Complete accounts of integrated drug discovery and development : recent examples from the pharmaceutical industry 9780841233966, 0841233969, 9780841234314, 0841234310, 9780841233980

Table of contents :
Content: ForewordPreface1. Discovery and Development of GDC-0994: A Selective and Efficacious Small Molecule Inhibitor of ERK1/22. Preclinical Identification and Development of AM-6138: An Inhibitor of BACE1 for the Treatment of Alzheimer's Disease3. Discovery and Chemical Development of Verubecestat, a BACE1 Inhibitor for the Treatment of Alzheimer's Disease4. Discovery and Chemical Development of JNJ-50138803, a Clinical Candidate BACE1 Inhibitor5. Discovery and Development of Ruzasvir: An Investigational Next Generation Pan-Genotype HCV Nonstructural Protein 5A (NS5A) Inhibitor for the Cure of Hepatitis C Virus Infections6. AZD6564, Discovery of a Potent 5-Substituted Isoxazol-3-ol Fibrinolysis Inhibitor and Development of an Enantioselective Large-Scale Route for Its Preparation7. Design and Development of the Glucokinase Activator AZD16568. Development of a Reversible P2Y12R Antagonist, AZD1283-Discovery Research and Large-Scale Chemistry9. Discovery, Process Development, and Scale-Up of a Benzoxazepine-Containing mTor InhibitorEditors' BiographiesAuthor IndexSubject Index

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Complete Accounts of Integrated Drug Discovery and Development: Recent Examples from the Pharmaceutical Industry Volume 1

ACS SYMPOSIUM SERIES 1307

Complete Accounts of Integrated Drug Discovery and Development: Recent Examples from the Pharmaceutical Industry Volume 1 Ahmed F. Abdel-Magid, Editor Therachem Research Medilab, LLC, Chelsea, Alabama, United States

Jaan A. Pesti, Editor EnginZyme AB, Stockholm, Sweden

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

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, issuing body. | American Chemical Society. Division of Organic Chemistry, sponsoring body. Title: Complete accounts of integrated drug discovery and development : recent examples from the pharmaceutical industry / Ahmed F. Abdel-Magid, editor, Therachem Research Medilab, LLC, Chelsea, Alabama, United States, Jaan A. Pesti, editor, EnginZyme AB, Stockholm, Sweden, Rajappa Vaidyanathan, editor, Bristol-Myers Squibb, Bangalore, India ; sponsored by the ACS Division of Organic Chemistry. Description: Washington, DC : American Chemical Society, [2018-] | Series: ACS symposium series ; 1307 | Includes bibliographical references and index. Identifiers: LCCN 2018048857 (print) | LCCN 2018050283 (ebook) | ISBN 9780841233966 (ebook) | ISBN 9780841233980 (print) Subjects: LCSH: Drug development. | Pharmaceutical industry. | Pharmaceutical technology. Classification: LCC RM301.25 (ebook) | LCC RM301.25 .C66 2018 (print) | DDC 615.1/9--dc23 LC record available at https://lccn.loc.gov/2018048857

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 © 2018 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

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.

ACS Books Department

Contents Foreword .......................................................................................................................... ix Preface .............................................................................................................................. xi 1.

Discovery and Development of GDC-0994: A Selective and Efficacious Small Molecule Inhibitor of ERK1/2 ...................................................................... 1 Xin Linghu, Nicholas Wong, James F. Blake, John J. Gaudino, and John G. Moffat

2.

Preclinical Identification and Development of AM-6138: An Inhibitor of BACE1 for the Treatment of Alzheimer’s Disease .............................................. 29 Steven M. Mennen, John Stellwagen, and Ryan D. White

3.

Discovery and Chemical Development of Verubecestat, a BACE1 Inhibitor for the Treatment of Alzheimer’s Disease ............................................................ 53 David A. Thaisrivongs, William J. Morris, and Jack D. Scott

4.

Discovery and Chemical Development of JNJ-50138803, a Clinical Candidate BACE1 Inhibitor ................................................................................. 91 Harrie J. M. Gijsen, Jinguang Lin, and Yannis Houpis

5.

Discovery and Development of Ruzasvir: An Investigational Next Generation Pan-Genotype HCV Nonstructural Protein 5A (NS5A) Inhibitor for the Cure of Hepatitis C Virus Infections ..................................................... 115 Ling Tong, Joseph A. Kozlowski, Louis-Charles Campeau, and Jingjun Yin

6.

AZD6564, Discovery of a Potent 5-Substituted Isoxazol-3-ol Fibrinolysis Inhibitor and Development of an Enantioselective Large-Scale Route for Its Preparation ........................................................................................................... 151 Staffan Karlsson, Daniel Pettersen, and Henrik Sörensen

7.

Design and Development of the Glucokinase Activator AZD1656 .................. 185 Darren McKerrecher and Alan Steven

8.

Development of a Reversible P2Y12R Antagonist, AZD1283—Discovery Research and Large-Scale Chemistry ................................................................ 221 Fredrik Zetterberg and Carl-Johan Aurell

9.

Discovery, Process Development, and Scale-Up of a BenzoxazepineContaining mTor Inhibitor .................................................................................. 249 James W. Leahy, Sriram Naganathan, Denise L. Andersen, Neil G. Andersen, and Stephen Lau

vii

Editors’ Biographies .................................................................................................... 287

Indexes Author Index ................................................................................................................ 291 Subject Index ................................................................................................................ 293

viii

Foreword For every drug that is sold on the market, there will be dozens of other drugs that failed somewhere in development, most often as the benefit/risk was simply not good enough to warrant commercialization. Every drug, but also all those which do not crown their development with commercialization, have a story. It starts with a biological hypothesis on the cause for a disease, the development of biological assays, and after the identification of the first hits, the development of these to leads. Then comes the further evolution of the leads to candidates and then finally their progression through the many phases of the development. And when all obstacles are overcome, ultimately we attain commercialization to serve the patient. Every step of the way is complex and involves the interplay of many disciplines, making the discovery and development of a new drug into the biggest and most challenging “science experiment” one can imagine. Each phase of the genesis of a new drug requires the combination of significant scientific insight with the ability to find the creative solutions to the problems and challenges that threaten to derail the process at every step of the way. Many scientific disciplines are involved, but it is the synthetic chemist that is the essential factor. A highly remarkable and insightful description of the uniqueness of chemistry amount the sciences was already suggested in the 19th century by the French scientist Marcellin Berthelot: “La chimie crée son objet. Cette faculté créatrice, semblable à celle de l’art lui-même, la distingue essentiellement des sciences naturelles et historiques.” (“Chemistry creates its object of study. Such a creative power is analogous to the power of art; it essentially distinguishes chemistry from natural and historical sciences.”) Marcelin Berthelot, La synthèse chimique Alcan, Paris, 1887 It clearly describes the central role that we play as chemists. We are the scientists that create. We are not just descriptive and find ourselves with an insurmountable problem, but we have the means to design the solution. This is the same creative process that gives us art, but we chemists are providing a beautifully conceived and prepared organic molecule. The beauty of the creative solution is a molecule with the right properties which ultimately provide the indispensable tool that cures a disease. And the complexity of the creative task, from initially designing the molecule to actually being able to make it safely and economically in large amounts, is covered and described for several examples ix

in this book. The topics encompass drugs that are covering the whole gamut of diseases that inflict mankind: they reach from infectious diseases, systemic disorders, over pain and CNS diseases to various metabolic malfunctions. The long list of diseases clearly demonstrates the strong need for better medicines, created and made by organic chemists. Anybody reading the book will realize the central role that organic chemistry plays in the discovery and development of new drugs. As organic chemists we should be proud of the central role of our science and hope that that the message of Berthelot from 1887 is being recognized again.

Kai Rossen Editor Organic Process Research & Development

x

Preface

As for yin and yang, the relationship between Drug Discovery and Process Chemistry in the pharmaceutical industry is both interdependent and complementary. Neither can produce viable pharmaceuticals in reasonable times and quantities without the other. Discovery Chemists produce small quantities of identified lead drug candidates. While these quantities are enough for early testing and selection, the development process would remain impractical without enough substance to first test the concept in human clinical trials and eventually treat the patients who need them worldwide. The skills of Process Chemists would not be of use with no guidance as to what to make. The fusion of these two major functions transfers the drug discovery from theory to application and reality; it helps produce nearly all the pharmaceutical entities known today. The eventual production or screening of candidates for a pharmaceutical activity begins with the Discovery Chemists. Organic synthesis has progressed dramatically since the pioneering work of Friedrich Wöhler and the synthesis of the first organic molecule: urea. Thanks to innovation both in academia and industry, today’s organic chemists have ever-growing arrays of reactions, reagents, catalysts and techniques at their disposal to handle the synthesis of increasingly complex drug candidates. The skills of organic chemists in drug discovery and development have contributed significantly to the advancement of the pharmaceutical industry and have helped introduce new effective life-preserving medicines. Indeed, most drugs found in the modern pharmacy arise from synthetic origins, entirely or partially. Nearly all drugs that populate the contemporary pharmacopeia required the skills of Discovery Chemists to prepare the first few grams. When a drug candidate displays efficacy and shows a promising improvement over the existing therapies, the expertise of process chemists is brought to bear to render the synthesis safe, scalable, and economical. A good example is Gilead’s Harvoni,® a drug that comprises two discrete active molecules (ledipasvir and sofosbuvir) with different mechanisms of action used to treat hepatitis C. Gilead’s scientists expended much effort to discover the efficacy of this paired drug, as well as to devise reasonable means to develop a scalable synthesis. As a result of these intense efforts, a serious previously uncontrolled disease is now curable. xi

The advent of advanced, ubiquitous computational tools to identify active sites and binding principles has led to the evolution of structure-based rational drug design concepts in Discovery Chemistry. Very exciting is the emergence of CRISPR technology – a number of companies have been established that may exploit this new knowledge to produce the next generation of drugs. Discovery Chemistry has as its raison d'être: the identification and synthesis of new drugs that improve upon existing therapies, and treat unmet medical needs. While the initial synthesis developed by the Discovery Chemists may be capable of producing hundreds of grams of a drug candidate with minimal tweaks, it is generally not designed to be the most synthetically efficient or economically viable approach. Similarly, Process Chemists have been at the forefront of innovating new technologies to efficiently assemble complex molecules, and have utilized advanced tools to gain a better appreciation of the ‘reaction space’ around a particular chemical transformation. The use of advanced computing and automated reaction arrays permits the examination of a significantly wider scope of reaction conditions. An interesting manifestation of this ability is the understanding of ‘cliffs’ – areas in the reaction space where a small change in a parameter (e.g. temperature) will cause a large change in the yield or purity. Needless to say, a robust process would not exist near such a cliff where a small inadvertent change might result in an unfortunate loss of yield or purity! Process Chemistry’s central role is to produce high quality drug substances at commercially acceptable costs on multi-ton scales in an environmentally responsible manner. The chapters in this book are written to accentuate the interdependency and synergy between drug discovery and process development disciplines to advance a new chemical entity into clinical trials and eventually to the market. Due to the success of our previous books (Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage Process Development, Volumes 1 and 2), we sought to further arm experienced modern synthetic organic chemists, and budding researchers perhaps still in universities with real world examples from the pharmaceutical industry. In addition, 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 hundreds of millions of patients and alleviate their suffering, improve their quality of life and possibly save their lives from diseases and disorders. The chapters presented in this book are written by a selected group of outstanding, highly 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 xii

these drug candidates on large scale. The story leading up to the discovery of each drug or drug candidate is presented by the Discovery Chemist(s), and then the Process Chemist(s) describe the development of the same drug to give the reader a complete story of drug discovery and development. The reader will experience a rare and unique opportunity to obtain 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 fewer limits on relevant details. Our special thanks to all the authors who are acknowledged in the chapters listed below. All have volunteered their efforts and time to sculpt this book. Their willingness to contribute, the demanding work expended in the writing, and the result at the finish bear witness to their outstanding contributions to this book. Some made this book their second experience with us editors. We cannot say enough about them. Xin Linghu of Ideaya Biosciences, Nicholas Wong and John G. Moffat of Genentech, and James F. Flake and John J. Gaudino of Array BioPharma, describe the discovery and synthesis of GDC-0994, a selective ERK1/2 inhibitor for the treatment of human cancers. The biological rationale for targeting ERK1/2, which is part of the Ras/Raf/MEK/ERK (MAPK) signal transduction pathway is discussed. Steven M. Mennen of the Department of Process Research, and John Stellwagen and Ryan D. White of the Department of Medicinal Chemistry, of Amgen describe the drug discovery effort to identify inhibitors of the β-site amyloid precursor protein cleaving enzyme-1 (BACE1) for the treatment of Alzheimer’s disease. They describe the discovery and development of AM-6138, a candidate for preclinical development. David A. Thaisrivongs and William J. Morris of the Process Research and Development Department, and Jack D. Scott of Discovery Chemistry, of Merck & Company discuss the discovery and development of verubecestat, a potent and selective BACE1 inhibitor that has been evaluated in late-stage clinical studies as a potential disease-modifying therapy for the treatment of Alzheimer’s disease. Ling Tong and Joseph A. Kozlowski of Discovery Chemistry, and LouisCharles Campeau and Jingjun Yin of Process Research & Development, of Merck and Company discuss the discovery, and early/late development of ruzasvir, a potent next generation HCV nonstructural protein 5A (NS5A) inhibitor with pangenotype activity that suppresses HCV replication. Ruzasvir displays optimized in vitro activity against known clinically relevant HCV genotype 1 resistanceassociated substitutions (RAS). Staffan Karlsson of Early Chemical Development in in Pharmaceutical Sciences, and Daniel Pettersen and Henrik Sörensen of Medicinal Chemistry, Cardiovascular, Renal and Metabolic Diseases, both of AstraZeneca’s IMED Biotech Unit, report the discovery and scale up of AZD6564 - an oral fibrinolysis inhibitor for treatment of heavy menstrual bleeding. It was discovered by focusing on improving potency, permeability and selectivity towards GABAA activity. xiii

Darren McKerrecher of Medicinal Chemistry, Oncology, IMED Biotech Unit, AstraZeneca and Alan Steven of Pharmaceutical Technology & Development, AstraZeneca discuss AZD1656, a potent, selective glucokinase activator for the treatment of Type II diabetes. The synthesis of the coupling partner for the SNAr reaction involved a highly selective decarboxylation and the control of impurities arising from the use of azetidine as a coupling partner. Downstream challenges included an amidation reaction with a pyrazine coupling partner made using a Curtius rearrangement performed in batch mode, and the development of crystallization conditions that allowed the reproducible isolation of a slow-growing polymorph of the API. The processes developed were tested as part of a 500 kg manufacture of AZD1656. Fredrik Zetterberg of Galecto Biotech AB and Carl-Johan Aurell of AstraZeneca R&D of Pharmaceutical Sciences, Early Chemical Development, Large Scale Chemistry, describe the selection of AZD1283 as a P2Y12R antagonist for clinical development. This was expected to be a novel treatment for thrombosis and related disorders as a backup candidate for the now marketed AstraZeneca drug ticagrelor. Discussed as well are the synthetic development efforts starting with the early procedure for the first few milligrams to the development of a multi kilogram synthesis. James W. Leahy of the Department of Chemistry at the Florida Center of Excellence for Drug Discovery & Innovation at the University of South Florida, Sriram Naganathan at Dermira Inc, Denise L. Andersen at Cytokinectics Inc, Neil G. Andersen at Achaogen and Stephen Lau at Gilead Sciences Inc relate work they performed while at Exelixis Inc. Out of a family of highly potent and selective inhibitors of a mammalian target of rapamycin, the lead compound (XL388) exhibited low nanomolar activity and >1000-fold selectivity over related PI3K kinases. This compound, chosen as a potential oral treatment of cancers, was rapidly scaled up to enable first-in-human studies. Harrie J.M. Gijsen and Yannis Houpis of Janssen Research & Development, Janssen Pharmaceutica NV and Jinguang Lin of STA Pharmaceuticals discuss the discovery and development of a BACE1 inhibitor, hypothesized to be a potential disease-modifying treatment for Alzheimer’s Disease. The need for a next generation of BACE1 inhibitors has led to the discovery of JNJ-50138803. The initial medicinal chemistry synthesis is presented, as well as the evolution to more scalable synthesis routes, incorporating multiple improvements. This has culminated into a synthesis route, proven suitable to prepare a multikilogram GMP batch. We are eternally grateful to this group of scientists who authored these chapters and whose willingness to tackle this difficult task, who contribute countless hours of writing and rewriting, and whose pharmaceutical/chemical/ engineering expertise allowed the creation of this book. We hasten to add that a Volume 2 with additional pharmaceutical stories in this genre will follow in 2019. We would be remiss is we did not acknowledge the many referees who made creative suggestions for improvement and correction. We must thank our colleagues at ACS Books who encouraged and facilitated the compilation of this book: Elizabeth Hernandez, Sara Tenney, Amanda Koenig and Arlene Furman. Special thanks goes to Kai Rossen whose foreword elegantly states the xiv

interlocking importance of pharmaceuticals and organic chemistry, and whose role as the Editor-in-Chief of Org. Process Res. Dev. has contributed significantly to the advancement of process chemistry. Finally, we thank the Division of Organic Chemistry of the American Chemical Society for their sponsorship of our biennial symposium from which this book sprung.

Ahmed F. Abdel-Magid Therachem Research Medilab, LLC Chelsea, AL 35043, USA

Jaan A. Pesti EnginZyme AB Stockholm 114 28, Sweden

Rajappa Vaidyanathan Bristol-Myers Squibb Bangalore 560099, India

xv

Chapter 1

Discovery and Development of GDC-0994: A Selective and Efficacious Small Molecule Inhibitor of ERK1/2 Xin Linghu,1 Nicholas Wong,*,2 James F. Blake,3 John J. Gaudino,3 and John G. Moffat2 1Ideaya

Biosciences, 7000 Shoreline Ct Suite 350, South San Francisco, California 94080-4990, United States 2Genentech Inc., 1 DNA Way, South San Francisco, California 94080-4990, United States 3Array BioPharma Inc, 3200 Walnut Street, Boulder, Colorado 80301, United States *E-mail: [email protected]

This chapter describes the discovery and synthesis of GDC-0994, a selective ERK1/2 inhibitor for the treatment of human cancers. The biological rationale for targeting ERK1/2, which is part of the Ras/Raf/ mitogen-activated extracellular signal-related kinase kinase (MEK)/extracellular-signalregulated kinases (ERK) mitogen-activated protein kinase (MAPK) signal transduction pathway, is discussed. We give a brief overview of the medicinal chemistry efforts that led to of the discovery of GDC-0994 and the original synthetic sequence. From the original medicinal chemistry synthesis, we address key limitations to further increase stereoselectivity and yields and the determination of the active pharmaceutical ingredients (API) final form to support Phase 1 clinical development.

Rationale for the Pursuit of Inhibitors of ERK1/2 The mitogen-activated protein kinase (MAPK) signal transduction pathway, mediated by sequential activation of the Ras, Raf, mitogen-activated extracellular signal-related kinase kinase (MEK), MAPK and extracellular-signal-regulated kinases (ERK) proteins, is a critical network controlling normal and cancer cell © 2018 American Chemical Society

proliferation. Constitutive activation of the MAPK pathway by oncogenic Ras or Raf mutations, or by overexpression of growth factors/growth factor receptors, is frequently observed in tumor types such as colon, lung, pancreatic, ovary, and kidney (1). Thus, different nodes of this pathway have attracted significant interest as a therapeutic target for cancer (2). Small molecule inhibitors of B-Raf and MEK have shown promising activities in the clinic for a variety of solid tumors. The approval of Raf inhibitors vemurafenib (3) and dabrafenib (4), alone and in combination with MEK inhibitors trametinib (5) and cobimetinib (5), as treatments for BRAF(V600E)-mutant driven metastatic melanoma validate the approach of targeting the MAPK pathway as an effective way of treating cancer. Beyond this indication, a much larger range of MAPK-dependent cancers have yet to be successfully treated with inhibitors of this pathway, for several reasons. First, due to the incompletely understood mechanistic complexity of the Raf kinase isoforms, inhibition of nonmutant Raf in the context of K-Ras mutation results in a “paradoxical activation” (6) of growth signals rather than inhibition. Second, single-agent MEK kinase inhibitor inhibition has not produced robust or durable clinical efficacy in K-Ras-mutant tumors, probably due to compensating feedback activation of the pathway. And last, as is the case for many oncogene-addicted types of cancer, a strong selective pressure for acquired resistance to single agents and inhibition pathway results in many different mechanisms of resistance, resulting in relapse. There is therefore a strong rationale for drugs to inhibit this critical pathway at multiple nodes, whether sequentially or simultaneously. For that reason, we and others have been pursuing discovery (7, 8) and development of inhibitors of ERK1 and ERK2. These two kinases, which share 89% sequence identity and are thus regarded for the purpose of drug development as equivalent and interchangeable, provide the central effector node upon which all the upstream activating mechanisms converge. Once activated, ERK1/2 phosphorylates serine/threonine residues of more than 50 downstream substrates and activates both cytosolic and nuclear proteins that are responsible for cell growth, proliferation, survival, angiogenesis, and differentiation, all hallmarks of the cancer phenotype (9–11). The utility of ERK inhibitors is exemplified by previous studies in which researchers at Genentech and Merck independently reported that dual inhibition of MEK and ERK by small molecule inhibitors acted to overcome acquired resistance to MEK inhibitors, providing further rationale for developing selective ERK1/2 inhibitors (7, 8). A more recent study also showed that combined MEK and ERK inhibition results in synergistic antitumor activity in preclinical models of k-Ras-mutant cancers (12). Thus, clinical evaluation of a potent and selective ERK inhibitor has been an important goal.

Discovery of GDC-0994 Our high throughput screening (HTS) campaign identified 1 (Figure 1) as an ATP competitive ERK1/2 inhibitor with good kinase selectivity and favorable physicochemical properties (13). Structure-based optimization of 1 led to a series of potent and selective inhibitors, as exemplified by 2 (13). Proof-of-concept 2

(POC) studies were carried out in nude mice bearing subcutaneous HCT116 colorectal cancer xenografts. Multiple doses of 2 were administered bis in die (BID, twice a day) by oral gavage (150 mg/kg), and resulted in tumor growth inhibition of 69% (95% colony inhibition [CI] (30, 88)) at 28 days. Although 2 demonstrated the necessary POC, it also possessed high clearance and low oral bioavailability in multiple species, a profile shared by most of the analogs from this series (13).

Figure 1. Structure of HTS lead and initial optimized compound. Reproduced with permission from ref. (14). Copyright 2015 American Chemical Society. The high clearance and low oral bioavailability were attributed the oxidative metabolism of the benzylic C-5 and C-8 positions on the piperidinopyrimidine ring. In addition to high clearance, 2 exhibited low permeability, limiting its oral absorption. These inherent pharmacokinetic (PK) liabilities of the piperidinopyrimidine urea template prompted us to search for an alternative motif. Utilizing structure-based design, we discovered 3 (Figure 2) (14). Given its combination of activity, selectivity, and mouse pharmacokinetics, 3 emerged as a promising lead. Additionally, the kinase selectivity profile of 3 showed no kinase inhibition (>70%) other than ERK1 and ERK2 (170 kinases screened at a test concentration of 100 nM).

Figure 2. Structure of pyridone core 3. Reproduced with permission from ref. (14). Copyright 2015 American Chemical Society. 3

Xenograft studies of 3 demonstrated significant antitumor activity. Dosed orally (PO) twice a day in nude mice bearing HCT116 colorectal cancer xenografts for 21 days, the 75 mg/kg dose produced significant tumor growth inhibition (70%) and the treatment was well tolerated. Body weight losses were within an acceptable range (95% since we obtained 4 in 96% e.e. (determined by chiral column chromatography). The absolute configuration of 4 was determined by X-ray crystallographic analysis, indicating that the N-alkylation to produce 14 occurred with clean inversion (Scheme 1). With a reliable supply of sulfide 14 available, oxidation with m-chloroperbenzoic acid (CPBA) resulted in the desired sulfone 15 allowing for an SNAr reaction with methyl-pyrazole-amine 16 with NaH in DMF at 150 °C to form 17. Purification via chromatography to remove impurities allowed access to clean protected GDC-0994 (4). Subsequent deprotection of the tertbutyldimethylsilyl (TBS) group with hydrochloric acid (HCl) allowed for the isolation of GDC-0994 freebase as an amorphous solid and facilitated material delivery for critical toxicological studies.

Scheme 1. Medicinal chemistry preparation of 4 (GDC-0994). Adapted with permission from ref. (14). Copyright 2015 American Chemical Society.

Process Development of GDC-0994 GDC-0994 cleared several internal milestones enabling resourcing for full process development to facilitate API supply for further preclinical and clinical studies. The original medicinal chemistry route was examined and contrasted with novel routes with different bond disconnection strategies. Ultimately, we decided to maintain the bond disconnection strategy, focusing on the optimization of key transformations. The medicinal chemistry route (Scheme 1) suffered from several drawbacks that needed to be addressed to ensure a robust manufacturing process for GDC-0994. Our goal was to 1) Determine the criticality of the tedious Soxhlet extraction in the purification of the Suzuki-Miyaura cross-coupling product and determine alternatives if required 5

2) Introduce an efficient and environmentally benign asymmetric synthesis of chiral diol, avoiding the use of osmium and further improving enantiomeric excess 3) Improve the regioselectivity of pyridone SN2 reaction to reduce purification burden and maximize material throughput 4) Develop a more atom economical, safe, and scalable sulfide oxidation 5) Understand and minimize impurity formation in the endgame process 6) Identify an appropriate polymorph of API for further formulation development 7) Completely eliminate the use of chromatographic purification through the development of streamlined isolations of key intermediates and API

Development of Kumada-Corriu Cross-Coupling The aryl-aryl bond formation between pyrimidine-bromide 5 and pyridine 6 was initially achieved via a Suzuki-Miyaura cross-coupling (Scheme 2) in reasonable yields. However, after hydrolysis to form 8, the subsequent SN2 reaction of crude 8 with 13 resulted in only 10% conversion after refluxing in THF for 30 h (Table 1, entry 1). A lengthy and cumbersome Soxhlet extraction was implemented to purge impurities present in crude 8 and restored previously observed conversion in the SN2 reaction (Table 1, entry 2). Comparing crude 8 by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) to a purified authentic sample revealed no significant differences to account for the loss in conversion. Further analysis by inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed and revealed that the boron content was reduced from 3000 ppm to 700 ppm after the Soxhlet extraction. This suggested that unknown boron-containing inorganic impurities may have had a detrimental effect on the downstream chemistry. Although the conversion could be improved by a dramatic increase in reaction temperature of crude 8 with 13 (Table 1, entries 3 and 4), the incomplete understanding and potential manufacturing liabilities associated with the Suzuki-Miyaura coupling were determined to be an undesired risk.

Scheme 2. Suzuki cross-coupling employed in the discovery chemistry synthesis. Adapted with permission from ref. (18). Copyright 2017 American Chemical Society. 6

Table 1. Effect of Purity of 8 on SN2 Reactivity

To address the concerns with the reproducibility of a Suzuki cross-coupling for the manufacture of biaryl 7, a Kumada-Corriu coupling mediated by iron, nickel, and palladium catalysts was envisioned as a viable alternative circumventing the detrimental formation of inorganic boron salts (19–25). (Scheme 2). 4-Pyridylmagnesium chloride 19 was generated through a magnesium-iodide exchange protocol employing iPrMgCl•LiCl (26). The Kumada-Corriu cross-coupling reaction was then initiated by introducing 19 into a mixture of 20 and a selected catalyst. A brief catalyst screen indicated that the Pd-catalyzed cross-coupling reactions resulted in overall better conversions and cleaner reaction profiles when compared to other transition metals (Table 2) and allowed for the substitution of the cheaper pyrimidine-chloride 20 for the previously used pyrimidine-bromide 5. The catalyst was then further narrowed to PEPPSI-IPr (Scheme 3), and process development to optimize other parameters was initiated.

Table 2. Catalyst Screen of Kumada-Corriu Cross-Couplinga,b Entry

Catalyst

% Conversion of 20

HPLC A % of 7

1

Fe(acac)3

500 MW, high hydrogen bond donor count and polar surface area (PSA)], which resulted in poor exposure in the CNS. Amgen design efforts to improve CNS exposure led to hydroxyethylamines (HEAs), exemplified by 1, capable of robust CNS exposure and Aβ reduction after oral dosing (Figure 1) (8). Unfortunately, advancement of HEAs was limited due to challenges in achieving favorable absorption, distribution, metabolism, excretion, and toxicological (ADMET) profiles in preclinical studies. Subsequent hit identification efforts employed a fragment-based drug design (FBDD) approach that led to aminoquinolines (2) (9). These designs utilized an amino-heterocycle to engage the catalytic aspartate residues of BACE1, and represented a novel approach in moving away from prior peptidomimetics; however, advancement was ultimately hindered by poor pharmacokinetic properties. 30

Figure 1. Select BACE1 inhibitor scaffolds investigated at Amgen leading up to AM-6138.

Improved success in balancing potency and CNS exposure was realized with aminooxazoline xanthenes (AOX), which also utilized an amino-heterocycle to engage the catalytic aspartates (10, 11). A candidate for preclinical development was ultimately identified from this series (3, AM-8718) but was subsequently shown to produce an adverse retinal pathology in preclinical toxicology studies. This finding was characterized by the accumulation of autofluorescent granules in the retinal pigmented epithelium (RPE) layer of the retina and was analogous to that observed by Lilly with LY2811376 (12). This pathology halted the clinical development of LY2811376 and was shown to be unrelated to BACE1 inhibition based on similar retinal findings with BACE1-knockout mice dosed with LY2811376. With uncertainty around translatability of these findings to humans, we also regarded this finding as a hazard that needed to be avoided and paused the development of AM-8718. We hypothesized that inhibition of cathepsin D (CatD), a lysosomal aspartyl protease structurally similar to BACE1, was the off-target driver of the observed RPE effects in rats (13). CatD is an important enzyme for normal phagolysosomal function in the RPE. Interestingly, while AM-8718 showed only modest inhibition in a biochemical CatD assay (IC50 = 2.3 μM), and unbound plasma exposures in preclinical safety studies failed to achieve meaningful coverage of this IC50, it is well-known from prior studies of cathepsin inhibitors that biochemical assays can underestimate relevant cellular activity (14). The often-large difference between biochemical and cellular IC50 is attributed to enrichment of basic compounds in the acidic lysosome compartment (i.e., lysosomotropism) that is observed only with an intact cellular or in vivo system. By considering the basicity of AM-8718 and the relatively low pH of the lysosome (pH 4–5) where CatD is expressed, we were able to account for theoretical levels of compound enrichment in the acidic compartment (i.e., lysosomotropism) and estimate a more relevant level of in vivo CatD inhibition 31

compared with the biochemical CatD assay. This approach showed a strong correlation between unbound plasma levels in toxicology studies and RPE effects for not only AM-8718, but also several other analogs from diverse series (15). Therefore, immediate design efforts were focused on maximizing intrinsic CatD selectivity. A later report from Pfizer described chemoproteomic studies that firmly implicated CatD inhibition to be the key off-target and supported our earlier conclusions (16). Engaging the S3 subpocket of BACE1 can deliver significant CatD selectivity by exploiting a key residue difference compared with BACE1 (specifically, Asp318 of CatD versus Ala335 in BACE1) (17). Starting from AM-8718 (3), we first examined the impact of incorporating a larger picolinamide P3 group to occupy the S3 pocket in combination with the xanthene core as a way of further improving CatD selectivity. Analogs were screened at a high concentration in our biochemical CatD assay (up to 400 μM) given the underprediction of cellular activity. We successfully identified potent BACE1 inhibitors with much improved biochemical CatD selectivity compared with AM-8718 (e.g., >10,000-fold CatD/BACE1 IC50), exemplified by AOX 4 (18). Importantly, 4 was shown to avoid retinal effects while affording robust Aβ lowering in rats. This result further substantiated our design approach to avoid effects in the retina by maximizing CatD selectivity. Unfortunately, the xanthene picolinamides generally showed low oral bioavailability that limited in vivo exposure and moderate hERG inhibition that limited cardiovascular safety. The relatively high MW and PSA in this series, coupled with the inability to improve bioavailability and hERG while maintaining robust CNS exposure, ultimately prevented advancement of the series. As a result, we sought a smaller, more ligand-efficient scaffold to more easily improve ADME properties while maximizing CatD selectivity. Truncation of the xanthene core to a smaller fluorophenyl ring, as originally described by Shionogi (19), and optimizing the warhead amino-heterocycle led to aminothiazine dioxides (5). Initial analogs showed promising properties as a flexible scaffold to balance potency, CNS exposure, and off-target activities (20).

Discovery of AM-6138 Aminothiazine dioxide 5 afforded significant CatD selectivity (>100,000fold), attributed to the P3 picolinamide exploiting the key residue difference in the S3 subpocket (Table 1). Although the aminothiazine dioxides generally showed robust inhibition of Aβ production in HEK293 cells expressing BACE1 and APP and good metabolic stability in liver microsomes, the series suffered from high P-glycoprotein (P-gp) mediated efflux ratio (ER >3). The aminothiazine dioxides consistently demonstrated modest CNS exposure in rats that was likely limited by P-gp, because it is the primary efflux transporter at the blood–brain barrier. The P-gp efflux was considered partially driven by the relatively high PSA of the sulfone moiety and corresponding basicity (pKa = 7.0), which was determined by potentiometric titration. 32

Table 1. Warhead Modifications

In addition, examination of multiple aminothiazine dioxide cocrystal structures with BACE1 revealed that, although the warhead nitrogens efficiently engaged the catalytic aspartates Asp93 and Asp289, the aminothiazine ring could adopt two binding conformations, with the sulfone puckered either toward or away from the S1 pocket. Therefore, we examined several warhead modifications to sterically shield the sulfone and/or moderate the pKa in a way that could also introduce additional conformational rigidity as a means of further improving the properties. We theorized that a [3.2.1] bridged-bicyclic warhead, formed by an ethylene linker connecting the carbons on either side of the sulfone, would fill the spaces occupied by the two puckered conformations and may shield the polar sulfone group. Two diastereomeric [3.2.1] bridged-bicyclic warheads were synthesized (compounds 8 and 9) and evaluated. Fortuitously, one isomer (8) demonstrated improved BACE1 potency, CatD selectivity, and metabolic stability. The P-gp 33

efflux ratio of 8 was considered high (i.e., >3), so we sought to further moderate the warhead pKa as a means of improving efflux, while retaining adequate basicity for desirable BACE1 biochemical and cellular potency. Introduction of a fluorine to either of the neighboring methyl groups resulted in decreased basicity (10, pKa = 6.0; 11, pKa = 6.3) compared with the unsubstituted warhead 8 (pKa = 7.1). Fortunately, the decreased basicity of 11 was accompanied by both improved permeability and reduced P-gp efflux without sacrificing significant BACE1 potency. The least basic isomer (10) showed decreased enzyme potency with a slightly higher cell shift. Given the favorable potency and efflux of 11, this warhead was selected to be combined with P3 amide optimization efforts. Analysis of BACE1 cocrystal structures with analogs related to 11 revealed the S3 binding pocket to be relatively narrow, formed between the 10s loop and Gly291, where the amide NH and the nitrogen ortho to the carboxamide interact with the backbone of Gly291. Analogs lacking either of these nitrogens were shown to lose >100-fold BACE1 potency. As previously discussed, given the key residue difference at the back of the S3 pocket (CatD Asp318 versus BACE1 Ala335), we aimed to exploit this steric and polarity difference by maintaining a substituent para to the carboxamide to create a disfavorable interaction with CatD and increase selectivity. We sought analog designs with modestly increased polarity in the heteroaryl ring to maintain or improve metabolic stability and lipophilic efficiency while minimizing efflux. Additionally, P3 group exploration from prior series structure–activity relationship (SAR) revealed an additional methyl substituent ortho to the carboxamide could add potency and minimize potential amide bond cleavage in vivo. Representative analogs are shown in Table 2. Pyridyl amides (11, 12, and 13) and pyrazinyl amides (6 and 14) showed near single-digit nanomolar potency in the BACE1 enzyme and cell assays and excellent selectivity against CatD. Although BACE1 potency could be improved with 14 by extending deeper into the S3 pocket, higher microsomal turnover accompanied the increased lipophilicity. Compounds with the best BACE1 cell potency, selectivity over CatD, and low P-gp efflux were evaluated in a pharmacodynamic (PD) assay using naïve SpragueDawley rats to assess Aβ40 reduction in the CSF and brain after oral dosing. For initial screening, CSF and brain Aβ40 levels were measured at a single timepoint (4 h) after dosing to increase assay throughput and provide rapid prioritization of compounds based on unbound plasma exposure at 4 h, and the corresponding Aβ40 levels compared with untreated control animals. In this assay, 6 gave 76% reduction of CSF Aβ and 66% reduction in the brain Aβ after a 10 mpk oral dose, corresponding to an estimated unbound plasma EC50 consistent with time-course PD studies of 66 nM. Importantly, no effects on the RPE layer of the retina of rats were observed after 4 days of treatment with AM-6138 (6), where average unbound plasma exposures achieved >150-fold the rat PD EC50 for effect in the brain. Given these results and favorable cardiovascular safety and PK properties, AM-6138 was selected as a candidate for preclinical development.

34

Table 2. P3 Structure–Activity Relationship

Medicinal Chemistry Route to AM-6138 For the initial medicinal chemistry route, a stereoselective approach was utilized that was designed to enable facile exploration of substitutions at both the [3.2.1] bridged-bicyclic warhead and the P3-substituent. The retrosynthetic analysis is shown in Scheme 1. Disconnection of the amide bond allows coupling of P3 acids with the P1 aniline to enable rapid screening of the S3 space through late-stage amidation. Assembly of the warhead relied on fragment 18 and a sulfone, where the critical benzylic chiral center was envisioned to be formed via a stereoselective alkylation of a chiral tert–butanesulfinimine (21). In addition, substitutions on the bicyclic warhead can be accessed through variation of the pendant groups on the sulfone (R-group).

Scheme 1. Retrosynthetic analysis to facilitate SAR studies. 35

Execution of the medicinal chemistry route is described in Schemes 2–4. Starting from commercially available 2-(methylsulfonyl)acetonitrile (15), allylation in the presence of potassium carbonate followed by condensation with formaldehyde and Deoxo-Fluor®-mediated fluorination of the resulting alcohol provided racemic methylsulfone 16 in 45% yield over three steps. Enantiomerically pure tert–butanesulfinimine 18 was prepared by condensation of (S)-2-tert–butanesulfinamide with commercially available acetophenone 17 in the presence of titanium tetraethoxide. For the stereoselective addition of the sulfone anion to the sulfinamide, a solution of 18 in toluene was cooled to –40 °C, activated with trimethylaluminum, and slowly added to a solution of the lithium anion of methylsulfone 16, generated in situ by addition of a slight excess of n-butyllithium at –78 °C. After workup and column chromatography, the desired adduct 19 was obtained in 80% yield as a 1:1 mixture of the diastereomers arising from the racemic sulfone, which could not be separated by normal-phase chromatography.

Scheme 2. Subunit synthesis and condensation. After removal of the tert–butanesulfinamide auxiliary with HCl, the aminothiazine dioxide ring was formed by treatment with copper chloride to effect intramolecular cyclization of the benzylic amine with the nitrile to form an amidine (Scheme 3). The amidine was then protected with two Boc groups, to provide intermediate 20 in good yield. The pendant allyl group was subsequently converted to an iodoethyl group via a three-step procedure. First, the olefin was converted to an aldehyde through ozonolysis at –78 °C, followed by treatment with triphenylphosphine. The resulting crude aldehyde was reduced to the corresponding alcohol with borane-THF at 0 °C. This two-step conversion to the alcohol was necessary to prevent side reactions observed with attempts to directly access the alcohol from a reductive workup of the ozonolysis. For example, use of sodium borohydride led to a significant impurity resulting from migration of one of the Boc groups from the amidine to the resulting alkoxide. After borane reduction, the hydroxyl group was converted to the iodide under standard conditions. At this point, the diastereomers could be separated by column chromatography to provide the desired isomer 21 in 45% yield over four steps. Formation of the [3.2.1] bridged-bicyclic warhead was accomplished by deprotonation at the carbon adjacent to the sulfone with lithium 36

hexamethyldisilazide at –78 °C and displacement of the primary iodide. The nitro group was then reduced in good yield to form aniline 22.

Scheme 3. Assembly of the [3.2.1] bridged-bicyclic warhead.

The synthesis was completed by HATU-mediated coupling of the aniline nitrogen to the pyrazine-acid 23, followed by deprotection of the Boc groups with TFA to reveal 6 in good yield. Overall, this 15-step route provided 6 in approximately 7% yield from commercially available starting materials and supported delivery of 8.5 g for 4 day rat toxicology and profiling studies.

Scheme 4. Completion of initial AM-6138 synthesis. 37

Interface between the Medicinal and Process Chemistry Groups To Expedite First-in-Human (FIH)-Enabling Toxicology Lot Delivery Several factors drove a desire to accelerate the program development timeline, including a compelling biological target rationale, the grievous impact of AD, the high therapeutic potential of a BACE1 inhibitor, and an intensely competitive landscape. To expedite the program, the process chemistry team closely interfaced with the discovery team during late lead optimization. As confidence emerged around a lead scaffold, based primarily on promising SAR and favorable shortterm screening toxicology results, the joint team aimed to: (1) Accelerate delivery of material to be used for candidate selection toxicology studies; and (2) Increase familiarity with the route to facilitate moving directly into clinical manufacturing. Transitioning a discovery program quickly and directly into clinical development required a close collaboration and focused on the most significant synthetic route liabilities instead of de novo process development for the first multikilogram delivery. The collaborative team was set up wherein our discovery scale-up and development (DSD) group first evaluated the medicinal chemistry route during the production of multigram (1–10 g) lots for screening toxicology studies. This allowed for the process chemistry group to quickly focus on issues critical to potential pilot plant tech transfer, where the DSD scale-up could then utilize any updated processes identified by the process group to supply any additional lots prior to the final tech transfer and production run. Retrosynthetic analysis of 6 was designed to ensure synthetic flexibility to accommodate potential changes to the P3 group, and to ensure that the process itself could be safely scaled without going through high-energy intermediates (Scheme 5). The allyl group used in the original discovery route was changed to a cinnamyl group to install a chromophore in the event that chiral column chromatography would be needed. This installation of an aryl group fortuitously afforded solid intermediates instead of oils, which simplified isolations on large scale. Evaluation of the intermediates used throughout the initial discovery route quickly highlighted that the presence of the aryl nitro throughout the synthesis resulted in multiple high-energy and potentially explosive intermediates (Scheme 5). For example, analysis by differential scanning calorimetry indicated that 24a had an exothermic decomposition greater than 1.6 kJ/g and was predicted to be shock-sensitive. Because of these concerns, the route was redesigned to start with aryl bromide 18b and incorporate an ammonia surrogate at a later stage of the synthesis. This change eliminated high-energy intermediates and increased the flexibility to accommodate potential changes from the ongoing medicinal chemistry efforts.

38

Scheme 5. Retrosynthetic analysis of AM-6138.

Process Chemistry Route to AM-6138 The forward synthesis began by condensation of cinnamaldehyde 26 and nitrile 27 followed by conjugate reduction with sodium borohydride (Scheme 6). Aldol reaction of 28 with formaldehyde provided the racemic alcohol 29 which could be further transformed into the key alkyl fluoride 25 in the presence of DAST. Attempts to synthesize intermediate 25 in an asymmetric fashion were all met without success. Enzymatic resolutions of both alcohol 29 and alkyl fluoride 25 were evaluated with various lipase and nitrilase enzymes. Extensive screening of enzymes and process conditions all suffered from significant retro-aldol reaction or lack of reactivity, respectively. An asymmetric synthesis of 25 was highly desired; however, given the time pressure of the program, we instead elected to carry the racemic alkyl fluoride to a point where it could be resolved by either chromatography or, more preferably, by crystallization.

Scheme 6. Synthesis of racemic alkyl fluoride fragment 25.

39

The two fragments were coupled by addition of the lithiated sulfone of 25 to tert–butanesulfinimine 18b at cryogenic temperature (Scheme 7). Scale-up of the discovery process proved to be challenging due to the cryogenic requirements. The original process required two cryogenic reactors where sulfone 25 was lithiated in the first reactor and the tert–butanesulfinimine was complexed with trimethylaluminum in a separate reactor. Cold transfer of the tert–butanesulfinimine complex into the lithiated sulfone completed the desired coupling to 24b. The process performed as expected on 3 kg scale, affording 74% yield; however, scale-up to 50 kg scale in the same reactor train resulted in >30% of an unknown impurity. Root cause analysis first identified the impurity as 31, which came from dimerization of tert–butanesulfinimine 18b. Careful analysis of the batch historian data revealed that the increased scale required a prolonged addition time to complex trimethylaluminum with 18b compared with the 3 kg batch. Reproduction of the 50 kg batch historian profile in a programmable automated lab reactor returned a comparable poor purity profile as seen in the 50 kg batch.

Scheme 7. Minimization of undesired condensation during anion coupling process. With the failure of the large batch, the team needed to quickly identify a fit-for-purpose solution to ensure that neither the preclinical toxicology studies nor the clinical timeline of the program were jeopardized. Because close collaboration with the discovery and process teams had been ongoing to enable just-in-time process development, we were able to rapidly design around the instability of 18b with the required trimethylaluminum activator. The lithiated anion of 25 was found to be stable at cryogenic temperatures for >24 h. The stability of 25 allowed us to leverage the cryogenic reactor with the highest cooling capacity and directly add trimethylaluminum to the lithiated sulfone. The resulting complex was stable for >24 h at temperatures below –50 °C and began to degrade as the temperature increased. The updated process was simple to control by setting a temperature limit during the addition of trimethylaluminum to the lithiated sulfone as not to 40

exceed –60 °C, followed by the addition of 18b to the cryogenic reactor. Discovery and process chemistry teams first demonstrated the modified process at laboratory scale and then directly transferred to the pilot plant for a 5 kg demonstration batch. The single cryogenic reactor process delivered 24b in three batches of 13 kg and an average 90% isolated yield, which was a significant improvement over the original 74% yield. By the simple concept of forming the trimethylaluminate of 25 initially instead of with 18b and combining them, we engineered around the instability of the aluminate of 18b and enabled a high-yield coupling. Over 80 kg of coupled intermediate 24b were manufactured as a 1:1 mixture of diastereomers resulting from racemic sulfone 25 (Scheme 7). Chromatography was originally used during discovery efforts, but the identification of crystalline intermediates was a major focus area for development due to the expense and slow throughput of multikilo-scale chromatography. Because there were several intermediates that have a basic amine present, we felt this could be an opportunity to separate the diastereomers as the corresponding salts. Examination of pharmaceutically acceptable acids did not identify a salt pair capable of diastereomer separation (22) and led us to utilize an extensive screen of sulfonic acids to identify crystalline intermediates. Our screen of sulfonic acids successfully identified two separate crystalline salts capable of the purification of 32, as well as the separation of diastereomers (33/34) by simple recrystallization (Figure 2).

Figure 2. Sulfonic acid screening kit. Symbol ○ indicates no solids observed; symbol ● indicates crystalline solids present. Typically executed on 0.2 mmol/vial scale with either 2 solvents per substrate (2×12 format) or as shown with 2 substrates (1×12 format). Thus, deploying two successive sulfonic acid salts used in series allowed us to avoid chromatography in the downstream process and ultimately resulted in a significant improvement in the campaign timeline. Deprotection 41

of tert–butanesulfinimine 24b with HCl followed by salt formation with p-biphenylsulfonic acid (PBSA) provided 32 as a 1:1 mixture of diastereomers, which was purified by crystallization (Scheme 8). Cyclization of 32 with methanesulfonic acid (MSA) afforded a 1:1 mixture of two diastereomeric salts (33 and 34) that had sufficiently different solubility in dichloromethane to allow selective crystallization of diastereomer 33. The procedure was executed successfully on large scale to provide a solution containing 30.9 kg of 33 in 47% yield and 97.8:2.2 d.r.

Scheme 8. Cyclization and separation of amidine diastereomer intermediates 33 and 34.

Further elaboration of the styrene olefin to form the [3.2.1] bridged-bicyclic warhead required a sequence of ozonolysis and reduction to the primary alcohol 38 to set up for a later cyclization. The ozonolysis/reduction sequence presented a significant challenge for scaling due to the hazardous nature of ozonolysis intermediates and substrate-specific challenges for reduction to alcohol 38. The ozonolysis was carefully studied to understand how the process progressed and what in-process controls would be required to ensure the reaction was complete. As shown in Scheme 9, the ozonolysis process was designed such that there was a participating solvent (methanol) to drive the intermediates from the primary ozonide 36 to methoxy hydroperoxide 37 as described by Kula, which could be more readily reduced than the precursor secondary ozonide (23).

42

Scheme 9. Ozonolysis and reduction process to deliver primary alcohol 38.

The design of the ozonolysis process was based on the hypothesis that ozone reacts instantly with olefin 35. Therefore, upon detection of ozone in the reactor, the reaction is complete and the system could then be purged of residual ozone and oxygen to safely sample for conversion. This hypothesis was studied on 5 L scale, wherein a ReactIR was used to follow conversion and an ozone detector was placed at the effluent of the reactor to detect when ozone was no longer being consumed (Figure 3). Generation of ozone was conducted with air diluted with nitrogen to stay below 5% oxygen concentration (24).

Figure 3. General equipment setup for ozone process; scrubber employed for production scale.

43

As shown in the lab scale trial, ReactIR indicated the generation of intermediate 37 and the simultaneous consumption of 35 (Figure 4). During this time, the ozone detector measured nondetectable levels of ozone. At approximately 440 min, the ReactIR trend plateaued, and ozone was detected in the effluent at low ppm levels. The generation of ozone was stopped and the reactor was purged with nitrogen before sampling to confirm reaction completion. This process design was then transferred to the plant and was safely executed on approximately 2×20 kg scale, using an explosion-proof ozone detector and a scrubber in the output of the reactor (25). As can be seen in the ozone trace, ozone was continuously consumed until the point where the increase in ozone appeared (Figure 5). At this time, generation of ozone was paused and the reaction was purged for safe sampling on plant scale and demonstrated completion of the ozonolysis portion of the process.

Figure 4. Normalized ReactIR trends from 5 L ozone process. + = 35; ▪ = 37. Quenching of the methoxy hydroperoxide 37 required careful control due to the nature of the substrate. Under high and low pH conditions, the intermediate aldehyde 39 was converted to the unsaturated aldehyde 40 through a retro conjugate addition (Scheme 10). Careful control of pH and temperature were critical for clean reduction to alcohol 38 while preventing migration of the Boc group to the primary alcohol to afford 41, which could not be readily processed, to the desired product. Attempts to use common reductants, such as triphenylphosphine or dimethylsulfide, resulted in a challenging isolation. As a result, we elected to perform a direct hydride reduction of the methoxy hydroperoxide 37 to the desired alcohol 38 using sodium triacetoxyborohydride [Na(OAc)3BH] because the by-products could be readily purged in the isolation. 44

Figure 5. Inline analysis of production scale ozonolysis. × = ozone production in kilograms; ● = analysis of unconsumed ozone at reactor exit in kilograms per hour.

Scheme 10. Prevention of undesired side reactions during ozonolysis workup. To ensure a tight process control, a portionwise addition of sodium triacetoxyborohydride was chosen. We monitored the progress of the reaction by ReactIR and observed that first addition of sodium triacetoxyborohydride was the most exothermic (Figure 6). This informed our plant scale production to break down the additions of sodium triacetoxyborohydride portions into eight charges, which increased in size as the reaction progressed to ensure tight temperature control in the production reactor. The ozonolysis sequence and sodium triacetoxyborohydride reduction delivered over 40 kg of alcohol 38 in 80% yield for the combined three-step process from 33. 45

Figure 6. Normalized ReactIR trends from 5 L reduction of alkoxy hydroperoxide 37. ▪ = 37; ▴ = 38; × = Na(OAc)3BH.

The formation of the [3.2.1] bridged-bicyclic warhead was performed on mesylate 42 instead of iodide 21, used in medicinal chemistry, to avoid the challenges of removing the triphenylphosphine oxide by-product by crystallization (Scheme 11). On production scale, it was observed that mesylate 42 was prone to elimination to 45 and side reaction with THF to 46 and ultimately 47 during solvent swap and azeotropic drying. The side-products were mitigated by concentration of DCM to low volume followed by addition of cold THF, instead of a complete solvent swap to THF at elevated temperatures. Deprotonation of 42 with LiHMDS smoothly generated 43. Aqueous workup and Boc deprotection afforded 13.7 kg of bromide 44, which served as a key intermediate to stage material for future deliveries.

Scheme 11. Formation of [3.2.1] bridged-bicyclic warhead. 46

The final steps of drug substance manufacturing required amination to access aniline 22. As describe previously in Scheme 5, the process had been redesigned to proceed through the aryl bromide instead of the aryl nitro to avoid high-energy intermediates. Aniline 22 could be directly accessed by a copper-catalyzed azide addition to 44 followed by trimethylphosphine-mediated reduction of aryl azide 48 (Scheme 12). Hazard analysis of the azidation process by accelerated rate calorimetry (ARC) identified a potential thermal runaway reaction initiating at 90 °C with an adiabatic temperature rise (ATR) of >100 °C. Because the 90 °C thermal onset temperature was so close to the reaction operating temperature of 50 °C, the priority was directed toward identifying alternative processes that avoided azide intermediates instead of attempting to engineer a safe process for handling 48 or to identify a nonpyrophoric alternative to trimethylphosphine.

Scheme 12. Cu-catalyzed azidation of aryl bromide 44. To bypass the azidation process in Scheme 12, we first evaluated C-N coupling techniques for ammonia (26, 27) and ammonia surrogates (28, 29). Benzophenone-imine and trifluoroacetamide were selected as the most viable surrogates based on minimal formation of the des-bromo product 49 (Scheme 13) (30). Palladium-catalyzed cross-coupling of both ammonia surrogates provided 22 (Paths 1 and 2); however, the trifluoroacetamide process was preferred due to the ease of removal of the trifluoroacetate by-product by crystallization.

Scheme 13. Metal-catalyzed cross-coupling of ammonia surrogates. Using trifluoroacetamide as a surrogate for ammonia proved to be efficient and the by-products could be easily removed by crystallization. Due to the propinquity of the C-N coupling to the drug substance, the copper-catalyzed process (Path 3) was selected over palladium (Path 2) to minimize the risk of palladium metal contamination. The process itself was fully developed to ensure minimal amount of by-product 49 formed due to the difficulty in its removal in downstream processing. In our specific case, we observed that a 1:1 ratio of ligand to catalyst minimized 49 to 0.6%, whereas a 2:1 ratio of ligand to catalyst 47

resulted in 4.5% of 49. An additional factor we observed was that minimizing the content of water in the reaction was key to ensuring low levels of 49. The C-N coupling was executed on kilo scale controlling side-product 49 to less than 0.5% (Scheme 14). Removal of residual copper was completed along with the simultaneous hydrolysis of trifluoroacetamide 50 by quenching with aqueous EDTA, followed by removal of the organic solvent. The drug substance was synthesized by amide bond formation between amine 22 and carboxylic acid 23. T3P® was chosen as the dehydrating reagent because it allowed for downstream isolation of high-quality material, although alternative coupling reagents, such as HATU, ECI, and so forth, also provided the desired product.

Scheme 14. Synthesis of AM-6138 methanesulfonic acid salt. Extensive form and salt-screening experiments were carried out in an effort to identify the appropriate solid form of the API. An anhydrous free form was initially targeted, but the compound formed solvates with all the solvents examined. Because the chemistry, manufacturing, and control (CMC) strategy targeted a solution formulation for early clinical trials, a salt form was also acceptable. The MSA salt was selected over other salts because it did not form solvates, and the solubility was sufficient to formulate as a solution for oral dosing. The synthetic process was completed to deliver 6 in 74% yield, which was converted to 6•MSA in 76% isolated yield. The overall process from materials commercially available in bulk quantities required 20 synthetic steps and afforded 6.6% isolated yield. This first delivery afforded 4.4 kg of 6•MSA in 98.7% purity by HPLC area percentage, 99.3% w/w, and contained 460 ppm residual copper.

Conclusions Inhibition of BACE1 has been a compelling target for the potential treatment of Alzheimer’s disease. Since the first characterization of BACE1 in 1999, significant effort at Amgen and across the industry has focused on identifying potent inhibitors capable of achieving meaningful exposure in the brain to reduce levels of Aβ, while maintaining a favorable off-target profile. Retinal effects observed in preclinical species treated with a variety of BACE1 inhibitors, 48

including AM-8718, led to a refined discovery strategy focused on minimizing CatD inhibition. This strategy relied on exploiting a key residue difference between BACE1 and CatD in the S3 subpocket and identifying a more efficient core and warhead to allow for desirable ADME properties. AM-6138 is a novel BACE1 inhibitor identified from this effort and has been shown to avoid off-target retinal effects. Rapid delivery of large quantities of AM-6138 for candidate selection and FIH-enabling toxicology studies was enabled through early engagement between the medicinal and process chemistry teams. Accelerated development timelines precluded de novo process development for the first multikilogram delivery. However, safety analysis indicated that several intermediates in the original synthesis presented an explosion hazard on scale and required a strategic redesign of the synthesis to avoid high-energy intermediates. Several challenges were overcome for kilogram production, including the development of a cryogenic, lithiated sulfone addition to a tert–butanesulfinimine, a safe ozonolysis, and the conversion of an aryl halide to aniline via an ammonia surrogate. The complete process was 20 synthetic steps and was executed on scale to deliver >4 kg of final drug substance.

Acknowledgments The authors would like to thank Richard Lewis, Karina R. Vaida, and Laurie Schenkel for input on early medicinal chemistry strategy and route optimization toward AM-6138, Jie Yan for discovery scale-up efforts, Matthew Potter-Racine for GMP analytical development, Deborah M. Choquette, Ashraf Wilsily, and Jason S. Tedrow for input on production strategy, and Norchim SAS for execution of the kilogram production of AM-6138.

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22. Aboul-Enein, H. Y. In CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation; Kozma, D., Ed.; CRC Press: Boca Raton, FL, 2002. 23. Kula, J. Safer ozonolysis reactions: A compilation of laboratory experience. Chem. Health Saf. 1999, 6, 21–22. 24. Osterberg, P. M.; Niemeier, J. K.; Welch, C. J.; Hawkins, J. M.; Martinelli, J. R.; Johnson, T. E.; Root, T. W.; Stahl, S. S. Experimental limiting oxygen concentrations for nine organic solvents at temperatures and pressures relevant to aerobic oxidations in the pharmaceutical industry. Org. Process Res. Dev. 2015, 19, 1537–1543. 25. Harling, A. M.; Glover, D. J.; Whitehead, J. C.; Zhang, K. The role of ozone in the plasma-catalytic destruction of environmental pollutants. Appl. Catal. B 2009, 90, 157–161. 26. Lee, D.-Y.; Hartwig, J. F. Zinc trimethylsilylamide as a mild ammonia equivalent and base for the amination of aryl halides and triflates. Org. Lett. 2005, 7, 1169–1172. 27. Huang, X.; Buchwald, S. L. New ammonia equivalents for the Pd-catalyzed amination of aryl halides. Org. Lett. 2001, 3, 3417–3419. 28. Wolfe, J. P.; Ahman, J.; Sadighi, J. P.; Singer, R. A.; Buchwald, S. L. An ammonia equivalent for the palladium-catalyzed amination of aryl halides and triflates. Tetrahedron Lett. 1997, 38, 6367–6370. 29. Lee, S.; Jorgensen, M.; Hartwig, J. F. Palladium-catalyzed synthesis of arylamines from aryl halides and lithium bis(trimethylsilyl)amide as an ammonia equivalent. Org. Lett. 2001, 3, 2729–2732. 30. Tao, C.-Z.; Li, J.; Fu, Y.; Liu, L.; Guo, Q.-X. Copper-catalyzed synthesis of primary arylamines from aryl halides and 2,2,2-trifluoroacetamide. Tetrahedron Lett. 2008, 49, 70–75.

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Chapter 3

Discovery and Chemical Development of Verubecestat, a BACE1 Inhibitor for the Treatment of Alzheimer’s Disease David A. Thaisrivongs,1,* William J. Morris,1 and Jack D. Scott2 1Process

Research and Development, Merck & Co., Inc., Rahway, New Jersey 07065, United States 2Discovery Chemistry, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States *E-mail: [email protected]

This chapter highlights the discovery and chemical development of verubecestat, a potent and selective beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor that has been evaluated in late-stage clinical studies as a potential disease-modifying therapy for the treatment of Alzheimer’s disease. The medicinal chemistry effort that culminated in the invention of verubecestat focused on the design, synthesis, and evaluation of various iminoheterocyclic BACE1 inhibitors in order to discover a compound with a profile that would enable clinical progression to evaluate the safety and efficacy of chronic administration in patients. Once verubecestat was identified as a suitable clinical candidate, a synthetic route was developed to supply multikilogram quantities of the compound to support preclinical safety studies and the early clinical program. As verubecestat progressed into Phase III studies, a short, efficient, and robust manufacturing process was developed.

Amyloid Hypothesis in Alzheimer’s Disease Alzheimer’s disease is a progressive neurodegenerative disease that accounts for 60–80% of the estimated 47 million cases of dementia worldwide; overall costs of this illness are expected to approach $1 trillion in 2018 (1, 2). It © 2018 American Chemical Society

has been predicted that by 2050, without additional health care intervention, there will be over 130 million people afflicted with dementia globally (1). The currently available therapies provide only modest symptomatic improvement for Alzheimer’s disease patients and do not target the underlying disease etiology. In the face of such a global human health crisis, the identification of a disease-modifying therapy to slow this neurodegeneration has become an urgent unmet medical need. According to the amyloid hypothesis, the buildup of neurotoxic amyloid beta (Aβ) peptides in the brain over a period of years, possibly decades, leads to the formation of the amyloid plaques and neuronal death that characterize Alzheimer’s disease (3). The formation of Aβ peptides arises from the sequential cleavage of amyloid precursor protein (APP), first by the membrane-bound aspartyl protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and then followed by the γ-secretase enzyme complex. This process provides Aβ peptides of varying lengths with Aβ40 predominating (4, 5). These Aβ peptides, especially Aβ42, have been shown to oligomerize, eventually leading to neurotoxicity (5). There are a number of other substrates processed by BACE1, and the biological understanding and implications of chronic inhibition of this enzyme continue to be areas of intense study (6–9). In addition, there is a structurally related aspartyl protease, BACE2, which is largely expressed in the periphery and whose function has also been extensively studied in the context of beta cell function and pigmentation (10–12). Genetic validation for the role of BACE1 in disease progression includes the identification of mutations in APP that lead to enhanced cleavage by BACE1, which account for a portion of familial Alzheimer’s disease cases (9). More recently, a study reported the identification of a mutation in APP (A673T) 2 residues toward the N-terminus of the BACE1 cleavage site that correlates with a protective effect against Alzheimer’s disease and a deceleration in cognitive decline. This mutation was shown in vitro to reduce BACE1 APP processing, leading to a 40% reduction of Aβ40 and reduced aggregation (13). On the basis of this evidence, among other compelling data, it has been hypothesized that BACE1 inhibition, which decreases the production of Aβ peptides, has the potential to be a disease-modifying therapy for Alzheimer’s disease.

Discovery of Verubecestat Verubecestat (1, Figure 1) represents the culmination of a multiyear research campaign at Merck & Co., Inc. (Kenilworth, NJ, United States) to invent potent, selective inhibitors of BACE1 for evaluation as a potential disease-modifying treatment for Alzheimer’s disease (14–19). The structure of verubecestat arose from a multifaceted discovery effort that began with the identification of weakly binding hits from an NMR-based fragment screen undertaken in parallel to more traditional approaches toward aspartyl protease inhibitors (19, 20). Enabled by an X-ray co-crystal structure of 2 bound to BACE1 and a robust collaboration with our structural biology and modeling colleagues, we designed several novel iminoheterocyclic aspartyl protease binding cores, including iminohydantoins 54

(e.g., 3), iminopyrimidinones (e.g., 4 and 5), and iminothiadiazine dioxides (e.g., 6 and verubecestat). With these cores, we explored a wide variety of substituents, including biaryls (e.g., 3, 4, and 6) and diaryl amides (e.g., 5 and verubecestat). Due to the high homology of BACE1 and BACE2 around their active sites, selectivity for BACE1 over BACE2 was difficult to achieve during the course of our discovery efforts. While the biological understanding of dual inhibition of BACE1 and BACE2 continues to evolve, the reported phenotypes did not preclude compound progression (10–12, 21).

Figure 1. Selected preclinical data of the isothiourea fragment hit and lead iminoheterocyclic BACE1 inhibitors.

Among the extensive characterization of our BACE1 inhibitors, two assays in particular provided important data to differentiate compounds during lead optimization. The first was an in vitro assay for Cathepsin D (CatD) inhibition. CatD is a widely expressed aspartyl protease with an important role in the lysosomal degradation of proteins (22). Loss of function of this enzyme through inhibition or by genetic knockout in preclinical species is not well tolerated (23, 24), and thus selectivity over CatD was assessed very early in our evaluation of 55

compounds (19). The second assay of note was an in vivo assessment of the pharmacodynamics of Aβ40 lowering in both the cerebral spinal fluid (CSF) and cortex of rats. This assay enabled assessment of the ability of an orally dosed BACE1 inhibitor to modulate the levels of Aβ40, a clinically relevant biomarker, in the central compartment of rats. Ultimately, the combination of our unprecedented iminothiadiazine dioxide core with a diaryl amide substituent distinctly provided the best overall combination of BACE1 potency and selectivity, as well as the pharmacokinetic, pharmacodynamic, and preclinical safety profile required to enable long-term clinical dosing in patients and thus test the amyloid hypothesis with a BACE1 inhibitor (18, 21). Verubecestat progressed into Phase I studies, where it was found to be generally well tolerated in both normal healthy volunteers and Alzheimer’s disease patients. A significant reduction in the levels of Aβ40 and Aβ42 in the CSF of both normal healthy volunteers and patients was observed over 14 and 7 days of dosing, respectively (21). Subsequently, verubecestat became the first BACE1 inhibitor to progress into Phase II/III studies. The first trial, EPOCH, evaluated the efficacy and safety of verubecestat in patients with mild to moderate Alzheimer’s disease. In the Phase II portion of EPOCH, the safety of verubecestat was evaluated over 3 months in 200 patients at doses of 12, 40, and 60 mg. The subsequent Phase III trial studied the safety and efficacy of verubecestat in approximately 1800 randomized patients over 78 weeks at doses of 12 and 40 mg (25). In the second Phase III trial, APECS, the safety and efficacy of verubecestat was evaluated in approximately 1500 patients with prodromal Alzheimer’s disease over 2 years at doses of 12 and 40 mg (21).

Medicinal Chemistry Synthesis of Verubecestat The route used by the medicinal chemistry team to prepare verubecestat (1) started by adding the lithium anion of methyl sulfonamide 10 to Ellman ketimine 9 to provide sulfinyl amine 11 in moderate yield and diastereoselectivity (Scheme 1); the major isomer could be readily isolated using silica chromatography (18). The chiral auxiliary was cleaved using hydrogen chloride to produce amine 12, and the para-methoxybenzyl group was subsequently removed upon treatment with trifluoroacetic acid in the presence of 1,3-dimethoxybenzene as a benzyl cation scavenger to afford the aminosulfonamide 13. We initially attempted a three-step method to assemble the thiadiazine core using a protocol that had been efficiently executed on an analogous substrate class (Scheme 2) (18). In that case, treatment of aminosulfonamide 14 with benzoyl isothiocyanate afforded thiourea 15. The benzoyl group was readily removed with sodium methoxide to afford thiourea 16. The thiadiazine ring was then formed via activation of the sulfur atom with iodomethane and subsequent intramolecular cyclization to furnish 17 in an overall 81% yield. 56

Scheme 1. Medicinal chemistry route to aminosulfonamide 13.

Scheme 2. Thiadiazine ring formation using benzoyl isothiocyanate in an analogous substrate class.

57

While the formation of analogous benzoyl isothiocyanate 18 proceeded in high conversion from aminosulfonamide 13, the subsequent benzoyl removal using sodium methoxide did not afford thiourea 19 (Scheme 3) (18). Instead, multiple side-products were detected that arose from nucleophilic aromatic substitution of the aromatic fluorine, a vulnerability that was heightened by the para-nitro group. The use of less basic sodium carbonate did, however, allow for the isolation of thiourea 19 in 70% yield. Thiadiazine ring formation using iodomethane at an elevated temperature also proved problematic given the continued susceptibility of the substrate toward unwanted nucleophilic aromatic substitution, and we were ultimately able to obtain only a modest yield of thiadiazine 20 (18). While this route did afford sufficient quantities of the target to enable initial medicinal chemistry efforts, investigations continued into alternative thiadiazine ring-closing conditions to further reduce the formation of nucleophilic aromatic substitution side-products.

Scheme 3. Initial route to thiadiazine core 20.

In short order, the use of cyanogen bromide in refluxing 1-butanol was identified as an efficient method for forming thiadiazine 20 in a single step from aminosulfonamide 13 with reasonable conversion (Scheme 4) (18). Crude 20 was treated with di-tert-butyl dicarbonate to afford protected thiadiazine 21 in moderate isolated yield over the sequence. The nitro group was then reduced under heterogeneous conditions to the corresponding aniline (22) in high yield. This functional handle allowed for extensive investigation of the structure–activity relationship of inhibitors with respect to the S3 pocket of BACE1. The medicinal chemistry synthesis of verubecestat was completed with the installation of the fluoropyridine amide and the cleavage of the thiadiazine protecting group to provide 1 in high yield over the final two steps.

58

Scheme 4. Completion of the medicinal chemistry synthesis of verubecestat (1).

First Generation Process Route The first generation process route to verubecestat, which was implemented to supply both the preclinical safety work and the early clinical studies, relied substantially on the synthesis developed by the medicinal chemistry team (Scheme 5) (14, 26). This route used the same bond formation sequence as the previous one, but the discovery of two new intermediate salts were key to enabling API production on a multikilogram scale. The synthesis began with the deprotonation of N-methyl sulfonamide 10 using n-butyllithium and the diastereoselective addition of the resulting anion to sulfinyl ketimine 9 to give the corresponding sulfinyl amine 11. This intermediate was treated with excess trifluoroacetic acid to remove both the para-methoxybenzyl protecting group and the chiral auxiliary, revealing the corresponding aminosulfonamide 13. The enantiopurity of this intermediate was upgraded via the preparation and crystallization of the mandelate salt 25. This treatment provided an added advantage of avoiding the need for tedious preparative chromatography to separate the stereoisomers introduced in the formation of 11.

59

Scheme 5. First generation supply route to verubecestat (1). After freebasing the mandelate salt 25, we performed the cyclization with cyanogen bromide to provide thiadiazine 20. Protection of the resulting free amine as the corresponding tert-butyl carbamate and subsequent catalytic hydrogenation of the aryl nitro group with palladium on carbon revealed aniline 22. The aniline 22 was coupled with 5-fluoro-2-picolinic acid (23) using propylphosphonic anhydride (T3P). Removal of the protecting group with para-toluenesulfonic acid allowed for the isolation of para-toluenesulfonic acid salt 26, which provided an essential point of API purity control, particularly given the ensemble of product-related impurities generated during the hydrogenation to make 22. Finally, treating salt 26 with potassium carbonate and crystallization of 1 as the freebase completed the first generation process route.

Scouting Route Alternatives An ideal manufacturing process is not only one that requires a minimum number of high-yielding chemical transformations, but one that is also 60

environmentally friendly, operationally safe, reproducibly robust, and low cost. Thus, despite the scalability and efficiency of the first generation process route, we initiated a wide-ranging exploration to identify alternative syntheses of verubecestat (1) that could form the basis of a commercial manufacturing process. The key challenge in considering how to best synthesize 1 was the enantioselective formation of the stereogenic α,α-dibranched amine. To this end, we considered a variety of retrosynthetic disconnections that offered distinct strategic approaches for targeting this central functional group (Figure 2). In particular we were highly interested in evaluating enantioselective methods for introducing the desired stereochemistry, and pursued many that incorporated C-H insertion, 1,2- and 1,4-addition, and aziridination chemistries, among others. In the end, few of these endeavors bore fruit, a reflection of the relative paucity of methods known for constructing a stereogenic α,α-dibranched amine in a highly enantioselective manner.

Figure 2. Some of the evaluated strategies for the synthesis of the core of verubecestat (1).

One notable innovation made in the course of work directed specifically at the development of an enantioselective Mannich-type reaction for the synthesis of 1 was the discovery of an enantioselective palladium-catalyzed synthesis of cyclic sulfamidates (27). In this reaction, an enantiometically pure substituted phosphinooxazoline ligand controls the stereoselectivity of a palladium-catalyzed arylation reaction between cyclic iminosulfates and arylboronic acids. Both electron-poor and ortho-substituted arylboronic acids can be engaged productively 61

in this process, including 5-bromo-2-fluorophenylboronic acid (27), which when treated with cyclic iminosulfate 28 provides cyclic sulfamidate 29 in high yield and enantioselectivity (Scheme 6). We were able to subsequently demonstrate that this intermediate can be converted in five steps to 1. Ultimately, however, this alternative route was not competitive economically with those that leveraged the diastereoselective methodology, which enabled the first synthesis of verubecestat (1) by the medicinal chemistry team.

Scheme 6. Palladium-catalyzed enantioselective synthesis of cyclic sulfamidate 29 and its application to the synthesis of verubecestat (1).

Having made the determination that a Mannich-type addition of a suitable methyl sulfonamide (10) to an Ellman sulfinyl ketimine (34) was the most efficient way of accessing the stereogenic α,α-dibranched amine of 1 (Figure 3), our retrosynthetic analysis was reduced to strategic decisions of how to order and execute the following transformations: (1) removal of both the sulfonamide protecting group and the chiral auxiliary, (2) intramolecular cyclization of the resulting diamine (30) with a source of cyanogen to provide the desired thiadiazine of 1, and (3) installation of the secondary amide. We recognized that the existing synthesis of this amide, which relied on a coupling of 5-fluoro-2-picolinic acid (23) and aniline 22 (Scheme 5), necessitated a four-step sequence that included the installation and removal of a protecting group (from 20 to 26, vide supra). From the outset of our commercial route scouting activities, we were confident that replacing this series of functional group interconversions with a single transition metal-catalyzed C–N coupling of commercially available 5-fluoro-2-picolinamide (31) with an aryl bromide (32) would substantially improve the efficiency of the overall route. All of these disconnections led back to compounds that are or can be readily prepared in one step from commercially available materials. 62

Figure 3. Retrosynthetic analysis of verubecestat (1).

Sulfinyl Ketimine Synthesis In the first generation process route to 1 (Scheme 5), exposing 1-(2-fluoro-5nitrophenyl)ethan-1-one (7) and a slight molar excess of (R)-2-methylpropane-2sulfinamide (8) to a superstoichiometric amount of the strong dehydrating agent titanium(IV) isopropoxide provided sulfinyl ketimine 7 in 67% yield and 96.7% purity on a multikilogram scale (Scheme 7). Though this process was capable of delivering suitable quantities of material to support the early clinical development program, it nevertheless required a series of very inefficient unit operations that we sought to avoid in the context of a commercial manufacturing route. Removal of the residual inorganics was achieved by adding water at the end of the reaction to precipitate them, largely as titanium dioxide. However, the physical properties of this amorphous solid made it exceedingly challenging to separate from the rest of the batch, so much so that for a pilot plant scale run, 15 separate centrifuge filtrations were necessary to recover the product solution from the paint-like mixture of inorganic salts. A subsequent carbon treatment of the filtrate at 30% w/w was still needed to further purge impurities before the product was crystallized. Even after a thorough optimization of this workup and isolation process, more than 80 volumes of solvent in total relative to 7 were required to isolate 9, and vessels used for this process required additional cleaning protocols above and beyond typical rinses between batches in a commercial manufacturing setting. This inefficiency is a general problem with the formation of such ketimines. In a recently published Organic Synthesis protocol on the same reaction using 63

acetophenone, which exemplifies the state of the art for these transformations, over 165 volumes of solvent are needed just to filter off the titanium oxide generated upon the addition of water (28).

Scheme 7. Synthesis of sulfinyl ketimine 9 in the first generation process route. There are a variety of efficient methods for the formation of sulfinyl aldimines, including dehydrating agents such as magnesium sulfate (29), copper sulfate (30), cesium carbonate (31), potassium bisulfate (32), and molecular sieves (33), strong bases such as sodium hydroxide and potassium tert-butoxide (34), and Lewis acids such as ytterbium triflate (35) and tris(2,2,2-trifluoroethyl) borate (36). The only reagent, however, that can be generally utilized in the synthesis of nearly all classes of sulfinyl ketimines is titanium(IV) isopropoxide (37). Although resins have been shown to scavenge such titanium species, an impractical loading is required since excess reagent is often necessary to drive the ketimine condensation to completion (38). Despite a wide survey of dehydrating agents, we did not identify a superior reagent for the synthesis of 36 (vide infra), both with respect to yield and bulk reagent cost. Efforts to crystallize 36 (Scheme 8) in the presence of stoichiometric metal salts were met with little success, and the addition of filtering aids like Celite did not substantially improve the characteristics of the workup. The key process development breakthrough was to simply avoid the precipitation of any inorganics altogether. This was achieved by quenching the crude reaction not with water but with a concentrated solution of excess aqueous potassium glycolate, which preferentially generated titanium glycolate over titanium oxide. Glycolic acid is a cheap, nonhazardous, and biodegradable alpha-hydroxyl acid that has low toxicity and broad metal-sequestering properties (39). Titanium glycolate is a reported inorganic complex (40) but to our knowledge has never been reported in the context of a synthetic application or as a method to simplify the aqueous workups of titanium-mediated reactions. While there are isolated reports of workup protocols for reactions employing titanium alkoxides that enable the residual metal salts to be washed away (41–43), despite the ubiquity of titanium(IV) isopropoxide-mediated formations of sulfinyl imines in the organic synthesis literature, we are not aware of such a reaction that avoids the filtration of titanium oxide. Additional optimization of the sulfinyl ketimine formation parameters revealed that at slightly elevated temperatures a much lower excess of titanium(IV) isopropoxide was required, further enhancing the robustness of the revised workup protocol. A 20% improvement in yield, an over 60% reduction in waste, and 64

the isolation of product in considerably higher purity in the commercial process compared with the first generation process route (Scheme 7) without changing a single reagent or solvent underscores how there are opportunities for development along the entire process train that can deliver substantial improvements to the overall transformation.

Scheme 8. Synthesis of sulfinyl ketimine 36 in the commercial process route.

Synthesis of the α,α-Dibranched Amine We experimented with alternative protecting groups for methyl sulfonamide 10 (Scheme 9) in Mannich-type reactions with sulfinyl ketimine 36, including the analogous tert-butyl carbamate and tert-butyldimethylsilyl derivatives, and even evaluated the unprotected variant (which necessitated the formation of the corresponding dianion), but none proved as effective as para-methoxybenzyl. In the commercial process route, the synthesis of 10 began with the addition of para-anisaldehyde (37) to a solution of methylamine in methanol, which spontaneously formed N-methyl imine 38 (Scheme 9) (44). The subsequent reduction was accomplished by adding the crude solution of 38 to a suspension of sodium borohydride in tetrahydrofuran. The reaction was worked up with aqueous sodium hydroxide and the resulting secondary amine extracted with toluene. The solution of 39 could then be azeotropically dried efficiently, at which point triethylamine and methanesulfonyl chloride were added. Once the formation of 10 was complete, the reaction was quenched with water and the final product crystallized by adding n-heptane to the organic layer. This through-process was highly optimized, and at scale provided 10 in 92% yield over the three reactions.

Scheme 9. Synthesis of methyl sulfonamide 10 in the commercial process route. 65

The reaction of methyl sulfonamide 10 with sulfinyl ketimine 36 behaved similarly to the analogous reaction with 9 in the first generation process route (Scheme 5). Excess nucleophile was still necessary for high conversion, and the addition of the lithium anion of 10 to sulfinyl ketimine 36 had to be conducted under cryogenic conditions (–60 to –65 °C). This temperature operating window is extremely energy-intensive to achieve and maintain on a large scale. It constrains the selection of vessels and even commercial manufacturing sites for such processes. Despite a substantial effort to optimize the original reaction parameters, including an exhaustive survey of organometallic bases, additives, solvents, and protecting groups, only marginal improvements were realized, and on a pilot plant scale only a 73% assay yield of sulfone adduct 40 could be obtained (Scheme 10).

Scheme 10. Synthesis of 40 using the protocol from the first generation process route. This result is typical of such reactions with ketimines. To our knowledge, every reported diastereoselective addition of an organometallic reagent to a chiral ketimine is performed under cryogenic conditions, and with rare exception, the isolated yield is moderate (below 75%) (45). Since there were no significant sideproducts that formed during the course of this process, we attributed our modest result to the basicity of both the nucleophile and the unquenched product. When a reaction was quenched with excess deuterated acetic acid and the remaining starting materials recovered, the methyl group of sulfinyl ketimine 42 had greater than 99% incorporation of a deuterium atom (Scheme 11) (46). This result is consistent with a kinetic competition between 1,2-addition and alpha-deprotonation when the anion of methyl sulfonamide 41 reacts with sulfinyl ketimine 36. In a similar mechanistic experiment, when anion 43 (prepared by deprotonating 40 with an equimolar amount of n-hexyllithium) was added to sulfinyl ketimine 36, the methyl group of the recovered 42 had 87% deuterium incorporation (Scheme 12). Both of these side reactions irreversibly reduce the amount of 36 available for reaction, which accounts for the observed incomplete conversion even when a large excess of nucleophile is employed. We hypothesized that when this process is conducted in batch mode, the actual kinetic selectivity of the desired 1,2-addition over the competing α-deprotonation, as judged by analysis of the product distribution, is masked by the mixing rate of the reactive species (47, 48). Since we had also observed that the reaction was extremely fast even at cryogenic temperatures (i.e., complete within seconds at most), we further postulated 66

that executing the same chemistry in a continuous mode with extremely fast micromixing (49–54) should limit the exposure of unreacted sulfinyl ketimine 36 with anion 43, concomitantly reducing the amount of undesired α-deprotonation caused by the unquenched product. To evaluate the potential for a continuous process to improve the α,α-dibranched amine synthesis, we assembled a lab-scale flow reactor using four high-performance liquid chromatography pumps, stainless steel and fluoropolymer tubing, and static mixing tees (Table 1). With each of the four pumps (delivering methyl sulfonamide 10, n-hexyllithium, sulfinyl ketimine 36, and an acetic acid quench solution) set at 1 mL/min, 55% conversion to α,α-dibranched amine 40 was observed with no significant byproducts (entry 1).

Scheme 11. Deuterium quenching experiment reveals deprotonation of 36 by nucleophile 41.

Scheme 12. Deuterium quenching experiment reveals deprotonation of 36 by unquenched product 43. 67

Table 1. Proof-of-Concept for Improved Reaction Performance in Continuous over Batch Mode for the Synthesis of 40

At these relatively low flow rates, the mixing quality at the static mixing tees was poor, resulting in a significant amount of deprotonated sulfinyl ketimine 36. In stark contrast, when all other variables were kept constant and the pumping rate was increased (e.g., to 20, 32, and 40 mL/min; entries 2, 3, and 4, respectively), 86 to 88% conversion was achieved. This simple experiment provided a wealth of insight into the potential of flow chemistry to improve this process. The correlation between total flow rate and reaction conversion was consistent with our hypothesis that the quality of mixing between lithium anion 41 and sulfinyl ketimine 36 was a critical parameter governing the extent of unwanted α-deprotonation of 36 by unquenched product 43. A static micromixer was essential for making these observations; with a simple 0.05-in. inner diameter tee, the conversion at a total flow rate of 20 mL/min was only 66%. In addition, that the conversion appeared to plateau once 68

a certain mixing characteristic was achieved suggested that the balance of the material represented the unwanted kinetic competition between 1,2-addition and α-deprotonation when sulfinyl ketimine 36 reacts with 41 (vide supra). Further, the data also demonstrated that there was a substantial efficiency gain by operating in a continuous instead of a batch mode, as these superior results were all obtained at a noncryogenic temperature (-10 °C). High flow rates also significantly shortened the residence time for both the deprotonation of 10 and the reaction of 36 with 41. At the fastest flow rate (entry 4), lithium anion 41 reacted with sulfinyl ketimine 36 after only 30 ms (τ1) of its formation, and the resulting product 43 was quenched with acetic acid after only an additional 2.5 ms (τ2), yet the product was obtained in 87% conversion (entry 4). In fact, we have been unable to execute a continuous process with a residence time that is too short for both the deprotonation and sulfinyl ketimine addition to achieve completion, which suggests that as long as the mixing characteristics are sufficiently good, both reactions have reached their maximal conversion when the respective streams leave the mixing chambers. Regardless of the residence time, however, the diastereomeric ratio of the product was the same. In the first generation process route, an excess of nucleophile was necessary to achieve optimal reaction performance, with the maximum conversion achieved at 1.7 equivalents of methyl sulfonamide 10 relative to the sulfinyl ketimine. In this respect, the continuous process mirrored the batch one. An evaluation of reagent stoichiometry in flow was straightforward to perform using our lab-scale equipment by simply adjusting the relative pumping rates and collecting quenched crude reaction samples once steady state had been established at each flow rate set point. Optimal reaction conversion was observed with 1.5 equivalents of nucleophile, but to ensure robustness on a larger scale, we choose to employ 1.7 equivalents of n-hexyllithium and 1.8 equivalents of methyl sulfonamide 10 relative to sulfinyl ketimine 36. Our proof-of-concept experiments demonstrated that, unexpectedly, only moderate cooling was required to achieve significant reaction conversion (Table 1), which enabled much of our subsequent optimization work to be performed by simply submerging the heat exchangers and micromixers in a –10 °C cooling bath (Table 2, entry 1). We were unable to thoroughly evaluate reactions below –30 °C, as precipitation of both methyl sulfonamide 10 and lithium anion 41 quickly clogged the flow path at those temperatures. To our further surprise, there was no significant difference between continuous reactions conducted at –10 °C and 1 °C (entries 1 and 2). Even at ambient temperature, only a small reduction in conversion was observed (entry 3). Remarkably at 38 °C, a temperature at which the half-lives of organolithium species in ethereal solvents are on the order of mere hours (55), the reaction performance was still not substantially altered (entry 4). To our knowledge, these data represent reaction conditions that are far removed from any reported ketimine addition with a hard nucleophile and underscore the extreme conditions that are both accessible and can be uniquely effective when operating in a continuous mode (56). 69

Table 2. The Synthesis of 40 in Continuous Mode at Extreme Temperatures

Maintaining steady-state performance of an organolithium-mediated reaction in flow for many hours can be a steep challenge (57), and subsequent experiments using kilogram-scale equipment were necessary to uncover additional processing risks that were not revealed when the continuous process was conducted for relatively short periods of time. For example, although the reaction performed just as well around 0 °C as it did at -20 °C, over the course of tens of minutes we observed a gradual increase in backpressure at each of the pumps, signaling that a system clog was building. Further increasing the temperature to 20 °C hastened the decline from steady-state operation. Since rigorously dried feed solutions caused the equipment to clog at approximately the same rate as wet feed solutions, adventitious moisture did not appear to be the primary source of fouling. Neither did the issue seem to be only the insolubility of methyl sulfonamide 10 or lithium anion 41, since the effect of either should be less pronounced at higher temperatures. Instead, we hypothesized that a decomposition event at the point of mixing of methyl sulfonamide 10 and n-hexyllithium was exacerbated for prolonged periods of time at temperatures substantially above -20 °C. We measured the heat of reaction for this deprotonation as -85.2 kJ/mol with an accompanying adiabatic temperature rise of 31.2 °C, and although the heat removal in flow is generally highly efficient, the precise point along the flow 70

path at which 10 interacts with n-hexyllithium likely reaches and maintains a much warmer temperature than the surrounding cooling liquid. Further evidence that is consistent with this hypothesis was obtained when lithium anion 41 was prepared in batch at low temperature and then warmed to 20 °C; decomposition and precipitation were observed (58). When the feed solutions were all delivered at -20 °C, we could execute the continuous process on a kilogram scale for an hour without event. Nevertheless, warning signs remained that it would not be possible to perform the continuous deprotonation for the even longer periods of time that would be required for commercial-scale batches. Specifically, the deposition of unidentified insoluble material at the point of contact between methyl sulfonamide 10 and nhexyllithium led, over time, to an increase in the pressure drop through the first mixer. No data pointed to significant risks of fouling at either the sulfinyl ketimine addition or quench mixers. A way to completely avoid this issue was to generate lithium anion 41 in a batch reactor and then flow the resulting solution into the reaction mixer (Figure 4) (59). This design eliminated the fouling event associated with the continuous deprotonation, but necessarily created trade-offs. First, the feed solutions for the fully continuous process design (methyl sulfonamide 10 in tetrahydrofuran, nhexyllithium in hexanes, and sulfinyl ketimine 36 in tetrahydrofuran) were very stable at an ambient temperature, while lithium anion 41 was not. Thus it was necessary to generate and maintain 41 at a low temperature (i.e., -20 °C) over the course of the entire batch.

Figure 4. Redesigned continuous process that incorporates a batch deprotonation of 10 to improve chemical robustness.

Second, temperature-controlled jacketed lines were also required to transfer lithium anion 41 from the deprotonation vessel to the reaction mixer. Critically, however, this strategy still realized the performance improvements of executing the sulfinyl ketimine addition in flow while ensuring the deprotonation of 10 was robust. Lab-scale tests of the redesigned process demonstrated that it enabled steady-state operation for multiple hours and that the changes did not diminish the overall reaction performance. 71

Nevertheless, in our first preparation of 40 in batch mode at pilot scale, we discovered a new crystalline phase of lithium anion 41 that was stable in our desired operating window (60). The resulting slurry of 41 in tetrahydrofuran was incompatible with the planned flow process, so our attention immediately focused on establishing modified reaction conditions that would solubilize this polymorph. Dilution did not appear promising, as when the concentration of 41 was halved, some solids remained. We could also not use a more polar solvent (e.g., dimethyl sulfoxide, dimethylformamide, or N-methyl-2-pyrrolidone), as these substantially reduced the reaction yield. Further, we had already demonstrated that lithium anion 41 was not sufficiently stable above -20 °C to allow us to overcome the insolubility of 41 by raising the temperature. Given the well-known tendency of organolithium species to form higher-order aggregates in solution (61), we hypothesized that additives able to perturb such species might increase the solubility of lithium anion 41. Such a group of structurally diverse and readily available additives (i.e., tetramethylethylenediamine, N,N′-dimethylpropyleneurea [DMPU], N,N′-dimethylethyleneurea, pyridine, trimethylamine, and dimethoxyethane) were tested, and DMPU was most effective in solubilizing 41 at the target concentration and temperature. Further optimization experiments demonstrated that about 1.0 equivalent of DMPU relative to lithium anion 41 was required to generate a homogeneous solution of 41 at -20 °C, which resisted crystallization even upon addition of crystalline 41 as a seed. The addition of DMPU did not significantly impact the performance of the reaction or the stability of the lithium anion feed solution. Having established that mixing efficiency was critical to high reaction conversion, one of the main objectives of our first pilot plant-scale batch was to evaluate the reaction’s performance at a range of flow rates through a commercial-scale mixer. A Y-shaped mixer was fabricated from 1/4-in. stainless steel Schedule 40 pipe with a Koflo static mixing element (62) installed in the downstream segment (Table 3). We measured the conversion and diastereoselectivity of the reaction over a range of flow rates (from 55.5 kg/h to 250.0 kg/h total solution), maintaining a constant flow rate ratio to ensure the delivery of 1.7 equivalents of lithium anion 41 relative to sulfinyl ketimine 36. Within this array of flow rates, we independently varied the temperature of the 36 feed stream (from 22 to -62 °C) while holding the temperature of the 41 feed solution between -10 and -20 °C. In all cases, the conversion monotonically improved as the total flow rate increased. This effect was most pronounced at the warmest temperature, where the conversion increased from 64.2 to 82.1% when the total flow rate was raised from 55.5 kg/h to 250.0 kg/h (entries 1 to 5). As the feed temperature of sulfinyl ketimine 36 was lowered, the overall reaction temperature was consequently reduced, leading to higher reaction conversions and a progressive decrease in the impact of the total flow rate on conversion. At the coldest temperature, the improvement in conversion increased from 80.5 to 85.6% when the total flow rate was raised from 55.5 kg/h to 250.0 kg/h (entries 16 and 20).

72

Table 3. The Impact of Flow Rate and Temperature on the Continuous Formation of 40 at Pilot Plant Scale

73

Modifications to the overall equipment train enabled rigorous temperature control at each point along the flow path. Improvements in our ability to execute the entire process rapidly enabled us to significantly shorten the length of time between the start of the n-hexyllithium charge into methyl sulfonamide 10 and the beginning of the flow reaction. Since solutions of lithium anion 41 slowly decrease in purity over time even at -20 °C, the best results are obtained with freshly prepared anion. Based on the data we had collected at pilot plant scale about the relationship between reaction conversion, diastereoselectivity, and temperature, we choose to deliver both reactants between -10 and -20 °C, a temperature range that did not necessitate cryogenic equipment. In addition, matching the feed solution temperatures enabled a single external chiller unit to supply cooling fluid to both flow heat exchangers, simplifying the engineering requirements. With these changes we were able to conduct the optimized process at steady-state conditions for hours at pilot plant scale to produce more than 100 kg of product in a single batch with consistent assay yields of 88 to 89% (Figure 5).

Figure 5. Optimized continuous process at pilot plant scale. We were only able to identify weakly crystalline polymorphs of 40, which ultimately precluded its isolation directly from the quenched reaction mixture. Instead, given our ultimate interest in cleaving the tert-butyl sulfinamide auxiliary as the next step to furnish the ideal C-N coupling substrate (vide infra), we chose to evaluate chiral acid salts of the corresponding primary amine as isolable crystalline intermediates. That substrate, amine 44, could be readily and quantitatively formed by quenching the continuous process reaction stream into an excess of aqueous hydrogen chloride (Scheme 13). We surveyed a wide array of commercially available chiral acids in the presence of 44 in a high-throughput manner; (S)-mandelic acid emerged as the best. In the optimized crystallization process, the desired salt (45) could be isolated in 98.5% yield (relative to the desired enantiomer present in the starting material), 99.9% purity, and 98.8% ee from toluene. Using a water-immiscible solvent like toluene for the crystallization after an aqueous deprotection of 43 provided a seamless connection between 74

an organic phase extraction of freebased amine 44 (the end of the continuous process) and the downstream salt isolation after freebasing.

Scheme 13. Optimized conditions for the crystallization of 45 from the sulfinyl ketimine addition reaction.

Copper-Catalyzed Amidation We initially discovered a copper-catalyzed C-N coupling between aryl bromide 40 and 5-fluoro-2-picolinamide (31), which afforded aryl amide 46 in 70% isolated yield (Scheme 14). While this reaction performed serviceably at pilot plant scale, the modest yield, very high catalyst loading, and excess of 31 required to achieve full conversion provided the impetus to further investigate the amidation.

Scheme 14. Copper-catalyzed amidation of aryl bromide 40. 75

Evaluation of alternative aryl bromide coupling partners unexpectedly revealed that the partially deprotected amine 44 also underwent efficient C–N coupling, achieving full conversion and providing the coupled adduct in 90% yield from mandelate salt 45 (Scheme 15). In addition, with this substrate we were able to reduce the catalyst loading to 20 mol%, and unlike the analogous reaction with 40, the revised C–N coupling protocol only required an equimolar amount of ligand relative to copper. The employment of aryl bromide 44 also enabled a reduction in the charge of 31 to only 1.2 equivalents.

Scheme 15. Improved amidation sequence employing aryl bromide 44.

To rationalize the difference in reaction performance between aryl bromides 40 and 44, we initially hypothesized that the free amine present in 44 coordinated to copper, favoring formation of the active catalytic species and retarding the formation of inactive cuprate complexes (63, 64). The observation that both the amounts of diamine ligand and amide 31 could be reduced in the coupling employing 44 is consistent with this proposal. In the reaction with aryl bromide 40, the excess of amide 31 was required at least in part due to a competing background hydrolysis that formed 5-fluoro-2-picolinic acid (23). Reaction monitoring by NMR revealed that this hydrolysis was promoted by copper iodide and suppressed by increasing the concentration of the diamine ligand. The availability of an additional amine from the starting material to occupy a coordination site on the metal further suppressed hydrolysis of 31, enabling the reduction of the number of equivalents required to achieve full conversion.

Removal of the para-Methoxybenzyl Group Following the optimization of copper-catalyzed amidation, we shifted our focus toward developing robust conditions for the removal of the para-methoxybenzyl protecting group. One issue we needed to address at the 76

outset was the exceedingly poor solubility of amine 47 in nearly all organic solvents. An expansive solvent screen revealed that acetic acid provided the requisite solubility to facilitate the deprotection. Methanesulfonic acid proved to be the optimal acid to effect the cleavage of the para-methoxybenzyl group, affording clean deprotection in 2 hours at 60 °C. During the course of our lab-scale development we found that once the reaction was complete, the batch could be cooled, diluted with water, and washed with toluene before carrying out a reverse addition into concentrated ammonium hydroxide to induce the crystallization of the product. We were surprised to discover that upon performing this process on a multikilogram scale, a large, insoluble mass formed in the reactor while cooling the batch from 60 °C upon reaction completion. We analyzed this insoluble material by mass spectrometry and observed multiple species consistent with polymerization of the para-methoxybenzyl cation generated during the deprotection. To block this undesirable side reaction, we conducted the deprotection in the presence of the cation scavenger 1,3-dimethoxybenzene, which prevented the formation of these polymers by trapping the para-methoxybenzyl cation. These adducts formed during the deprotection could readily be removed during the toluene wash prior to isolation of 48 from ammonium hydroxide and provided robust conditions that could be scaled without issue (Scheme 16).

Scheme 16. Removal of the para-methoxybenzyl group.

Thiadiazine Formation and Completion of the Synthesis As in the earlier syntheses of 1 (Schemes 4 and 5), cyanogen bromide proved to be the best reagent to generate the thiadiazine heterocycle in a single chemical step. We initially observed that when amine 48 was treated with cyanogen bromide in a mixture of isopropyl acetate and acetonitrile, the hydrogen bromide salt of verubecestat (49) crystallized spontaneously (Scheme 17). This reactive crystallization provided a direct isolation of 49 in high chemical purity and was advantageous as it eliminated the need for an aqueous workup and subsequent isolation. In addition, the discovery of this reactive crystallization was fortuitous as similar thiadiazine formation conditions, which employed bases, resulted in the over-cyanation of 1 to cyanamide 50 (Scheme 18). This process was demonstrated successfully on a multikilogram scale, affording 49 in 88% yield. 77

Scheme 17. Thiadiazine formation and isolation of verubecestat (1).

Scheme 18. Over-cyanation of 1 to 50 in the presence of cyanogen bromide under basic conditions.

Surprisingly, during a pilot plant batch of this process, we observed a strikingly atypical reaction profile. When we analyzed the solid isolated from this batch, we observed a mixture of the desired product (49) along with unreacted amine 48. In addition, the supernatant contained greater than 40% of cyanamide 51, an intermediate measured to be less than 1% in the supernatant of previous batches (Figure 6).

Figure 6. Cyanamide intermediate 51 formed during the conversion of 48 to 49. The solid isolated from this batch was subjected to rigorous characterization to unambiguously determine the composition of the material. X-ray powder diffraction showed a mixture consisting of the desired crystalline phase of 49 and 78

an unknown phase (65). Scanning electron microscopy revealed that the isolated solid was composed of particles displaying two distinct morphologies. One of the morphologies was consistent with the previously characterized batches of 49, and a second, granule-like aggregate was significantly larger in size (Figure 7). The granule-like aggregates were sieved using a range of screen sizes, and the isolated solids were found to be a single pure phase by powder indexing (66).

Figure 7. Scanning electron microscope images showing two distinct morphologies in the isolated unknown solid.

Further characterization of these aggregates by high-performance liquid chromatography and inductively coupled plasma mass spectrometry revealed the purified granules were a mixture of amine 48, verubecestat (1), and two equivalents of hydrogen bromide (Table 4). This analysis demonstrated conclusively that the isolated solid from the batch was a mixture of desired product 49 and a co-crystal of the hydrogen bromide salts of both 48 and 1. This finding was also supported by single-crystal X-ray diffraction analysis of the co-crystal, which revealed a structure consistent with the component analysis via high-performance liquid chromatography (Figure 8).

Table 4. Component Analysis of the Unknown Impurity Phase Component

Fraction in impurity phase

Mol % of component

Mol % normalized to API

Amine (48)

40.1

23.6

1.01

Verubecestat (1)

42.4

23.5

1.00

Hydrogen bromide

18.6

52.8

2.25

79

Figure 8. Single-crystal X-ray diffraction of the co-crystal formed between the hydrogen bromide salts of 48 and 1. Thermal ellipsoids are shown at the 50% probability level.

The formation of the co-crystal provided insight into the mechanism of the reaction. The co-crystallization event removed hydrogen bromide from the solution phase of the reaction, which, when coupled with the observation that cyanamide remained in the supernatant, suggested that soluble hydrogen bromide was necessary for intramolecular ring closure of the thiadiazine. Indeed, when cyanamide 51 was isolated and treated with hydrogen bromide in a mixture of acetonitrile and isopropyl acetate, 49 was readily formed. The poor solubility of the co-crystal across all reaction solvents rendered the existing process untenable. Our initial efforts to modify the endgame sought to employ basic conditions in order to prevent the accumulation of hydrogen bromide salts that would lead to subsequent co-crystal formation. While this approach did generate 1, we were unable to identify conditions that suppressed the formation of the over-cyanation impurity 50, an issue that we had previously addressed using the isolation of 49 (vide supra). A more significant endgame redesign was subsequently undertaken in which we focused on a stepwise approach to initially convert amine 48 to cyanamide 51 followed by intramolecular ring closure to form the thiadiazine. This sequence would eliminate the undesired co-crystallization by preventing the formation of 1 in the presence of the amine 48 and hydrogen bromide. A stepwise approach would also address the over-cyanation issue by preventing the formation of 1 in the presence of cyanogen bromide. Our preliminary evaluation of bases revealed that amine bases weaker than N,N-diisopropylethylamine provided 51 as the exclusive product, but only in prohibitively low levels of conversion (Table 5, entries 1 to 3). Inorganic bases such as potassium phosphate monobasic or sodium bicarbonate, however, led to improved conversions across a broad range of solvents. We ultimately chose to advance sodium bicarbonate as the base and 2-methyltetrahydrofuran as the 80

solvent as that combination provided optimal conversions and streamlined the aqueous workup given its use of a water immiscible solvent.

Table 5. Survey of Solvents and Bases for the Conversion of 48 to 51

Once we had a solution of cyanamide 51, we quenched the residual cyanogen bromide present by adding aqueous sodium hydroxide. Interestingly, we found these conditions promoted intramolecular cyclization to form the thiadiazine, but the ring-closed adduct was once again accompanied by over-cyanation byproduct 50. We then hypothesized that a reductive workup (67, 68) would be necessary to completely destroy residual cyanogen bromide, and to that end sodium thiosulfate was identified as a suitable reducing agent. When the process stream containing 51 and residual cyanogen bromide was washed with 10% aqueous sodium thiosulfate prior to treatment with sodium hydroxide, the formation of 50 was almost completely suppressed. The resulting 2-methyltetrahydrofuran stream containing 51 was treated with aqueous sodium hydroxide to promote intramolecular cyclization to verubecestat (1), which was first isolated as para-toluenesulfonate salt 26 to ensure API purity specifications were met. A final salt break of para-toluenesulfonic acid salt 26 with potassium carbonate afforded 1 in 92% isolated yield from 48 (Scheme 19). 81

Scheme 19. Synthesis of verubecestat (1) using the revised endgame.

Emergence of a New API Polymorph Following the completion of a clinical resupply campaign, routine release testing revealed the presence of extraneous matter within a single batch of verubecestat (1), which necessitated reprocessing. The recrystallization of this particular batch was carried out using the same conditions from which the API was routinely isolated, however, X-ray powder diffraction analysis revealed a new phase of the API, labeled Form II, which had a higher melting point than the previously isolated phase (Form I). Equilibration studies conducted by slurrying Form I in a range of solvents showed conversion to Form II under all conditions evaluated. Together these data demonstrated that Form II was a more thermodynamically stable phase of verubecestat (1). The spontaneous formation of new API polymorphs in late development or even after product registration is not without precedent. The well-known example of ritonavir, which was temporarily withdrawn from the market when a new polymorph of the API was discovered, provided a cautionary tale of the potential for commercial disruption related to an unexpected API phase change (69). Based on preliminary data, we expected that our ability to robustly produce Form I would be compromised now that a more thermodynamically stable Form II had emerged. In an effort to avoid complications related to metastable polymorphs, our development team decided to target Form II in all future clinical supply deliveries and for the commercial launch. Despite its higher melting point and reduced solubility in biorelevant media, Form II was still Class I under the U.S. Food and Drug Administration guidelines for the classifications of biopharmaceutics, like Form I, and both polymorphs had a similar dissolution 82

profile across a broad pH range. This finding was significant as it assuaged concerns that the new polymorph would impact the bioavailability of verubecestat (1) and simplified the regulatory hurdles for changing the API polymorph during clinical development. The emergence of Form II forced us to reinvestigate our endgame process. First, a new API isolation that reliably produced Form II would need to be established. Second, the process that generated verubecestat prior to the formation of para-toluenesulfonic acid salt 26 (Scheme 19) would require revisions to ensure no additional complications would arise due to the reduced solubility of Form II. Given the subtle differences in solubility between the two polymorphs, we suspected the process changes would be minor. The API isolation of Form I involved concentrating an ethyl acetate solution of 1 to approximately 18 wt % and heating to 50 °C before charging 15 vol % of n-heptane and seeding with Form I. The batch was aged for 2 hours before n-heptane was added to reach 60 vol % n-heptane followed by a reduction in batch temperature to 20 °C. In order to access Form II we found that the n-heptane charge prior to seeding could be adjusted to 5 vol % and the use of Form II seeds would allow for reliable production of the Form II polymorph. Most importantly from a processing perspective, we found that Form II was equally capable of rejecting the typical impurities produced during the salt break of para-toluenesulfonic acid salt 26. We also found the processability of both forms to be similar as both reduced to similar particle size distributions during wet milling, avoiding the need for additional process development for particle size control. The endgame process, which proceeded through 1, also required only minor revisions. A solubility assessment of the newly discovered Form II in 2-methyltetrahydrofuran revealed only a subtle decrease in solubility. To prevent the undesired crystallization of 1 prior to the formation of 26, we adjusted the target volumes during distillation and were pleased to find that despite the increase in the amount of 2-methyltetrahydrofuran, we did not observe meaningful increases in product losses to the mother liquors.

Phase III Clinical Status In 2017, the EPOCH trial was stopped early following the recommendation of the external Data Monitoring Committee, which noted that while the observed safety signals did not warrant terminating the study, there was virtually no chance of finding a positive clinical effect with respect to the slowing of cognitive or functional decline (25, 70). Patients who received verubecestat did show an up to 80% reduction of Aβ peptides in their CSF compared to patients on placebo. In addition, a subset of patients had a reduction in amyloid load in their brains according to amyloid imaging using positron emission tomography (25). In February 2018, it was announced that the APECS trial would be discontinued based on the external Data Monitoring Committee recommendation that it was unlikely that a positive benefit/risk could be established if the trial continued (71). 83

Scheme 20. The commercial manufacturing process for verubecestat (1).

Summary The sum of this work constitutes the discovery of verubecestat (1) and the ultimate development of the commercial manufacturing process (Scheme 20). The overall yield of 67% from the coupling of methyl sulfonamide 10 and sulfinyl ketimine 36 through to 1 improves significantly on the first generation process route, for which the same sequence delivers a 28% overall yield. Additionally, substantial gains in efficiency across the entire process produced more than an 80% reduction in waste (72). These results were enabled by the invention of a continuous process for the addition of 41 to 36 to form the stereogenic α,αdibranched amine, the identification of a new chiral salt (45) to provide a key 84

point of stereochemical purity upgrade, a C-N coupling that takes advantage of an unexpected boost in reactivity gained by employing a substrate (44) that has been partially deprotected, and a new orchestration of the thiadiazine synthesis, which entirely circumvents the risk of co-crystallization of the starting material and product for that reaction. Finally, a revised API crystallization has been developed to deliver Form II, the recently identified API polymorph, in a robust and efficient manner.

Acknowledgments We thank the many colleagues from the Research Laboratories and the Manufacturing Division of Merck & Co., Inc., Kenilworth, NJ, United States, who contributed to this work, many of whose names are included in the references below.

References 1.

Alzheimer’s Disease International. World Alzheimer’s Report 2015. https://www.alz.co.uk/research/WorldAlzheimerReport2015.pdf (accessed Sept. 26, 2018). 2. Alzheimer’s Association. 2015 Alzheimer’s Disease Facts and Figures. Alzheimer’s Dement. 2015, 11, 332–384. 3. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353–356. 4. Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J. C.; Collins, F.; Treanor, J.; Rogers, G.; Citron, M. Science 1999, 286, 735–741. 5. Haass, C.; Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 2007, 8, 101–112. 6. Willem, M.; Garratt, A. N.; Novak, B.; Citron, M.; Kaufmann, S.; Rittger, A.; DeStrooper, B.; Saftig, P.; Birchmeier, C.; Haass, C. Science 2006, 314, 664–666. 7. Hu, X.; Hicks, C. W.; He, W.; Wong, P.; Macklin, W. B.; Trapp, B. D.; Yan, R. Nat. Neurosci. 2006, 9, 1520–1525. 8. Cheret, C.; Willem, M.; Fricker, F. R.; Wende, H.; Wulf‐Goldenberg, A.; Tahirovic, S.; Nave, K.‐A.; Saftig, P.; Haass, C.; Garratt, A. N.; Bennett, D. L.; Birchmeier, C. EMBO J. 2013, 32, 2015–2028. 9. Vassar, R.; Kuhn, P.-H.; Haass, C.; Kennedy, M. E.; Rajendran, L.; Wong, P. C.; Lichtenthaler, S. F. J. Neurochem. 2014, 130, 4–28. 10. Esterházy, D.; Stützer, I.; Wang, H.; Rechsteiner, M. P.; Beauchamp, J.; Döbeli, H.; Hilpert, H.; Matile, H.; Prummer, M.; Schmidt, A.; Lieske, N.; Boehm, B.; Marselli, L.; Bosco, D.; Kerr-Conte, J.; Aebersold, R.; Spinas, G. A.; Moch, H.; Migliorini, C.; Stoffel, M. Cell Met. 2011, 14, 365–377. 11. Rochin, L.; Hurbain, I.; Serneels, L.; Fort, C.; Watt, B.; Leblanc, P.; Marks, M. S.; De Strooper, B.; Raposo, G.; van Niel, G. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 10658–10663. 85

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43. Reeves, J. T.; Tan, Z.; Han, Z. S.; Li, G.; Zhang, Y.; Xu, Y.; Reeves, D. C.; Gonnella, N. C.; Ma, S.; Lee, H.; Lu, B. Z.; Senanayake, C. H. Angew. Chem., Int. Ed. 2012, 51, 1400–1404. 44. Thaisrivongs, D. A.; Morris, W. J.; Tan, L.; Song, Z. J.; Lyons, T. W.; Waldman, J. H.; Naber, J. R.; Chen, W.; Chen, L.; Zhang, B.; Yang, J. Org. Lett. 2018, 20, 1568–1571. 45. Robak, M. T.; Herbage, M. A.; Ellman, J. A. Chem. Rev. 2010, 110, 3600–3740 and references therein. 46. Thaisrivongs, D. A.; Naber, J. R.; McMullen, J. P. Org. Process Res. Dev. 2016, 20, 1997–2004. 47. Rys, P. Acc. Chem. Res. 1976, 9, 345–351. 48. Rys, P. Angew. Chem., Int. Ed. Engl. 1977, 16, 807–817; Angew. Chem. 1977, 89, 847−857. 49. Yoshida, J.; Nagaki, A.; Iwasaki, T.; Suga, S. Chem. Eng. Technol. 2005, 28, 259–266. 50. Nagaki, A.; Togai, M.; Suga, S.; Aoki, N.; Mae, K.; Yoshida, J. J. Am. Chem. Soc. 2005, 127, 11666–11675. 51. Yoshida, J. Flash Chemistry: Fast Organic Synthesis in Microsystems; John Wiley & Sons, Ltd.: West Sussex, U.K., 2008. 52. J. Yoshida, J.; Nagaki, A.; Yamada, T. Chem.– Eur. J. 2008, 14, 7450–7459. 53. Yoshida, J. Chem. Rec. 2010, 10, 332–341. 54. Kim, H.; Nagaki, A.; Yoshida, J. Nat. Commun. 2011, 2, 264 and references therein. 55. Schlosser, M., Ed. Organometallics in Organic Synthesis; Wiley: New York, 1994; p 172. 56. For a related example of the energy savings and enhanced reaction performance at industrial scale when a continuous process is used instead of a stirred tank process, see: Hessel, V.; Hofmann, C.; Löwe, H.; Meudt, A.; Scherer, S.; Schönfeld, F.; Werner, B. Org. Process Res. Dev. 2004, 8, 511–523. 57. Laue, S.; Haverkamp, V.; Mleczko, L. Org. Process Res. Dev. 2016, 20, 480–486 and references within. 58. Attempts to characterize the nature of this solid material did not provide a single structure, but instead point to fragmentation of lithium anion 8. 59. Thaisrivongs, D. A.; Naber, J. R.; Rogus, N. J.; Spencer, G. Org. Process Res. Dev. 2018, 22, 403–408. 60. We have not been able to determine why previous lab-scale experiments failed to elucidate this polymorph. Subsequent to this observation in the pilot plant, we were able to reproduce the crystallization event on a lab scale. We believe this phenomenon is complementary to that of a “disappearing polymorph”; see Dunitz, J. D.; Berstein, J. Acc. Chem. Res. 1995, 28, 193–200. 61. Reich, H. J. Chem. Rev. 2013, 113, 7130–7178 and references therein. 62. Model number: ¼-40-3-12L-1, purchased from Koflo Corporation, 309 Cary Point Drive, Cary, IL 60013, United States. 63. Klapars, A.; Huang, X.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 7421–7428. 88

64. Strieter, E. R.; Blackmond, D. G.; Buchwald, S. L. J. Am. Chem. Soc. 2005, 127, 4120–4121. 65. Artino, L.; Varsolona, R.; Brunskill, A. P. J.; Morris, W. J.; Thaisrivongs, D. A.; Waldman, J. H.; Lyons, T. W.; Xu, Y. Org. Process Res. Dev. 2018, 22, 385–390. 66. Maguire, C. K.; Brunskill, A. P. J. Mol. Pharmaceutics 2015, 12, 2061–2067. 67. Bailey, P. L.; Bishop, E. J. Chem. Soc., Dalton Trans. 1973, 9, 917–921. 68. Shang, C.; Qi, Y.; Xie, L.; Liu, W.; Yang, X. Water Res. 2005, 39, 2114–2124. 69. Chemburkar, S. R.; Bauer, J.; Deming, K.; Spiwek, H.; Patel, K.; Morris, J.; Henry, R.; Spanton, S.; Dziki, W.; Porter, W.; Quick, J.; Bauer, P.; Donaubauer, J.; Narayanan, B. A.; Soldani, M.; Riley, D.; McFarland, K. Org. Process Res. Dev. 2000, 4, 413–417. 70. Merck & Co., Inc. Merck Announces EPOCH Study of Verubecestat for the Treatment of People with Mild to Moderate Alzheimer’s Disease to Stop for Lack of Efficacy, February 14, 2017. http://investors.merck.com/news/pressrelease-details/2017/Merck-Announces-EPOCH-Study-of-Verubecestatfor-the-Treatment-of-People-with-Mild-to-Moderate-Alzheimers-Diseaseto-Stop-for-Lack-of-Efficacy/default.aspx (accessed Sept. 26, 2018). 71. Merck & Co., Inc. Merck Announces Discontinuation of APECS Study Evaluating Verubecestat (MK-8931) for the Treatment of People with Prodromal Alzheimer’s Disease, February 13, 2018. http:// investors.merck.com/news/press-release-details/2018/Merck-AnnouncesDiscontinuation-of-APECS-Study-Evaluating-Verubecestat-MK-8931-forthe-Treatment-of-People-with-Prodromal-Alzheimers-Disease/default.aspx (accessed Sept. 26, 2018). 72. The process mass intensity for the first generation process route was over 1200, while that for the commercial manufacturing process was only 267.

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Chapter 4

Discovery and Chemical Development of JNJ-50138803, a Clinical Candidate BACE1 Inhibitor Harrie J. M. Gijsen,1 Jinguang Lin,2 and Yannis Houpis*,1 1Janssen

Research & Development, Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium 2STA Pharmaceuticals, A WuxiApptec Company, 589 N. Yulong Rd., Changzhou, 213127, China *E-mail: [email protected]

BACE1 inhibition is hypothesized to be a potential disease-modifying treatment for Alzheimer’s Disease (AD). Multiple BACE1 inhibitors have progressed into clinical trials, but several have been terminated due to off-target side effects or lack of therapeutic efficacy, presumably due to targeting an AD population progressed too far into the disease. The need for a next generation of BACE1 inhibitors has led to the discovery of JNJ-50138803. The initial medicinal chemistry synthesis is presented, as well as the evolution to more scalable synthesis routes, incorporating multiple improvements. This has culminated into a synthesis route, proven suitable to prepare a multikilogram GMP batch.

Introduction Alzheimer’s Disease (AD) is the most common form of dementia and leads to neurodegeneration of the brain, causing progressive problems with memory, thinking, and behavior. It is a devastating disease with enormous impact, not only on the patient but also on family and caregivers. With an aging population, its prevalence is increasing and, currently, no medication is available that can prevent, slow, or stop the disease progression. A defining pathological characteristic of AD is the presence of amyloid plaques and tau tangles in the brain. Data regarding both

© 2018 American Chemical Society

genetic causes and sporadic disease support targets and pathways associated with amyloid pathology as a key therapeutic focus. The β-amyloid cleaving enzyme-1 (BACE1) is the first and rate-limiting step in the formation of β-amyloid peptides and therefore has been a prime target for drug development since its discovery in 1999 (1). The evolution of BACE1 inhibitors toward brain-penetrant drugs able to significantly reduce β-amyloid production in the brains of animals and humans has been covered in multiple reviews (2, 3). Several compounds have moved into advanced Phase 2b or 3 clinical trials, including verubecestat (4), lanabecestat (5), elenbecestat (6), and atabecestat (7) (Figure 1).

Figure 1. Lead BACE inhibitors in the clinic. AD drug development has proven to be extremely challenging, with disappointing results in all late-stage clinical trials thus far (8). Among those, the studies of amyloid interventions have mostly targeted relatively late stages of disease, which may have been too late to successfully intervene in the disease process. For BACE, the recent termination of the Phase 3 trials of verubecestat (9, 10) as well as lanabecestat (11) based on futility analyses can be seen as examples of this. BACE1 inhibition has been shown to more effectively suppress initiation of β-amyloid pathology than progression, stressing the need for early intervention to be therapeutically efficacious (12). In line with this, several ongoing trials with BACE inhibitors are targeting an earlier, even asymptomatic population (13). Considering the need for long-term treatment in a relatively healthy but elderly population, compounds will require a very safe side-effect profile. With multiple BACE compounds already terminated due to off-target side effects, atabecestat being the most recent example (14), the need for next-generation BACE inhibitors remains strong.

Discovery of JNJ-50138803 By the time we initiated our BACE1 inhibitor program, researchers had identified several key pharmacophoric elements required for the design of an optimal BACE1 inhibitor (2, 3), as evidenced by the structural similarities of 92

the inhibitors in Figure 1. These key pharmacophoric elements play important roles in the optimal interactions between the inhibitor and the enzyme. For example, the presence of an amidine-containing heterocycle in the part of the BACE1 inhibitor known as the warhead achieves an optimal hydrogen-bond interaction with the two aspartate residues in the catalytic site of the aspartic protease. A tertiary carbon connected to the amidine endocyclic nitrogen atom, as depicted with an asterisk in Figure 2, allows for efficient filling of the enzyme pockets by positioning the aryl substituents directly into the S1 and S3 pockets and the methyl substituent into the S2′ pocket. The amide linker between the two (hetero)aromatic rings also helps direct the heteroaryl ring deeply into the S3 pocket, with the amide-NH additionally making a hydrogen bond to the Gly230 backbone carbonyl of the protein. The requirement of central penetration necessitates modulation of the amidine pKa to a value of 6–8, which will still allow protonation at the site of actual enzyme inhibition. In addition, the modulation of pKa has been proven to be key in mitigating liabilities such as cardiovascular issues via inhibition of the human ether-à-go-go-related gene (hERG) potassium ion channel inhibition and drug–drug interactions (especially cytochrome P450 2D6 inhibition).

Figure 2. Pharmacophore of BACE1 inhibitors.

Our initial work (15) targeted a combination of all of these characteristics and resulted in amino-piperazinone 1 (Figure 3). Suboptimal brain penetration prompted further modification of the warhead, with a 1,4-oxazine providing a template with an intrinsically reduced amidine basicity, as well as carbon atoms, which would allow for modification via further substitution (16). While 1,4-oxazine analog 2 was a potent BACE1 inhibitor in vitro (IC50 22 nM), the compound was not able to reduce central amyloid beta levels in mice due to poor brain penetration, which could be attributed to strong P-glycoprotein (PgP)-mediated efflux. 93

Figure 3. Medicinal chemistry evolution toward JNJ-50138803. Further reduction of the amidine pKa by substitution of the 1,4-oxazine with electron withdrawing groups (EWGs) initially led to 2-fluoro-1,4-oxazine 3 (16). This compound displayed a reduced PgP-mediated efflux and resulted in highly effective reduction of β-amyloid levels in the brain and cerebrospinal fluid (CSF) of mice and dogs, respectively. However, progression of 3 was halted due to a still significant hERG inhibition observed in a functional electrophysiology experiment, the hERG patch clamp assay (56% at 3 µM). Subsequent in vivo studies in an anesthetized guinea pig showed that this hERG inhibition translated 94

into an unacceptable QTc prolongation in the electrocardiogram, leading to an insufficient safety margin for 3 (17). In addition, there were concerns around the chemical stability of the fluoro substituent. A number of variations were synthesized bearing alternative EWGs, providing warheads covering a range of pKa’s, as exemplified by compounds 4–7. CF3-substituted 4 had a pKa similar to 3 and maintained enzymatic and cellular potency. The less electron-withdrawing CHF2 substituent resulted in a pKa of 8.4 for 5, translating into an increased PgP-mediated efflux. Adding multiple EWGs as in 6 reduced the amidine pKa to 6.8, which led to a reduced cellular potency, as measured by inhibition in cells of the formation of the highly amyloidogenic peptide of 42 amino acids long: Aß42. The olefinic CF2-substituted 7 resulted from a side reaction in the synthesis route toward 4 (vide supra) and was an intermediate in the synthesis of 5. This compound showed a significantly reduced enzymatic potency, probably related to an altered and suboptimal conformation of the 1,4-oxazine ring. An optimal balance of potency, minimal efflux, and reduced hERG inhibition was found in the CF3-substituted 1,4-oxazines, and multiple analogs of 4 with various S3 pocket–targeting heteroaryl groups were prepared, including compounds 8 and 9 (Figure 3). Compound 8 was co-crystallized with the BACE1 enzyme, and the crystal structure of 8, solved at 1.94 Å resolution, is shown in Figure 4 (PDB code 6E3Z). This structure confirmed a binding mode similar to that seen for other BACE1 inhibitors, with a strong network of interactions of the catalytic aspartate dyad and the amidine moiety in 8 and optimal filling of the S1 and S3 pockets.

Figure 4. Crystal structure of 8 in BACE1 (amino acids 1 - 454) Protein Data Bank (PDB) code 6E3Z. 95

Further in vitro and in vivo profiling of the most promising compounds led to the selection of CF3-substituted 1,4-oxazine 9, or JNJ-50138803, as the preferred candidate (17). The PK/PD relationship of 9 in dog is shown in Figure 5, and these data were modeled to provide an estimated EC50 to reduce ß-amyloid peptide Aβ42 levels in dog CSF of 105 ng/mL (18). The minimal hERG patch clamp inhibition for 9 of 22% at 3 µM resulted in a significantly improved cardiovascular safety margin for 9 compared with 3, and toxicity studies rendered 9 sufficiently safe to progress to GMP synthesis of JNJ-50138803 in order to prepare for GLP toxicity studies and clinical trials (17).

Figure 5. Beagle dog plasma levels of 9 and effect on CSF Aβ 1-42 levels on day 1 and 6, after repeated oral dosing (fasted state, once daily dosing). Avg + SEM, n = 6/dose group. MS = MesoScale.

Medicinal Chemistry Synthesis of JNJ-50138803 A key structural feature of all leading BACE1 inhibitors, including JNJ-50138803, is a tertiary amine chiral center (2, 3). In our case, this is introduced by the amino acid intermediate 12 (Scheme 1), which was readily obtained from acetophenone 10 via a Strecker reaction and subsequent hydrolysis of the resulting α-aminonitrile 11. The acid group of 12 provided a versatile entry toward multiple variations of substituted BACE1 inhibitor warheads in the early phase of lead optimization, including all variations present in compounds 1–7 (Figure 3). At this point in the program, the desired stereochemistry of the tertiary carbon center was already established, and racemic 12 was resolved easily on a 100 g scale via chiral supercritical fluid chromatography (SFC) to obtain the desired enantiomer 13. Cyclization of amino acid 13 to morpholinedione 14 was achieved in moderate yield by acylation of the amino group with chloroacetyl 96

chloride followed by lactonization. This intermediate proved to be a useful precursor to prepare a range of substituted morpholino warheads.

Scheme 1. Initial medicinal chemistry synthesis route of JNJ-50138803. The trifluoromethyl group was introduced by the addition of the Ruppert−Prakash reagent (TMS-CF3) (19) in the presence of a catalytic amount of tetra-n-butylammonium difluorotriphenylsilicate (TBAT) to 14, to afford 15 as an inseparable and unassigned 3:1 diastereomeric mixture. The hydroxyl group was replaced with hydrogen by chlorination and subsequent reductive dechlorination using metallic zinc in acetic acid to provide 18 in 4:1 diastereomeric ratio, favoring the desired stereochemistry. The reproducibility of this reaction was poor, forming various amounts of chlorofluoro-eliminated 17 as a side product depending on the zinc source. While this allowed us to explore -CHF2- and =CF2-substituted analogs such as 5 and 7, respectively (Figure 3), a more reproducible outcome was clearly desirable. After careful analysis of the reaction conditions and zinc sources, the presence of copper impurities was found to be responsible for the formation of 97

17. Thus, using fresh zinc dust in acetic acid at 100 °C resulted in the selective reductive dehalogenation to 18, whereas using zinc-copper couple in acetic acid at room temperature resulted in almost quantitative formation of the chloro-fluoro elimination product 17. Conversion of amide 18 into amidine 20 was achieved by sequential thionation with P2S5, followed by aminolysis of the intermediate thioamide 19 with ammonia in methanol in a closed, pressurized vessel at elevated temperature. Aniline 21 was obtained in a moderate yield (40–60%) via amination of the bromoarene 20 under copper-catalyzed, Buchwald-type conditions using benzophenone imine as the nitrogen source and 1,2-dimethylethylenediamine (DMEDA) as metal chelator. A significantly higher yield (90%) was achieved using a copper-catalyzed reaction employing sodium azide, in which the intermediate aryl azide was immediately reduced to the corresponding aniline through the use of stoichiometric Cu(I)I. In the last step, acylation of the aniline 21 with 5-cyanopicolinic acid was achieved selectively using the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) (20) to produce JNJ-50138803.

Alternative Routes to JNJ-50138803 While the initial medicinal chemistry route enabled the synthesis of gram quantities of JNJ-50138803 as well as the exploration of other EWG-substituted analogs, the number of steps (11) and the low overall yield (1000

0.002

0.001

6

10.4

0.017

0.007

Process Chemistry of Ruzasvir Given the tight integration of process research and development in medicinal chemistry functions, the transition of ruzasvir from discovery to development was greatly expedited. A group of process and analytical chemists worked closely with medicinal chemists to identify opportunities to streamline SAR by enabling and inspiring design. For example, an early synthetic route to Z-group SAR is outlined in Scheme 1. Various aryl groups can be installed into core 53 via dialkylation of indole phenol 52 with α,α-dihalotoluene analogs. Subsequent introduction of sidechains via borylation-Suzuki would give the target 54. This original approach was instrumental in the discovery of elbasvir (17). However, the scarce availability of α,α-dihalotoluene analogs, especially those of heterocyclic scaffolds, made this approach impractical and tedious for the broader exploration of the Z-group introduction. The first attempt to make ruzasvir using this approach was unsuccessful due to the initial difficulty in preparing dihalomethyl cyclopropylthiazole from the aldehyde. Ideally, direct 133

aminal formation from readily available aldehydes is desirable to enable rapid investigation of design elements at this point in time.

Scheme 1. Key intermediates and routes developed during lead optimization. Disappointingly, the reaction of aldehydes with 55 resulted in undesired product 56 caused by cyclization at the C-3 position of the indole, not at nitrogen. Inspired by our previous efforts on the commercial processing of elbasvir (34, 35), we targeted a new intermediate 57 (Scheme 1) for SAR. The indoline 57 reacted with aldehydes to provide the desired cyclic aminal intermediate 58 which could be readily oxidized into indole 59 with DDQ. With this new approach, a very broad set of aldehydes was used to fully explore the SAR of the Z group, ultimately leading to the identification of the cyclopropylthiazole group in ruzasvir. We also incorporated a differentiated halogenation pattern (Cl, Br vs. Br, Br) which allowed medicinal chemists to explore both symmetrical and unsymmetrical designs for the proline and amino acid capping group via successive borylation/Suzuki. This synthetic strategy enabled and inspired the design of analogs which could not be prepared efficiently via the original route, further underscoring the importance that synthetic chemistry approaches play in the discovery of novel therapeutics. Intermediate 59 became a central part of our supply strategy for early delivery of API on a g scale. However, chiral SFC was required to provide the active diastereomer at the hemiaminal center of the API. As API demands started increasing, we noted that an enantioselective entry into the key benzoxazino-indole core 59 would really enable our team to commit to this approach for an API supply route for pre-clinical and clinical needs of ruzasvir. Clinical Supply Route to Ruzasvir Given that chiral relay of the indoline stereochemistry of 57 analogs to the aminal analogs 59 had previously been established (34, 35), our first priority was to devise a method for procuring the enantiopure indoline 57. The racemic 134

indoline 57A can be prepared very efficiently in four steps from 3-bromo-5-fluoro phenol 60 (Scheme 2) (30). We surmised that if an efficient classical resolution could be implemented on 57, we could avoid investing resources to develop an asymmetric route early in development and defer this investment for a commercial manufacturing route. We therefore screened several chiral acids in a number of solvents to try to identify a suitable resolving agent for 57. Gratifyingly, we identified (+)-DTTA (di-p-tolyltartaric acid) as a suitable acid, resulting in high recovery of the enantiopure indoline in ethanol. With this proof, we had confidence in being able to develop a chiral relay strategy that would enable access to the enantioenriched benzoxazino-indole core. We committed to further process development on this route as our supply path.

Scheme 2. Asymmetric synthesis of core 59A via chiral relay strategy. We started by developing a more robust synthesis of racemic 57A. Instead of a two-step sequence (30), direct heating of the phenol 61 and hydrazine HCl salt 62 in MsOH gave the desired indole product 55A in a 75% yield. Reduction of the indole 55A to racemic indoline 57A using Fe-TFA gave inconsistent results, and significant debromination was determined when Sn powder was used. However, we found that the slow addition of Sn powder to a solution of the indole 55A in MsOH gave a reduction product in the 66% yield with 99.9% ee. As expected, the chiral relay of the stereocenter in 57A through to the aminal occurred with high diastereoselectivity (>99.9% de). This was achieved by treating 57A with the thiaozle aldehyde 63 in MeCN with a catalytic amount of TFA. The desired diastereomer was crystallized directly out of the reaction mixture and similar to the elbasvir process (35); this crystallization-induced equilibrium of product diastereomers led to isolation of the desired isomer 58A in 88% yield and >99.9% de. However, the oxidation of the indoline 58A back to the indole 59A was suprisingly problematic. As observed with previous analogs, this reaction can 135

result in significant erosion of the aminal stereochemistry during the oxidation process (36). DDQ was used during SAR evaluation, and we found it gave almost complete epimerization at the aminal center even if an enantiopure starting material was used. Fortunately, as was the case for elbasvir (34, 35), the use of potassium permanganate, in combination with NaHCO3 as an oxidant, translated the high dr in 58A into a high ee for the indole core 59A with 66% yield. This unique selectivity may be explained by the possible ability of the permanganate anion to act as a hydrogen atom abstractor for the benzylic C–H bond (37, 38), whereas other oxidants might have resulted in formation of a radical cation, which could be susceptible to β-scission and racemization of the center (36). The Cl, Br-core 59A was readily converted to the bis-boronate 64 with Pd(OAc)2-XPhos as a catalyst. From here, we considered two options to complete the synthesis of razusvir (Scheme 3). First, there is a more linear approach, similar to the elbasvir approach, is the double-Suzuki of 64 with two Boc-protected bromo-imidazole fragments 66 followed by deprotection and final amide coupling. The advantage of this approach is it provides two more opportunities after Pd-catalyzed reactions to remove Pd, which was critical for elbasvir process due to a lack of API crystalline phase to remove Pd and other impurities.

Scheme 3. End game strategies to ruzasvir.

We preferred a second, more convergent approach via the double Suzuki reaction of 59A with fully elaborated bromo-imidazole sidechain 65 to provide ruzasvir in just one single step instead of three steps as in the linear approach. Saving two steps in the overall linear synthetic sequence could provide higher overall yields and shorter cycle times for API synthesis and production. While the double Suzuki with 65 worked well, with two sequential Pd-catalyzed reactions as the last two steps, removal of Pd in the API presented a significant challenge on a large scale. This was exacerbated by the lack of a crystalline form of the API at the time. To solve this problem, identifying a highly crystalline phase of ruzasvir or its salt became critical. 136

Ruzasvir itself did not have a crystalline form and only formed a dioxane solvate as a free base in dioxane solvent. However, dioxane is highly toxic and its purity upgrade was unsatisfactory. Turning our attention to acid salt formation, we surveyed over 20 acids and found that (S)-mandelic acid formed a crystalline salt of ruzasvir, which provided a great chemical purity upgrade in high recovery as well as efficient Pd removal. This discovery was key to committing to the convergent approach end game. The double Suzuki reaction with bromo-imidazole sidechain 65 was interrogated via high-throughput experimentation using various catalysts, solvents, and bases. Preformed catalyst Pd(AmPhos)2Cl2 in DME-water and K2CO3 gave the best results. After the reaction and work-up, the (S)-mandelate salt of ruzasvir was isolated in 85% yield and high purity with 50% waste. As the drug development continued and more API was required, an asymmetric route was highly desirable. One option was to develop an asymmetric route to indoline 57A via asymmetric transfer hydrogenation of imine 70 followed by copper-catalyzed cyclization to the indoline (34). We quickly prepared the imine substrate 70 and then established proof of concept of this approach (Scheme 4).

Scheme 4. Asymmetric synthesis of indoline core 57A via asymmetric transfer hydrogenation. Any chiral relay approach inherently suffers from redox inefficiencies during the sequence of indole synthesis, reduction to indoline, and reoxidation to indole. An ideal manufacturing route would avoid such redundant redox sequences to improve Process Mass Intensity (PMI), which is calculated by total kg of all reagents and solvents used in the entire synthesis to make 1 kg of API, as well as supply chain velocity and cost of goods. To take advantage of the efficient synthesis of indole phenol 55A, we explored a few asymmetric cyclization approaches to access benzoxazino-indole 59A (Scheme 5). First, we attempted (chiral) acid-catalyzed condensation with aldehyde, but only C,O cyclization occurred after extensive screening due to the high C3-reactivity of the indole under acidic conditions. We achieved a moderate yield of the desired N, O cyclization under basic conditions with stoichiometric 137

LiHMDS and ClP(O)(OEt)2, but a catalytic asymmetric version presented a significant challenge.

Scheme 5. Approaches to prepare core 59A from indole phenol 55A.

We were also able to prepare the gem-dichloromethyl derivative from the corresponding aldehyde and explored an asymmetric phase-transfer-catalyzed process. Much to our delight, we achieved ~40% yield and 70% ee via the dimeric intermediate 74. However, due to competing N, O, and C3-alkylation, further optimization of this reaction did not lead to significant improvement despite extensive efforts. At this point, any asymmetric approach involving the cyclization of the indole phenol was deemed overly difficult due to the inherent limitations of the indole reactivity. To address the redox inefficiencies and the difficulty in accessing the enantio-enriched benzoxazino-indole core from the indole 54, we imagined a retrosynthetic disconnect that builds the indole ring last and obviates the redox steps via first formation of the hemiaminal, followed by the C–N bond formation reaction. Even though the two steps have scarce precedents even as racemic reactions, we were particularly attracted by this possibility since it offered two potential opportunities to develop asymmetric catalytic processes for setting the key hemiaminal stereocenter (Scheme 6). We chose the dichloro-substrate (70B, X = Cl) to avoid potential halide selectivity issues in the subsequent indole formation step. The racemic hemiaminal formation with aldehyde 63 proceeded smoothly in the presence of TFA to form 75. While precedents for similar asymmetric transformations are rare, they mostly rely on chiral Bronsted acid-catalyzed reactions. We carried out exhaustive high-throughput experimentation with the limited commercially available chiral Bronsted acid catalysts under a variety of conditions. However, we were disappointed with not being able to identify conditions that could surpass 20% ee selectivity. It was clear that a significant catalyst design and synthesis campaign was needed to achieve acceptable results. 138

Scheme 6. Retrosynthetic design for commercial route to ruzasvir. Prior to initiating this effort, we elected to study the stereochemical integrity of benzoxazine 75 in the subsequent C–N bond-forming reaction. Through chiral SFC, we obtained 75 in >90% ee. When a small suite of Cu- and Pd-catalyzed reactions was attempted on 75, we were gratified to see that we could get very high conversion to the indole 59B for this unprecedented reaction. However, to our surprise, products from all reactions were obtained in complete racemic form. This seemed to be the end of this approach as we could not achieve high ee in the first step or maintain the ee in the second step (Scheme 7).

Scheme 7. C-N coupling to form indole 59B. Before moving away from this approach, we decided to investigate the cause of this unexpected racemization. We knew that the product was stable under the reaction conditions, and we suspected the starting material racemized under the basic conditions, despite the proton at the hemiaminal center not appearing to be acidic enough for base-induced racemization. When treating the starting material with K3PO4, we indeed observed the ring-opening intermediate 76 by NMR, which is likely the cause of the racemization of the starting material. This pivotal result inspired us to take a completely different approach to setting the hemiaminal stereocenter. Given the stereochemical liability of the benzoxazine 75 under reaction conditions and that the product retained complete stereochemical integrity, we envisioned enantioselective formation of the hemiaminal with concomitant arylation of the nitrogen, directly yielding the benzoxazino-indole present in 139

ruzasvir. In our design, a transition metal catalyst (such as a palladium/chiral phosphine complex), capable of catalyzing the Buchwald−Hartwig C−N coupling, would also control the formation of the stereogenic center at the α-positon of the incipient C–N bond, thereby setting the hemiaminal stereocenter starting from racemic hemiaminal 75 (Scheme 8).

Scheme 8. Design plan for enantioselective synthesis of hemiaminals via Pd-catalyzed C−N coupling. Another key advantage of this approach was the very large number of readily available chiral phosphines that we could investigate to achieve high ee. This is in stark contrast to the rather limited number of chiral Bronsted acid catalysts currently commercially available. We quickly investigated this design by using benzoxazine 75 under palladium catalysis (10 mol%), with potassium phosphate as the inorganic base, against a suite of 192 chiral phosphines in our collection. For this C–N coupling with no α-hydrogen on the amine to reduce Pd(II) to Pd(0), usually either a Pd(0) source is used or Pd(II) needs to be pre-reduced by other reductants such as the phosphine ligand. With the hope for asymmetric transformation, we did not intend to use chiral ligands as the reductant, which could potentially modify these ligands. We initially chose Pd2dba3 as the palladium source with limited success. (R,R)-QuinoxP* (Scheme 9) did show promise in ee but had very poor conversion. Quite importantly, with the ability to efficiently screen hundreds of reactions at a time, we ran the same screen with Pd(OAc)2 as with a palladium precursor. We were surprised and delighted to identify several ligands which provided excellent levels of enantioselectivity for our desired indole 59B. QuinoxP* turned out to be the best at 100% conversion and 92% ee (Scheme 9) (39). 140

Scheme 9. Pd-catalyzed C–N coupling to form indole 59B. The increased ee and conversion from a Pd(0) source to a Pd(II) source with QuinoxP* made us wonder whether it was possible that the active catalyst may bear a bisphosphine monoxide, which could form in situ by the oxidation of the ligand by Pd(II). We undertook independent synthesis of the monoxide of the best ligand (QuinoxP*) and confirmed that this new ligand did give 100% conv and 92% ee with Pd2dba3, the Pd (0) precursor now. Reexamination of our data set also provided additional evidence for this hypothesis. It was quite clear at this point that the active ligand was QuinoxP* monoxide, which was generated in situ by oxidation with Pd(OAc)2. This represents a quite efficient double activation of both the Pd(II) source and the ligand to the active catalyst; this was further confirmed by subsequent mechanistic studies (40). With the promising screening results at the mg scale, we started process development of this reaction but encountered many challenges. When we ran this reaction at the g scale with mechanical stirring, the conversion often stalled at ~50–70%. Based on prior knowledge in Pd-catalyzed C–N coupling in toluene with an inorganic base such as K3PO4, we attributed the issue to possible loss of water during the reaction under non-sealed conditions (41). We discovered previously that the addition of water could help accelerate the cross-coupling reaction by solubilizing the inorganic base. We were then glad to see that the addition of 4 equiv of water in portions during the course of this reaction reestablished >99% conversion on scale. However, we still found the reaction to be inconsistent. After an extensive study of various reaction parameters, we again narrowed it down to the effect of water, although on this occasion, the water played a detrimental role in the activation of the catalyst. Pd(OAc)2 and QuinoxP* were typically mixed in toluene to form a Pd(II)QuinoxP* complex 77, which was then added to the starting material 75 and base in toluene. We found that the longer the catalyst solution was aged, the lower the conversion in the subsequent reaction. When the water content in the toluene solvent (as measured by a Karl-Fischer titrator) increased from 25 ppm to 100 ppm, weaker conversions occurred. We reasoned that water accelerated the catalyst dual activation to form 78, which is 141

highly active and decomposes in the absence of an aryl bromide to trap it. As a result, controlling the water content in toluene is important for catalyst activity. For a more robust process that can tolerate a slightly higher water content, we added portions of the starting material during the catalyst preparation to trap any activated catalyst 78, and indeed the catalyst activity was restored. Addition of all of the starting material upfront, however, led to slightly lower conversion, possibly due to competitive binding of the starting material to the Pd(II) precursor (Figure 9).

Figure 9. Impact of water content during the catalyst preparation (2% Pd).

By minimizing the amount of water during catalysis and conversely adding water during the reaction, we were able to consistently run this reaction in multi-kg scale in the plant to support the program. However, due to the high cost of QuinoxP* ($45,000/kg), we then turned our attention to reducing the catalyst loading. It was clear that in order to lower the catalyst loading from the 2.5% to 5% mol required for scale-up, we would need to study both catalyst activation and deactivation to gain an in-depth understanding and design for our process. We first identified four catalyst deactivation pathways (Scheme 10): •

Once active catalyst 78 forms, it needs to be trapped by an aryl halide to avoid decomposition. Under the current reaction system, the starting material traps the active catalyst to form the oxidative addition complex 79, but it then quickly continues to react to form the product and HBr/ KBr byproduct, which react with unactivated catalyst complex 77 to form complex 81, a much less active catalyst. 142

•

•

•

The catalyst can be deactivated to complex 84 via either the hydrolysis of complex 85 or trapping of the activated catalyst 78 by imine 83 from the hydrolysis of the starting material open form 76. Due to a small amount (~0.3%) of dibromo-analog in the starting material 75 carried over from its precursor 70B, the active catalyst can be trapped by the more active bromide to form an inactive complex 82. We have also seen evidence of product inhibition via oxidative addition into the aryl chloride with increased levels of complex 82 (X = Cl) toward the end of the reaction when the product concentration is much higher than that of the starting material 75.

Scheme 10. Catalytic cycle and decomposition pathways. To address these issues, we needed: • • •

a more efficient catalyst activation/trapping to avoid formation of the less active catalyst 81, a way to reactivate inactive catalyst complex 82 from either impurity trapping or product inhibition, and conditions to slow down the hydrolysis of either complex 85 or ring-open form 76.

To trap the active catalyst more efficiently without forming any HBr/KBr byproduct that could deactivate the Pd(II) catalyst precursor 77, we chose an activated aryl halide such as iodobenzene or para-bromobenzonitrile. The oxidative addition of complex 86 can be prepared in >80% yield as an air-stable compound that is even stable to silica gel chromatography. This complex can then 143

be activated by 2-tolylboronic acid via a Suzuki reaction under the same reaction conditions to generate active catalyst 78 in situ. Very importantly, 2-tolylboronic acid also reactivates the deactivated Pd complex 82 from di-bromo-impurity and product inhibition via Suzuki. To minimize the hydrolysis, we used 10% of pivalic acid as a soluble proton shuttle and only 2.5 equivalents of K3PO4 to increase the reaction rate instead of excess amounts of water and K3PO4. Combining all these improvements, we were able to lower the catalyst loading to ~0.5%, a 5- to 10-fold reduction from the previous system (Scheme 11). This mode of activation of bisphosphine ligand to its monoxide and use of an arylboronic acid to insert these stable oxidative addition complexes into the catalytic cycle can potentially have broader applications for other bisphosphine ligands, especially in asymmetric catalysis.

Scheme 11. New catalyst design.

In summary, the novel Pd-catalyzed asymmetric dynamic C–N cyclization reaction enabled the synthesis of ruzasvir in six linear steps (Scheme 12). A high yielding three-step sequence was developed for the synthesis of starting material 75 (42). The asymmetric C–N cyclization gave the di-chloro product 59B in 92% ee, which was then upgraded to >99% ee by salt formation with (S)-CSA (camphorsulfonic acid). Double borylation then provided bisboronate 64 using a Pd(OAc)2-XPhos catalyst just like the Cl,Br-analog in the clinical supply route. The final double Suzuki reaction of the bisboronate 64 with the fully elaborated side chain 65 produced ruzasvir in high yield and purity (Scheme 12). After extensive process development to improve process efficiency and robustness, this route was successfully scaled up in yields consistent to lab scale to provide multikg quantities of ruzasvir for late stage clinical development.

144

Scheme 12. Manufacturing route to ruzasvir.

Figure 10. Mean RNC reduction during a 5-day monotherapy with 60 mg MK-8408 in HCV-infected patients-preliminary data (n = 3/arm). (see color insert)

145

Figure 11. Efficacy of ruzasvir + grazoprevir + uprifosbuvir in HCV patients who previously failed therapy with either Harvoni or Zepatier (43). (see color insert)

Conclusions Ruzasvir was evaluated in phase I and II clinical trials for the treatment of HCV. In a 5-day monotherapy trial, 60 mg of ruzasvir demonstrated potent antiviral activity and achieved a >3 log mean maximal viral load reduction in HCV patients with GT1, 2, and 3 (Figure 10) (43). Ruzasvir was also efficacious when combined with grazoprevir and uprifosbuvir in patients who failed with Harvoni® or Zepatier,® as shown in Figure 11 (44). Ruzasvir was evaluated in clinical trials as part of an all-oral, interferon-free FDC regimen with grazoprevir and uprifosbuvir; this trial was administered once-daily for the treatment of chronic HCV infection. On September 29th, 2017, Merck announced its decision to discontinue the development of the investigational combination regimens MK-3682B (grazoprevir/ruzasvir/ uprifosbuvir) and MK-3682C (ruzasvir/uprifosbuvir) for the treatment of chronic HCV infection. This was based on the review of available phase II efficacy data, when considering the evolving marketplace and the growing number of treatment options available for patients with chronic HCV infection (45).

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Ingravallo, P.; Nomeir, A.; Asante-Appiah, E.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2016, 26, 5132–5137. Yu, W.; Coburn, C. A.; Nair, A. G.; Wong, M.; Tong, L.; Dwyer, M. P.; Hu, B.; Zhong, B.; Hao, J.; Yang, D.-Y.; Selyutin, O.; Jiang, Y.; Rosenblum, S. B.; Kim, S. H.; Lavey, B. J.; Zhou, G.; Rizvi, R.; Shankar, B. B.; Zeng, Q.; Chen, L.; Agrawal, S.; Carr, D.; Rokosz, L.; Liu, R.; Curry, S.; McMonagle, P.; Ingravallo, P.; Lahser, F.; Asante-Appiah, E.; Nomeir, A.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2016, 26, 3800–3805. Dwyer, M. P.; Keertikar, K. M.; Chen, L.; Tong, L.; Seluytin, O.; Anilkumar, G. N.; Yu, W.; Zhou, G.; Lavey, B. J.; Yang, D.-Y.; Wong, M.; Kim, S. H.; Coburn, C. A.; Rosenblum, S. B.; Zeng, Q.; Jiang, Y.; Nomeir, A. A.; Liu, R.; Agrawal, S.; Xia, E.; Kong, R.; Zhai, Y.; Ingravallo, P.; Asante-Appiah, E.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2016, 26, 4106–4111. Yu, W.; Zhou, G.; Coburn, C. A.; Zeng, Q.; Tong, L.; Dwyer, M. P.; Chen, L.; Mazzola, R.; Kim, J.-H.; Sha, D.; Selyutin, O.; Rosenblum, S. B.; Lavey, B.; Nair, A. G.; Kim, S. H.; Keertikar, K. M.; Masse, F.; Agrawal, S.; Liu, R.; Xia, E.; Zhai, Y.; Curry, S.; McMonagle, P.; Ingravallo, P.; Asante-Appiah, E.; Chen, S.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2016, 26, 4851–4856. Tong, L.; Yu, W.; Coburn, C. A.; Chen, L.; Selyutin, O.; Zeng, Q.; Dwyer, M. P.; Nair, A. G.; Shankar, B. B.; Kim, S. H.; Yang, D. Y.; Rosenblum, S. B.; Ruck, R. T.; Davies, I. W.; Hu, B.; Zhong, B.; Hao, J.; Ji, T.; Zan, S.; Liu, R.; Agrawal, S.; Carr, D.; Curry, S.; McMonagle, P.; Bystol, K.; Lahser, F.; Ingravallo, P.; Chen, S.; Asante-Appiah, E.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2016, 26, 5354–5360. Yu, W.; Coburn, C. A.; Nair, A. G.; Wong, M.; Tong, L.; Dwyer, M. P.; Hu, B.; Zhong, B.; Hao, J.; Yang, D. Y.; Selyutin, O.; Jiang, Y.; Rosenblum, S. B.; Kim, S. H.; Lavey, B. J.; Zhou, G.; Rizvi, R.; Shankar, B. B.; Zeng, Q.; Chen, L.; Agrawal, S.; Carr, D.; Rokosz, L.; Liu, R.; Curry, S.; McMonagle, P.; Ingravallo, P.; Lahser, F.; Asante-Appiah, E.; Nomeir, A.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2016, 26, 3800–3805. Yu, W.; Coburn, C. A.; Nair, A. G.; Wong, M.; Rosenblum, S. B.; Zhou, G.; Dwyer, M. P.; Tong, L.; Hu, B.; Zhong, B.; Hao, J.; Ji, T.; Zan, S.; Kim, S. H.; Zeng, Q.; Selyutin, O.; Chen, L.; Masse, F.; Agrawal, S.; Liu, R.; Xia, E.; Zhai, Y.; Curry, S.; McMonagle, P.; Ingravallo, P.; Asante-Appiah, E.; Lin, M.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2016, 26, 3414–3420. Yu, W.; Tong, L.; Hu, B.; Zhong, B.; Hao, J.; Ji, T.; Zan, S.; Coburn, C. A.; Selyutin, O.; Chen, L.; Masse, F.; Rokosz, L.; Liu, R.; Curry, S.; McMonagle, P.; Ingravallo, P.; Asante-Appiah, E.; Chen, S.; Kozlowski, J. A. J. Med. Chem. 2016, 59, 10228–10243. Tong, L.; Yu, W.; Chen, L.; Selyutin, O.; Dwyer, M.; Nair, A.; Mazzola, R.; Kim, J.; Sha, D.; Yin, J.; Ruck, R.; Davies, I.; Hu, B.; Zhong, B.; Hao, J.; Ji, T.; Zan, S.; Liu, R.; Agrawal, S.; Xia, E.; Curry, S.; McMonagle, P.; Bystol, K.; Lahser, F.; Carr, D.; Rokosz, L.; Ingravallo, P.; Chen, S.; Feng, K.; Cartwright, M.; Asante-Appiah, E.; Kozlowski, J. J. Med. Chem. 2017, 60, 290–306. 148

31. Neau, E.; Dansette, P.; Andronik, V.; Mansuy, D. Biochem. Pharmacol. 1990, 39, 1101–1107. 32. McMurtry, R.; Mitchell, J. Toxicol. Appl. Pharmacol. 1977, 42, 285–300. 33. Dansette, P.; Amar, C.; Smith, C.; Pons, C.; Mansuy, D. Biochem. Pharmacol. 1990, 39, 911–918. 34. For a chiral relay strategy to elbasvir, see: Mangion, I.; Chen, C. Y.; Li, H.; Maligres, P.; Chen, Y.; Christensen, M.; Cohen, R.; Jeon, I.; Klapars, A.; Krska, S.; Nguyen, H.; Reamer, R. A.; Sherry, B. D.; Zavialov, I. Org. Lett. 2014, 16, 2310–2313. 35. Li, H.; Chen, C. Y.; Nguyen, H.; Cohen, R.; Maligres, P.; Yasuda, N.; Mangion, I.; Zavialov, I.; Reibarkh, M.; Chung, J. J. Org. Chem. 2014, 79, 8533–8540. 36. Yayla, H. G.; Peng, F.; Mangion, I. K.; McLaughlin, M.; Campeau, L. C.; Davies, I. W.; DiRocco, D.; Knowles, R. R. Chem. Sci. 2016, 7, 2066–2073. 37. Garder, K. A.; Kuehnert, L. L.; Mayer, J. M. Inorg. Chem. 1997, 36, 2069–2078. 38. Mayer, J. M. Acc. Chem. Res. 1998, 31, 441–450. 39. Li, H.; Belyk, K. M.; Yin, J.; Chen, Q.; Hyde, A.; Ji, Y.; Oliver, S.; Tudge, M. T.; Campeau, L. C.; Campos, K. R. J. Am. Chem. Soc. 2015, 137, 13728–13731. 40. Ji, Y.; Li, H.; Hyde, A. M.; Chen, Q.; Belyk, K. M.; Lexa, K. W.; Yin, J.; Sherer, E. C.; Williamson, R. T.; Brunskill, A.; Ren, S.; Campeau, L. C.; Davies, I. W.; Ruck, R. T. Chem. Sci. 2017, 8, 2841–2851. 41. Yin, J.; Zhao, M. M.; Huffman, M. A.; McNamara, J. M. Org. Lett. 2002, 4, 3481–3484. 42. For a general method, see: Qi, J.; Oliver, S. F.; Xiao, W.; Song, L.; Brands, J. K. M. Org. Process. Res. Dev. 2017, 121, 1547–1556. 43. Asante-Appiah, E.; Marshall, W.; Gane, E.; Popa, S.; McMonagle, P.; Curry, S.; Maganti, L.; Gao, W.; Garrett, G.; Cilissen, C.; De Lepeleire, I.; Black, T.; Hazuda, D.; Butterton, J. J. Hepatol. 2016, 64, S401–S402. 44. Wedemeyer, H.; Wyles, D.; Reddy, R.; Luetkemeyer, A.; Jacobson, I.; Vierling, J.; Gordon, S.; Nahass, R.; Zeuzem, S.; Wahl, J.; Barr, E.; Nguyen, B.; Robertson, M.; Joeng, H.; Liu, H.; Jumes, P.; Dutko, F.; Martin, E. J. Hepatol. 2017, 66, S85. 45. Merck & Co., Inc. Merck Discontinues MK-3682B and MK-3682C Development Programs; Press Release; Merck & Co., Inc: Kenilworth, NJ, September 29, 2017. http://investors.merck.com/news/press-release-details/ 2017/Merck-Discontinues-MK-3682B-and-MK-3682C-DevelopmentPrograms/default.aspx (accessed June 6, 2018).

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Chapter 6

AZD6564, Discovery of a Potent 5-Substituted Isoxazol-3-ol Fibrinolysis Inhibitor and Development of an Enantioselective Large-Scale Route for Its Preparation Staffan Karlsson,1 Daniel Pettersen,2 and Henrik Sörensen*,2 1Early

Chemical Development, Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca, SE-431 83 Gothenburg, Sweden 2Medicinal Chemistry, Cardiovascular, Renal and Metabolic Diseases, IMED Biotech Unit, AstraZeneca, SE-431-83 Gothenburg, Sweden *E-mail: [email protected]

We report the discovery and scale up of AZD6564, an oral fibrinolysis inhibitor for treatment of heavy menstrual bleeding. From virtual screening hits, our target compound AZD6564 was discovered by focusing on improving potency, permeability, and selectivity toward GABAa activity. AZD6564 was selected as a candidate for development into clinical studies. The first-generation synthesis of AZD6564 to prepare milligram to gram quantities for in vitro screening relied on a tedious multistep linear synthesis in which chromatography was employed in several steps. Moreover, since no asymmetric approach was identified to install the chiral centers, enantiopure material was obtained after chiral column chromatography. The development of a practical route to the target molecule without recourse to chromatography is described.

Introduction The clotting process, coagulation, is essential in preventing massive blood loss (1). Clotting factors and platelets clump together to form a plug at the injured blood vessel, forming a fibrin clot that stops bleeding and prevents substantial © 2018 American Chemical Society

blood loss. After wound repair, the fibrinolysis mechanism results in dissolution of the fibrin clots to restore normal blood vessel function. Any irregularity in this process can lead to a bleeding disorder. Symptoms of bleeding disorders may include heavy menstrual bleeding (menorrhagia), frequent nosebleeds, excessive bleeding from small cuts, and easy bruising. For example, heavy menstrual bleeding affects 1 in every 5 women; and as such it is one of the most common gynecological complaints (2). There are several medical options available to treat or prevent bleeding disorders and their complications, including iron supplements, nonsteroidal anti-inflammatory drugs (NSAIDs), blood transfusion, contraceptives, and hormonal treatment. One of the most considered treatments of bleeding disorders is the use of fibrinolysis inhibitors, usually represented by ε-aminocaproic acid (EACA) and tranexamic acid (TXA) (Figure 1) (3). They act by blocking the lysine binding site in plasminogen, a key protein in the fibrinolysis mechanism (4). Tranexamic acid (Lysteda©, Cyklo-F©, and Femstrual©) was approved in the United States in 2009 for the treatment of heavy menstrual bleeding, and since 2011 has also been available in the United Kingdom (5). Because its treatment is considered to be cost-effective in many countries, it has been added to the WHO list of essential medicines (6). However, both EACA and TXA suffer from the need of high daily doses (7, 8). High doses of TXA have been reported to lead to gastrointestinal side effects, and there are some rare reports of seizures, hypothesized to be mediated by GABAa activity (9).

Figure 1. Fibrinolysis inhibitors acting by binding to the lysine binding site in plasminogen.

Despite attempts by several companies to find alternative treatments in this area, no other plasminogen lysine binding site inhibitors have reached the market. Therefore, we set out on an internal program to search for novel oral fibrinolysis inhibitors with reduced daily dose and without GABAa-mediated side effects.

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Discovery of AZD6564 TXA, being a relatively small and polar compound with a distinct dipole moment, binds to the lysine binding pocket in the kringle 1 domain of plasmin (10). TXA, a zwitterion, fits the characteristics of the binding pocket very well, as the pocket is quite shallow and heavily polar; thus, it resembles the shape of a typical flashlight battery, with a positive end (piperidine nitrogen) and a negative end (carboxylic acid). Using TXA as the model ligand, we initiated a virtual screen from which the known compound 5-(4-piperidinyl)-1,2-oxazol-3(2H)-one (4-PIOL ) 1 and the pyrazolol 2 were identified as lysine pocket binders (Table 1) (11).

Table 1. Data for Compounds 1 to 3 and Reference Compounds

Key for the interaction of compounds 1 and 2 with the lysine binding site was the positively charged nitrogen in the piperidine interacting with two aspartates (ASP54, 56) and the polar heterocycle’s (i.e., for compound 1 represented by the isoxazol-3-ol moiety) interaction with two arginines (ARG34, 70) (Figure 2).

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Figure 2. Docking overlay of TXA (brighter area) and 4-PIOL 1 (darker area ) in the binding site of plasmin(ogen). Reprinted with permission from reference (10). Copyright 2013 American Chemical Society. Compounds 1 and 2 displayed encouraging potencies, with 1 displaying a 4fold higher potency compared to TXA in a clotlysis assay in human plasma (Table 1). Because of their relatively low molecular weight, they were promising hit molecules from a ligand efficacy (LE) perspective. However, both 1 and 2 suffered from increased activity toward the GABAa receptor compared to TXA (12). In a GABAa binding assay, 1 displayed an IC50 of 35 µM compared to 1600 µM for TXA. Furthermore, both 1 and 2 also suffered from low permeability, as measured in Caco-2 cells (A to B 340:1 cis/trans ratio (Scheme 10). Due to a tight time schedule, we could not evaluate asymmetric hydrogenations, but rather relied on finding a traditional resolution in a downstream step.

Scheme 10. Hydrogenation of 20 to give 21.

Thus, 400 g of pyridine hydrochloride 20 was hydrogenated at ~ 20 bar in methanol with platinum oxide as catalyst. It is important that removal of the spent catalyst takes place under inert conditions as the platinum black formed during the reaction is very pyrophoric. This problem was solved by connecting the bottom outlet of the reactor directly to a homemade filter made from an HPLC column (diameter = 47 mm) with the packing material removed and a Celite® filter cut into shape at the bottom of the column. Analytical samples of the reaction mixture were removed from the top inlet of the reactor. When HPLC-MS and NMR spectra indicated complete reaction, the inlet was closed and the bottom valve to the filter was opened. Pressurizing the reactor with 20 bar of nitrogen forced the mixture through the homemade filter with no back pressure issues to yield a clear filtrate. After line washes with methanol, the reactor and the HPLC filter were then cleaned with water to quench the catalyst. After the water treatment, the removal of the catalyst was a safe procedure.

Eventful Hydrogenations After the trip to our plant in Södertälje, we had 4.4 kg of 20. The factory did not have a working reactor for the hydrogenation and thus the material was transported to our facility in Mölndal for this step. The maximum volume of our dedicated reactor was 5 L, allowing us to run the hydrogenation in three batches of ~ 1.5 kg of 20 in each batch. During our first run at this scale, we used 1.7 mol% of platinum oxide and a starting pressure of 38 bar. Hydrogenation overnight displayed complete consumption of starting material. Thus, the conclusion was drawn that we had probably used an unnecessary excess of catalyst. Consequently, for our second batch on this scale, only 1.05 mol% platinum oxide was used. However, this time hydrogenation overnight still indicated 25% of starting material remained. 166

We decided to add more platinum oxide in order to complete the hydrogenation expediously; that required the addition of the oxide under inert conditions since our reactor contained about 13 g of very fine pyrophoric platinum black. The attempted addition of the finely powdered platinum oxide via a funnel with a back flow of nitrogen resulted in an unfortunate cloud of catalyst geysering above the funnel. Now, accidently 20 bar of nitrogen was blown into the reactor which was virtually full of reaction mixture containing methanol and platinum black. A fair amount of the contents burst out through the addition funnel and sprayed the wall. Instantaneously, the ejected reaction mixture caught fire. The fumes set off the fire alarm and the entire building, with about 200 staff, was evacuated. After the fire brigade had switched off the alarm and the material that remained in the reactor was fully hydrogenated, the third hydrogenation was initiated. But another challenge arose. We were fully aware that as a consequence of the extraordinary diffusion rate of hydrogen and working with a hydrogen pressure of ~ 38 bar, there is no room for imperfections in seals and gaskets. All nuts and bolts had to be tightened carefully and even the tiniest leak would either mean a consumption of hydrogen or a worse scenario. Still, after these precautions, a 3 cm flame was visible standing straight up from the reactor, most likely due to ignition of a minor leak of hydrogen in the presence of platinum black. It was indeed stressful to have an open flame during a hydrogenation operation, but after discussions with our safety officer, we decided that we could tolerate the small flame. Finally, the three batches were combined and after crystallization, the pure (340:1 cis:trans) hydrochloride (82% assay yield) was isolated. Considering the losses due to the fire, we were very pleased with the overall yield.

Resolution of the Piperidine: Enzymatic or Salt Resolution For the reasons previously described, and since very little time was available for identifying an asymmetric route, the chosen route was based on the production of racemic piperidine cis-(±)-21 followed by resolution. Classical and enzymatic resolution approaches were investigated in parallel and evaluated for a large-scale campaign (Scheme 11). For a classical resolution, we screened 21 against an inhouse library of homochiral acids (Figure 3).

Scheme 11. Resolution of cis-(±)-21.

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Figure 3. Commercially available chiral acids used for quick-screen resolution of racemic amines.

Thus, the homochiral acids (0.5–0.7 equiv) were added to the racemic amine in two different solvents (e.g., EtOH and EtOAc), followed by stirring for 24 h. If precipitation occurred, the mixture was filtered and the solid collected was analyzed by chiral HPLC. In this case, it was found that the phosphoric acid 22 gave an almost diastereomerically pure precipitate just after one crystallization. After free-basing/extraction with Na2CO3/EtOAc, (2R,4S)-21 was obtained in >97% ee (as determined by its Mosher amide) and 35% yield (70% of theory). After some experimentation, we found the best conditions to be a 1:2 ratio of methanol:ethyl acetate as solvent. The resolution gave a consistent result also at 200 g scale. However, we were concerned about the accessibility of the chiral phosphoric acid in larger amounts and at a reasonable cost. As a backup plan, we considered the possibility to recycle the phosphoric acid which could be done through extractions. However, we first wanted to await the result from the enzymatic resolution approach. Among all classes of available enzymes, the lipases are known to be straightforward to apply in resolutions. This is especially true of the immobilized 168

enzymes, which can be removed from reaction mixture after completion just through a simple filtration. The immobilized Candida antarctica B enzyme (e.g., Cal B, Novozyme 435) is a well-known lipase that has found wide applicability in various organic transformations. We decided upon using it as a starting point to evaluate the enzymatic resolution of cis-(±)-21. Thus, in a 0.66 M KH2PO4 buffer system and NaOH solution adjusted to pH 8, the substrate was dissolved and Novozyme 435 was added. We were happy to discover that enantioselective hydrolysis did indeed occur; however, luck was not completely on our side since it was the undesired enantiomer that was hydrolyzed. The enantioselectivity, however, was excellent furnishing the unreacted ester (2R,4S)-21 in 34% assay yield and 97% ee. Acid (2S,4R)-23 was removed, together with the immobilized enzyme, by a fairly time-consuming filtration, followed by extraction of our desired ester (2R,4S)-21 using MTBE. The resolution was scaled up and run on 13.8 mol (3.6 kg) scale with similar results. Having established two good approaches for the resolution of (±)-21, we had to choose one of them for further scale up. Both of these approaches were considered good enough to be used on multikilogram scale, but as Novozyme 435 is more readily available at a low cost compared with chiral phosphoric acid 22, it was a straightforward decision in favor of the enzymatic resolution (Scheme 12).

Scheme 12. Resolution of cis-(±)-21 using two different routes. Adapted with permission from reference (23). Copyright 2014 American Chemical Society. Protection of Piperidine (2R,4S)-21 as Carbamate 24 With the establishment of the chiral centers, protection for the amine was next required. As mentioned, protecting groups requiring hydrogenolysis for their removal (e.g., a benzyl carbamate group) were not feasible since 5-substituted isoxazol-3-ols are known to cleave under such conditions (24). Furthermore, 169

we speculated that a larger and more malleable protective group was likely to jeopardize crystallizations of the analog for intermediate (2R,4S)-13, which were important in order to avoid chromatographic purifications. Owing to time constraints, for the initial small deliveries we settled for the established methyl carbamate group as a protecting group (Scheme 13). The carbamate was introduced in quantitative yield using 2-MeTHF, DIPEA, and methyl chloroformate at 0 °C. During an 80 g initial scale up of this step to supply the project with material for the early pre-clinical studies, an incident occurred which is worth some attention. After completing the carbamate formation, (2R,4S)-24 was isolated as a rock-hard solid. Naturally, repeated attempts to dissolve the solid in CH3CN by scraping the inside of the flask with a spatula resulted in an unexpected hole in the flask and the contents ended up in a dirty, hot silicone oil bath. Repeated extractions of the silicone oil with CH3CN selectively extracted our material and in the end gave us back most (> 95%) of the compound in good quality, except for some minor silicone oil. Since the subsequent step was an ester hydrolysis, residual silicone oil was easily removed by extraction. Time constraints required us to keep the methyl carbamate as the protecting group and thus we continued.

Scheme 13. N-protection of piperidine (2R,4S)-21. Hydrolysis of the Ester The subsequent hydrolysis of the methyl ester was a priori considered a simple task. Standard hydrolysis using NaOH or LiOH resulted in 5–20% epimerization of the 4-position of (2R,4S)-24, resulting in acid (2R,4R)-10 as an impurity. To avoid the epimerization, different order of addition of reagents, more dilute conditions, and lower loading of the hydroxides were attempted unsuccessfully. In a previous project, we had experienced the same problem of epimerization during hydrolysis. In that project, in order to minimize epimerization, we developed a new, mild hydrolysis method in which triethylamine was used in combination with lithium salts in wet solvents (30). Probably due to the low basicity of triethylamine compared to alkali hydroxides normally employed in hydrolyses, we could avoid most of the epimerization. Most common solvents could be employed for the hydrolysis, such as CH2Cl2, CH3CN, 2-MeTHF, and, surprisingly, also ester solvents, such as iPrOAc. This gave us the flexibility to choose a solvent which could be used in the previous and subsequent steps, permitting telescoping. By applying the LiBr/Et3N method 170

on ester (2R,4S)-24 in 2-MeTHF, we obtained carboxylic acid (2R,4S)-10 in 99% yield with at most 1% of the trans-isomer (2R,4R)-10 (Scheme 14). Accordingly, by running the protection of (2R,4S)-21 to give carbamate (2R,4S)-24 in 2-MeTHF, it was possible, after an aqueous wash, to use the 2-MeTHF organic layer directly in the hydrolysis step. Using the resulting carboxylic acid (2R,4S)-10 contained in the organic phase in the subsequent β-keto ester formation step was also successful (this will be discussed in the next section). The hydrolysis gave us consistent results also on a larger scale.

Scheme 14. Mild conditions were required in order to maintain the stereochemistry at position 4 in piperidine (2R,4S)-24. Preparation of the β-Keto Ester Next, installation of the β-keto ester was planned as a precursor to building the isoxazole ring. In the first-generation synthesis, this was achieved by pre-activation of the carboxylic acid with carbonyldiimidazole (CDI) followed by reaction with ethyl potassium malonate to give keto ester (2R,4S)-11. For the scale-up, it would be desirable if acid (2R,4S)-10, (which was obtained as a solution in 2-MeTHF), could be telescoped directly to the β-keto ester (2R,4S)-11. The reaction between CDI and acid (2R,4S)-10 to give the intermediate imidazolide was fast and monitored by 1HNMR spectroscopy. Completion was indicated by disappearance of the signal from the starting acid (2R,4S)-10 at 2.63–2.70 ppm. The imidazolide was then treated with a preformed complex of magnesium chloride and ethyl potassium malonate; 30–70% conversion occurred after 2 h. This step was followed by 1HNMR spectroscopy and worked as expected at scales up to 8 g, but caused us a serious headache on going from small lab scale with magnetic stirring to larger scale with overhead stirring. Thus, 80 g of carboxylic acid (2R,4S)-10 was quantitatively converted to the corresponding imidazolide by the straightforward addition of CDI. The resulting suspension was subsequently added to the prestirred mixture of magnesium chloride and ethyl potassium malonate. We were horrified when we did not see any conversion at all after 2 h of reaction time. Heating the mixture was not an option since we were concerned about the stability of both the intermediate imidazolide and the resulting β-keto ester (2R,4S)-11. It was reasoned that we had most likely run into a problem with the potassium magnesium complex. However, we had no idea how to analyze the state of the potassium magnesium complex. 171

Our first thoughts were that we had used poor quality magnesium chloride. After some consideration, a second theory appeared—that the particle surface area of the ingoing magnesium chloride and ethyl potassium malonate was considerably increased by the grinding effect of the stirring bar against the glass walls of the flask used in our small scale reactions. In a reactor equipped with an overhead stirrer, there is quite obviously no grinding. A small scale test was performed with some of the mixture from the reactor mixed with magnesium-malonate complex produced in a flask with a magnetic stirrer. To our relief, our theory was confirmed and a larger portion of the potassium magnesium malonate was prepared in a separate flask with a magnetic stirrer. The resulting suspension/complex was added to the reaction mixture and full progress resulted after overnight reaction. The extra malonate that had been added during our first attempt was removed by washes with aqueous sodium bicarbonate. After this ordeal, keto ester (2R,4S)-11 was isolated in 96% yield as an oil after removal of the solvent (Scheme 15).

Scheme 15. Synthesis of β-keto ester (2R,4S)-11.

As the project progressed onto the next batch, it became apparent that different particle sizes of the magnesium chloride charged gave different outcomes in the reaction. Consequently, the slow conversion was most likely due to low solubility of the magnesium chloride in 2-MeTHF. For the reactor scale batches that followed, we solved the problem by refluxing the magnesium chloride with the ethyl potassium malonate for 5 h in order to make the complex. However, long reaction times were still a serious bottleneck and later batches starting with up to 760 g of carboxylic acid (2R,4S)-10 required as much as five days and additional magnesium chloride/potassium ethyl malonate complex to reach completion. Unfortunately, we could see that epimerization took place during this lengthy procedure and we now had 6% of the wrong epimer at the 4-position in our material. After having finished our final batches (3 kg AZD6564, 5), we discovered that the reaction could be completed in less than 4 h at 25 °C in THF rather than Me-THF, with 12 volumes of solvent instead of 20, and with the same yield/purity outcome. This test reaction gave 76% yield on a 10 g scale. 172

Isoxazol-3-ol Formation The most commonly used route to isoxazol-3-oles commences with the reaction between a β-keto ester and hydroxylamine (Scheme 16) (31). Hydroxylamine may attack the β-keto ester in more than one way, and also equilibrate between the different species formed before cyclization to one of the heterocycles (Scheme 17).

Scheme 16. Formation of the isoxazol-3-ol (2R,4S)-13.

Scheme 17. Simplified mechanistic view. Two isomeric compounds, i.e., the byproduct 2H-isoxazol-5-one (e) and isoxazol-3-ol (f), may result from acid catalyzed cyclization of the possible intermediates formed by reaction between β-keto ester and hydroxylamine. Normally the hydroxamic acid intermediate (b) is not isolated and the reaction mixture is acidified directly. Fast acidification favors formation of desired isoxazol-3-ol (f), while slow acidification has been found to favor formation of the isomeric and undesired 2H-isoxazol-5-one (e). The isomeric structure (e) is frequently formed in alarming amounts even if acidification 173

is fast. A detailed mechanistic study was published in 1986 in which several of the intermediates were identified based on the 13CNMR spectra (32). The formation of the byproduct 2H-isoxazol-5-one (e) during slow acidification has been attributed to equilibration of the hydroxamic acid (b) and the oxime (c) via catalytic amounts of free hydroxylamine. It is well documented that the oxime (c) cleanly gives the 2H-isoxazol-5-one (e) upon acidification. It has also been established that an overly high pH during the reaction between hydroxylamine and β-keto ester (a) will lead to formation of the 2H-isoxazol-5-one byproduct (e) upon acidification (33). It was speculated that at above pH 10 (in aqueous systems), the anion of hydroxylamine (34) will start playing a role in the reaction with the β-keto ester (a). The anion of hydroxylamine may give rise to d. Intermediate d has, to our knowledge, not been positively identified as an intermediate in these reactions but is included in the scheme because its existence and the subsequent reaction path to e was already theorized (32). The mechanisms of these reactions have been studied and discussed in several publications and appear rather complicated (35–37). To make matters worse, early publications in the area contain errors due to confusion of isoxazol-3-ol with the 2H-isoxazol-5-one. Since the synthesis of isoxazol-3-ols from β-keto esters had been extensively studied by several groups, we gathered that we were unlikely to improve the step. Our LO group had used this particular method successfully. Therefore, our keto ester from the previous step was treated with one equivalent of sodium hydroxide in methanol at - 40 °C, followed by one equivalent of hydroxylamine (38). As an extra precaution for avoiding hydroxylamine breakdown and to maintain reproducibility, the reaction was performed in the presence of 0.05 mol% Na2EDTA, which complexes the iron salts. Soluble iron salts are known to be very efficient catalysts (in the ppm range) for the breakdown of hydroxylamine (39). With a heat of reaction of ~ 4 kJ/g, the reaction may be violent at high concentrations. Typical decomposition products are N2, N2O, NO, and NH3. The sampling of the hydroxamic acid formation reaction (2R,4S)-25 was carried out by withdrawing aliquots with a liquid nitrogen cooled pipette followed by immediate HPLC analysis. When conversion of ester (2R,4S)-11 was confirmed (usually after a period of about 4 h at –40 °C), the mixture was added in one portion to 6 M HCl maintained at 80 °C. After a reaction time of 15–30 min, complete consumption of starting material could be confirmed by HPLC-MS. Apart from our desired product, (2R,4S)-13, about 20–30% undesired isomeric isoxazol-5-one, (2R,4S)-12, was also formed as determined by 1HNMR spectroscopy. The mixture was cooled and brought to pH 10.5 with NaOH in order to convert the product to its hydrophilic anion. After removal of lipophilic impurities (among which byproduct (2R,4S)-12 is one) by an extraction with MTBE, the aqueous phase was again acidified with hydrochloric acid to pH 1-2 and extracted with MTBE. Product (2R,4S)-13 was isolated with a purity of 99.9% (by HPLC-MS with detection at 210 nm) in 65–70% yield after removal of the solvent and crystallization from methanol/water.

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Deprotection of Carbamate To Give AZD6564 The final step to produce 5 was the removal of the methyl carbamate protective group from (2R,4S)-13 by treatment with HBr in HOAc (Scheme 18). Our initial experiment at 4.6 g scale first produced material that was brown. Because of the discoloration, the material was purified using preparative HPLC. To our disappointment, the 1HNMR assay was still unsatisfactory and we realized that our compound had passed through the HPLC as the HBr salt. The material was redissolved in pure water and neutralized to pH 6.5 with aqueous ammonia. During the neutralization, the zwitterion precipitated as a crystalline solid monohydrate, which was filterable and gave a 93% assay and a 99.8 area % HPLC purity. The material was identified as a monohydrate, explaining the missing 7% as water. This first deprotection experiment gave a 56% assay yield.

Scheme 18. Removal of the methyl carbamate group to give final product 5 along with byproduct 26. We proceeded with the next 500 g and 280 g starting material campaigns yielding 72% and 68%, respectively, of API. Deprotection of the 500 g material lot displayed a slower reaction rate than the deprotections run on smaller scales in round-bottomed flasks. Most likely, this was an effect of HBr escaping into the head space of the reactor in our large-scale reactions, resulting in a lower concentration of HBr in solution and consequently a slower reaction as compared to the small-scale runs. A small sample of the reaction mixture was removed from the reactor and was subjected to forcing conditions by microwave heating at 120 °C. It was seen that some methylation took place upon this treatment in parallel with the deprotection but it was assumed that the methylation was caused by the high temperature. A short delivery deadline made us take the decision to heat the mixture to a gentle 50 °C, which soon gave rise to serious concerns when the methylation of the API appeared to be about 5%, as determined by HPLC-MS.

175

We had grave concerns that the side product would end up in our final material. Our workup at this point consisted of removal of the HOAc/HBr in vacuo followed by dissolution of the residue into 50% aqueous 2-propanol, polish filtration, and precipitation at the isoelectric pH by neutralization with ammonia. To our relief, the precipitated material was 99.9% pure by HPLC-MS and did not contain any detectable methylated material. By extracting the filtrate at pH 14, the pure methylated material was isolated and characterized as byproduct 26 (by NMR analysis) (40). The structure of the byproduct also provided an explanation for its unexpectedly easy removal during workup (Scheme 19).

Scheme 19. Addition of base to the acidic solution of 5 (AZD6564) and 26 precipitates zwitterionic 5. Impurity 26 will stay in solution in its protonated form. The pKa values for 5 have been measured experimentally.

Since the nitrogen in byproduct 26 is N-methylated to give an isoxazol-3-one, tautomerism is impossible and the acidity of the hydroxy group in isoxazol-3-ols will not be displayed. We speculate that the methylation is a simple N-methylation by bromomethane which in turn would be expected to form from the cleavage of the methyl carbamate group. It is thus logical that byproduct 26 does not form a zwitterion and it should consequently not cocrystallize with AZD6564. Our final route for producing up to 500 g of AZD6564 (5) is shown in Scheme 20. The salt resolution using acid 22 was used for producing some of the (2R,4S)-21 and is thus included in the scheme.

176

Scheme 20. Summary of the synthesis route for the first 500 g AZD6564, 5. Adapted with permission from reference (23). Copyright 2014 American Chemical Society. 177

GMP Multikilogram Scale Campaigns General Observations in the GMP Campaign Scaling up to the next level (i.e., through production of 3 kg of 5 for phase 1 studies) required starting with ~ 10 kg of 19 and necessitated larger reactors. Therefore, the preparation was transferred to our site in Södertälje. Some small changes were made but the route remained essentially unchanged. The complete route is summarized in Scheme 21.

Scheme 21. GMP route for production of AZD6564 (5). Adapted with permission from reference (23). Copyright 2014 American Chemical Society. During further investigation of the first step in the synthesis, we discovered that PEPPSI-IPr (41) (Figure 4) is a suitable catalyst for the Negishi coupling between 19 and neopentylzinc bromide, requiring only 0.5 mol% of catalyst. The PEPPSI-IPr catalyzed reaction showed a cleaner reaction profile than neopentylmagnesium bromide/Fe(acac)3. Neopentylzinc bromide was also more accessible on larger scale than the equivalent Grignard reagents. 178

Figure 4. Structure of PEPPSI-Ipr. The pyridine coupling partner 19 was not a bottleneck. Starting with 10 kg methyl 2-chloroisonicotinate 19 and 0.5 mol% PEPPSI-IPr in MTBE, followed by the addition of 1.05 equiv of neopentylzinc bromide at < 40 °C, gave an 166 kJ/mol exothermic reaction and a maximal adiabatic temperature rise of 58 °K. After 12 h, the reaction was worked up with citric acid and EDTA. Concentration, acidification with hydrogen chloride in 2-propanol, and precipitation with MTBE gave the hydrochloride salt 20 in 66% yield. For the next step, hydrogenation at a slightly elevated temperature (40 °C) instead of ambient temperature gave cis-(±)-21 hydrochloride in 93% yield, after concentration and precipitation from MTBE. Resolution of cis-(±)-21 was accomplished by enzymatic resolution as in the GLP campaign, but with small alterations. The hydrolysis was simplified by dissolving cis-(±)-21 (13.1 kg) with 1.57 equiv of dipotassium phosphate in 80 L of water and with no further adjustment of pH to give a pH 8 solution. Immobilized Candida antarctica lipase (CALB-T3-150, 3.95 kg) was added, followed by stirring for 40 h at 35 °C. Cooling, addition of 2-MeTHF, and basification with KOH solution gave, after filtration, the desired epimer as its methyl ester retained in the organic phase. The extract was directly diluted with more 2-MeTHF, followed by addition of DIPEA and then methyl chloroformate. The reaction mixture, now containing carbamate (2R,4S)-24, was washed with water followed by addition of triethylamine and LiBr, and heating at 85 °C for 41 h in order to accomplish the hydrolysis to acid (2R,4S)-10. Crystallization was effected after acidic and aqueous workup, concentration, and heptane addition, and gave pure acid (2R,4S)-10 in 31% yield over three telescoped steps (enzymatic resolution, hydrolysis, and carbamate formation). Improvements to the resolution steps were: •

•

For the enzymatic hydrolysis, dipotassium phosphate was used as the single pH regulator in larger excess and without adjustment of pH at any point during the reaction. The enzymatic hydrolysis was carried out at 35 °C instead of 20 °C. 179

•

•

Filtration after the enzymatic step was performed after addition of MTBE and pH adjustment. This removed (2S,4R)-23 by extraction rather than by a slow and tedious filtration. Chiral acid (2R,4S)-10 was crystallized from the concentrated extract by addition of n-heptane.

The resulting acid (2R,4S)-10 was then ready for conversion to keto ester (2R,4S)-11. In a similar manner to the GLP campaign, a 3-fold excess of the magnesium chloride complex with ethyl potassium malonate was prepared under reflux in 2-MeTHF for 6 h. The acid (2R,4S)-10 was activated by adding a solution of the acid to a 1.2-fold excess solution of CDI in 2-MeTHF. By this order of addition, the reaction became more practical as well as consuming less CDI (42, 43).The imidazolide was then added to the previously prepared magnesium chloride complex and the mixture was stirred for 61 h at 25 °C and then for 48 h at 35 °C to give 97% conversion. Acidic workup and assay showed a 77% yield. Improvements in conversion of 10 to 11 were: • •

Reverse order for preparation of imidazolide; acid (2R,4S)-10 was added to CDI in 2-MeTHF. The reaction mixture was heated at 35 °C for 48 h to complete the reaction.

During one of the optimization efforts for the subsequent step, that is, conversion of the β-keto ester (2R,4S)-11 to protected isoxazol-3-ol (2R,4S)-13, the sodium salt of (2R,4S)-25 precipitated and as a result, the reaction failed. We discovered that the reaction could be improved by using triethylamine instead of sodium hydroxide and also running the reaction at –10 °C instead of –40 °C. These operations avoided solid salt formation. Acid treatment in a similar manner as in the GLP route was followed by using MTBE both for workup and crystallization, yielding the isoxazol-3-ol (2R,4S)-13 in 55% yield. Improvements in the conversion of 11 to 13 were: • •

Hydrolysis and reaction with hydroxylamine using triethylamine instead of sodium hydroxide and at –10 °C instead of –40 °C. Crystallization and workup of (2R,4S)-13 with MTBE.

The API formation step was carried out along the lines of the GLP route, with the change that a scrubber with ethylenediamine, sodium thiosulfate, sodium hydroxide, and water was connected to the reactor in order to trap bromomethane, bromine, and hydrogen bromide gases. Thus, carbamate (2R,4S)-13 was heated in 33% HBr/HOAc at 30 °C for 16 h. Evaporation, eventually followed with the addition of water by dissolution in 2-propanol filtration and neutralization using aqueous ammonium hydroxide, gave the crude API. The material was finally stirred in cold water for 20 h to give the final API (5), in 3 kg as a monohydrate in 89% yield and with an ee of 99.9%. The assay of this material was 89.8% and the water content was 10.2%. Table 3 summarizes the first-generation synthesis with the final GMP route. 180

Table 3. Comparison of First-Generation Synthesis with the GMP Route Parameter

First-generation synthesis

GMP route

Number of steps

8

8

Chromatography steps

4

0

Telescoped steps

No

Yes

Chiral column chromatography

Yes

No

0.3%

7%

Yes

No

Difficult

Medium

Overall yield Cryogenic conditions Ease of operation

Conclusions In summary, a first-generation synthesis of the fibrinolysis inhibitor AZD6564, involving four chromatographic purifications and an overall yield of 0.3%, was improved to a chromatography-free synthesis with a 7% overall yield. Two different cross-couplings were demonstrated for the attachment of a neopentyl side chain on pyridine, starting from neopentylmagnesium chloride or neopentylzinc bromide and easily accessible methyl 2-chloroisonicotinate. Hydrogenation of the resulting 2,4-disubstituted pyridine gave rise to racemic 2-(2,2-dimethylpropyl)piperidine-4-carboxylic acid, which was resolved using either chemical or enzymatic means. The enzymatic resolution was selected for our GMP route. After protection of the nitrogen as a methyl carbamate, a particularly mild ester hydrolysis with triethylamine and LiBr was required in the step to follow in order to preserve the stereochemistry. The resulting acid was activated using carbonyldiimidazole and reacted with the preformed complex between magnesium chloride and ethyl potassium malonate to give a β-keto ester. Reaction with hydroxylamine at –10 °C in the presence of triethylamine followed by fast acidification gave a mixture of two isomeric isoxazoles, which were separated by extractions and crystallization. The final step, removal of the methyl carbamate protecting group, gave rise to some N-methylated byproduct, which was easily removed by precipitation and isolation of the end product as its zwitterion.

Acknowledgments A number of chemists have been involved in solving the chemical problems associated with developing the synthesis of AZD6564. In particular, the following people have made important contributions: Leifeng Cheng, Martin Bollmark, 181

Peter Schell, Søren M. Andersen, Robert Berg, Christofer Fredriksson, Catarina Liljeholm, Angéle Cruz, and Fritiof Pontén.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Triplett, D. A. Clin. Chem. 2000, 46, 1260–1269. Bruinvels, G.; Burden, R.; Brown, N.; Richards, T.; Pedlar, C. PLoS One 2016, 11, e0149881. Okamoto, S.; Okamoto, U. Keio J. Med. 1962, 11, 105–115. Castellino, F. J.; Ploplis, V. A. Thromb. Haemostasis 2005, 93, 647–654. Wilton, J. M. Nursing for Women’s Health 2012, 16, 146–150. WHO Model List of Essential Medicines. http://www.who.int/medicines/ publications/essentialmedicines/en/. (Jan 2018). Leminen, H.; Hurskainen, R. Int. J. Women’s Health 2012, 4, 413–421. Wellington, K. J.; Wagstaff, A. J. Drugs 2003, 63, 1417–1433. Furtmüller, R.; Schlag, M. G.; Berger, M.; Hopf, R.; Huck, S.; Sieghart, W.; Redl, H. J. Pharmacol. Exp. Ther. 2002, 301, 168–173. Boström, J.; Grant, A. J.; Fjellström, O.; Thelin, A.; Gustafsson, D. J. Med. Chem. 2013, 56, 3273–3280. Schmidt, T. C; Eriksson, P-O; Gustafsson, D.; Cosgrove, D.; Frølund, B.; Boström, J. J. Chem. Inf. Model. 2017, 57, 1703–1714. Cheng, L.; Pettersen, D.; Ohlsson, B.; Schell, P.; Karle, M.; Evertsson, E.; Pahlén, S.; Jonforsen, M.; Plowright, A. T.; Boström, J.; Fex, T.; Thelin, A.; Hilgendorf, C.; Xue, Y.; Wahlund, G.; Lindberg, W.; Larson, L-O.; Gustafsson, D. ACS Med. Chem. Lett. 2014, 5, 538–543. Byberg, J. R.; Labouta, I. M.; Falch, E.; Hjeds, H.; Krogsgaard-Larsen, P.; Curtis, D. R.; Gynther, B. D. Drug Des. Delivery 1987, 1, 261–274. Frydenvang, K.; Matzen, L.; Norrby, P.-O.; Sløk, F. A.; Liljefors, T.; Krogsgaard-Larsen, P.; Jaroszewski, J. W. J. Chem. Soc., Perkin Trans. 2 1997, 17, 1783–1792. Ballatore, C.; Huryn, D. M.; Smith, A. B. ChemMedChem 2013, 8, 385–395. Woodcock, S.; Green, D. V. S.; Vincent, M. A.; Hillier, I. H.; Guest, M. F.; Sherwood, P. J. J. Chem. Soc., Perkin Trans. 2 1992, 12, 2151–2154. Boulton, A. J.; Katritsky, A. R.; Majid Hamid, A.; Øksne, S. Tetrahedron 1964, 20, 2835–2840. Comins, D. L.; Brown, J. D. Tetrahedron Lett. 1986, 27, 4549–4552. Comins, D. L. J. Heterocycl. Chem. 1999, 36, 1491–1500. Comins, D. L.; Joseph, S. P.; Goehring, R. R. J. Am. Chem. Soc. 1994, 116, 4719–4728. Oldenziel, O. H.; van Leusen, D.; van Leusen, A. M. J. Org. Chem. 1977, 42, 3114–3118. Frølund, B.; Kristiansen, U.; Brehm, L.; Hansen, A. B.; KrogsgaardLarsen, P.; Falch, E. J. Med. Chem. 1995, 38, 3287–3296. Andersen, S. M.; Bollmark, M.; Berg, R.; Fredriksson, C.; Karlsson, S.; Liljeholm, C.; Sörensen, H. Org. Process Res. Dev. 2014, 18, 952–959. Oster, T. A.; Harris, T. M. J. Org. Chem. 1983, 48, 4307–4311. 182

25. Whelligan, D. K.; Solanki, S.; Taylor, D.; Thomson, D. W.; Cheung, K.-M. J.; Boxall, K.; Mas-Droux, C.; Barillari, C.; Burns, S.; Grummitt, C. G.; Collins, I.; van Montfort, R. L. M.; Aherne, G. W.; Bayliss, R.; Hoelder, S. J. Med. Chem. 2010, 53, 7682–7698. 26. Frølund, B.; Jørgensen, A. T.; Tagmose, L.; Stensbøl, T. B.; Vestergaard, H. T.; Engblom, C.; Kristiansen, U.; Sanchez, C.; Krogsgaard-Larsen, P.; Liljefors, T. J. Med. Chem. 2002, 45, 2454–2468. 27. Wang, D.-S.; Chen, Q.-A.; Lu, S.-M.; Zhou, Y.-G. Chem. Rev. 2012, 112, 2557–2590. 28. Fürstner, A.; Leitner, A.; Méndez, M.; Krause, H. J. Am. Chem. Soc. 2002, 124, 13856–13863. 29. Whelligan, D. K.; Solanki, S.; Taylor, D.; Thomson, D. W.; Cheung, K.M. J.; Boxall, K.; Mas-Droux, C.; Barillari, C.; Burns, S.; Grummit, C. G.; Collins, I.; van Montfort, R. L. M.; Aherne, G. W.; Bayliss, R.; Hoelder, S. J. Med. Chem. 2010, 53, 7682–7698. 30. Mattsson, S.; Dahlström, M.; Karlsson, S. Tetrahedron Lett. 2007, 48, 2497–2499. 31. Sørensen, U. S.; Krogsgaard-Larsen, P. Org. Prep. Proced. Int. 2001, 33, 515–564. 32. Katritzky, A. R.; Barczynski, P.; Ostercamp, D. L.; Yousaf, T. I. J. Org. Chem. 1986, 51, 4037–4042. 33. Jacobsen, N.; Kolind-Andersen, H.; Christensen, J. Can. J. Chem. 1984, 62, 1940–1944. 34. The pKa of the hydroxylamine-hydroxy group is 13.7 while the hydroxylamine-amino group was measured to 5.94. See Kashima, C.; Konno, Y.; Yoshiwara, N.; Tajima, T. J. Heterocycl. Chem. 1982, 19, 1535–1536. 35. Jacquier, R.; Petrus, C.; Petrus, F.; Verducci, J. Bull. Soc. Chim. Fr. 1967, 3003–3004. 36. Jacquier, R.; Petrus, C.; Petrus, F.; Verducci, J. Bull. Soc. Chim. Fr. 1970, 2685–2690. 37. Jacquier, R.; Petrus, C.; Petrus, F.; Verducci, J. Bull. Soc. Chim. Fr. 1971, 3664–3665. 38. Excess of hydroxylamine, higher temperatures or prolonged reaction times for the generation of the hydroxamic acid gave, upon the subsequent acid catalyzed cyclization, more of undesirable 2H-isoxazol-5-one. 39. Cisneros, L. O.; Rogers, W. J.; Mannan, M. S.; Li, X.; Koseki, H. J. Chem. Eng. Data 2003, 48, 1164–1169. 40. This is concluded both by the 13C chemical shift of the methyl group (32.8 ppm) and the 1H-15N HMBC peaks between protons in the isoxazol-3-one and the methyl group and the nitrogen in the isoxazol-3-one. 41. Hadei, N.; Kantchev, E. A. B.; O’Brien, C. J.; Organ, M. J. Org. Lett. 2005, 7, 3805–3807. 42. In the GLP process, 2-3 equiv of CDI were used. We had no method of determining the purity of this material, so whether the higher amount of CDI used in the GLP campaign is a result of the order of addition is uncertain. 183

43. Engstrom, K. M.; Sheikh, A.; Ho, R.; Miller, R. W. Org. Process Res. Dev. 2014, 18, 488–494.

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Chapter 7

Design and Development of the Glucokinase Activator AZD1656 Darren McKerrecher1 and Alan Steven2,* 1Medicinal

Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Darwin Building, Unit 310, Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom 2Pharmaceutical Technology & Development, AstraZeneca, Charter Way, Macclesfield, SK10 2NA, United Kingdom *E-mail: [email protected]

AZD1656 is a potent, selective glucokinase (GK) activator that progressed to Phase IIb trials for the treatment of type 2 diabetes. This compound was identified by in-depth optimization of a high-throughput screening (HTS) hit while resolving challenges concerning selectivity, toxicology, physicochemical properties, and potency. The development of a route to AZD1656 that was amenable to scale-up and deemed to be commercially viable retained the same building blocks as the medicinal chemistry synthesis. The α-resorcylate core was functionalized using a Mitsunobu reaction followed by an SNAr reaction in a sequence that differentiated the two resorcylate hydroxyl groups by protecting one of them. The synthesis of the coupling partner for the SNAr involved a highly selective decarboxylation and the control of impurities arising from the use of azetidine as a coupling partner. Downstream challenges included an amidation reaction with a pyrazine coupling partner made using a Curtius rearrangement performed in batch mode and the development of crystallization conditions that allowed the reproducible isolation of a slow-growing polymorph of the active pharmaceutical ingredient (API). The processes developed were tested as part of a 500 kg manufacture of AZD1656.

© 2018 American Chemical Society

Role of Glucokinase in Glucose Homeostasis Glucokinase (GK) is an enzyme that plays a central role as a glucose sensor in the regulation of glucose homeostasis, catalyzing the conversion of glucose to glucose-6-phosphate (1). In both the liver, where the action of GK promotes glycogen synthesis, and the pancreas, where the action of GK results in glucosesensitive insulin release, GK is the rate-limiting enzyme in glucose utilization. Accordingly, and alongside others (2–6), we initiated a program to identify GK activators (GKAs) as a strategy to improve glycemic control by modulating hepatic glucose balance and decreasing the threshold for insulin secretion.

Identification of Pyridine Acid Lead Series A high-throughput screen (HTS) of AstraZeneca’s compound collection identified 1, mediating GK activation with an EC50 of 3.2 µM. Removal of the double bond (prone to isomerization and a potential Michael acceptor) and the methylthio-moiety (a potential metabolic liability), alongside structure–activity relationship (SAR) exploration of the aryl ring led to the identification of 3,5-disubstituted amide derivatives such as 2 (Figure 1), which showed promising in vivo activity in an oral glucose tolerance test (OGTT) (7, 8).

Figure 1. Identification of HTS hit 1 and initial optimization to pyridine acid leads 2–4. Focusing on improvements in unbound clearance (9) led to the incorporation of an α-branch adjacent to the aryloxy group. The combination of a (1S)-1-methyl2-phenylethoxy side chain (which had delivered excellent in vitro potency) with a 186

(1S)-2-methoxy-1-methylethoxy ether side chain (which had delivered excellent solubility and low plasma protein binding) led to the design of 3 (10), which showed excellent dose-dependent efficacy in the high-fat-fed female Zucker rat OGTT model. We also established that replacing the α-branched alkoxy side chain with a substituted phenyl ether gave a further reduction in unbound clearance while retaining good potency, solubility, and acceptable plasma protein binding. This yielded an additional short-list candidate 4 with a significantly increased pharmacokinetic half-life in rats (11).

Switch to Neutral Series and Identification of AZD1092 The progress of 3 and 4 was halted when it was revealed that both compounds caused epididymal and testicular toxicity in rat- and dog-safety studies (12). Further investigations into the observed toxicological findings revealed that the compounds were potent RAR-α antagonists, a finding consistent with the observation of testicular toxicology (13). Modeling the binding of the compounds to GK and RAR-α showed that the carboxylic acid moiety formed a clear interaction with RAR-α but not with GK. Thus, we postulated that compounds without the carboxylic acid functionality might be free of the RAR-α associated testicular toxicity and sought to identify a series of neutral GKAs (13). The replacement of the pyridine acid with neutral heterocycles was tolerated in terms of GK potency, but suffered from solubility-limited absorption. Polar substituents, such as the 4-methanesulfonyl derivative, were tolerated, but these compounds also failed to show oral exposure in pharmacokinetic studies, presumably due to poor permeability (14). Balancing these observations with compounds such as 5 achieved these goals, demonstrating in vivo activity at 3 mg kg-1 in a rat OGTT. Profiling of 5 in safety studies confirmed that the compound was free of RAR-α antagonism (IC50 >30 µM) and the testicular toxicology observed with 3 and 4 (15). Further optimization led to the identification of 6, which met all criteria for progression as a development candidate (Figure 2), was well-tolerated in rat and dog preclinical toxicology studies, and progressed toward clinical development as AZD1092 (15).

Figure 2. Development of neutral lead 5 and optimization to 6 (AZD1092).

187

Risk Mitigation and Optimization Toward AZD1656 In parallel, an assessment of risks associated with the profile of AZD1092 (6) highlighted the potential for suboptimal permeability and an observation that 1-methyl-3-aminopyrazole (an intermediate in the synthesis of AZD1092 [6]) and a putative metabolite) generated a positive response in the Ames genotoxicity assay. We demonstrated that switching the 1-methyl-3-aminopyrazole for a 5methylpyrazine derivative retained GK activity and that the fragment 2-amino-5methylpyrazine was inactive in the Ames assay. In addition, replacement of the hydroxy group of AZD1092 (6) with a methoxy group improved permeability as a result of removing a hydrogen bond donor. The resultant increase in lipophilicity needed to be offset, due to the known relationship between hERG liability and lipophilicity, prompting the replacement of the pendant benzene ring of AZD1092 (6) with another pyrazine ring. The combination of these findings resulted in the identification of the pyrazine derivative 7, which showed excellent oral absorption and physicochemical, pharmacokinetic, and in vivo efficacy data compared to AZD1092 (6) (Table 1). Compound 7 showed a benign safety profile and was renamed AZD1656 as it progressed into clinical development, where it rapidly overtook AZD1092 (16).

Table 1. Data Comparison Between AZD1092 (6) and AZD1656 (7)

188

Medicinal Chemistry Route to AZD1656 (7) The medicinal chemistry route to the synthesis of AZD1656 (7) is outlined in Scheme 1 (17). The ether side chain was introduced by a Mitsunobu reaction of the requisite phenol 8 with (2R)-1-methoxypropan-2-ol (9). Ester hydrolysis as part of the same step then yielded acid 10. This was subsequently engaged in an amide coupling with aminopyrazine 11 to form amide 12. Finally, the removal of the benzyl group followed by an SNAr reaction with chloropyrazine 13 completed the synthesis (17). A key attribute of this approach during the discovery phase of the program was that the order of steps could be readily altered to allow the synthesis of diverse analogs.

Scheme 1. Summary of medicinal chemistry route to AZD1656 (7).

Evaluation of the Strategic Merits of the Medicinal Chemistry Route from a Development Perspective When AZD1656 (7) was handed over to development, the process chemistry team considered synthesis strategies that could potentially deliver up to 150 metric tons of active pharmaceutical ingredient (API) on an annual basis in order to meet projected peak year sales. The retention of the α-resorcylate core 14, pyrazines 11 and 13, and a chiral side chain (such as alcohol 9) as the building blocks of the synthesis was appealing, given what were anticipated to be efficient reactions for 189

their assembly and the synthetic accessibility of the materials involved (Scheme 2). The use of acid 15 as a synthetic precursor to the API was attractive on the grounds that it is a highly crystalline material whose isolation could be used to reject impurities late in the synthesis.

Scheme 2. Principal disconnections of AZD1656 (7). One briefly considered variant, related to the strategy shown in Scheme 2, used a 3,5-dihalobenzonitrile starting material. Successive nucleophilic displacements of a material such as 16 (Scheme 3) should differ greatly in their respective rates, allowing the 3- and 5- positions to be successfully differentiated. This approach was tested on material associated with AZD1092 (6), namely alcohol 17 (Scheme 3) (18). While the initial SNAr to give ether 18 was successful, the ensuing downstream introduction of pyrazinone 19 was undone by the preference for this ambidentate nucleophile to react with aryl bromide 20 through nitrogen, rather than the pyrazinone oxygen atom so as to afford a mixture composed of compounds 21 and 22 (19). This ended this approach.

Initial α-Resorcylate Functionalization Studies Given the intention to use the amidation of aminopyrazine 11 with acid 15 for the API bond-forming step, efforts turned to the identification of an efficient synthesis of 15 (20). The retention of the medicinal chemistry approach (Scheme 1), using a benzyl-protecting group to form ether 8, was unattractive due to its failure to selectively afford access to the monoprotected derivative (≤44% isolated yield). Early investigations into differentiating the hydroxyl groups of a starting material related to an α-resorcylic acid derivative focused on the introduction of the chiral side chain (21). It soon became clear that this approach was inherently 190

flawed. As Scheme 4 shows, the pKa values of a starting material like methyl α-resorcylate (23) (which reacts via anion 24) and a monoalkylated derivative such as 25 are similar. This means the latter is likely to react again, through anion 26, with an electrophile such as tosylate 27, before all of the methyl α-resorcylate (23) has been consumed.

Scheme 3. Attempted derivatization of a 3,5-dihalobenzonitrile.

Scheme 4. Calculated pKas (aqueous, T=25 °C, zero ionic strength) relevant to monoalkylation approach (22). (Reproduced from reference (20). Copyright 2018, American Chemical Society.) 191

Scheme 5. Sequential elaboration of methyl α-resorcylate (23).

A monoarylation approach was studied in parallel with the alkylation studies described above and started with the use of potassium carbonate as base and chloropyrazine 13 as electrophile (Scheme 5). In addition to being subject to overreaction (as with the alkylations) so as to form the product of double arylation 28, these arylations were also subject to the intermolecular migration of a newly installed pyrazine group, under the basic conditions of the reaction. Thus the desired conversion of methyl α-resorcylate (23) to ether 29 was characterized by the incomplete consumption of the former (due to its regeneration as part of the 192

migration process) and the formation of the product of double arylation 28 by a second pathway. The methyl α-resorcylate (23) was prestirred with 2.5 molar equivalents of cesium carbonate, prior to the introduction of chloropyrazine 13, so as to access meaningful amounts of dianion 30 (Scheme 5) (23). If the analogous pKa data to that shown in Scheme 4 translated into reactivity toward a carbon electrophile (as opposed to a proton), 30 should have been significantly more reactive toward chloropyrazine 13 than the conjugate base of phenol 29, the species that could overreact to form bisether 28 (Scheme 5). Reproducible access to a mixture that consisted predominantly (95% area by HPLC [LCAP]) of 29 occurred and was able to be exploited by the project (Scheme 5) when this prestir was married with the dosed addition of a DMSO solution of a slight deficiency (0.97 molar equiv) of 13. With the monoarylation product 29 in hand, its remaining resorcylate hydroxyl was then pursued through a Williamson etherification with tosylate 27, prepared from commercially available (2R)-1-methoxypropan-2-ol (9) (Scheme 5) (23). Multiple olefin byproducts, attributed to the elimination of tosic acid from 27, formed in initial attempts. The screening of solvents as well as carbonate bases and strong organic bases determined that the use of cesium carbonate in DMSO was effective at forming ether 31 (Scheme 5), without any detectable erosion in the integrity of the stereogenic center. Because such a combination was also effective at facilitating the initial coupling with chloropyrazine 13, there was the opportunity to telescope the two transformations into a single process without the isolation of intermediate 29. The selectivity observed in the initial arylation (Scheme 5) was partly undone, however, by the intermolecular pyrazine migration that continued to take place once the tosylate electrophile 27 had been added. Thus, the formation of the desired product of the telescope, 31, was accompanied by the formation of more bisether 28, pushing its level to ca. 12 LCAP. Another bisether 32, which arose from the methyl α-resorcylate (23) liberated in the migration process, was also detected. Further drawbacks to this approach, albeit ones that were accepted given the comparatively early stage of development, included:

• • •

The relative expense of cesium carbonate; Concerns over its suspension in a reactor when scaling up further; and The negative impact on process mass intensity and throughput of washing out the DMSO solvent from a product-rich organic fraction using multiple water washes.

Ether 31 was purified by chromatography because it is an oil, and it was isolated in 59% yield from methyl α-resorcylate (23). The use of sodium hydroxide in N-methyl-2-pyrrolidone successfully hydrolyzed ester 31 to acid 15 in 78% yield (Scheme 6). Strict control of the reaction time was necessary to control the formation of impurities arising from the hydrolysis of the azetidine amide and the pyrazine ether bonds. 193

Scheme 6. Hydrolysis of ester 31.

Completion of the Early Development Route to AZD1656 The approach taken by the medicinal chemists to the synthesis of chloropyrazine 13 was not subject to significant modification as part of early development studies. Potassium carbonate in wet tetrahydrofuran was used to hydrolyze ester 33 to acid 34 (Scheme 7), so as to avoid the methyl chloride byproduct generated by the medicinal chemists’ use of dimethylformamide and lithium chloride. Toluene replaced dichloromethane as the solvent in the conversion of 34 into acid chloride 35 with thionyl chloride. That said, early attempts at telescoping a toluene solution of 35 into its coupling with azetidine hydrochloride were unsuccessful in the absence of dichloromethane. Thus, after solvent swapping into dichloromethane, the solution of 35 was added into a chilled solution of azetidine hydrochloride and triethylamine in the same solvent. This mode of combining the two solutions better controlled impurities arising from N-acyl azetidine ring opening by chloride than did an azetidine addition to the acid chloride solution. The chloropyrazine produced by this process was used in the coupling described in Scheme 5 to form ether 29.

Scheme 7. Synthesis of chloropyrazine (13). (Reproduced from reference (52). Copyright 2018, American Chemical Society.) 194

The medicinal chemistry approach to the aminopyrazine 11 building block, which involved converting 5-methylpyrazine-2-carboxylic acid (36) to carbamate 37, was retained for the purpose of early development deliveries of API (Scheme 8). The accommodation of this chemistry in a pilot plant environment provides another example of the use of the Curtius rearrangement for the delivery of multikilogram quantities of material, either in semi-batch mode (24–29) or using flow (30–32). Attempts to convert 5-methylpyrazine-2-carboxylic acid (36) to acyl azide 38 via the acid chloride, mesylate, or isobutyl- mixed anhydride were unsuccessful, either from the point of view of conversion or byproduct formation, leading to the continued use of 36 with diphenylphosphoryl azide (DPPA) (33–36).

Scheme 8. Completion of the synthesis of AZD1656 (7) as part of the first scale-up delivery. In an alternative take on the process safety control strategy that was adopted for its large-scale preparation later on in the project life cycle (vide infra), the inventory of the unstable acyl azide 38 was limited by using a controlled addition of DPPA to a hot solution of the other reaction components, such that 38 should have rearranged rapidly as soon as it formed (24). Given the only modest ability of tert-butyl alcohol at trapping the isocyanate intermediate 39 arising from the rearrangement of 38, it was used as the bulk solvent. In order to allow the tert-butyl alcohol to be handled in a pilot plant setting, it was melted prior to charging and 195

warm (>30 °C) water used in the reactor’s condenser in order to prevent blockages arising from its freezing. The neat trifluoroacetic acid hitherto used to deprotect carbamate 37 was successfully reduced to a 20% molar excess, and water was introduced as a bulk solvent (Scheme 8). The appreciable water solubility of aminopyrazine 11 was managed using multiple (4) extractions with n-propyl acetate when working up the deprotection reaction, in order to minimize losses to the aqueous phase. This facilitated the production of 11, which was passed through a plug of silica gel in order to remove colored impurities, allowing 72.5 kg to be delivered as part of the first scale-up campaign. An extensive reagent screen by the development team produced few hits for the coupling of acid 15 with aminopyrazine 11 due to the poor nucleophilicity of the latter. The use of n-propylphosphonic anhydride (T3P) and N-methylmorpholine in 2-methyltetrahydrofuran was one of the few hits and was successfully accommodated. It thus allowed the synthesis of 3.4 kg of AZD1656 (7), completing the first scale-up delivery of the API (Scheme 8).

A Re-Evaluation of α-Resorcylate Desymmetrization Approaches The attractiveness of compound 31 (Scheme 5), a key intermediate in the early development approach, as an intermediate for the commercial route used to supply AZD1656 (7) was diminished by its being an oil. Thus, our investigation of alternative route options remained ongoing while the chemistry shown in Schemes 5–8 was used to satisfy early clinical demands. It was recognized that a suitable intermediate arising from the monoprotection of methyl α-resorcylate (23) would offer the opportunity to reject impurities through its crystallization, as well as being primed for the introduction of the chiral side chain through alkylation. Although a synthetic strategy that uses protecting groups has inherent inefficiencies, we hoped these could be mitigated by the selection of a group that could be removed alongside the hydrolysis of the resorcylate methyl ester functionality, in order to unmask acid 15 (37). These considerations led to the targeting of the monobenzoylation of methyl α-resorcylate (23), a transformation known to generate a solid, namely ester 40 (38). Gratifyingly, the monobenzoylation could indeed be achieved with a high level of selectivity by maintaining the pH within a range of 7.8–8.2 (Scheme 9). The insolubility of 40 in the aqueous reaction medium was no doubt responsible for the limited levels of bisbenzoylation observed in this slurry-to-slurry transformation. Maintaining a near-constant pH was critical for the selectivity and achieved by codosing the benzoyl chloride with an aqueous solution prepared by mixing separate solutions of lithium hydroxide and potassium carbonate (20). This procedure provided 40 with levels of methyl α-resorcylate (23) and the overreaction product 41 that were both below 2 LCAP. 196

Scheme 9. Synthesis of acid 45 using a benzoyl protecting group strategy. (Reproduced from reference (20). Copyright 2018, American Chemical Society.) With the α-resorcylate 3- and 5-hydroxyls successfully differentiated using an alternative to the monoarylation used in the early development studies, there was now the need to etherify the remaining hydroxyl of phenol 40 with the chiral side chain. A Williamson etherification option based on the use of tosylate 27 (Scheme 5), cesium carbonate, and DMSO and analogous to the conditions used in the early development route was rapidly developed and scaled up, allowing access to ether 42. However, these basic conditions inevitably brought with them some unwanted migration of the recently installed benzoyl substituent to a second molecule of 40 (vide supra). The need to identify a long-term solution meant reconsidering the use of a Mitsunobu reaction, the reaction used by the medicinal chemists, as a possible means of installing the side chain (39–44). As previously mentioned, we were attracted by the option of telescoping the product of the etherification into a step that would simultaneously remove the benzoyl protecting group. Using liquid–liquid partitioning, the resorcylate hydroxyl unveiled in this manner could be used to draw phenol 43 into an alkaline phase, away from the redox byproducts of the Mitsunobu reaction. After some experimentation with the solvent and the order of addition of the reagents, the addition of diisopropyl azodicarboxylate (DIAD) to a toluene solution of the other components led to the rapid and clean conversion of phenol 40 to ether 42 (Scheme 9). The reaction proved to be faster and higher-yielding than the Williamson etherification conditions against which it was being compared. By using equimolar amounts of triphenylphosphine and DIAD, no residual DIAD tracked through into the workup. DSC analysis was used to establish that there were no thermal stability issues with any of the process or waste streams associated with the Mitsunobu reaction. Routing via ether 42 potentially posed the challenge of cleanly separating intermediates of interest from the benzoic acid byproduct arising from the hydrolytic removal of its benzoate ester functionality on downstream processing. With this in mind, a transesterification approach that generates an organic-soluble 197

alkyl benzoate byproduct was instead targeted for the debenzoylation. Gratifyingly, once the redox byproducts of the Mitsunobu reaction had been filtered off, the addition of methanolic sodium methoxide smoothly converted ester 42 to phenol 43 (Scheme 9). Careful extraction with dilute (0.25 M) potassium hydroxide solution left the methyl ester of 43 intact, allowing its potassium salt to be partitioned away from the methyl benzoate coproduct of the transesterification. This operation also served to partition phenol 43 away from residual organic-soluble redox byproducts from the Mitsunobu reaction and the bisetherification product 32 (Scheme 5). It also largely separated 43 from any lingering 41, an impurity which would reform α-resorcylic acid (44) (Scheme 9) on downstream processing (vide infra), but was found to transesterify more slowly than 42. The addition of more potassium hydroxide further basified the aqueous phase containing ester 43, allowing its hydrolysis to acid 45 (Scheme 9). The initial process generated 45 that was subject to oiling on attempted crystallization from a mixture of toluene and heptane. This was attributed to the low melting point (42 °C) of 45 and the presence of impurities. In order to rectify the latter, the aqueous phase arising from the hydrolysis was washed with tert-butyl methyl ether to remove small quantities of residual triphenylphosphine oxide and diisopropyl hydrazine-1,2-dicarboxylate. After acidification, 45 was extracted into tert-butyl methyl ether, away from a third impurity, residual α-resorcylic acid 44 (vide supra), whose levels were consequently reduced to ≤0.2 LCAP. The incorporation of these operations allowed 45 to be reproducibly isolated as a crystalline solid, providing the API synthesis with a valuable isolation point.

Route Selection The coupling of acid 45 and chloropyrazine 13 was discovered to be high-yielding when using cesium carbonate in DMSO. While the retention of these particular conditions would bring the same issues as to their use elsewhere on the project (vide supra), we were confident that a more in-depth re-evaluation of alternatives and process development studies could mitigate these issues. Consequently, the route initiated with the monobenzoylation of methyl α-resorcylate (23) was deemed to have yielded a viable means of accessing acid 15, the precursor to the API. The existence of monobenzoylation and monoarylation approaches for the monoprotection of methyl α-resorcylate (23) meant one option had to be discarded prior to initiating development for commercial scale production of the API. The route options are delineated in Scheme 10. In spite of the monobenzoylation approach involving two more transformations (five vs three) than the option that routed via ether 29, the selection of the former was straightforward. The monobenzoylation approach featured a crystalline precursor (acid 45) to acid 15 and a series of transformations that had proved to be high-yielding, even with minimal development. Although the use of the benzoyl protecting group is inefficient in terms of the number of reaction steps, processing is streamlined by the use of liquid–liquid partitioning to remove the intermediates of interest away 198

from related substances and reaction byproducts, such that the conversion of ester 40 to 45 can be telescoped.

Scheme 10. Summary of approaches to acid 15.

Although the monoarylation approach featured fewer chemical transformations than the monobenzoylation approach, it did not route via any highly crystalline solids that could be exploited in isolations. The hydrolysis of the ester functionality of 31, to afford acid 15, was also always likely to be accompanied by some hydrolysis of the azetidine amide and Cpyrazine–O bonds. This also ruled out converting 43 of the monobenzoylation approach to 15 via 31 (dotted arrow in Scheme 10). It should also be noted that, in spite of development efforts, the Williamson etherification process for the introduction of the side chain of ether 42 (Scheme 10) had a much less favorable process mass intensity (207.4 kg/[kg 45]) at the point where it was supplanted by the Mitsunobu conditions (69.6 kg/[kg 45]). Much 199

of the waste associated with the former process can be attributed to water washes used to remove DMSO from a product-rich organic phase.

Scale-Up of Manufacture of Acid 45 Not unexpectedly, given the presence of multiple phases and the potential for bisbenzoylation, the successful scale-up of the monobenzoylation reaction was envisaged as being challenging (45). Indeed, initial pilot plant batches produced ester 40 with up to 20 LCAP of bisbenzoylation product 41 (Scheme 9). This was partly attributed to the pH probe measurements, used to control the reagent additions, not aligning with the local pH at the site of the reaction, and addressed after different locations for the pH probe had been tested. Dosing the benzoyl chloride as a solution in toluene attenuated its background hydrolysis, making the control of pH easier. This had to be balanced against the increasing tendency of 40 to coalesce from fine particles into a thick, unstirrable mixture as the amount of toluene used was increased. A path forward was achieved by limiting the amount of toluene used (46% wt/wt benzoyl chloride) and the length of time (30 min) after the end of the addition before the product was filtered off. With these modifications in place, a normalized conversion of methyl α-resorcylate (23) to 40 of 96 LCAP could typically be achieved while restricting the amount of bisbenzoyl product 41 to about 2 LCAP. Control of the water content of 40, to well below the ~0.5% wt/wt associated with its initial isolation and drying, would ensure it was reproducibly consumed in the ensuing Mitsunobu reaction and limit the amount of α-resorcylic acid equivalent that could potentially track through the rest of the synthesis. This was achieved, and the purity improved, by treating a solution of the initially isolated 40 with activated charcoal and a powdered cellulose filter aid before it was recrystallized. In this way, 1.02 metric tons of 40 with a water content of 180 °C) (54, 55). The use of the phase-transfer catalyst Aliquat 336 in sulfolane lowered the temperature requirements, presumably by partially solubilizing the starting material. This allowed the decarboxylation to cleanly proceed over the course of an hour without excessive darkening of the reaction mixture. Controlling the water content of the mixture prior to heating to the reaction temperature helped to further control the color of the Na•53 produced during the decarboxylation and was achieved through azeotropic distillation with toluene. None of the regioisomeric 2,6-disubstituted product Na•54 arising from decarboxylation at the 3-position of the pyrazine ring was detected during the conversion of sodium salt Na•50 to Na•53. Calculations performed using dimethyl sulfoxide as a surrogate solvent for sulfolane (Scheme 13) indicate that the decarboxylation to form Na•53 proceeds via a lower energy pathway than Na•54, supporting the observed regioselectivity of the decarboxylation. 202

Scheme 13. Calculations performed at IEFPCM//B3LYP/6-31+G(d,p) level of theory to assess the regioselectivity of the decarboxylation. (Reproduced from reference (52). Copyright 2018, American Chemical Society.) Sodium salt Na•53 (Scheme 12) was isolated by filtering it off at the end of the reaction at 70 °C, a condition above the melting point of the sulfolane solvent. Initial scale-up batches displayed agglomeration during the subsequent drying operation, which was attributed to the presence of residual sulfolane in Na•53. The discovery that the yield for the downstream conversion to acid 34 could be increased from 60–68% to 85–90% by switching from Na•53 to the corresponding free acid as the starting material guided the decision not to dry Na•53 but to elaborate it to its free acid form. This was conveniently started by dissolving Na•53 off the filter with an alkaline solution and washing with toluene to remove any residual Aliquat 336 and the products of its decomposition. Subsequent acidification with hydrochloric acid precipitated the free acid 53 in a form that filtered easily (Scheme 12). This delivered 831 kg of high strength (98% wt/wt) 53 that was free of sulfolane, in an average yield of 77% from sodium salt Na•50. Laboratory studies showed that stepwise heating, at 50 °C (to form the acid chloride) and then 80 °C (to chlorinate the pyrazinone ring), of a toluene solution of acid 53 with phosphorus pentachloride efficiently formed acid chloride 35 (Scheme 14). Although 35 is an immediate precursor to chloropyrazine 13, in order to maximize the quality of the latter, the formation and isolation of acid 34 was targeted. After the phosphorus oxychloride byproduct of the chlorinations had been distilled off, the solution of 35 was treated with charcoal before it was hydrolyzed to 34, whereupon it crystallized out of the aqueous reaction medium. 203

This sequence allowed 698 kg of 34 to be isolated with a strength of >99.5% wt/wt and in a yield of 60% relative to the diaminomaleonitrile (47) input.

Scheme 14. Completion of the late-stage manufacture of chloropyrazine 13.

Sourcing azetidine free base, the coupling partner of acid 34, was ruled out due to its tendency to oligomerize (56), necessitating the use of the hydrochloride salt (57, 58). Accelerating rate calorimetry (ARC) of this salt indicated an onset of exothermic activity from ~71 °C, although further testing indicated that it was not a detonation or deflagration risk (59). 3-Chloropropylamine (55) is a typical impurity in azetidine hydrochloride and gives rise to impurities 56 and 57 (Figure 4). 3-Chloropropylamine (55) was shown to react faster than azetidine with acid chloride 35, so any increase in the charge of azetidine relative to 35 would increase levels of alkyl chloride 56. Fortuitously, it proved possible to control levels of 55 in an aqueous azetidine solution by washing it with toluene prior to use in the coupling with 35. The coupling performed well when the toluene solution of 35, formed this time from 34, was added to an alkaline solution of azetidine (Scheme 14). The success of these biphasic conditions meant the dichloromethane solvent used in the early development iteration, with its toxicity and incineration-related liabilities (60), was no longer required for the coupling. As with previous versions of the coupling, a reverse addition minimized opening of the azetidine ring of chloropyrazine 13 by chloride. A disadvantage of this protocol was the formation of impurity 58 that it encouraged (Figure 4), although 58 was conveniently purged in the mother liquors from the crystallization of 13. The procedure was used to deliver 647 kg of 13 in an average yield of 75% from 34. With further development, there may well be the opportunity to access 13 directly from acid 53 without the need to isolate 34.

204

Figure 4. Impurities associated with the formation of chloropyrazine 13. (Reproduced from reference (52). Copyright 2018, American Chemical Society.)

SNAr Reaction Development A large amount of process development was performed on the stages associated with the conversion of methyl α-resorcylate (23) to acid 45 in the period leading up to the selection of the best way of manufacturing ether 15. By contrast, the development of the conversion of 45 to AZD1656 (7) had only been subject to a series of incremental improvements, over the same period, in order to derisk the continual manufacture of development supplies. With the decision to route via 45 confirmed, more wide-ranging improvements to its coupling with chloropyrazine 13 to form 15 were considered. The success achieved with carbonate bases was thought to be due to the presence of an equilibrium quantity of dianion arising from the partial deprotonation of its phenolic functionality together with the deprotonation of the carboxylic acid of 45 (Scheme 15). The assessment of potassium carbonate as a cheaper and more easily suspended alternative to the cesium carbonate used in the initial version of the process was initially characterized by excessive reaction times. This was addressed through the addition of a small quantity of exogeneous water, which presumably helped to solubilize the base. As with all heterogeneous reactions, there was the risk of the offline analysis of the reaction being performed with a sample that was not representative of the whole batch. With this in mind, in-line Raman data was used to follow the conversion of 45 to ether 15 (61).

205

Scheme 15. Transformations associated with SNAr reaction. A small amount (0.1–0.2 LCAP) of ester 59 always formed in the reaction, presumably through the intermediacy of an activated carbonyl species 60 (Scheme 15). Ester 59 could be hydrolyzed conveniently to acid 15 by further basifying the system at the end of the reaction through the addition of more water. After gathering solubility data for 15 as a function of temperature and solvent (DMSOwater mixtures), a final solvent composition of 56% v/v dimethyl sulfoxide was targeted and achieved by acidifying with concentrated hydrochloric acid. The potassium chloride generated by the neutralization was screened off from the hot solution in order to control the sulfated ash content of the isolated ether 15, as well as the mobility of the slurries from which it was isolated. After subsequent cooling to 20 °C and seeding, a slurry of high-quality 15 was formed. The alternative processing option that was actually implemented, due to time constraints, involved acidifying with dilute hydrochloric acid and extracting the ether 15 from the slurry that formed using liquid–liquid extractions with warm isopropyl acetate. A water wash rid the extracts of adventitiously extracted dimethyl sulfoxide, before azeotropically drying and a cooling crystallization yielded 15 that was readily filtered, washed, and dried. This protocol allowed 742 kg of 15 to be produced in an overall yield of 93%. One of the factors that favored the adoption of the workup process based on an extractive workup was confidence that crystallization would retain residual chloropyrazine 13 in its mother liquors. If residual levels of this material in 15 were not controlled, it had the capability of tracking downstream and disfavoring the crystallization of the desired polymorph of the API. 206

Redevelopment of Aminopyrazine 11 The medicinal chemistry route to aminopyrazine 11, while concise, was not without its drawbacks. The need for a large excess of tert-butyl alcohol, in order to fully trap isocyanate 39 (Scheme 8), brought with it the contamination of the carbamate 37 with related substances. These arose from the more efficient trapping of isocyanate by small quantities (0.1–0.2% wt/wt) of alcohol congeners in the tert-butyl alcohol. The thermal lability of carbamate 37 under the conditions of the Curtius rearrangement led to the premature formation of a small quantity of 11, which was unproductively trapped as a urea impurity 61 (Scheme 16) by the remaining isocyanate intermediate. The multiple extractions used to extract 11 after the deprotection of carbamate 37 were especially onerous (Scheme 8).

Scheme 16. Curtius approach to aminopyrazine 11. As demands for the drug substance increased, and in light of the above caveats, it became apparent that the route to aminopyrazine 11 shown in Scheme 8 was eligible for re-evaluation (62). The hydrolysis of isocyanate 39 generated over the course of the Curtius rearrangement of acid 36 was not progressed due to concerns over the recovery of the water-soluble 11 from a medium with an appreciable water content. Routing via an intermediate that protected the amino group as a benzyl carbamate, which could be removed by hydrogenolysis catalyzed by a supported metal catalyst, was targeted because it would avoid the need for an aqueous workup. Gratifyingly, and as might have been expected, the substitution of the tert-butyl alcohol by the more reactive benzyl alcohol led to the formation of the corresponding carbamate product 62 in a cleaner fashion (Scheme 16). Revisiting the route to aminopyrazine 11 (Scheme 16), and the ever increasing scale of its manufacture, required an updating of the control strategy used to manage the hazards posed by the azide-containing process streams produced by the Curtius rearrangement (63). Self-heating due to decomposition of the acyl azide solution could occur at ambient plant temperatures, with an initial temperature of 21–23 °C corresponding to an adiabatic time to maximum rate of 24 h. Consequently, the addition of the DPPA was performed over at least 2 h and 207

at no more than 17 °C (64). In the event of a failure of either cooling or agitation, a fast dump into aqueous sodium hydroxide solution was kept as an alternative, should the batch temperature have exceeded 30 °C. In order to eliminate the risk of DPPA hydrolysis to volatile (bp 37 °C) and explosive hydrazoic acid, as well as to prevent any isocyanate hydrolysis, toluene solutions of acid 36 and benzyl alcohol were azeotropically dried prior to their use. The controlled disengagement of the nitrogen generated by the Curtius rearrangement itself was achieved by the regulated addition of the acyl azide solution to a solution of benzyl alcohol in toluene kept at 86–88 °C, a condition above the thermal trigger point for the Curtius rearrangement. As shown in Figure 5, when this addition took place over 2 h, gas generation was pro rata.

Figure 5. Heat and gas generation during the addition of the acyl azide solution to a solution of benzyl alcohol. (Reproduced from reference (62). Copyright 2018, American Chemical Society.) The hydrogenolysis of benzyl carbamate 62 to aminopyrazine 11 using a palladium on charcoal catalyst proved relatively straightforward to develop (Scheme 16). The exothermicity of the reaction was controlled by limiting the hydrogen pressure to just one atmosphere. Trace quantities of acidic impurities that could protonate the pyrazine ring were speculated as giving rise to small amounts of overreduction to dihydropyrazine 63 (Scheme 17). It was duly discouraged by hydrogenolyzing in the presence of a catalytic amount (1 mol%) of sodium hydroxide. The small quantities (0.10–0.15 LCAP) of 63 that did form were oxidized back to 11 by passing a stream of 1% oxygen in nitrogen through the batch. These processes were used in two separate manufacturing campaigns, each of which produced ca. 200 kg of aminopyrazine 11. By routing via a benzyl carbamate instead of a tert-butyl carbamate, the overall yield for the 208

manufacture of 11 was improved from 43% in early scale-up deliveries to 68%. The corresponding reduction in process mass intensity was 56%, largely due to the removal of an aqueous workup from the step used to form 11.

Scheme 17. (Reversible) overreduction side reaction.

Amidation To Form AZD1656 The continued use of n-propylphosphonic anhydride (T3P) for the final bondforming step used to form the API on a large scale posed a number of drawbacks. These included the need and expense associated with the use of a ~70% mol/mol excess of the reagent to completely consume acid 15 (65). As a chemical weapons precursor, the storage, use, and disposal of T3P also needs to be fully documented. The n-propylphosphonic acid waste generated from its use also requires treatment prior to discharge into watercourses. Finally, impurities formed in a process using T3P retarded the rate of crystallization of the desired polymorph of the API (vide infra), necessitating its later recrystallization. With the need to develop a long-term solution for the amidation step, attention turned to the activation of acid 15 using thionyl chloride, so as to form acid chloride 64 (Figure 6). The initial examination of these conditions resulted in the observation of significant quantities of the corresponding acid anhydride 65 (Figure 6), a material that only slowly turned over to AZD1656 (7) once aminopyrazine 11 had been added. Acid chloride 64, although more reactive toward 11 than acid anhydride 65, still reacted sluggishly. In addition, inadequate scavenging of the hydrogen chloride generated as a byproduct of the use of thionyl chloride led to partial opening of the azetidine ring, so as to form alkyl chloride 66. As Scheme 18 shows, unscavenged hydrogen chloride was also implicated in the formation of other impurities, including amide 67 and 68 (formed via phenol 69).

Figure 6. Intermediates associated with the activation of acid 15 using thionyl chloride. 209

Scheme 18. Transformations associated with amidation reaction.

A screen of different base additives present during the acid chloride formation, as well as solvents, focused on the use of pyridine as the additive and acetonitrile as solvent. Under these circumstances (Scheme 19), the level of alkyl chloride 66 in isolated AZD1656 (7) was only about 200 ppm and the level of the refractory anhydride intermediate 65 was lower compared with the level obtained using other basic additives. Further advantages included short reaction times for the activation of acid 15 and the subsequent amidation (98%

98

99.5

Quality

GLP

GMP

GMP

Pre-tox in rat and dog

Tox in rat and dog FIH/SAD Formulation work

MAD

B

A

A

Amount (kg)

Purpose

Polymorph

0.004

Campaign 1

PD model

References 1.

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Chapter 9

Discovery, Process Development, and Scale-Up of a Benzoxazepine-Containing mTor Inhibitor James W. Leahy,1,* Sriram Naganathan,2,* Denise L. Andersen,3 Neil G. Andersen,4 and Stephen Lau5 Exelixis, Inc., 1851 Harbor Bay Parkway, Alameda, California 94502, United States *E-mail: [email protected]; [email protected] 1Current Address: Department of Chemistry, The Florida Center of Excellence for Drug Discovery & Innovation, University of South Florida, 3720 Spectrum Boulevard, Suite 305, Tampa, Florida 33612, United States 2Current Address: Dermira, Inc., 275 Middlefield Road, Suite 150, Menlo Park, California 94025, United States 3Current Address: Cytokinetics, Inc., 280 E. Grand Avenue, South San Francisco, California 94080, United States 4Current Address: Achaogen, 1 Tower Place, Suite 300, South San Francisco, California 94080, United States 5Current Address: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404, United States

A family of novel, highly potent, and selective inhibitors of the mammalian target of rapamycin (mTOR) containing the benzoxazepine core were identified. The lead compound (XL388), which exhibited low-nanomolar activity and >1000-fold selectivity over related PI3K kinases, was chosen for further development as a potential oral treatment of several different tumor types. It inhibited cellular phosphorylation of substrates of mTOR complexes 1 and 2. It also showed good pharmacokinetics, moderate oral bioavailability, and dose-dependent antitumor activity in mice implanted with human breast and colon tumor xenografts. The synthesis of the clinical candidate was rapidly scaled up to enable first-in-human studies. A safe and scalable route and process were developed for the tetrahydrobenzo[f][1,4]oxazepine core fragment. The constituent fragments of XL388 were assembled © 2018 American Chemical Society

using a telescoped sequence to manufacture 7.8 kg of active pharmaceutical ingredient under current Good Manufacturing Practice (cGMP), in 99.7 HPLC area % purity and a 21% overall yield over eight synthetic steps.

Introduction When Sehgal and coworkers at Ayerst Research Laboratories reported the isolation of rapamycin (1, Figure 1) from a strain of Streptomyces hygroscopicus in 1975 (1, 2), it is hard to imagine that they could have foreseen the implications that their discovery would have nearly half a century later (3). Not only is 1 (aka sirolimus) an approved immunosuppressant drug, but its hydroxyethyl derivative 2 (i.e., everolimus) is also approved for use in both immunosuppressant and oncologic indications (4). However, the approval of 1 as a drug really only scratches the surface of the impact of its discovery.

Figure 1. Rapamycin, Everolimus, and FK-506. When a team at Fujisawa Pharmaceutical Co. isolated the related macrolide 3 (FK-506, i.e., tacrolimus) from a strain of Streptomyces tsukubaensis found in a Japanese soil sample and demonstrated that it too possessed immunosuppressive properties (5), it sparked the subsequent investigation of the unique pipecolinate/ tricarbonyl moieties that each of these compounds possesses. This led to the discovery that they bind to an endogenous family of peptidyl-prolyl isomerases called immunophilins and, specifically, to FK-506 binding proteins, or FKBPs (Figure 2) (6). While it was quickly established that the complex of FKBP12 and 3 led to the inhibition of the calcium-dependent protein phosphatase calcineurin (7), the discovery of the biological objective of FKBP12 and 1 took a more convoluted pathway (8). Ultimately, it became known as the Target of Rapamycin (TOR), and 250

the mammalian orthologue (mTOR) has proven to be a fascinating molecule with myriad therapeutic ramifications (9, 10).

Figure 2. Biochemical Targets of 1 and 3.

Subsequent study of mTOR revealed that it plays a complex role in cellular regulation. To date, it has been shown to be a critical component of two different protein complexes known as mTORC1 and mTORC2, and these complexes help control very different cellular processes (Figure 3) (11). The mTORC1 complex includes mTOR and the regulatory-associated protein of mTOR (RAPTOR). It is activated through a series of signaling pathways, such as the Akt or the MAPK/ ERK pathways, triggering translation (via the phosphorylation of 4E-BP1 and S6K), cholesterol and fatty acid biosynthesis (via phosphorylation of SREBPs), and inhibiting autophagy (via phosphorylation of Ulk1) (12).

Figure 3. mTORC1 and mTORC2 signaling pathways. 251

This complex can be controlled with rapamycin and is presumed to be the therapeutic target of 1 as a drug. Meanwhile, mTORC2 is a complex with the rapamycin-insensitive companion of mTOR (RICTOR) which, as the name implies, is considerably less prone to rapamycin control. mTORC2 has been implicated in a number of cellular processes including, among other things, regulation of the cytoskeleton and autophagy. Insulin-like growth factor 1 receptor (IGF1R), protein kinase C, RhoA, and Akt are among the downstream targets of mTORC2, the latter of which may serve to regulate mTORC1. Fortunately, for purposes of understanding the implications of abrogating these pathways, both of these complexes can be controlled by a single ligand called Torin 1 (13). Unlike calcineurin, which is a phosphatase (14), mTOR is a kinase (15). Furthermore, it has been shown to be a member of the phosphoinositol-3-kinase (PI3K) family (Figure 4), which phosphorylate phosphatidylinositols—intracellular carriers of phosphates that can subsequently phosphorylate other targets and are composed of a number of different members (e.g., PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ, Vps34, ATM, DNA-PK, and mTOR) (16). The PI3Ks have attracted considerable interest as potential drug targets, primarily for the treatment of cancer (17).

Figure 4. PI3K family of kinases.

Idelalisib (4) (18) and copanlisib (5) (19) are both PI3K inhibitors that have been approved by the U. S. Food and Drug Administration for oncological indications (Figure 5). Each of these compounds possesses some isoform selectivity (i.e., selective inhibition of one of the members of the family over other members), where 4 is selective for PI3Kδ, whereas 5 is a dual inhibitor of both PI3Kα and PI3Kδ. However, the prospects of inhibiting other isoforms or even all of the isoforms also attracted considerable attention from a drug discovery perspective. For example, alpelisib (6) (20) and taselisib (7) (21) are selective for the inhibition of PI3Kα and duvelisib (8) is a dual PI3Kγ/PI3Kδ inhibitor (22), whereas buparlisib (9) (23) and voxtalisib (10) (24) are pan-selective PI3K inhibitors. Notably, dactolisib (11) is an inhibitor of mTOR as well as of all of the isoforms of PI3K (25).

252

Figure 5. Some of the known inhibitors of PI3K kinases.

Initial Research Studies As part of our own program aimed at developing PI3K inhibitors, we sought to identify small molecules that would be selective for the inhibition of mTOR, which we felt could be a useful approach to the treatment of cancer that might limit side effects that could arise via full inhibition of other PI3K isoforms. The drug discovery program at Exelixis is predicated on the high-throughput screening (HTS) of the in-house library of nearly 5 million individual compounds. This library of compounds was based in part on the practical generation of scaffolds that contained multiple points for diversification. This strategy could be exemplified in Scheme 1. A core scaffold such as 12 could be designed that contained two orthogonally functionalized moieties that would allow derivatization using well-established reactions that are amenable to high-throughput synthesis. In the example shown, a large-scale preparation of core 12 allows for both the installation of a variety of different groups at the bromide (SNAr substitution as well as a number of palladium-mediated transformations) followed by the subsequent deprotection of the amine and installation of a variety of additional diverse groups such that thousands of versions of 14 could be prepared. For this particular project, core 15, a benzoxazepine, proved to be our starting point.

Scheme 1. Diversity/density library design and core 15.

253

Using a single-point HTS mTOR assay to initially identify potential actives, we were able to find ~8,000 compounds that warranted further investigation. Based on an enzyme-linked immunosorbent assay designed to measure the phosphorylation of 4E-BP1 (the downstream target of mTORC1), we conducted a full IC50 evaluation of these compounds and counter-screened them against PI3Kα to identify compounds that would provide selective leads. Among the hits that were identified from this campaign was 16 (Figure 6), which had been added to our library via the aforementioned diversification strategy.

Figure 6. Initial HTS hit. We were intrigued by 16 for a variety of reasons, including that the benzoxazepine scaffold was virtually unexplored from a drug discovery perspective (26) and that the quinoline functionality was well-known as a key component of kinase inhibitors (27). Furthermore, our modular synthetic approach to the synthesis of 16 allowed for the rapid exploration of the structure–activity relationship (SAR) around this lead. Initially, we had synthesized 16 from the benzoxazepine 15, which we had prepared from chromone via Schmidt rearrangement, bromination, and reduction of the lactam (Scheme 2 – yield on largest scale shown) (28). Because we had copious amounts of this core on hand, we were also able to prepare analogs 20 and 21 in order to confirm that both of the appendages to our initial hit were required for activity (Figure 7) (29).

Scheme 2. Initial synthesis of core 15.

Figure 7. Follow-on SAR via functional group deletion. 254

By confirming that deletion of either substituent rendered the compounds effectively inactive, we felt confident that we would be able to rapidly explore the SAR about this scaffold and hone in on an optimized compound. As stated above, given the modular approach to this core structure, we also were confident that we could readily and rapidly access a large number of analogs with great flexibility. Furthermore, with an initial hit that falls within traditional Lipinski parameters (30), we were also optimistic about the prospects of identifying lead compounds with acceptable physicochemical properties. Even in the face of this optimism, we had considerable internal debate about the binding orientation of benzoxazepine 16 with the kinase binding region of mTOR. There is ample evidence that quinoline kinase inhibitors bind to the hinge region via a hydrogen bond with the quinoline nitrogen such as with 22 (Figure 8) (31). However, removal of the quinoline nitrogen of 16 via the synthesis of naphthalene derivative 23 did not diminish mTOR inhibition (in fact, it increased the selectivity over PI3Kα), effectively ruling out this binding orientation.

Figure 8. Hypothesis for binding orientation of 16. We therefore began to suspect that the quinoline was instead interacting with the DFG loop (an activation loop found in kinases that contains a conserved AspPhe-Gly, or DFG, sequence) similar to the way it does in 11 (32). If that were to be the case, it would suggest that the hinge region would instead be binding to the benzoxazepine oxygen. Although such an interaction would be unprecedented, it 255

was not hard to imagine given the number of morpholine derivatives such as 9 that have been demonstrated to inhibit PI3K (33). We therefore sought to conduct early SAR studies by modifying the region we suspected of interacting with the DFG region (Figure 9). A number of heterocyclic substituents were well-tolerated in this region, but we were struck by the increased potency and PI3Kα selectivity observed with a 5-benzimidazolyl substituent.

Figure 9. Activity of benzimidazole analogs and benzamide analog. In fact, 24 was the first sample we had that effectively pegged our mTOR assay and demonstrated a subnanomolar IC50. It also increased our observed selectivity over PI3Kα to nearly 200-fold. We therefore initiated a cellular assay looking at the phosphorylation of S6K in a human prostate cancer cell line (PC-3) and were pleased to see reasonable inhibition in this assay as well.

Lead Discovery With a viable (and readily available) DFG-loop binder in hand, we set out to explore the amide region of the benzoxazepine. Although we had confirmed that complete removal of the benzamide resulted in concomitant complete loss of activity, we did have enough samples from our initial HTS collection to observe that the p-trifluoroacetyl substituent increased the activity. For example, 26 was also an HTS hit, and although it was not as active as benzoxazepine 16, the activity drop was small enough that we felt there was room for optimization. We therefore set out to optimize this region of the molecule. We also began focusing on some of the potential ADME liabilities of our lead structure. Specifically, we had observed that our lead compound (at this point, 25) was only moderately stable in mouse liver microsomes, with only ~ 50% of the compound remaining after only 30 min. This would obviously portend a short and undesirable half-life when we initiated in vivo studies. Furthermore, it also was quite active at inhibiting several cytochrome P450 isoforms, so we examined our new analogs in each of these assays in parallel to try to rapidly hone in on a potential lead compound. 256

Finally, we utilized a crude method for the relatively rapid assessment of potential pharmacokinetic viability by performing what we referred to as “HTS PK,” wherein we would dose mice with our compounds (in this case at 100 mg/kg PO) and observe how much of the material could be found in their blood at 1 h and 4 h timepoints. In this manner, we could tell whether the compound was likely to have any oral bioavailability as well as get an appreciation for how fast it was being cleared. We quickly settled on replacing the trifluoroacetyl group with a sulfone, which did not have a dramatic impact on the mTOR activity of our lead compound, but did show an appreciable improvement with respect to the compound’s stability in mouse liver microsomes as well as its proclivity for inhibiting the various cytochrome P450s (CYPs) (Figure 10). Furthermore, we were able to observe feasible mouse HTS PK with 27, which gave us confidence that we could identify a lead compound that not only retained the activity/selectivity profile we were seeking, but also would be orally bioavailable.

Figure 10. Sulfone leads and PC-3 xenograft efficacy of 28. 257

We recognized that we had focused exclusively on the para position of the benzamide, so we initiated a more advanced SAR campaign that would explore other positions around this benzene ring. We were delighted to find that substituting ortho to the carbonyl imparted dramatically enhanced selectivity for mTOR over PI3Kα, a feature that did not extend to meta-substitution. We felt that 28 was a viable candidate for demonstration of a proof of concept given its >500-fold selectivity for mTOR, decent cellular activity, reasonable HTS PK, and ADME parameters. We were therefore pleased to observe a marked pharmacodynamic effect, with observable knockdown of p-Akt (S473), p-p70S6K (T389), p-S6 (S240/244), and p-4E-BP1 (T37/46) at 100 mg/kg. Furthermore, we saw 98% tumor growth inhibition with this compound in a PC-3 mouse xenograft model at 300 mg/kg dosing, suggesting that an mTOR selective compound could indeed prove effective. However, we also recognized that we still needed to improve on our lead compound. Using the ortho-alkylated sulfone as our starting point, we initiated another SAR campaign at the meta-position. We quickly established that fluorination at the meta-position not only led to a moderate increase in mTOR activity, but had a dramatic impact on its behavior in the metabolism panel. Specifically, 29 was effectively untouched in mouse liver microsomes and had a >1,000-fold selectivity profile for inhibition of mTOR over any of the PI3K isoforms in our panel (Figure 11).

Figure 11. Optimized amide.

We made a variety of additional analogs and found that replacing either or both of the methyl groups with ethyl groups typically improved activity against mTOR but resulted in a less desirable ADME profile, so much of our final work involved making each of these permutations. Content that we had significantly improved 258

this region of our lead and that there was little room for additional enhancement, we returned to further optimization of the DFG-loop heterocycle instead of the benzimidazole.

Lead Optimization Ultimately, we prepared a myriad of different analogs that were modified exclusively on the heterocycle, with an eye toward identifying a candidate with optimal biochemical, cellular, and pharmacodynamic activity against mTOR that also had excellent ADME and pharmacokinetic parameters. Not surprisingly, these efforts revealed a number of compounds that were superior in one or more of these categories, but fell short in others. A full discussion of all of these compounds extends far beyond the scope of this chapter (29), but we ultimately settled on the three “final” candidates shown in Figure 12.

Figure 12. Top development candidates.

Each of these compounds displayed attractive features. Analog 32, for example, was among the most potent compounds we found against mTOR with nearly 17,000-fold selectivity over PI3Kα. However, the ADME and PK characteristics of 30 and 31 were dramatically superior. We therefore examined each of these candidates in a mouse xenograft MCF-7 human breast cancer efficacy study (Figure 13). It was clear that both 30 and 31 were superior to 32 at equal doses, so we based our final decision on the cleaner metabolic profile and thus selected 30, which became known as XL388, as our development candidate. Development candidate 30 (XL388) is active against both mTORC1 (IC50 = 9.8 nM) and mTORC2 (IC50 = 166 nM), meaning it is active against both the rapamycin-sensitive and rapamycin-resistant complexes. It is inactive against each of the isoforms of PI3K (PI3Kα IC50 > 3 μM; PI3Kβ, PI3Kγ, and PI3Kδ IC50’s all > 5 μM), and the only other PI3K family of kinases for which it showed any observable activity was DNA-PK (IC50 = 8.8 μM). In fact, it was inactive against an extensive (> 140) panel of kinases. 259

Figure 13. MCF-7 xenograft efficacy of 30, 31, and 32. We therefore examined the effects of 30 in both MCF-7 human breast cancer cells and Colo-205 human colon cancer cells against several of the markers for mTOR inhibition, including both S6 and 4E-BP1 (downstream targets of mTORC1) and Akt (mTORC2). In MCF-7, the IC50’s for these were 303 nM, 448 nM, and 350 nM, respectively, while in Colo-205, these IC50’s were 162 nM, 611 nM, and 421 nM. From an ADME perspective, the plasma protein binding fell within an acceptable 84–90% range in all species tested (mouse, rat, dog, monkey, and human) and the compound was quite stable on exposure to liver microsomes for each of these species as well. From a CYP inhibition perspective, it did not show substantial inhibition of any of the isoforms of human CYPs tested at 5 μM, including inhibition of both midazolam and testosterone oxidation by CYP3A4. It also did not show PXP-mediated CYP induction. Furthermore, benzoxazepine 30 was determined not to inhibit P-glycoprotein-mediated transport in a calcein AM assay (34) and showed acceptable permeability (Papp = 151 nm/s) in MDCK cells. The potential for inhibition of the hERG potassium channel is a concern for any kinase inhibitor, so we were pleased to see that the IC50 of our lead compound in a patch clamp assay was greater than 15 μM. We also evaluated it in multiple genotoxicity assays and observed no induction of mutations. We were thus convinced that it was safe for full pharmacokinetic analysis. The development candidate, formulated as an HCl salt with ethanol, PEG400, and water, is orally bioavailable in mice, rats, dogs, and monkeys. Furthermore, we were able to establish that an oral dose of 100 mg/kg of 30 in a nude mouse xenograft model knocked down phosphorylation of each of our downstream markers (S6, 4E-BP1, and Akt) by more than 80% in each of the cell lines we examined for at least 8 h, with the inhibition being almost completely gone by 24 h. This definitive pharmacodynamic effect provided us with ample confidence that we could move forward with efficacy models. In separate 14 d efficacy studies 260

using human tumor xenografts in nude mice dosing benzoxazepine 30 at 100 mg/ kg bid, we were able to observe 31% regression against MCF-7 and 92% tumor growth inhibition against Colo-205, both of which were significantly higher than a 5 mg/kg IP dose of 1 in the same models. We were thus confident of moving forward with our nomination of 30 (XL388) as a viable clinical candidate.

Process Development When we embarked on process development for the scale-up, we recognized the need to quickly prepare approximately 0.5 – 1 kg of benzoxazepine 30 (XL388) for Investigative New Drug (IND) Application-enabling toxicology studies and approximately 5 kg for first-time-in-human clinical studies, as well as for development of a suitable clinical formulation.

Scheme 3. Discovery synthesis of XL388. The discovery synthesis was simple and modular (Scheme 3); yield on largest scale shown. We decided to follow the same approach with some modifications. The final coupling to form the amide bond using the acid chloride led to significant quantities of the bis-amide impurity 39 (Figure 14), due to lower selectivity between the secondary amine and the aromatic amine in the reaction with the acid chloride 38. It was decided early in the program to pursue the direct coupling of the acid of 38 to the amine partner 37, where the amide coupling was more selective, thus avoiding the formation of the bis-amide. The amide coupling using the acid chloride was nevertheless an attractive option, but the decision to utilize the acid was based on limited available data and a tight development timeline.

Figure 14. Bis-amide impurity. 261

As discussed in the SAR effort leading to the discovery, the nominated benzoxazepine candidate (30) is made up of three recognizable fragments, allowing for modular assembly of the final molecule (Figure 15).

Figure 15. Retrosynthetic analysis.

Thus, the process development effort was divided into three areas: production of the methylsulfonyl benzoic acid (40), production of the Boc-protected bromobenzoxazepine (33), and the assembly of the final active pharmaceutical ingredient. The required 5-bromo-aminopyridine (35) is commercially available in substantial quantities from a variety of commercial suppliers. The target amount of the drug substance translated into requirements of about 5 – 10 kg of 40 and about 12 – 15 kg of 33. The process development for the large-scale manufacture of the two intermediates and the drug substance are discussed in the following sections (35).

Process Development of Bromobenzoxazepine The initial route for the production of bromobenzoxazepine to enable the discovery effort was based on the ring expansion of 4-chromanone (17) (36, 37) using a Schmidt rearrangement (38, 39) to arrive at the benzoxazepine ring (Scheme 4). Aromatic bromination using conventional methods afforded the brominated lactam 19. If the bromination was attempted on the chromanone prior to ring-expansion using the Schmidt rearrangement, it had the opposite regioselectivity. Reduction of the lactam 19 to the amine 15 was accomplished using LAH complexed with Grignard reagents (40, 41) to avoid debromination (42). Protection of 15 with Boc-anhydride (43), to form the Boc-protected bromobenzoxazepine 33, completed the production of the required starting material. 262

Scheme 4. Early synthesis of bromobenzoxazepine. There were several areas of this synthesis that proved to be problematic. The two main concerns were regarding the safety of the Schmidt rearrangement (associated with the handling of sodium azide on a large scale and inconsistency in the timing and rate of nitrogen gas evolution during rearrangement) and the amount of debrominated product 41 (Figure 16), which was formed during the hydride reduction of the lactam.

Figure 16. Structure of debrominated product 41. The level of debrominated product increased with increasing scale of the reaction, resulting in up to 40% of this byproduct on a 500 g scale. Although small amounts of the debrominated product could be purged during the final process, the majority of the 41 had to be separated from the desired Boc-protected bromobenzoxazepine 33 by careful chromatography. This approach was not feasible to produce sufficient quantities of 33 required to support the needs of the development program. Interim Approach to Bromobenzoxazepine An alternative approach was pursued by the discovery group, one which avoided the use of aluminum-based reducing agent, thereby mitigating conditions that lead to debromination. In this approach, the benzoxazepine ring was constructed using a Mitsunobu reaction (Scheme 5) (44–46). The precursor to the ring-forming reaction was obtained following the reductive amination reaction between ethanolamine and 5-bromosalicylaldehyde (42). Protection of the resulting amine with Boc-anhydride in aqueous base yields the key primary 263

alcohol 43. Cyclization of 43 under the conditions of the Mitsunobu reaction yields Boc-protected bromobenzoxazepine 33.

Scheme 5. Mitsunobu route to bromobenzoxazepine. This strategy proved valuable for providing material for further development needs, albeit only on an interim basis. It was not suitable as a long-term solution due to safety concerns, high cost, and the complex isolation procedures required to obtain the product in high purity.

Imide Route to Bromobenzoxazepine Although the issue of debromination during the reduction could be solved by using alternative reducing agents, the establishment of the benzoxazepine ring was the major concern due to the safety concerns of the Schmidt rearrangement. We chose to solve both problems in the same approach by first constructing the seven-membered ring as an imide and then reducing it with borane (Scheme 6). The use of borane as a reducing agent avoids the debromination associated with LAH (47, 48).

Scheme 6. Imide route to bromobenzoxazepine. The required starting materials for this approach, ethyl bromoacetate and 5-bromosalicylamide (44), are available commercially. Reaction of ethyl bromoacetate and amide 44 in the presence of potassium carbonate and dimethylacetamide yielded the ether 45. This intermediate was not isolated, but directly converted to the imide 46 using the commercially available THF solution of sodium t-amylate. Isolation of the imide by filtration was accomplished 264

following its precipitation by addition of aqueous citric acid. The reduction of 46 to the cyclic amine was conducted using an excess of borane-THF solution. The excess borane was neutralized with methanol, but the resulting intermediate amine-borane complex was stable under the conditions of the reaction. The amine could be liberated from the complex by treatment with concentrated hydrochloric acid under refluxing conditions, allowing for the convenient isolation of the amine as a hydrochloride salt (15•HCl). Treatment of 15•HCl with Boc-anhydride in ethanolic aqueous potassium carbonate yielded the required Boc-protected bromobenzoxazepine 33. The imide-based approach worked well on small scale, but several significant problems were encountered when the process was scaled up. The filtration of the scaled-up imide was very slow, taking several days instead of the few hours expected based on small-scale experience. The yield of the isolated product was also much lower, 23% versus the typical yield of 70–80% obtained on the small-scale experiments. Upon analyzing the reaction streams and filtrates, we discovered the major product was the acid-amide 47 formed from hydrolysis of the intermediate ester 45 (Figure 17).

Figure 17. Structure of the intermediate hydrolysis product 47.

During the in-process analysis for reaction completion, no evidence for the formation of 47 was observed. Thus, the most probable origin of this hydrolysis product was during the quench. Because the neutralization of the reaction mixture was highly exothermic, the aqueous citric acid was added rather slowly, and this led to the presence of the product in a highly alkaline aqueous milieu leading to a significant amount of the hydrolysis product (49). It became apparent that an alternative method of forming the imide 46 would be necessary.

Borane Reduction of the Imide Reduction of imide 46 with borane was explored as a way of mitigating the debromination observed when employing aluminum-based reducing agents (50, 51). Because the initial reaction between the borane and the imide was accompanied by liberation of one mole of hydrogen, we carried out calorimetric evaluation to determine the rate of gas evolution (Figure 18) and any related exotherm (Figure 19). 265

Figure 18. Hydrogen evolution during addition of BH3•THF to 46. ( + = Hydrogen evolution; o = amount of BH3•THF added.). (Reproduced with permission from ref. (35). Copyright 2015 American Chemical Society.)

Figure 19. Heat evolution during addition of BH3•THF to imide 46. (Reproduced with permission from ref. (35). Copyright 2015 American Chemical Society.) As expected, the hydrogen evolution was practically instantaneous and was confined to the initial part of the addition. Similarly, most of the heat evolution was also observed only during the initial 10% of the addition, and the exotherm was essentially limited to the first third of the addition. Based on the calorimetric data, the BH3•THF was added carefully to a slurry of imide 46 in THF while maintaining the temperature below approximately 35 °C. Due to concerns about the risk of forming diborane during the reduction, the reaction was not heated above approximately 50 °C (52, 53). Because neutralization of the excess borane by the addition of methanol also liberated hydrogen gas, calorimetric analysis was performed to study the characteristics of the gas evolution and related exotherm for the neutralization. Data from calorimetric studies showed that both the hydrogen evolution (Figure 20) and the exotherm (Figure 21) were limited to the first 10% of methanol charge, and both could be controlled by the addition rate. Fortunately, there was no delayed onset of the hydrogen generation or heat evolution. 266

Figure 20. Hydrogen evolution during neutralization of excess borane. (+ = Hydrogen evolution; o = amount of methanol added.). (Reproduced with permission from ref. (35). Copyright 2015 American Chemical Society.)

Figure 21. Heat evolution during neutralization of excess borane. (Reproduced with permission from ref. (35). Copyright 2015 American Chemical Society.) After about 24 h at 50 °C, and when in-process analysis showed complete consumption of the imide, the excess borane was quenched by slow addition of methanol. Because the air-handling system in the laboratory was limited in its ability to sweep away the liberated hydrogen gas at a sufficient rate, we were restricted to both the rate of methanol addition (over about 16–24 h) as well as the scale of the reaction. For initial material supply, the reduction was carried out on 500 g per batch. However, this was not seen as a long-term concern because of the presence of appropriate equipment at contract manufacturers where the scale-up would be conducted. At the conclusion of the quench, the resulting amine-borane complexes from the multiple batches were combined and treated with concentrated HCl followed by heating to reflux to obtain the amine hydrochloride salt (15·HCl) as an easily filterable solid. This isolated product was converted to Boc-protected bromobenzoxazepine 33 by treatment with Boc-anhydride and aqueous potassium 267

carbonate in ethanol. In our kilo labs, we manufactured 2.9 kg of 33, sufficient to prepare material for IND-enabling toxicology studies. Alternative Synthesis of Imide An alternative synthesis, one which is robust and scalable, of the imide 46 was needed for viable long-term supply of 33. Because the hydrolysis of the reaction intermediate to form the acid-amide 47 was facile, we decided to exploit this property and investigate an alternative ring closure strategy from isolated 47. We found that 47 could be cyclized to imide 46 using thionyl chloride (54) by refluxing in toluene. Thus, using the same starting materials as the existing protocol, we could alkylate 5-bromosalicylamide with ethyl bromoacetate and instead of the cyclization with an alkoxide base, the intermediate was hydrolyzed to acid-amide 47 using aqueous sodium hydroxide (Scheme 7) at 50 °C. The acid was isolated by filtration following cooling to room temperature and acidification to pH