Inhibitors of HIV-1 Attachment: The Discovery and Development of

Drug Annotation Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/jmc

Inhibitors of HIV‑1 Attachment: The Discovery and Development of Temsavir and its Prodrug Fostemsavir Nicholas A. Meanwell,*,† Mark R. Krystal,‡,○ Beata Nowicka-Sans,‡,¶ David R. Langley,∥ David A. Conlon,⊥ Martin D. Eastgate,⊥ Dennis M. Grasela,# Peter Timmins,∇,◆ Tao Wang,† and John F. Kadow†,○ †

Departments of Discovery Chemistry and Molecular Technologies, ‡Virology, and ∥Computer-Assisted Drug Design, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States ⊥ Chemical and Synthetic Development, Bristol-Myers Squibb Research and Development, 1 Squibb Drive, New Brunswick, New Jersey 08903, United States # Innovative Medicines Development, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, New Jersey 08543-4000, United States ∇ Drug Product Science and Technology, Bristol-Myers Squibb, Reeds Lane, Moreton, Merseyside CH46 1QW, United Kingdom

ABSTRACT: Human immunodeficiency virus-1 (HIV-1) infection currently requires lifelong therapy with drugs that are used in combination to control viremia. The indole-3-glyoxamide 6 was discovered as an inhibitor of HIV-1 infectivity using a phenotypic screen and derivatives of this compound were found to interfere with the HIV-1 entry process by stabilizing a conformation of the virus gp120 protein not recognized by the host cell CD4 receptor. An extensive optimization program led to the identification of temsavir (31), which exhibited an improved antiviral and pharmacokinetic profile compared to 6 and was explored in phase 3 clinical trials as the phosphonooxymethyl derivative fostemsavir (35), a prodrug designed to address dissolution- and solubility-limited absorption issues. In this drug annotation, we summarize the structure−activity and structure− liability studies leading to the discovery of 31 and the clinical studies conducted with 35 that entailed the development of an extended release formulation suitable for phase 3 clinical trials.

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INTRODUCTION The global epidemic associated with human immunodeficiency virus-1 (HIV-1) virus infection continues to present a significant health burden for the estimated 37 million people who are infected, 1 million of which reside in the United States (U.S.).1 Deaths attributable to HIV-1 infection amount to 1.2 million per year worldwide, which equates to one person approximately every 30 s.1 Combinations of antiretroviral drugs are critical to controlling viremia and mortality due to HIV-1 infection. Death rates in the U.S. fell sharply in 1995 after the introduction of the first protease inhibitors (PIs), which were combined with nucleoside reverse transcriptase inhibitors (NRTIs) and later non-nucleoside reverse transcriptase inhibitors (NNRTIs).2a These drug combinations provided effective control of viral replication by slowing the rapid selection of resistant virus that had been observed with monotherapy. With further additions and improvements to antiretroviral drug combinations, HIV-1 infection has become a © XXXX American Chemical Society

chronic disease for those with access to drug therapy although the health burden imposed on individuals by a long-term infection and the associated drug therapy is still being adjudicated.2b,3 First line therapy currently relies upon combinations of NRTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) and HIV-1 integrase strand transfer inhibitors.4 The combination of tenofovir disoproxil (1), emtricitabine (FTC, 2), and efavirenz5 (3) was the first drug combination to be co-formulated into a single pill for once daily dosing and set the stage for others to follow, including Stribild,6 which combines 1 and 2 with the integrase active site inhibitor elvitegravir (4) and a pharmacokinetic enhancer agent.7 However, in the U.S., the emergence of resistance in patients taking first line regimens occurs at a rate of approximately 50000 per year, a number that has been Received: September 8, 2017

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DOI: 10.1021/acs.jmedchem.7b01337 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Drug Annotation

of the cDNA into the host cell genome, and subsequent transcription and translation, which results in the expression of the luciferase protein. Screening of more than 100000 compounds under biosafety level 3 laboratory conditions led to the identification of multiple inhibitors that sampled a variety of chemotypes, with biochemical pharmacological analyses revealing that the majority were NNRTIs, an established inhibitor class recognized for its structural promiscuity. However, the indole glyoxamide derivative 6, a member of a purely prospective library of amides obtained from a commercial source, was determined to be mechanistically unique and viewed as a tractable lead structure.12 Resynthesis of 6 confirmed that the antiviral activity was associated with the designated structure, which exhibited an EC50 value of 153 nM in the pseudotyped virus assay. In contrast to the restricted spectrum of antiviral activity associated with a CCR5 antagonist like 5, indole 6 also inhibited infection by a pseudotyped envelope derived from the T-tropic LAI virus, which is dependent on a CXCR4 coreceptor. Moreover, 6 had no effect on a pseudotyped virus infection in which the HIV-1 virions were encapsidated with the vesicular stomatitis virus envelope G protein, data that collectively were consistent with 6 inhibiting an aspect of the virus entry process. This notion was confirmed by time-of-addition experiments, which demonstrated that 6 inhibited events early in the HIV-1 replication cycle and by its inhibition of the fusion of cells expressing the HIV-1 envelope protein with those expressing both the CD4 receptor and the CXCR4 coreceptor, a “virusfree” cell-based fusion assay. In virus infectivity assays, 6 exhibited potent antiviral activity against both M-tropic, T-cell line-adapted and primary HIV-1 strains, with EC50 values ranging from 0.09 to 5.9 μM.12 The compound demonstrated therapeutic indexes (TIs) that were dependent on cell type and ranged from 33 to 1600 based on measured CC50 values of 100 to >200 μM. The specificity of 6 for HIV-1 inhibition was established by screening against a panel of RNA and DNA viruses that included HIV-2 and simian immunodeficiency virus (SIV), where the EC50 values were all >200 μM.12 In addition, 6 was inactive in 13 microbial assays and 82 primary receptor screens while evaluation in a panel of human cell lines revealed low in vitro cytotoxicity.

comparable to the number of new infections annually, and these patients rely upon second line therapeutics that extends the drug repertoire to include protease inhibitors and the CCR5 antagonist maraviroc (5).7−9 Later line therapeutics for those that are heavily treatment-experienced (HTE) are limited, and patients in this class typically assemble a cocktail of drug regimens that they can tolerate.8 To address this problem, the development of new inhibitors of HIV-1 replication that are mechanistically orthogonal to current therapies has been advocated.10

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SCREENING FOR MECHANISTICALLY UNIQUE HIV-1 INHIBITORS To identify mechanistically novel HIV-1 inhibitors, a high throughput screen was developed that relied upon cotransfecting HEK293T cells (virus producer cells) with a plasmid containing proviral DNA comprised of HIV-1 LAI-Δenv-luc, in which firefly luciferase replaced the native virus envelope gene, and a plasmid expressing the env gene from the JR-FL virus, a clade B, M-tropic, CCR5-dependent virus for which expression was driven by the HIV-1 long terminal repeat.11 This system produced infectious pseudotyped virions where the env proteins had been supplied in trans in order to facilitate the initial infection but which, because of the absence of the env gene, were capable only of a single round of replication. HeLa cells expressing the CD4 protein, the primary attachment receptor for HIV-1, and the chemokine CCR5 coreceptor, were infected with the pseudotyped virus in the presence of test compounds, and the extent of infection was determined by measuring the level of expression of the luciferase reporter gene. The assay was designed to harvest inhibitors of a significant fraction of the HIV-1 viral lifecycle beginning with the entry and capsid uncoating process and proceeding through reverse transcription of the viral RNA to complementary DNA (cDNA), integration

Indole glyoxamides have been explored as pharmacophoric elements for a range of biological targets, including inhibitors of phospholipase A2, and the only initial concern with the structure of 6 revolved around the potential of the α-dicarbonyl moiety to act as an electrophile.13 However, both of the carbonyl moieties of 6 possess amide character with that proximal to the indole ring a vinylogous amide or enamide. It has been our experience that the α-dicarbonyl element is chemically quite robust, with no overt reactivity that would compromise its developability detected throughout the lifetime of the preclinical program.12,14 While the initial structure− activity survey work focused on probing the requirements of the benzamide element based on facile synthetic accessibility, the early results were less than encouraging, with simple B



Drug Annotation

been sought previously; however, small drug-like molecules acting at this step of the viral lifecycle had proven to be elusive.15 While the in vitro and in vivo pharmacokinetic and safety profiles of 8 were supportive of advancing the molecule into clinical trials, development of the compound was halted after a phase 1 dose escalation study in normal healthy volunteers (NHVs) revealed that plasma concentrations of the drug were below those targeted for efficacy studies in HIV-1 infected individuals.16 However, 8 was evaluated as a potential microbicide in a macaque model of HIV-1 transmission where intravaginal application of a 5.5 mM solution 30 min in advance of a vaginal challenge with the simian-HIV SHIV162P3 protected 6 out 8 animals from infection.17 Moreover, the combination of 8 with a peptide-based inhibitor of gp41 6helix bundle formation with or without a CCR5 antagonist provided complete protection in 3 and 6 animals, respectively.17 Antiviral potency was improved with the 4,6-dimethoxysubstituted indole 9, which was one of the more potent of the simply substituted analogues prepared.14a However, this compound was subject to O-demethylation at both of the methoxy substituents when incubated in liver microsomes (LM), raising the specter of the formation of the chemically reactive indoloquinone 11 in vivo.18 A solution to this problem was found in the C-6 azaindole 10 (BMS-488043), which largely preserved the antiviral properties of 9 while introducing a mildly basic nitrogen atom to the heterocyclic nucleus that obviates quinone formation.19 Importantly, the in vitro profile of 10 addressed the poor metabolic stability and low membrane permeability associated with 8. The half-life of 10 in HLM was >100 min, which contrasted with a t1/2 of 37 min for 8, while Caco-2 permeability was improved from 51 nm/s for 8 to 178 nm/s for 10, although the measured Log D values at pH = 6.5 were similar for both compounds, 1.6 for 8 and 1.5 for 10.19,20 The binding of 10 to human plasma proteins was higher (95%) than for 8 (73%), while the solubility of 10 (∼0.04 mg/mL at pH = 6.5) was somewhat lower than for 8 (∼0.2 mg/mL at pH = 6.5) despite similar melting points (236 °C for 8 and 229.5− 232 °C for 10). Notably, the solubility of both molecules was improved at high and low pH, reflecting the amphoteric nature of the compounds and the measured pKa values (2.9 and 9.6 for 8; 2.9 and 9.3 for 10). The pharmacokinetic profile of 10 was improved in the rat, dog, and cynomolgus monkey when compared to 8, with a 6−12-fold increase in the plasma AUC following oral dosing.20 Importantly, when dosed to rats as a suspension at 5 mpk, the plasma AUC and Cmax values for 10 were 62% and 75% of that observed with the solution formulation, respectively, reflecting acceptable dissolution properties. More extensive formulation studies demonstrated that both nanosized particles and an amorphous dispersion of the drug in polyvinylpyrrolidone provided significant 5- and 9fold improvements in exposure, respectively, following oral administration to dogs, when compared to the micronized clinical tablet formulation.21

substituents systematically installed around the phenyl ring singly and in combination typically having a deleterious impact on antiviral potency.14b In contrast, the effect of introduction of substituents to the indole ring proved to be much more promising, with small substituents at the 4-position providing a significant boost in potency which quickly led to an enhanced focus on this aspect of the pharmacophore.12,14a The 4-fluoro analogue 7 was over 50-fold more potent (EC50 = 2.6 nM) than prototype 6 in the pseudoptype assay, a profile that extended to several additional HIV-1 strains where the advantage ranged from 7- to 260-fold.12,14 Antiviral potency was also enhanced by Cl, Br, MeO, and EtO substituents at C-4, while iPrO, OH, or OAc substituents were less well tolerated and NO2 had an essentially neutral effect, demonstrating specific steric and polarity limitations that were largely ambivalent toward electronic properties. Although still early in the program, the antiviral profile of 7 was sufficiently promising that it was examined as a sentinel compound designed to probe and identify potential issues that might be encountered in advancing this chemotype.12,14 In the rat, the pharmacokinetic (PK) profile of 7 revealed modest oral bioavailability of 17% at a dose of 25 mpk and 9% at 5 mpk with moderate clearance, although the terminal half-life after oral administration was longer than the IV half-life.12,14 This observation was indicative of slow absorption that was attributed to problems associated with solubility or dissolution in the gut because the permeability coefficient of 7 across a confluent layer of Caco-2 cells was predictive of good absorption. This was indeed the case in the dog and monkey, where the oral bioavailability after administration of 7 as a solution in polyethylene glycol (PEG 400)/ethanol at a dose of 10 mpk was ∼100%. However, the poor intrinsic physical properties of 7 became clearly apparent during dose escalation studies in both the rat and the dog where nonlinear exposure profiles were observed, while the plasma levels in rats following suspension dosing of particles with an average diameter of 46 μm were essentially unmeasurable. This circumstance was improved only marginally by dosing 7 as a nanosuspension, where the oral bioavailability was 4% in the rat and 36% in the dog following a dose of 1 mpk. The solubility of 7 in water was low (5 μg/mL) but much higher in PEG 400 (4.6 mg/mL), properties reflected in the high melting point of the compound (236 °C). In an effort to address the pharmaceutics issue, the next phase of drug design focused on the synthesis of compounds incorporating nitrogen atoms in the core heterocycle, initially explored in the context of 7-azaindoles but ultimately examined in a systematic fashion. BMS-378806 (8) emerged as the first clinical candidate, and although the antiviral activity of 8 was insensitive to coreceptor tropism, there was a 30-fold variability in potency across the strains of HIV-1 that were evaluated, with a median EC50 toward a panel of 11 clinical isolates of 12 nM, a value that shifted to 61.5 nM when screening was extended to a broader range of isolates.12 Resistance mapping studies using the NL4-3 and LAI HIV-1 strains identified M426L, M434I/V, and M475I of gp120 as the primary resistance mutations, of which residues 426 and 475 of the protein are, interestingly, located in the binding pocket that accommodates F43 of the CD4 receptor. Biochemical studies demonstrated that 8 competed with the binding of soluble CD4 to a monomeric form of gp120 in an ELISA assay, IC50 ∼ 100 nM.12 Thus, 8 was believed to function by binding to the viral gp120 envelope protein and preventing the initial attachment of virus to host cell CD4. Inhibitors of the HIV-1 gp120-CD4 interaction had C



Drug Annotation

In a Phase 1 clinical trial conducted in NHVs, the systemic exposure of 10 was dose-proportional from 400 to 800 mg when the drug was administered with a light meal and from 400 to 1200 mg when dosed with a high-fat meal, but there were limited increases in exposure with higher doses in the absence of a high-fat meal.25 Consequently, doses of 800 and 1800 mg of 10 administered on a bis in die (BID) schedule with a high fat meal to maximize drug exposure were selected for an 8 day monotherapy study in HIV-1 infected subjects (n = 12 in each drug arm and n = 6 in the placebo control) that would provide proof-of-concept for the AI mechanism. The mean decline in plasma viral load measured on day 8 was 0.72 and 0.96 log10 copies/mL at the 800 and 1800 mg doses, respectively, which compared to 0.02 log10 copies/mL for the placebo control group. The antiviral response was predicted by baseline sensitivity to the compound, with lower EC50 values associated with greater efficacy, and while absolute exposure did not correlate with efficacy, the Ctrough/EC50 value had predictive value. Four subjects experienced a more than 10-fold reduction in viral susceptibility to 10 by the end of the drug dosing period, reflecting the emergence of resistant virus.24,25 Five gp120 substitutions at four loci were observed in these emergent viruses. The five substitutions, V68A, L116I, S375I/ N, and M426L, were observed by population-based sequencing of the envelope genes, with the most common substitution occurring at S375. Reverse genetic engineering of these substitutions into the background of highly susceptible envelopes confirmed their effect on reducing sensitivity to 10 although they retained full sensitivity to other viral envelope inhibitors.24a Although 10 was well tolerated in these clinical studies, the need to dose the drug with a high fat meal in order to achieve targeted plasma exposure and the excessive pill burden associated with the high doses and excipient loading presented a significant challenge to further development. Administration of a 200 mg dose of 10 in solution to NHVs afforded 2-fold higher plasma levels than the capsule formulation for which exposure plateaued at a dose of 800 mg, suggestive of dissolution- or solubility-limited absorption for this compound.26 A solution to these pharmaceutics problems was found in the phosphonooxymethyl-based prodrug 12, which was designed to solubilize the drug in the gastrointestinal (GI) tract and release the parent drug by a presystemic cleavage mechanism mediated by alkaline phosphatase expressed on the brush border membrane.26 This prodrug strategy took advantage of prior experience in the context of an antifungal drug candidate and taxane derivatives and envisaged that the prodrug would be dephosphorylated presystemically in the GI tract to release a N-hydroxymethyl derivative that, based on the pKa value of the indole N−H, would degrade to the parent drug 10 with release of formaldehyde.27,28 The pKa value for the pyrrole N−H of 10 was determined experimentally to be 9.3, and literature precedent indicated rapid collapse of Nhydroxymethyl derivatives when the pKa value of the parent indole was 8.3 μM (n = 3).22 However, there were poorly sensitive isolates that were also observed within each subtype, reflecting subtle structural differences within HIV-1 gp120 that conferred a spectrum of sensitivity even within closely related envelope proteins of the same subtype. Compound 10 was an effective inhibitor of the replication of HIV-1 viruses resistant to NRTIs, NNRTIs, and protease inhibitors (PIs) but was inactive toward HIV-2 and other RNA viruses and was noncytotoxic toward multiple mammalian cell lines.19 Gel filtration analyses indicated that 10 bound reversibly to gp120 with a 1:1 drug:protein stoichiometry and a KD value of 19.1 nM, reflective of a fast associative t1/2 of 2−3 min and a slower dissociative t1/2 of 43 min.22 The compound inhibited soluble CD4 binding to 11 distinct gp120 envelope proteins in a competitive fashion, and three independent biochemical analyses suggested that 10 acted by stabilizing a conformation of gp120 not recognized by CD4.23 Sequencing of the envelope genes of the T-tropic HIV-1 strains LAI and NL4−3 that were passaged in the presence of increasing concentrations of 10 revealed amino acid substitutions located predominantly within gp120, with mutations V68A, M426L, M434I, S440R, and M475I observed in selected viruses. Other mutations observed with this class of inhibitor included F423Y, which is in the CD4 binding pocket, and I595F and K655E, which are in the gp41 ectodomain. All of these substitutions were shown to reduce sensitivity to 10.24a While these mutated viruses remained competent for virus entry with normal kinetics, some were found to be more sensitive to the broadly neutralizing antibodies 2F5 and 4E10, attributed to an allosteric effect on epitope presentation.24b D



Drug Annotation

Figure 1. Synopsis of the SAR studies associated with HIV AIs.

developing more potent compounds that had emerged from the continuing SAR studies. A summary of the results of extensive structural modifications conducted around this chemotype is provided in Figure 1, which also captures some of the design concepts examined by other laboratories.32 Substitution of the benzamide phenyl ring was generally poorly tolerated, although 2-pyridyl and some five-membered heterocycles, particularly thiophenes and furans, were acceptable replacements.14b The introduction of small substituents to the piperazine ring proximal to the glyoxamide moiety could be beneficial; however, this was context-dependent and small alkyl moieties were often prone to metabolic modification.33 The glyoxamide element was found to be optimal for the indole-based series, and while there was tolerance for limited substitution at C-4 of the heterocycle, substitution at the C-5 and C-6 positions were poorly tolerated as was replacing C-5 with a N atom. However, replacing C-6 with a N atom was more readily accommodated, associated with a modest 5-fold reduction in antiviral activity when compared to the parent indole.34 Further SAR studies showed that there was considerable scope for the introduction of substituents at C-7, with many amides and heterocycles conferring enhanced antiviral potency that also broadened the functional spectrum of inhibition.35,36 Replacements for the benzamide moiety were also explored extensively because this turned out to be a labile metabolic site in humans. Many structural elements that preserved the overall topology of the benzamide element demonstrated potent antiviral activity, with the microbicide candidate 13 (BMS-599793, DS-003) and the tetrahydroisoquinoline 14 being representative examples, although compounds with a profile suitable for development proved to be elusive.37,38

cleavage and the rate of absorption of the parent drug, parameters that need to be carefully matched in order to avoid precipitation of the released parent drug prior to absorption.28 While the in vitro permeability data categorized 10 as a biopharmaceutics classification system (BCS) class 2 compound, given the complexity of the prodrug release process, it was recognized that the potential of this approach would need to be explored experimentally.26,28,30 Prodrug 12 exhibited much improved physical properties, with solubility determined to be in excess of 12 mg/mL at pH = 5.4, which compared to 0.04 mg/mL for the parent drug 10 at a pH ranging from 4 to 8.26 Indeed, the solubility properties of 12 allowed for formulation in H2O at concentrations higher than 100 mg/mL for drug administration in preclinical toxicology studies. Intravenous administration of 12 to rat, dog, and cynomolgus monkey revealed a rapid conversion to parent 10, while no or only very low levels of prodrug were detected in the plasma of animals administered 12 orally. The absolute bioavailability of 10 after oral dosing of 12 to rats, dogs, and cynomolgus monkey was 62%, 93%, and 67%, respectively. Most importantly, in an oral dose escalation study conducted in rats, the plasma exposure of 10 increased in an almost linear fashion following administration of doses of 16, 72, and 267 mpk of 12, with the plasma area under the curve (AUC) and Cmax of 10 approximately 2- and 3-fold higher, respectively, than that achieved after administering the parent drug as a suspension at a dose of 200 mpk. In clinical PK studies conducted in NHVs, administration of 12 resulted in a dose-proportional increase in the plasma exposure of 10 over doses ranging from 25 to 800 mg.26 At a dose equivalent of 800 mg of 10, the plasma Cmax after administering 12 was 6-fold higher than that from parent drug in a capsule dosed with a high fat meal while the plasma AUCINF improved by 3-fold.26 However, the tmax of the parent drug after dosing 12 was much earlier, between 0.5 and 1 h, while the t1/2 was 1.5 ± 0.2 h, much shorter than the t1/2 of ∼10 h observed after dosing 10 as a capsule formulation. The absorption profile of 10 following dosing in the capsule formulation was indicative of prolonged absorption, leading to a flip-flop pharmacokinetic profile that can be attributed to slow dissolution and/or the poor solubility of the drug in the gastrointestinal tract.31

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STUDIES LEADING TO THE DISCOVERY OF TEMSAVIR (BMS-626529, 31) AND FOSTEMSAVIR (BMS-663068, 35) While these studies established the value of the phosphonooxymethyl prodrug to overcome the pharmaceutics issues with 10, further clinical studies with 12 were deferred in favor of E



Drug Annotation

With a focus on identifying compounds with higher intrinsic antiviral potency toward a broader spectrum of HIV-1 isolates, a target projected Ctrough value was set that would, at a minimum, theoretically inhibit 90% of the contemporary B clade virus isolates that were available from the CASTLE clinical study that had been conducted with the PI atazanavir (15).39 This necessitated identifying a compound displaying a minimum of a 10-fold improvement in its overall profile compared to 10, an objective that was envisioned as being achievable by combining enhanced antiviral potency with improved intrinsic PK properties. Many of the seminal SAR studies were conducted with an indole core based on commercial availability of starting materials and an abundance of established synthetic methodology.35 The learnings from indole SARs were instructive and readily transferred to the 6-azaindole series, although with some adjustments in order to accommodate the effects of the presence of the nitrogen atom in the core heterocycle. Substitution at the C-7 position of the indole core with functionality rich in sp2-hybridized atom content (carboxamide, aromatic and heteroaromatic rings) provided many compounds with improved potency. A particularly important SAR observation associated with C-7 substitution was provided by the carboxamides 16−18, from which 17 was the most potent antiviral agent, 4-fold improved compared the primary amide 16, while the dimethylamide 18 was the least potent HIV-1 inhibitor in this subseries, with an EC50 value 800-fold higher than that for 17 (Table 1).35 This result was rationalized in the

Figure 2. Single crystal X-ray structure of the C-7-substituted monomethylamide 17.

Although C-7 substituted indoles were explored broadly, the conundrum presented by trying to marry antiviral potency with an acceptable PK profile could not be resolved satisfactorily although these studies did confirm the promise of C-4 fluoro and C-4 methoxy core substituents that were adopted for subsequent examination of the 6-azaindole series.35 An extensive series of five- and six-membered heteroaryl groups attached to the core azaindole via a carbon atom linker were surveyed that, in general, possessed high intrinsic antiviral potency, providing that nonbonding interactions between the azaindole core and attached heterocycle favored a planar topography. Analogues 22−26 (Figure 3) provide a synopsis of the SARs observed in the 4-fluoro series, where favorable and unfavorable intramolecular interactions are marked by green and red arrow markers, respectively. Some of these analogues are believed to rely upon H-bonding interactions between the C-6 nitrogen atom of the azaindole and a C−H of the appended heterocycle to stabilize a coplanar arrangement.41 While several strategies were attempted in an effort to balance the permeability and metabolic stability in the 6-aza, 7carbon-linked heteroaryl series, including the introduction of substitution to the C-7 heterocycle, variation in the substitution pattern of the pyridine ring element of the azaindole core, or replacement of the benzamide with a 2-pyridylamide, none of these provided a compound that was considered suitable for advancement. Indeed, the metabolic stability trends were generally found to be diametrically opposed to those observed for membrane permeability, with the relatively low level of overlap suggesting that while identifying a molecule in this series with a suitable balance of properties might be possible, it would require considerable finesse. As a consequence, attention began to focus on C-7 substituents in which the heterocycle was attached to a 4-fluoro- or 4-methoxy-6-azaindole core via a nitrogen atom linker. Two key compounds to emerge from this initiative were the 1,2,4-triazole derivative 27 and its 1,2,3triazole isomer 28 (BMS-585248), for which key profiling data are compiled in Table 2 along with comparative data for 10.36 The EC50 values for these compounds in the initial screening assay were improved by almost 10-fold when compared to 10 but, most importantly, inhibitory activity was maintained toward a much broader range of clinical isolates. The rat PK profiles of both compounds were promising, with IV clearance 10-fold lower compared to 10, while the AUC after oral dosing was improved by approximately 13-fold for each analogue (Table 2). More detailed studies with 28 led to its selection as a development candidate, and the molecule was advanced into IND-enabling toxicology studies as a prelude to conducting

Table 1. Antiviral Activity toward JR-FL Virus and Cytotoxicity Associated with a Series of 4-Fluoro-, 7Substituted Indole-Based HIV-1 Attachment Inhibitors (AIs)

compd

R

EC50 (nM)

CC50 (μM)

16 17 18 19 20 21

CONH2 CONHCH3 CONH(CH3)2 2-pyridyl 3-pyridyl 4-pyridyl

2.03 0.52 407 0.65 9.09 3.37

>300 >300 >300 69 132 >300

context of topographical arguments in which amides 16 and 17 are able to adopt a coplanar arrangement with the indole heterocycle, favored by a H-bonding interaction between the amide oxygen atom and the indole N−H. This conformation was observed in the single crystal X-ray structure determined for 17 (Figure 2) and suggested that the poor antiviral activity associated with the dimethylamide 18 was a function of allylic 1,3-strain between the indole C-6 hydrogen atom and the transmethyl N-substituent of the amide causing a distortion from coplanarity.35,40 A similar premise is believed to contribute, in part, to the potency advantage associated with the 2-substituted pyridine 19 compared to its 3- and 4-substituted isomers 20 and 21, respectively, where only the nitrogen atom of 19 can establish a productive H-bonding interaction with the indole N−H.35 F



Drug Annotation

Figure 3. A synopsis of the SARs associated with a series of C-7-substituted, 4-fluoro-6-azaindole-based HIV AIs. Green arrows reflect a productive intramolecular interaction, while the red arrows indicate an unfavorable intramolecular effect.

Table 2. Antiviral and Rat PK Profiles of 10, 27, and 28

1,2,4 triazole derivative 31 (BMS-626529, temsavir) also exhibited improved PK properties in the rat when compared to progenitor 29.22 More importantly, the rat PK profile of 31 represented an improvement over 10, with clearance an order of magnitude lower and a half-life that was nearly doubled, data that were reflected in an approximate 8-fold higher plasma AUC following oral dosing. While studies in dogs and cynomolgus monkeys using solution-based formulations confirmed the improved intrinsic PK properties, the relative oral bioavailability of 31 when dosed as a suspension was 52% of that of the solution-based formulation, attributed to the low crystalline aqueous solubility of 0.022 mg/mL at pH = 7.4 and suggesting the potential for dissolution- and/or solubilitylimited absorption. Nevertheless, acceptable safety margins were obtained in preclinical toxicology species using PEG400/ 0.1N NaOH as the formulation vehicle and 31 was advanced into IND-enabling studies as a prelude to clinical evaluation. Because both 28 and 31 were profiled as BCS class 2 molecules, and it was anticipated that high clinical doses would need to be explored in order to inhibit the more recalcitrant clinical isolates, their phosphonooxymethyl prodrugs 34 and 35, respectively, were evaluated. Considerable experimentation with a series of amines was required in order to identify the lysine salt 34 (BMS-663747) and the tris(hydroxymethyl)-

phase I clinical trials where the molecule was dosed as a spray dried dispersion formulation.36 Doses of 100, 200, 400, 800, and 1200 mg of 28 administered to NHVs revealed less than dose-proportional increases in plasma exposure over the range evaluated, and the Ctrough value measured 12 h after the 1200 mg dose was just under 2-fold the median protein bindingadjusted EC90 value for B-clade viral isolates.36 This exposure profile was considered to be inadequate to conduct an efficacy study in HIV-1 infected patients and development of the compound was halted. Several N-linked azole derivatives were explored in the 4methoxy series, where it was found that substitution at the 3- or 4-position of the appended rings was well tolerated as exemplified by the representative examples 29−33 that are compiled in Table 3. The rat PK profiles of several N-linked 1,2 pyrazole, 1,2,3-triazole, and 1,2,4-triazole derivatives were determined to be promising, particularly for the unsubstituted 1,2,4 triazole analogue 29 which was advanced into dog PK studies, where it demonstrated superiority to 10. Unfortunately, further in vitro profiling of 29 revealed a risk for cardiac complications based on hERG inhibition and the potential for drug−drug interactions (DDIs) because the molecule inhibited CYP 3A4 with an IC50 value of ∼1.0 μM.42 Substitution of the 1,2,4 triazole ring overcame these liabilities, and the 3-methylG



Drug Annotation

Table 3. Antiviral Activity of C-4 Methoxy, C-7 Heteroaryl, 6-Azaindoles and Comment on the Origin of Nonbonded Intramolecular Interactions between the C-7 Heterocycle and the Core Azaindole

Prodrug 35 possessed high aqueous solubility (>11 mg/mL in the pH range of 1.5−8.2 at room temperature) and was stable under both acidic and neutral conditions for at least 24 h at 37 °C, as well as in its solid state. The high oral bioavailability (80−122%) of 31 observed after administration of prodrug 35 to rats, dogs, and monkeys reflected efficient prodrug release and absorption of parent drug, while the systemic exposure of 35 was very low. Similar behavior was anticipated in clinical studies based on the observation that 35 was hydrolyzed to form 31 in the presence of human placental alkaline phosphatase and in human hepatocyte preparations in vitro. In rats and dogs, the AUC of 31 was similar after administration of either the parent or prodrug at lower dosages (e.g., ≤ 25 mg/ kg), but at higher doses (200 mg/kg), administration of a solution of the prodrug provided superior exposure of 31 compared to dosing of the parent drug. In dogs, the prodrug 35 could be given as either an aqueous solution or as a solid in dry filled capsules without compromising its exposure advantage at the higher doses.

aminomethane (mono tromethamine, tris) salt 35 (BMS663068, fostemsavir) as advantageous crystalline forms.

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THE MODE OF ACTION OF HIV ATTACHMENT INHIBITORS AND MODELING STUDIES The mechanism of HIV-1 virus entry is a complex and carefully choreographed multistep process that presents a number of distinct opportunities for antiviral intervention, and studies have identified inhibitors targeting the major steps.43 In its most rudimentary form, the HIV-1 entry process is comprised of three discrete steps: virus attachment, coreceptor binding, and membrane fusion. The initial step is the binding of the HIV-1 envelope protein gp120 subunit of the gp120/gp41 coat

While 34 exhibited improved pharmaceutical properties that translated into dose-related increases in plasma exposure following oral administration, in investigational new drug (IND)-enabling toxicology studies, the low aqueous solubility associated with 28 (7 μg/mL at pH = 7.4) resulted in a propensity for the parent molecule to crystallize in the tissues of animals at targeted exposure margins and further development of the compound was suspended. The higher intrinsic solubility of 31 (∼20 μg/mL at pH = 2−9) facilitated progress of its prodrug 35 through IND-enabling toxicology studies. H



Drug Annotation

strategy for the virus by eliciting antibodies that do not result in protective immunity because this conformation is exposed only when engaging CD4 where it is inaccessible to antibodies. Computer-aided modeling studies with AIs have relied upon the construction of homology models of gp120 in the unliganded state, a pre-CD4 binding intermediate conformation and a CD4-bound conformer that, when combined with information taken from SARs and the positions of key resistance mutations, have been used to predict a potential binding pocket and mode of action.50,51 These studies have suggested that AIs bind within the conserved outer domain of unliganded gp120 adjacent to the CD4 binding loop and under the β20−β21 sheet. The model developed suggests a mechanism for inhibition of both CD4-dependent and CD4independent virus entry because transitions that lead to the formation of the four-stranded gp120 β3−β2−β21−β20 bridging sheet associated with the open state are a prelude to exposure of the co-receptor binding site. In the model of the unliganded form of gp120, the CD4 binding site is only partially assembled but forms more fully when the β20−β21 sheet rearranges to form the water channel and the binding pocket that recognizes F43 of CD4. Collectively, these models suggest that HIV-1 AIs stabilize the closed, unliganded form of gp120 by preventing disruption of the bridging sheet and rearrangement of the β20−β21 sheet into a state that is competent for recognition of CD4, a mechanism that also applies to CD4-independent viruses which are believed to more effectively sample the CD4-bound state.,22,23,48,51−53 The activity of 31 toward CD4-independent viruses can be rationalized by a model in which the compound binds to a conformation of gp120 that is not competent to undergo the conformational changes necessary to expose the coreceptor binding sites and/or release gp41 to initiate membrane fusion.53 Recently, additional mechanistic studies that include the determination of cocrystal structures of 10 and 31 bound to a complex of trimeric BG505 SOSIP gp120, the glycandependent antibody PGT122, and the gp120-gp41 interface antibody 35O22 have been described.54 The structures are consistent with the mechanism of action (MOA) proposed from the modeling studies and confirm that the AIs bind to gp120 at the interface between the inner and outer domains under the β20−β21 loop in a fashion that places the benzoyl moiety within the W427 binding site of the open state (Figure 4). The benzoyl ring engages gp120 in a parallel π-stacking interaction with Phe382 and an offset π-stacking interaction with W427. While the AIs bind to gp120 mainly through hydrophobic interactions, H-bonds between the backbone NH of W427 and the oxamide CO that is distal to the core heterocycle and the azaindole NH and the side chain of D113 were observed. The structures also reveal that the binding mode is slightly different from that predicted because the AI molecules bind within a surface-accessible induced pocket adjacent to the C terminus of the α1-helix of the inner domain and adjacent to but under the β20−β21 loop of the outer domain. However, this binding pocket is on the opposite side of the β20−β21 loop to that predicted, with the consequence that this loop, particularly W427, is pushed into the binding pocket that recognizes F43 of CD4. The ability of AIs to stabilize the closed ENV trimer state taken together with the X-ray structures of the bound complex suggest a dual MOA. At higher concentrations, the AIs form a stoichiometric (3:3) complex with the trimeric gp120 and block

protein complex to the cellular receptor CD4 that is expressed on T-cells. Complex formation between gp120 and CD4 triggers a conformational change in the viral protein, exposing the co-receptor binding site in the V3 loop that is recognized by the chemokine receptor CCR5 in M-tropic viruses and CXCR4 in T-tropic viruses. Engagement of a chemokine receptor instigates a second conformational change in gp120 that leads to the dissociation of the protein from gp41. Once free from constraint, gp41 undergoes a substantial conformational rearrangement in a fashion that projects its hydrophobic amino terminus into the host cell membrane, the first of a series of events that are still poorly defined. A critical step is the selfassociation of the carboxyl terminus heptad repeats of gp41 with the assembled, trimeric amino terminus heptad repeats of gp41, ultimately leading to fusion of the virus and host cell membranes. Once the virus and host cell membranes have fused, the viral capsid enters the cytosol, where dismantling occurs to release the two copies of viral RNA and the process of viral replication is initiated. It is the first step of this process, the initial attachment of virus to CD4+ cells, that AIs interfere with, thereby preventing all downstream virus replication events. Detailed biochemical studies with 10 have indicated that the compound binds to HIV-1 gp120 and stabilizes a conformation that is not competent to engage the CD4 receptor, although it has been suggested that under some conditions a ternary complex forms whereby 10 interferes with CD4-induced conformational changes that lead to the exposure of gp41.23,44 Considerable progress has been made in solving crystal structures of the HIV-1 gp120 trimer in its various forms over the last two decades, and these are complementing biochemical studies that collectively are providing more detailed insights into the choreography and mechanism of the process of HIV-1 entry.45,46 The HIV-1 gp120 protein is conformationally plastic, an important property that contributes to evasion of surveillance by the host immune system and which is augmented by genetic variation in the protein and diversity in the glycan shield that typically comprises half of the molecular weight of the mature protein.47,48 Conformational dynamics studies of HIV-1 envelope trimers labeled with small molecule fluorophores introduced into different loops of gp120 are illuminating key aspects of the structural changes associated with the native protein. These studies have revealed that native, unliganded Env protein is structurally dynamic, sampling three conformations that have been designated as low-, intermediate-, and high-FRET states based on the strength of the observed signal. The low-FRET state is populated most frequently, leading to its designation as the closed ground-state conformation, a thesis confirmed by analyzing ground-statestabilizing mutations. The intermediate- and high-FRET states are stabilized to differing extents by soluble CD4 and the 17b antibody, which mimics the co-receptor, but the combination of both proteins favor the intermediate-FRET state, suggesting this might be the state stabilized by the co-receptor. The conformational mobility observed with the unliganded HIV-1 Env protein is consistent with the large negative entropic change associated with the binding of gp120 to CD4.46 The HIV-1 AI 31 was found to stabilize the closed, ground-state of the Env protein, which is also the state recognized by broadly neutralizing antibodies.49 These observations provide insight into the design of broadly neutralizing antibodies which would require presenting the closed, ground-state as the immunogen.56 However, the open states of gp120 that are sampled appear to be more immunogenic and act as an effective decoy I



Drug Annotation

Table 4. In Vitro Activity of 31 against Laboratory Strains of HIV-1 EC50 ± SDa (nM) co-receptor tropism

virus

host cells

10

31

CCR5

JR-FL SF-162 Bal

PM1 PM1 PM1

3.0 ± 0.9 4.7 ± 0.6 23.0 ± 0.6

0.4 ± 0.1 0.5 ± 0.2 1.7 ± 0.5

CXCR4

LAI NL4−3 MN IIIb RF

MT2 MT2 MT2 MT2 MT2

4.1 ± 1.8 17.4 ± 8.5 >1000 160 ± 78 >2000

0.7 ± 0.4 2.2 ± 0.6 14.8 ± 5.2 16.2 ± 1.7 >2000

89.6

PM1

≥871

57.6 ± 11.4

dual a

Figure 4. X-ray structure of the cocrystal of the complex of gp120 with 31 showing that the AI binds between the inner (orange) and outer domain (green) of gp120 and under the β20−β21 loop (mauve). The AI predominantly interacts with gp120 via hydrophobic interactions and forms H-bonds with D113 from the α1-helix of the gp120 inner domain and W427 of the outer domain. The benzamide moiety occupies the site of gp120 that is occupied by W427 in the open state such that W427 and the β20−β21 loop are pushed toward the CD4 binding loop (red), thereby blocking CD4 binding.

SD = standard deviation.

CD4 binding and virus attachment to host CD4+ T-cells. However, at lower concentrations, the gp120 trimer is not saturated (2:3 or 1:3 AI:gp120 stoichiometry), and while CD4 is still able to bind, the association of the one or two bound AIs stabilizes the trimer toward the conformational rearrangement that leads to the activation of the fusion machinery and, hence, virus entry, is blocked.

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THE ANTIVIRAL PROFILE OF 31 The 10-fold higher potency of 31 compared to 8 and 10 correlated well with its improved biochemical properties, particularly the Kd of 0.83 nM for JR-FL gp120 measured at room temperature, which is 23-fold lower than that for 10, Kd = 19 nM.22 In a gp120/CD4 binding enzyme-linked immunosorbent assay (ELISA), 31 exhibited inhibition that was 6-fold more potent than 10 (IC50 of 14 vs 87 nM, respectively).22 The increased affinity for gp120 and the slower off-rate of 31 (t1/2 = 458 min) translated into a better overall antiviral profile against a spectrum of laboratory strains, validating the approach of optimizing potency toward a sentinel virus. This is illustrated by the data presented in Table 4, where the EC50 of 31 against almost every strain is significantly lowered compared to 10 and is independent of coreceptor tropism, with the lone exception of the HIVRF strain, which appears to be resistant to both compounds. Similar results were obtained when 12 clinical isolates were examined in peripheral blood mononuclear cell (PBMC) infection assays, with the susceptibilities of each of the viruses to 31 improved compared to those of 8 and 10 (Figure 5). The lone outlier in this cohort for which 31 demonstrates a high EC50 is a CRF01_AE virus, a subtype that exhibits reduced susceptibility against all the known AIs due to the presence of two polymorphisms that each reduce sensitivity to this mechanistic chemotype, as detailed below.37 Importantly, 31 exhibited no cytotoxicity (CC50 > 200 μM) toward MT-2 (T lymphocytes), HEK293 (kidney), HEp-2 (larynx), HepG2 (liver), HeLa (cervix), HCT116 (colorectal), MCF-7 (breast), SK-N-MC (neuroepithelium), HOS (bone), H292 (lung), and

Figure 5. Comparison of susceptibilities of 12 clinical strains of HIV-1 to each of the three attachment inhibitors 8, 11, and 31. Each symbol represents a different clinical isolate. The filled circle with the lowest susceptibility is a CRF01_AE strain, which exhibits resistance against all AIs.

MDBK (bovine kidney) cell lines, while CC50 values of 105 and 192 μM were recorded in the T-cell line PM1 and in PBMCs, respectively. Adding to its attractive preclinical profile, 31 exhibited low human serum protein binding (85%), which translated to a modest 1.5- to 2.1-fold effect on antiviral activity when evaluated in the cell culture assay in the presence of 40% human serum.22 To address concerns about the spectrum of HIV-1 inhibition associated with 31, an extensive analysis of the potency against clinical isolates was performed in PBMCs and also against virus pseudotyped with clinical envelopes in the PhenoSense EntryAssay performed at Monogram BioSciences. While the anticipated variability in susceptibilities to 31 was observed across 40 subtype B viruses, with EC50s ranging from 0.01 to >2000 nM, the majority of isolates were highly susceptible to 31. It was calculated that a concentration of 0.65 nM of 31 would exceed the EC50 against 50% (50EC50) of subtype B viruses (n = 40), which translated to a trough concentration of 25 nM in order to exceed the EC90 value for 90% of the clinical isolates.55 A comparative evaluation of 48 other clinical isolates from different HIV-1 subtypes (A, C, B, D, CRF01_AE, F, CRF_BF, G, and group O) demonstrated J



Drug Annotation

that 31 was potent toward isolates from most subtypes, with the exception of the CRF01_AE subtype and group O viruses.22,37,55 In addition, the antiviral activity of 31 was insensitive to coreceptor tropism, illustrated by the median EC50 values of 4.41 nM toward CCR5-tropic isolates (n = 73), 20.2 nM for CXCR4-tropic (n = 8), and 2.38 nM for dualtropic (n = 7) viruses. Excluding the resistant CRF01_AE subtype (n = 8) and group O (n = 2) viruses, the antiviral potency across the cohort was similar to median EC50 values of 3.65 nM (CCR5), 3.66 nM (CXCR4), and 2.38 nM (dual tropic). Additional evidence for the lack of effect of co-receptor tropism on the susceptibility to 31 was obtained through an analysis of individual clones obtained from a subject infected with a dual-tropic virus. A nearly identical IC50 geometric mean was observed when comparing 12 independent CCR5-tropic versus 11 independent CXCR4-tropic envelopes from the same individual.22,55 The anti-HIV-1 spectrum of 31 was also investigated using viral envelopes cloned from clinical samples (PhenoSense Entry assay). A total of 202 clinical envelopes were assayed, of which the majority were subtype B isolates (133) with additional representatives from subtypes A, C, CRF01_AE, F, F1, CRF_BF, and CRF01_AG (Table 5). As with the whole

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PHASE 1 CLINICAL TRIALS AND THE DEVELOPMENT OF AN EXTENDED RELEASE FORMULATION FOR ORAL DELIVERY The promising in vitro antiviral profile of 31 was explored in clinical studies that took advantage of the phosphonooxymethyl prodrug 35 as the delivery vehicle; however, the PK data obtained for 31 proved to be less than encouraging.56 In a single ascending dose (SAD) phase 1 clinical study, 35 was administered at dosages equivalent to 100, 200, 400, 600, 800, and 1000 mg of 31, where it was observed that the exposure of 31 was found to increase more than proportionally with dose.56 The exposure of 31 from a 200 mg dose of 35 was similar to that seen from a 1.8 g dose of 31 administered in the traditional clinical capsule, representing a significant improvement. However, although very good bioavailability of 31 was achieved after dosing of 35, with virtually undetectable levels of prodrug found in plasma, the half-life of 31 was short at just 1.5 h.56a This profile meant that the drug would have to be administered at least every 8 h in order to achieve minimum drug plasma concentrations sufficient to ensure robust antiviral activity. Inspired by the observation that oral administration of the poorly soluble parent drug in animal studies led to a slow terminal administration phase consistent with slow in vivo dissolution of the compound in the GI tract and its consequent prolonged absorption, the development of an extended release formulation of 35 was pursued. However, this approach, which would rely upon unmasking of the prodrug only immediately before absorption throughout the gastrointestinal tract, was unprecedented. A viable extended release dosage form of a drug with such a short half-life would need to deliver a significant amount of prodrug from the dosage form in the colon while balancing GI transit times with an extended delivery time from the dosage form in order to ensure once or twice daily dosing. In addition, it was anticipated that the level of alkaline phosphatase activity in the colon would influence the bioconversion of the prodrug released in the lower regions of the GI tract. Because the level of alkaline phosphatase activity in the colon was unknown, an extended release delivery of the prodrug appeared to be a high-risk strategy. To examine the feasibility of an extended release formulation, the human pharmacokinetics of 31 when dosed orally as the tromethamine salt of 35 was modeled in silico.57,58 The model was applied to a series of drug release profiles typical of those achievable with nondisintegrating, hydrophilic matrix, extended release tablets (drug inputs over the range of 4−20 h) to provide simulations of the pharmacokinetics expected from doses of 600 mg of 35. These simulations demonstrated the potential for at least a 2-fold reduction in Cmax and a 4-fold increase in Cmin for the delivery of parent drug 31 from the dosage form over 5 h or more with Cmax:Cmin ratios of less than 20, which were within the desirable target range. To test this hypothesis experimentally, a human site of absorption study was undertaken by employing Intellisite capsules containing 100 mg of 35 in powder form along with an indium-111 radiolabel adsorbed onto to a mannitol carrier. The capsules were dosed with 240 mL of H2O containing a different radiolabel (Tc-99m DTPA). The solution radiolabel allowed gamma scintigraphy to outline the anatomy of the GI tract, while the radiolabel on the mannitol powder allowed gamma

Table 5. Distribution of IC50 Values for 31 against Subtype B (A) and Nonsubtype B (B) Envelopes Determined in the PhenoSense Entry Assay with the Number of Isolates Designated by na subtype

n

median EC50 (nM)

EC50 range (nM)

A B C CRF01_AE F F1 CRF_BF CRF02_AG

33 133 36 5 3 4 15 3

2.24 0.34 1.14 >100 0.10 3.75 3.09 1.77

0.38 to >100 0.05 to >100 0.07 to >100 >100 0.06−0.40 0.84−16 0.26 to >100 1.67−2.98

a

The median IC50 and the range of IC50 values for each cohort are listed.

virus (clinical isolates tested in PBMCs), a broad spectrum of susceptibilities was observed for these envelopes, with IC50 values ranging from 0.05 to >100 nM, while again none of the 5 CRF01_AE-derived pseudotype viruses were sensitive to 31.22,37 The median EC50 values for subtype B (n = 133), A (n = 41), and C (n = 36) viruses were generally low (0.34, 2.26, and 1.30 nM, respectively, Table 5), as were the 90EC90 values (4.59, 87.9, and 7.09 nM for subtypes B, A, and C, respectively). Compound 31 retained activity toward isolates from drugexperienced subjects (n = 50) that had developed resistance to various antiretroviral regimens (NRTIs, NNRTIs, and PIs).55 Most importantly, 31 also retained activity against viruses that had become resistant to other entry inhibitors, including 5 and the peptide-based gp41 inhibitor enfuvirtide 32 as well as the CD4-targeting monoclonal antibody ibalizumab that is currently undergoing clinical evaluation, suggesting an absence of cross-resistance with other HIV-1 entry inhibitors.22,55 Finally, two-drug combination studies of 31 with seven agents representing different classes of ARVs showed that there was no antagonism of these inhibitor classes, data that suggest that AIs can be safely used in any combination regimen.22 K



Drug Annotation

scintigraphic tracking of the fate of the contents of the capsule within the GI tract. Assessing its position from the gamma scintigraphy images, the capsules were triggered to release the contents in the proximal small intestine, the distal small intestine, and the ascending colon in an eight-subject crossover study. As a reference, a 100 mg dose of 35 formulated as powder in a hard gelatin capsule that would deliver drug into the stomach was administered to the subjects as part of the crossover study design. The levels of 31 in plasma postdrug release were followed for 24 h, and delivery to the ascending colon indicated an AUC reduction to around 40% relative to the reference, which was within the range where extended delivery was considered feasible.57,58 Three initial prototype extended release formulations based on parameters suggested by the model were then prepared and tested in humans, all of which exhibited good pharmacokinetic profiles and from which a viable prototype for clinical testing was identified. Uniquely, the studies showed that there was adequate expression of alkaline phosphatase in the lower GI tract to ensure both conversion of the prodrug 35 on release from the dosage form and adequate, reliable absorption and delivery of the active parent 31 into the bloodstream. More recently, the extended release formulation has been further developed to offer potential for a range of dosage strengths. By developing drug release profiles for tablets with a common hydrophilic matrix composition but with a varied drug load (to yield different drug to polymer ratios (DPRs)) and different physical sizes (to yield a range of surface area to volume ratios (SAVRs), a surface plot could be constructed and validated with a limited number of in vivo data points. By aligning this plot with the in silico model, the properties (SAVR, DPR) for a particular dose could quickly be inferred to provide pharmacokinetic properties equivalent to the product already established in the clinic (Figure 6). With this approach, alternate strength products delivering pharmacokinetic profiles equivalent to those achieved with the 600 mg prototype have been rapidly and successfully confirmed.

Figure 6. Relationship between tablet SAVR, DPR, and predicted area under plasma concentration−time curve for 31 based on in silico modeling. Vertical lines with spot and cross markers indicate data from human in vivo studies. The model overestimates the area under the curve a little but correctly predicts the shape of the response surface and so remained useful in predicting formulation direction.

key component of developing an overall synthesis of 35. While retrosynthetic analysis suggested two complementary approaches based on the order in which the azaindole ring was constructed, fundamental issues associated with a pyridine starting point led to attention being focused on developing a strategy that originated with pyrrole despite the fact that there was no obvious precedent for this approach.60d,e The final process relied upon a regioselective Friedel−Crafts acetylation of N-benzenesulfonyl pyrrole (36) and was followed by chlorination of the ketone to afford intermediate 37, which was amidated with the sodium salt of N-formyl tosylamide to yield the protected 3-ketopyrrole derivative 38.60d,e Following formation of ketal 39, a Pictet−Spengler cyclization afforded 40, providing a rapid assembly of the core bicyclic ring system. The challenge presented by forming the fully aromatic azaindole heterocycle and installing the 4-methoxy substituent was completed using an unprecedented radical-mediated aromatization promoted by cumene hydroperoxide that was developed to provide access to the N-protected 4-methoxy-6azaindole 41 (Scheme 1). A novel bromination process for the preparation of the C-7 bromo azaindole 44 that relied upon an efficient three-step telescope sequence was developed. Noxidation of 41 was followed by regioselective bromination of the N-oxide 42 to afford the C-7 bromide 43.60f Deprotection of the benzenesulfonyl protecting group of 43 followed by isolation of the hydrochloride salt provided the 7-bromoazaindole 44 in 62−69% yield over the multiple transformations. While procedures for the introduction of the triazole ring to 44 to provide 45 were developed, the timing of triazole incorporation was reassessed and a strategy was pursued that relied upon appending the oxalyl amide side chain to the azaindole core prior to the triazole installation.60g,h This approach overcame several potential disadvantages due to the

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DEVELOPMENT OF A LARGE-SCALE SYNTHESIS OF 35 In addition to the need to develop an extended release formulation, access to substantial quantities of 31 and 35 to supply advanced clinical trials presented a considerable challenge.59,60 The route used to support early pharmaceutical development was based on the synthesis originally used to identify 35 as a clinical candidate and, with some optimization, was used to prepare over 1 metric ton of drug substance.60b Once positive results were observed from the clinical studies, it became apparent that, owing to the high projected dose and the challenging synthesis, a more efficient, environmentally appropriate, and economically viable route to 35 would be required if this compound were to progress to a commercial launch. It was quickly determined that additional optimization of the original process would not be able to meet these criteria, and evaluation and development of a new synthetic route was pursued, the results of which have recently been described in detail.60 However, designing an efficient synthesis of such a complex molecule proved to be extremely challenging, and a wide-ranging strategic interrogation of the structure was critical to developing a holistic understanding of the underlying intricacies within the molecule. In particular, the interplay between the electronics and reactivity within the heterocyclic core presented a significant challenge. Overcoming these to deliver a concise synthesis of the azaindole ring system was a L



Drug Annotation

Scheme 1. Optimized Synthetic Approach to 31 and Its Prodrug 35

isolated yield and purity from CH3CN/H2O. This salt displayed excellent physical properties that resulted in outstanding filtration rates. In addition, its low solubility in CH3CN (10-fold resistance within 8 days in 4 out of 24 subjects and suggest the possibility of a higher genetic barrier for 31 which may be related to the enhanced potency and wider antiviral spectrum in vitro.24,62 These results paved the way for the initiation of a phase 2b clinical trial which compared the antiviral efficacy of 35 in combination with 1 and the integrase inhibitor raltegravir (48) with ritonavir-boosted 17 combined with 1 and 48 in treatment-experienced patients.63 In this study, doses of 400 and 800 mg BID and 600 and 1200 mg QD of 35 performed similarly to the control drug combination. At week 24, 80% of the patients receiving the 400 mg twice daily dose of 35 and 69% in the 800 mg twice daily group, achieved an HIV-1 RNA viral load of less than 50 copies per mL, while 76% in the 600 mg once daily group and 72% in the 1200 mg once daily group achieved this target compared with 75% of patients in the arm receiving ritonavir-boosted 17, data summarized in Table 6. In

alkylation with di-tert-butyl (chloromethyl) phosphate, necessitating the development of a salt metathesis process that converted the Li salt to the K salt, providing access to a more soluble and reactive alkylation partner. Overall, the final processing conditions resulted in the isolation of 47 in 70% yield and with high purity. The final step in the API process is a simple solvolysis of the two tert-butyl groups from the phosphate portion of 47 that occurs in aqueous CH3CO2H and is followed by salt formation with tromethamine and addition of the antisolvent acetone to precipitate the monotromethamine salt 35 as a highly crystalline solid while providing excellent rejection of multiple impurities.

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CLINICAL TRIALS WITH THE EXTENDED RELEASE FORMULATION OF 35 With the identification of a slow release formulation of 35 that enabled the delivery of targeted plasma concentrations of parent drug 31 following BID or quaque die (QD) oral dosing, the stage was set for a phase 1b proof-of-concept study conducted in HIV-1-infected participants.56 In 8-day monö and treatmenttherapy trials conducted in 50 treatment-naive experienced subjects infected with HIV-1 subtype B, 35 was administered once or twice daily with or without ritonavir and good overall efficacy was demonstrated.61 All variants with a baseline IC50 value of 100 nM in the PhenoSense HIV-1 Entry Assay) showed a reduced response. Of the seven subjects with an IC50 value of >100 nM, 5/7 were classified as nonresponders with a viral load decline of

Inhibitors of HIV-1 Attachment: The Discovery and Development of

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