Antiviral Drugs

From Basic Discovery Through Clinical Trials

Wieslaw M. Kazmierski

• Publisher: Wiley
• Year: 2011
• Print ISBN:
  9781118251225
  9780470455630
• eBook ISBN:
  9780470934685
• Edition: 1
• Topic: Medicine
• Subtopic: Pharmacology

This book focuses on new small molecule approaches to combat viral infections. The chapters describe the discovery and development from bench through the clinic of relatively recently-approved antiviral drugs and compounds in advanced clinical development. Organized by a virus (such as HIV, HCV, RSV, influenza, HBV and CMV) and written by top academic and industrial authorities in the field, the book provides a unique opportunity to study, understand and apply discovery and development principles and learning without the need for an individual to research, analyze and synthesize all immense sourcing references.  Topics showcase challenges and solutions of issues encountered, offering tremendous experience accumulated over many years of research that will be particularly useful to basic and bench scientists as well as clinicians as they continue discovering and developing new drugs and therapies.

PART I

HUMAN IMMUNODEFICIENCY VIRUS

1

DISCOVERY AND DEVELOPMENT OF ATAZANAVIR

Awny Farajallah

Bristol-Myers Squibb Company, Plainsboro, New Jersey

R. Todd Bunch

Bristol-Myers Squibb Company, Evansville, Indiana

Nicholas A. Meanwell

Bristol-Myers Squibb Company Research and Development, Wallingford, Connecticut

INTRODUCTION

Atazanavir is an azapeptide inhibitor of HIV-1 protease that prevents the formation of mature virions in HIV-1-infected cells by inhibiting the cleavage of gag and gag-pol polyproteins. Atazanavir was developed to address a number of concerns with existing protease inhibitors: high pill burden, high frequency of dosing, and metabolic side effects such as hyperlipidemia. The development program focused initially on dosing atazanavir without coadministering a pharmacokinetic enhancer such as ritonavir. Ritonavir inhibits hepatic CYP3A4 and the metabolism of drugs (such as atazanavir) utilizing this pathway, thereby increasing the blood levels of these drugs. The emerging practice of “boosting” with low-dose ritonavir led to the evaluation of this alternative dosing strategy, initially in treatment-experienced subjects who had previously received and failed multiple antiretroviral regimens, then in antiretroviral-naive patients.

DISCOVERY

Atazanavir (BMS-262362, Scheme 1) was identified from studies of a series of pseudosymmetric azapeptide substrate analogs of HIV-1 protease in which fundamental inhibitory activity relies on replacement of the hydrolyzable amide bond by a hydroxyethylene isostere [1–3]. Although the initial examples of this chemotype demonstrated favorable in vitro and in vivo properties [1,2], further optimization for in vitro HIV-1 inhibitory potency and in vivo exposure was required to identify atazanavir [3,4]. As shown in Scheme 1, the two closely related lead compounds 1 and 2 demonstrated disparate properties: The isoamyl derivative (1) is a potent HIV-1 protease inhibitor, IC50 = 16 nM, with good antiviral activity in cell culture, EC50 = 2.7 nM, but exhibits poor oral bioavailability, while the cyclohexylmethyl analog (2) is a 10-fold weaker protease inhibitor, IC50 = 177 nM, and antiviral agent, EC50 = 55 nM, that demonstrates good bioavailability following oral administration to mice [3]. Based on x-ray crystallographic information of the early lead CGP-53820 (3) cocrystallized with HIV-1 protease [5], the potential of introducing larger P1′ substituents was examined, with the anticipation that potency would be enhanced while preserving the physicochemical properties conferring good absorption. From a drug design perspective, the effort sought to establish additional favorable contacts between the inhibitor and protease residues Arg8, Phe153, Gly148, and Gly149 located at the periphery of the S1′ pocket [4]. The synthesis and evaluation of a derivative in which the cyclohexylmethyl substituent of 2 was replaced by a biphenyl moiety established the validity of the hypothesis since the new compound potently inhibited HIV-1 protease, IC50 = 35 nM, demonstrating tolerance for a large P1′ element, while expressing excellent antiviral activity in cell culture, EC50 = 1.8 nM. Replacing the two valine residues with tert-Leu resulted in CGP-75355 (4), which showed a further improvement in cell culture potency, EC50 = 0.7 nM. More important, protease inhibitors that incorporated tert-Leu residues were generally well absorbed in mice following oral administration.

SCHEME 1 Discovery of atazanavir.

With the structural elements critical for potent antiviral activity established, attention was directed toward improving pharmaceutical properties, with a focus on increasing solubility [4]. To this end, heterocycles designed to increase hydrophilicity were introduced to replace the distal phenyl of the biphenyl moiety. This exercise identified the 2-pyridyl as the optimal element, providing atazanavir (5, BMS-262632, CGP-73547) as a potent HIV-1 protease inhibitor, IC50 = 26 nM, that retained potency toward HIV-1 protease resistant to saquinavir. Atazanavir (5) exhibits excellent antiviral properties in cell culture, EC50 = 1.4 nM and EC90 = 3 nM, with a good therapeutic index, CC30 = 21.8 μM and CC90 = 31.8 μM. Following oral administration to mice and dogs, atazanavir (5) showed good plasma bioavailability, establishing blood levels in excess of the EC90 [4].

SYNTHESIS

The strategic approach to the preparation of atazanavir developed by the discovery chemistry team, summarized in Scheme 2, has largely been followed by the process synthesis depicted in Scheme 3 [4], with several modifications designed to facilitate the preparation of multikilogram quantities of the active pharmaceutical ingredient [6]. The discovery synthesis was satisfactory for the preparation of material for preliminary toxicological evaluation but was deemed to be less than optimal for the synthesis of the significantly larger quantities of drug required for longer-term toxicological studies and clinical trials. The process chemistry strategy was focused on improving the synthesis of the two key intermediates, hydrazine (9) and epoxide (11), that were used in the discovery synthesis. With access to these molecules in hand, optimization of the reagents and conditions used for their coupling, followed by improvements in the final elaboration to the product, were completed.

SCHEME 2 Discovery synthesis of atazanavir.

SCHEME 3 Optimized process for the large-scale preparation of atazanavir.

In the discovery synthesis (Scheme 2), a nickel-catalyzed cross-coupling reaction between 2-bromopyridine and the Grignard reagent (7) derived from a protected form of 4-bromobenzaldehyde (6) was used to prepare the aldehyde (8) [4]. This procedure required the use of diisobutylaluminum hydride, a problematic reagent when used in large-scale reactions. Access to (9) was improved considerably by coupling 2-bromopyridine with commercially available 4-formylbenzeneboronic acid (13) under Suzuki conditions (Scheme 3), a process mild enough to obviate the inefficiencies associated with the tandem protection/ deprotection of the formyl group used by the discovery group. Condensation of (8) with tert-butyl carbazate followed by Pd-catalyzed hydrogenation then afforded the tert-butoxycarbonyl (Boc)-protected hydrazine (9) in high yield.

SCHEME 4 Alternative synthesis of atazanavir.

An optimized approach to the preparation of epoxide (11) relied upon ring closure of a vicinal diol, a process that avoided the racemization-prone L-Boc-phenyalaninal as an intermediate. The secondary mesylate (16) was prepared in quantitative yield in a one-pot process comprising silyl protection of the primary alcohol of (15) followed by mesylation with methanesulfonyl chloride. Desilylation occurred upon exposure of the product to ammonium fluoride in acetic acid, a much less expensive reagent than the more commonly used tetrabutylammonium fluoride. Epoxide formation was completed by treating the liberated alcohol with potassium tert-butoxide in isopropanol, determined to be the optimal base after screening several candidates, to provide (11) in good overall yield. The coupling of the two key structural elements (9) and (11) was effected by heating equimolar quantities in isopropanol at reflux for 24 h. Isolation of (17) was straightforward, accomplished by precipitation upon diluting the reaction mixture with water and recrystallization from a mixture of acetonitrile and water which removes small quantities of by-products. The two Boc moieties of (17) were removed under acidic conditions and the exposed amines coupled simultaneously with N-methoxy- carbonyl-L-tert-leucine using a water-soluble carbodiimide [6].

A more recent approach to the synthesis of atazanavir (5), depicted in Scheme 4, relies upon a diastereoselective reduction of the ketone precursor (22) to install the tertiary alcohol with the correct absolute configuration, allowing a different retrosynthetic disconnection [7]. Reduction of ketone (22) with lithium tri-tert-butoxyaluminum hydride provided the syn-1,2-amino alcohol moiety found in atazanavir (5), with the high (28 : 1) Felkin–Anh type of diastereoselectivity controlled by the chirality of the proximal N-(methoxycarbonyl)-L-tert-leucinyl moiety.

The final issue to resolve with atazanavir was the bioavailability of the free base, which is poorly soluble in water, <1 μg/mL, leading to poor bioavailability due to dissolution-limited absorption. The bisulfate salt was identified as the optimal form of atazanavir for development after an extensive survey which identified this salt as possessing excellent aqueous solubility, 4 to 5 mg/mL. The bisulfate salt was found to exist as two crystalline forms with form 1, formed with good reproducibility in acetone, acetonitrile, or ethanol, displaying good crystallinity [8]. Comparison of the 1H-NMR spectra of the free base and bisulfate salt indicated that protonation had occurred on the pyridine ring, as might be anticipated.

PRECLINICAL PHARMACOKINETICS

The nonclinical safety profile of atazanavir has been eva...

Table of contents

• Cover
• Title Page
• Copyright
• Contributors
• Preface
• Part I: Human Immunodeficiency Virus
• Part II: Hepatitis C Virus
• Part III: Respiratory Syncytial Virus Inhibitors
• Part IV: Influenza, Hepatitis B, and Cytomegalovirus Inhibitors
• Plates
• Index