Comprehensive accounts of pharmaceutical research and development : from discovery to late-stage process development v1 9780841231887, 0841231885, 9780841231894, 9780841231917

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Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1239.fw001

Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage Process Development Volume 1

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-2016-1239.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 1239

Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery

Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1239.fw001

to Late-Stage Process Development Volume 1 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-2016-1239.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 9780841231887 (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.

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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

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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

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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

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Discovery and Chemical Development of Suvorexant - A Dual Orexin Antagonist for Sleep Disorder Debra J. Wallace,*,1 Ian Mangion,1 and Paul Coleman2 1Department

of Process Chemistry, Merck and Co, Rahway, New Jersey, 07065, United States 2Department of Medicinal Chemistry, Merck and Co, Westpoint, Pennsylvania, 19486, United States *E-mail: [email protected]. Phone1-732-594-3041.

This chapter outlines the discovery and chemical development of the dual orexin antagonist, suvorexant, which was recently approved as Belsomra® in the US and Japan for treatment of sleep disorders. The biological evidence that orexin signaling plays a central role in maintaining wakefulness and hence was a viable target for developing therapeutic entities is outlined, followed by details of the medicinal chemistry efforts to identify hit-molecules and ultimately the development candidate. The requirements of an ideal process chemistry synthesis, suitable for generating kilogram-, and ultimately manufacturing- scale quantities are explained. We then show how a staged approach to the synthetic development of suvorexant was employed focusing on an appropriate synthesis based on the scale of implementation and stage of development. We discuss how a number of alternative routes were evaluated before the final manufacturing route was chosen based on our guiding process chemistry principles. In particular, the importance of complete mechanistic understanding to allow for final reaction optimization for a key transformation is illustrated.

© 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.

Introduction

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Suvorexant (Belsomra®) is a first-in-class orexin receptor antagonist approved for the treatment of insomnia in the U.S. and Japan (1). Suvorexant 1 (Figure 1) has demonstrated efficacy in helping patients fall asleep and maintain sleep and is the first approved therapeutic agent that selectively blocks wake-promoting orexin signaling in the brain (2). This novel mechanism of action differs from other commonly used hypnotic agents that promote sleep through general suppression of central nervous system (CNS) activity. In this account, we describe the discovery and initial synthesis of suvorexant as well as the eventual conception and implementation of a large scale manufacturing route for this molecule.

Figure 1. Structure of suvorexant.

Role of Orexin Peptides Orexin peptides are central regulators of CNS arousal and vigilance and were discovered by two independent research teams in 1998 (3, 4). Orexin neuropeptides, orexin-A (OX-A) and orexin-B (OX-B) are generated in the hypothalamus from a common pre-pro-peptide precursor. OX-A and OX-B bind to and activate two transmembrane G-protein coupled receptors (OX1R and OX2R). Both OX1R and OX2R transactivate cells primarily through a Gq-mediated signaling leading to an increase in intracellular Ca2+ levels in neuronal cells (5–8). There are reports that both receptors can also alter intracellular cyclic adenosine monophosphate levels through a Gs/Gi/o-dependent mechanism. Orexin receptors are highly conserved across mammalian species with greater than 90% sequence identity between rodents and humans (9). Neurons that secrete excitatory orexins are restricted within the CNS and highly localized to the hypothalamus. The orexin-secreting neurons activate centers of the brain involved in arousal and wakefulness. The nerve terminals of orexin neurons project into the regions of the CNS that govern wakefulness and ascending arousal. Both OX2 and OX1 receptors are found in the laterodorsal tegmentum (LDT), pedunculopontine tegmentum (PPT), and dorsal raphe (DR) while the histaminergic tuberomammillary nucleus (TMN) and noradrenergic locus coeruleus (LC) have preferential expression of OX2R and OX1R, respectively (10–13). 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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A landmark event in the field of sleep research was the discovery that loss-of-function mutations in canine OX2R were the primary cause of inheritable narcolepsy and cataplexy in dogs (14). Following this discovery, it was learned that humans with narcolepsy, a sleep disorder marked by excessive daytime sleepiness and rapid transitions between sleep and wake, have significant loss of orexin-producing neurons resulting in depleted levels of excitatory orexins in the cerebrospinal fluid (CSF) (15, 16). Further corroboration for the role of orexins in maintaining wakefulness was established by the generation of rodent genetic strains. In mice, the knockout of the OX2R gene produces a narcolepsy-like phenotype similar to orexin peptide knockouts whereas OX1R knockouts present a milder phenotype with limited increases in sleep fragmentation (17, 18). Orexin peptides are secreted in the brain and their expression oscillates in a circadian manner peaking during the daytime hours in primates with levels falling during normal sleep (19, 20). Expression of orexins is thought to offset normal homeostatic sleep drive which builds during the day and provide stabilization of the wake state in the presence of increasing sleep drive. Aberrant signaling of this system during the normal sleep phase could drive undesired wakefulness and disrupted sleep. The discovery of this new neuropeptide signaling system, its anatomical restriction within the CNS, and its clear role in driving wakefulness provides a compelling rationale for the design of orexin receptor antagonists that selectively target this system. Orexin receptor antagonism represents a novel pharmacotherapy for the treatment of insomnia that has generated broad interest (21). Since a small molecule antagonist would presumably specifically target arousal-promoting signaling without attenuating other CNS functions, it is anticipated that a therapeutic molecule might have an improved tolerability profile and differentiated effects on sleep efficacy.

Discovery of Suvorexant We recognized the compelling evidence that orexin signaling plays a central role in maintaining wakefulness and, as part of a broader effort in developing therapeutic entities for treating wake/sleep dysregulation, we initiated a program to discover potent orexin antagonists with clinical utility. We embarked on a high-throughput (HTS) campaign of our sample collection to identify novel chemotypes that could function as antagonists of both OX1R and OX2R. We chose to focus on dual orexin antagonists (DORAs) due to the more robust sleep phenotype observed in dual receptor knockouts as well as the recognition that orexin tone is significantly reduced in human narcoleptics. This screening campaign was productive and identified multiple, diverse chemical series with promising attributes (Figure 2) (22). One of the hits of interest from our screen was diazepane amide 2 which bound with good affinity to both OX1R and OX2R and blocked orexin-A excitatory signaling in a cell based assay. This ligand while potent, lacked favorable drug-like properties. For example, compound 2 suffered from poor physicochemical properties (low solubility and high cLogP) and high rates of 3

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hepatic metabolism. Our initial campaign focused on identifying molecules with reduced lipophilicity and improved metabolic stability. Extensive SAR exploration efforts were rewarded by the discovery of 3 which was found to maintain good receptor potency on both OX1R/OX2R and favorable brain penetration, while having reduced lipophilicity (23). Orexin antagonist 3 was active in vivo reducing spontaneous locomotor activity and promoting sleep in rats.

Figure 2. Hits from HTS.

While compound 3 displayed many favorable attributes, it had a poor pharmacokinetic profile with low oral bioavailability (F < 5%) and high clearance in the rat and dog. Significant reductions in plasma clearance could be achieved with further alteration of the diazepane structure and heteroaryl substitution to provide analogs such as 4 (24). Detailed metabolic studies on orexin antagonist 4 indicated a strong propensity for this molecule to generate electrophilic, reactive metabolites following metabolic activation. Specifically, we hypothesized that oxidative metabolism at methylene sites on the diazepane ring or on the heteroaryl moieties could generate reactive species that would be trapped by glutathione (GSH). Subsequent metabolic studies revealed that the fluoroquinazoline in 4 was a major site for bioactivation and trapping by glutathione (GSH) after incubation of 4 in microsomes treated with GSH. Replacement of the fluoroquinazoline in 4 with a chlorobenzoxazole suppressed the formation of undesired glutathione adducts and provided an overall favorable balance of potency, physicochemical properties, and pharmacokinetic profile. These efforts produced 1 (OX1R Ki = 50 nM; OX2R = 56 nM) which was subsequently advanced into preclinical development as MK-4305 and later named suvorexant. Suvorexant (1) maintains excellent potency against both OX1R and OX2R, has improved oral bioavailability and pharmacokinetics, and demonstrates potent in vivo activity in promoting sleep in preclinical species. As predicted by earlier conformational studies (25, 26), suvorexant adopts a U-shaped bioactive conformation when bound to OX2R and OX1R. When bound to the transmembrane helices in OX2R and OX1R, this conformation of suvorexant allows it to maximize key hydrogen bond and van der Waals contacts with critical residues within the ligand binding site thereby blocking neuropeptide binding and 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.

preventing transmembrane helix motions required for activation. High resolution protein-ligand crystal structures have been solved for suvorexant bound to OX1R and OX2R (27, 28).

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Medicinal Chemistry Synthesis of Suvorexant The synthesis of suvorexant began with heteroconjugate addition of N-Boc-1,2-diaminoethane 5 to methyl vinyl ketone followed by in situ trapping of 6 with benzyl chloroformate to provide the ketone adduct 7. Selective removal of the Boc group followed by intramolecular reductive amination and reprotection of the secondary amine as a Boc carbamate provides the racemic diazepane 8 in 38% overall yield from 5. At early stages of medicinal chemistry exploration, both the R- and S- antipodes were independently studied. Subsequently, it was found that the R-isomer had superior potency for orexin receptor binding, and this isomer of 8 could be resolved by chiral stationary phase HPLC to produce 9 after deprotection. Separately, the 2-(2H-1,2,3-triazol-2-yl)-5-methylbenzoic acid 10 was prepared via a microwave mediated amination of 2-iodo-4-methylbenzoic acid 11 with triazole/CuI to afford a 55:45 mixture of the 2-triazolyl and 1-triazolyl regioisomers 10 and 12, respectively in good yield. The two regioisomers were separated chromatographically from each other. Standard amide coupling of diazepane 9 with acid 10 provided intermediate 13 in good yield. Hydrogenolysis of the CBz group in 13 with Pd(OH)2 on carbon under 1 atm of hydrogen provided the unpurified diazepane 14 which could be subsequently treated with 2,5-dichloro-1,3-benzodioxazole 15 to afford suvorexant 1 (Scheme 1).

Scheme 1. Medicinal Chemistry Synthesis of Suvorexant (MK-4305) 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.

Based on suvorexant’s favorable profile, it was selected to enter preclinical development. Suvorexant showed robust dose-dependent promotion of sleep in rodents, dogs, and nonhuman primates (29). Further evaluation of this molecule showed that it was not genotoxic and was well-tolerated in preclinical toxicology assessments. Suvorexant had a safe cardiovascular profile in dogs and it showed good tolerability in longer duration toxicology studies in both rats and dogs. Given this promising preclinical profile, suvorexant was rapidly accelerated into clinical development.

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Initial Kilogram Scale Synthesis Once MK-4305 (suvorexant) was chosen as the compound to enter development, attention turned to evaluation of the chemical synthesis and in particular its suitability for larger scale implementation. The requirements for a manufacturing scale synthesis differ from those ideal for basic research purposes, where a modular and flexible approach is preferred allowing for many analogues to be prepared from common intermediates. However as scale increases other considerations become key as outlined below. Requirements of an ideal manufacturing route • • • • • • • • • • •

Control of impurity profile and physical properties Robust and reproducible reactions Appropriate isolations and purifications (no chromatography) Substrates and reagents must be available on large scale Cost of goods Maximize yield, minimize number of steps and operations, minimize costly reagents Safety concerns (for example, exotherms are more significant on scale) Minimal environmental impact (green chemistry targets) Freedom to operate Awareness of large-scale equipment and capabilities Regulatory considerations

Synthesis Assessment In reviewing the medicinal chemistry approach to MK-4305 (Scheme 1) a few areas were flagged as concerns for immediate or longer term use. 1.

2. 3.

Fairly lengthy linear synthesis with extensive use of protecting groups, in particular operations to afford the differentially protected and enantiomerically pure diamine 8 appeared inefficient Racemic synthesis relying on chromatographic separation of racemic 8 to afford a single enantiomer Final API isolated as a weakly crystalline form with low melting point 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.

4.

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5.

Difficulty preventing over addition in reaction of Boc-ethylene diamine 5 with methyl vinyl ketone leading to an inseparable mixture of products (see Scheme 2). Triazole acid 10 formation was non-selective and involved chromatographic purification

However the team was aware that the resources and time required to address all of the challenges above might not be appropriate in early development, and hence initial efforts focused on bond forming improvements, but accepted that some form of enantiomer separation would still be used. The inspiration for a shorter route came from observing the undesired over addition product 16, which led us to suggest that the desired conjugate addition to methyl vinyl ketone should be possible starting from a secondary amine, and that secondary amine could include the desired benzoxazole western portion of the molecule, rather than a temporary protecting group. As such we postulated that addition of 17 to methyl vinyl ketone would afford the desired 18, avoiding the use of one of the protecting group, significantly shortening the route and giving higher yield in the conjugate addition step, Scheme 2. In turn 17 should be accessible by reaction of the previously used Boc-ethylenediamine 5 with an activated benzoxazole fragment.

Scheme 2. Proposed Shorter Synthesis for Reductive Amination Precursor 18

Synthesis of 22 With this proposed change the preparation of compound 18 was explored. At time of project initiation, a reliable source of an activated benzoxazole such as chloride 15 was not available so preparation from 2-amino-4-chlorophenol 19 was evaluated. Direct formation of the benzoxazole chloride 15 from 19 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.

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proved challenging, but the corresponding thiol 20 could be prepared by reaction with thiophosgene and isolated directly from the reaction mixture in high yield, (Scheme 3).

Scheme 3. Preparation of 18

In the medicinal chemistry route, the benzoxazole chloride 15 was prepared from thiol 20 by reaction with PCl3/POCl3, however the team rapidly identified oxalyl chloride as a milder alternative to these conditions. The initially formed chloride 15 was not isolated, but on treatment with triethylamine and Boc-ethylene diamine, the desired 17 was formed. Although an isolation of 17 was initially used, ultimately a through process was developed whereby after aqueous quench and solvent swap to acetonitrile, straightforward addition of DBU and methyl vinyl ketone allowed for excellent conversion to 18 in 75% isolated yield over the three step sequence from 20 (chlorination, amidation, conjugate addition) (30). The desired Boc-deprotection/intramolecular reductive amination sequence (Scheme 4) appeared to have much in common with the medicinal chemistry approach, however the presence of the benzoxazole in 18 vs the stable CBz group in 7 led to some challenges.

Scheme 4. Proposed Intramolecular Reductive Amination

Deprotection of the Boc group in 18 could be achieved with HCl or TFA, and the associated salts were stable as solids or in solution, but did not participate directly in the reductive amination reaction. Neutralization of the salts to the free amine 21 allowed for reductive amination promoted by sodium triacetoxyborohydride (STAB), however decomposition of both starting amine 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 product 22 was seen on the time scales that would be associated with such large scale operations. Switching the deprotection reagent to methanesulfonic acid (MSA) allowed for the resulting MSA salt solution to be used directly in the reductive amination in modest yields. Alternatively the bis-MSA salt could be isolated by direct filtration, giving further purity upgrade and this salt was used in the reductive amination, again accompanied by formation of a number of impurities, including 23, 24 and 25 (31) (Figure 3).

Figure 3. Impurities formed in the reductive amination reaction.

Impurities 23 and 24 appear to result from hydrolysis of the benzoxazole moiety, suggesting that pH control in the mixture might be needed. Indeed, better results were obtained when the bis-MSA salt 26 was converted in situ to the monoMSA salt by addition of 1 equivalent of sodium acetate prior to addition of the STAB. In this way ring opened impurities were minimized and a near quantitative solution yield of amine racemic 22 was obtained (Scheme 5).

Scheme 5. Optimized Reductive Amination Procedure

With racemic diamine in hand, attention turned to separation of the enantiomers. In the medicinal chemistry route this was achieved by chiral column chromatography of the differentially protected diamine racemic 8. However for a target delivery of around five kilograms of final material, the time, solvent and stationary phase requirements to separate enantiomers of racemic 22 were clearly impractical and instead a classical resolution approach was pursued. A screen of readily available chiral acids and common solvents led to identification of a 1:1 L-dibenzoyltartaric acid salt from THF as the best lead, giving about 76% ee in the initial salt, and further work concentrated on this system. The two diastereomerically pure salts 27 and 28 were prepared from samples of each enantiomer of amine 22 (32) to study solubilities of the pure compounds. This confirmed that THF would be the optimum solvent for resolution based on solubility differences (Table 1). 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.

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Table 1. Solubility Data for 1:1 and 2:1 L-DBT Salts

However the process was further complicated by competing formation of a 2:1 amine:acid salt 29 leading to racemic material. Solubility of this hemi-salt 29 in THF was comparable to the desired 1:1 salt 27 and an addition protocol to minimize its formation was developed. Hence the amine racemic 22 was added slowly to 1.5 equivalents of L-di-benzoyltartaric acid thereby maintaining an excess of acid to suppress formation of the hemi-salt. In this way a 38% yield of salt 27 containing 74% ee amine (87% ds salt) was obtained on multi-kilogram scale. Based on the modest ee obtained in the initial salt formation, an upgrade of the optical purity of the isolated salt was sought via re-crystallization or re-slurry protocols. Surprisingly the previously employed THF did not offer any further improvement. This led to the proposal that the enantiopurity of the product was compromised by partial formation of the hemi-salt 29, rather than the diastereomeric 28. 1H NMR confirmed the presence of excess amine in these isolated salts giving further credence to this theory. Given the similar solubility of these two species in THF (3.25 mg/mL vs 4.1 mg/mL, Table 2) an efficient upgrade in this solvent appeared unlikely and attention switched to use of methanol which offered both reasonable absolute solubility, and a significant difference between the desired mono salt 27 and hemi-salt 29. Ultimately the use of IPAc as co-solvent with methanol attenuated the solubility and reduced losses to the filtrate. The final upgrade procedure was carried out by re-slurrying the solids in a 3:1 IPAc:MeOH mixture proceeding in 70% yield to afford 96% ee amine (Scheme 6). 10

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Scheme 6. Resolution and Upgrade of Amine 22

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Triazole Acid Synthesis The medicinal chemistry route to triazole acid 10 involved a microwave promoted amination of iodide 11 with 1,2,3-triazole in NMP at 120 °C, using a diamine promoter which proceeded to give a 55:45 ratio of regioisomers 10 and 12. A lengthy extraction of the water soluble products and chromatographic separation using high solvent volumes was then required to afford the desired isomer in around 40% final yield (Scheme 7). For large scale processing both the use of microwave and the chromatographic purification needed to be addressed.

Scheme 7. Medicinal Chemistry Preparation of Triazole Acid 10 After an initial screen of all reaction variables, we found that complete reaction and higher selectivity could be achieved using an excess of 1,2,3-triazole, copper iodide, without the use of microwave or a diamine promoter, at 65 °C in THF/DMF with potassium carbonate as base. Under these conditions the reaction typically reached >98% conversion and produced an 81:19 ratio of regioisomers with 99.5% regioisomeric purity.

Scheme 8. Optimized Triazole Acid Preparation

Amide Coupling and API Form In preparation for the final amide bond formation, the L-DBT salt 27 was converted to the free amine form 22 with aqueous sodium hydroxide and following extraction with dichloromethane, was used as a solution in the subsequent step. Coupling of the two penultimate fragments was initially attempted using traditional coupling reagents (EDC, DCC, etc), however rate of reaction was very low, likely due to steric constraints. Additionally an isomeric impurity 31 was seen to form in the final compound which probably results from impurity 25 (see Figure 3). Ultimately it was found that coupling via the acid chloride gave a faster reaction rate and cleaner reaction profile. The acid chloride 32 was prepared by treatment of acid 10 with oxalyl chloride and DMF in dichloromethane, to which was added a solution of amine 22 and trimethylamine. This produced clean conversion to the final compound within half an hour. After an extractive work up and concentration of the CH2Cl2 solution, suvorexant 1 was isolated by the slow addition of heptane to the dichloromethane solution (Scheme 9) in 88% yield and 99.5% purity with 96.4% ee.

Scheme 9. Coupling To Give Crude MK-4305 12 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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MK-4305 isolated from the above reaction was shown to have the same crystal form (form 1) as that obtained in Medicinal Chemistry. This form was confirmed to be anhydrous and weakly crystalline, but had a low, broad melting point of 123 °C (+/- 3°C), and poor physical properties leading to slow filtration. High throughput polymorph screens revealed one new form (form 2) which was obtained by heating MK-4305 in water at close to 100 °C. This provided material which was more crystalline as assessed by the sharper peaks in the XRPD spectrum (Figure 4).

Figure 4. XRPD spectrum of the original (form 1) and new form (form 2). The melting point of the new form 2 was 154 °C. It was non-hygroscopic, absorbing only 0.06 wt% water at 95% RH and 25 °C, stable after aging overnight at 30, 40, 50 and 60 °C, and TGA confirmed it to be anhydrous. This form was considered appropriate for further development. Somewhat surprisingly stability studies showed conversion of form 2 to the less crystalline form 1 after stirring at room temperature in most solvents, with water being an exception. On large scale, the form turnover was achieved by slurrying the initially isolated form 1 in water at just below 100 °C, and after cooling, form 2 solid was isolated by filtration in 97% yield, 99.6% chemical purity and 97.4% ee.

Summary of Initial GMP Delivery Using the chemistry as described above, a rapid delivery of over 3.0 kg of material in around 12% overall yield from the commercial chloro-aminophenol starting material 19 was achieved (Scheme 10). The process generated material with very high chemical purity (99.6%) and moderate enantiomeric purity (97.4%) which was sufficient to support the program through initial toxicology studies, early formulation development and into PhI clinical studies (33). Key successes of the approach were as follows: 13 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.

1.

2.

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3. 4.

Use of the benzoxazole fragment in place of the temporary protecting group on the ethylene diamine gave a much shorter and more convergent synthesis avoiding a number of protecting group manipulations A classical resolution of racemic 22 replaced the time consuming chiral separation A new and highly crystalline form of MK-4305 was discovered Higher selectivity and more scalable conditions for the triazole acid synthesis, and a method to remove the undesired regioisomer via salt formation rather than chromatography.

Scheme 10. Summary of First GMP Delivery of MK-4305

PhII Synthetic Approach As clinical data became available from PhI studies, it became clear that larger amounts of API would be needed to support the project moving forward into PhII and beyond. In reviewing the approach used for the initial kilogram scale delivery, the team felt that the synthesis offered a convergent approach and the bond forming reactions were efficient and high yielding. For the most part only minor modifications to these reactions would be necessary. However the classical resolution to afford a single enantiomer of 22 was low yielding, afforded only moderate ee material even after an upgrade step, and required kinetic control for good results due to facile formation of the thermodynamically favored 2:1 29. This would not be appropriate for API deliveries in excess of a few kilograms. As such, preparation of diamine 22 in high enantiomeric purity and high yield became the main objective for the next stage of development with a view to supporting API deliveries of 10-100 kg scale. 14

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Asymmetric Reductive Amination

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While other synthetic approaches to a single enantiomer of 22 could be envisaged, given the success of the intramolecular reductive amination from the first delivery and the high yielding process to prepare the precursor, our efforts focused on an asymmetric version of this reaction. The value of high throughput experimentation for such an endeavor quickly became apparent as a number of reagent combinations were evaluated, as outlined in Scheme 11.

Scheme 11. Screening for Asymmetric Version of the Reductive Amination

Low ee’s and/or conversions were obtained using chiral phosphoric acid reagents and chirally modified sodium borohydride derivatives. Some encouraging selectivities were seen with rhodium- or iridium-catalyzed hydrogenations, but the most promising lead was using a ruthenium-catalyzed transfer hydrogenation promoted by complex 33. After further optimization of the ligand system, we were able to achieve 90% conversion to give 22 in 85% ee. Thiswould be appropriate for the next stage of development, especially as it was anticipated an upgrade in enantiopurity should be possible via salt formation with the previously used resolving agent di-benzoyl tartaric acid. The reductive amination proceeded smoothly on multi-kilogram scale however unexpected challenges were encountered during the salt formation to upgrade the enantiomeric purity. Use of L-di-benzoyl tartaric acid led to material with very poor filtration properties. As with the previous delivery, upgrade to 15 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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better than around 97% ee proved elusive. Additionally impurity 25 was seen to form over time during the upgrade, which was further exacerbated by slow filtration rates. Screening for other salt forms led to the discovery that a chiral acid was not needed. Formation of the acetate salt 34 gave more efficient upgrade of enantiomeric purity with better filtration properties. However, the longer cycle times associated with large-scale processing once again led to significant formation (>10%) of the isomeric 25. Further operations were then required to remove this impurity and improve the ee, which in turn led to further degradation, reducing the overall yield. Despite these limitations, this process allowed a 62% yield of the acetate salt in 98.5% ee on 10-50 kg scale, supporting API deliveries to initiate PhII studies (Scheme 12).

Scheme 12. Initial Asymmetric Reduction Route

While the discovery of the asymmetric reductive amination was a breakthrough step and allowed for rapid generation of larger amounts of material on the 10-50 kg scale, further optimization would be required before implementation of 100s kilogram scale. In particular, conversion, enantiomeric excess, stability and purity upgrade of the product all required attention. Efforts initially focused on optimization of the asymmetric transformation. Extensive screening confirmed the triisopropyl ligand 37 and p-cymene aryl group as optimum, however changes to the base, formic acid equivalents, reduction in reaction temperature and change of solvent to dichloromethane all led to an improvement in both conversion and enantiomeric excess (Table 2). Under these optimal conditions, the reaction proceeded in 98% yield to give diamine 22 of 94% ee. With a higher yielding process in place, attention turned to the best way to stabilize and isolate the product. The impurity formed during the previous process was confirmed to be the amine isomer 25, presumably generated by transient formation of the bicyclic compound 38 (Scheme 13), and was common to an impurity previously seen in the achiral version of the process (see Figure 3). 16

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Table 2. Optimization of Reductive Amination Conditions

Scheme 13. Isomerization of Amine 22 17 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.

To understand conditions under which this isomerization would be problematic, a stability study of amine 22 was carried out under conditions and pH which could feasibly be encountered during the reductive amination reaction, work-up, and subsequent amide coupling. Somewhat surprisingly the amine showed good stability under strongly acidic or basic conditions, however at neutral pH, and particularly in weak acids (with pKa in range 3-6) extensive isomerization was seen giving what appeared to be an equilibrium ratio of 22:25 60:40, (Table 3).

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Table 3. Stability of Amine 22 at Various pHs Temperature Time

10 °C 8 h 48 h

pH1

1

45 °C 8 h 48 h

60 °C 8 h 48 h

Amount of 25 (based on HPLC) (%)

12

0.3

0.2

0.2

0.2

0.2

1.7

10

0.4

0.6

1.6

3.6

1.6

4.4

8

0.5

1.2

3.5

12

8.2

16

5

4.5

11.3

37

41

41

41

2

0.5

0.7

1.5

6.9

5.1

21

target pH obtained by addition of dilute HCl or NaOH.

These results explain the rapid isomerization during the previous salt formation procedures. Both acetic acid and di-benzoyltartaric acid have pKa’s in the range which the desired product is the least stable. Additionally the isomerized product 31 seen in early EDC promoted couplings (see Scheme 9) is thought to arise from prior isomerization of the amine under the mildly acidic pH conditions in that reaction, which was mitigated by switching to the acid chloride prepared under a more acidic regime. As such to ensure good yields, operating conditions in the pH range 3-6 would need to be avoided and this was addressed in two ways: 1) Modification of work-up conditions: Once the target conversion was obtained, the reaction was quenched with aqueous sodium hydroxide solution to ensure strongly basic conditions were maintained during the subsequent extractions and solvent reduction. 2) Alternative salts for ee upgrade and isolation: To ensure pH remained below 2 during salt formation, stronger acids were evaluated. In the presence of HCl, degradation was seen to be minimal. Appropriate ee upgrade was not seen in many common extraction solvents, however a mixture of DMAc:toluene produced the HCl salt 39 in excellent enantioselectivity after a single isolation, with minimal loss to the liquors. 18

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With the above changes to the reaction conditions and isolation procedure, the asymmetric transfer hydrogenation now proceeded in almost quantitative conversion, and 94% ee as measured in the reaction mixture. After standard extractive work-up, HCl salt formation, and a single isolation allowed a 90% yield of the amine HCl salt in essentially complete enantiomeric purity (Scheme 14).

Scheme 14. Optimized Reductive Amination and Isolation Conditions

Early and End-Game Steps Modifications As MK-4305 continued to move through PhII trials and successively larger synthetic campaigns were required, improvements to the other synthetic steps were also sought to address robustness, yields and environmental (Green Chemistry) concerns. To avoid the use of thiophosgene on large scale, an alternative synthesis of thiol 20 was developed using potassium ethylxanthate as reagent. Initially pyridine was employed as solvent for this transformation, but ultimately the less toxic ethanol was suitable, providing a 96% yield. In the next sequence, dichloromethane was replaced with THF for the chlorination of thiol 20, K2CO3 was used in place of triethylamine for the amination to give 17, and DBU was replaced with catalytic sodium hydroxide to promote the conjugate addition reaction. These changes had a beneficial impact on the amount and type of waste generated for preparation of 18 and moreover improved the yield and robustness (Scheme 15).

Scheme 15. Improved Synthesis of 18 In reviewing the final coupling and form conversion steps for MK-4305, a number of issues from the initial kilogram scale delivery required further development, as outlined below. 19 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.

1. 2. 3.

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4.

High volumes of dichloromethane used for coupling reaction. An off-line salt break and extraction with dichloromethane was needed to prepare the free amine for coupling. A form turnover step is required. Although in some cases a “purification step” is desirable, in this case essentially no purity improvement is seen due to the very low solubility in water. Lack of understanding of the API phase, and conversion to the undesired form 1 at room temperature in most organic solvents.

Initial efforts focused on optimization of the coupling reaction. Although coupling via the acid chloride was high yielding, both the acid chloride 32 and free amine 22 needed preparation in separate operations, with dichloromethane being used to efficiently extract the amine. Moreover despite screening for other suitable solvents, the very thick nature of the slurry seen during the reaction, likely due to formation of triethylamine hydrochloride, necessitated use of high volumes of dichloromethane as reaction solvent to retain mobility and good reaction rates. An alternative approach was explored using a bi-phasic system to allow for reaction of the acid chloride with the amine HCl salt 39 in the presence of an aqueous inorganic phase. After some experimentation, optimum conditions involved preparation of the chloride with oxalyl chloride in isopropyl acetate (IPAc) with catalytic DMF leading to a homogeneous solution. This was added to a two phase mixture of the amine salt 39 (no separate salt break needed) in more IPAc and aqueous potassium carbonate with vigorous stirring. Coupling to form the desired final compound 1 was essentially complete by the end of the acid chloride addition and after aqueous work up a 98% solution yield was obtained. At this stage crude isolation and form turnover in water at 100 °C as previously described would afford the desired form 2, however to minimize operations direct isolation of the desired form was now evaluated. To aid this, further characterization and understanding of the relationship between form 1 and 2 was undertaken. Although form 2 has the higher melting point and thus was initially assumed to be more stable, conversion to form 1 was seen at room temperature in a range of solvents, especially if form 1 seed was added. Further studies indicated that the two forms were enantiotropic with a transition temperature between 35 and 40 °C and form 1 was more stable at 25 °C. Despite the potential challenges of isolating the less stable form at room temperature, the favorable morphology of form 2 led to its selection for longer term use. In the solid state no conversion of form 2 to form 1 was seen during storage at 25 °C/60% RH for up to 36 months. With the new knowledge of the relationship between form 1 and 2, generation of form 2 would need to occur above 40 °C. Isolation either above that temperature, or in a solvent in which conversion to form 1 is very slow was sought. The previously used process involved heating in water at 100 °C which converted all material to form 2. Although the slurry was then cooled to below the transition temperature for isolation, the very low solubility of final material in water prevented any significant conversion to form 1 under the time cycles employed. Similar behavior was seen in other solvents where the intrinsic 20

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solubility of MK-4305 was low, such as heptane and MTBE, whereas in a 1:1 IPA:water mixture, form 2 converts to form 1 below 40 °C and re-converts to form 2 above 40 °C.

Figure 5. Stability of Form 2 in 90:10 and 85:15 Heptane:IPAc based on XRPD.

Figure 6. Stability of Form 2 in 90:10 and 85:15 Heptane:IPAc based on melting point. To allow for direct isolation of form 2 from the final IPAc stream, solubility and form stability studies of various IPAc:anti-solvent mixtures were carried out. This led to identification of 85:15 n-heptane:IPAc mixture as suitable to give 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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high recovery, good impurity rejection, and form stability. In this solvent ratio, conversion of the desired form 2 to form 1 was essentially negligible even at room temperature in the absence of any form I seed as assessed by both XRP and melting point (Figure 5 and 6). With 10% of form 1 remaining in the initial mixture, further change was only observed after 48 h at room temperature and hence a robust operating window was assured. Ultimately an isolation process was designed whereby the final IPAc layer containing product was reduced in volume above 40 °C, leading to crystallization of form 2 upon further addition of heptane. Filtration was also carried out at above 40 °C, leading to a 96% isolated yield of >99.6% purity final API (Scheme 16).

Scheme 16. Optimized coupling and isolation of MK-4305 Form 2

Summary of PhII Synthetic Approach With the improvements detailed in the above sections for the asymmetric hydrogenation, early steps, and end game process, the suvorexant synthesis became suitable for implementation on several hundred kilogram scale and was reliable and reproducible to support the project through PhII clinical trials (Scheme 17) (34).

Scheme 17. Summary of PhII Synthetic Approach to Suvorexant 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.

Commercial Synthesis Development To this point a robust and readily scalable process had been developed that was amenable to the synthesis of hundreds of kilograms of MK-4305. However, the team now faced the question of whether this synthesis was appropriate for execution on the commercial scale, in which metric ton quantities might be required. Although the discovery of an asymmetric reductive amination had greatly improved overall yield and reduced waste, there remained two considerations for that reaction that merited further evaluation of the process:

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1.

2.

The asymmetric reduction used a high loading (3 mol %) of a ruthenium catalyst that impacts cost of goods and long term sustainability of the process This reaction is run preferentially in dichloromethane, which is disfavored as a process solvent due to its environmental impact.

Therefore the team embarked on two parallel efforts to solve the perceived liabilities of the existing synthesis. First we will discuss efforts to revise the synthesis to remove the metal-catalyzed asymmetric reductive amination in favor of an enzymatic approach, which entailed an entirely new sequence to the chiral diazepine core. Second we will review investigations into the mechanism of the existing reductive amination, in order to understand what factors might be limiting to catalyst loading and solvent selection. Development of an Enzymatic Approach The key structural feature of MK-4305 is the core chiral diazepane ring 22, which had been assembled using the aforementioned ruthenium-catalyzed asymmetric reductive amination. This method achieved high levels of enantioselectivity (94% ee), but required the use of a transition metal catalyst and dichloromethane as solvent, both of which we hoped to eliminate to lessen the environmental impact of the process. Therefore an alternative bond disconnection was envisioned taking advantage of biocatalytic transamination technology (Figure 7) (35–37). Specifically, an asymmetric transamination of a ketone 40 that bears a suitable leaving group could set the stage for a cascade transamination (TA)/medium ring annulation, completing the diazepane system (38). By this time there were already some reports demonstrating the utility of enantioselective biocatalytic reactions in pharmaceutical applications, in particular an improved synthesis of sitagliptin using this same enabling transamination reaction (Scheme 18), which inspired us in our thinking (39). The hope was that this enzymatic approach might offer advantages both in reaction performance and environmental sustainability. Furthermore, if the leaving group for the annulation could be derived from activation of an alcohol, then compound 40 could be constructed from inexpensive starting materials including ethanolamine (41) and the previously employed methyl vinyl ketone. This would then offer some advantage over the existing process, where the analogous intermediate was made using N-Boc ethylene diamine 5, a more expensive raw material. 23

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Figure 7. Proposed cascade transamination/ring annulation strategy.

Scheme 18. Enantioselective Transamination Approach to Sitagliptin To effectively compete with the ruthenium-catalyzed asymmetric reductive amination process, we needed a rapid and scalable approach to ketone 40 that would also allow us to evaluate several possible leaving groups to optimize the ring annulation, presuming a suitable transamination could be achieved. We were able to apply a strategy similar to the existing process, in which an acid catalyzed condensation of commercially available phenol 19 with trimethyl orthoformate yielded benzoxazole 42 (Scheme 19) (40, 41). The reaction stream of 42 was then partially distilled to remove methanol and used directly in a subsequent lithiation/ bromination sequence to furnish bromide 43. This activated benzoxazole was then treated with ethanolamine to provide alcohol 44 without the use of chromatography or intermediate isolations in 90% overall yield from phenol 19 (42–46).

Scheme 19. Chromatography-Free Synthesis of Ketone 46 The next step to the targeted ketone substrate required an aza-Michael addition to methyl vinyl ketone. While this was similar to an analogous reaction for the existing process, there is one critical distinction – whereas in the existing process 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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the conjugate addition can effectively occur only on one nitrogen due to the use of a Boc protecting group on the diamine backbone. Here chemoselectivity between nitrogen and oxygen addition was targeted in the absence of protecting groups. An additional complexity is that the Michael addition itself is reversible, and while chemoselectivity may be high at low conversions, it would invariably worsen as the reaction progressed. Additionally, under basic conditions the benzoxazole heterocycle itself is subject to hydrolysis over time. To overcome this problem, a wide variety of bases and solvents were surveyed in a high-throughput multi-well format, including organic and inorganic bases with varying pKas. These initial screens identified alkali carbonates in polar solvents (e.g. DMF) as being near optimal displaying >10:1 regioselectivity. However results on scale were capricious, likely due to the low solubility of the carbonates under the conditions studied, with most of the activity ascribed to a dilute soluble population of base. We soon realized the variability of results tracked roughly with residual water in our laboratory solvents, and that the actual active base was likely to be likely trace hydroxide from adventitious water. We evaluated concentrated sodium hydroxide as a catalyst and found that it effectively promoted the aza-Michael in 95% yield and 42:1 selectivity (N to O) as a 1 mol % additive. Gratifyingly, this aza-Michael addition could be telescoped with functionalization of free alcohol 45 as the corresponding mesylate (46) without any intermediate workup or isolation. This was a principal benefit of the low loading of base in the aza-Michael addition, such that only a small excess of mesyl chloride was required to consume the sodium hydroxide present to enable a through process. These conditions were then applied to a one step synthesis of mesylate 46 from alcohol 44, following the aza-Michael with mesylation and crystallization of the product in 81% yield (Scheme 19). We now have an efficient method to access sulfonate esters, such as mesylate 46, from inexpensive starting materials. This put us in a position to evaluate the crucial transamination reaction with an opportunity to match or improve the economics of the existing process. However, we came to quickly realize that a highly selective transamination of the ketone (40 to 47, Scheme 20) is only the first of a number of outcomes that might occur in the course of a tandem transamination and ring annulation sequence. We had seen from earlier research on the asymmetric reductive amination process that the benzoxazole is labile to nucleophilic attack and postulated that the transamination event might initiate an intramolecular ring opening (47 to 48). These same studies had demonstrated the propensity of diazepane 22 to undergo an unusual rearrangement of the diazepane ring (22 to 25), which in our studies occurred in a pH range of 3 to 12. We reasoned that at a low pH (< 3) diazepane 22 predominantly exists in a doubly protonated form, and that above pH 12 it exists as a free base. As such, extreme pH regimes suppress intermediate 49, which arises from a singly protonated species, and therefore the undesired rearrangement of the diazepane ring is also suppressed under these conditions. However, based on literature precedent and our own experiences, we expected that the enzymatic transamination would perform poorly outside of near-neutral pH ranges, due in part to instability of the enzyme itself. 25

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In addition we expected that the sulfonate ester leaving group in the annulation reaction would be susceptible to hydrolysis under basic reaction conditions, however non-acidic conditions would be required for the annulation step to proceed. A successful process would have to balance these competing factors.

Scheme 20. Competing Pathways during the TA/Ring Annulation Sequence The transamination of ketone 46 was evaluated with a panel of commercially available enzymes, though unfortunately most provided no significant conversion. One exception was ATA-117 (Codexis), which gave a modest 8% conversion of the ketone. However, this encouraged us to evaluate the (R)-selective transaminase developed for the aforementioned sitagliptin process, as this was an evolved variant of ATA-117 developed specifically to accommodate a sterically larger substrate than the natural enzyme (47–49). Indeed, this enzyme provided both good conversion and high enantioselectivity in the transamination of 46 (Table 4, entry 1, >99% ee). To our delight, we also established proof of concept that the desired ring annulation to form 22 could occur in situ without further manipulations. However, we observed significant amounts of guanidine 48 (nonproductive path a depicted in Scheme 20) under a variety of conditions, suggesting that competition between SN2 attack on the sulfonate ester and ring opening of the benzoxazole was finely poised. In addition we also observed the diazepane rearrangement product 25, which was expected in light of observations previously discussed, but which imposed limits on the yield of the reaction. Finally, yield was also impacted by hydrolysis of the sulfonate ester activating group. To mitigate these side reactions, a range of sulfonate esters was examined under identical conditions (Table 4, entries 2-5), but in the end mesylate 46 was still superior in overall profile. Interestingly, the tosylate variant displayed better selectivity against the formation of guanidine 48 (X = OTs) (entry 2), but nevertheless slower reaction kinetics and poorer substrate stability provided diminished yield. Replacing the sulfonate ester with an alkyl chloride eliminated the substrate stability issue and indeed provided clean reactivity in the transamination (entry 6). But only the undesired guanidine 48 (X = Cl) arose as a product because the chloride was insufficiently reactive to compete with the benzoxazole ring opening pathway. 26

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Modulating the pH of the reaction to encompass more basic conditions did lessen the formation of 48 and 25 (entries 7-10), but a practical upper limit of pH 10 was encountered due to stability issues for both the sulfonate ester and indeed the enzyme itself (entries 9-10). The final conditions chosen in this study leveraged both a moderate pH and a slow addition protocol for 46 to minimize substrate decomposition pathways, ultimately delivering diazepane 22 in 71% yield and greater than 99% ee (entry 11) (50).

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Table 4. Optimization of the Transamination Protocol

With this unusual tandem transamination/medium ring annulation approach to produce the core diazepane in hand, we now had a new standard by which to judge the existing supply route. That is, with the use of halogenated solvents or heavy metal catalysts eliminated in this alternative route, a business case for a more sustainable large-scale commercial-supply based on the ruthenium-catalyzed

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.

asymmetric reductive amination would require greener solvent choice and a lower loading of the catalyst. We will next discuss our efforts to achieve these goals.

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Revisiting the Asymmetric Hydrogenation: Mechanism and Optimization We have earlier described efforts to identify and improve the rutheniumcatalyzed asymmetric reductive amination (Scheme 21), which had proven competent for furnishing clinical supply during development. However, the focus for a potential commercial route using this approach relied on lowering the amount of the ruthenium catalyst required under the existing conditions, perhaps by improving reaction rate or by eliminating unproductive reaction pathways of the catalyst. In so doing, we hoped to then demonstrate applicability of the optimized chemistry in non-halogenated solvents. To accomplish this, we began a series of studies meant to examine the kinetic behavior of the reaction network (33).

Scheme 21. Reductive Amination Approach

A general mechanism for the reductive amination based on analogy to literature is shown in Figure 8. Briefly, we thought of the reaction as occurring in three stages, from insertion of formic acid into catalyst 33 to yield Ru-formate 49 to extrusion of carbon dioxide forming Ru-H species 50, and ultimately reduction of the substrate (21) regenerating the catalyst. We hoped that a kinetic analysis would reveal the rate order of substrate and reagents while providing insight into what reaction features might play into the rate-limiting step of this process. Two observations became immediately apparent upon conducting the reaction under standard conditions: • •

the overall reaction displayed first-order kinetics. the reaction appeared to be zero-order in the substrate (21).

This would require that regeneration of the ruthenium hydride were rate limiting rather than the reduction of the substrate. There was precedence using related tethered ligand derivatives of 33 in which it was shown that the reaction may be zero-order in substrate at low conversions when the substrate is at a high concentration, but then become first-order at higher conversions when the reduction of the substrate becomes rate-limiting rather than catalyst regeneration (51). In this system, reaction rate increases with a decreasing initial concentration of substrate, a phenomenon that is consistent with product inhibition. 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.

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Figure 8. Initially proposed catalytic cycle for asymmetric transfer hydrogenation.

However, it was quickly shown that this was not the case, and in general secondary amines had no impact on the performance of the reaction. The kinetic phenomena might also be explainable by reversibility in the transfer hydrogenation; however enantiopure product resubjected to reaction conditions did not racemize. Finally, it was considered that the extrusion of carbon dioxide (compound 49 to compound 50) might be reversible. This was in contrast to the general understanding from the literature at that time (52). However by adding carbon dioxide into the reaction system, a substantial loss in reactivity could be induced seemingly validating our last standing hypothesis. The apparent equilibrium between formate 49 and hydride 50 was studied further by 1H NMR spectroscopy, in which it was revealed that in the absence of substrate they quickly formed in a 95:5 ratio (49:50) that did not change even upon extended aging. Again, this was counterintuitive as it had been anticipated that the entropic gain realized by extrusion of carbon dioxide would render formation of 50 irreversible in practical conditions, but empirical results continued to suggest otherwise. Conclusive evidence for the equilibrium process was established when the known RuH(p-cymene)(TsDPEN) (51) (53) was generated and put in a CD2Cl2 solution that was then exposed to CO2 (Scheme 22). 1H NMR immediately showed resonances fully consistent with the Ru-formate complex (52) together with complete consumption of the Ru-hydride species. 29

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Scheme 22. Conversion of Ru-Hydride 51 to Ru-Formate 52 under CO2.

If we then incorporate this finding into the initially proposed catalytic cycle, we arrive at a modified scheme shown below (Figure 9). Here the concentration of CO2 will influence the rate of the reverse reaction indicated as k-2, and indeed this was verified experimentally. As such, the team implemented a simple engineering solution in order to improve reaction rate: by purging CO2 continuously with nitrogen throughout the process, regeneration of the catalyst is no longer limiting at higher conversions.

Figure 9. Modified catalytic cycle reflecting CO2 catalyst inhibition.

30 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 the improved reaction rate a number of other reaction benefits were realized (Scheme 23):-

1. 2.

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3.

Reduction of the catalyst loading to 2 mol %, reducing cost and material consumption. An alternative solvent system of acetonitrile and toluene rather than dichloromethane can be used. The improved fundamental reactivity allowed us to select solvents that were slightly inferior with regard to overall reaction rate but substantially superior with regard to environmental impact. No requirement to isolated the MSA salt of 21, instead Boc removal from 18 was carried out using HCl in the desired reaction solvent, toluene, and a solution of 21 taken directly into the hydrogenation reaction after phase separation.

Scheme 23. Final Hydrogenation Conditions

With an improved asymmetric transfer hydrogenation in hand that addressed both catalyst loading and solvent choice concerns, we were now prepared to select this approach as a commercial route to MK-4305. Also, the observations that arose from this mechanistic study appear more broadly applicable to this class of Noyori-type formic acid-driven transfer hydrogenations (54), exemplifying the unexpected value that can come from a detailed reaction study.

Summary of Process Development Efforts Herein we have described a series of efforts to design and optimize syntheses of MK-4305, in order to reduce waste and cost of goods, eliminate chromatography and reduce isolations, increase yield and process robustness, and ultimately identify the best route to supply the API commercially with minimal environmental impact (Scheme 24). 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.

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Scheme 24. Final Manufacturing Synthesis of MK-4305

As MK-4305 advanced from Medicinal Chemistry to early clinical trials, then to larger clinical studies, and finally to commercialization, the challenges and basic requirements of a suitable synthesis evolved. However, as we continued to study the chemistry, our knowledge evolved with the synthesis and we were able to deliver improvements that overcame the needs of each phase of development. Critical milestones in the development of the commercial process include the following: 1. 2. 3. 4. 5.

6.

Replacing chiral chromatography from the Medicinal Chemistry synthesis with a classical resolution. Streamlining step count and protecting groups by using the benzoxazole heterocycle as the starting point for a new synthesis of the diazepine ring. Doubling yields of the triazole acid while eliminating chromatographic isolation. Installing a direct isolation of the desired form of MK-4305 in the final step, removing an intermediate form turnover operation. Discovering and optimizing an unprecedented catalytic asymmetric reductive amination, and continually improving the process through mechanistic investigation. Developing alternative synthetic approaches, including an asymmetric, enzymatic transamination to deliver the chiral diazepine.

The path to development of the final manufacturing process involved several unexpected discoveries, unusual diversions, and dedicated efforts to continue to improve the chemistry to its maximum even when a workable solution was already in hand. It is the defining challenge to the process chemist to devise a synthesis that will withstand the test of time; that will continue to serve as the benchmark for efficiency and cost even as chemical technology evolves and new chemists in different parts of the world consider alternative approaches (55). A manufacturing process is never perfect, and we still wonder whether this reaction could have 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.

been more selective or if that reaction could give a better yield. However, these questions are just one facet of the joy of chemical synthesis, and we hope the journey of the MK-4305 process will inspire others who engage with the demands of contemporary chemistry.

Acknowledgments

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We would like to thank our many colleagues in the Merck Research Labs and Merck Manufacturing Division who contributed tirelessly to this work, in addition to those colleagues named in references below.

References 1. 2.

3.

4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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15. Nishino, S.; Ripley, B.; Overeem, S.; Lammers, G. J.; Mignot, E. Lancet 2000, 355, 39–40. 16. Peyron, C.; Faraco, J.; Rogers, W.; Ripley, B.; Overeem, S.; Charnay, Y.; Nevsimalova, S.; Aldrich, M.; Reynolds, D.; Albin, R.; Li, R.; Hungs, M.; Pedrazzoli, M.; Padigaru, M.; Kucherlapati, M.; Fan, J.; Maki, R.; Lammers, G. J.; Bouras, C.; Kucherlapati, R.; Nishino, S.; Mignot, E. Nat. Med. 2000, 6, 991–997. 17. Chemelli, R. M.; Willie, J. T.; Sinton, C. M.; Elmquist, J. K.; Scammell, T.; Lee, C.; Richardson, J. A.; Williams, S. C.; Xiong, Y.; Kisanuki, Y.; Fitch, T. E.; Nakazato, M.; Hammer, R. E.; Saper, C. B.; Yanagisawa, M. Cell 1999, 98, 437–451. 18. Willie, J. T.; Chemelli, R. M.; Sinston, C. M.; Tokita, H.; Williams, S. C.; Kisanuki, Y. Y.; Marcus, J. N.; Lee, C.; Elmquist, J. K.; Kohlmeier, K. A.; Leonard, C. S.; Richardson, J. A.; Hammer, R. E.; Yanagisawa, M. Neuron 2003, 38, 715–730. 19. Fujiki, N.; Yoshida, Y.; Ripley, B.; Honda, K.; Mignot, E.; Nishino, S. NeuroReport 2001, 12, 993–997. 20. Gotter, A. L.; Winrow, C. J.; Brunner, J.; Garson, S. L.; Fox, S. V.; Binns, J.; Harrell, C. M.; Cui, D.; Yee, K. L.; Stiteler, M.; Stevens, J.; Savitz, A.; Tannenbaum, P. L.; Tye, S. J.; McDonald, T.; Yao, L.; Kuduk, S. D.; Uslaner, J.; Coleman, P. J.; Renger, J. J. BMC Neurosci. 2013, 14, 90–106. 21. Roecker, A. J.; Cox, C. D.; Coleman, P. J. J. Med. Chem. 2015, 59, 504–530. 22. Coleman, Paul J.; Cox, C. D.; Roecker, A. J. Curr. Top. Med. Chem. 2011, 11, 696–725. 23. Whitman, D. B.; Cox, C. D.; Breslin, M. J.; Brashear, K. M.; Schreier, J. D.; Bogusky, M. J.; Bednar, R. A.; Lemaire, W.; Bruno, J. G.; Hartman, G. D.; Reiss, D. R.; Harrell, C. M.; Kraus, R. L.; Li, Y.; Garson, S. L.; Doran, S. M.; Prueksaritanont, T.; Li, C.; Winrow, C. J.; Koblan, K. S.; Renger, J. J.; Coleman, P. J. ChemMedChem. 2009, 4, 1069–1074. 24. Cox, C. D.; Breslin, M. J.; Whitman, D. B.; Schreier, J. D.; McGaughey, G. B.; Bogusky, M. J.; Roecker, A. J.; Mercer, S. P.; Bednar, R. A.; Lemaire, W.; Bruno, J. G.; Reiss, D. R.; Harrell, C. M.; Murphy, K. L.; Garson, S. L.; Doran, S. M.; Prueksaritanont, T.; Anderson, W. B.; Tang, C.; Roller, S.; Cabalu, T. D.; Cui, D.; Hartman, G. D.; Young, S. D.; Koblan, K. S.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. J. Med. Chem. 2010, 53, 5320–5332. 25. Cox, C. D.; McGaughey, G. B.; Bogusky, M. J.; Whitman, D. B.; Ball, R. G.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2009, 19, 2997–3001. 26. McGaughey, G.; Bayly, C. L.; Cox, C. D.; Schreier, J. S.; Breslin, M. J.; Pitzenberger, S.; Ball, R.; Coleman, P. J. J. Comput. Aided. Mol. Design 2014, 28, 5–12. 27. Yin, J.; Mobarec, J. C.; Kolb, P.; Rosenbaum, D. M. Nature 2015, 519, 247. 28. Yin, L.; Babaoglu, K.; Brautigam, C.; Clark, L.; Shao, Z.; Scheuermann, T.; Harrell, C. M.; Gotter, A. L.; Roecker, A. J.; Winrow, C. D.; Renger, J. J.; Coleman, P. J.; Rosenbaum, D. M. Nat. Struct. Mol. Biol. 2016, 23, 293–299. 34

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29. Winrow, C. J.; Gotter, A. L.; Cox, C. D.; Doran, S. M.; Tannenbaum, P. L.; Breslin, M. J.; Garson, S. L.; Fox, S. V.; Harrell, C. M.; Stevens, J.; Reiss, D. R.; Cui, D.; Coleman, P. J.; Renger, J. J. J. Neurogenet. 2011, 25, 52–61. 30. Stewart, G. W.; Baxter, C. A.; Cleator, E.; Sheen, F. J. J. Org. Chem. 2009, 74, 3229–3231. 31. We will discuss a possible mechanism for the formation of impurity 25 in a later part of this chapter. 32. The enantiomerically pure samples were prepared by small scale chiral separation of racemic 22. 33. Baxter, C. A.; Cleator, E.; Brands, K. M. J.; Edwards, J. S.; Reamer, R. A.; Sheen, F. J.; Stewart, G. W.; Strotman, N. A.; Wallace, D. J. Org. Process Res. Dev. 2011, 15, 367–375. 34. Strotman, N. A.; Baxter, C. A.; Brands, K. M. J.; Cleator, E.; Krska, S. W.; Reamer, R. A.; Wallace, D. J.; Wright, T. J. J. Am. Chem. Soc. 2011, 133, 8362–8371. 35. For a review on biocatalytic transformations see: De Wildeman, S. M. A.; Sonke, T.; Schoemaker, H. E.; May, O. Acc. Chem. Res. 2007, 40, 1260–1266. 36. Moore, J. C.; Pollard, D. J.; Kosjek, B.; Devine, P. N. Acc. Chem. Res. 2007, 40, 1412–1419. 37. Reetz, M. T. Ang. Chem., Int. Ed. 2011, 50, 138–174. 38. Mangion, I. K.; Sherry, B. D.; Yin, J.; Fleitz, F. Org. Lett. 2012, 14, 3458–3461. 39. Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J.; Devine, P. N.; Huisman, G. W.; Hughes, G. J. Science 2010, 329, 305–309. 40. For a related heterocyclic condensation see: Vechorkin, O.; Hirt, N.; Hu, X. Org. Lett. 2010, 12, 3567–3569. 41. For a related heterocyclic condensation see: Cioffi, C. L.; Lansing, J. J.; Yüksel, H. J. Org. Chem. 2010, 75, 7942–7945. 42. The synthesis of aminobenzoxazoles from benzoxazoles has also been reported via direct oxidative methods, see: Guo, S.; Qian, B.; Xie, Y.; Xia, C.; Huang, H. Org. Lett. 2011, 13, 522–525. 43. Froehr, T.; Sindlinger, C. P.; Kloeckner, U.; Finkbeiner, P.; Nachtsheim, B. J. Org. Lett. 2011, 13, 3754–3757. 44. Wertz, S.; Kodama, S.; Studer, A. Angew. Chem., Int. Ed.. 2011, 50, 11511–11515. 45. Li, Y.; Xie, Y.; Zhang, R.; Jin, K.; Wang, X.; Duan, C. J. Org. Chem. 2011, 76, 5444–5449. 46. Lamani, M.; Prabhu, K. R. J. Org. Chem. 2011, 76, 7938–7944. 47. For other applications of (R)-selective transaminases see: Truppo, M. D.; Turner, N. J.; Rozzell, D. Chem. Commun. 2009, 2127–2129. 48. Koszelewski, D.; Clay, D.; Rozzell, D.; Kroutil, W. Eur. J. Org. Chem. 2009, 2289–2292. 49. Koszelewski, D.; Tauber, K.; Faber, K.; Kroutil, W. Trends Biotechnol 2010, 28, 324–325. 35

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50. The mass balance consists of 45 from hydrolysis of the sulfonate and oligomers derived from intermolecular alkylation of amine 47. 51. Cheung, F. K.; Lin, C.; Minissi, F.; Criville, A. L.; Graham, M. A.; Fox, D. J.; Wills, M. Org. Lett. 2007, 9, 4659–4662. 52. Fujii, A.; Hashiguchi, S.; Uematsu, N.; Ikariya, T.; Noyori, R. J. Am. Chem. Soc. 1996, 118, 2521–2522. 53. Noyori, R.; Hashiguchi, S. Acc. Chem. Res. 1997, 30, 97–102. 54. Applying the CO2 purging approach to the transfer hydrogenation of acetophenone using this catalyst system produced a nearly 10-fold rate increase for a sealed reaction. 55. Zhang, T. Y. Chem. Rev. 2006, 106, 2583–2595.

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Chapter 2

Discovery of Novel Protein Fucosylation Inhibitors and Development of a Manufacturing Process To Prepare Inhibitor 6,6,6-Trifluorofucose Seb Caille1 and John G. Allen*,2 1Process

Development – Drug Substance Technologies, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States 2Therapeutic Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States *E-mail: [email protected].

In the manufacture of therapeutic monoclonal antibody proteins the level of fucosylated glycan in the product can affect efficacy and is a critical product attribute. Particularly for biosimilar antibodies, the fucosylation level may need to substantially match that of the reference product. 6,6,6-Trifluorofucose (1, fucostatin I), is a metabolic inhibitor of fucosylation that when added to cell culture reduces the fucosylation of recombinantly expressed antibodies in a concentration-dependent manner. A robust process for the synthesis of 1 from D-arabinose in 11% overall yield and >99.5/0.5 diastereomeric ratio was developed based on a key diastereoselective ruthenium-catalyzed tandem ketal hydrolysis-transfer hydrogenation reaction.

Introduction Antibody-dependent cell-mediated cytotoxicity (ADCC) is an innate immune response in which natural killer (NK) cells and oesinophils are targeted to pathogen-infected or cancer cells by IgG antibodies. The antibody binds to the target cell via its variable region and to the FcγRIIIa receptor of the effector cells via its constant fraction crystallizable (Fc) region. Therapeutic IgG1 antibodies bearing Asn297 glycans with lowered levels of fucosylation have been shown © 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.

to exhibit enhanced ADCC due to increased binding of the Fc region to fraction crystallizable γ receptor IIIa (FcγRIIIa, Figure 1). Afucosylated Fc glycan forms stronger interactions with FcγRIIIa glycan and favors a high affinity conformation around the Fc residue Tyr296 (1). Increased ADCC has been associated with improved in vivo efficacy in animal oncology models and in the clinic (2, 3), and therefore fucosylation level is an important product attribute to control in the manufacturing of mAb products (4). Protein fucosylation may be reduced by: • •

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• •

Manipulation of cell growth and production conditions. Screening for cell lines or clones producing protein with low fucosylation levels. Use of engineered cell lines in which a key enzyme involved in protein fucosylation is knocked out. Addition of selective or non-selective chemical inhibitors of fucosylation (5, 6).

Figure 1. Fucostatins and the ADCC response. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

Chemical inhibition is an appealing approach to reduce fucosylation during antibody manufacture in as much as other protein or cell growth attributes remain undisturbed. In addition, this method permits fucose levels to be controlled based on inhibitor dosage using cell lines that had been previously optimized. Inhibitors of fucosylation (7, 8), and other glycosylations (9–11) have been previously described, however, reported potencies are in the micromolar range. In addition to weak potency, many glycosylation inhibitors carry the risk of incorporation into the antibody product (12–14). 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.

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6,6,6-Trifluorofucose (1, fucostatin I, Figure 2) was discovered to be a fucosylation inhibitor and validated in our laboratories along with several other potent fucosylation inhibitors (15). The potential of fucostatin I was demonstrated in improved cell lysis activity of afucosylated anti-mesothelin (16) IgG1 monoclonal antibodies produced in the presence of this inhibitor. The wide-scale use of 1 in mAb manufacturing was anticipated and mandated the development of an efficient manufacturing process to prepare this molecule on several hundred gram scale. The target material (1) had previously been synthesized by Tokokuni and coworkers (17), however this route to the molecule did not meet our production requirements due to the use of stoichiometric toxic metals and the use of multiple chromatography steps, and due to the overall length of the synthetic route including several protection/deprotection steps. This chapter will focus on: • •

The identification and evaluation of 6,6,6-trifluorofucose (1) and other fucosylation inhibitors. A description of the manufacturing process developed to prepare 1 on several hundred gram scale.

Figure 2. Furanoside and pyranoside forms of 6,6,6-trifluorofucose (1).

Discovery of the Fucostatin Inhibitors of Fucosylation. Inhibition of GMD by GDP-Fucose and GDP-1 (1D) 6,6,6-Trifluorofucose (1, fucostatin I) was designed to function as a mechanism targeted inhibitor of fucosyltransferase wherein the oxocarbenium character of the transition state of the α-(1,6)-fucosyltransferase FUT8 would be destabilized by the electron withdrawing fluorine atoms (18). Fucose occurring in endogenous glycoproteins is generated starting from D-glucose by the de novo pathway shown in Figure 3. It is proposed that guanosine diphosphate-1 (GDP-1, 1D) is formed by the salvage pathway depicted in Figure 3 and in fact serves as an allosteric inhibitor of GDP-mannose 4,6-dehydratase (GMD), thus blocking the production of fucosylated glycoproteins. In the following sections we suggest that allosteric inhibition of GMD (19) by 1D rather than inhibition of fucosyl transferases is the primary mechanism of action. The acetylated carbohydrate 2 39

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was also prepared and evaluated as a fucosylation inhibitor. It is proposed that this material (2) diffuses into cells as a prodrug, undergoes deprotection to generate 1, and leverages the fucose salvage pathway to form the active metabolite 1D (Figure 3). Compound 1 on the other hand likely enters the cell by active transport (20).

Figure 3. Formation of 1D and Inhibition of GMD. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

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Inhibition and Rescue of Cell Surface Fucosylation Inhibitors were initially screened for their ability to inhibit cell surface fucosylation in Chinese hamster ovary (CHO) cells. In an automated assay, cell surface fucosylation was determined by Lens Culinaris A (LCA) lectin binding to fucose-containing glycans as detected with a fluorescein isothiocyanate (FITC)-antibody conjugate and measured by fluorescence assisted cell sorting (FACS, Figure 4a). Cell surface glycans could be completely defucosylated by treatment with 2. In treated cells, cell surface fucosylation could be restored (rescued) by addition of L-fucose or L-fucose per-O-acetate in a dose-dependent manner. Complete rescue resulted at 100 µM of L-fucose or L-fucose per-O-acetate, suggesting that FUT8 is not inhibited in cells treated with 2, although studies on purified FUT8 were not performed. Importantly, D-fucose, which is not a substrate for fucose transporters nor a substrate for fucose kinase, failed to rescue inhibition by 2 (Figure 4b).

Cocrystallization of GDP-Fucose and 1D with GMD Cocrystal structures of GDP-fucose (PDB 5IN5) and 1D (PDB 5IN4) with GMD were obtained, and for the first time showed the occupied allosteric fucose binding site of the enzyme (Figure 5). The crystal structure asymmetric unit contained four GMD monomers in a homotetramer with an overall appearance of a dimer of dimers. Each GMD monomer consists of an N-terminal cofactor binding domain containing an NADP/H cofactor and a C-terminal substrate binding domain containing GDP. The NADP/H and GDP binding sites formed at the interfaces between two GMD-GMD symmetrical dimers. Each GMD-GMD dimer also contains two molecules of 1D at allosteric sites formed at the dimer interface. 1D forms simultaneous direct and water-mediated hydrogen bond contacts with two different monomers, likely stabilizing this interface. 1D is bound in a horseshoe conformation, and the nucleobase and trifluoromethyl group of the inhibitor interact with one GMD monomer while the phosphate and ribose groups interact with the other monomer. A loop of residues 69-77 near the allosteric site is disordered in a known apo GMD structure (PDB 1T2A, unpublished) and in our other structures lacking inhibitors (data not shown). In the presence of 1D this loop becomes ordered and packs above the inhibitor. The guanine nucleobase forms a base stacking interaction between His75 in the allosteric loop and Phe60. The trifluoromethyl group of 1D is buried in a small pocket and compared to GDP-fucose forms more extensive Van der Waals (vdw) interactions with near-by hydrophobic residues. These increased vdw interactions are in all likelihood responsible for the gain in potency over the natural inhibitor GDP-fucose (KD 11 vs. 35 µM respectively) as both molecules bind in identical conformations with a slight 0.5 Å shift of the larger trifluoromethyl group.

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Figure 4. (a) Dose-response inhibition of cell surface fucosylation by 2. (b) Rescue of cell surface fucosylation by L-fucose and L-fucose per-O-acetate, LCA lectin binding. 20 µM 2 gives 14.0 ± 3.3% cell surface fucosylation. Error bars represent the standard deviation. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

In order to explain the unexpectedly high potency of 2, the molecule was initially proposed to covalently bind to GMD (21–23), but biophysical data were not consistent with that hypothesis. The high potency of 2 and low dissociation constant of 1D were instead explained by contacts revealed in the 1D/GMD cocrystal structure as described above. Overlap with GDP-fucose was perfect, but for the exception of a noted deeper incursion of the trifluoromethyl group into a pocket coated with alanine and leucine. The additional vdw interactions result in a reduced dissociation constant for 1D compared to GDP-fucose. The activity of 2 is remarkable compared to the related difluoro (3) and monofluoro (4) analogs (Figure 6). These analogs were less potent in the cell surface assay (4.7±1.9 µM and 6.6±5.5 µM, respectively), underscoring the importance of the trifluoromethyl group in potently binding GMD. 42 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. (a) 1D (PDB 5IN4) cocrystallized with GMD. Four molecules of 1D bind to the GMD tetramer at the interface of a dimer pair as indicated by the arrows. (b) 1D in the GMD binding pocket, partial space-filling model. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society. 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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Figure 6. Difluoro and monofluoro analogs of 2.

Metabolite Analysis and Design of Non-Incorporating Fucosylation Inhibitors As shown in Figure 3, 2 diffused into cells upon dosing and was de-esterified to generate 1. This material (1) was subsequently converted to 1C by fucose kinase, and then to 1D by GDP-β-L-fucose pyrophosphorylase (GFPP). Both metabolites 1C and 1D were identified by selective reaction monitoring – mass spectrometry (SRM-MS) in cell lysate (Figure 7a for 2). 1D was synthesized by chemo-enzymatic methods and was shown to bind GMD by surface plasmon resonance (SPR, KD 11 µM, Figure 7d). GMD as the target of inhibition was consistent with the accumulation of GDP-mannose (Figure 7b for 2), as well as the disappearance of GDP 4-keto-6-deoxy-mannose (data not shown) and GDP-fucose in the cell. These data together with the rescue data vide supra suggested FUT8 inhibition is not a significant contributor to the overall inhibition of fucosylation. As will be discussed in more detail in an upcoming section, dosing CHO cells with 2 led to low levels of 1 being incorporated into the glycans of expressed proteins by replacement of fucose. Replacing the exoanomeric oxygen of 1 and 2 with a methylene group was expected to block inhibitor incorporation by preventing the corresponding GDP sugars from being substrates in glycosylation reactions. By preparing these methylene analogs as phosphonates we hoped they would enter the salvage pathway as substrates of GFPP. Thus fucose phosphonate analogs 5 and 6, and 6,6,6-trifluoromethylfucose phosphonate analogs 7 and 8 were prepared as suitably protected prodrugs (24) (Figure 8). We were pleased to find that the phosphonate prodrug 5 was deprotected in the cell and gave rise to a phosphonate GDP metabolite 5B as demonstrated by identification of these compounds in cell lysate by SRM-MS (Figure 7a for 5). The GDP-phosphonate 5B was synthesized and was found to bind to immobilized GMD by surface plasmon resonance (SPR, Figure 7d, KD 9 µM). Protein incorporation was not observed for phosphonate 5, and this inhibitor blocked protein and cell surface fucosylation (IC50 18.1±6.5 µM). The non-native α-anomer 6 is not considered to be a substrate for the fucose salvage pathway and it was inactive in the cell surface assay. Another analog of 5 that combined the phosphonate strategy and the 5-CF3 group of 1 and 2, β-trifluoromethylphosphonate 7, formed the corresponding GDP metabolite 7B but failed to inhibit fucosylation. This observation may have been due to inactivity at GMD as evidenced by a lack of GDP-mannose accumulation (Figure 7b for 7).

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Figure 7. (a) Log(AUC) of metabolites observed by SRM-MS in lysate from cells treated with fucosylation inhibitor analogs 2, 5, 7 and 8. (b) Relative concentrations of GDP-mannose and GDP-fucose observed by SRM-MS in lysate from cells treated with fucosylation inhibitor analogs 2, 5, 7 and 8, and normalized to 100%. (c) Chromatographic data for metabolites observed by SRM-MS in cells treated with 2 and arbitrarily normalized to 100% intensity. (d) Derived steady-state SPR dissociation curves for 1D (circles, KD 11 µM), 5B (triangles, KD 9 µM), GDP-mannose (diamonds, KD 51 µM), GDP-fucose (open triangles, KD 35 µM),. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

Figure 8. Exoanomeric CH2 fucosylation inhibitor analogs and metabolites. 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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By contrast, the corresponding non-native α-anomer 8 was a potent inhibitor of cell surface (IC50 7.0±7.0 µM) and protein fucosylation. Surprisingly, the glycans of protein produced in the presence of 8 showed some incorporation of 1. SRM-MS analysis of lysates from these cells identified the corresponding deprotected phosphonate (8A), but relatively little of the GDP-phosphonate (8B). Instead, the phosphate metabolite was observed in this case (1D). Cellular (25) hydrolysis of the α-phosphonate 8 to reducing sugar 1 would explain the similarity in metabolic data provided by 8 and 2, where both 8 and 2 give rise to the same metabolite (1) (26). This interesting hypothesis has some precedent in the cleavage of C-glycosides in bacterial cell-free lysate (27) and in phosphonate hydrolysis by bacterial hydroxyethylphosphonate dioxygenase and the protein product of the bacterial PhpD gene (28, 29), and deserves further study.

Blocking Fucosylation of Expressed Proteins In order to study the effect of the fucostatins on fucosylation of expressed proteins, anti-TRAIL2 receptor (TR-2) immunoglobulin G1 monoclonal antibodies (IgG1 mAbs) (30) and anti-mesothelin (MSLN) IgG1 mAbs (16) were prepared in CHO cells using a single dose of 2 in DMSO on day 0. Protein fucosylation could be titrated with different doses of 2, with an EC50 of ~4 µM (Figure 9a). No changes were observed up to the top concentration of 20 µM of 2 in the cumulative growth, viability or productivity behavior of treated cells, or in other protein characteristics (Figure 9b). Selective inhibition of fucosylation was a design of the targeted fucostatins. As was seen for cell surface fucosylation (Figure 4a), fucosylation of produced protein could also be rescued by a single dose of fucose on day 0 via the GMD-independent salvage pathway (Figure 9d). Hydrophilic interaction liquid chromatography (HILIC) analysis found 1 incorporated in lieu of fucose in about 0.5% of total glycans (white triangles in Figure 9e). Both compounds 1 and 2 gave similar results in these studies. Although compound 2 slowly hydrolyzed to 1 in cell culture medium, this did not affect the results observed as both 1 and 2 are cell penetrant. ADCC activity was tested in vitro for anti-MSLN antibodies targeting natural killer (NK) cells from human donors to MSLN expressing cancer cells. Target cell lysis was improved for Abs produced in the presence of increasing amounts of 2 (Figure 9c), an observation consistent with reduced fucosylation levels. Additionally, NK cells expressing low affinity V158F fraction crystallizable γ receptor IIIa (FcγRIIIa, V158F polymorphism) showed good efficacy on activation by afucosylated anti-MSLN antibodies. The grey bars represent cell lysis EC50’s for lower antigen expression CAPAN2 (pancreatic adenocarcinoma) cells treated with natural killer (NK) cells isolated from a human donor harboring the low affinity FcγRIIIa allele. Efficacy is significantly reduced for this system compared to high antigen expressing N87 (gastric carcinoma) treated with high affinity Val158-FcγRIIIa allele NK cells (black bars). However, using antibodies expressed in the presence of 2, less than 0.1 µg/mL of anti-MSLN antibodies with 20% afucosylated glycans was needed to lyse 50% of CAPAN2 target cells 47

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by low affinity NK cells. The 80% fucosylated antibodies resulted from a single 2 µM dose of 2 on day 0 of a 10 day production run. Patients with high and low affinity FcγRIIIa receptor alleles show different responses to ADCC therapeutics (31). One advantage of afucosylated therapeutic IgG1 antibodies is improved responses in all patient populations (32, 33), as modeled in this assay.

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Glycan Incorporation Comparison for Fucosylation Inhibitors Besides reduced fucosylation, HILIC analysis of proteins expressed in the presence of 2 also displayed low levels of incorporation of 6,6,6-trifluorofucose in place of fucose (Figure 9e). No other aspects of cell growth including cumulative viable cell density, mAb titer, and specific productivity in the expression system were impacted by dosing with 2. Low incorporation of 2 suggests that 1D is a weak substrate of fucosyltransferase FUT8, possibly due to inductive electron withdrawing destabilization of the glycosylation transition state (34). Higher levels of inhibitor incorporation occurred with difluorofucose (3) and monofluorofucose (4, Figure 6 and Table 1), compounds that bear fewer electron-withdrawing fluorine atoms and may not be as effective in destabilizing the cationic FUT8 transition state. Functional enzymatic inhibition of fucosyl transferases by the fucostatins or their metabolites was not directly tested in this study, but seems unlikely, or at least less significant, since fucosylation was fully rescued in treated cells with the addition of fucose. Phosphonate sugars 5, 6, 7, and 8 (Figure 8) were designed to eliminate glycan incorporation by substituting a methylene group for the exoanomeric oxygen. This was expected to prevent the corresponding GDP sugars from being substrates in glycosylation reactions. The phosphonates were prepared as acetate/pivaloyloxymethyl (POM) esters that diffuse into the cells as prodrugs (24) and be cleaved by intracellular esterases. When prepared and tested, β-fucosephosphonate 5 (fucostatin II) u deprotection in cells, form the GDP derivative, 5B and, similar to 2, accumulate GDP-mannose and bind GMD (KD 9 µM, SPR, Figure 7). Unlike 1, 5 was only cell penetrant in its prodrug form. In protein production experiments, due to hydrolytic instability of the prodrug 5 in the production growth medium, 5 was added as a DMSO stock to fresh medium every day starting on day 3. Potent inhibition of mAb fucosylation occurred with an EC50 of ~30 µM. By this protocol (35) mAbs were produced with varying amounts of fucosylation and showed no indication of incorporated inhibitor (Figure 10a). Despite showing no influence on cell viability in a propidium iodide assay up to 100 µM, in the production assay, inhibition of cumulative cell density, titer and specific production was apparent at >40 µM due to loss of cell viability (Figure 10b). The non-native α-anomeric stereoisomer 6 was inactive in the cell surface assay and was not further tested.

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Figure 9. (a) 2 was added on day 0 of production at the levels indicated on the x-axis to an anti-TR-2 mAb -expressing CHO cell-line grown under fed-batch production conditions. Glycan analysis was performed on the produced antibodies (circle = percent fucosylated glycans, square = percent afucosylated glycans, triangle = percent high mannose glycans; open circle = percent glycans with incorporated 6,6,6-trifluorofucose). (b) Cumulative viable cell density, titer and specific productivity obtained under different concentrations of 2 in three different cell lines (each data point represents an average of duplicates). (c) Anti-MSLN mAb antibodies produced in the presence of varying amounts of 2 and with varying levels of fucosylated glycans (filled circles) were purified and assayed in a calcein-based ADCC assay with cells containing high (N87; black bars) and lower (CAPAN2; gray bars) target expression. EC50 values represent the amounts of anti-MSLN mAb needed to lyse 50% of target cells at the indicated anti-MSLN mAb fucosylation levels. (d) CHO cells expressing anti-TR-2 mAb were grown under fed-batch production conditions. Compound 2 was added to the cells on day 0 at 20 µM concentration. Production cultures were also dosed with 1 mM fucose on either day 0 or day 6. The glycan profile of the produced anti-TR-2 mAb antibodies was measured (black bars = percent fucosylated glycans, gray bars = percent afucosylated glycans, white bars = percent high mannose). Each bar represents and average of duplicates. (e) HILIC Asn297 glycan analysis of Ab produced in the presence of 250 µM 2 showing 98% afucosylated glycans and 0.7% incorporation. Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

Although 5 effectively blocked fucosylation with the advantage of undetectable incorporation, it was about ten-fold less potent than 2 in the cell surface assay (FITC-LCA IC50 18.1±6.5 µM). To improve potency we attempted to take advantage of additional vdw contacts about a 5-CF3 group as for 1/1D. Compound 7, the trifluoromethyl analog of 5, was inactive, even though it did give rise to the required GDP metabolite 7B. This suggests that 7B is not a ligand of GMD, the combination of both exo-anomeric methylene and 5-CF3 perhaps being too dissimilar from native GDP-fucose to bind. Further mechanistic studies could not be completed due to the chemical instability of 7B.

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Figure 10. (a) The impact of repeated additions of 5 on cell culture performance using medium exchanges initiated on day 3. Compound 5 was added to the cultures daily at the levels indicated on the x-axis. (b) Cumulative viable cell density, titer and specific productivity obtained in the presence of different concentrations of 5 (each data point represents an average of duplicates). Reproduced with permission from ref. (15). Copyright 2016, American Chemical Society.

Considering that compound 7 was inactive, it was surprising to observe potent inhibition of cell surface and protein fucosylation for the α-isomer of 7, compound 8 (FITC-LCA IC50 7.0±7.0 µM). An analysis of the metabolites from cells treated with 8 found deprotected 8A, but less of the 8B GDP derivative was detected. In addition to the phosphonate metabolite 8A, phosphate metabolite 1C was also observed. Consistent with the formation of the same metabolite, low levels of incorporation of 1 were observed in expressed proteins. These results suggested the phosphonate group of 8 may be hydrolyzed in the cell to give 1 in situ (27–29). Rigorous purification and characterization of 8 eliminated the consideration of contamination of the testing compound. Since similar metabolites were not found in cells treated with 7, it is proposed that hydrolysis of the phosphonate α-isomer may be accelerated compared with hydrolysis of the β-isomer 7 due to the anomeric effect. Similar in situ hydrolysis of the α-isomer 6 would not have been detected in these studies. 51

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Table 1. Phosphate and Phosphonate Metabolite Data

Data for fucostatins and other analogs are summarized in Table 1. As described above, 6,6,6-trifluorofucose peracetate (2) was a potent inhibitor of protein fucosylation as measured by FITC-LCA staining of cell surface glycoproteins. Compound 2 also inhibited the fucosylation of the glycans of expressed proteins as determined by hydrophilic interaction chromatography (HILIC) analysis. Metabolite identification from the lysate of treated cells displayed that 2 was processed through the fucose salvage pathway as evidenced by formation of 1C and 1D as metabolites. The accumulation of intracellular GDP-mannose and loss of GDP-fucose, as well as the rescue of fucosylation by added fucose, suggests the inhibition of GMD by 1D. As corroborating evidence, 1D was found to bind to GMD (KD 11 µM, SPR) and was cocrystallized in 52 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 GMD allosteric site (19). It’s possible fucosyl transferases are also weakly inhibited by 1D as has been reported for other fucosylation inhibitors (7, 8), but inhibition of purified FUT8 was not determined for the fucostatins or their metabolites. The improved potency of the fucostatins compared to the previously reported fucosylation inhibitor, 2-deoxy-2-fluorofucose (2FF) is also shown in Table 1. GDP-2FF has been reported to have ~50 µM inhibition of GMD and ~200 µM inhibition of FUT8 (8). In the FITC-LCA assay 2FF had >100 µM activity in inhibiting cell surface fucosylation. A second reported fucosylation inhibitor, peracetylated 5-thiofucose (7) (5T-Fuc) inhibits sialyl-Lewisx on HepG2 cells with EC50 = 21 µM. Fucostatin potency of 0.87-18.1 µM compares favorably to previously reported inhibitors.

Development of a Process To Manufacture 6,6,6-Trifluorofucose. Published Route and a Novel Strategy. Among the several analogs characterized as potent and selective inhibitors of protein fucosylation, 6,6,6-trifluorofucose (1) was particularly attractive due to its chemical stability under cell culture conditions and high water solubility (>50 mg/mL). In contrast, DMSO was used to solubilize 2 but addition of DMSO can impact cell culture parameters (36). Besides improved solubility, compound 1 was the most potent of the inhibitors evaluated (FITC-LCA IC50 0.87 μM) and could be administrated via single dosing at the start of the cell culture cycle. In consideration of the factors above, 6,6,6-trifluorofucose (1) was pursued as a reagent for large-scale monoclonal antibody production. Wide ranging application of the fucostatin I inhibitor 6,6,6-trifluorofucose (1) for manufacture of protein therapeutics demanded a robust process to deliver several hundred grams of the inhibitor. The synthetic route to prepare 1 reported by Tokokuni and co-workers (17) was not suitable in this regard. The Tokokuni route involved: • • • • •

A nine step synthesis from expensive D-lyxose (37) (Scheme 1). The use of stoichiometric quantities of toxic mercury, lead, and chromium reagents. Non-crystalline synthetic intermediates. Multiple challenging silica gel chromatographies. Use of nine protecting groups.

Moreover, the Tokokuni approach is not stereoselective and yields a 50/50 mixture of epimers at C5. In planning the development of a novel approach to 1, multiple objectives were outlined. It was conceived that the new route would sustain the anomeric carbon of the starting material in 1 and would involve crystalline synthetic intermediates for purification. Furthermore, chromatographic separations and the extensive use of protecting groups would be avoided. Finally, the approach would be diastereoselective at C5 (38). 53 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. Tokokuni Route to 6,6,6-Trifluorofucose (1). Reproduced with permission from ref. (38). Copyright 2016, American Chemical Society.

Initial Factors D-(−)-Arabinose provided the advantages of low cost (39) and convenient availability on kilogram-scale and was selected as a starting material for an improved process to manufacture 1. The anomeric carbon of D-(−)-arabinose would be preserved in 1, thus providing an opportunity to carry out the sequence using rigid five-membered ring furanosides and significantly improving the odds of uncovering crystalline intermediates that might be used for purification. The enantioselective hydrogenation of trifluoromethyl ketones is precedented using hydrogen gas (40) or via transfer hydrogenation (41), and we planned to carry out such a diastereselective hydrogenation of ketone 9 (Scheme 2) using substrate control or by employing a chiral catalyst. Ketone 9 would be prepared by addition (42) of a trifluoromethyl group starting with known carboxylic acid 54 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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10 (43). A procedure to prepare 10 from D-(−)-arabinose on small scale has been reported (43), however this process would need to be developed to enable manufacture of 10.

Scheme 2. Retrosynthetic Analysis of a Novel Approach to 1. Reproduced with permission from ref. (38). Copyright 2016, American Chemical Society.

Process To Prepare Furanoside Ester 12a The formation of methyl furanoside intermediates 11a/11b is the first step of our process (Scheme 3). This reaction must be quenched after ~4 h by the addition of ammonium bicarbonate at 20 °C due to isomerization of the product over longer reaction times. The methyl furanosides 11a/11b appear to be the kinetic product of the reaction while the thermodynamic pyranoside products 11c/11d are formed more slowly. Once formed, one of the pyranoside anomers (11d) crystallized out of the reaction medium. Thirty hours were required to convert more than 50% of the material to pyranosides 11c and 11d. This period of time was long enough to allow for robust control upon preparation of kilogram batches of 11a/11b which contained less than 10% of the undesired 11c/11d. Upon quenching the reaction mixture with ammonium bicarbonate and filtration of the ammonium chloride by-product, the bulk of the methanol was distilled and the remainder was dissolved in water in preparation for the next chemical transformation. The methyl furanosides 11a/11b underwent a selective Heyns oxidation (44) of the primary alcohol group in the presence of the secondary alcohols (Scheme 3). The oxidation was performed using platinum black and oxygen at elevated temperature in water. The rate of reaction was optimal between pH 8 and 9. 55

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Addition of sodium bicarbonate (NaHCO3) base in portions throughout the process was effective to maintain the ideal pH. Despite a high metal catalyst loading (20%) and moderate yield of 10a/10b (60-63%), the downstream treatment consisted of filtration of the catalyst and use of the aqueous product solution directly in the next step, thus ensuring process practicality. Platinum black was recycled via reduction with hydrogen.

Scheme 3. Preparation of Benzyl Ester 12a Benzyl ester 12a was identified as an intermediate that had the capacity to be crystallized as a single diastereomer (>98/2 dr) and provided a valuable opportunity for purification. Crude acids 10a/10b were esterified using benzyl bromide in DMF with tetrabutylammonium bromide as a phase transfer catalyst. 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.

Crude compounds 12a/12b were crystallized from t-butylmethylether (MTBE), resulting in almost complete rejection of furanoside diastereomer 12b in the mother liquors. Unfortunately, anomer 12b is a productive intermediate to prepare 1 and thus the overall yield of the synthetic sequence is reduced by the selective isolation of anomer 12a. However, the study of the subsequent diastereoselective ketone hydrogenation was simplified by the use of a single furanoside species. Overall, starting from inexpensive D-(–)-arabinose material, 12a was isolated in 16% yield (45).

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Process To Manufacture Trimethylsilyl Ketal 14 Trifluoromethyl ketones commonly form hydrates and thus are unreliable synthetic intermediates. Consequently, a strategy was devised involving the conversion of ester 12a into trimethylsilyl ketal 14 (46, 47). In situ hydrolysis of ketal 14 would subsequently provide the ketone substrate for the planned diastereoselective hydrogenation. The two hydroxyl groups of 12a were expected to interfere with the formation of the trimethylsilyl ketal from ester 12a, which prompted their transient protection. It was decided to protect these hydroxyl groups as trimethylsilyl ethers. Removal of the trimethylsilyl ether groups was expected during the acidic work-up and isolation of the transfer hydrogenation product.

Scheme 4. Preparation of Trifluoromethyl Furanoside 14 The 12a diol was silylated using TMSCl and imidazole in DMF (Scheme 4) to provide crude ester 13, that was used in the next step without further isolation. Trifluoromethyl ketal 14 was obtained by treatment of 13 with TMSCF3 in the presence of catalytic tetrabutylammonium fluoride (85-90% yield from 12a). Crude 14 was not submitted to an aqueous work-up due to its poor stability in the presence of water. Instead, excess fluorinating agent was quenched by 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.

addition of silica gel to the reaction mixture. The silica gel was filtered off, and the product solution containing stable intermediate 14 was used in the subsequent step without further purification.

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Tandem Ketal Hydrolysis-Transfer Hydrogenation Process The in situ formation of the trifluoromethylketone hydrogenation substrate 15 from the trimethylsilyl ketal starting material 14 was carried out using KOH. This base also enabled the formation of the freebase (active form) of the transfer hydrogenation catalyst from the commercially available HCl salt. Both of these processes proceeded well in 2-propanol, a fitting solvent to conduct the transfer hydrogenation reaction. Upon treatment of 14 with KOH in 2-propanol, a four step desilylation sequence takes place resulting in the unstable hydrogenation substrate 17. In the absence of the catalyst, under the reaction conditions, 17 was found to decompose. Consequently, the timing of addition of the base and catalyst is important for designing a robust process to prepare the target secondary alcohol product 18. One possible sequence for the desilylation cascade is depicted in Scheme 5.

Scheme 5. A Proposed Base Catalyzed Desilylation Cascade. Reproduced with permission from ref. (38). Copyright 2016, American Chemical Society. In this proposal the desilylation starts with the hydrolysis of trimethylsilyl ketal 14 to form ketone 15. Ketone 15 is in equilibrium with the corresponding hydrate 16a. Spatial proximity of the ketone hydrate group and the C2 silyl ether enables a base catalyzed trimethylsilyl group transfer from the silyl ether function at C2 providing trimethylsilyl hemi-ketal 16b. This hemi-ketal then hydrolyzes 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.

under the reaction conditions to afford ketone 17. Consistent with this mechanistic proposal, alcohol 18 is the product of the hydrogenation process (48) prior to HCl quench of the reaction mixture. In the absence of transfer hydrogenation catalyst, the decomposition of 17 leads to the formation of multiple products.

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Table 2. Optimization of the Diastereoselective Hydrogenation of Ketal 14

The transfer hydrogenation conditions reported by Noyori and co-workers in their pioneering report served as a starting point for the optimization of the synthesis of 19 from 14 (Table 2) (49). Using 2% RuCl[(R,R)-TsDPEN](mes) and 0.3 equivalents of KOH at 20 ºC in IPA (entry 1) gave 90% assay yield of 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.

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desired alcohol 19 in 2 h after acidic quench. Interestingly, no attrition of the diastereomeric ratio (dr) of alcohols (19/20) occurred upon longer reaction times (14 h). Triethylamine-formic acid has been shown to give a kinetically controlled (50) mixture of products irrespective of the starting ketone’s structure. Although this reductant produced alcohols 19 and 20 in slightly higher dr (entry 2) compared to isopropyl alcohol, the use of this solvent conferred practicality to the process and these conditions were selected for further development. A slower reaction rate was observed using 24 h was necessary to reach the pre-filtration crystallization end-point. This was presumably caused by a correspondingly low equilibration rate of the four isomeric species of 1 in the absence of water. This caused the concentration of the single crystallizing species in solution to be low at any point 61

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during the process. To allow a productive crystallization rate, one equivalent of water was added in order. Filtration of the crystallized product, cake wash with n-heptane, and drying of the solid provided 1 as a crystalline material in 74% corrected yield from ketal 14. The C5 epimeric diasteromer of 1 (see Scheme 1) was present in 7% in the crude hydrolysis reaction mixture but was largely reduced to 9/1 dr from a trimethylsilylketal intermediate is the key transformation of the sequence. A ruthenium catalyzed tandem ketal hydrolysis-transfer hydrogenation step effected this transformation in excellent yield and diastereoselectivity. The potent and selective fucosylation inhibitor 6,6,6-trifluorofucose (1) and the process to access quantities of this reagent enables the preparation of mAbs displaying improved ADCC and improved in vivo efficacy.

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HER-2/neu-positive metastatic breast cancer. J. Clin. Oncol. 2008, 26, 1789–1796. Ihara, H.; Ikeda, Y.; Taniguchi, N. Reaction mechanism and substrate specificity for nucleotide sugar of mammalian alpha 1,6-fucosyltransferase-a large-scale preparation and characterization of recombinant human FUT8. Glycobiology 2006, 16, 333–342. Pande, S.; Rahardjo, A.; Livingston, B.; Mujacic, M. Monensin, a small molecule ionophore, can be used to increase high mannose levels on monoclonal antibodies generated by Chinese hamster ovary production cell-lines. Biotechnol. Bioeng. 2015, 112, 1383–1394. Allen, M. J.; Boyce, J. P.; Trentalane, M. T.; Treiber, D. L.; Rasmussen, B.; Tillotson, B.; Davis, R.; Reddy, P. Identification of novel small molecule enhancers of protein production by cultured mammalian cells. Biotechnol. Bioeng. 2008, 100, 1193–1204. The cost of D-lyxose is roughly $10/gram and the material is hard to procure on kilogram scale. Achmatowicz, M. M.; Allen, J. G.; Bio, M. M.; Bartberger, M. D.; Borths, C. J.; Colyer, J. T.; Crockett, R. D.; Hwang, T. –L.; Koek, J. N.; Osgood, S. A.; Roberts, S. W.; Swietlow, A.; Thiel, O. R.; Caille, S. Telescoped process to manufacture 6,6,6-trifluorofucose via diastereoselective transfer hydrogenation: scalable access to an inhibitor of fucosylation utilized in monoclonal antibody production. J. Org. Chem. 2016, 81, 4736–4743. D-(−)-Arabinose can be purchased for less than $100/kilogram. Kuroki, Y.; Sakamaki, Y.; Iseki, K. Enantioselective rhodium(I)-catalyzed hydrogenation of trifluoromethyl ketones. Org. Lett. 2001, 3, 457–459. Sterk, D.; Stephan, M.; Mohar, B. Highly enantioselective transfer hydrogenation of fluoroalkyl ketones. Org. Lett. 2006, 8, 5935–5938. Wiedemann, J.; Heiner, T.; Molston, G.; Prakash, G. K. S.; Olah, G. A. Direct preparation of trifluoromethyl ketones from carboxylic esters: trifluoromethylation with (trifluoromethyl)trimethyl silane. Angew. Chem., Int. Ed. 1998, 37, 820–821. Wu, J.; Serianni, A. S. D-Penturonic acids: solution studies of stableisotopically enriched compounds by 1H and 13C NMR spectroscopy. Carbohydr. Res. 1991, 210, 51–70. Heyns, K.; Heinemann, R. Oxydative umwandlungen an kohlenhydraten. III. Katalytische oxydation von D-glukose. Justus Liebigs Ann. Chem. 1947, 558, 187–192. Multiple reasons account for the low isolated yield of 12a from 11a, including loss of 12a in the aqueous phase containing residual N,N-dimethylformamide as well as loss of 12a in the mother liquors during the crystallization and the recrystallization of the ester product (12a). Walter, M. W.; Adlington, R. M.; Baldwin, J. E.; Schofield, C. J. Reaction of (trifluoromethyl)trimethylsilane with oxazolidin-5-ones: synthesis of peptidic and nonpeptidic trifluoromethyl ketones. J. Org. Chem. 1998, 63, 5179–5192.

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47. Singh, R. P.; Leitch, J. M.; Tawnley, B.; Shreeve, J. M. Diketo compounds with (trifluoromethyl)trimethylsilane: double nucleophilic trifluoromethylation reactions. J. Org. Chem. 2001, 66, 1436–1440. 48. The transfer hydrogenation screening results are detailed below. 49. Hashiguchi, S.; Fujii, A.; Takehara, J.; Ikariya, T.; Noyori, R. Asymmetric transfer hydrogenation of aromatic ketones catalyzed by chiral ruthenium(II) complexes. J. Am. Chem. Soc. 1995, 117, 7562–7563. 50. Fujii, A.; Hashiguchi, S.; Uematsu, N.; Ikariya, T.; Noyori, R. Ruthenium(II)catalyzed asymmetric transfer hydrogenation of ketones using a formic acid−triethylamine mixture. J. Am. Chem. Soc. 1996, 118, 2521–2522. 51. Sterk, D.; Stephan, M.; Mohar, B. Highly enantioselective transfer hydrogenation of fluoroalkyl ketones. Org. Lett. 2006, 8, 5935–5938. 52. GC FID analysis: Rtx®-5 Amine, 30 m x 0.32 mm ID, 1.0 µm df; Inlet 10/1 split ratio, 300 °C; Oven 120 °C for 2 min, 15°C/min to 240°C for 8 min; 14 at 6.98 min, 15 at 7.35 min. 53. The rejection of ruthenium via the crystallization of 1 was not surveyed, however it was considered unlikely that levels of metal of 35-45 ppm in solution of 14 would result in amounts of Ru in crystalline 1 of 99.90% purity (no impurity > 0.06%) to support the clinical trials. Further medicinal chemistry investigations led to the discovery of BIIB028, an inhibitor with improved stability, enhanced solubility that enabled an intravenous formulation, and greater potency for in-hospital chemotherapy trials. Improvements to the three-stage medicinal chemistry route permitted the synthesis of 12 kg of API at 99.6% purity in support of the phase I clinical trials.

Discovery of BIIB021 Heat shock proteins (Hsps) are a class of molecular chaperones whose primary function is to maintain the proper conformation of proteins. Hsps are abundant and normally provide routine regulation of client protein folding in the cell. However when cells are under stress, from temperature, oxidative or radiological challenges, they overexpress Hsps in order to preserve cellular function (1–4). Inhibition of a specific 90 kDa chaperone, known as Hsp90, results in the generation of misfolded client proteins that are eliminated from the cell by © 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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ubiquitination and proteasome degradation. Of the many Hsp90 clients, 48 are associated with cellular signaling cascades and cell growth mechanism. Among these are a large number of oncogenic proteins including HER2, mutant EGFR, Raf-1, Akt, mutant BRAF and EGFR (5–8). The belief that cancerous cells were more sensitive to Hsp90 inhibition than healthy cells encouraged the development of numerous pharmacologically active molecules designed to block the protective effects of Hsp90. Early clinical work focused on the development of 17-allylamino-17- desmethoxygeldanamycin (17-AAG), a derivative of natural product geldanamycin (Figure 1), but the low oral bioavailability, poor solubility, and toxicological profile led investigators to look for alternatives with improved pharmaceutical properties. Investigations at Memorial Sloan-Kettering Institute and Conforma identified PU3 from optimization of a purine-based scaffold obtained from structure-based drug design (Figure 1) (9).

Figure 1. Purine-based scaffold lead identification.

As part of the SAR development a pharmacophore model was proposed. This model described interactions of the N1 and 6-amino groups of the pyrimidine ring with the purine binding pocket of Hsp90, and another interaction between the aromatic ring situated 5 Å away at the C-8 position and an aryl binding region (Figure 2).

Figure 2. Pharmacophore model development. Reproduced with permission from ref. (9). Copyright 2010 American Chemical Society. 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.

A flip of the purine ring system provided an alternative scaffold that was amenable to rapid optimization because the N9 position was a good partner for simple alkylation reactions (Figure 3).

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Figure 3. Ring-flip approach led to a new class of purine scaffolds. Reproduced with permission from ref. (9). Copyright 2010 American Chemical Society.

Screening of 6-chloro-2-aminopurine analogs with a variety of substituted benzyl rings led to compounds with greatly improved potencies. The most potent analog (BIIB021) contained a substituted pyridyl system at N9, which was also part of the structure of the proton pump inhibitor omeprazole (Figure 4). The efficacy of BIIB021 in xenograft models containing the human stomach carcinoma N87 tumor cell line was evaluated. Oral dosing of BIIB021, on a 5days/week schedule for 5 weeks, was conducted on mice in which tumor fragments were implanted subcutaneously and allowed to grow until the tumor size reached 80-100 mm3. Dose-dependent tumor growth inhibition was observed over 36 days and BIIB021 demonstrated no treatment-related toxicity at these doses other than a 3-5% weight loss (Figure 5). At this point, BIIB021 was selected for development as an oral Hsp90 inhibitor for the treatment of solid tumors. The program was transitioned into process development in order to identify a robust manufacturing process.

Figure 4. Substitution patterns on the N9 benzylic portion increased potency. 71 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. In vivo efficacy of orally administered BIIB021 in a tumor xenograft model. Growth of N87 tumor xenografts in athymic mice treated with BIIB021 5 days/week. Reproduced with permission from ref. (9). Copyright 2010 American Chemical Society. Process Development of BIIB021

Introduction The first generation GMP process, a medicinal chemistry route, was scaled up by a Contract Manufacturing Organization (CMO) to produce ~12 kg of BIIB021 (4) in three batches for the initial clinical trials (Scheme 1) (10, 11). In the synthesis, benzylpurine 3 was synthesized by alkylation of chloropurine 1 (12, 13) with 2-chloromethylpyridine hydrochloride 2 in the presence of NaI and K2CO3. The product 3 was isolated from the filtrate after removal of insoluble inorganics by precipitation with water and then converted into BIIB021 by reaction with ethanolic methanesulfonic acid (MeSO3H).

Scheme 1. First Generation Process for BIIB021. Reproduced with permission from ref. (10). Copyright 2015 American Chemical Society. 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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Data obtained from the GMP runs indicated that control of BIIB021 quality and yield was inconsistent. One of the batches failed to meet the specification for residual content of 5 (Figure 6), an impurity derived from N7-alkylation of chloropurine 1. The yields for the salt formation step ranged from 55% to 80%.

Figure 6. Structures of impurity 5 and 6.

The presence of the 4-ethoxy impurity 6 (Figure 6) in the API indicated that an SNAr reaction (14) had occurred (Scheme 2) and may have caused the yield variability. HPLC monitoring of the salt formation reaction revealed that the side reaction was alarmingly fast, consuming 20% of the free base 3 in 30 min (Figure 7) (15).

Scheme 2. Reaction of Benzylpurine 3 with EtOH during the Salt Formation. Reproduced with permission from ref. (10). Copyright 2015 American Chemical Society.

Alcohols are known to react with MeSO3H to form alkyl sulfonate esters (MeSO3R), a class of potentially mutagenic impurities (16, 17). Low level of the ester MeSO2OEt (0.8-1.4 ppm) was detected in the API. Due to these side reactions of the alcohols with the substrate and MeSO3H, they were excluded as solvents for the MeSO3H salt formation and recrystallization. The synthetic route was short and the starting materials were available from multiple vendors; therefore, the development aimed to improve the process by understanding and resolving the key issues, i.e., control of process impurities, improving robustness of the MeSO3H salt formation reaction, and developing a MeSO3H salt recrystallization process. 73 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 7. Free base 3 loss and impurity 6 increase in the salt formation in EtOH. Reproduced with permission from ref. (10). Copyright 2015 American Chemical Society.

Results and Discussion Development of the Alkylation Reaction for the Synthesis of Benzylpurine 3 The reaction of 1 with 2 was investigated using HPLC to obtain quantitative composition of the reaction mixture. The results (Scheme 3) revealed significant amounts of impurities: ~17% of the N7 alkylation product 5, ~6% of the 2-NH2 alkylation product 7 and ~1% of the bis-alkylation product 8. The impurities 7 and 8 were isolated, purified and fully characterized on the basis of 1H and 13C NMR analysis and high resolution mass spectroscopy data. The main side product 5 had low solubility in DMF, precipitated during the reaction and was mostly removed by filtration of the reaction mixture. Crude 3 was isolated from the filtrate by precipitation with H2O in ~55% yield and ~95% purity. Investigations indicated NaI had a negligible effect on the impurity profile, yield, and the reaction rate. Solvents and bases affected the selectivity modestly, but the rate dramatically. Some representative results are shown in Table 1. For example, the yield of free base 3 increased modestly from ~73% in DMF (entry 1) to ~81% in DMSO (entries 3 and 4) due to decrease of impurity 5. In DMF, with K2CO3, the reaction went to completion within 1 h at 40 °C (entry 1); however, with iPr2NEt it was only 90% complete even after 7 h at 70 ºC (entry 2). With NaOH (granular), the reaction went to completion in DMSO within 30 min at 22 ºC (entry 5), but no reaction occurred in H2O at 60 ºC in 4 h (entry 7). 74

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Scheme 3. Reaction Profile for the Alkylation of Chloropurine 1. Reproduced with permission from ref. (10). Copyright 2015 American Chemical Society.

Table 1. Effect of Solvents, Bases, Temperature. Reproduced with permission from ref. (10). Copyright 2015 American Chemical Society. Entry

Solvent

Base (equiv)

Temp (°C)

Time (h)

Conv. of 1

Ratio of 3:5

1

DMF

K2CO3 (3.2)

40

0.5

> 97

4−5 : 1

2

DMF

iPr2NEt (2.5)

70

7

90

6−7 : 1

3

DMSO

K2CO3 (1.4-1.8)

40

0.5

> 99

6−7 : 1

4

DMSO

DBU (2.2)

22

1

> 98

6−7 : 1

5

DMSO

NaOH (2.0)

22

0.5

> 98

5−6 : 1

6

IPA

iPr2NEt (2.5)

60

4

0

-

7

H2O

NaOH (3.0)

60

4

< 10

-

These results were comparable to the generally modest yields and selectivity of the N9 alkylation reactions of 1 (12, 13). Therefore, efforts were focused on the reaction in DMSO with NaOH, DBU and K2CO3 as bases due to their slightly improved selectivity. With granular NaOH, the initial scale-ups required a higher temperature to complete the reaction and resulted in a high level of impurity 8 (up to 3%), a new impurity 9 (Figure 8), and a low quality of isolated 3. With DBU, the N7 isomeric impurity 5 remained soluble in the reaction mixture and an extra step was needed for its removal. However the use of K2CO3 as the base proved successful. The 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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reaction with K2CO3 was optimized and scaled up to 1 kg successfully providing 3 in ~70% yield and 95-98% purity. The conditions are summarized in Scheme 4. The 35-55 ºC temperature range provided a good reaction rate and purity profile. At higher temperature, (e.g. 65 ºC), the levels of impurities 8 and 9 increased. Use of 6 volumes of DMSO provided satisfactory volume efficiency for the reaction.

Figure 8. Impurity 9.

Scheme 4. Synthesis of 3 in DMSO

Interestingly, the mode of the addition of the starting materials into the reactor had an impact on the reaction rate. Adding 2 in greater than three equal portions into a mixture of 1 and K2CO3 in DMSO at 15-40 min time intervals depending on scale and the cooling rate resulted in immediate CO2 off-gassing, a temperature rise and formation of product 3. The reaction went to completion within an hour after the final addition. However, when 2 was added all at once, the reaction exhibited an induction time, a period when product 3 was not formed, and took up to 8 h to reach acceptable conversion. The induction time might be explained by the unavailability of CO32- needed for the deprotonation of N9-H of 1 to generate the nucleophile, which became available only after the HCl in 2 was neutralized. K2CO3 with different particle sizes varied in performance for the scale-ups (18, 19). In the initial scale-ups using granular K2CO3 (>70 mesh), the reaction stalled and required additional K2CO3. With powdered K2CO3 (-325 mesh), the reaction performed reproducibly, despite a slightly higher level of impurity 8 (0.7% vs. 98% purity and ~75% yield. Purification of 3 was pursued to avoid BIIB021 recrystallization, which was considered difficult due to its instability in solution. Removal of 5 in 3 by recrystallization was unattractive due to either product loss, insufficient purging of 5, or both. In some instances, this impurity was actually enriched. The difficulty associated with removing 5 was exemplified by the results obtained with DMF-EtOAc, which typically afforded 3 in acceptable purity, but low yield (50-65%, up to 1 kg scale). In a 10 kg pilot plant campaign, the recrystallization yield was typical, but 3 contained impurity 5 (1.2%). MeSO3H salt formation in DMSO of this lot followed by precipitation with EtOAc gave BIIB021 with a new impurity 10, a compound derived from 5 by hydrolysis of the –Cl (Scheme 5) (14). This impurity was difficult to purge.

Preparation of BIIB021 Most of the common solvents were excluded for preparation of BIIB021 due to low solubility of 3, except for the polar non-protic solvents DMSO, dimethylacetamide (DMAc), and DMF. Investigation of the stability of BIIB021 in these solvents at different temperatures showed the degradation rate followed the order of DMSO>>DMF>DMAc (see Figure 9 for data obtained at 40 ºC) and increased with a rise in temperature. In the best solvent, DMAc, the BIIB021 degradation rate was still significant at 40 ºC, ~2% in 2 h. However, it was negligible at 22 ºC due to the precipitation of BIIB021 and its removal from solution degradation. Therefore, DMAc was deemed appropriate for the reaction at 22 ºC. The major degradation products in these solvents, particularly in 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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DMSO, were 11 and 12, generated from hydrolysis of aromatic chloride and de-methylation, respectively (Scheme 6).

Figure 9. Stability of in situ prepared BIIB021 in DMF, DMAc and DMSO at 40 ºC. Reproduced with permission from ref. (10). Copyright 2015 American Chemical Society.

Scheme 6. Major Impurities 11 and 12 in the MeSO3H Salt Formation. Reproduced with permission from ref. (10). Copyright 2015 American Chemical Society. Ideally, this salt formation step would provide BIIB021 with the required impurity profile and desired physical properties. Only one polymorphic form was found; therefore, its control was not an issue. With this consideration, ICH class 3 anti-solvents (acetone, MTBE, EtOAc, and anisole) were investigated for crystallization of crude BIIB021 (Scheme 7). MTBE provided the best yield, purity, and volume efficiency, increasing the yield by ~10% as compared to DMAc alone. 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.

Scheme 7. Salt Formation in DMAc

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The salt formation procedure included the following steps: • • • • • • •

add 1.0 equivalents of MeSO3H into a mixture of 3 and DMAc at ~22 ºC. add MTBE. add seed crystals. stir. add MTBE. cool. filter.

BIIB021was typically obtained in 80-85% yield with the main impurity 5 at ~ 0.20%. Reducing impurity 5 to ≤ 0.15% to meet the specification was achieved in some of the experiments by running the crystallization under more dilute conditions. However, this approach was inefficient and unreliable. Therefore, a new recrystallization process using DMSO-EtOAc (see Recrystallization of BIIB021 section) was developed to remove the impurities (Scheme 8).

Scheme 8. Three-Stage Process for BIIB021 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.

Although this three-stage process was demonstrated at kilogram scale in the lab, in a 20 kg pilot plant campaign, the filtration of 3 took more than a week and the product was obtained in significantly lower yield and purity. Therefore, our efforts were directed to telescoping the alkylation and salt formation steps to circumvent the isolation of 3 (21).

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Telescoping the Alkylation and Salt Formation Steps Since the synthesis of 3 in DMSO and recrystallization of BIIB021 in DMSOEtOAc was demonstrated, telescoping both steps in DMSO was examined. Not surprisingly, hydrolysis impurities 10 and 11 formed quickly in the salt formation in the DMSO filtrate containing 3 and were present in the BIIB021 at > 0.1% levels. Substitution of DMAc for DMSO had a negligible effect on the alkylation reaction rate and impurity profile; crude BIIB021 was readily obtained from the DMAc filtrate containing 3 in satisfactory yield and purity, compared with the results from the stepwise process. With this result, this process (Scheme 9) was systematically optimized.

Scheme 9. Telescoped Process in DMAc

The stoichiometry of 1 and 2 was adjusted to ensure a low level of 2 in the final alkylation reaction mixture, since 2 was difficult to remove in this telescoped process. The maximum amount of 2 that could be reliably purged in the downstream process was ~ 0.5% and this level was obtained consistently by using 1.05 equivalents of 1. 80 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 calculated charge of DMAc (7.5 vol), which was initially based on the concentration of 3 in the salt formation, was increased to avoid loss of 3 during the filtration (3 in solution). Product 3 was supersaturated in the filtrate, as indicated by the slow formation of large crystals upon storage at ambient temperature for several days. This result also indicated the nucleation rate was very slow and suggested a negligible yield loss would occur during the filtration operation. However, there was a significant yield loss in the first run of a 20 kg pilot plant campaign that was attributed to precipitation of 3 during the hold time before filtration. Increasing the DMAc charge to 10-12 volumes delayed onset time of precipitation to ~16 h and diminished the product loss in the filtration operation. This was demonstrated by ~80% in situ assay yield of 3 in subsequent 50 and 100 kg campaigns. Reduction of the K2CO3 equivalents was studied since BIIB021 from the first 20 kg pilot campaign contained high levels of potassium based inorganic impurities (as indicated by the residue on ignition values, ROI (22)), even after recrystallization. Efforts to remove these impurities by filtration through a variety of media had no effect on lowering the ROI, suggesting the inorganic impurities were dissolved salts. Eventually BIIB021 having a low ROI (< 0.06%) was obtained by decreasing the amount of K2CO3 from 1.8-2.2 to 1.1-1.3 equivalents. The results can be explained by elimination of the contribution of K2CO3, which has a higher solubility (7.8 mg/100 g) than KHCO3 (3.3 mg/100 g) and KCl (2.3 mg/100 g) in DMAc at 22 ºC. Decreasing the K2CO3 equivalents reduced the filtration time by ~50%, however, it increased the reaction time to 7-8 h from ~1 h. This reaction time increase had no negative impact on the reaction impurity profile or the quality of the isolated BIIB021. In fact, the HPLC profile of the reaction mixture showed excellent stability after a reaction time of ~72 h at 55 °C. Serendipitously, the N7 side product 5 precipitated from the reaction mixture in DMAc faster and more completely than in DMSO. At ~22 ºC, residual 5 in the filtrate decreased to 99.90% purity. Table 2 shows the data from the three batches of the last campaign.

Table 2. Recrystallization Batch Data batch

BIIB021 output (kg)

Yield (%)

Purity (%)

09430F0001

45.2

75.3

99.9

09430F0002

51.4

81.6

99.9

09430F0003

52.0

82.6

99.9

Scheme 10. Key Parameters of the Final API Process To Prepare BIIB021. Reproduced with permission from ref. (10). Copyright 2015 American Chemical Society. 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.

This process, despite use of rapid crystallization from solution produced the API with high crystallinity (mp > 250 ºC), good stability, and desired physical properties for drug product production.

Conclusions for BIIB021

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This telescoped two-stage process exhibited satisfactory robustness, scalability, and reliability during pilot scale production runs and enabled preparation of recrystallized BIIB021 in high purity and in good overall yield. The API showed good stability and was used to support Phase II clinical trials. Scheme 10 summarizes the key process parameters of this improved synthesis.

Discovery of BIIB028 BIIB021 had three limitations as a drug candidate: • • •

Because of its lower potency, higher doses were required to see positive biological effects and the margin for safety was negatively impacted. The methanesulfonic acid salt was relatively unstable and great care was needed to manufacture it at a reasonable commercial scale. It could not be formulated into an acceptable intravenous formulation that the clinical team required for in-hospital chemotherapy trials.

Therefore, as BIIB021 progressed through early phase clinical trials, a follow-on Hsp90 inhibitor program was initiated to improve upon the potency and tolerability as well as the physical properties (solubility, stability, etc.) of the first generation compound. The 6-chloropurine scaffold was probed for positions amenable to further modification. Unfortunately many of the substitutions around the pyrimidine and pyridyl ring systems led to a loss in potency. An X-ray structure of BIIB021Hsp90 complex, however, revealed that the N7 position was oriented out toward a solvent exposed area and might be amenable to analog development (Figure 11) (25).

Figure 11. X-ray structure of BIIB021 complexed with Hsp90. Reproduced with permission from ref. (25). Copyright 2012 American Chemical Society. 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.

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Replacement of the N7 nitrogen with an alkenyl carbon produced a series of pyrrolo[2,3-d]pyrimidines (Figure 12) that retained their binding affinity for Hsp90. The 5-alkynyl derivative was similar in potency to BIIB021 and extension of the terminus of the alkyne with alcohols gave more potent Hsp90 inhibitors.

Figure 12. Optimization of 5-alkynylpyrrolo[2,3-d]pyrimidines. Reproduced with permission from ref. (25). Copyright 2012 American Chemical Society.

The alkynol analogs had improved efficacy in xenograft models at oral doses ~20-fold lower than BIIB021. In rodents, the oral dose-limiting toxicity of BIIB021 was gastrointestinal in nature (diarrhea, bleeding) so in an effort to improve tolerability and minimize local exposure of the Hsp90 inhibitor in the GI tract intravenous formulations were examined (26). Unfortunately, the alkynols possessed poor aqueous solubility (~5 μg/mL at pH 7) so a series of solubility enhancing pro-drugs, for example, replacing the –OH with -HNR2+, -CO2-, or –OCOCH2NH3+, were investigated (26). Synthesis and solubility screening efforts eventually pointed to the phosphate prodrug of the butynyl alcohol, BIIB028 (solubility > 1 mg/mL at pH 7) and it was selected as the second-generation clinical candidate. Process development of BIIB028 follows.

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.

Process Development of BIIB028 Introduction

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The first generation GMP process for BIIB028 (27, 28) is shown in Scheme 11. In this route, iodide 13 was converted into alcohol 14 of ~97% purity by a Sonogashira coupling with 3-butyn-1-ol and K2CO3, in aqueous DMSO–ACN (25). The alcohol 14 was then converted to BIIB028, a more soluble prodrug form of the active drug substance 14, by reaction with excess POCl3 in OP(OEt)3 at 0–5 °C followed by an aqueous quench and pH adjustment with aqueous NaOH to precipitate the product. BIIB028 of ~98% purity was then subjected to purification by: • • •

basification to produce an aqueous solution of its disodium salt. MTBE and EtOAc washing of the aqueous phase to reduce levels of organic soluble impurities. acidification, isolation and drying to give BIIB028 of ~99% purity.

This route provided BIIB028 for the early clinical trials.

Scheme 11. First GMP Process for BIIB028

The process generated two main impurities of concern (Scheme 12). The diol 15 (≤ 2%), produced in the Sonogashira reaction, proved difficult to remove during the isolation of 14 and was carried forward to produce the bis-phosphorylated impurity 16 in BIIB028. The chloride 17 (12.5%) was generated in the phosphorylation reaction. Although this potentially mutagenic impurity was purged during isolation of BIIB028, its formation represented an undesired yield loss. 87 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 12. Origin of Impurities of Concern in BIIB028 GMP Synthesis

Scheme 13. Synthesis of Starting Material 13 88 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.

Development Focus Given that a process for making multi-kilogram quantities of intermediate 13 was available (Scheme 13) (25), development work targeted improvement of the API route. This was achieved by: • •

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•

simplification of operational aspects for each reaction. improvement of yields, process robustness, and quality by better control of process impurities (15, 17, residual Pd and Cu). reduction of the time required for BIIB028 precipitation and reprecipitation filtrations.

Results and Discussion Process Evaluation of the Sonogashira GMP Reaction Conditions The preparation of 14 under the original conditions was evaluated using HPLC to monitor reaction progress, to determine product losses to filtrates and washes during the work up, and for analysis of the isolated product. The analytical results and operational observations are summarized below: • • • • •

the reaction proceeded to completion (< 0.1% 13) after 3.5 h. product 14 was isolated in two crops: 1st crop in 27% yield and 98.1% purity with 0.38% 15 and 2nd crop in 22% yield and 96.5% purity. eight different organic solvents, four filtrations and eight filter cake washes were used. 14 was crystallized from a ternary solvent system. drying of a THF stream containing 14 with Na2SO4 only minimally reduced the solution’s water level (from 4.7% to 4.0%).

Although the conversion proceeded well, the isolation of 14 needed improvement to better control impurities and to reduce the number of unit operations and solvents.

Sonogashira Reaction: Process Improvement Studies The initial focus was to identify an alternative solvent(s) to minimize the formation of 15 and other impurities while maintaining high conversion to 14. The results from these studies are compiled in Table 3. The most preferable results were obtained from the use of aqueous THF. An important observation was that the formation of 15 could be nearly avoided by stopping the reaction with ~ 1% 13 remaining (entries 10 and 11). Additional work identified 1.0-1.6% 13 as an appropriate reaction endpoint. An in-process control HPLC method with a short run time and prompt analysis contributed to elimination of 15 during the scale up run. 89 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. Effect of Reaction Solvents on the Preparation of 14

a

Entry

Solvents (v/v)

Product 14 (%)

Impurity 15 (%)

1

ACN-DMSO-water

94

1.5

2

THF

58

0

3

1,2-DME

78

0.33

4

1,4-dioxane

86

0.25

5

MTBE

51

0.05

6

THF-water (4:1)

94

0.24

7

1,2-DME-water (4:1)

78

0.33

8

1,4-dioxane-water (4:1)

86

0.25

9

MTBE-water (4:1)

51

0.05

10

THF-water (1:1)

92

2.2

11

(1:1)a

98

0.03

THF-water

Reaction stopped with < 1% 13 remaining.

Several alternative catalysts were screened for use in the reaction, including Pd(OAc)2 with n-Bu4NOAc (“ligandless” Pd conditions, Table 4) (29). The use of “ligandless” Pd conditions in either THF or 1-methyl-2-pyrrolidinone (NMP) were abandoned, as the reactions were prone to stalling and could not be driven to completion. From this work, the use of 10% Pd/C, CuI and PPh3 was pursued.

Table 4. Effect of Catalyst on the Preparation of 14 Entry

Catalyst

Solvents

14 (%)

1

10% Pd/C, CuI, PPh3

THF-water (1:1, v/v)

98

2

Pd(OAc)2 (2 mol%) n-Bu4NOAc (1.5 equiv)

NMP

80

3

Pd(OAc)2 (4 mol%) n-Bu4NOAc (1.5 equiv)

THF

49

4

Pd(dppf)Cl2

NMP

0

5

Pd(PPh3)2Cl2

NMP

0

A brief study was performed to optimize the charge of CuI (Table 5). A charge of 3.7 mol% CuI was found to be sufficient, and was chosen for further development.

90 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. Effect of CuI Equivalents on the Preparation of 14 Entry

CuI (mol%)

Product 14 (%)

Impurity 15 (%)

1

0

4

0

2

2.0

83

0.29

3

3.7

91

0.17

4

5.5

91

0.29

A brief study was performed to determine the necessary equivalents of 3butyn-1-ol (corrected for purity), and 1.05 equivalents was optimal. In the original procedure, the solution of 14 in THF was washed multiple times with aqueous brine solutions containing NH4OH for removal of residual Cu and Pd species. It was verified that washing of the organic stream in triplicate with 15% brine containing concentrated NH4OH (92:8, w/w) reduced levels of Cu to < 2 ppm (ICP-MS) in 14. Additional work identified a modified aqueous wash composition (15% aqueous brine, concentrated NH4OH and L-cysteine (91.6/7.5/0.9, w/w/w)) for reducing Pd to levels of below 4 ppm (ICP-MS). This solved our metal removal requirements.

Table 6. Selected Results for Recrystallization of 14 Entry

Solvents (v/v)

Volumes

Recovery (%)

Purity of 14 (%)

1

THF

6

70

98.6

2

MeOH

12

84

98.4

3

EtOH

8

90

98.6

4

IPA

12

86

98.5

5

ACN

15

85

98.5

6

THF-MeOH (1:2.8)

17

76

98.6

7

THF-MeOH (1:3.2)

19

51

99.4

8

THF-MeOH (1:9)

20

76

97.4

9

THF-MeOH (1:15)

16

73

98.8

10

THF-heptane (1:11); THF-MeOH (~1:1)

12

55

99.2

Attention was then turned to improving isolation conditions for alcohol 14. The alcohol was isolated from THF-EtOAc-heptane as per the original conditions and was of 98.4% purity. Recrystallization of 14 was explored as a means of 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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upgrading its purity. Use of most solvent systems showed no improvement in purity (Table 6). Purity upgrades occurred, unfortunately, with low yields of 51–55% (entries 7 and 10). Additional work therefore focused on elimination of the recrystallization in favor of direct precipitation of 14 from the THF stream with a primary goal of increasing the recovery while keeping the purity reasonable. By concentrating the stream (to ~2.5 vol) and adding MeOH (5 vol), 14 was isolable in 88–90% yield and in purities of 98.5–98.6%. This isolation protocol was adopted for further scale up. Using the aforementioned process improvements, a scale up run was performed as summarized in Scheme 14. From this scale up run, 14.7 kg (85%) of 99.0% purity 14 was isolated. Residual volatiles and metals were well controlled (0.9% H2O; 372 ppm MeOH; < 0.4 ppm Cu; 3.3 ppm Pd); and impurity 15 was not detected.

Scheme 14. Improved Process for Preparation of 14

Development of Improved Conditions for the Phosphorylation Reaction The preparation of BIIB028 under the original conditions (cf. Scheme 11) was evaluated on lab scale using HPLC to monitor reaction progress, to determine product losses to filtrates and washes during the work up, and to analyze the isolated product. The results and operational observations are summarized below: • • • •

reaction was complete after ~5 h at 0-5 °C; a significant amount of chloride 17 (12.5%) occurred. product slurry filtration time was unacceptably long (overnight). BIIB028 was isolated in 73% yield and 98.2% purity, and contained 17 (1.6%) and several other low level impurities (≤ 0.07%). product losses to the filtrate and wash occurred

Initial work focused on studying the effect of reaction temperature on formation of the undesired chloride 17 in 13 volumes of PO(OEt)3 (Table 7).

92 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.

Table 7. Effect of Temperature on the Conversion of 14 and Formation of 17

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a

Entry

POCl3 (equiv.)

Temp. (°C)

BIIB028 (%)

17 (%)

1

4

0-5

98.2

1.6

2

4

-10 to -15

97.6

0.84

3

4

-30

99.0

0

4a

2

-50

97.5

0.3

A very slow reaction rate at -50 °C was observed; 0.63% 14 at 24 h.

The amount of 17 produced was found to decrease as the temperature was lowered. A reaction temperature of -30 °C was selected for scale up. Use of -50 °C was not pursued due to a very slow reaction rate, formation of 17 possibly due to the long reaction time, and the difficulty in transferring the viscous product slurry into the water quench. The product yield and quality were determined from the equivalents of POCl3 used. The results indicated that a significant yield increase was achievable with reduced equivalents of POCl3; 2.5 equivalents was selected as the preferred charge. At reaction completion, the mixture was very thick and precluded its transfer into water for quenching without dilution. Previous experiments suggested that charging of the product slurry into water was the preferred quench method. Further studies established that an increase of PO(OEt)3 solvent to 20 vol produced a slurry transferable into water for smoother quenching. Formation of a new impurity 20 (1.7–3.9%), a diphosphate identified by LCMS, occurred during the final NaOH pH adjustment (Scheme 15). This impurity was very difficult to remove during BIIB028 isolation in this or the following step. Additional work revealed that formation of 20 occurred if the final pH adjustment was initiated prior to full conversion of the intermediates (18, 19) into BIIB028 during the quench. Therefore, an HPLC in-process control method was developed to determine the completion of this conversion by determining the level of 20 produced in the pH adjustment. When an aliquot of the quenched reaction solution was treated with NaOH to the appropriate pH and HPLC analysis showed ≤ 0.5% 20, the conversion was considered complete and pH adjustment of the batch was initiated. Implementation of this simple HPLC in-process control test eliminated formation of 20 during the scale-up. BIIB028 precipitated from solution as very fine particles during the final pH adjustment. In laboratory studies, this solid was not effectively captured during isolation by centrifugation and filtrations were unacceptably slow (several hours). Therefore, BIIB028 precipitation conditions were investigated to reduce the isolation time. Table 8 lists the variables studied and the preferred parameters identified. 93

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 15. Formation of Impurity 20

Table 8. Precipitation Variables Assessed and the Parameters Identified Variable

Values Studied

Preferred Value

H2O quench (vol)

30, 46

46

aq NaOH (M)

0.5, 1, 10

0.5

NaOH charge time (h)

1, 3

3

Agitation intensity

slow, medium, fast

slow

(°C)a

20, 30, 40, 50

40

decantationb

yes, no

yes

Temperature supernatant

Prior to the start of the caustic charge. to product slurry filtration.

a

b

Agitation halted and supernatant drawn off prior

These preferred conditions included the atypical practice of halting agitation, allowing solids to settle and thereafter drawing off the supernatant prior to product slurry filtration, which significantly reduced the volume to be filtered. Settling versus filtration of the batch was superior for product isolation due to its unique physical characteristics. These process changes resulted in a 75–80% reduction in BIIB028 isolation time. Using the aforementioned process improvements, a scale up run was performed in duplicate (Scheme 16). Since the following step was water based, BIIB028 was not dried in vacuo to constant weight; both batches (99.1% and 99.3% purity) were combined to produce one lot of reprecipitated BIIB028.

94 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. Improved Process for the Preparation of BIIB028

Development of Improved Conditions for the Reprecipitation The reprecipitation of BIIB028 was included as a final GMP step in order to insure < 0.05% content of 14 in the drug substance. Meeting this residual level was necessary for obtaining a successful IV drug product formulation. The reprecipitation under the original conditions (cf. Scheme 11) was evaluated using HPLC to determine product losses to washes and filtrates and to analyze the isolated product. The analytical results and operational observations are summarized below: •

• • •

• • • •

charging of caustic to an aqueous suspension of BIIB028 at ambient temperature to its target pH proceeded through the intermediacy of a very viscous solution. flow of the BIIB028 disodium salt aqueous solution stopped during the filtration. MTBE and EtOAc washes were effective in lowering levels of organic soluble impurities. during the acidification operation to pH 3-4, the aqueous phase had the consistency and appearance of a beaten egg between pH 5–7 and agitation of the slurry was problematic. the rate of product slurry filtration needed further improvement. BIIB028 was isolated in 76% yield and 99.1% purity as a beige solid with numerous low level impurities (≤ 0.28%). product losses to the EtOAc washes, filtrate and filter cake wash occurred. a new impurity formed whose levels increased during drying of the wet cake at 35–40 °C in vacuo.

To avert formation of the highly viscous phase during BIIB028 disodium salt aqueous solution preparation, aqueous 50%NaOH was added after the slurry reached 50 °C. With this change, the operation proceeded smoothly during scale up and without any noticeable agitation issues. To improve the rate of the polish filtration of the aqueous disodium salt solution, we performed an empirical study of pre-washing the filtration apparatus with solvents and found THF wash was an effective remedy. While the reason 95 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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for this is unclear, we presumed that the THF modified the surface property and its interaction with the disodium salt. In the original procedure, sequential washing of the BIIB028 disodium salt aqueous solution at 30–35 °C with MTBE and EtOAc was used to lower levels of impurities. Additional work determined that duplicate EtOAc washes were sufficient. Of importance was circumventing the undesirable slurry characteristics within the pH 5-7 range during the acidification of the basic disodium salt solution to pH 3-4. Eventually we achieved this by reversing the quench process, i.e., adding the disodium salt solution to an excess of aqueous HCl. During this process, the BIIB028 remained an homogeneous solution as its HCl salt. Thereafter, addition of caustic to pH 3-4 resulted in precipitation of BIIB028. This protocol effectively resolved the precipitation issue. Reprecipitation conditions were investigated to reduce the BIIB028 isolation time. Table 9 lists the variables studied and preferred values identified. A 75–80% reduction in BIIB028 isolation time was achieved using the preferred conditions.

Table 9. BIIB028 Reprecipitation Variables Assessed and Preferred Values Variable

Values Studied

Preferred Value

H2O (vol)

15, 25

15

aq HCl (M)

0.1, 0.2, 1

1

Aq NaOH charge time (h)

0.75, 1

1

Agitation rate (rpm)

200, 350

350

10, 20, 30

30

yes, no

yes

Temperature (°C)

a

supernatant decantationb Prior to the start of the caustic charge. to product slurry filtration.

a

b

Agitation halted and supernatant drawn off prior

A new impurity 21 (Figure 13), formed by demethylation of the OMe under acidic conditions, occurred during the drying operation in vacuo at 35–40 °C. Additional studies identified two operations to help control the level of this impurity to 0.07% in reprecipitated BIIB028: •

•

The pH of the aqueous filtrate from the cake washing operation was monitored. Upon reaching a pH of ≥ 5, washing of the filter cake was terminated. The drying temperature was lowered from about 40 °C to 25 °C. 96

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Figure 13. Impurity 21.

Scheme 17. Improved Process for Reprecipitation of BIIB028

Using this improved procedure, 12.6 kg of BIIB028 (82% yield, 99.6% purity) was produced (Scheme 17). Two major impurities in reprecipitated BIIB028 were 22 (0.09%) and 24 (0.25%), both originating from impurities in the starting material 13 (Figure 14). Impurity 24 was derived from impurity 23 present in 13. Levels of residual volatiles also met specification limits during release testing (PO(OEt)3, 338 ppm).

Figure 14. Main impurities in the final BIIB028 API. 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.

Conclusions for BIIB028

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The improved process allowed a multi-kilogram quantity synthesis of BIIB028 in a single run, in higher overall yield (70%) and purity (99.6%) (30, 31). The control of impurities was achieved through the use of additional HPLC in-process controls (Sonogashira and phosphorylation reactions) and lowering of the phosphorylation reaction temperature. The use of six organic solvents was eliminated and the use of CuI, PPh3, 10% Pd/C, and POCl3 was reduced. Precipitation condition optimizations achieved a 75-80% reduction of filtration times of crude and reprecipitated BIIB028. Unfortunately, development of BIIB021 and BIIB028 was discontinued due to change of the company focus.

Acknowledgments The authors would like to thank Dr. Albert Kwok, Biogen, for helpful discussions during the course of BIIB021 work, and Dr. Yiqing Lin of Biogen for supporting analytical work on the BIIB028 project. The authors would also like to thank Dr. Thomas Clifford, Dr. Aarti Joshi and Dr. Shubham Chopade for their important contributions to the BIIB028 work while at IRIX Pharmaceuticals, Florence, SC. Dedicated to Professor Lanny Liebeskind of Emory University on the occasion of his 65th birthday.

References 1.

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Seo, Y. H. Small Molecule Inhibitors to Disrupt Protein-protein Interactions of Heat Shock Protein 90 Chaperone Machinery. J. Cancer Prev. 2015, 20, 5–11. Butler, L. M.; Ferraldeschi, R.; Armstrong, H. K.; Centenera, M. M.; Workman, P. Maximizing the Therapeutic Potential of HSP90 Inhibitors. Mol. Cancer Res.: MCR 2015, 13, 1445–51. Ren, J.; Yan, B. B.; Shi, F.; Xiong, B.; Shen, J. K. Progress in the Study of Small Molecule Inhibitors of HSP90. Acta Pharmacol. Sin. 2015, 50, 640–9. Stefan, T. G., W. , Anti-Hsp90 Therapy in Autoimmune and Inflammatory Diseases: a Review of Preclinical Studies. The Infona portal; https:// www.infona.pl/resource/bwmeta1.element.springer-doi-10_1007-S12192016-0670-Z. Solarova, Z.; Mojzis, J.; Solar, P. Hsp90 Inhibitor as a Sensitizer of Cancer Cells to Different Therapies (review). Int. J. Oncol. 2015, 46, 907–26. Seo, Y. H. Organelle-specific Hsp90 Inhibitors. Arch. Pharm. Res. 2015, 38, 1582–90. Jhaveri, K.; Taldone, T.; Modi, S.; Chiosis, G. Advances in the Clinical Development of Heat Shock Protein 90 (Hsp90) Inhibitors in Cancers. Biochim. Biophys. Acta, Mol. Cell Res. 2012, 1823, 742–755. Menezes, D. L.; Taverna, P.; Jensen, M. R.; Abrams, T.; Stuart, D.; Yu, G. K.; Duhl, D.; Machajewski, T.; Sellers, W. R.; Pryer, N. K.; Gao, Z. The Novel 98

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Oral Hsp90 Inhibitor NVP-HSP990 Exhibits Potent and Broad-spectrum Antitumor Activities in vitro and in vivo. Mol. Cancer Ther. 2012, 11, 730–9. Biamonte, M. A.; Van de Water, R.; Arndt, J. W.; Scannevin, R. H.; Perret, D.; Lee, W. C. Heat Shock Protein 90: Inhibitors in Clinical Trials. J. Med. Chem. 2010, 53, 3–17. Shi, X.; Chang, H.; Grohmann, M.; Kiesman, W. F.; Kwok, D.-I. A. Process Development of an N-Benzylated Chloropurine at the Kilogram Scale. Org. Process Res. Dev. 2015, 19, 437–443. Shi, X.; Chang, H.; Kiesman, W. F. Process Development of the Synthesis of BIIB021, A Potent Hsp90 Inhibitor for Cancer Treatment. The 13th International Conference and Exhibition: The Scale Up of Chemical Processes; Lake Maggiore, Italy, July 9−12, 2012. For N9 alkylation reactions of 6-chloro-9H-purin-2-amine (1) with a variety of alkylating agents under different conditions, see references 5 in ref. 10. Brik, A. W. C.-Y.; Best, M. D.; Wong, C.-H. Tetrabutylammonium FluorideAssisted Rapid N9-Alkylation on Purine Ring: Application to Combinatorial Reactions in Microtiter Plates for the Discovery of Potent Sulfotransferase Inhibitors in situ. Bioorg. Med. Chem. 2005, 13, 4622–4626. The hydroxy analogous impurities 11 and 10 discussed below are formed via the same mechanism. For general discussion about the SNAr displacement reactions of 6-halopurines nucleosides, see Liu, J; Robins, M. J. SNAr Displacements with 6-(Fluoro, Chloro, Bromo, Iodo, and Alkylsulfonyl)purine Nucleosides: Synthesis, Kinetics, and Mechanism. J. Am. Chem. Soc. 2007, 129, 5962–5968. All mention of purity or impurity level will refer to HPLC area % unless it is otherwise indicated. Coordination Group for Mutual Recognition-Human Committee (CMDh) home page. http://www.moh.gov.cy/moh/phs/ phs.nsf/All/5CB059619BF1ED85C225740C0035BBFF/$file/ CDMh_Letter_for_all_MAHs_for_MPs_containing_active_ingredients_in _the_form_of_Mesilates__Isetionates__Tosilates_or_Besilates.pdf? OpenElement. Robinson, D. I. Control of Genotoxic Impurities in Active Pharmaceutical Ingredients: A Review and Perspective. Org. Process Res. Dev. 2010, 14, 946–959and the references cited therein. Forryan, C. L.; Klymenko, O. V.; Wilkins, S. J.; Brennan, C. M.; Compton, R. G. Experimental and Theoretical study of the Surface-controlled Dissolution of Cylindrical Particles. Application to Solubilization of Potassium Hydrogen Carbonate in Hot Dimethylformamide. J. Phys. Chem. B 2005, 109, 20786–20793. Forryan, C. L.; Klymenko, O. V.; Brennan, C. M.; Compton, R. G. Heterogeneous Kinetics of the Dissolution of an Inorganic Salt, Potassium Carbonate, in an Organic Solvent, Dimethylformamide. J. Phys. Chem. B 2005, 109, 8263–8269.

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20. Fedorynski, M. W. K.; Matacz, Z.; Makosza, M. Reactions of Organic Anions. 86. Sodium and Potassium Carbonates: Efficient Strong Bases in Solid-liquid Two-phase Systems. J. Org. Chem. 1978, 43, 4682–4684. 21. Isolation of free base 3 (~99.5% purity) from the filtrate of the alkylation reaction in DMAc was demonstrated by crystallization at -10 °C followed by addition of MeOH-H2O anti-solvent at 2 kg scale. Due to the easy filtration, this isolation was deemed scalabe. 22. U.S. Pharmacopeia home page. http://www.pharmacopeia.cn/v29240/ usp29nf24s0_c281.html. 23. Crystallization onset time is dependent on the level of supersaturation and presence of seeds; see Mullin, J. W. Crystallization, 4th ed.; Elsevier Butterworth Heinemann: Amsterdam, 2001; p 206. 24. Due to the instability of BIIB021 in DMSO solution, a polish filtration was not performed in the recrystallization step. Instead, in the GMP productions a polish filtration of the solution of free base 3 in DMAc was performed prior to the salt formation. In addition, the solvents MTBE for crystallization and isolation of crude BIIB021, and DMSO and EtOAc for recrystallization were charged into the reactors through in-line filters. 25. Shi, J.; Van de Water, R.; Hong, K.; Lamer, R. B.; Weichert, K. W.; Sandoval, C. M.; Kasibhatla, S. R.; Boehm, M. F.; Chao, J.; Lundgren, K.; Timple, N.; Lough, R; Ibanez, G.; Boykin, C.; Burrows, F. J.; Kehry, M. R.; Yun, T. J.; Harning, E. K.; Ambrose, C.; Thompson, J.; Bixler, S. A.; Dunah, A.; Snodgrass-Belt, P.; Arndt, J.; Enyedy, I. J.; Li, P.; Hong, V. S.; McKenzie, A.; Biamonte, M. A. EC144 Is a Potent Inhibitor of the Heat Shock Protein 90. J. Med. Chem. 2012, 55, 7786–7795. 26. Lundgren, K.; Biamonte, M. A. The Discovery of BIIB021 and BIIB028. In Inhibitors of Molecular Chaperones as Therapeutic Agents; Machajewski, T. D., Gao, Z., Eds.; RSC Drug Discovery Series No. 37; The Royal Society of Chemistry: Cambridge, U.K., 2014; pp 158−179. 27. Zhang, H.; Burrows, F. J. Anti-tumor Methods Using Multi Drug Resistance Independent Synthetic Hsp90 Inhibitors; Int. Pat. Appl. 2007035963 A2, March 29, 2007. 28. Kashibatla, S. R.; Biamonte, M. A.; Shi, J.; Boehm, M. F. Pyrrolopyrimidine Derivatives Used as Hsp90 Inhibitors; Int. Pat. Appl. 2006105372 A2, October 5, 2006. 29. Urgaonkar, S. V. J. G. Ligand-, Copper-, and Amine-Free Sonogashira Reaction of Aryl Iodides and Bromides with Terminal Alkynes. J. Org. Chem. 2004, 69, 5752–5755. 30. Walker, D. G.; Humora, M. J.; Kiesman, W. F.; Joshi, A.; Clifford, T.; Chopade, S. Abstract of papers, 244th National Meeting of the American Chemical Society, Philadelphia, PA, Aug 19−23, 2012; American Chemical Society: Washington, DC, 2012; ORGN-209. 31. Walker, D. G. Process Improvements to the Synthesis of BIIB028, A Potent Hsp90 Inhibitor; The 14th International Conference and Exhibition: The Scale Up of Chemical Processes, La Jolla, CA, July 15−17, 2013.

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Chapter 4

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Discovery of the Novel Oxadiazole-Containing 5-Lipoxygenase Activating Protein (FLAP) Inhibitor BI 665915 Hidenori Takahashi,*,1 Alessandra Bartolozzi,1 and Thomas Simpson2 1Small Molecule Discovery Research, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877, United States 2Department of Chemistry, West Chester University, 700 South High Street, West Chester, Pennsylvania 19383, United States *E-mail: [email protected].

This is a brief review of the discovery of FLAP inhibitor BI 665915 (Takahashi, H., et al. J. Med. Chem. 2015, 58, 1669−1690). The chapter summarizes the discovery efforts including structure-activity relationship (SAR), drug metabolism and pharmacokinetics (DMPK) profile and medicinal chemistry synthetic route towards oxadiazolecontaining 5-lipoxygenase-activating protein (FLAP) inhibitors. A knowledge-based lead generation followed by lead optimization using a structure-based drug design provided compounds that demonstrated excellent FLAP binding potency (IC50 < 10 nM) and potent inhibition of LTB4 synthesis in human whole blood (hWB) (IC50 < 100 nM). Optimization of the binding, functional potencies and physicochemical properties resulted in the identification of a amino-pyrimidinyl molecule (BI 665915) that significantly inhibited LTB4 production in a murine ex vivo whole blood study in a dose-dependent manner. This also significantly inhibited atherosclerosis progression in a rabbit disease model. Based on the high quality of its overall profile and in vivo activity, the compound was advanced into preclinical development.

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Introduction 5-Lipoxygenase-activating protein (FLAP) was discovered in the early 1990s as an essential accessory protein that is involved in the cellular biosynthesis of leukotrienes (LTs) in the 5-lipoxygenase (5-LO) pathway (1, 2). FLAP and 5-LO are primarily expressed in neutrophils, monocytes, macrophages, eosinophils and mast cells. LTs are a family of eicosanoid pro-inflammatory mediators that are biosynthesized from arachidonic acid (AA) (3–6). The LT pathway is initiated upon inflammatory stimuli which activates phospholipase A2 (PLA2) which hydrolyzes phospholipid to release AA from the cell membrane. AA then binds to membrane-attached FLAP and interacts with 5-LO, leading to the oxidation of AA to the unstable intermediate leukotriene A4 (LTA4) (7). LTA4 is the common precursor for the biosynthesis of leukotriene B4 (LTB4) and the cysteinyl leukotriene C4 (LTC4) that can be further transformed into leukotrienes D4 and E4 (i.e., LTD4 and LTE4, respectively). These lipid mediators activate G protein-coupled transmembrane receptors (GPCRs). For example, LTB4 activates BLT1 and BLT2 while CysLTs stimulate CysLT1, CysLT2, and CysLTER that further trigger the pro-inflammatory signaling pathway (Figure 1). Through the course of research on LTs (8–10). it has become clear that they play important pathophysiological roles across a wide range of respiratory, allergic (11, 12), and cardiovascular diseases (13–17) such as asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD), arthritis, inflammatory bowel disease, psoriasis, liver fibrosis, cancer, endothelial dysfunction, intimal hyperplasia, atherosclerosis, myocardial dysfunction, ischemic stroke and aortic aneurysms. This chapter describes the structure-guided design, structure-activity relationships (SAR) and biological evaluation of a novel class of oxadiazole-containing FLAP inhibitors, as well as their divergent synthesis for the SAR development.

Discovery and SAR of the Novel Oxadiazole Containing Flap Inhibitors The first FLAP inhibitor, MK-886, was reported by Merck-Frosst in 1989 (18). In addition to MK-886, four other selective FLAP inhibitors have advanced to clinical trials (Figure 2) (19–21). These compounds share a common structural feature that comprises a carboxylic acid moiety that is tethered to a heteroaryl (i.e., an indole in MK-886, MK-591 and AM-803) or an aryl (i.e., a phenyl in BAY X1005 and ABT-080) scaffold via an alkyl linker (22, 23). The clinical progression of ABT-080 was stopped after phase I. MK-886 (24), MK-591 (25) and BAY X1005 (26) showed efficacy in asthmatic patients without adverse hepatotoxic events in contrast to the 5-LO inhibitor Zileuton© (27). Further development of MK-591 was discontinued because the degree of improvement observed in the clinical studies was not as good as expected given the biochemical potency (28), and deCODE stopped the development of BAY X1005 due to formulation issues. Finally, Amira’s FLAP inhibitor AM-803 (GSK-2190915) displayed good safety and tolerability, as well as efficacy on 102

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the allergen-induced asthmatic response in patients with mild asthma in phase II clinical trials (29, 30). Genetic, pharmacological, and clinical studies support the use of LT modulators beyond the treatment of asthma. Specifically, pre-clinical studies have shown that LTs play pathophysiological roles in cardiovascular diseases (31). The FLAP inhibitors MK-886 and BAY X1005 have demonstrated statistically significant reduction of the aortic atherosclerotic lesions in the apoE/LDLR double knockout mice model (32–34).

Figure 1. Leukotriene pathway. (Reproduced from reference (41). Copyright 2015 Americam Chemical Society). 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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In a Swedish nationwide population-based analysis of a large number of patients, the occurrence of cardiovascular endpoints in asthmatic patients treated with the CysLT1 antagonist Montelukast© revealed a modest decrease in the risk of recurrent stroke and myocardial infarction (35). Additionally, the 5-LO inhibitor VIA-2291 (Atreleuton©) has displayed a statistically significant reduction in noncalcified plaque volume at 24 weeks in the treated patients as compared to the placebo group (36). Recently, the drug discovery efforts on LT pathway inhibitors have been directed towards demonstrating a proof of clinical concept in cardiovascular diseases. For instance, AstraZeneca recently advanced FLAP inhibitor, AZD5718, into phase I clinical trials for cardiovascular disorders (37).

Figure 2. FLAP inhibitors advanced into clinical trials. (Reproduced from reference (41). Copyright 2015 Americam Chemical Society).

Based on the data in support of the therapeutic value of FLAP inhibitors, we decided to pursue FLAP as a target for the treatment of atherosclerosis. Although we initially applied high throughput screening for identifying lead chemical series, the method didn’t provide robust lead series. Then, we implemented a knowledge-based drug design approach to identify new lead chemical series using the X-ray co-crystal structure of human FLAP/MK-591 complex (PDB ID: 2Q7M) (38) in combination with published SAR on bis-aryl FLAP inhibitors (34, 39). The X-ray co-crystal structure of human FLAP/MK-591 complex revealed that FLAP crystallized as a homotrimer with MK-591 bound in a groove located at the interface of adjacent monomers and exposed to the lipid bilayer. Through computer-assisted drug design, we identified a series of substituted oxadiazoles, which were predicted by docking studies to occupy the same binding pocket as MK-591 (Figure 3). Indeed, the initial FLAP inhibition SAR of these oxadiazoles supported the predicted binding mode. 104

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Figure 3. Proposed binding mode for the oxadiazole series of FLAP inhibitors: (A) Overlay of a docking pose generated for 6 (dark gray sticks) with the crystal structure of FLAP in complex with MK-591 (2) (light gray sticks; PDB ID: 2Q7M). Key amino acids are labeled in the binding site and the inhibitors are shown as sticks. (B) The docking pose of 6 (dark gray sticks) in the FLAP binding site shown on semi-transparent rendering of solvent accessible protein surface for the binding site defined by labeled amino acids that are within 3.5 Å sphere of the inhibitor. (Reproduced from reference (41). Copyright 2015 Americam Chemical Society).

The SAR development in this series was initially driven by: • •

The FLAP binding assay that measured the ability of a test compound to displace the radio-labeled ligand [125I]-L-691831 (PE NEX084) (40). The FLAP functional assay that determined the inhibition of LTB4 synthesis in human whole blood (hWB) (41). 105

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The SAR summary of a select group of oxadiazole substituents is shown in Table 1. The amino-containing compound 7 showed a robust FLAP binding potency (IC50 = 20 nM) and served as a good starting point for further SAR development. The strong binding activity of 7 translated into potent inhibition of LTB4 synthesis in the hWB assay (IC50 = 440 nM). Although alkylation of the amino group (e.g., -NHEt) retained the binding and cellular potency, polar substituents on the amine moiety such as the sulfonyl group were not tolerated (data not shown). To explore the available space, a phenyl derivative 8 was synthesized and tested, which showed > 10-fold boost in the FLAP binding potency (IC50 = 1.4 nM) as compared to 7. Unfortunately, the improved binding affinity of 8 did not translate into higher potency in the hWB assay (IC50 = 470 nM). Compound 8 has high clogP (4.07) (42) and low topological polar surface area (TPSA: 91 Å2) (43). We hypothesized that highly lipophilic compounds such as 8 may be highly protein bound in human plasma (i.e., low free fraction) leading to low activity in hWB. To test this hypothesis, the phenyl substituent was systematically replaced with more polar 5- and 6-membered hetero-aromatic groups. Generally, the FLAP binding SAR of these analogs was relatively flat. For instance, the 3-pyridine 9, the 4-imidazole (11), the 3-pyrazole (12) and the 4-pyrazole (13) derivatives showed comparable FLAP binding potency (IC50 < 10 nM) to the phenyl analog 8. However, in line with our hypothesis, these less lipophilic hetero-aromatic compounds were two- to four-fold more potent in the hWB assay (9, 11-13: IC50 =110-270 nM) than 8. In contrast, pyridone derivative 10 displayed a good binding potency (IC50 = 2.9 nM) but did not inhibit the LTB4 production in the hWB assay at the highest tested concentration of 5 μM. Our analysis of a larger set of compounds in this series had revealed a relationship between the FLAP binding or hWB potency and TPSA (Figure 4). Although no clear correlation between TPSA and FLAP binding IC50 was observed, generally, compounds with high TPSA (> 120 Å2) showed diminished hWB activities, which could be attributed to lower cell penetration. Indeed, the lower activity of 10 (TPSA = 124 Å2) in the hWB assay was consistent with the findings from this analysis. Based on its attractive binding and functional potencies, 13 was selected for further optimization. Table 2 shows the SAR summary of the 4-pyrazole substitutions. Methyl substituted compound 14 showed comparable binding potency (IC50 = 1.9 nM) to 13 and approximately two-fold improvement in hWB activity (IC50 = 45 nM). Although 13 and 14 showed good FLAP binding and functional potencies, they were both highly crystalline and displayed low aqueous solubility at pH 6.8 (0.4 and 0.03 μg/mL, respectively).

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Table 1. SAR of Substituted Oxadiazoles

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Figure 4. Correlation between TPSA (Å2) and FLAP binding IC50 (nM) (A) or hWB IC50 (nM) (B).

To improve the aqueous solubility, we decided to substitute the methyl group of 14 with polar solubilizing groups. The N,N-dimethylethylamine analog 15 showed good binding and functional potencies, as well as high aqueous solubility (270 μg/mL at pH 6.8). However, 15 has an embedded basic moiety (i.e., the –NMe2 group) that was believed to be the source of cytochrome P450 (CYP450) enzymes 2C9 and 2D6 inhibition (IC50 = 7 and 10 μM, respectively). In contrast, compound 16, which was substituted with a neutral N,N-dimethylacetamide group, did not inhibit the CYP450 enzymes at the highest tested concentrations (30 μM) while maintaining the FLAP binding and hWB activities (IC50 = 3.6 and 81 nM, respectively). The carboxymethyl derivative 17, de-protonated and negatively charged under physiological and assay conditions, was five-fold less potent in FLAP binding than 16, and did not inhibit the LTB4 production in the hWB assay. However, a hindered and neutral hydroxyl containing analog 18 showed good binding potency and functional activity (IC50s of 3.5 and 69 nM, respectively). Apparently, removal of the charged group (i.e., -COO– → -OH) and shielding of the polar hydroxyl moiety with the methyl groups provided a good balance of functional activity and aqueous solubility (e.g., 18: hWB IC50 = 69 nM, pH 6.8 sol = 8.1 μg/mL). Docking studies with N-substituted 4-pyrazoles suggested that the N-1 substituent pointed out of the FLAP binding pocket towards the lipophilic center of the phospholipid bilayer (Figure 3B). The observed loss of binding potency associated with 17 (i.e., an analog bearing a charged group) and potent binding activity of 18 (i.e., a derivative containing a neutral and shielded polar functional group) was consistent with this modeling hypothesis.

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Table 2. SAR of N-Substituents on 4-Pyrazole

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Table 3 shows the SAR at the benzylic carbon group. The unsubstituted compound 19 showed lack of FLAP binding and hWB potencies at the highest tested concentrations (1 μM and 5 μM, respectively). However, the dimethyl analog 20 showed good FLAP binding activity (IC50 = 6.7 nM) and a robust functional inhibition in the hWB assay (IC50 = 93 nM). This SAR suggests that the substitution on the C-8 position is crucial for the FLAP binding activity. Interestingly, replacing the gem-dimethyl group with a cyclopropane ring, as in 21, resulted in a 15-fold loss of binding potency (cf. 21 binding IC50 = 100 nM vs. 20 binding IC50 = 6.7 nM) while increasing the ring size to cyclobutane ring restored the binding and functional activities (e.g., 14: binding IC50 = 1.9 nM; hWB IC50 = 45 nM). We calculated the torsional angle between the bis-aryl groups for various R1 and R2 substituents. These calculations suggested that the cyclopropane ring of 21 caused the maximal distortion of the bis-aryl angle (119.0°). In contrast, the geminal methyls of compound 20 (104.2°) and the cyclobutane ring in compound 14 (109.0°) affected smaller deviations from the tetrahedral bond angle. In view of the observed binding potencies of these compounds, it appears that the bond angle between the two aryl groups has a profound impact on the FLAP binding potency. A bond angle of < 119° seemed to be required for achieving a good binding potency (IC50 < 100 nM). Docking studies indicated that the benzylic carbon occupied a small hydrophobic space that was at the interface of the α4 and α1 helices of two adjacent FLAP monomers and lined with the side chains of a collection of hydrophobic amino acid residues such as V20, V21, I119, L120, and F123 (Figure 3A and B). The binding pose showed that the linker substituents pointed towards the inner part of this small pocket. A closer examination of the docking model indicated that the R2 substituent occupied a smaller hydrophobic cavity closer to the I119, L120 and F123 cluster of residues, while R1 was pointing towards a slightly larger space near the V20, and the V21 residues of the two adjacent monomer (Figure 3B), which suggested a potential stereochemical preference. To validate the modeling prediction and to assess the impact of various substituents on the FLAP binding and functional potencies, additional SAR on the benzylic carbon group was performed. The racemate of the iso-propyl/methyl analog 22 showed similar binding potency as compared to the germinal dimethyl compound 20 (cf. IC50 = 2.0 nM vs. IC50 = 6.7 nM, respectively). Compound 22 was resolved to provide the (R) and the (S) enantiomers, (23 and 24, respectively). The (R)-enantiomer 23 showed potent FLAP binding and functional activities with IC50 values of 1.6 and 33 nM, respectively. In fact, the (R)-enantiomer 23 was approximately five-fold more potent in FLAP binding and ten-fold more potent in hWB activity than the corresponding (S)-enantiomer 24. A similar pattern was also observed with enantiomers 25 and 26. These data confirmed that the substitution on the benzylic carbon of the bis-aryl core required an optimal size and chirality for maximal FLAP inhibition.

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Table 3. SAR of Substitutions on the Methylene Linker

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Table 4. SAR of Left Hand Side Phenyl Substitutions

The docking studies also suggested that the substituents on the left-hand side phenyl occupied a small cavity inside of the FLAP helices (Figure 3B); hence, it was predicted that groups occupying this binding pocket would have size limitation. Our initial SAR in this area had indicated that para-amino substituted nitrogen-containing 6-membered heteroaryls were preferred. Table 4 shows the SAR of a select group of these 6-membered heteroaromatic groups. For example, the amino-pyridyl analog 27 and the amino-pyrimidinyl analog (28: BI 665915) 112 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.

displayed good binding potencies (IC50 = 1.1 and 1.7 nM, respectively) and hWB activities (IC50 = 66 and 45 nM, respectively). Introduction of an alkyl chain on the amino substituent did modulate both the binding and the functional potencies. For instance, the aminomethyl analog 29 retained both binding and functional activities as compared to the non-alkylated derivative 28. However, larger substituents such as iso-propyl amine (e.g., 29) eroded the FLAP binding and cellular potencies.

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Table 5. Overall Profile of BI 665915

Overall, the SAR indicated several favorable structural features to achieve potent FLAP binding and functional inhibition. For instance, the most favorable effects were obtained when the C-8 benzylic carbon was a part of cyclobutane ring or carries (R)-cyclopropyl/methyl groups. Also, N,N-dimethylacetamide, N,N-dimethylaminoethyl and 1,1-dimethyl-ethanol moieties were preferred 113 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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as the substituents on the pyrazole ring. Thus, an array of compounds that combined these preferred groups were synthesized and evaluated. Compound 28 that featured the N,N-dimethylacetamide substituted pyrazole and the (R)-cyclopropyl/methyl central substituents showed good FLAP potencies and a favorable off-target profile (Table 5). This compound was also very selective for FLAP as compared to other enzymes and receptors in the leukotriene pathway such as 5-LO, cycloxygenease (COX)-1 and -2, PLA2, LTA4 hydrolase, BLT1, and BLT2 (< 50 % inhibition at 10 μM). Based on its overall profile, 28 was selected for in vitro and in vivo DMPK assessment. Compound 28 showed a modest human hepatocyte clearance (41 percent of hepatic blood flow (% Qh)) and relatively high plasma protein binding (unbound fraction = 4.7 %). It displayed high membrane permeability in Caco-2 cell line with a low efflux ratio (AB = 34 x 10-6 cm/s, efflux ratio = 1.9). Weak CYP450 3A4 induction was detected (~2-fold at 30 µM) for 28; however, there was no evidence of time-dependent inhibition (highest tested concentration = 100 µM) suggesting a low risk for potential drug-drug interactions. The pharmacokinetic properties of 28 were evaluated in rat, dog, and cynomolgus monkey. It showed low iv plasma clearance in all three species (CL = 7 % Qh in rat, 2.8 % Qh in dog, and 3.6 % Qh in cynomolgus monkey). The volumes of distribution (Vss) across the tested species were in the range of 0.5 to 1.2 L/kg, and the bioavailabilities were moderate to good (F = 45 to 63 %). The overall DMPK profile of 28 was very attractive and qualified it for advancement into the ex vivo stimulated LTB4 production in mice whole blood study. Compound 28 demonstrated a dose-dependent inhibition of an ex vivo stimulated LTB4 production in mice whole blood after a single oral dose (LTB4 production inhibition = 94.9 % at 100 mg/kg dose, 77.8 % at 30 mg/kg dose and 9.0 % at 10 mg/kg dose as compared to the vehicle treated group) (41). Compound 28 was also found to significantly inhibit atherosclerosis progression in a rabbit disease model (44, 45), that qualified it as a pre-clinical development candidate.

Medicinal Chemistry Synthetic Route The general synthetic strategy that was implemented for the SAR development of the oxadiazole-containing FLAP inhibitors is shown in Scheme 1. According to this approach, dialkylation of 4-bromophenylacetonitrile 31 would generate a quaternary nitrile of formula 32, which could be converted to the amidoxime 33 by treatment with hydroxylamine. Cyclization of 33 with a compound bearing an activated carboxylic acid moiety could produce an oxadiazole of formula 34, which could be further functionalized via Suzuki-Miyaura cross-coupling reaction to generate the FLAP inhibitors 35. For some examples, the synthetic protocol was modified as follows: the phenyl group of 32 was functionalized via SuzukiMiyaura cross-coupling reaction and followed by an oxadiazole cyclization and installation of oxadiazole functional group. The representative synthetic route for 23 and 24 using the later synthetic protocol is summarized in Scheme 2. 114

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Scheme 1. General synthesis of oxadiazole-containing FLAP inhibitors. (Reproduced from reference (41). Copyright 2015 Americam Chemical Society).

Sequential dialkylation of 4-bromophenylacetonitrile 31 with 2-bromopropane and methyl iodide generated a racemic mixture of structure 36. Compound 36 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester via Suzuki-Miyaura cross-coupling reaction afforded 37. Then 37 was reacted with hydroxylamine to form the amidoxime, and the amidoxime intermediate was coupled with 1-methyl-1H-pyrazole-4-carboxylic acid to afford the racemate 22; which was separated into pure enantiomers 23 and 24 using chiral supercritical fluid chromatography (SFC). The absolute configuration at the C-8 of 23 and 24 were assigned based on single-crystal X-ray diffraction studies of representative compounds.

Scheme 2. Representative synthesis of chiral compounds 23 and 24 using chiral SFC separation

In conclusion, a series of oxadiazole-containing FLAP inhibitors was identified from the knowledge-based drug design. The series was successfully optimized for FLAP binding and funcational hWB activities, selectivity and in vivo efficacy through the structure-based rational drug design. The compound 28 demonstrated desired potency and overall drug-like properties, and 28 was nominated for a preclinical development candidate. 115 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 the SAR development, we applied the SFC chiral separation to synthesize enatiomercally pure compounds. The asymmetric synthetic route for large scale preparation of 28 (BI 665915) was successfully developed by the chemical development, and the details are discussed in the following chapter.

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16. Radmark, O. 5-Lipoxygenase-derived leukotrienes: mediators Also of Atherosclerotic Inflammation. Arterioscler., Thromb., Vasc. Biol. 2003, 23, 1140–1142. 17. Riccioni, G.; Zanasi, A.; Vitulano, N.; Mancini, B.; D’Orazio, N. leukotriene in atherosclerosis: New target insights and future therapy perspective. Mediators Inflammation 2009, 2009, 1–6. 18. Gillard, J. W.; Ford-Hutchinson, A. W.; Chan, C.; Charleson, S.; Denis, D.; Foster, A.; Fortin, R.; Leger, S.; McFarlane, C. S.; Morton, H.; Piechuta, H.; Rendeau, D.; Rouzer, C. A.; Rokach, J.; Young, R.; MacIntyre, D. E.; Peterson, L.; Bach, T.; Eiermann, G.; Hopple, S.; Humes, J.; Hupe, L.; Luell, S.; Metzger, J.; Meurer, R.; Miller, D. K.; Opas, E.; Pacholok, S. L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio5-isopropylindol-2-yl]-2,2-dimethylpropionic acid), a novel, oraly active leukotriene biosynthesis inhibitor. Can. J. Physiol. Pharmacol. 1989, 67, 456–464. 19. Evans, J. F.; Ferguson, A. D.; Mosley, R. T.; Hutchinson, J. H. What’s all the FLAP about?: 5-lipoxygenase-activating protein inhibitors for inflammatory diseases. Trends Pharmacol. Sci. 2007, 29, 72–78. 20. Sampson, A. P. FLAP inhibitors for the treatment of inflammatory diseases. Curr. Opin. Invest. Drugs 2009, 10, 1163–1172. 21. Pettersen, D.; Davidsson, O.; Whatling, C. Recent advances for FLAP inhibitors. Bioorg. Med. Chem. Lett. 2015, 25, 2607–2612. 22. Kolasa, T.; Gunn, D. E.; Bhatia, P.; Basha, A.; Craig, R. A.; Stewart, A. O.; Bouska, J. B.; Harris, R. R.; Hulkower, K. I.; Malo, P. E.; Bell, R. L.; Carter, G. W.; Brooks, C. D. W. Symmetrical bis(heteroarylmethoxyphenyl)alkylcarboxylic acids as inhibitors of leukotriene biosynthesis. J. Med. Chem. 2000, 43, 3322–3334. 23. Stock, N. S.; Bain, G.; Zunic, J.; Li, Y.; Ziff, J.; Roppe, J.; Santini, A.; Darlington, J.; Prodanovich, P.; King, C. D.; Baccei, C.; Lee, C.; Rong, H.; Chapman, C.; Broadhead, A.; Lorrain, D.; Correa, L.; Hutchinson, J. H.; Evans, J. F.; Prasit, P. 5-Lipoxygenase-Activating Protein (FLAP) Inhibitors. Part 4: Development of 3-[3-tert-butylsulfanyl-1-[4-(6ethoxypyridin-3-yl)benzyl]-5-(5-methylpyridin-2-ylmethoxy)-1H-indol-2yl]-2,2-dimethylpropionic acid (AM803), a potent, oral, once daily FLAP inhibitor. J. Med. Chem. 2011, 54, 8013–8029. 24. Friedman, B. S.; Bel, E. H.; Buntinx, A.; Tanaka, W.; Han, y. H.; Shingo, S.; Spector, R.; Sterk, P. Oral leukotriene inhibitor (MK-886) blocks allergeninduced airway responses. Am. Rev. Respir. Dis. 1993, 147, 839–844. 25. Diamant, Z.; Timmers, M.; van der Veen, H.; Friedman, B. S.; De Smet, M.; Depre, M.; Hilliard, D.; Bel, E. H.; Ster, P. J. The effect of MK-591, a novel 5-lipoxygenase activating protein inhibitor, on leukotriene biosynthesis and allergen-induced airway responses in asthmatic subjects in vivo. J. Allergy Clin. Immunol. 1995, 95, 42–51. 26. Dahlen, B.; Kumlin, M.; Ihre, E.; Zetterstrom, O.; Dahlen, S. E. Inhibition of allergen-induced airway obstruction and leukotriene generation in atopic asthmatic subjects by the leukotriene biosynthesis inhibitor BAYx 1005. Thorax 1997, 52, 342–347. 117

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27. DuBuske, L. M.; Grossman, J.; Dube, L. M.; Swanson, L. J.; Lancaster, J. F. Randomized trial of zileuton in patients with moderate asthma: effect of reduced dosing frequency and amounts of pulmonary function and asthma symptoms. Zileuton Study Group. Am. J. Manage. Care 1997, 3, 633–640. 28. Brooks, C.D.W.; Summers, J.B. Modulators of leukotriene biosynthesis and receptor action. J. Med. Chem. 1996, 39, 2629–1654. 29. Follows, R.M.A.; Snowise, N.G.; Ho, S.Y.; Ambery, C.L.; Smart, K.; Mcquande, B.A. Efficacy, safety and tolerability of GSK2190915, a 5-lopoxygenase activating protein inhibitor, in adults and adolescents with persistent asthma: a randomized dose-ranging study. Respir. Res. 2013, 14, 54–63. 30. Kent, S.E.; Boyce, M.; Diamant, Z.; Singh, D.; O’Connor, B.J.; Saggu, P.S.; Norris, V. The 5-lipoxygenase-activating protein inhibitor, GSK2190915, attenuates the early and late responses to inhaled allergen in mild asthma. Clin. Exp. Allergy 2013, 43, 177–186. 31. Hakonarson, H.; Thorvaldsson, S.; Helgadottir, A.; Gudbjartssson, D.; Zink, F.; Anderesdottir, M.; Manolescu, A.; Arnar, D. O.; Andersen, K.; Sigurdsson, A.; Thorgeirsson, G.; Jonsson, A.; Agnarsson, U.; Bjornsdottir, H.; Gottskalksson, G.; Einarsson, A.; Gudmundsdottir, H.; Adalsteinsdottir, A. E.; Gudmundsson, K.; Kristjansson, K.; Hardarson, T.; Kristinsson, A.; Topol, E. J.; Gulcher, J.; Kong, J.; Gurney, M.; Thorgeirsson, G.; Stefansson, K. Effects of a 5-lipoxzygenase-activating protein inhibitor on biomarkers associated with risk of myocardial infarction. J. Am. Med. Assoc. 2005, 293, 2245–2256. 32. Jawien, J.; Gajda, M.; Rudling, M.; Mateuszuk, L.; Olszanecki, R.; Guzik, T. J.; Cichocki, T.; Chlopicki, S.; Korbut, R. Inhibition of five lipoxygenase activating protein (FLAP) by MK-886 decreases atherosclerosis in apoE/ LDLR-double knockout mice. Eur. J. Clin. Invest. 2006, 36, 141–146. 33. Jawien, J.; Gajda, M.; Olszanecki, R.; Korbut, R. Bay x 1005 attenuates atherosclerosis in apoE/LDLR-double knockout mice. J. Physiol. Pharmacol. 2007, 58, 583–588. 34. Macdonald, D.; Brideau, C.; Chan, C. C.; Falgueyret, J. P.; Frenette, R.; Guay, J.; Hutchinson, J. H.; Perrier, H.; Prasit, P.; Riendeau, D.; Tagari, P.; Therien, M.; Young, R. N.; Girard, Y. Substituted 2,2-bis-arylbicycloheptanes as novel and potent inhibitors of 5-lipoxygenase activating protein. Bioorg. Med. Chem. Lett. 2008, 18, 2023–2027. 35. Ingelsson, E.; Yin, L.; Back, M. Nationwide cohort study of the leukotriene receptor antagonist montelukast and incident or recurrent cardiovascular disease. J. Allergy Clin. Immunol. 2012, 129, 702–707. 36. Tardif, J.C.; L’Allier, P.L.; Ibrahim, R.; Grégorie, J.C.; Noza, A.; Cossette, M.; Kouz, S.; Lavoie, M.A.; Paguin, J.; Brotz, T.M.; Taub, R.; Pressacco, J. Treatment with 5-lipoxygenase inhibitor VIA-2291 (Atreleuton) in patients with recent acute coronary syndrome. Circ. Cardiovasc. Imaging 2010, 3, 298–307. 37. AstraZeneca website; https://www.astrazeneca.com/our-science/ pipeline.html (May 1, 2016) 118

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38. Ferguson, A. D.; McKeever, B. M.; Xu, S.; Wisniewski, D.; Miller, D. S.; Yamin, T. T.; Spencer, R. H.; Chu, L.; Ujjainwalla, F.; Cunningham, B. R.; Evans, J. F.; Becker, J. W. Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein. Science 2007, 317, 510–512. 39. Ogawa, A.; Ujjainwalla, F.; Vande Bunte, E. K.; Chu, L.; Ondeyka, D.; Kopka, I.; Li, B.; Ok, H. O.; Patel, M.; Sisco, R. (Merck & Co., INC.) WO2008156721 A1, 2008. 40. Charleson, S.; Prasit, P.; Léger, S.; Gillard, J. W.; Vickers, P. J.; Mancini, J. A.; Charleson, P; Guay, J.; Ford-Hutchinson, A.W.; Evans, J.F. Characterization of a 5-lipoxygenase-activating protein binding assay: Correlation of affinity for 5-lipoxygenase-activating protein with leukotriene synthesis inhibition. Mol. Pharmacol. 1992, 41, 873–879. 41. Takahashi, H.; Riether, D.; Bartolozzi, A.; Bosanac, T.; Berger, V.; Binetti, R.; Broadwater, J.; Chen, Z.; Crux, R.; DeLombaert, S.; Dave, R.; Dines, J.A.; Fadra-Khan, T.; Fleggs, A.; Garrigou, M.; Hao, M.H.; Huber, J.; Hutzler, J.M.; Kerr, S.; Kotey, A.; Liu, W.; Lo, H.Y.; Loke, P.L.; Mahaney, P.E.; Morwick, T.; Napier, S.; Olague, A.; Pack, E.; Padyana, A.; Thomson, D.S.; Tye, H.; Wu, L.; Zindell, R.M.; Abeywardane, A.; Simpson, T. Synthesis, SAR and Series Evolution of Novel OxadiazoleContaining 5-Lipoxygenase Activating Protein Inhibitors: Discovery of 2-[4-(3-{(R)-1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (BI 665915). J. Med. Chem. 2015, 58, 1669–1690. 42. clogP values were calculated by 4.1 BioByte using MOE. 43. Ertl, P.; Rhode, B.; Selzer, P. Fast calculation of molecular polar surface as a sum of fragment-based contributions and its application to the prediction of drug transport. J. Med. Chem. 2000, 43, 3714–3717. 44. Bylock, L.A. (Boehringer Ingelheim Pharmacueticals, Inc.) WO2013113799 A1, 2013. 45. Manuscript in preparation

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Chapter 5

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Development of an Efficient Asymmetric Synthesis of the Chiral Quaternary 5-Lipoxygenase Activating Protein Inhibitor Keith Fandrick,* Jason Mulder, Jean-Nicolas Desrosiers, Nitin Patel, Xingzhong Zeng, Daniel Fandrick, Carl A. Busacca, Jinhua J. Song, and Chris H. Senanayake Department of Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06778, United States *E-mail: [email protected].

The rapid pace of the development program along the structurally complex 5-lipoxygenase activating protein (FLAP) inhibitor required a dual strategic approach within process development. In order to advance the program forward, the Discovery synthesis was rendered safe and scalable while eliminating the non-scalable chromatographic chiral separation. This approach allowed the advancement of the target while offering the necessary development time to discover an efficient asymmetric process for the synthesis of the challenging drug target. Multiple approaches were explored experimentally for an asymmetric synthesis; the ultimate route was derived from a dual boronate rearrangement process that was rendered robust and efficient for large-scale operations.

© 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.

Introduction Process development resides at the interface between medicinal chemistry and manufacturing. In this capacity, process development is charged with two main tasks:

•

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•

The delivery of high quality drug substance to advance the drug candidate through development. The development of a practical and economical process for manufacturing.

For the cases where a drug candidate is particularly synthetically challenging, process development teams invoke a strategy where they quickly modify and render safe the medicinal chemistry approach to supply the required drug substance to advance the candidate. This strategic approach provides the necessary time to devise innovative solutions that are required to develop an efficient chemical process or route for manufacturing. The complexity of compound 1, a 5-lipoxygenase activating protein (FLAP) inhibitor, necessitated such an approach. The compound represents a significant challenge for development (1–3) as it contains diverse functionalities and at its core is an all-carbon quaternary stereogenic center. Retrosynthetically the target can be reduced to the aryl-aldehyde 5 that contains the crucial quaternary sterogenic center (Figure 1). A classical approach to the synthesis of 5 involves the construction of the benzylic quaternary center via ionization of the tertiary alcohol followed by trapping with a suitable soft nucleophile (4). Due to the formation of the benzylic carbocation, the route is racemic and a chiral separation/resolution is required to produce the single enantiomer FLAP inhibitor. Unfortunately, this also sacrifices at least half of the overall yield. As highlighted in a recent review article (5), the stereoselective construction of all-carbon quaternary centers embedded in acyclic systems has proved to be particularly challenging. Although there are several asymmetric methodologies for the synthesis of acyclic all-carbon stereocenters in the literature (4, 6–14), these methods require cryogenic temperatures and/or have limited substrate scope. Thus the development of a new practical synthesis was required. The resolution approach was implemented initially to supply the necessary drug substance for development. We then explored the validity of three approaches (Figure 1) for the asymmetric synthesis of 5 including:

• • •

The nitro olefin approach employing the asymmetric copper catalyzed conjugate addition with a suitable nitro olefin substrate (15). A route employing the stereospecific pinacol rearrangement. A stereospecific boronate rearrangement approach (16, 17).

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Figure 1. Racemic and Asymmetric Strategies Experimentally Explored.

Modified Discovery Route The synthesis of 5 presented significant challenges for large-scale operations and required two main objectives, (Scheme 1). The first was to eliminate the bottleneck and low yielding chiral SFC separation in the Discovery synthesis. A resolution strategy toward the quaternary center would avoid the chiral SFC separation and be amenable to large scale synthesis. Despite the aforementioned liabilities of the Discovery synthesis, nitrile 6 was a strategic intermediate as it could be readily converted to the corresponding carboxylic acid, which in turn could serve as an effective handle for resolution via diastereomeric salt formation (18). The second main objective was to render the conversion of nitrile 6 to the corresponding amidoxime safe for large scale operations. The highly convergent end-game required extensive optimization for both the optimal reaction sequence and the high catalyst loading Suzuki-Miyaura cross-coupling (19, 20). The overall objectives (Scheme 2) are to render the original discovery approach robust and safe in order to deliver the initial material needs for early development. 123

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Scheme 1. Discovery Approach

Scheme 2. Resolution Strategy

Racemic Acid Synthesis A robust and efficient synthesis of the requisite tertiary alcohol 15 was targeted first. The Discovery route to 15 involved methyl Grignard addition to the relatively expensive ketone 7 (Scheme 1). Two alternative approaches were explored involving either the cyclopropyl Grignard (21) addition to an acetophenone precursor, or the aryl Grignard addition to cyclopropylmethyl ketone 21. Our efforts focused on optimization of the latter process (Scheme 3) as the starting materials were widely available. The requisite 4-bromophenyl Grignard reagent was generated in situ via metal halogen exchange of 1,4-dibromobenzene using the iPrMgCl-LiCl complex (22, 23). The aryl Grignard addition to the ketone proceeded in high yield to provide tertiary alcohol 15 that was used directly in the subsequent cyanation reaction. It was suspected that the low purity for nitrile 16 in the Discovery route was caused by prolonged holding of the mixture containing the tertiary alcohol and BF3•OEt2 before before the TMSCN addition, thus generating a number of impurities. It was found that the carbocation was very effectively trapped by premixing the tertiary alcohol 15 with excess TMSCN and slowly adding the BF3•OEt2 (24) reagent to the mixture at low temperature, thus improving the yield of 16 to nearly quantitative, with excellent purity. Compounds 15 and 16 were both oils making their isolation and purification challenging. Purification had to be postponed until isolation of a downstream solid could be achieved. Thus it was imperative that the two steps (to form 15 and 16) were high yielding and generated minimal impurities. 124

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Scheme 3. Synthesis of 20-DCA Salt The racemic nitrile 16 was converted to acid 20 via hydrolysis with potassium hydroxide. A high concentration of potassium hydroxide in 1-propanol was required in order to drive the hydrolysis of the nitrile moiety fully to the carboxylic acid. In subsequent optimization, performing the chemistry in a pressure vessel (facilitating temperatures > 120 °C, compared to a reflux temperature of 107 °C at atmospheric pressure) reduced the reaction time by a factor of 2-3. Acid 20 could not be isolated as a solid directly, but the corresponding dicyclohexylammonium (DCA) salt was readily formed and provided for a clean isolation of 20-DCA.

Resolution of Racemic Acid 20 After screening an extensive library of chiral amines, a robust process was identified for the resolution using the chiral amine (1R,2R)-1,3-dihydroxy-1(4-nitrophenyl)propan-2-amine (DNP) utilizing a single solvent (IPA) system (Scheme 4). Consistent good overall yield (28-32%) and high enantiomeric purity (>99% ee) were achieved with good crystallization control through temperature cycling. In order to meet the >99% enantiomeric purity requirement, the process required two enrichments. Efforts to isolate the crude carboxylic acid directly after the hydrolysis as the 23-DNP salt (avoiding the intermediate DCA isolation) led to lower overall recoveries (20% overall yield).

Scheme 4. Resolution of 20

Amidoxime Formation With an efficient synthesis of the enantiomerically pure carboxylic acid, the conversion to the key chiral nitrile 6 was achieved via an efficient telescopic sequence (Scheme 5). The reaction sequence could be monitored by React-IRTM. The implementation of this process analytical technology (PAT) monitoring ultimately allowed the reduction of the reagent charge of thionyl chloride, since no change was observed during further excess addition (Figure 2), which ultimately simplified the processing and subsequent operations. 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.

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Scheme 5. Formation of Nitrile 6

Figure 2. React-IRTM trace of acid chloride formation.

Conversion of this rather hindered nitrile to amidoxime 26 presented a significant safety challenge for large scale operations. In the Discovery route, this transformation was accomplished thermally from the corresponding nitrile and hydroxylamine. This approach required thermally unsafe refluxing conditions in ethanol and 20-30 fold excess of hydroxylamine. For large scale synthesis, the amidoxime synthesis was accomplished in a step-wise process via the oxime intermediate 25 (Scheme 6). A DIBAL-H reduction of nitrile 6 to the aldehyde was followed by oxime formation. A rapid exotherm was observed during the HCl quench with local hot spots, which led to the undesired reduction of oxime 25 to the corresponding primary amine. The presence of the amine complicated the work-up significantly by causing emulsions during the phase separations. A CeliteTM filtration resolved the immediate problem. The amidoxime was formed by a two-step one-pot process via chloro-oxime formation using NCS and catalytic acid (17), followed by ammonia treatment (25, 26). After recrystallization from a toluene and heptane mixture, amidoxime 26 was isolated in 75% yield based on the assay of crude 25, (98.9% purity, 97.3 wt%, 99.4 %ee) (overall yield from solid 23-DNP to solid 26 over 3 steps was 59%). 126

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Scheme 6. Formation of Amidoxime 26

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Oxadiazole Formation The oxadiazole 27 was formed via reaction of amidoxime 26 and the acyl imidazole derived from activation of acid 3 with CDI (Scheme 7). A higher reaction temperature (100 °C) was required to drive the final hydroxyl elimination forming the 1,2,4 oxadiazole. The charge of CDI was found to be optimal at 1.05 equivalents and the pyrazole-carboxylic acid 3 at 1.1 equivalents. Charging either of these materials in greater excess was detrimental to the reaction (vida infra). THF was used as a carrier solvent for reagent addition and was then removed by distillation until the reaction temperature reached 100 °C. As a result, only 2.5 volumes of DMF were used in the reaction obviating the need for any back extractions during work-up (27). Oxadiazole 27 was obtained as an oil (not isolated) in an average yield of 95% (assay corrected) and 90% purity.

Scheme 7. Synthesis of Oxadiazole 27 Significant inconsistencies were seen in initial Suzuki-Miyaura coupling studies using nitrile 6 (Discovery Route) since 6 could not be purified. In order to achieve a consistent and a practical palladium catalyst loading for the Suzuki-Miyaura coupling, it was advantageous to have a crystalline solid starting material to insure consistent quality. Fortunately oxadiazole 27 (Scheme 7), whose free base is non-crystalline, could be crystallized as its mesylate salt in 80% yield (95% purity, 99.3%ee). No other acids screened provided such a well-behaved, isolable salt. Several reaction intermediates were identified in the cyclization reaction in route to 27 (Scheme 8). The excess amount of (1H-imidazol-1-yl)(1Hpyrazol-4-yl)methanone C, formed from CDI and pyrazole acid, reacted with the intermediate amide A to form B which may inhibit the final dehydration to form the oxadiazole. Through the course of the reaction, intermediates A and B are converted to E, which is dehydrated to provide oxadiazole 27. Impurity D, apparently formed directly from a reaction of amidoxime 26 with excess CDI is a non-productive pathway. This impurity could be controlled via using an excess of acid 3 as compared to CDI. 127

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Scheme 8. 1,2,4-Oxadiazole Mechanistic Insights

Initial Boronate Synthesis and Suzuki-Miyaura Cross-Coupling A palladium-catalyzed borylation protocol using di-t-butylphosphinoferrocene tetrafluoroborate (FcP(t-Bu)2-HBF4) (28) as the ligand was developed into an efficient process for the synthesis of highly pure boronate 4 (Scheme 9). Following the reaction, the mixture was diluted with THF, the inorganics were removed by filtration, and the solvent was switched to EtOH. The boronate was isolated as a white crystalline solid from EtOH directly after an activated carbon treatment of the solution, which was implemented to remove residual palladium species which could have a deleterious effect on the subsequent Suzuki-Miyaura coupling. The scale-up of the Suzuki-Miyaura coupling after preliminary optimization proceeded smoothly to provide 28 (Scheme 9). An activated carbon treatment of the EtOAc solution of the crude product was used to remove color and to reduce residual palladium level to < 10 ppm.

Scheme 9. Completion of the Synthesis of 1 128 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.

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The final alkylation step was modified from the initial solvents of THF/water to DMF to improve throughput and the reaction rate, most likely due to the improved solubility of the starting materials. In addition, an organic base (DBU) was used instead of poorly soluble K2CO3. After reaction completion, the crude API (1) was crystallized from the reaction mixture. The crude API contained 0.53% of an unexpected impurity which was not purged in the final crystallization. This impurity was identified as compound 29 resulting from the 1,4-addition of 27 to dibenzylideneacetone and subsequent alkylation analogous to the API (Figure 3). Extensive work to remove 29 resulted in two effective options: • •

Crystallization of HCl salt of the API / salt break. Anisole recrystallization.

However, since the dimethylamide moiety is sensitive to strong acid and base, resulting in partial amide hydrolysis, the anisole crystallization was chosen to remove the impurity. After a final crystallization from ethanol/water, 1 was obtained with < 0.20% of the impurity which met the acceptance criteria. This route was sufficient to provide the first early development batches of 1 up to 1.5 kg.

Figure 3. dba adduct impurity 29.

Alternative Suzuki-Miyaura Coupling The above process was effective to provide drug substance for early development work packages. However, there was a motivation to change the endgame to address the high Pd loading needed for the Suzuki-Miyaura coupling and to eliminate the potential of the formation of the dba (dibenzylideneacetone) adduct impurity altogether. A strategic decision was made to move the Suzuki-Miyaura coupling one step earlier in the synthetic sequence. This would change the Pd source to address the issues discussed above and provide more opportunities for palladium removal prior to isolation of the final API. The isolable solid amidoxime 26 was a viable alternative coupling partner (Scheme 10). The Suzuki-Miyaura coupling of 26 with boronate 4 was a clean and efficient reaction which could be run with low catalyst loadings of 0.05 to 0.2 mol% Pd(TFA)2 (29) with the FcP(t-Bu)2-HBF4 ligand. Upon reaction completion, N-acetylcysteine was added to the reaction mixture as a palladium scavenger. The product precipitated from the reaction mixture directly and was isolated in 95% yield with > 99A% (220 nm HPLC purity). The formation 129

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of 1,2,4-oxadiazole 28 utilized a process analogous to that used for 27 (vide supra) yielding an ethyl acetate solution of 28. During our development work, a crystalline form of 28 free base or salt could not be found despite substantial effort, (regardless of material chemical purity or solvent composition). While in storage at room temperature for 6 months, a very concentrated IPA solution of 28 produced a few crystals. With some scoring of the flask, the entire 70 g sample crystallized. This fortuitous discovery provided an additional purity control point prior to isolation of the final API to remove organic and inorganic impurities. Using these crystalline seeds, 28 could be consistently isolated in high yield and purity as a crystalline solid. The palladium level in 28 was controlled to < 10 ppm with a carbon treatment/crystallization strategy. The final alkylation was conducted under conditions analogous to that shown above, heating the mixture of 28, 19, and DBU at 45 °C for 2 h. Following work-up, the crude 1 was isolated from IPAc, and a final recrystallization from ethanol/water provided the target compound 1 (83% for 2 steps, >99% purity) with < 5 ppm palladium.

Scheme 10. Second Generation Racemic Synthesis of 1

Asymmetric Pinacol Rearrangement Approach With the objective to develop an alternative robust and scalable enantioselective synthesis of the chiral quaternary aryl-aldehyde 5, we decided to explore the stereospecific pinacol rearrangement of a tri-substituted epoxide (Scheme 11) (30–32). Jung et al. reported the pinacol rearrangement of 2-aryl-3-ethynyloxiranes for the synthesis of ibuprofen (33). Similarly, Eisai demonstrated the efficiency of this methodology to synthesize biologically active emopamil (13). In this present case, such an approach would allow the formation of the all-carbon stereocenter via a Lewis acid-induced migration of the cyclopropyl substituent (34). Considering that multiple methodologies are known to provide tri-substituted enantioenriched epoxides (35–37), this strategy would alleviate the more challenging direct enantioselective synthesis of the quaternary stereocenter. 130

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Scheme 11. Pinacol Rearrangement Approach

In order to study this route, E-trisubsituted alkene 11 was first prepared on 220 gram scale (Scheme 12). 4-Bromo-phenethylalcohol was brominated with PBr3 to provide the corresponding secondary bromide in 94% yield, which upon treatment with triphenylphosphine, provided access to the corresponding triphenylphosphonium bromide (38, 39). A subsequent Wittig olefination with cyclopropanecarboxaldehyde using LHMDS afforded the -tri-substituted alkene in a 6:1 E/Z ratio (40). The crystalline alkene was then enriched to 99.8:0.2 E/Z after a recrystallization from MeOH at 5 °C.

Scheme 12. Synthesis of E-Olefin 11

With alkene 11 in hand, the enantioselective epoxidation was explored. After screening of various enantioselective catalytic epoxidations, Shi’s methodology turned out to be the most promising system in terms of enantiomeric excess, yield, and affordability of the catalyst (Scheme 13) (14, 41, 42). After optimization of reaction conditions, an addition of OxoneTM (potassium peroxomonosulfate) (43, 44) and buffer over 4 h at 0 °C in acetonitrile provided the desired epoxide with 82% yield, over 99:1 dr and 92% ee on 15g scale. These reaction conditions are very reproducible and straightforward to scale-up (50 g) since an inert atmosphere or anhydrous conditions are not required.

Scheme 13. Asymmetric Pinacol Synthesis of Aldehyde 5 131 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.

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The stereospecific pinacol rearrangement to afford the quaternary stereocenter was studied. An extensive screening of a wide range of Lewis and Brønsted acids was performed. Strong Lewis acids, such as BF3•OEt2 led to complete epoxide opening (45), but with a significant amount of ketone originating from a [1,2]-shift of the aryl substituent instead of the cyclopropyl migration. The optimal reagent found was methylaluminum bis(2,6-di-tert-butyl-4-methylphenoxide) (MAD) which was formed in situ from Me3Al and BHT (46–50). Complete stereospecificity generated the desired aldehyde 5 with 94% yield and 92% ee. Overall, this route where the quaternary center is built through a stereospecific pinacol rearrangement can be achieved on multi-gram scale without the need of purification by flash chromatography. There are a number of limitations to this asymmetric approach that precluded its implementation on large scale, and these include:

• • • •

Lower level of enantioselectivity (92% ee). High volume of water required to solubilize Oxone.TM Limited availability of the catalyst’s enantiomer. High catalyst loading (30 mol%) needed to reach full conversion due to catalyst de-activation during the epoxidation.

Asymmetric Conjugate Addition Approach One of our strategies to generate the all-carbon quaternary stereogenic center was to utilize a Cu-catalyzed Asymmetric Conjugate Addition (ACA) of dimethylzinc to a (Z)-nitroalkene substrate (15). In the past 15 years, the Cu-catalyzed ACA of enones, nitroalkenes and Meldrum’s acid derivatives with organozinc, aluminum, magnesium or boronic acid reagents has attracted considerable attention owing to its capability to construct chiral molecules with all-carbon quaternary stereogenic centers in high enantioselectivity (51–58). Hoveyda’s system (6) is of particular interest due to organozinc reagents’ high reactivity, excellent enantioselectity and high yield observed on the challenging acyclic nitroalkene substrates. This renders this methodology particularly attractive for the targeted compound 1. However, under optimized Hoveyda conditions on our substrate, only moderate enantioselectivity (65%) was obtained initially in the ACA of Me2Zn to the (E)-nitroalkene catalyzed by (CuOTf)2•PhH - Hoveyda dipeptide ligand complex. After intensive research, it was found that Me2Zn’s relatively lower reactivity and nitroalkene isomerization, coupled with use of less reactive (E)-nitroalkene and use of (CuOTf)2•PhH as Cu-precatalyst, were the main reasons for the moderate enantioselectivity observed. To solve this problem, we modified the reaction to use the more reactive (Z)-nitroalkene under conditions that minimize the nitroalkene isomerization.

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.

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Scheme 14. Asymmetric Conjugate Addition Approach (Z)-Nitroalkene 9 is a yellow crystalline solid, readily prepared from 4-bromobenzoic acid via a three-step protocol in 72% overall yield (Scheme 14). Thus, under our modified conditions, enantioselective conjugate addition of dimethylzinc to the (Z)-nitroalkene with 4% [(MeCN)4Cu]PF6 – Hoveyda ligand complex in toluene at -30 °C smoothly delivered the corresponding nitroalkane 8 in 91% yield and 95% ee. The crude nitroalkane was converted via a Nef reaction (59, 60) into enantiomerically enriched aldehyde 5, thus demonstrating this approach for the synthesis of the key quaternary intermediate. However, this approach was discontinued in preference to the boronate rearrangement approach described below.

Asymmetric Boronate Rearrangement Approach After evaluating several synthetic strategies for the construction of the key all-carbon stereocenter, Aggarwal’s approach was attractive with respect to both the availability of the key starting materials and overall cost. Aggarwal and co-workers reported a stereoretentive transformation of secondary carbonates to tertiary boronates (61–64). Furthermore, the authors demonstrated that the resulting boronic esters could then be utilized to introduce a formyl functionality and thus have an effectively asymmetric synthesis of all-carbon quaternary stereocenters (Scheme 15) (65, 66). The stereochemistry of the process was ultimately generated from a scalable asymmetric catalytic reduction of a prochiral ketone (67). The reported Aggarwal processes required cryogenic temperatures (both boronate rearrangements: -78 °C and -100 °C) and could lead to the accumulation of thermally unstable intermediates (Scheme 15). Although the cryogenic temperatures could be managed with specialized reactors, the accumulation of these unstable intermediates with long processing times (1 h or more) called for significant development work before this approach could be employed for large scale operations. As the chemistry was developed and optimized with the aryl bromide for the key Suzuki-Miyaura cross-coupling (20, 68–70), it was necessary to avoid the use of alkyl lithium bases which are incompatible with this halogen substitution. As this approach was short and all the reagents were relatively inexpensive, we undertook efforts to render the process safe, robust and amenable to large scale synthesis (17, 18). 133

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Scheme 15. Aggarwal’s Approach for the Construction of Chiral Quaternary Stereocenters from Secondary Chiral Carbamates

The required chiral secondary alcohol 37 was prepared by a Noyori asymmetric transfer hydrogenation (71, 72) of bromoacetophenone 13 (91% ee, Figure 4). Purging of the reaction with dry nitrogen (73) allowed the catalyst loading to be reduced to 0.1 mol% by removing the by-product carbon dioxide that is generated in the reaction. The intermediate alcohol 37 (not isolated) was treated with carbamyl chloride 38 to form solid carbamate 39, whose enantiomeric purity could be enriched to 99.9% ee with a single recrystallization.

Figure 4. Synthesis of chiral benzylic carbamate 39.

The reported Aggarwal boronate rearrangement process (66–69) was shown to be quite general with respect to both the boranate substitution and the migratory group. The reported process is performed in three separate parts (Figure 5). The benzylic carbamate is initially deprotonated with an alkyl lithium base (74, 75) and the resulting lithiated carbamate is trapped with the boronic ester to form the unstable boronate complex. The key 1,2-alkyl rearrangement was promoted with the addition of a Lewis Acid to minimize racemization. On large scale, the unit operations would require considerable more time to complete, and thus the unstable lithiated carbamate 41 and the boronate complex 43 would be held for long periods rendering the current process unsuitable for large scale synthesis. If the deprotonation of the benzyl carbamate 40 is conducted in the presence of the boronic ester 42 the in situ formed lithiated carbamate 41 would be instantly trapped by the electrophile to form the boronate complex 43 (Figure 5). Thus, 134 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 accumulation of the configurationally unstable lithiated carbamate 41 would be avoided and would allow the process to be conducted at non-cryogenic temperatures. In order to accomplish the in situ deprotonation a suitable base that is compatible with the electrophilic boronic ester needed to be found.

Figure 5. Boronate rearrangement mechanisms.

Attempts to perform the in situ deprotonation of benzylic carbamate 39 with the reported conditions (sec-butyllithium) provided no product. It was postulated that the alkyl lithium base traps the boronic ester at a much faster rate than the deprotonation of the benzylic carbamate. A base survey found that LDA (76) is compatible with the boronic ester in the in situ deprotonation and boronate formation. Furthermore, high conversion and low ee erosion were obtained for the desired system 39 even at -10 °C (Scheme 16) (77).

Scheme 16. First Generation Non-cryogenic Boronate Rearrangement

Further examination of the reaction variables revealed that the use of methanolic magnesium bromide was not needed as the desired tertiary boronate ester 12 was formed with this LDA process in near perfect yield (99%, Scheme 17 and high stereoretention (98.6% ee). The process was demonstrated to produce 24 kg of the chiral non-racemic tertiary boronate 12 in a single batch (99% yield, 98.6% ee). 135 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 17. Second Generation Non-cryogenic Boronate Rearrangement Based on the results in the in situ deprotonation, an identical strategy was employed with the deprotonation of DCM (78) for the enantiospecific conversion of the boronate 12 to the enantiomerically enriched aldehyde intermediate 5 containing the key all-carbon quaternary stereogenic center. It was postulated that the in situ deprotonation would avoid the problematic accumulation of the highly unstable lithiated DCM-species (76, 79). Attempts at the in situ deprotonation of DCM with LDA proved to be successful as high yields were obtained for the aldehyde product even at -15 °C (Figure 6). However, through a series of stress testing experiments it was found that at these elevated temperatures (-15 °C), the LDA base would need to be added at a fast rate (90%) and upon the hydrogen peroxide treatment any remaining tertiary boronic ester 12 would be converted to the tertiary alcohol 48 (Scheme 20). The residual tertiary alcohol 48 would then serve as an acid scavenger via ionization of the benzylic alcohol 48, thus inhibiting the acid-promoted chlorination of the oxime. This indeed proved to be the case as the addition of catalytic amounts of HCl at the onset of the chlorination was sufficient to eliminate the unpredictable exothermic induction period and rendered the process robust and reproducible. This modified process was demonstrated with the crude formylation product (90% molar conversion) which was directly processed to the solid amidoxime (10 kg scale) intermediate 26 in 99% ee and 52% overall yield for the entire sequence from boronic ester 12 (Scheme 21).

Scheme 19. Initial Investigation of the NCS Chlorination of Oxime 26 138 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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Scheme 20. Formation of Tertiary Alcohol 48 from Incomplete Formylation of Boronic Ester 12

Scheme 21. Synthesis of Amidoxime 26 from Tertiary Boronate 12

Non-Precious Metal Borylation The literature protocol for the synthesis of the pinacol boronate ester 4 involved the Suzuki-Miyaura borylation of 2-amino-5-bromopyrimidine with bis(pinacolato)diboron in presence of palladium-based catalysts (84–86). Employing the known process from the literature using PdCl2(dppf)-CH2Cl2 (5 mol%) in the presence of potassium acetate (3 equiv) in 1,4-dioxane at 100 °C, inconsistent results were obtained due to difficulties in achieving complete conversion, and due to hydrolysis of the pinacol boronate ester 4 during the aqueous workup. After a catalyst screening was conducted, we discovered that the use of 0.1 mol% of Pd2(dba)3 and 0.2 mol% Fc(PtBu2)HBF4 in the presence of KOAc (2 equiv) in 2-MeTHF at 80 °C afforded the pinacol boronate ester 4 in 80% yield with high purity on a multi-kilogram scale (Figure 9).

Figure 9. Pilot Plant Synthesis of 4. 139 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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However, the use of expensive palladium metal coupled with the high cost and low atom economy associated with bis(pinacolato)diboron provided us the impetus to develop a more cost-effective and efficient process. Initial efforts on the formation of the target boronic acid 52 using 2-amino-5-bromopyrimidine and B(Oi-Pr)3 with n-BuLi/THF/-78 °C gave inconsistent and poor yields (11-40%). While boronic acid can be prepared from heteroaryl bromide containing an amino group utilizing an amine protection (87–96), most of those methods suffered from modest yields and required two or three isolation steps as well as a deprotection operation. Our attention turned to easily accessible and removable silyl based protecting groups such as tetramethyldisilylazacyclopentane (STABASE) (97–99) and trimethylsilyl chloride (TMSCl) (100, 101). Use of STABASE for the in-situ protection of the amino functionality followed by typical boronic acid synthesis and its purification (Figure 10) afforded (2-aminopyrimidin-5-yl) boronic acid 52 in 77% overall yield (102) in 85 wt% purity, with the remaining mass balance composed of water. While the STABASE process was suitable for the preparation of kilogram quantities of 52, it became apparent that the STABASE reagent was not economically viable on large scale.

Figure 10. Synthesis of 52 using STABASE group. To our delight, replacement of STABASE with the readily available TMSCl was successful using similar conditions (LiHMDS/toluene/0 °C) (Scheme 22). At this point, a screening was performed using different bases for the amine protection and NaH in presence of catalytic amount of 2-propanol (7 mol%) afforded bisTMS adduct 53 in quantitative yield. The crude bis-TMS adduct was subjected to n-BuLi/THF/-78 °C conditions in the presence of B(OiPr)3 to afford boronic acid 52 after aqueous acid treatment and workup. The crude boronic acid 52 was re-slurred in water at 85 °C to afford purified (2-aminopyrimidin-5-yl) boronic acid 52 in overall 80% yield in high purity (>99 area% and 90 wt% with the remainder being simply water).

Scheme 22. Optimized Route to Boronic Acid 52 140 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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Asymmetric Synthesis of FLAP Inhibitor 1 With a safe and scalable processes established for the key quaternary stereocenter in >99% ee (amidoxime 26) and the achiral pinacol boronate ester 52 these intermediates were then in position to intercept the optimized key Suzuki-Miyaura cross coupling (19, 20, 103) and the end-game of the synthesis (Scheme 23). Although the cross-coupling could be performed with Pd2(dba)3 and the tetrafluoroborate salt of P(t-Bu)3 (1 mol% Pd), the catalyst loading could be reduced to 0.2 mol% (Pd) with the di-tert-butylphosphinoferrocene (t-Bu2PFc-HBF4) (104–106) ligand. The Suzuki-Miyaura product 2 precipitated directly from the reaction mixture in high yield and purity. Following the synthesis of the oxadiazole (107) from the CDI-activated carboxylic acid 3, a final alkylation of the pyrazole completed the synthesis. This sequence was employed to produce multi-kilogram quantities of the target compound 1 to support preclinical development.

Scheme 23. Synthesis of 1 from Amidoxime 26

Conclusion The FLAP program and subsequent development of the target compound 1 highlights the roles of both Discovery and Development in bringing a target compound from the initial discovery hit to the development of safe and robust scalable processes for the manufacture of the drug candidate. Once the compound transitions to the development phase, the process group is charged with the rapid delivery of high quality drug substance. Two main issues needed to be resolved quickly in the discovery approach. The first was the elimination of the bottleneck and low yielding chiral SFC separation that was accomplished by the strategic introduction of a carboxylic handle for a chiral base resolution. The second and most pertinent problem was rendering the conversion of the nitrile to the corresponding amidoxime safe for large scale operations. A two-step operation that avoided the thermal addition of hydroxylamine to the nitrile was accomplished by first reducing the nitrile to the corresponding aldehyde that could then be converted in situ to the oxime. Significant development was required 141

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to derive the ambient conditions for the oxidation and amidooxime formation. Although the first generation process was longer than than the original discovery approach, it accomplished the two main task of a process group; which is to render the route safe and scalable to provide the necessary multikilogram of material that was required to advance the compound in development. This strategic approach offered the necessary development time to discover an efficient and economical asymmetric process for the synthesis of the challenging drug target. Multiple approaches were explored experimentally for an asymmetric synthesis. A pinacol rearrangement based approach was synthetically feasible for the synthesis of the challenging all carbon quaternary core of the compound, but due the limited availability of the epoxidation catalyst’s enantiomer coupled with high catalyst loading and lower enantioselectivity this approach was not pursued for further development. An improved process employing the Hoveyda asymmetric Cu-mediated conjugate addition to nitro-olefins was developed. Due to the low reactivity observed with the (E)-nitroalkenes, the reaction was modified to employ the more reactive (Z)-nitroalkenes with conditions that minimized the nitroalkene isomerization. The corresponding non-racemic chiral adduct was converted to the key quaternary aldehyde intermediate via a Nef reaction. The asymmetric conjugate addition approach was discontinued in preference to the third approach that was derived from a dual boronate rearrangement process which the asymmetry of the process was ultimately derived form a well precedented and scalable asymmetric catalytic reduction of a prochiral ketone. The discovery of the compatibility of hindered amide bases for both the benzylic carbamate and formylation deprotonations was instrumental in allowing both processes to be performed in situ and thus rendered robust and efficient for large scale operations. Overall, the Discovery and Process Development groups were able to accomplish the two aspects of producing new therapeutics: the initial discovery of the drug target and the subsequent development of an efficient, safe, economical and robust process for the synthesis of the drug candidate on large scale.

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Chapter 6

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The Discovery and Process Chemistry Development of GDC-0084, a Brain Penetrating Inhibitor of PI3K and mTOR Timothy P. Heffron,1 Andrew McClory,2 and Andreas Stumpf*,2 1Department

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

Aberrant signaling of the PI3K pathway has been implicated in the majority of cases of glioblastoma multiforme (GBM), a malignant brain tumor with an associated poor prognosis. In order for a PI3K/mTOR inhibitor to inhibit PI3K pathway signaling where GBM tumors reside, such molecules must be capable of penetrating the blood-brain barrier (BBB). This chapter describes the medicinal chemistry efforts that led to the discovery of GDC-0084, a BBB penetrating inhibitor of PI3K and mTOR, followed by the process chemistry development that enabled its advancement to clinical studies.

The Discovery of GDC-0084 Glioblastoma multiforme (GBM) is an aggressive form of primary brain tumor with more than 20,000 new diagnoses each year (1). Unfortunately, there is significant room for improvement upon the limited chemotherapeutic treatment options for GBM as the two-year survival rate ranges from 4-29% (2). Inhibition of PI3Kα is a compelling potential approach to treating GBM as aberrant PI3K signaling is implicated in more than 80% of cases (3, 4). With the apparent medical need and biological rationale in mind, we sought to realize a blood-brain barrier (BBB) penetrating PI3Kα inhibitor to reach the © 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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brain where GBM tumors reside. At the onset of this program, we had extensive experience in the discovery of PI3K inhibitors as we had recently concluded a multi-year effort pursuing inhibitors of PI3Kα for the treatment of peripheral disease (5–14). However, there was still a need for a PI3K inhibitor that achieved brain penetration as, at the time, we were not aware of any PI3K inhibitors capable of penetrating the BBB. To achieve free BBB penetration, we anticipated that we would need to minimize efflux transport mediated by P-gp and Bcrp, two transporters highly expressed at the BBB which actively limit brain penetration of small molecules (15). Achieving brain penetration with a PI3K inhibitor, then, presented a significant challenge due to the need to avoid the P-gp and Bcrp mediated efflux normally experienced by kinase inhibitors. Indeed, when we studied the extent to which our previous clinical PI3Kα inhibitor GDC-0941 (5) and clinical PI3K/mTOR inhibitor GDC-0980 (10) were capable of penetrating the BBB in mice, we found that essentially no penetration was achieved (Table 1). While the [brain]/[plasma] ratios were low, the more meaningful measure of free BBB penetration, [brain]u/[plasma]u also indicated that no free penetration was achieved. Furthermore, the lack of BBB penetration was consistent with in vitro permeability assay results using Madin-Darby canine kidney (MDCK) cells transfected to highly express P-gp or Bcrp (Table 1). In these assays, both GDC-0941 and GDC-0980 experience high B-A/A-B efflux ratios, demonstrating that they are substrates of both P-gp and Bcrp.

Table 1. In Vitro Permeability Efflux Ratios from MDR1 and Bcrp1 Transfected MDCK Cell Line Assays and in Vivo Brain-to-Plasma Ratios for Compounds GDC-0941 and GDC-0980

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That GDC-0941 and GDC-0980 were each substrates of P-gp and Bcrp was not unexpected given their physicochemical properties are inconsistent with the median values of 119 marketed CNS drugs (Table 2), for which low transporter mediated efflux is expected (16). Particularly noteworthy, the molecular weights of GDC-0941 and GDC-0980 are significantly greater than those of marketed CNS drugs. We thought that attempts to harmonize the physical properties of our PI3K inhibitors with the median value of marketed CNS drugs might increase the probability that the PI3K inhibitors would lack efflux mediated by P-gp or Bcrp.

Table 2. Comparison of Calculated Physicochemical Properties of Compounds GDC-0941 and GDC-0980 with Marketed CNS Targeting Drugsa

In an effort to reduce the MW, to realize PI3K inhibitors with physical properties more consistent with marketed CNS drugs, we evaluated 1 (Table 3) in which the piperazine sulfonamide moiety of GDC-0941 is absent. With the reduction of MW from 513 to 337 when compared to GDC-0941, we were pleased to see a dramatic reduction in P-gp and Bcrp mediated efflux (Table 3). Unfortunately, 1 also had much weaker potency than GDC-0941 and accordingly was deemed inadequate. Encouragingly however, 2, an analog of GDC-0980 in which the piperazine amide has been eliminated, retained most of the potency of GDC-0980, but with substantially reduced MW. This encouraging result demonstrated that adequate PI3Kα potency could be achieved in a comparatively low MW molecule that, most excitingly, was not a substrate of either P-gp or Bcrp (Table 3). Despite this important discovery, however, 2 was not by itself adequate as it was rapidly metabolized in in vitro microsomal incubations. Nevertheless, this initial result gave us the encouragement to pursue further morpholinopyrimidine-based PI3K inhibitors. 149

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Table 3. A Comparison of the Potency, Permeability Efflux Ratios, and in Vitro Metabolic Stability of GDC-0941 and GDC-0980 with Analogs That Do Not Have Substitution in the Solvent Exposed Region (1 and 2)

From our previous studies, we knew that substitution of the thiophene of the thienopyrimidine at the 6-position (numbering in Table 3) had allowed for us to achieve molecules with reasonable in vitro and in vivo ADME properties (e.g. GDC-0980) and so we began to design and synthesize analogs of 2 that were substituted at the 6-position. A substantial number of analogs were generated in this effort that were evaluated in permeability assays to determine if they were P-gp and/or Bcrp substrates. From this effort we rapidly identified that hydrogen bond donor (HBD) count had a dramatic effect on transporter-mediated efflux. For example, the pairs of molecules 36 with 4 and 5 with 6 (Table 4) differ in each case by the presence of a hydroxyl group or a methyl ether. In each case, elimination of the hydrogen bond by alkylation to the methyl ether results in a molecule that is a much weaker efflux transporter substrate. In the end, 4 and 6 also had reasonable exposure in mice (Table 5) with evident free BBB penetration (Table 5) (17, 18). With these molecules in hand we were prepared for in vivo studies to verify that they could achive potent inhibition of PI3K/mTOR signalling behind the BBB as intended. In order to demonstrate inhibition of our kinase target by 4 and 6 in the intended compartment we conducted experiments to evaluate pharmacodynamic response in the brain. Oral doses of 4 or 6 to healthy mice inhibited pAKT, a downstream marker of PI3K signaling, in normal brain tissue (i.e. behind a fully intact BBB, Figure 1). Additionally, 4 and 6 were shown to inhibit PI3K signaling (pAKT, pS6) in a U87 (human primary glioblastoma tumor cell line) tumor model in mice (17) that led to a corresponding inhibition of tumor growth (Figure 2). Furthermore, 6 also demonstrated inhibition of growth of tumors implanted in mouse brains, providing even further evidence of target inhibition in the brain that suggests such a molecule might provide benefit to patients with glioblastoma (18).

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Table 4. Efflux Ratios for Select Thienopyrimidines

Table 5. Potency, Efflux Ratios, Mouse PK and Brain Exposure for 4 and 6

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Figure 1. Inhibition of pAKT in female CD-1 mice brain tissue after oral administration of 4 and 6 as MCT suspension. Normalized pAKT was measured 1 h and 6 h post dose and are indicated by the bars in the chart and error bars indicate ± standard error of the mean. The percentage reduction in pAKT compared to the untreated group is indicated where applicable. The mean pAKT for the untreated groups are based on samplings of brain tissue from 3 animals per time point. Sampling of 1 animal per time point was analyzed for compound 4. This Figure is reproduced from Reference (17), copyright 2012, American Chemical Society.

Figure 2. In vivo efficacy of 4 and 6 versus U87 MG/M human glioblastoma xenografts. Female NCr nude mice bearing subcutaneous tumors were administered escalating doses of 4 or 6 orally as a suspension in vehicle (0.5% methylcellulose/0.2% Tween-80) or vehicle once daily (QD) for 24 days. Changes in tumor volumes over time by dose for each compound are depicted as cubic spline fits generated via Linear Mixed Effects analysis of log-transformed volumes. This Figure is reproduced from Reference (17), copyright 2012, American Chemical Society. 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.

While excellent preclinical tool compounds, 4 and 6 were determined to be not suitable for advancement to human clinical study as human liver microsomal incubations indicated that they were likely to have rapid clearance in humans (Table 6). We, therefore, extended our studies from 4 and 6 in the hopes of improving upon human metabolic stability while also maintaining potency and continuing to limit transporter efflux to maintain CNS penetration.

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Table 6. MDR1 and Bcrp1 Transfected MDCK Cell Permeability Efflux Ratios and Human Liver Microsomal Stability for Select Compounds

Many efforts to modify the substitution at C-6 of the thiophene within 4 and 6 (numbering in Table 3) were attempted to achieve improved human metabolic stability. Unfortunately, those efforts were not successful or, when human metabolic stability improved, either reduced CNS penetration or potency was also encountered. During these efforts we also returned to alternative morpholinopyrimidine cores that we had evaluated during studies that led to the discovery of GDC-0980. One such core was a purine, instead of thienopyrimidine, and 7 was a molecule we had identified that had excellent PI3K potency and metabolic stability. We now hoped to modify 7 so that it could also achieve BBB penetration (Table 6). We had previously demonstrated the importance of minimizing HBD count to minimize efflux and so from 7, which contains four HBD, we recognized we would need a molecule with reduced HBD count. Previously we alkylated hydroxyl groups to their corresponding methyl ethers to reduce HBD count (e.g. 3 to 4, 5 to 6). In this case, with two hydroxyl groups, we decided to hydrolyze one hydroxyl group in the course of a cyclization to realize a new tricyclic core (19). Compound 8 (Table 7), containing this new tricyclic core, 153

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was found to have low transporter mediated efflux, suggesting it should penetrate the BBB, and exhibits good metabolic stability. However, its cellular potency was modest. To improve the potency of 8, we used the same aminopyridine group found in GDC-0980, known to improve mTOR potency relative to the aminopyridine in 8 (6). Gratifyingly, the resultant molecule, GDC-0084 (Table 7), is a potent PI3K/mTOR inhibitor with excellent human metabolic stability and low transporter efflux in vitro (19).

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Table 7. Potency, Efflux Ratios and Human Metabolic Stability of Tricyclic Purine-Based PI3K Inhibitors 8 and GDC-0084

Figure 3. Inhibition of p-AKT by GDC-0084 in normal mouse brain tissue along with corresponding brain and unbound brain concentrations. *Significantly different from untreated control. p17 L/kg). Compound (7) was determined to have significantly improved pharmacokinetics relative to canertinib (44). Compound (84) however was considerably more variable across species, showing a volume of distribution (24.8 L/kg) and oral bioavailability that was acceptable in rat (60%), but with considerably poorer parameters in dog and particularly in monkey, where plasma half-life following intravenous administration (0.7 h) and oral bioavailability (4%) were disappointing. The superior preclinical PK profile led to the selection of dacomitinib, (PF-00299804, 7), for further human clinical studies.

Conclusions We have described the discovery of dacomitinib, an irreversible inhibitor of erbB1, 2 and 4 with an improved pharmacokinetic and toxicity profile relative to canertinib. We counter-screened analogues against JAK3 to avoid the undesirable thrombocytopenia observed during human trials of canertinib. The irreversible nature of dacomitinib’s, erbB1 inhibition was confirmed in an erbB1 T790M X-ray co-crystal structure at 1.8 Å resolution, clearly showing the 224 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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presence of dacomitinib covalently linked to the target cysteine (58). Dacomitinib was found to be highly selective for the erbB family with only Lck, Src and JAK3 showing IC50’s < 10 μM (IC50 = 0.094, 0.110 and 3.57 μM, respectively) when screened against a panel of 38 kinases (59). Dacomitinib has shown excellent efficacy in human tumor xenograft models in nude mice and excellent pharmacokinetic properties across species (44). Tumor regression was observed with dacomitinib in the wild-type erbB1 over-expressing A431 epidermoid carcinoma xenograft, the erbB2/3 expressing H125 non-small-cell lung cancer xenograft and the erbB2 over-expressing SKOV3 ovarian carcinoma xenograft (44). Both in vitro and in vivo dacomitinib has been shown to be a potent inhibitor of erbB1-activating mutations (L858R, exon 19 deletions) as well as the erbB1 T790M resistance mutation. Dacomitinib has also shown robust anti-tumor activity in the NCI-H1975 and engineered HCC827-del/T790M NSCLC xenograft models that are resistant to erlotinib and gefitinib (44, 59). In clinical trials the dose-limiting toxicities of dacomitinib include rash, diarrhea, paronychia, dehydration and stomatitis. The maxium tolerated daily oral dose has been established to be 45 mg and the mean human plasma half-life (t1/2) was 59 – 85 h (60–62). The clinical efficacy of dacomitinib has been most widely assessed in NSCLC (63, 64). Phase II studies evaluating dacomitinib as salvage therapy in advanced NSCLC patients who progressed following chemotherapy and treatment with erlotinib demonstrated preliminary evidence of activity (61, 65). Phase II evaluation of dacomitinib as a second-line therapy for NSCLC versus erlotinib, successfully met the primary endpoint of a statistically significant improvement in PFS (66). Unfortunately in subsequent phase III double-blind, randomised trials (BR-26) (ARCHER 1009) dacomitinib did not meet its selected endoints (67, 68). A subanalysis of the ARCHER 1009 trial demonstrated superiority of dacomitinib over erlotinib among NSCLC patients with erbB1 mutations (69). while phase II evaluation of dacomitinib in a first-line setting in this patient population has demonstrated significant activity with an objective response rate of 74% (70). A similar randomised phase III study of dacomitinib versus gefitinib (ARCHER 1050) is on-going (NCT01774721). Dacomitinib has also demonstrated efficacy in a phase II trial as a first-line treatment in recurrent and/or metastatic SCCHN where 12.7% of patients achieved a partial response and 57.1% of patients experienced stable disease (71). In summary, dacominitib, (PF-00299804, 7) has demonstrated clinical efficacy in cancers driven by EGFR dysregulation (69–72). A phase III trial to further evaluate dacomitinib is currently in progress. The role of this irreversible pan-erbB inhibitor for the treatment of cancer has yet to be determined.

Acknowledgments The authors would like to thank Alexander Bridges, Haile Tecle, Helen Lee, Karen Sexton, Kevin Schlosser, R. Thomas Winters, Stephen Fakhoury, Julie A. Spicer, Andrea J. Gonzales, Irene W. Althaus, Tong Zhu, Shannon L. Black, 225 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.

Adrian Blaser, Amy Delaney, William A. Denny, Paul A. Ellis, Patricia J. Harvey, Ken Hook, Florence O. J. McCarthy, Brian D. Palmer, Freddy Rivault, Teresa Spoon, Andrew M. Thompson, Erin Trachet, Simon Planken, Olivier Dirat and Shu Yu.

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Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127–132. Rewcastle, G. W.; Palmer, B. D.; Bridges, A. J.; Showalter, H. D. H.; Sun, L.; Nelson, J.; McMichael, A.; Kraker, A. J.; Fry, D. W.; Denny, W. A. Tyrosine kinase inhibitors. 9. Synthesis and evaluation of fused tricyclic quinazoline analogs as ATP site inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor. J. Med. Chem. 1996, 39, 918–928. Solca, F.; Dahl, G.; Zoephel, A.; Bader, G.; Sanderson, M.; Klein, C.; Kraemer, O.; Himmelsbach, F.; Haaksma, E.; Adolf, G. R. Target binding properties and cellular activity of afatinib (BIBW 2992), an irreversible ErbB family blocker. J. Pharm. Exp. Ther. 2012, 343, 342–350. Sanderson, K. Irreversible kinase inhibitors gain traction. Nat. Rev. Drug Discovery 2013, 12, 649–651. Erlichman, C.; Hidalgo, M.; Boni, J. P.; Martins, P.; Quinn, S. E.; Zacharchuk, C.; Amorusi, P.; Adjei, A. A.; Rowinsky, E. K. Phase I study of EKB-569, an irreversible inhibitor of the epidermal growth factor receptor, in patients with advanced solid tumors. J. Clin. Oncol. 2006, 24, 2252–2260. Rewcastle, G. W.; Palmer, B. D.; Thompson, A. M.; Bridges, A. J.; Cody, D. R.; Zhou, H.; Fry, D. W.; McMichael, A.; Kraker, A. J.; Denny, W. A. Tyrosine Kinase Inhibitors. 10. Isomeric 4-[(3-Bromophenyl)amino] pyrido[d]pyrimidines Are Potent ATP Binding Site Inhibitors of the Tyrosine Kinase Function of the Epidermal Growth Factor Receptor. J. Med. Chem. 1996, 39, 1823–1835. Gajiwala, K. S.; Feng, J.; Ferre, R.; Ryan, K.; Brodsky, O.; Weinrich, S.; Kath, J. C.; Stewart, A. Insights into the aberrant activity of mutant EGFR kinase domain and drug recognition. Structure 2013, 21, 209–219. Engelman, J. A.; Zejnullahu, K.; Gale, C.-M.; Lifshits, E.; Gonzales, A. J.; Shimamura, T.; Zhao, F.; Vincent, P. W.; Naumov, G. N.; Bradner, J. E.; Althaus, I. W.; Gandhi, L.; Shapiro, G. I.; Nelson, J. M.; Heymach, J. V.; Meyerson, M.; Wong, K.-K.; Jänne, P. A. PF00299804, an irreversible pan-erbB inhibitor, is effective in lung cancer models with EGFR and erbB2 mutations that are resistant to gefitinib. Cancer Res. 2007, 67, 11924–11932. Jänne, P. A.; Schellens, J. H.; Engelman, J. A.; Eckhardt, S. G.; Millham, R.; Denis, L. J.; Britten, C. D.; Wong, S. G.; Boss, D. S.; Camidge, D. R. Preliminary activity and safety results from a phase I clinical trial of PF-00299804, an irreversible pan-HER inhibitor, in patients (pts) with NSCLC. J. Clin. Oncol. 2008, 26, 15SAbstract 8027. Park, K.; Heo, D. S.; Cho, B.; Kim, D.; Ahn, M.; Lee, S.; Millham, R. D.; Campbell, A.; Zhang, H.; Kim, J. PF-00299804 (PF299) in asian patients (pts) with non-small cell lung cancer (NSCLC) refractory to chemotherapy (CT) and erlotinib (E) or gefitinib (G): A phase (P) I/II study. J. Clin. Oncol. 2010, 28, 15SAbstract 7599. Takahashi, T.; Boku, N.; Murakami, H.; Naito, T.; Tsuya, A.; Nakamura, Y.; Ono, A.; Machida, N.; Yamazaki, K.; Watanabe, J.; Ruiz-Garcia, A.; Imai, K.; Ohki, E.; Yamamoto, N. Phase I and pharmacokinetic study of dacomitinib (PF-00299804), an oral irreversible, small molecule inhibitor 231

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of human epidermal growth factor receptor-1, -2, and -4 tyrosine kinases, in Japanese patients with advanced solid tumors. Invest. New Drugs 2012, 30, 2352–2363. Brzezniak, C.; Carter, C. A.; Giaccone, G. Dacomitinib, a new therapy for the treatment of non-small cell lung cancer. Expert Opin. Pharmacother. 2013, 14, 247–253. Mok, T.; Lee, K.; Tang, M.; Leung, L. Dacomitinib for the treatment of advanced or metastatic non-small-cell lung cancer. Future Oncol. 2014, 10, 813–822. Campbell, A.; Reckamp, K. L.; Camidge, D. R.; Giaccone, G.; Gadgeel, S. M.; Khuri, F. R.; Engelman, J. A.; Denis, L. J.; O’Connell, J. P.; Jänne, P. A. PF-00299804 (PF299) patient (pt)-reported outcomes (PROs) and efficacy in adenocarcinoma (adeno) and nonadeno non-small cell lung cancer (NSCLC): A phase (P) II trial in advanced NSCLC after failure of chemotherapy (CT) and erlotinib (E). J. Clin. Oncol. 2010, 28, 15S Abstract 7596. Ramalingam, S. S.; Blackhall, F.; Krzakowski, M.; Barrios, C. H.; Park, K.; Bover, I.; Seog Heo, D.; Rosell, R.; Talbot, D. C.; Frank, R.; Letrent, S. P.; Ruiz-Garcia, A.; Taylor, I.; Liang, J. L.; Campbell, A. K.; O’Connell, J.; Boyer, M. Randomized phase II study of dacomitinib (PF-00299804), an irreversible pan–human epidermal growth factor receptor inhibitor, versus erlotinib in patients with advanced non-small-cell lung cancer. J. Clin. Oncol. 2012, 30, 3337–3344. Ellis, P. M.; Shepherd, F. A.; Millward, M.; Perrone, F.; Seymour, L.; Liu, G.; Sun, S.; Cho, B. C.; Morabito, A.; Leighl, N. B.; Stockler, M. R.; Lee, C. W.; Wierzbicki, R.; Cohen, V.; Blais, N.; Sangha, R. S.; Favaretto, A. G.; Kang, J. H.; Tsao, M-S.; Wilson, C. F.; Goldberg, Z.; Ding, K.; Goss, G. D.; Bradbury, P. A. Dacomitinib compared with placebo in pretreated patients with advanced or metastatic non-small-cell lung cancer (NCIC CTG BR.26): a double-blind, randomized, phase 3 trial. Lancet Oncol. 2014, 15, 1379–1388. Ramalingam, S. S.; Jänne, P. A.; Mok, T.; O’Byrne, K.; Boyer, M. J.; Von Pawel, J.; Pluzanski, A.; Shtivelband, M.; Docampo, L. I.; Bennouna, J.; Zhang, H.; Liang, J. Q.; Doherty, J. P.; Taylor, I.; Mather, C. B.; Goldberg, Z.; O’Connell, J.; Paz-Ares, L. Dacomitinib versus erlotinib in patients with advanced-stage, previously treated non-small-cell lung cancer (ARCHER 1009): a randomized, double-blind, phase 3 trial. Lancet Oncol. 2014, 15, 1369–1378. Ramalingam, S. S; O’Byrne, K.; Boyer, M.; Mok, T.; Jänne, P. A.; Zhang, H.; Liang, J.; Taylor, I.; Sbar, E. I.; Paz-Ares, L. Dacomitinib versus erlotinib in patients with EGFRmutated advanced nonsmall-cell lung cancer (NSCLC): pooled subset analyses from two randomized trials. Ann. Oncol. 2016, 3, 423–429. Kris, M. G.; Mok, T.; Ignatius Ou, S.-H.; Martins, R.; Kim, D.-W.; Goldberg, Z.; Zhang, H.; Taylor, I.; Letrent, S. P.; Jänne, P. A. First-line dacomitinib (PF-00299804), an irreversible pan-HER tyrosine kinase inhibitor, for patients with EGFR-mutant lung cancers. J. Clin. Oncol. 2012, 30, 487S Abstract 7530. 232

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71. Abdul Razak, A. R.; Soulieres, D.; Laurie, S. A.; Hotte, S. J.; Singh, S.; Winquist, E.; Chia, S.; Le Tourneau, C.; Nguyen-Tan, P. F.; Chen, E. X.; Chan, K. K.; Wang, T.; Giri, N.; Mormont, C.; Quinn, S.; Siu, L. L. A phase II trial of dacomitinib, an oral pan-human EGF receptor (HER) inhibitor, as first-line treatment in recurrent and/or metastatic squamous-cell carcinoma of the head and neck. Ann. Oncol. 2013, 24, 761–769. 72. Jänne, P. A.; Ou, S. H.; Kim, D. W.; Oxnard, G. R.; Martins, R.; Kris, M. G.; Dunphy, F.; Nishio, M.; O’Connell, J.; Paweletz, C.; Taylor, I.; Zhang, H.; Goldberg, Z.; Mok, T. Dacomitinib as first-line treatment in patients with clinically or molecularly selected advanced non-small-cell lung cancer: a multicentre, open-label, phase 2 trial. Lancet Oncol. 2014, 13, 1433–1441.

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Chapter 9

Early and Late Stage Process Development for the Manufacture of Dacomitinib Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1239.ch009

Shu Yu1 and Olivier Dirat*,2 1Chemical

Research and Development, Pfizer Global Research and Development, Groton, Connecticut 06340-8156, United States 2Chemical Research and Development, Pfizer Global Research and Development, Sandwich, Kent, CT13 9NJ, United Kingdom *E-mail: [email protected].

The process chemistry efforts to support the development of dacomitinib, a potent irreversible epidermal growth factor receptor inhibitor designed for the treatment of non-small cell lung cancer, are described. Early development routes that enabled the delivery of the first ten’s of kilograms of API are first discussed, followed by a more detailed account of the development of the commercial route, an efficient three steps with two isolations process using, as a key transformation, a low temperature Dimroth rearrangement. The commercial route has been used in Pfizer’s manufacturing facilities to produce over 800 kg of API in 58% overall yield.

Introduction Dacomitinib is a potent irreversible epidermal growth factor receptor (Pan erbB) inhibitor designed for the treatment of non-small cell lung cancer (NSCLC). The discovery and medicinal chemistry synthesis of dacomitinib were described in the preceding chapter. This chapter will describe the process chemistry efforts to support the pre-clinical studies, clinical development program and the potential commercial requirements.

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Evaluation of the Medicinal Chemistry Route Since quality, speed and scalability were the focus of the early stage of development, our work started by evaluating the discovery chemistry route (Scheme 1). The synthesis started with the condensation of 2-amino4-fluorobenzoic acid 2, with amidine acetate 3 to afford intermediate 4. Regioselective nitration gave a mixture of two isomers 5 and 6 in ~5:1 ratio, favoring the desired isomer 5, the undesired isomer 6 being successfully purged during isolation. Treatment of 5 with POCl3 afforded chloro-quinazoline 7, which after nucleophilic aromatic substitution with 8 gave 9. This was followed by a second nucleophilic aromatic substitution with a stronger nucleophile NaOMe under more forcing conditions to give intermediate 10. Nitro reduction afforded arylamine 11, which reacted with acid chloride 13 formed in situ, affording the desired API, dacomitinib 1 (1).

Scheme 1. Medicinal Chemistry Route to Dacomitinib 236 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.

A critical evaluation of the discovery route in the laboratory revealed that the synthesis was well designed as all the chemical transformations afforded the desired products in good to excellent yield. However, two major issues existed for the route: •

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•

although the in situ yield for the formation of 9 was good, the isolation of the product was challenging causing extensive loss of product to the mother liquor. the reduction of the nitro group via catalytic hydrogenation was capricious: when the conditions were not controlled perfectly, the hydrogenation could afford a small amount of a des-chloro impurity, which could not be purged in the downstream steps.

The formation of the des-chloro impurity was extremely sensitive to palladium catalysis, as a trace amount of leftover catalyst from the hydrogenator was enough to cause de-chlorination. The product, amine 11, also exhibited poor solubility in organic solvents. A mixture of DME and DMAc was required to enable catalyst removal by solubilizing 11. DMAc was subsequently removed via distillation, which was an undesirable operation. In order to meet the API demand for preclinical and clinical studies, the medicinal chemistry route was adapted to adress the issues identified and allow scale-up.

Enabling of the Medicinal Chemistry Route for Scale-Up Since large amounts of intermediate 5 were readily available, the initial process development work started from 5. The formation of intermediate 9 was initially performed as a telescoped process but later found to be better executed in two separate steps. In the telescoped process, the isolated yield was modest, often in the 50-60% range due to product losses to the mother liquor. It soon became clear that the culprit was toluene, as even relatively small amounts resulted in large losses. The formation of intermediate 7 in the absence of toluene was therefore sought. It was found that 5 could also be chlorinated with 6 equivalents of SOCl2, and gratifyingly, when n-heptane was added to the mixture at the end of reaction, the desired product 7 crystallized, affording a direct drop process. The isolated 7 underwent nucleophilic aromatic substitution smoothly with amine 8, to afford 9 in excellent isolated yield. The two-step process was successfully performed in the pilot plant on a 70 kg scale. The displacement of the fluoride with methoxide proceeded smoothly, however the filtration was very slow due to the presence of small particles. This problem was solved by ripening the particles with heat-cool cycles. As the catalytic hydrogenation of 10 suffered from a number of issues (vide supra, undesired de-chlorination and solubility of product), alternative conditions were sought. Platinum catalysts proved to be a good alternative to palladium catalysts as they provided much lower ratio of the undesired de-chlorination. Profiling of the reaction indicated that the reaction occurred in two stages: 237

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• •

a fast reduction of the nitro group to hydroxylamine 14. a slower reduction of the hydroxylamine 14 to amine 11 (Figure 1).

The slow second stage was detrimental, as long reaction times increased the risk of des-chloro impurity formation. A broad screen of solvents revealed that methanol was a good solvent for this reduction as the reaction could be complete within an hour. The low solubility of amine 11 in methanol did not allow for the possibility of catalyst removal by filtration. Through extensive experimentation, it was discovered that a mixed solvent system that contained 70% THF and 30% methanol could achieve fast hydrogenation and keep the product in solution for catalyst removal via a hot filtration. In combination with vigorous vessel cleaning, the process was successfully implemented in the pilot plant and produced 32 kg of 11 in 94% yield.

Figure 1. Nitro reduction reaction profiling.

Compound 11 contains two nucleophilic nitrogens, and owing to the differences in the stereoelectronic effects, the nitrogen at the 6-position was more reactive towards acylation, affording the desired API. The final amide coupling reaction performed well when a two-vessel process was used. The acid chloride 13 was prepared in one vessel and added into a THF solution of amine 11 in a second vessel. The two-vessel process ensured that the concentration of 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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acid chloride 13 was low at all stages of the amide formation reaction, thereby minimizing the chances of bis-amide formation. This modified medicinal chemistry route was successfully executed three times in the pilot plant (Scheme 2). As the clinical program continued to show promise, alternative, more efficient routes were investigated.

Scheme 2. Modified Medicinal Chemistry Route

N-Arylation Route to Dacomitinib Oxidation state and functional group manipulations are inefficient operations as they typically do not add structural complexity. The N-arylation route shown in Scheme 3 introduces the nitrogen at the 6-position at the correct oxidation state (hence no need for a nitro reduction); the methoxy group was also introduced through the starting material thereby not requiring functional group manipulations. Finally, the new starting material 15 was commercially available, whereas the previous starting material 5 required in-house preparation from 2 in 2 steps and 59% yield (Scheme 1) Iodination of 15 using NIS produced 16 in excellent yield and regioselectivity, however, the by-product succinimide was hard to remove when dichloromethane was employed as solvent (2). However when 2-methoxy-ethanol was used as the solvent, the iodination performed equally well to give 16 and provided an excellent purge of succinimide during the isolation of 17. Accordingly, the two steps were telescoped without isolating 16. Condensation of 16 with amidine acetate 3 yielded intermediate 17, which crystallized from the reaction mixture affording a direct drop process. The chlorination of 17 was best performed in a mixture of toluene and acetonitrile. This mixture of solvents also performed well during the nucleophilic aromatic substitution leading to 19. The resulting telescoped process from 17 to 19 achieved greater than 95% yield over two steps. At this point, the stage was set for the pivotal palladium catalyzed N-arylation (3). 239

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Scheme 3. N-Arylation Route to Dacomitinib

High throughput screening was used to rapidly evaluate palladium sources, ligands, bases and solvents. The combination of Pd2(dba)3, Xantphos, the dual bases Cs2CO3/Hunig’s base and mixed solvents toluene/water afforded the desired API efficiently in 75% isolated yield. The crude API prepared this way contained varying amounts of residual palladium. However, activated carbon and 1,1,2,2-tetramethylethylenediamine (TMEDA) treatment allowed consistent reduction of the palladium content to 100 L/kg) owing to the insolubility of intermediates 10 and 11. For this reason, this route was deemed not optimal for commercial manufacture and therefore was not pursued further.

Scheme 5. Approaches to Dacomitinib Using a Dimroth Rearrangement

Route B started with a nitro reduction which proceeded very well in high yields. The Dimroth rearrangement on the aniline 24 proceeded smoothly with 71% yield at 55 °C, again within one hour. The amide bond formation also proceeded well. Similarly to route A, the aniline quinazoline intermediate 11 was very insoluble and required processing volumes greater than 100 L/kg and therefore route B was also deemed not optimal. Clearly the later the introduction of the quinazoline ring the more efficient the process would be. 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.

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Route C solved the solubility issue as the quinazoline ring was formed in the last step with the amide already installed, which provided increased solubility. Indeed, all intermediates in this sequence had solubilities that require a dilution of less than 10 L/kg. While the first two steps were straightforward, the Dimroth rearrangement initially appeared challenging owing to a thermal side reaction of the product. The formation of lactam 26 had been observed under acidic, basic and/or thermal conditions. Lactam 26 readily hydrolyzed to the corresponding hydroxyl lactam 28 in the presence of water, or reacted with aniline 8 to give compound 27 (Scheme 6). It was therefore crucial to find milder reaction conditions for the Dimroth rearrangement than the typical conditions of refluxing acetic acid. To our delight, the Dimroth rearrangement proceeded smoothly at lower temperatures giving 80% yield at only 30 °C in 16 h. Accordingly, route C was selected as the commercial route to dacomitinib and the process was developed further.

Scheme 6. Side Reactions of Dacomitinib

Development of the Nitro Reduction Step The nitro group reduction reaction performed well with many palladium on carbon catalysts in several solvents. We chose acetonitrile as solvent as it enabled telescoping with the next step. For many aromatic nitro group reductions, the rate limiting step is the final reduction of the hydroxyl amine to aniline. In our case, profiling studies showed that the rate limiting steps were the initial reduction of nitro 23 to nitroso 29 and the reduction of the nitroso 29 to hydroxyl amine 30, the hydroxyl amine 30 being so reactive that it has never been observed during the reaction (Scheme 7, Figure 2). The main side reaction is the reduction of the nitrile which immediately cyclises to form quinazoline 31. This impurity purges readily in the downstream process, so did not pose a concern. 242 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. Nitro Reduction Intermediates

Figure 2. Nitro reduction reaction profiling.

Development of the Amide Bond Formation Step The first task was to identify a suitable coupling agent for the amidation reaction between aniline 24 and acid 12. We focused on reagents that do not require a separate activation step to improve efficiency of the manufacturing process (one vessel required and no activation IPC required). After screening 6 reagents (cyanuric chloride, CDMT, DCC, EDC, EEDQ and T3P®) with a range of additives using the acetonitrile solution of aniline 24 obtained after nitro reduction, propanephosphonic acid anhydride (T3P®) was clearly the reagent of choice for this transformation. T3P® was also conveniently available as a solution in acetonitrile. Acid 12 was isolated as an HCl salt, so one equivalent of a non nucleophilic base was required for the reaction. Pyridine-derived bases (pyridine and 2,6-lutidine) gave superior impurity profiles compared with aliphatic amines (Hunig’s base and triethylamine), carbonates and alkoxides. Therefore, 2,6-lutidine was selected as base (Scheme 8), and the reaction was profiled (Figure 3). 243

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Scheme 8. Amidation Reaction Scheme

The reaction profiling led to interesting observations on the side reactions. Firstly, the most prevalent impurity (1,4-adduct 34) formed very rapidly at the start of the reaction. We initially thought that a 1,4 addition of aniline 24 onto the α,β-unsaturated amide was occurring. However we have failed to form any trace of adduct 34 after examining many conditions to react the product with aniline 24. We serendipitously observed that if the cis-acid 37 was used instead of the trans-acid 12, it formed adduct 34 exclusively under the reaction conditions, which led us to postulate the mechanism shown in Scheme 9. Adduct 34 can be envisaged to form via activation of cis-acid 37 to form a highly reactive electophile 38 (the trans-acid cannot form the ring) that reacts with aniline 24 to form ketene 39. This in turn also reacts with aniline 24 to form adduct 34. The postulated mechanism fits with observed levels of 34 and the reaction profiling, but we have not been able to obtain proof of the existence of the reactive intermediates (38 and 39).

Scheme 9. Postulated Mechanism of Formation of Adduct 34

The rate of formation of bis-amide 35 was particularly intriguing as its levels keep increasing, even past reaction completion. The hydrolysis of the formamidine group was ruled out as the root cause as the levels of aniline 33 remained constant throughout the reaction. The formation of aniline 36, where the formamidine group has been replaced by an amide, led us to speculate the mechanism shown in Scheme 10 where the formamidine nitrogen in 24 is first acylated and the resulting formanidinium species 40 is transferred by the attack of another molecule of starting material 24. An indirect proof of this mechanism was obtained by submitting pure amide product 25 to the reaction conditions and observing elevated levels of bis-amide 35. This minor side reaction pathway explains the constant growth of the bis-amide 35 impurity post reaction completion. 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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Figure 3. Amidation reaction profiling.

Scheme 10. Postulated Mechanism for the Formation of Bis-amide 35 245 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. Amidation processes.

The initial work-up and isolation process was extremely simple as a direct drop after quench with an aqueous solution of sodium hydroxide. This provided an efficient process from nitro 23 to amide 25 with no solvent changes, distillations or phase separations. Upon scale up, this process proved to be problematic owing to two issues: 246 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 start of the quench operation the reaction mixture was exposed to aqueous conditions at low pH which led to the hydrolysis of the formamidine group. The pH swing-induced crystallization lacked robustness and led to a poor purge of impurities and inorganics.

Accordingly, a new quench, work-up, and crystallization processes were designed. To suppress the instability of 25 under aqueous acidic conditions, a reverse quench was implemented whereby the reaction mixture was added into a basic aqueous solution of sodium hydroxide. To improve the robustness of the crystallization, a seeded cooling crystallization from a mixture of acetonitrile and toluene was designed. The quench and crystallization operations were linked by an aqueous work-up using toluene. The new process, whilst requiring more unit operations, proved to be very robust on scale up and delivered significantly pure product (Table 1). A flow diagram comparing the initial and the new processes and the corresponding isolated solids is shown in Figure 4.

Table 1. Comparison of Initial and Final Amidation Processes Assay

Yield

Residue on Ignition (ROI)

Phosphorous content (ppm)

Initial process

87-91%

63-71%

0.4-1.8%

1500-10000

Final process

95-96%

73-77%

0.10-0.18%

200-300

Development of the Dimroth Rearrangement Reaction A successful Dimroth rearrangement was unlikely to be feasible for this reaction as both starting material 25 and product 1 are unstable towards high temperatures, and typical Dimroth rearrangement conditions are refluxing acetic acid. We therefore focused on identifying low temperature conditions for this reaction. A wide range of acids was screened at 30 °C in acetonitrile at high concentrations. This screen identified salicylic and acetic acid as the best compromise between conversion and impurity formation. Cost and simplicity of operations led us to select acetic acid. A solvent screen performed with several concentrations identified that neat acetic acid was optimal. Balancing impractically long reaction time when low temperatures were used with impurity generation at higher temperatures, a compromise was found at 30 °C using two equivalents of aniline 8 to increase the reaction rate (Scheme 11). Compounds 25 and 43 were observed during the reaction while compound 42 was not during reaction profiling experiments. This indicated that the rate limiting steps are both the initial formamidine exchange and the rearrangement itself, since 25 will be converted slowly to 42 which itself will be converted quickly to 43 but 43 will rearrange slowly to product. 247

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Scheme 11. Dimroth Rearrangement Reaction

The initial work-up and isolation process was simple and consisted of the addition of IPA followed by a pH induced crystallization via the addition of an aqueous solution of sodium hydroxide. The yield was excellent (85%) and this process performed well on pilot scale (20 kg), but calculations predicted that filtration time on commercial equipment could take up to 10 days, which was unacceptable. The root cause for this slow filtration could be the physical properties of the solid, the viscosity of the solvent, or a combination of both. To answer this question, we tested typical small particles of 1 in the filtration, alongside a slurry made of the typical solvent composition with engineered large particles of 1 (Figure 5). Both filtrations were equally slow, pointing to the solvent system being the root cause for the slow filtration.

Figure 5. Particles tested in the filtration. 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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The work-up and isolation process was therefore re-designed to avoid filtration of a viscous aqueous/organic mixture. An extractive process was sought using 2-MeTHF as organic solvent. The poor solubility of dacomitinib 1 in 2-MeTHF led to volumeous dilutions and volume swing as the reaction was performed in low volumes of acetic acid. We next examined a work-up where the volume of 2-MeTHF was capped at 10 L/kg and studied the addition of sodium hydroxide carefully. This showed that two liquid phases were observed until less than 5.7 equivalents of acetic acid remained. Between 5.7 equivalents and 2 equivalents, a three liquid phase system existed and below 2 equivalents the product 1 precipitated. We therefore designed a process that partially quenched acetic acid, leaving enough acetic acid in the system to ensure a two phase system with the product still in solution. The phases were then separated. The remaining acetic acid needed to be quenched to allow the product to crystallize. To achieve this, an organic base in conjunction with acetonitrile was used in order to keep the mixture monophasic. Several tertiary amines were investigated and all performed well. The lipophilicity of the amine appeared to correlate with yield loss as liquid amines appeared to be acting as solvents as well as bases (Table 2). To balance yield, boiling point of the amine and cost, we selected triethylamine as the organic base.

Table 2. Tertiary Amines Screening for Work-Up Me2NEt

Et2NMe

Et3N

Et2N(i-Pr)

nBu2NEt

Log P

0.7

1.1

1.5

2.4

4.6

Boiling point

36 °C

64 °C

89 °C

105 °C

181 °C

Yield

82%

80%

77%

69%

48%

With a practical extractive process in place, we were able to design a robust seeded crystallization that delivered a fast filtration (Figure 6). Whilst the yield range was slightly lowered to 75-80% from 80-85%, it was worth the trade for the improvement on the purge of inorganic and organic impurities. Finally, a dramatic improvement in predicted filtration time on commercial equipment from ten days to 14 h was realized (Table 3).

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Figure 6. Dimroth rearrangement processes.

Table 3. Comparison of the Dimroth Rearrangement Processes Yield

Total impurities

Filtration K value

Predicted filtration speed

Initial process

80-85%

1.2-1.6%

700

10 days

Final process

75-80%

NMT 0.050.07%

70

14 h Actual: Less than 10 h

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Conclusion Several processes were developed during the project lifecycle to meet the clinical and projected commercial demands. The initial medicinal chemistry route was significantly improved to enable the delivery of the first tens of kilos of API by changing reagents, solvents and conditions. The N-arylation route was developed to avoid oxidation state and functional group manipulations, but was ruled out based on economic and practical considerations. Finally, an efficient three-step, two-isolation process has been developed to manufacture dacomitinib on commercial scale. The commercial process comprises of a nitro group reduction reaction telescoped with an amidation reaction, followed by a Dimroth rearrangement to install the quinazoline ring motif. Key to the development of this process was the ability to perform the Dimroth rearrangement under unprecedented low temperature conditions, which provided a practical operating window for the desired reaction without the degradation of starting material and product. Extractive work-ups followed by seeded controlled crystallizations for the two isolations were critical to ensure high purity and fast filtrations. Finally, the use of acetonitrile in steps 1 and 2 allowed an efficient telescope between the hydrogenation of the nitro group and the amidation reaction. The commercial route has been used in Pfizer’s manufacturing facility and produced over 800 kg of API to date in 58% overall yield (Scheme 12).

Scheme 12. The Commercial Process to Dacomitinib

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Acknowledgments The authors would like to thank Mark Barrila, Weiling Cai, Juan Colberg, Mark Delude, Shane Eisenbeis, Kevin Girard, Arun Ghosh, Mark Maloney, Jason Mustakis, Mike Waldo and Greg Withbroe for their contributions to the early development part of this project; Ruth Boetzel, Claudio Brunelli, Ciaran Byrne, Steve Challenger, Yaling Cheng, Steve Collins, Doug Critcher, Rob Crook, Andrew Davidson, Niamh Dennehy, Andrew Derrick, Stuart Field, Andy Fowler, Denise Harris, Mike Hawksworth, Dave Henderson, James Hogbin, Ricky Jones, Phil Levett, Neil McDowall, Ivan Marziano, Jinu Mathew, Suju Mathew, Phil Peach, Sam Prior, Chris Stoneley, Roman Szucs, Steve Twiddle, Steve Yeo and David Walker for their contributions to the late development part of this project. Special thanks to David Daniels, Rob Singer, Karen Sutherland and Steve Twiddle for proof reading the manuscript.

References 1.

2. 3. 4.

5. 6. 7.

Smaill, J. B.; Gonzales, A. J.; Spicer, J. A.; Lee, H.; Reed, J.; Sexton, K.; Althaus, I. W.; Zhu, T.; Black, S. L.; Blaser, A.; Delaney, A.; Denny, W. A.; Ellis, P. A.; Fakhaury, S.; Harvey, P. J.; Hook, K.; McCarthy, F. O. J.; Palmer, B. D.; Rivault, F.; Schlosser, K.; Spoon, T.; Thompson, A. M.; Trachet, E.; Winters, R. T.; Tecle, H.; Bridges, A. J. Med. Chem. Submitted for publication. Arnold, L. D.; Sobolov-Jaynes, S. B. Eur. Pat. Appl. 1998, 837063. Yin, J.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 6043–6048. Flahive, E. J.; Ewanicki, B. L.; Sach, N. W.; O’Neill-Slawecki, S. A.; Stankovic, N. S.; Yu, S.; Guinness, S. M.; Dunn, J. Org. Proc. Res. Dev. 2008, 12, 637–645. Chandregowda, V.; Rao, G. V.; Reddy, G. C. Org. Proc. Res. Dev. 2007, 11, 813–816. For the synthesis of erlotinib: Asgari, D.; Aghanejad, A.; Mojarrad, J. S. Bull. Korean Chem. Soc. 2011, 32, 909–914. Foucourt, A.; Dubouilh-Benard, C.; Chosson, E.; Corbiere, C.; Buquet, C.; Iannelli, M.; Leblond, B.; Marsais, F.; Besson, T. Tetrahedron 2010, 66, 4495–4502.

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Chapter 10

Discovery and Development of the Natural Product Derivative SPI-1865 as a Gamma Secretase Modulator for Alzheimer’s Disease: Part I Jed L. Hubbs,*,1 Nathan O. Fuller,2 Wesley F. Austin,3 Ruichao Shen,4 and Brian S. Bronk5 Satori Pharmaceuticals Inc., 281 Albany Street, Cambridge, Massachusetts 02139, United States 1Laboratory of Organic Chemistry, ETH Zürich, HCI G336, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland. 2Rodin Therapeutics, 300 Technology Square, Cambridge, Massachusetts 02139, United States 3Celgene, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140, United States 4Enanta Pharmaceuticals, 500 Arsenal Street, Watertown, Massachusetts 02472, United States 5Sanofi, 640 Memorial Drive, Cambridge, Massachusetts 02139, United States *E-mail: [email protected].

This chapter describes the discovery of the gamma-secretase modulator SPI-1865 by Satori Pharmaceuticals and is followed by a chapter on its development. Satori was a 15-person company based in Cambridge, MA with all of its resources devoted to discovering and developing this compound. The project began by isolation of highly active phytosterol gamma secretase modulator (GSM) identified by fractionation of black cohosh extract. Exploratory SAR work established tolerable structural changes and improved the stability and physicochemical characteristics of the molecule. Efforts were then devoted to decrease liver metabolism and improve the blood brain penetration of the series. In the final push, © 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.

cytochrome P450 inhibition was reduced to minimize risk of drug-drug interactions. This work gave rise to a viable gamma-secretase modulator: SPI-1865.

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Introduction Gamma secretase modulators (GSMs) continue to be a promising drug class for disease modifying treatment of Alzheimer’s disease (AD). The peptide amyloid-beta (Aβ) is implicated in onset and progression of AD and is produced by the enzymes beta-secretase (BACE) and gamma-secretase (1). The most advanced BACE inhibitor verubecestat (Merck) is currently in two Phase 3 clinical trials, one for prodromal AD and one for mild to moderate AD. The results of these trials should be available in 2018. A positive outcome would be fundamental advance in AD treatment and would validate a tremendous amount of research and development efforts focused on BACE inhibitors. It would also encourage the development of other amyloid-targeting small molecules such as GSMs, the object of our work. Gamma secretase modulators change the composition of Aβ produced by neurons (2). This is in contrast to BACE inhibitors and gamma secretase inhibitors (GSIs), which stop Aβ production. This is important because evidence suggests that longer Aβ peptides, such as Aβ42, are more neurotoxic than shorter peptides such as Aβ40 (3). GSMs shift the ratio of Aβ42/ Aβ40 towards Aβ40 by binding to an allosteric site on the presenilin component of gamma secretase (4). It has unfortunately proven quite challenging to find robust clinical candidates to test gamma secretase modulation in humans. The compounds have suffered from poor efficacy and/or negative preclinical toxicity issues. When we started our program, the existing GSM leads belonged to two compounds classes: • •

those based on non-steroidal anti-inflammatory drugs.. those based on an arylimidazole pharmacophore (5).

It was therefore highly desirable to find new leads which may overcome the limitations of the known GSMs. In this chapter, we describe the discovery of a new class of gamma secretase modulators based on a phytosterol natural product that led to a viable candidate, SPI-1865.

Activity-Guided Fractionation of Black Cohosh Extract and Isolation of Lead Compound 1 A screen of natural product extracts was conducted to search for compounds that lowered Aβ42 production in a cell-based screen while sparing Aβ40 production (6). Only one extract tested from the black cohosh (Actaea racemosa) was found that selectively lowered Aβ42. Fractionation by normal phase chromatography gave 10 fractions (Figure 1) that were tested for their ability to inhibit Aβ42 production (Table 1). Fractions 5 and 6 were selected for further 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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fractionation based on their Aβ42 IC50 values (6.3 and 2.7 µg/mL respectively) and the size of the fractions (238 and 475 mg respectively).

Figure 1. TLC plates showing normal phase fractionation of black cohosh extract.

Table 1. Aβ42-Lowering Potency for Black Cohosh Fractions Fraction

Amount (mg)

IC50 (µg/mL)

3-1

239

n/a

3-2

86

36

3-3

51

77

3-4

140

7.4

3-5

238

6.3

3-6

475

2.7

3-7

67

8.8

3-8

43

15

3-9

242

7.9

3-10

38

6.4

This further fractionation afforded 10 pure triterpene glycosides (Scheme 1) (Figure 2) that were identified via a combination of NMR and mass spectrometry. This was expected based on the compounds that had been previously characterized from black cohosh root (7). Compound 1 was 10-fold more active (Aβ42 IC50 = 255 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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100 nM) than the next most active compounds (6 and 8, Aβ42 IC50 = 1000 nM) and consisted of our initial hit (Table 2). The structures of these compounds reveal the likely origin for 1, which could be formed by dehydration of the C16 hemiketal in sterol 5.

Scheme 1. Triterpene Glycosides Isolated from Black Cohosh Extract F3-6 and F3-5 These natural products also reveal interesting structure-activity relationships (SAR). For example, 8 (Aβ42 IC50 = 1000 nM) only differs by one stereocenter 256 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.

by being substituted with an arabinose rather than a xylose sugar. Similarly, the stereocenter at C24 with its acetate substituent appears critical as inversion from its configuration in 1 (Aβ42 IC50 = 100 nM) to that in 6 (Aβ42 IC50 = 1000 nM) also leads to a ten-fold decrease in potency. The presence of compounds 3 and 4 is also noteworthy as these can be formed by removal of the acetate from 1 and 8 followed by cyclization.

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Table 2. Pure Compounds Isolated from Black Cohosh with Aβ42 IC50 and Aβ40 IC50 IC50 (nM)

Compound #

Aβ40

Aβ42

1

6,300

100

2

>50,000

>50,000

3

>50,000

>50,000

4

>50,000

>50,000

5

50,000

5,600

6

>50,000

1,000

7

>50,000

32,000

8

16,000

1,000

9

16,000

3,000

With 1 as an initial screening hit, we set out to explore chemical modifications and SAR (Scheme 2) (8). This initial compound suffered from a number of limitations as a potential oral CNS therapeutic. Specific structural features of concern include:

• • •

the enol ether at C16-C17 is chemically unstable. the acetate at C24 is expected to be chemically and metabolically unstable. the sugar contributes to high polar surface area (PSA) and therefore the molecule is expected to be poorly CNS penetrant.

Enol Ether Reduction Under acidic conditions, 1 converted to ketal 11, which was significantly less active (Aβ42 IC50 = 3000 nM). However, Lewis acidic conditions using catalytic ZrCl4 in the presence of trace amounts of water, 1 converted to ketone 12. This compound was more active (Aβ42 IC50 = 600 nM) than the other isolated natural 257 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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products and was a promising intermediate for the synthesis of additional analogs as it was much more stable under acidic conditions. Even more promising, ketone 12 could be stereoselectively reduced to even more potent 13 (Aβ42 IC50 = 60 nM). This compound was Satori’s original lead molecule and was used to raise venture capital money for an expanded lead optimization program.

Scheme 2. Reactions and Replacement of the C16-C17 Enol Ether in 1

Replacement of the C3 Xyloside Group After removing the chemically unstable enol ether by two-step reduction, we turned our attention to the acetate and sugar moieties (Scheme 3). The xylosyl group in 13 was removed under acidic conditions to give 15 (Aβ42 IC50 = 600 nM), which was 10-fold less potent. The acetyl group in 13 was removed under basic conditions to give 14 (Aβ42 IC50 = 3100 nM), which was 50-fold less potent. When both the xylose and acetyl groups were removed the loss of potency was additive (Aβ42 IC50 > 10,000 nM). Since potency loss upon xylosyl removal was less significant, we chose to focus our initial efforts on finding a replacement of this group that would retain the potency found in 13 while improving the chances the compound would be a successful CNS drug. One strategy we pursued was to modify the sugar (8). This was accomplished by double oxidative cleavage with sodium periodate or lead tetraacetate to give dialdehyde 17, which was used without purification (Scheme 4). Dialdehyde 17 could be converted to the corresponding diol 18 (Aβ42 IC50 = 100 nM ) with sodium borohydride, diamine 19 (Aβ42 IC50 = 2400 nM) with dimethylamine and sodium cyanoborohydride, or to morpholine 20 (Aβ42 IC50 = 130 nM) by 258

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treatment with methylamine and sodium cyanoborohydride. We believed that morpholine 20 was quite promising for further optimization because it had a significantly lower PSA (98 Å2) than 18 and 19

Scheme 3. Acetate and Xylose Removal of 13

Scheme 4. Oxidative Cleavage of 13 and Further Reactions We replaced the methyl group on the nitrogen of the morpholine ring of compound 20 with H as well as a number of other substituents and examined their effects on its potency and whether they would improve its pharmaceutical properties. Examples of morpholine substituents tested are shown in Table 3. The unsubstituted morpholine 21 (Aβ42 IC50 = 70 nM) showed slightly improved potency relative to the methyl-substituted morpholine 20 (Aβ42 IC50 = 130 nM). An inductively electron withdrawing oxetanyl group resulted in a 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.

neutral compound at physiological pH and modestly decreased potency (22, Aβ42 IC50 = 170 nM). Amine or carboxylic acid substituents were also tolerated (23, Aβ42 IC50 = 110 nM; 24, Aβ42 IC50 = 370 nM), as were sulfonamides (25, Aβ42 IC50 = 80 nM) and amides (Aβ42 IC50 = 90 nM). In summary, we found that Aβ42 lowering potency was retained with a variety of substituents on the morpholine nitrogen atom, which allowed us to use this position to later fine-tune the physicochemical properties of our compounds or optimize other paramaters such as off-target pharmacology.

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Table 3. SAR of Representative N-Substituted Morpholine Derivatives

In addition to replacing the sugar moiety on C3 with a morpholine ring, we examined other possible xyloside replacements (9). We primarily focused on esters (Table 4, 27-31) and carbamates (32-36) since introduction of other groups such as ethers at the sterically encumbered neopentylic alcohol proved very challenging. The range of substituents affording potent Aβ42-lowering compounds was significantly narrower than with morpholines. For example, esters with basic substituents were generally potent (28, Aβ42 IC50 = 95 nM and 31, Aβ42 IC50 = 55 nM), but those with neutral (27, Aβ42 IC50 = 760 nM) and acidic (29, Aβ42 IC50 = 1600 nM) substituents showed only modest potency. In addition, the specific pyridyl ester 30 (Aβ42 IC50 = 260 nM) showed moderately 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.

good potency. Carbamates showed a similar trend where basic substituents resulted in potent compounds (34, Aβ42 IC50 = 85 nM and 35, Aβ42 IC50 = 47 nM). Other substituents were better tolerated than with esters. For example, carbamates 33 (Aβ42 IC50 = 340 nM) and 36 (Aβ42 IC50 = 450 nM) showed moderately good potency.

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Table 4. SAR of Representative Ester and Carbamate Substituted Analogs

Replacement of the C24 Acetyl Group To Improve Liver Metabolic Stability Now that we had established trends in SAR at the C3 position we turned our attention to replacing the acetyl group at the C24, which we believed was resulting in poor metabolic stability (10). Our hypothesis was that liver mediated carboxylesterase hydrolysis was responsible for this poor stability and turned to testing compounds in human liver microsome (HLM) stability assays to probe stability. The HLM assays were run in the absence of added NADPH to eliminate the effects of cytochrome P450-mediated metabolism. 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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Removal of the C25 hydroxyl group from tetraol 16 gave a compound with similar potency (Table 5, 37, Aβ42 IC50 = 780 nM) and as expected, HLM stability was poor (0% remaining at 60 min). When the ester was replaced with a dimethylamino carbamate, the Aβ42 IC50 was dramatically reduced (~20,000 nM) however the microsomal stability was now good (>100%) remaining at 60 min). We were surprised to find that removal of the hydroxyl group from 38 to give analog 39 restored most of the potency relative to the acetate (Aβ42 IC50 = 1000 nM) since the presence or absence of this group was inconsequential for the acetate analog (Table 5, 16 vs. 37). Compound 39 also maintained most of the HLM stability (81% remaining at 60 min).

Table 5. Aβ Lowering Activity and HLM Stability of C24-C25 Analogs

Finally, we examined C24 ether analogs. Replacement of the acetate in tetraol 16 with an ethyl ether gave ether 40 that was more potent (Aβ42 IC50 = 350) and which had excellent microsomal stability (100% remaining at 60 min). We were also surprised to find that removal of the hydroxyl group from 40 gave dramatically decreased Aβ42 lowering potency (41, Aβ42 IC50 = 4400 nM). Other ester and carbamate analogs were tested and the ethyl ether (40, Aβ42 IC50 = 350) and 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.

azetidinyl carbamate (data not shown) proved optimal and were utilized in further optimization.

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Analogs with Improved CNS Penetration With potency and metabolic stability optimized, we turned our attention to testing our compounds for their ability to enter the central nervous system. Our approach towards this was to examine combinations of C3 and C24 groups that afforded potent analogs. A subset of the compounds tested is shown in Table 6. These data demonstrated that a combination of a morpholine at C3 and an ether at C24 were necessary to get acceptable (>0.5) brain:plasma (B:P) ratios. For example, morpholine 42 with an unsubstituted morpholine and ethyl ether had B:P = 1.67. Carbamates at the C3 position (44-47) and at the C24 position (compounds 43, 45 and 47) all had poor B:P (≤0.13).

Table 6. Examination of C3 and C24 Substitution on Activity

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With confidence that we could achieve good brain exposure levels with our compounds, we prepared a set of C24 ether and C3 morpholine analogs with a variety of substituents on the morpholine nitrogen. A subset of these compounds is listed in Table 7. In preparing these analogs, our goal was to prepare analogs with diverse properties that may be suitable for selection as development candidates after further profiling. As shown, compounds with the most basic nitrogen atoms were the most potent (42, Aβ42 IC50 = 91 nM; 52, Aβ42 IC50 = 100 nM), while those with attenuated bacisity were approximately 2- to 3-fold less potent (48, Aβ42 IC50 = 250 nM ; 49, Aβ42 IC50 = 270 nM; 50, Aβ42 IC50 = 260 nM; 51, 270 nM).

Table 7. Potency of C24 Ether and C3 Morpholine Analogs with a Variety of Substituents on the Morpholine Nitrogen

While greater Aβ42-lowering was desirable, we thought that this might be counterbalanced by an increased toxicology risk associated with the more basic compounds. We found that both 49 and 52 lowered Aβ42 in mouse. At 100 mg/kg IP (single dose) methoxyethyl morpholine 49 lowered Aβ42 by 46% (±5%) at a brain exposure of 23 µM and plasma exposure of 12 µM. At 100 mg/kg PO (single

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dose) methylazetidinyl 52 lowered Aβ42 by 26% (±11%) at a brain exposure level of 20 µM and a plasma exposure level of 15 µM. Further characterization of 49, 52 and others revealed that several compounds were moderate to strong inhibitors of CYP3A4 and other cytochrome P450 isoforms (11). For example, 49 had a CYP3A4 IC50 = 1.1 µM and 52 had a CYP3A4 IC50 = 12 µM. Unsubstituted morpholine 42 was the strongest CYP3A4 inhibitor with 99% inhibition at 10 µM.

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Modeing CYP3A4 Inhibition In order to understand the reason for this strong inhibition, we turned to modeling. Using the structure of CYP3A4 bound to ketoconazole as a starting point (12), we found that the strong inhibition was likely due to coordination of the morpholine nitrogen atom to the iron atom in the heme moiety. Docking studies with other molecules suggested that additional steric bulk and a positive charge on the morpholine substituent would likely reduce CYP3A4 binding and inhibition.

Figure 2. Model of compound 42 bound to CYP3A4.

Based on these results we prepared a series of additional morpholine analogs with azetidine substituents either directly attached to the morpholine or attached through a methylene linker (Table 8). The least potent CYP3A4 inhibitors carried a bulky group on an azetidine moiety directly attached to the morpholine group (53, CYP3A4 IC50= 37 µM; 54, CYP3A4 IC50= >100 µM). The most potent CYP inhibitors bore an inductively electron withdrawing group on the azetidine group attached to the morpholine moiety (55, CYP3A4 IC50= 5.3 µM; 56, CYP3A4 IC50= 3.0 µM). Analogs with a methylene-linked azetidine group (57-60) had similar potencies (CYP3A4IC50= 13-22 µM). We ruled out 55 and 56 as potential preclinical development candidates based on their CYP3A4 inhibition, and 59 based on time dependent inhibition (CYP3A4IC50= 7.8 µM after preincubation with NADPH). 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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Table 8. CYP3A4 Inhibition for Several Azetidinyl Morpholine Analogs

As a next step in candidate selection, 53, 54, and 60 were selected for testing in rat PK/PD experiments at dose levels expected to give similar plasma exposure based on their individual rat PK profiles (Table 9). Consistent with our expectations, all compounds gave good brain exposure (B:P = 0.69 to 2.4; 6.3 to 33 µM) and statistically significant lowering of Aβ42 (23 to 50%). The compounds were examined in a 14-day exploratory safety study in the same strain of rats. Compound 53 (SPI-1865) provided the best therapeutic window based on amaximum tolerated dose of >90 mg/kg and >40µM average plasma concentration, and from this assessment and was chosen as a candidate for preclinical development. 266 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 9. PK/PD Experiments with Compounds 53, 54, and 60

Figure 3. SPI-1865 mult-dose oral PK/PD experiment in Sprague Dawley rats. 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.

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SPI-1865 was further profiled in PK/PD experiments to confirm it has sufficient gamma secretase modulating properties to justify further development (13). These studies included wild-type mouse and rat studies, and studies in Tg2576 mice that overexpress human amyloid precursor protein (APP). Figure 3 shows the results form a multi-dose PK/PD experiment in wild type rats. SPI-1865 shows a clear decrease in Aβ42 at 10, 30 and 60 mg/kg. The 10 mg/kg dose lowered Aβ42 by approximately 25% with 4.4 µM brain exposure and 8.0 µM plasma exposure. At 30 mg/kg, Aβ42 was lowered by approximately 49% with 16 µM brain exposure and 13 µM plasma exposure. The 60 mg/kg dose lowered Aβ42 by 61% at exposure levels of 45 µM in brain and 19 µM in plasma.

Conclusions Our discovery effort and lead optimization program took us from an active and Aβ42-selective extract to a CNS development candidate. Along the way challenges were encountered with chemistry, poor metabolic stability, CNS penetration, and cytochrome P450 inhibition. The following chapter will describe our company’s success in crafting a process to prepare SPI-1865 on large scale.

Acknowledgments The authors sincerely thank all coworkers at former Satori Pharmaceuticals, and colleagues at Carbogen-Amcis and WuXi AppTec for their contributions in this project.

References 1. 2. 3. 4.

5. 6.

7. 8.

Sastre, M.; Steiner, H.; Fuchs, K.; Capell, A.; Multhaup, G.; Condron, M. M.; Teplow, D. B.; Haass, C. EMBO Rep. 2001, 2, 835–841. Golde, T. E.; Koo, E. H.; Felsenstein, K. M.; Osborne, B. A.; Miele, L. Biochim. Biophys. Acta, Biomembranes 2013, 1828, 2898–2907. Weggen, S.; Beher, D. Alzheimers Res. Ther. 2012, 4, 9. Takeo, K.; Tanimura, S.; Shinoda, T.; Osawa, S.; Zahariev, I. K.; Takegami, N.; Ishizuka-Katsura, Y.; Shinya, N.; Takagi-Niidome, S.; Tominaga, A.; Ohsawa, N.; Kimura-Someya, T.; Shirouzu, M.; Yokoshima, S.; Yokoyama, S.; Fukuyama, T.; Tomita, T.; Iwatsubo, T. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 10544–10549. Crump, C. J.; Johnson, D. S.; Li, Y.-M. Biochemistry 2013, 52, 3197–3216. Findeis, M. A.; Schroeder, F.; McKee, T. D.; Yager, D.; Fraering, P. C.; Creaser, S. P.; Austin, W. F.; Clardy, J.; Wang, R.; Selkoe, D.; Eckman, C. B. ACS Chem Neurosci 2012, 3, 941–51. Shao, Y.; Harris, A.; Wang, M.; Zhang, H.; Cordell, G. A.; Bowman, M.; Lemmo, E. J. Nat. Prod. 2000, 63, 905–910. Fuller, N. O.; Hubbs, J. L.; Austin, W. F.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M.; Tate, B.; Xia, W.; Ives, J.; Findeis, M. A.; Bronk, B. S. ACS Med. Chem. Lett. 2012, 3, 908–913. 268

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9.

10.

11.

12.

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13.

Austin, W. F.; Hubbs, J. L.; Fuller, N. O.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M. B.; Findeis, M. A.; Tate, B.; Ives, J. L.; Bronk, B. S. MedChemComm 2013, 4, 569–574. Hubbs, J. L.; Fuller, N. O.; Austin, W. F.; Shen, R.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M.; Tate, B.; Xia, W.; Ives, J.; Bronk, B. S. J. Med. Chem. 2012, 55, 9270–9282. Hubbs, J. L.; Fuller, N. O.; Austin, W. F.; Shen, R.; Ma, J.; Gong, Z.; Li, J.; McKee, T. D.; Loureiro, R. M.; Tate, B.; Dumin, J. A.; Ives, J.; Bronk, B. S. Bioorg. Med. Chem. Lett. 2015, 25, 1621–1626. Ekroos, M.; Sjögren, T. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13682–13687. Loureiro, R. M.; Dumin, J. A.; McKee, T. D.; Austin, W. F.; Fuller, N. O.; Hubbs, J. L.; Shen, R.; Jonker, J.; Ives, J.; Bronk, B. S.; Tate, B. Alzheimer’s Res. Ther. 2013, 5, 1–12.

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Chapter 11

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Discovery and Development of the Natural Product Derivative SPI-1865 as a Gamma Secretase Modulator for Alzheimer’s Disease Ruichao Shen,*,1 Nathan O. Fuller,2 Jed L. Hubbs,3 Wesley F. Austin,4 and Brian S. Bronk5 Satori Pharmaceuticals Inc. Cambridge, Massachusetts 02139, United States 1Present address: Enanta Pharmaceuticals, 500 Arsenal Street, Watertown, Massachusetts 02472, United States 2Present address: Rodin Therapeutics, 300 Technology Square, Cambridge, Massachusetts 02139, United States 3Present address: Laboratory of Organic Chemistry, ETH Zürich, HCI G336, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland 4Present address: Celgene, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140, United States 5Present address: Sanofi, 640 Memorial Drive, Cambridge, Massachusetts 02139, United States *E-mail: [email protected].

This chapter describes the scale-up synthesis of the natural product derivative SPI-1865, employing a semi-synthetic approach. The challenge of securing a good quantity of starting material was solved by preparation of two cycloartenol glycosides in multikilogram scale via extraction/isolation from biomass and a ZrCl4-catalyzed reaction. By developing a protecting group-free ether formation reaction and a significantly improved five-step/3 pot process, we achieved kilogram-scale synthesis of SPI-1865 in good overall yield.

As SPI-1865 (1) was identified as the development candidate of our GSM program (1–5), it became our initial task to scale up the synthesis to kilogram scale for clinical studies.

© 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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The medicinal chemistry (Scheme 1) synthesis to 1 commenced with a double oxidative cleavage of the sugar moiety of cycloartenol triterpenoid glycosides 2 and 3 (6). Double reductive amination of the resulting dialdehyde 4 with 1-Boc-3-aminoazetidine hydrochloride provided N-Boc-azetidinyl morpholine compound 5. This compound was reduced with NaBH4 to produce C15 alcohol 6 with high diastereoselectivity (>95:5 dr). Protection of the C15 hydroxyl with TESCl followed by hydrolysis of the C24 acetate produced the diol 7. Selective alkylation of the C24 hydroxyl using NaH/EtI in DMF provided C24 ethyl ether 8. Finally, a double deprotection of the N-Boc-carbamate and the C15 TES ether using HCl in 1:1 aq MeOH at 50 °C, followed by reductive amination with acetone, provided 1. This route, requiring eight synthetic and four chromatography steps, was employed to supply 1 on multi-gram scale for in vivo PK/PD studies.

Scheme 1. Initial Synthetic Route to 1. (Reproduced with permission from reference (16). Copyright 2014 American Chemical Society).

As shown in Scheme 1, cycloartenol triterpenoid glycosides 2 and 3 served as required starting materials for the synthesis of 1. Therefore, before any optimization of the synthetic route, the first challenge we were facing was to obtain sufficient amount of 2 and 3; a non-trivial task. 272 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. Preparation of Compound 2/3

Our method to prepare 2 and 3 started from extraction and isolation of 24-O-acetylhydroshengmanol 3-O-β-D-xyloside (10) (7) and 24-Oacetylhydroshengmanol 3-O-α-L-arabinoside (11) (1) from the roots and rhizomes of Actaea racemosa, commonly known as “black cohosh” (Scheme 2). Glycosides 10 and 11 share the same aglycone, having the isomeric xylose and arabinose at the C3 position, respectively. Treatment of 10 and 11 with a catalytic amount of ZrCl4 in dichloromethane stereoselectively afforded 2 and 3, respectively, in 80% yield (2–4). Compounds 2/3 possessed a stable tetrahydropyran E-ring and a C15-ketone. We propose that a pinacol-type 1,2-hydride shift pathway is likely involved in this transformation (8). To meet the goal of producing one kilogram of GMP quality 1 for development studies, 10 kg of combined cycloartenol glycosides 2 and 3 would be needed. In consideration of the structural intricacy and very limited effort in total synthesis of cycloartenol natural products, the most practical approach would be an optimized process of extraction and separation of compounds 10 and 11 followed by the ZrCl4-catalyzed reaction. We set out to do so. Our original process commenced with methanol extraction of ground powder of black cohosh (Scheme 3). The concentrated methanol solution was precipitated by slow addition of 5% aqueous KCl solution. The collected solid, containing 10 and 11 in 10-15% combined HPLC purity was purified by reverse phase (RP) C-18 chromatography. A mixture of 10 and 11 in 32% combined purity was obtained. Treatment of this mixture with catalytic ZrCl4 in dichloromethane provided 2 and 3 as major products. Precipitation from aqueous ethanol further upgraded the purity of 2 and 3 to 96 HPLC %. Despite working well at the discovery stage, two major limitations prevented this process from serving as a practical one for developmental quantities. First, solids obtained from precipitation with 5% aqueous KCl had varying particle sizes. This made it difficult to maintain consistently a satisfactory recovery from filtration. Second, C-18 chromatography is obviously not practical for large scale preparations. During an effort to optimize the extraction process, a liquid/liquid extraction of the alcoholic extract of black cohosh was upgraded from 2.5% to a combined purity of 10 / 11 of 13-15%. This was accomplished using dichloromethane and 11.6% NaCl solution. Other major components in the extract were either assigned by comparison with authentic samples on HPLC or by previous analysis in the 273

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literature (9). These compounds include actein 12 (two C26 epimers) (10, 11), 23-epi-26-deoxyactein (13) (12), 23-O-acetylshengmanol 3-O-β-D-xyloside (14) (13), cimigenol 3-O-β-D-xyloside (15) (14), cimigenol 3-O-α-L-arabinoside (16) (15), and 25-O-acetylcimigenol 3-O-β-D-xyloside (17) (16) (Figure 1).

Scheme 3. Flow Chart of Original Process To Prepare 2/3

Figure 1. Structures of the identified compounds in DCM extract. (Reproduced with permission from reference (8). Copyright 2014 American Chemical Society).

At the ZrCl4 step of the original process, we found that in addition to 10 / 11, additional components in the black cohosh extract participated in the reaction to produce 2 / 3. Among the above identified components, compound 14 could be one of the productive components, as it may transform to 10 under acidic conditions (9, 13). On the other hand, other major components in the extract were not expected to interfere with the ZrCl4 reaction. Based on this analysis, it was reasonable to treat the DCM extract directly with ZrCl4 to generate compounds 2 / 3. In a typical batch, ZrCl4 (7% w/w) was added to a DCM solution obtained from liquid/liquid extraction containing 13.6% of 10 and 11. The water content of the DCM solution was pre-adjusted to an appropriate level (0.4% relative to solids) 274 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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as it is critical for initiating the reaction (8). The mixture was stirred at 23 °C for 70 min and quenched with triethylamine. An aqueous work up with 4% NaHCO3 solution followed by filtration of the organic layer through Celite® provided a DCM solution containing 10.7% of 2 and 6.4% of 3 (HPLC). As predicted, actein (12) and cimigenol compounds (15, 16) were not affected by the ZrCl4 treatment. Compounds 2 / 3 were present as the major components in the crude product. Although purification remained as a challenge, encouraged by reasonable resolution between 2 / 3 and other major components, we identified a fractionation/precipitation process. The DCM solution from above was charged on a silica gel column (3 times of the solid weight) and eluted with a gradient of 0-12% MeOH/DCM. Collection of the desired fractions provided a solution containing 17.8% of 2 and 10.5% of 3. This solution was loaded on silica gel column (8 times of the solid weight) and eluted with a gradient of 2-15% MeOH/DCM. Combination of desired fractions provided 2 and 3 in 64% combined purity (HPLC), with actein as the major remaining impurity. Similar to our original process, precipitation from an aqueous ethanol solution produced additional purification by removal of the actein compounds. In addition, we found it crucial to add Celite® in the precipitation process to speed up the filtration. The precipitated solid together with Celite® was redissolved in DCM and filtered. Concentration of the filtrate afforded a pale brown solid containing 2 and 3 in over 90% HPLC purity.

Scheme 4. Flow Chart for the Production of 1 and 2 Using the Optimized Process A production campaign at multikilogram scale of 2 and 3 was initiated with the optimized process, (see Scheme 4 for the flow chart for this process). This campaign originated with 1330 kg of dried solids obtained from ethanol extractoin of 7 metric tons of black cohosh biomass. Using the optimized process, 11 kg of combined compounds 2 and 3 were produced (8). Overviewing the whole process, one can promptly point out that the silica gel fractionation step is the rate-limiting step, consuming the most time and effort. Although this is an inevitable step before a synthetic route is developed, it can be improved by investigating additional precipitation methods, reusable absorbents, and more efficient eluents. Having solved the supply issue of starting material 2 / 3, we focused our attention on optimizing the synthetic route (Scheme 1). In this route, three normal phase and one reverse phase column chromatographies were 275

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employed as purification methods. In addition, NaH and DMF were used for the C24-O-alkylation, which generated a safety concern for scale-up. These aspects required us to improve this route before starting the kilogram-scale synthesis of 1. First we endeavored to find a safe process for the C24 hydroxy alkylation by investigating bases/alkylating reagents/solvents combinations. Treatment of diol 7 with NaOtBu (5.0 eq) and diethyl sulfate (2.5 eq), afforded a 93% conversion to 8 after only 30 min at 0 °C (Equation 1 ).

On the other hand, we were additionally encouraged by a separate reaction to explore the relative reactivity of the alcohols in tetrol 18. When the unprotected aglycone 18 was treated with NaH (4.0 eq) and EtI (1.5 eq) in THF at room temperature for 15 h, the favored product was the C24 mono-O-alkylation product 19 (41%), (Equation 2). While there was a significant amount of unreacted starting material (42%) and C15,C24 bis-ether 20 formed (17%), this experiment suggested that C24 alcohol is the most active one for alkylation. It might be possible to selectively alkylate the C24 hydroxyl without protecting the C15 hydroxyl, saving significant time. This result inspired us to apply the above optimized alkylation conditions to 21 which doesn’t have a protecting group at C15 alcohol. With NaOtBu (5.0 eq) as base, diethylsulfate (1.1 eq) as alkylating agent, and more eco-friendly toluene/DMF (3:1) as a mixed solvent, the alkylation conducted at 0 ºC for 2 h provided a 79% conversion to 22 and only 2% of bis-ether 23 (Equation 3).

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In the original route, Boc was used to protect the azetidine, which was designed to synthesize a series of analogs with different substitutions at the azetidine (4). The promising result from alkylation of 21 encouraged us to further explore whether the carbamate protecting group for the azetidine was necessary. In previous experiments we found that the N-methyl azetidine compound could form the quaternary ammonium salt 24 (Figure 2) under alkylation conditions with strong base (NaH / EtI). We hoped that a weaker base (NaOtBu) along with the added steric bulkiness by the isopropyl group could suppress the formation of quaternary salts.

Figure 2. Structure of 24.

Therefore, we investigated the alkylation conditions on the functionalized N-isopropyl azetidine 25 (Table 1). When using 2.7 eq of NaOtBu, 1.1 eq of Et2SO4, and toluene/DMF (3:1) as solvent, we were delighted to see that the reaction gave 72% of C24-O-alkylation product 1 with no quaternization of the azetidine nitrogen (entry 1). Increasing the amount of base to 5 eq with a lower substrate concentration (0.1 M) improved the conversion of 1 to 80%, but also increased the formation of diethyl ether 26, in spite of being performed at lower temperature (entry 2). Further increasing the substrate concentration to 0.2 M provided a 91% conversion to SPI-1865, indicating the reaction is sensitive to substrate concentration (entry 3). Increasing the amount of diethylsulfate (1.25 eq) with a lesser amount of DMF as solvent slightly improved the conversion to 1 (93%), while keeping di-ethyl ether 26 at about the same level (entry 4). These highly selective alkylation results are exciting because they allowed us to skip the use of both protecting groups and therefore to eliminate three steps (protection/deprotection/reductive amination) from the original sequence. Applying these improvements, the optimized synthesis of the candidate SPI1865 (1) is shown in Scheme 5. Following oxidative cleavage of the 1,2,3-triol on the sugar of 2 / 3, the crude dialdehyde 4 was used directly after work-up in the double reductive amination with amine 27. Using NaBH3CN as the reducing agent is critical for a clean reaction as we found incomplete reduction products formed when NaBH(OAc)3 was used. The fact that we were now running the reaction in EtOH allowed us to add NaBH4 directly to the reaction mixture to reduce the C15 ketone to furnish the C15 alcohol, upon completion of the reductive amination. Since C15 alcohol protection is no longer required for the alkylation step, the C24 acetate was hydrolyzed in the same reaction vessel by subsequently adding aqueous NaOH upon the completion of the ketone reduction. Therefore, three reactions were efficiently telescoped in one pot to yield triol 25. 277

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Table 1. Selective Alkylation of Compound 25

Scheme 5. Optimized Route for Kilogram-Scale Synthesis of Compound 1. Reproduced with permission from reference (16). Copyright 2014 American Chemical Society. In light of the excessive amount of boron reagent used, it is critical to monitor the boron level during purification of triol 25. A surprising result came when subjecting a solid obtained from precipitation of crude 25 in EtOAc to the alkylation conditions, which afforded the C-15 ethyl ether product 28 instead of 1. We proposed a cyclic boronate ester 29 formed at the C24,25-diol could be the major component in the precipitated solid in EtOAc. The boronate ester 278 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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would act as a protecting group for the C24 alcohol and leave only the C15 alcohol available for alkylation (Equation 4). To ensure complete degradation of the proposed cyclic boronate and consistent performance on scale, we decided to add diethanolamine to the reaction mixture after the hydrolysis of the C24 acetate. The new work up procedure was followed by filtration through a plug of silica gel, eliminating both lingering boron species and other impurities. At last crystallization in acetone/H2O further upgraded the purity of triol 25. This system provided triol 25 in 34% yield over four steps from glycoside 2 / 3.

In a typical alkylation batch, triol 25 (1.842 kg) was dried by aceotropic distillation and dissolved in toluene (11.6 L) and DMF (2.31 L). This solution was cooled to -1 °C and NaOtBu (1.32 kg, 5 eq) was added before further cooling to -20°C. Diethylsulfate (0.85 kg, 2 eq) was added over 15 min. The reaction was stirred at -20 °C until HPLC indicated the reaction had progressed a sufficient amount. After 205 min, the reaction was quenched with water (5.5 L) over 15 min, and warmed to 2 °C. A solution of 3:1 toluene:THF (9.2 L) was added and the mixture was warmed to 40 °C, whereupon the layers were separated. The aqueous layer was re-extracted twice with 3:1 toluene:THF (2 × 9.2 L) at 40 °C. The combined organic layer was washed with brine at 40 °C and concentrated under reduced pressure at 44 - 60 °C to provide crude 1 (1.88 kg, 66% purity by HPLC-CAD). Initial purification of crude 1 was achieved by plug chromatography on aminosilica gel (19.4 kg), using gradient elution (heptane/toluene/EtOAc). The fractions containing clean product were combined and concentrated to provide 1.24 kg of 1 (88.5% purity, HPLC-CAD). Next, crystallization of 1 (1.24 kg) was carried out for final purification by adding TBME (12.5 L), toluene (5.6 L) and water (0.063 L), and heating the mixture to reflux (~70 °C) to produce a clear solution. Seeding with crystalline 1 was necessary to initiate the crystallization process. The formed white suspension was cooled to 10 °C over 90 min, stirred for 15 min, and filtered to collect the crystallized product. The filter cake was washed with cold TBME (2.5 L) and dried under vacuum with heating to provide 0.99 kg of SPI-1865 (1, 95.3% purity HPLC-CAD). This purity was sufficient for our purposes. In summary, through development of a highly selective alkylation using a protecting group-free substrate and substantial optimization, we condensed the original route to a five step / three pot synthesis that involved two silica gel plugs and two crystallizations. These enhancements enabled us to accomplish kilogram scale syntheses of the gamma-secretase modulator 1 (SPI-1865) (17). 279

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Acknowledgments The authors sincerely thank all coworkers at former Satori Pharmaceuticals, and colleagues at Carbogen-Amcis for their contributions in this project.

References 1.

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Findeis, M. A.; Schroeder, F.; McKee, T. D.; Yager, D.; Fraering, P. C.; Creaser, S. P.; Austin, W. F.; Clardy, J.; Wang, R.; Selkoe, D.; Eckman, C. B. ACS Chem. Neurosci. 2012, 3, 941–951. Fuller, N. O.; Hubbs, J. L.; Austin, W. F.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M.; Tate, B.; Xia, W.; Ives, J.; Findeis, M. A.; Bronk, B. S. ACS Med. Chem. Lett. 2012, 3, 908–13. Austin, W. F.; Hubbs, J. L.; Fuller, N. O.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M. B.; Findeis, M. A.; Tate, B.; Ives, J. L.; Bronk, B. S. MedChemComm 2013, 4, 569–574. Hubbs, J. L.; Fuller, N. O.; Austin, W. F.; Shen, R.; Creaser, S. P.; McKee, T. D.; Loureiro, R. M.; Tate, B.; Xia, W.; Ives, J.; Bronk, B. S. J. Med. Chem. 2012, 55, 9270–82. Hubbs, J. L.; Fuller, N. O.; Austin, W. F.; Shen, R.; Ma, J.; Gong, Z.; Li, J.; McKee, T. D.; Loureiro, R. M.; Tate, B.; Dumin, J. A.; Ives, J.; Bronk, B. S. Bioorg. Med. Chem. Lett. 2015, 25, 1621–6. Du, M.; Hindsgaul, O. Synlett 1997, 395–397. Sakurai, N.; Kimura, O.; Inoue, T.; Nagai, M. Chem. Pharm. Bull. 1981, 29, 955–960. Shen, R.; Fuller, N. O.; Osswald, G.; Austin, W. F.; Hubbs, J. L.; Haag, D.; Kovacs, J.; Creaser, S. P.; Findeis, M. A.; Ives, J. L.; Bronk, B. S. Org. Process Res. Dev. 2014, 18, 676–682. He, K.; Pauli, G. F.; Zheng, B.; Wang, H.; Bai, N.; Peng, T.; Roller, M.; Zheng, Q. J. Chromatogr. A 2006, 1112 (1–2), 241–254. Kusano, A.; Takahira, M.; Shibano, M.; In, Y.; Ishida, T.; Miyase, T.; Kusano, G. Chem. Pharm. Bull. 1998, 46, 467–472. Jamróz, M. K.; Bąk, J.; Gliński, J. A.; Koczorowska, A.; Wawer, I. J. Mol. Struct. 2009, 933 (1–3), 118–125. Chen, S.-N.; Li, W.; Fabricant, D. S.; Santarsiero, B. D.; Mesecar, A.; Fitzloff, J. F.; Fong, H. H. S.; Farnsworth, N. R. J. Nat. Prod. 2002, 65, 601–605. Sakurai, N.; Inoue, T.; Nagai, M. Chem. Pharm. Bull. 1979, 27, 158–165. Sakurai, N.; Nagai, M.; Inoue, T. Yakugaku Zasshi 1975, 95, 1354–1360. Shao, Y.; Harris, A.; Wang, M.; Zhang, H.; Cordell, G. A.; Bowman, M.; Lemmo, E. J. Nat. Prod. 2000, 63, 905–910. Takemoto, T.; Kusano, G.; Kawahara, M. Yakugaku Zasshi 1970, 90, 64–67. Fuller, N. O.; Hubbs, J. L.; Austin, W. F.; Shen, R.; Ives, J.; Osswald, G.; Bronk, B. S. Org. Process Res. Dev. 2014, 18, 683–692.

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Chapter 12

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Telaprevir: From Drug Discovery to the Manufacture of Drug Substance Gerald J. Tanoury,*,1 Stephen Eastham,2 Cristian L. Harrison,1 Benjamin J. Littler,1 Piero L. Ruggiero,1 Zhifeng Ye,2 and Anne-Laure Grillo 1Process

Chemistry, 50 Northern Avenue, Boston, Massachusetts 02210, United States 2Technical Operations, and 50 Northern Avenue, Boston, Massachusetts 02210, United States 3Chemistry Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, Massachusetts 02210 *E-mail: [email protected].

This chapter describes the discovery of telaprevir as a HCV NS3•4A protease inhibitor and development of a process for commercial manufacture of the drug substance. The first section of the chapter covers the drug discovery efforts, covering the SAR that identified the essential structural requirements for the protease inhibitor and the final optimization that led to the discovery of telaprevir. The remainder of the chapter describes, in detail, the efforts required to develop a commercial manufacturing process for telaprevir, a tetrapeptide possessing a pyrazine cap on the P4 residue and a cyclopropyl amide on the P1 residue. The development efforts were divided into several stages: process development for the commercial manufacture of the P1 and P2 residues (aminoalcohol 20 and bicycloproline ester 12, respectively), amine deprotections (debenzylation) and amide couplings, and TEMPO-mediated oxidation of hydroxyamide 19 to give telaprevir. Although discussion of the development of 20 and 12 is brief, detailed descriptions of the development activities for the remaining stages of the commercial process are provided. The final commercial process © 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.

provided >60 metric tons of drug substance over 150 batches, with a total impurity level of ≤ 0.03%, providing telaprevir in ≥99.97% purity.

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Introduction Infection with hepatitis C virus (HCV) represents a major medical burden worldwide, with over 185 million people affected by the disease (1). Following the acute infection phase, 80% of individuals progress to a chronic infection stage, associated with significant morbidity and mortality. Long-term HCV infection has been shown to lead to liver disease, such as fibrosis, cirrhosis, and hepatocellular carcinoma (2, 3). Extrahepatic manifestations of HCV infection are also common and burdensome (4). The hepatitis C virus was identified as the causative agent of non-A and non-B hepatitis in 1989 (5, 6). The first approved therapy consisted of a weekly administration of interferon-alpha (IFN-α) for 48 weeks, and achieved a sustained viral response (SVR) in 12-15% of patients (7). In 1991, ribavirin (RBV) was introduced (8). Its administration in combination with pegylated interferon-alpha (PegIFN-α) resulted in SVR rates of about 50% across HCV genotypes (9). This combination of PegIFN-α and RBV remained the standard of care therapy until 2011, when the first direct-acting antiviral therapies (DAAs), boceprevir and telaprevir, were approved to be used in combination with pegIFN-α and RBV (10–13). Since then, more than 30 DAAs and host-targeting agents (HTAs) have been investigated in the clinic, and all-oral therapy combinations are now available that can achieve SVR rates greater than 90% (14, 15). The introduction of DAAs achieved a paradigm shift in HCV treatment, and was made possible by key discoveries that furthered our knowledge of HCV. These included molecular virology advances that uncovered the HCV replication cycle, and identified druggable targets for drug discovery (14). Additionally, the introduction of the HCV subgenomic replicon assay in 1999, an in vitro viral replication system that allowed the evaluation of the cellular activity of investigational compounds, further bolstered drug discovery efforts (16). The hepatitis C virus is a single-stranded flaviviridae virus. Following infection, the virus circulates in the blood as a complex with lipoproteins, and enters the hepatocyte through interaction with a number of cell-surface receptors and subsequent endocytosis (17). The positive-stranded viral RNA is released into the cytosol and translated by cellular ribosomes into a single 3,000-amino acid polyprotein chain that contains all the structural and non-structural viral proteins required for replication. Cleavage of this polyprotein chain by cellular and viral proteases generates structural proteins Core, E1 and E2, as well non-structural proteins p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. The non-structural proteins assemble into the multi-protein replicase complex that is responsible for the production of progeny of HCV RNA. Final steps in the HCV lifecycle are the packaging and maturation of HCV RNA into infectious virus that is eventually released into the bloodstream (18, 19). 282 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 non-structural proteins NS3, NS4A, NS5A and NS5B are all essential to HCV RNA replication, and have been the focus of intense efforts aimed at discovering small molecule inhibitors of the HCV lifecycle (20). A number of small molecule DAAs targeting these proteins have been approved or are in development for HCV treatment (15). Telaprevir, approved by the FDA in 2011 (12, 13), targets the non-structural protein NS3•4A. NS3 is a bi-functional protein comprising a catalytic serine protease domain in its N-terminal portion and a helicase domain in its C-terminal portion. NS3 associates tightly and non-covalently with NS4A to form the NS3•4A complex. This complex is responsible for the cleavage of the viral polyprotein between NS3 and NS4A, NS4A and NS4B, NS4B and NS5A, and NS5A and NS5B (21–23). NS3•4A also plays a role in helping HCV evade the immune system through its cleavage of host innate proteins MAVS and TRIF, eventually suppressing interferon synthesis (24). Thus NS3•4A is an attractive target for therapeutic intervention. This chapter describes the discovery of the NS3•4A inhibitor telaprevir, as well as its chemical development and manufacture.

The Discovery of Telaprevir Publication of the crystal structure of the HCV NS3•4A protease in 1996 sparked intense research efforts in pharmaceutical companies toward the identification of inhibitors of this enzyme for therapeutic intervention (25, 26). As of today, more than a dozen of HCV NS3•4A protease inhibitors have entered the clinic, with a few being approved for the treatment of HCV. These are part of multi-drug regimens designed to suppress the emergence of resistance (14, 15). However, early on in the discovery process, the identification of a therapeutically useful inhibitor of this enzyme presented several challenges: •

• • •

while enzyme assays were quickly developed to drive medicinal chemistry efforts, there was initially no cellular assay available to establish compound activity in a cellular environment. there was no existing animal model to evaluate inhibitor efficacy in vivo and establish PK/PD relationships. crystal structures of the enzyme revealed a flat hydrophobic active site presenting few pockets where binding affinity could be gained. early high throughput screens to identify inhibitor starting points yielded no hits.

Hence the Vertex approach to enable the discovery of an HCV protease inhibitor relied on: •

developing and implementing enzymatic and cellular assays to drive compound optimization. Early on, the subgenomic replicon assay developed by Bartenschlager et al. was implemented (16). This assay 283

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•

was later supplemented by an infectious HCV replication assay in primary human hepatocytes and by an HCV protease mouse model that allowed the measurement of protease activity in the liver, thus enabling PK/PD evaluation (27). using a natural substrate of the enzyme as a starting point and utilizing structure-based drug design iteratively as an integral part of the optimization cycle.

An additional element of the strategy included the profiling of compounds in rodents early in the assay cascade to assess their exposure at the intended site of action—the liver. This approach was justified by the observation that upon liver transplantation, HCV infected patients underwent a dramatic drop in HCV RNA levels, suggesting that the liver was the primary site of HCV replication. Vertex Pharmaceuticals entered the HCV drug discovery field in 1997 in collaboration with Eli Lilly. Joint research efforts over the next three years resulted in the discovery of telaprevir, which has been published (28–30). A summary is presented in this chapter. Due to the lack of hits from high throughput screening efforts, decapeptide 1 (Scheme 1, Ki = 0.89 uM), derived from the natural NS5A-NS5B substrate of the NS3•4A protease, served at the starting point for inhibitor optimization. This decapeptide spanned the S6 to S4′ sites of the enzyme binding site. Early work focused on identifying the optimal inhibitor size that would provide enough binding affinity, with the goal of achieving a molecular weight range in-line with a drug-like profile. Truncation was also supported by the observation that the NS3•4A protease was inhibited by its own cleavage products (31, 32). Truncation studies showed that removal of the P4′ amino acid resulted in significant loss of activity, but that additional truncations at P2′ and P3′ had little additional negative effect on enzyme affinity. Also, removal of the acidic residues at P5 and P6 resulted in significant loss of activity. Furthermore, removal of the P3 and P4 hydrophobic residues was also deleterious to binding to the enzyme, suggesting that productive hydrophobic interactions between enzyme and inhibitor at these sites could be found (33). Taken together these data supported pursuing inhibitors spanning up to S4 on the non-prime side. To compensate for the resulting loss in affinity, a covalent reversible inhibitor containing an electrophilic warhead at the C-terminus was pursued—an approach supported by a number of publications in the literature (34). Reversible and covalent inhibitors of serine proteases have been shown to be 10- to 1,000-fold more potent than non-covalent inhibitors, and aldehydes are the simplest functionality that can serve as electrophilic moieties. Hence, aldehyde warhead incorporation and optimization of the P2 substituent to a proline-based residue, yielded inhibitor 2 (Scheme 1) (35). Replacement of the P5 and P6 residues with a heteroaryl cap yielded tetrapeptide aldehyde 3 (Ki = 12 uM) as a starting point for further optimization (35). X-ray analyses of aldehyde-containing HCV NS3•4A inhibitors show that the C-terminal electrophilic warhead undergoes nucleophilic attack by Ser139, resulting in the formation of a covalent, reversible bond between the protease and the inhibitor. The resulting tetrahedral complex is stabilized through additional 284

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ionic interactions with two additional residues, His57 and Asp81, which together with Ser139 form the catalytic triad (29).

Scheme 1. From Decapeptide Substrate 1 to Tetrapeptide Aldehyde 3 Both sequential and parallel explorations were conducted to optimize tetrapeptide aldehyde 3. The aldehyde warhead served as an excellent tool for the parallel synthesis of a number of inhibitors with variations at P1 and P2 (36). P1 Residue The S1 specificity pocket of the HCV NS3•4A protease is defined by the side chains of Leu135, Phe154 and Ala157, and is responsible for selectivity versus the clotting cascade of serine proteases such as thrombin. The consensus sequence for substrates resulting in trans cleavage all have a cysteine at P1, a nucleophilic residue incompatible with an electrophilic warhead. Examination of SAR at this position showed a preference for small hydrophobic side chains such as ethyl, propyl and trifluoroethyl. Polar atoms such as oxygen and disubstitution alpha to the aldehyde were not tolerated. P2 Residue The S2 pocket had been recognized as important for inhibitor recognition and potency, and incorporation of bulky P2 substituents had been shown to result in increased affinity. These findings repeated for this series of tetrapeptide aldehydes. 285

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For example, replacement of the benzyl ether in 3 with a tetrahydroisoquinolyl carbamate (4) led to a gain of about 13-fold due to optimized contacts of the inhibitor in the S2 pocket.

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From Aldehydes to α-Ketoamides While the aldehyde warhead was a great tool to explore SAR at various subsites of the molecule, the moiety was easily oxidized in vivo and hence needed to be replaced. Investigation of a number of warheads such as carboxylic acids, trifluoromethyl ketones, chloromethyl ketones, etc., all commonly used in serine protease inhibitors, met with failure. A key discovery was the finding that the aldehyde could be replaced with an α-ketoamide, which resulted in up to 40-fold improvements in binding affinity (37). X-ray structure analysis of α-ketoamides such as 5 (Scheme 2) showed an unexpected arrangement of the tetrahedral intermediate, where the oxyanion hole defined by Ser139 and Gly137 was occupied by the non-electrophilic carbonyl of the warhead, and the negatively charged oxygen was pointing out toward solvent (37). An additional ten-fold improvement in potency could be gained via the introduction of a carboxylic acid moiety on the prime side (compound 6, Scheme 2). However, the charged nature of this compound and related analogs prevented entry into the cell resulting in loss of cell potency in the replicon assay.

Scheme 2. Comparison of Aldehydes and Ketoamide Warheads 286 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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P2: From 4-Hydroxyprolines to 3-Alkylprolines α-Ketoamides based on 4-hydroxyproline at P2, such as 5 and 7 (Schemes 2 and 3), showed good potency against the enzyme. However, due to their high molecular weights, they were generally associated with poor metabolic stability and poor exposure in the liver—the target organ. A key advance toward telaprevir was the replacement of the 4-hydroxyproline residue at P2 with a 3-alkylproline residue. This finding originated with the structure-based design hypothesis that a proline-based P2 bearing a 1- to 4-carbon substituent at the 3-position on the α face could result in displacement of a putative water molecule in the crystal structure of compound 7 bound to HCV NS3•4A, potentially resulting in improved binding affinity (38). Compound 8 was prepared and showed a Ki of 1.4 µM. Subsequent crystal structure examination disproved the water molecule displacement hypothesis, yet the reasonable activity of 7 led to further exploration of P2 proline substituents at the 3-position (38). Further exploration of 3-alkylproline-based inhibitors was also supported by their improved PK profile and liver exposure. Final Optimization to Telaprevir Key compounds that led to the synthesis of telaprevir are shown in Scheme 3. Extension of the 3-methylproline substituent in 8 (Ki = 1.4 µM) to ethyl, and matrix exploration of substituents at P1′, P3 and P4, led to compounds with improved potencies, 9 (Ki = 0.22 µM) and 10 (Ki = 0.15 µM). The observed potency improvement in 9 led to the synthesis of bicyclic ketone 11 (Ki = 0.040 µM). Finally, reduction of the ketone moiety to the bicyclic carbocycle, coupled with the incorporation of optimal residues at P1′, P3 and P4 eventually led to telaprevir (Scheme 3) (39). Telaprevir was selected for advancement based on its potency and its good liver exposure in preclinical animal species (27–29).

Development of a Chemical Process for the Manufacture of Telaprevir Telaprevir is a polypeptide composed of four amino acid residues and a pyrazine cap. Scheme 4 shows a retrosynthetic disconnection. Retrosynthetic analysis by amide bond cleavages reveals residues B – E and the pyrazine carboxylic acid A as appropriate building blocks for the construction of telaprevir. Pyrazine carboxylic acid A, N-protected cyclohexylglycine B and N-protected t-butyl glycine C are available commercially, but D and E require development of a commercial process. Scheme 5 depicts the processing steps to manufacture telaprevir from the residues shown in Scheme 4. Apart from the manufacture of D and E, the process can be divided into four sections: • •

amide coupling and Cbz-deprotection to provide H-t-Leu dipeptide 14. a second amide coupling and Cbz-deprotection to provide H-Chg tripeptide 16. 287

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•

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•

capping the N-terminus of H-Chg tripeptide 16 with pyrazine carboxylic acid with subsequent t-butyl ester cleavage to give P-cap acid 18. amide coupling of P-cap acid 18 with 20 followed by oxidation of the hydroxyamide 19 to provide telaprevir.

Scheme 3. Final Optimization Toward Telaprevir

This section of the chapter will discuss the activities required to develop the manufacturing route in Scheme 5, as well as a brief discussion of the processes to manufacture bicycloproline ester 12 and 20 (40–43).

Manufacture of Bicycloproline Ester 12 The initial manufacture of the key starting material 12 began with the reaction sequence shown in Scheme 6. Conversion of t-butyl glycine to the chiral imine with the unnatural enantiomer of camphor gave imine ester 21. A stereoselective Michael addition of imine ester 21 to cyclopentenyl ester 22 proceeded in low yield but gave Michael adduct 23 in high diastereomeric and chemical purities. Deprotection with hydroxylamine gave lactam 24, which was protected and then reduced with borane to give Z-protected bicycloproline ester 25 in high 288 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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enantiomeric purity. Removal of the Cbz group and crystallization as the oxalate salt provided bicycloproline ester 12 in 10 steps with high enantiomeric and chemical purities, and manageable yield. While this process was fit-for-purpose to manufacture up to 100 kg, we had tremendous interest in developing an alternative route to lower the cost of manufacture and to reduce material sourcing risks, especially for the unnatural enantiomer of camphor.

Scheme 4. Retrosynthetic Analysis of Telaprevir

Scheme 5. Processing Route for the Manufacture of Telaprevir 289 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 new process (Scheme 7) utilized a stereoselective lithiation/carboxylation sequence (44). In a fashion similar to Beak’s stereoselective α-lithiation of Boc-protected amines (45–47), our in-house-developed achiral ligand dipropylbispidine (DPBP) (see Scheme 7) induced excellent diastereoselectivity to provide the exo isomer in 95:5 diastereoselectivity. Direct, same-pot resolution with (S)-THNA (see Scheme 7) and recrystallization provided 27 (S)-THNA salt in >99.5:0.5 e.r. and d.r. Further processing of 27 (S)-THNA salt required the following:

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1. 2. 3.

conversion to the t-butyl ester with Boc2O, DMAP, and t-BuOH. chemoselective removal of the Boc group with MsOH/THF. crystallization of 12 as the oxalate salt.

The process was accomplished in an overall yield of 27% from Boc-protected 3-azabicyclo[3.3.0]octane 26 (based on total molar charge of 26), and was used for commercial manufacture of metric tons of bicycloproline ester 12.

Scheme 6. Initial Process for Bicycloproline Ester 12

Manufacture of Aminoalcohol 20 The development of a process for manufacture of aminoalcohol 20 used an epoxidation and subsequent nitrogen nucleophilic ring-opening protocol to establish the correct relative stereochemistry (Schemes 8 and 9). Racemic epoxidation of trans-2-hexenoic acid was performed with Oxone™ to give 290 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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racemic trans-epoxide 28, which was converted to the corresponding racemic epoxy amide 29 (Scheme 8). Enantioselective epoxidation of hexenoic amide 30 was accomplished the Shibasaki protocol (48) to give trans-epoxyamide 31 in high yield and high enantiomeric purity. Ring-opening of epoxy amide 29 with benzylamine followed by deprotection gave racemic aminoalcohol rac-20 (Scheme 9), which was subsequently resolved to give aminoalcohol 20 in high enantiomeric purity. Using the identical ring-opening/deprotection strategy for trans-epoxyamide 31 gave aminoalcohol 20 in equally high enantiomeric purity, without the loss to a resolution process.

Scheme 7. Commercial Route to Bicycloproline Ester 12

Scheme 8. Synthetic Routes to trans-Epoxy Amides 29 and 31 291 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 9. Synthetic Routes to Aminoalcohol 20

Development of a Process for the Manufacture of Dipeptide H-t-Leu Dipeptide 14 from Bicycloproline 12 The first stage for the manufacture of the polypeptide chain of telaprevir comprises the coupling of Cbz-protected tert-leucine (Z-t-Leu-OH) with 12 to afford Z-t-Leu dipeptide 13, and subsequent hydrogenolysis of the Cbz group to give H-t-Leu dipeptide 14. Cbz-protected tert-leucine was obtained commercially as the dicyclohexylamine (DCHA) salt, and 12 was obtained as the oxalate salt by custom commercial manufacturing, vide supra. In this section the development of the commercial manufacturing process shown in Scheme 10 is described. The first operations for development were the salt breaks to afford solutions of 12 and Z-t-Leu-OH in the same solvent. Next, amide coupling to manufacture Z-t-Leu dipeptide 13 was improved with a particular focus on identifying the best way to remove the excess Z-t-Leu-OBt ester from the reaction mixture. Finally the conditions for the Cbz removal and isolation of dipeptide H-t-Leu dipeptide 14 were screened which ultimately led to a decision to telescope a solution of H-t-Leu dipeptide 14 into the next stage rather than isolate H-t-Leu dipeptide 14 as a solid. 292

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Scheme 10. Commercial Manufacturing Process for H-t-Leu Dipeptide 14

Optimization of 12 Oxalate Salt Break A DoE study was setup to optimize the amount of K2CO3 and water used in the 12 oxalate salt break. Choosing the factors shown in Table 1 with their corresponding ranges, and setting a limit of 1% loss of 12 to the aqueous layer gave an acceptable operating range. In this study, conditions at 3 equivalents K2CO3 and 3 volumes of water minimized the loss of 12, but precipitated a high level of salts that complicated the phase separation. However, increasing the amount of water to four volumes kept all salts in solution with minimal loss of 12 to the aqueous phase. A water wash was used to ensure removal of dissolved base in the isopropyl acetate (i-PrOAc) organic phase. 293 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. Factor Ranges in DoE Study of 12 Oxalate Salt Break K2CO3 (equiv)

Water (vol)

Time

% 12 in aqueous layer

1

5

15 min

11.9

3

3

3h

0.3

1

3

20 min

5.1

3

5

1h

2.2

In the course of investigating the stability of the i-PrOAc solution of 12, the N-acetyl impurity 32 formed when stored at room temperature for several days. Figure 1 shows the rate of growth of 32 in an i-PrOAc solution of 12 at various temperatures. Based on these results, recommendations were made to store the i-PrOAc solution of 12 at room temperature for less than two days, or at lower temperature for a longer time, if necessary.

Figure 1. Stability of 12 in i-PrOAc.

Optimization of the Coupling Work-up Process In the previous procedures, histamine•2HCl as well as a 25% K2CO3 solution were utilized to remove the excess Z-t-Leu-OBt ester from the reaction mixture after the formation of Z-t-Leu dipeptide 13. Histamine is efficient, but considering its toxicity (49), a replacement was needed for commercial manufacturing. Potassium carbonate is inexpensive and nontoxic. However, we noticed that increased levels of impurities in the manufacturing of Z-t-Leu dipeptide 13 were related to the incomplete quenching of the activated ester by the 25% K2CO3 wash. This was also complicated by poor detection of the activated ester by HPLC analysis, which was solved by derivatization with benzylamine. 294 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 series of nitrogen-based nucleophiles was screened to quench the excess activated ester in the reaction solution. The combination of an amine base, Et3N or N-methylmorpholine (NMM), and amino acids, L-glycine, L-lysine, or L-cysteine, were examined. Based on efficiency of the reaction (complete conversion of HOBt-activated ester Z-t-Leu-OBt) and the time required for completion and commercial availability, L-lysine (1 equiv) with NMM (2 equiv) was chosen as the quench reagent combination. The mono-Lys adduct 33 (Scheme 11) was completely removed from the organic phase after the K2CO3 and HCl aqueous solution washes, and bis-Lys adduct (34) typically remained at 0.1 - 0.5 %.

Scheme 11. Amide Products from L-lysine/NMM Quench

Optimization of CBz-removal from Z-t-Leu Dipeptide 13 To Manufacture H-t-Leu Dipeptide 14 Catalyst Loading The legacy process for the hydrogenation step used a 10 wt% loading of Pd(OH)2/C as catalyst. A lower catalyst loading was investigated in order to improve the process efficiency on a production scale. The study, ranging from 10 wt% to 1 wt% catalyst loading, was complicated by the deleterious effect of residual acetyl impurity 32 on the reaction efficiency. With typical levels of acetyl impurity 32 at ~0.5% in the plant, experiments demonstrated 8 wt% catalyst loading was optimal. Unfortunately, lower loadings did not provide an efficient reaction.

Stability of H-t-Leu Dipeptide 14 After completion of the hydrogenolysis, and work-up, the process required a solvent switch from i-PrOAc to n-heptane. The stability of H-t-Leu dipeptide 14 in i-PrOAc at various temperatures was investigated (Figure 2). Two impurities, diketopiperidine 35 and N-acetyl dipeptide 36 (Scheme 12) were formed when the H-t-Leu dipeptide 14/i-PrOAc solution was stored at various temperatures for a certain period. To limit formation of these impurities, a solvent swap temperature of ≤ 40 °C was chosen. 295 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 12. Impurities from Decomposition of H-t-Leu Dipeptide 14 in i-PrOAc

Figure 2. Stability of H-t-Leu Dipeptide 14 in i-PrOAc Solution at Various Temperature. In summary, a scalable and robust isolation process for H-t-Leu dipeptide 14 was developed. H-t-Leu dipeptide 14 was taken forward to the next step as a solution in i-PrOAc.

Development of Manufacturing Process for GMP Starting Material H-Chg Tripeptide 16 from H-t-Leu Dipeptide 14 Scheme 13 shows the process for the conversion of H-t-Leu dipeptide 14 to H-Chg tripeptide 16. However, the legacy process used DMF as solvent for the amide coupling (H-t-Leu dipeptide 14 to Z-Chg tripeptide 15). The optimization of this sequence therefore started with adjusting the protocol for charging the amide coupling reagents to the reactor because H-t-Leu dipeptide 14 was now being introduced as a solution in i-PrOAc rather than a solid. Other key developments included an improved workup procedure to completely quench the activated esters after the amide coupling, and development of the crystallization 296 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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of H-Chg tripeptide 16. A number of stability, and spike and purge experiments were also performed to verify that tripeptide H-Chg tripeptide 16 could be obtained with a consistent purity profile because it is the designated GMP starting material for telaprevir.

Scheme 13. Conversion of H-t-Leu Dipeptide 14 to H-ChgTripeptide 16

Optimization of the Process to Synthesize Z-Chg Tripeptide 15 Optimization of the EDCI/HOBt Coupling Optimization studies began with a screen of various conditions and equivalents of reagents, resulting in the following procedure (Scheme 13) : 1. 2.

3. 4.

A reactor is charged with EDCI•HCl (1.05 equiv), HOBt•H2O (1.05 equiv), and NMP (2.5 vol). This suspension is cooled to 0 °C. A solution of Z-Chg-OH (1.05 equiv) in NMP (3.0 vol) is then added to the cooled suspension, maintaining the internal temperature at 0 ± 5 °C. After stirring for 1.5 h, the reaction mixture is treated with a solution of H-t-Leu dipeptide 14 (1.0 equiv) in i-PrOAc (10 vol), at 0 ± 5 °C. Once the addition is complete, the mixture is warmed to 20 ± 5 °C, after which the reaction is stirred for 5 - 17 h. 297

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Compound Z-Chg tripeptide 15 is stable to the reaction conditions if left overnight, and complete conversion was obtained in these instances.

Development of a New Method for Quenching Z-Chg-OBt

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In light of the impurity issues associated with activated ester Z-t-Leu-OBt, a derivatization method for Z-Chg-OBt was developed using benzylamine to generate the corresponding benzyl amide 37 (Scheme 14). This provided a means to accurately analyze the amount of Z-Chg-OBt remaining at reaction completion.

Scheme 14. Derivatized Product from Z-Chg-OBt With an improved analytical method in hand, a more efficient quench of the Z-Chg-OBt was investigated to avoid generation of the Chg-dimer impurity 38 from reaction with the deprotected amine H-Chg tripeptide 16 (Scheme 15).

Scheme 15. Impurity Resulting from Incomplete Quench of Z-Chg-OBt The original process used histamine in order to quench any unreacted activated esters of Z-Chg-OH, rendering them as water-soluble histamine amides. However, due to health issues surrounding histamine (49) as well as incomplete quenching of the activated ester, another work-up procedure was sought. A variety of reagents was examined. Screening experiments were carried out as follows: the coupling of Z-Chg-OBt and H-t-Leu dipeptide 14 to form Z-Chg tripeptide 15 was performed, followed by treatment of the reaction mixture with an amine base (Et3N or NMM) and/or an amino acid. The most desirable trapping combination would rapidly generate the desired amide, which would be stable to hydrolysis and readily be removed in the subsequent aqueous washes. L-Lysine and L-cysteine were capable of efficiently trapping the activated ester, but emulsions formed during basic, acidic, and neutral aqueous washes. However, 298

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glycine (1.0 equiv) with NMM (2.0 equiv) efficiently trapped Z-Chg-OBt as glycyl amide 39 (Scheme 16) and no epimerization of Z-Chg tripeptide 15 was observed. An additional water wash was incorporated in order to avoid a thick residue forming on the walls of the vessel.

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Scheme 16. Glycine Adduct of Z-Chg-OBt Optimization of the workup procedure showed that after a single wash with 5% K2CO3, a second wash with 25% K2CO3 was needed to effectively quench Z-Chg-OBt, and a subsequent acid wash (1N HCl) was required to remove the urea by-product formed from the EDCI reagent, as well as any unreacted H-t-Leu dipeptide 14 and other basic impurities. A final water wash was included in order to remove any excess acid present before beginning the hydrogenolysis of the Cbz group. Hydrogenolysis proceeded to completion in less than 35 min rather than the previously typical 1.5 h, suggesting that the additional water wash may provide an opportunity to further reduce the catalyst loading from 5 wt % Pd(OH)2/C. Final isolation and purification of H-Chg tripeptide 16 was achieved by solvent exchange to n-heptane and crystallization. The robustness of the crystallization process was verified by confirming the ability to purge intermediates Z-t-Leu dipeptide 13, H-t-Leu dipeptide 14, Z-Chg tripeptide 15, and impurities 40-44 (Scheme 17) from a spiked crude i-PrOAc solution of H-Chg tripeptide 16. Intermediate Z-Chg tripeptide 15 and ethyl ester 44 were the only two compounds that were not completely purged in the spike/purge studies (0.08 and 0.15%, respectively). Implementing the solvent exchange and n-heptane crystallization process during manufacture provided H-Chg tripeptide 16 in 85% recovery and 100% purity. Development of a Process for the Manufacture of P-cap Acid 18 From H-Chg Tripeptide 16 Chemical Development of the Conversion of H-Chg Tripeptide 16 to P-cap Acid 18 The initial process for converting H-Chg tripeptide 16 to P-cap acid 18 is a two-step process which required precipitation of the desired product by addition of water to a DMF solution (Scheme 18). The major disadvantage of this approach was that P-cap ester 17 filtered poorly in the plant and was very time-consuming to dry. So, the initial focus of the commercial process development was to telescope the steps directly from H-Chg tripeptide 16 to P-cap acid 18. 299 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 17. Structures of Spiked Impurities for Purging Studies of H-Chg Tripeptide 16

Scheme 18. Original Process for Conversion of H-Chg Tripeptide 16 to P-cap Acid 18 300 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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Once the most process-friendly set of reaction conditions (reagents, solvents, catalysts) were established for the formation of P-cap acid 18 from H-Chg tripeptide 16, the limits of the process were defined. In particular, a process characterization was performed and the acceptable limits of impurities were established. In order to evaluate the impact and safe limits of impurities in the starting material or product, a series of spike and purge experiments were performed. Impurities were spiked at the start of the process for P-cap acid 18. The spiked and residual impurities were tracked throughout the normal operation of the process and are described in the subsections below.

Solvent and Coupling Reagent Selection A qualitative solvent screen for the conversion of H-Chg tripeptide 16 and P-cap acid 18 in solvents of medium to high polarity which were likely to be compatible with the reaction conditions was performed initially. P-cap acid 18 had low solubility in most of the solvents examined except for, which provided good solubility and was expected to be inert to strong acid. However, due to the high solubility of P-cap ester 17 in CH2Cl2, and the low likelihood of crystallization, development of a telescoped process from H-Chg tripeptide 16 to P-cap acid 18 was pursued. The solubility studies also identified toluene as the preferred solvent for crystallization of P-cap acid 18. The next area of development was the activation of the pyrazine carboxylic acid as its acid chloride. We prepared a sample of the pure acid chloride by sublimation to afford a colorless solid, but discoloration occurred after a week. This prompted the decision to generate the acid chloride in situ and use it immediately. The initial screening studies for the telescoped acid chloride formation and coupling indicated that oxalyl chloride gave a consistently homogenous solution, the best conversion and the least discoloration. A solvent-dependent impurity was seen in the reactions with oxalyl chloride, with CH2Cl2 showing minimal impurity formation. Attempts with EDCI/HOBt or CDI as the coupling agent produced results inferior to oxalyl chloride. A final study to examine the preferred amine base showed NMM to be superior to i-Pr2NEt. Optimization of the reaction stoichiometry was pursued next. An initial screen showed that 1.4 equivalents of pyrazine carboxylic acid activated with 1.2 equivalents of oxalyl chloride in the presence of 5.0 equivalents of NMM produced the cleanest conversion to P-cap ester 17. Subsequent development of the reaction showed formation of the acid chloride was rapid (