with anomerization of the α-anomer α-30a in the solution phase was responsible for the overall β-enrichment witnessed (Scheme 16). Glycosylation reversibility, presumably via oxocarbenium ion 11a, had already been implied in the isomerization of the N-7 (31) to the N-9 (30) regioisomers, and in the corresponding glycosylation in the preparation of decitabine (vide supra) and literature precedents (54, 66, 88, 89). Apparently the combination of TfOH, TMSOTf or TBSOTf with BSTFA was essential for this process to occur under the conditions tested. It is probable that the higher β-selectivity observed when using 20 mol% (Table 10, entry 6) as compared to when using 10 mol% (entry 7) was the result of more rapid anomerization and therefore conversion of α-30a to β-30a. We believe that the α-selectivity observed in tests using the weaker silylating agent BSA and/ or weaker glycosylation catalysts results from an absence of anomerization and therefore indicate that the α-anomer is kinetically favored over the β-anomer in the glycosylation of 29 with 21. 309

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Scheme 16. Precipitation-Driven β-Enrichment via Anomerization. (Reproduced with permission from reference (119). Copyright 2013, ACS Publications)

Aging Step With anomerization being true, it followed that extending the reaction time would enhance selectivity and increase the isolatable yield of β-30a. Indeed, while immediate filtration upon consumption of deoxyribofuranose 21 (1 h at 60 °C) provided a 45% isolated yield of β-30a (β/α 28:1; Table 11, entry 1), leaving the product mixture to ‘age’ at 60 °C (entries 2-8) provided improved yields (53–68%) and greater β-enrichment (β/α 44–71:1), especially when the aging time was not less than 8 h. At 20 °C, glycosylation was complete within 19 h but proceeded in lower yields (39–42%; entries 9 and 10) along with relatively low β-enrichment that was, in fact, identical to that following filtration immediately upon consumption of 21 (entry 1). Notably, when the glycosylation product produced at 20 °C was subsequently aged at 60 °C for another 8 h (entry 11), improved β-selectivity and yield were obtained. This showed that while the glycosylation operated at ambient temperature, a discrete aging step at a raised temperature was necessary to produce higher yields and β-enrichment of the filtered solids. When the glycosylation/aging steps were conducted over an extended period at 80 °C, a low yield (38%) was obtained (entry 12). This displayed essentially no benefit over that of the exploratory phase of the project when the glycosylation was conducted for 30 min at 80 °C (Table 7, entry 1). We suspect that at 80 °C the solubility of β-30a is too high for effective precipitation to drive the equilibrium in the desired direction. Given these results, a target temperature of 60 °C was deemed suitable for both the glycosylation and aging steps. 310

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Table 11. Study of the Aging Stepa

Using potency assay, the absolute amounts of the two anomers (Figure 12) present in the solution phase throughout the aging step at 60 °C were determined over 1 d. While the amount of the β-anomer in solution was constant at a 2–4% assay-determined yield (based on 21), the α-anomer fell from 29% to 9% yield. The isolated yields of β-30a from nine aging experiments for different periods (plotted in Figure 12) clearly show the correlation between the isolated yield of β-30a, aging time and the decreasing level of the α-anomer in the solution phase. At the end of the test period, 97.3% HPLC-pure β-30a (2.1% α-30a) in 68% yield was isolated by filtration. Including the β-anomer present in solution, the total reaction yield of β-30a was just over 70%. The remainder of the mass balance was accounted for by the ca. 10% total yield of α-anomer and 20% decomposition. 311 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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We believe that more rapid anomerization, and therefore increased yields and process efficiency, is possible by further optimization of the conditions. The addition of more silylating agent and/or catalyst after completion of the glycosylation step would probably achieve this.

Figure 12. Solution phase assay of α-30b and β-30b and isolated yields of β-30a over the aging step. (Reproduced with permission from reference (119). Copyright 2013, ACS Publications)

Work-up As per the azacitidine process (vide supra), the relatively low catalyst loading meant that an aqueous work-up could be avoided altogether. In fact, the glycosylation slurry was simply filtered and washed (with MeCN) giving crude, albeit relatively high purity (>94%) β-30a. The silylated α-anomer α-30a, spent reagents and impurities were conveniently purged into the filtrate. Anhydrous conditions were essential, however, otherwise desilylation, co-precipitation and contamination of β-30a with the non-silylated α-anomer α-30b occurred.

Deprotection Deprotection of crude β-30a was effective using catalytic amounts of NaOMe in MeOH. Despite being heterogeneous as a result of the low solubility of β-30a and cladribine in MeOH, deprotection was rapid at 20 °C and furnished crude cladribine of 98.3–99.7% HPLC area % purity in up to 92% yield. Notably, on laboratory scales the crude product was contaminated with typically 80% yield that was contaminated with