Discovery of the Human Immunodeficiency Virus Type 1 (HIV-1

Article Cite This: J. Med. Chem. 2018, 61, 6308−6327

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Discovery of the Human Immunodeficiency Virus Type 1 (HIV-1) Attachment Inhibitor Temsavir and Its Phosphonooxymethyl Prodrug Fostemsavir

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Tao Wang,*,† Yasu Ueda,† Zhongxing Zhang,† Zhiwei Yin,† John Matiskella,† Bradley C. Pearce,† Zheng Yang,‡ Ming Zheng,‡ Dawn D. Parker,§ Gregory A. Yamanaka,∥,# Yi-Fei Gong,∥ Hsu-Tso Ho,∥ Richard J. Colonno,∥,○ David R. Langley,⊥ Pin-Fang Lin,∥ Nicholas A. Meanwell,† and John F. Kadow†,◆ Departments of †Discovery Chemistry and Molecular Technologies, ‡Pharmaceutical Candidate Optimization, §Discovery Pharmaceutics, ∥Virology, and ⊥Molecular Structure and Design, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States S Supporting Information *

ABSTRACT: The optimization of the 4-methoxy-6-azaindole series of HIV-1 attachment inhibitors (AIs) that originated with 1 to deliver temsavir (3, BMS-626529) is described. The most beneficial increases in potency and pharmacokinetic (PK) properties were attained by incorporating N-linked, sp2-hybridized heteroaryl rings at the 7-position of the heterocyclic nucleus. Compounds that adhered to a coplanarity model afforded targeted antiviral potency, leading to the identification of 3 with characteristics that provided for targeted exposure and PK properties in three preclinical species. However, the physical properties of 3 limited plasma exposure at higher doses, both in preclinical studies and in clinical trials as the result of dissolution- and/or solubility-limited absorption, a deficiency addressed by the preparation of the phosphonooxymethyl prodrug 4 (BMS-663068, fostemsavir). An extended-release formulation of 4 is currently in phase III clinical trials where it has shown promise as part of a drug combination therapy in highly treatment-experienced HIV-1 infected patients.

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INTRODUCTION Mortality from acquired immune deficiency syndrome (AIDS) declined significantly following the introduction of combination antiretroviral therapy (ART) directed toward the underlying cause, human immunodeficiency virus-1 (HIV-1) infection.1 Patients infected with HIV-1 typically realize suppression of replicating virus to levels below the limits of detection with contemporary drug combinations and can resume a productive life while on chronic therapy.2 However, new infections continue to be diagnosed, estimated at more than 37 000 in the United States (U.S.) in 2014, with the result that by the end of 2015 more than 1.1 million people in the U.S. were believed to be living with an HIV-1 infection. However, while AIDS-related deaths have declined worldwide, from 1.9 million in 2005 to ∼1 million in 2016, the fatality rate remains significant and is not restricted to the third world since AIDS-related deaths in the U.S. in 2014 were estimated to exceed 6700 individuals.3,4 Despite these statistics, HIV-1 infection is considered by many in the general populace to be a disease requiring routine although life-long drug therapy.2 © 2018 American Chemical Society

Unfortunately, this mistaken premise threatens the vigor of both the application of resources and the implementation of research efforts directed toward developing improved treatment options. Despite the availability of a variety of classes of antiretroviral agents that enable a range of treatment choices, many as part of convenient, once-daily drug regimens, treatment failures continue to occur.5 High viral heterogeneity, drug-associated toxicity, tolerability problems, and poor adherence can all lead to treatment failure and may result in the selection of viruses with mutations that confer resistance to one or more antiretroviral agents or even multiple drugs from an entire class.5,6 As a result, future therapeutic options for some patients may become significantly limited and there remains a need for safe, later-line regimens that can provide potent, durable therapy toward resistant viruses in the absence of significant side effects.7 In summary, despite current perceptions, it is still far better to prevent HIV-1 infection Received: May 11, 2018 Published: June 19, 2018 6308

DOI: 10.1021/acs.jmedchem.8b00759 J. Med. Chem. 2018, 61, 6308−6327

Journal of Medicinal Chemistry

Article

Chart 1

Scheme 1. Synthetic Approach to 7-Substituted 4-Methoxy-6-azaindole Derivatives

than to treat it, and until this situation is remedied or a cure, either functional or complete in nature, is discovered, infected patients will continue to need improved treatment options, which include new drug classes free of cross resistance to viruses that are no longer sensitive to existing therapies.5−7 The discovery of 1 (Chart 1) from a cell-based phenotypic screen as the initial member of a class of indole 3-glyoxamide derivatives that interfered with the attachment of HIV-1 gp120 to the CD4 protein expressed on human T-cells has been described previously.8 Mechanistic studies with this class of antiretroviral agent have shown that at low concentrations, these compounds bind to the viral gp120 protein and stabilize a conformation that is not recognized by CD4 while at higher concentrations these compounds may form a ternary complex that compromises release of the gp41 fusion protein from the gp120-gp41 complex.9−12 Initial structure−activity relationship (SAR) studies were focused on optimizing indole-based AIs, but as the challenges of developing this class of molecule became apparent, these studies evolved to encompass molecules with azaindole and diazaindole core heterocycles.13−15 The SARs associated with 1 and the optimization process that led to the identification of the 6-azaindole derivative BMS-488043 (2) have been described in detail.14a

This compound established clinical proof-of-concept for the HIV-1 AI mechanism in an 8 day, phase 2a monotherapy study that also revealed some of its deficiencies.16 Administration of 2 to HIV-1-infected patients at twice-daily doses of 800 and 1800 mg resulted in mean reductions in plasma viral load measured on day 8 of 0.72 and 0.96 log10 copies/mL, respectively, which compared to a decline of 0.02 log10 copies/ mL in the placebo control group. The mean maximal viral RNA decline was 1.5 log10 and 1.32 log10 copies/mL following administration of the 800 mg and 1800 mg doses of drug, respectively. However, the spectrum of viruses responding to drug therapy was less than ideal, with only 58% of patients in the 800 mg cohort and 67% of subjects in the 1800 mg arm experiencing a greater than 1 log10 copies/mL reduction in viral load, while four patients experienced more than a 10-fold reduction in virus susceptibility to 2 over the course of the dosing period.16 Moreover, in order to achieve targeted plasma exposure, concomitant dosing of 2 with a high fat meal was required as a means of overcoming dissolution- and/or solubility-limited absorption of the drug. While the antiviral effects associated with 2 were encouraging, significant improvements in the overall profile would be essential in order to achieve a clinically viable compound. The objective 6309



Article

Scheme 2. Alternative Synthetic Approach to 7-Substituted 4-Methoxy-6-azaindole Derivatives

pling of methoxide with 6 mediated by a copper(I) species, such as CuBr or CuI, gave 4-methoxy-7-chloro-6-azaindole (7).19 A Friedel−Crafts-type reaction of 7 with methyl or ethyl chlorooxoacetate under the influence of AlCl3 installed the 2oxoacetate group at the C-3 position of the 6-azaindole heterocycle to provide ester 8.20 The ester moiety of 8 was hydrolyzed using K2CO3 or NaOH in aqueous MeOH to yield the potassium or sodium salt 9 which was coupled with a monoaroyl piperazine 10 using either DEBPT, TBTU, HATU, or EDC as the coupling agent to generate the key intermediate diamide 11.21 A Stille or Suzuki coupling of 11 with an aryltin reagent or an arylboronic acid, respectively, formed the Clinked 7-aryl-4-methoxy-6-azaindole diamide 12, while a Cu(0)-mediated coupling of 11 with an azole was employed to synthesize N-linked analogues of 12.22−24 Alternatively, when necessary and feasible, aryl groups were installed at the C-7 position of 7 prior to the Friedel−Crafts acylation at C-3. As shown in Scheme 2, Pd- or Cu-catalyzed aryl C- or N-coupling of 7 gave 7-aryl-4-methoxy-6-azaindole derivatives 13 which were acylated with methyl or ethyl chlorooxoacetate in CH2Cl2 using AlCl3 as the mediator to afford esters 14. Hydrolysis of the ester moiety under basic conditions (K2CO3 or NaOH in MeOH/H2O) provided the potassium or sodium salt 15 which was coupled with a monoaroyl piperazine derivative 10 under the amide coupling conditions described in Scheme 1 to form 12. In general, the chemistry route described in Scheme 1 provided an expedient, parallel synthesis of multiple final products that facilitated SAR exploration, while the chemistry approach shown in Scheme 2 possessed an advantage when a larger quantity of material was required for advanced profiling or in specific cases where the diamide moiety was found to be incompatible with the conditions associated with installation of the C-7 substituent.

for a refined molecule was to increase the percentage of subjects responding to the AI and to eliminate the requirement for administration of the drug with a high fat meal. However, while a lower drug dose would be preferable, this was viewed as a particular challenge given the variation of antiviral potency shown by AIs toward a spectrum of HIV-1 strains. Nevertheless, in the face of these challenges it was believed that the targeted profile could be achieved by a combination of higher intrinsic antiviral potency that would extend inhibition to encompass a broader range of HIV-1 clinical isolates and improved pharmacokinetic properties. Thus, the objective became the identification of a compound that, at a minimum, represented a 10-fold advance over 2 based on a composite of these two factors. While 2 was advancing into clinical trials, the search for inhibitors with superior profiles had continued uninterrupted based on studies with five independent heterocyclic series: indoles, 4-azaindoles, 4-F-6-azaindoles, 4-MeO-6-azaindoles, and 4,6-diazaindoles.13−15 Studies with the indole series provided a seminal SAR observation that the antiviral potency was increased considerably when specific substituents linked by sp2-hybridized atoms were directly attached to the C-7 position of the heterocycle.13d,e As a consequence of this finding, substitution at position 7 of the 4- and 6-azaindole series with heteroaryl moieties was pursued in parallel. While the introduction of an sp2-hybridized heteroaromatic substituent at the C-7 position of indoles and 6-azaindoles could lead to increased potency, each series displayed unique SARs with respect to optimal potency and preclinical profiling.14 In this report, we describe the optimization and preclinical profiling of the 4-methoxy-6-azaindole series which provided a path to the most promising analogs, leading to the synthesis and selection of 3 (BMS-626529, temasavir) as a clinical candidate and its ultimate formulation as the phosphonooxymethyl prodrug 4 (BMS-663068, fostemsavir).17

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RESULTS AND DISCUSSION

The criteria for advancing compounds into in vivo PK studies during the campaign to find a next-generation development compound were based on in vitro data that focused on selecting compounds with superior anti-HIV-1 inhibitory activity compared to the prototype 1, high metabolic stability that preferably reflected a t1/2 of >100 min in both human and rat liver microsomal incubations under the conditions employed for these assays at the time, a high membrane permeability coefficient with Pc values of >100 nm/s in a Caco-

CHEMISTRY The initial synthetic approach used to support the medicinal chemistry efforts around the study of 7-substituted 4-methoxy6-azaindole derivatives is depicted in Scheme 1. A Bartoli-type reaction in which commercially available 5-bromo-2-chloro-3nitropyridine (5) was exposed to a 3- to 4-fold excess of vinylmagnesium bromide at low temperature (−78 to −20 °C) furnished 4-bromo-7-chloro-6-azaindole (6).18 Radical cou6310



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Table 1. HIV-1 (JRFL) Inhibition, Cell Cytotoxicity, HLM Stability, and Caco-2 Cell Permeability Data for a Series of C-7Substituted, C-Linked Heteroaryl, 4-Methoxy-6-azaindole Derivatives

a

Antiviral data are the average of two separate test occasions with the individual data in parentheses except where the standard deviation is included. bt1/2 in rat liver microsomes (RLMs).

tetrazolium-5-carboxanilide (XTT) used as an indicator of cell respiration and viability. 4-Methoxy-6-azaindole Derivatives with C-Linked Heteroaryl Substituents at C-7. The initial survey of C-7 substituted compounds focused on unsubstituted oxygen-, sulfur-, and nitrogen-containing five- and six-membered heterocycles attached to C-7 of the azaindole core through a carbon atom linkage, with the antiviral activity, cytotoxicity, stability in human liver microsomes (HLM), and membrane permeability data compiled in Table 1.25 The five-membered heterocycles examined encompassed furans 16 and 17, thiophenes 18 and 19, thiazoles 20−22, the oxazole 23, and the pyrazole 24, while six membered heteroaryl substituents included pyridines 25−27, the pyrazine 28, the pyridazines 29 and 30, and pyrimidines 31−33. The survey revealed that, in general, this series of compounds displayed excellent intrinsic antiviral potency, with all except the 2-substituted pyrimidine 32 more potent than 2. However, it is apparent from the data in Table 1 that antiviral potency is sensitive to the position of the heteroatoms of the C-7 substituent relative to the core azaindole heterocycle. For example the 2-substituted furan 16 was 5-fold more potent than its 3-substituted isomer 17 while the 2- and 5-substituted thiazoles 20 and 22 were 3- to 5-fold

2 cell assay, and an in vitro liability profile that predicted for an absence of significant drug−drug interactions and safety issues at the targeted exposures.25 The HIV-1 inhibitory activity of test compounds was determined using a pseudotype assay that relied upon an engineered virus in which the env gene was replaced with a firefly luciferase gene to provide a convenient readout of the extent of virus infection, measured by luciferase activity 3 days after virus inoculation. To allow infection of host cells, either a JRFL (M-tropic, CCR5-specific) or LAI (T-tropic, CXCR-4specific) virus envelope was provided to the pseudovirus by transfection. However, the absence of the env gene restricts these pseudoviruses to a single round of infection, and thus, the assay encompasses virus entry, reverse transcription, integration, and expression of viral proteins up to and including assembly of viral-like particles that are devoid of gp160 and, therefore, noninfectious. The concentration of drug inhibiting 50% of virus infectivity (EC50 value) was determined while the therapeutic index was assessed by determining the concentration at which the test drug was half-maximally cytotoxic toward MT-2 cells (CC50 value) after 3 days of incubation, with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H6311



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Table 2. HIV-1 (JRFL) Inhibition, Cell Cytotoxicity, HLM Stability, and Caco-2 Cell Permeability Data for a Series of C-7Substituted, C-Linked Heteroaryl 4-Methoxy-6-azaindole Derivatives

a

Antiviral data are the average of two separate test occasions with the individual data in parentheses except where the standard deviation is included. 6312



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Table 3. Antiviral Potency (HIV-1 (JRFL) Inhibition), Cell Cytotoxicity, HLM Stability, and Caco-2 Permeability Associated with 4-Methoxy-6-azaindole Derivatives 46−48

compd

R

antiviral EC50 (nM) (JRFL)a

cytotoxicity (MT-2 cells) CC50 (μM)

% remaining at 10 min of incubation in HLM (RLM)b

Caco-2 permeability Pc (nm/s)

46 47 48

H (R)-CH3 (S)-CH3

0.11 ± 0.01 (n = 3) 0.40 ± 0.16 (n = 4) 1.04 (1.00, 1.08)

>300 (n = 2) >300 (n = 2) 267, >300.00

100% (27%), t1/2 > 100 min 95% (80%) n/a

n/a 125 n/a

a

Antiviral data are the average of two separate test occasions with the individual data in parentheses except where the standard deviation is included. bAmount of parent compound remaining after 10 min of incubation in HLM or RLM (3 μM compound concentration).

Table 4. Rat PK Parameters for 2, 38, 46, and 47a compd

F (%)

AUC po 24 h (μM·h)

CL iv (mL min−1 kg−1)

Vss iv (L/kg)

t1/2 iv (h)

2 38 46 47

90 0.43 85 61

15 ± 6.3 0.13 ± 0.006 14.6 ± 2.8 7.8 ± 1.7

13 ± 4.0 5.6 ± 0.2 12 ± 5.5 8.8 ± 3.7

1.1 ± 0.22 0.18 ± 0.01 1.9 ± 0.64 1.1 ± 0.35

2.4 0.8 4.0 2.4

± ± ± ±

0.33 0.09 0.92 0.2

a

iv dose = 1 mg/kg; po dose = 5 mg/kg.

cycle of the survey are compiled in Table 2. The substituents examined included simple alkyl and alkoxy groups and amines, all of which were generally well tolerated with respect to maintaining potent antiviral activity since the EC50 values in the pseudotype assay were less than 1 nM. The introduction of a basic amino group, as exemplified by the pyridine 36, pyrazines 37, 39, and 40, and the thiazole 45, generally led to retention of potency when compared to the progenitors 25, 28, and 22, respectively. In contrast, alkyl and alkoxy substituents failed to provide clear SAR trends, leading to either retention (compare 44 and 22) or a reduction in antiviral potency (compare 34 and 25). Other substituents examined included NHSO2R, NHAc, and COOH all of which led to decreased antiviral potency (data not shown). While the potency of the compounds compiled in Table 2 met targeted criteria, in vitro profiling data revealed that only 38 and 41 achieved acceptable stability in HLM and only 35 met the criterion for membrane permeability that would qualify a compound for advancement into in vivo studies. Another approach toward increasing the overall polarity of molecules as a means of enhancing metabolic stability involved exploring the effect of introducing a heteroatom to the benzamide element in which the phenyl ring was replaced with a 2-pyridyl moiety.13b This structural modification was examined in the context of the pyrazine derivative 28 and included an evaluation of the methylated piperazine homologues, with the results compiled in Table 3.13c This approach was successful in the context of improving metabolic stability, with the t1/2 of 46 in HLM significantly enhanced to >100 min when compared with the t1/2 of 19 min recorded for the benzamide 28. However, antiviral potency was compromised by 3-fold so the addition of an (R)-methyl group to the piperazine proximal to the benzamide moiety was explored with the evaluation of 47 since this modification had proven to be beneficial in the indole series.13c Unfortunately, against this structural backdrop, methyl substitution led to a further 3-fold decrease in potency although 47 was associated with targeted

more active than the 4-substituted thiazole 21. Among the azines 25−33, the 3-pyridine 26, the 2-pyrazine 28, and the 4pyrimidine 31 offered the highest antiviral potency. A wide variation in membrane permeability, as measured in the Caco2 cell assay, was also observed for the different C-7 substituents. For example, the 4-thiazole 21 was predicted to be highly permeable with a Pc value of 217 nm/s, while conversely, both the 2- (20) and 5-substituted (22) thiazole isomers displayed low membrane permeability, with Pc values of ≤15 nm/s. Similarly, the two C-7 pyrimidine derivatives 31 and 32 exhibited high Pc values of 378 and 106 nm/s, respectively, while the 5-substituted isomer 33 was much poorer, with Pc values of 46 and 100 min in HLM, necessitating further optimization in order to meet the overall objective of the initiative. In an effort to achieve a balance between acceptable membrane permeability and optimal metabolic stability in HLM, several avenues of structural modification were explored. One approach toward overcoming the low permeability of metabolically stable compounds was to introduce lipophilic substituents to the periphery of the more polar heterocycles in an effort to mask the polarity. A second approach attempted to improve the metabolic stability of a subseries that exhibited high membrane permeability, such as the 4-thiazoles and pyrazines, by substituting the heterocyclic ring in an effort to judiciously increase local polarity. Select examples from this 6313



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Figure 1. (a) Noncovalent and nonbonding interactions between the furan ring of 16 and 17 and the azaindole core heterocycle. (b) Noncovalent and nonbonding interactions between the pyrimidine rings of 31, 32, and 33 and the azaindole core heterocycle.

α- and α′, respectively, in Figure 1a, in interactions that favored coplanarity. With such a structural arrangement, an intramolecular H-bond could be formed between a heteroatom at the α-position of the C-7 heteroaryl ring and the N−H of the pyrrole ring of the core heterocycle, while at the α′ position of the heteroaryl ring, H-bond donors that could engage the N6 azaindole nitrogen atom were preferred, as illustrated in Figure 1.26,27 This hypothesis is supported by comparison of the antiviral activity of the furans 16 and 17 (illustrated in Figure 1a), the thiophenes 18 and 19, the pyridines 25 and 27, and the pyrimidines 31 and 33 (illustrated in Figure 1b). In the case of the furan 16, a H-bond interaction between the ring oxygen atom and the pyrrole N−H would stabilize a planar arrangement potentially supported by a C−H H-bonding interaction with the azaindole nitrogen atom, as depicted in Figure 1a.26,27 In the isomeric furan 17, not only is the ring oxygen atom not able to engage the azaindole N−H, but if the furan ring was to approach a coplanar arrangement, it would introduce allylic 1,3-strain between the H atom at the 2position of the ring and the N−H of the core heterocycle (Figure 1a).28 The antiviral potency of the thiophene 18 is similar to the 3-substituted furan 17 and weaker than 16, reflecting the poorer H-bond accepting properties of the thiophene sulfur atom. A topological transposition of the thiophene ring of 18 would establish an attractive interaction between the low lying σ* orbital of the sulfur atom and the lone pair of electrons on the nitrogen atom of the azaindole heterocycle that would stabilize a planar conformation; however, this arrangement would introduce allylic 1,3-strain between the thiophene 3-C-H and azaindole N−H.26,29 The 15-fold potency difference between the thiazole 20 and the oxazole 23 can be explained by nonbonded interactions, with the favorable profile of 20 a function of dual interactions between the thiazole and azaindole rings that favor coplanarity. The thiazole sulfur atom of 20 can engage the azaindole nitrogen atom via a nitrogen lone pair to sulfur σ* interaction that is supported by a concomitant H-bonding interaction between the ring nitrogen atom and the azaindole N−H. For the oxazole 23 to adopt a similar coplanar conformation based on a favorable alignment between the azole nitrogen atom and azaindole N−H, a repulsive electronic effect would be established between the azaindole nitrogen and the oxazole oxygen atoms that would favor a distortion from planarity.

metabolic stability and Caco-2 permeability. In contrast, the (S)-methyl isomer 48 exhibited lower antiviral activity, a SAR result similar to that observed previously in the indole series.13c On the basis of their overall profiles, compounds 38, 46, and 47 were selected for rat PK studies, with the results presented in Table 4 along with data for 2 for the purpose of comparison. Compound 38 provided reduced exposure compared to 2, attributed to its moderate membrane permeability (Caco-2 Pc = 66 nm/s). However, 46 offered a comparable rat PK profile to 2, with a similar oral bioavailability of 85% compared to 90% for the prototype and plasma AUCs recorded over 24 h that were essentially identical at 14.6 and 15 μM·h, respectively. The installation of an (R)-methyl group reduced the exposure of 47 by approximately 50% based on the AUC value, which could be rationalized by the higher rate of metabolism predicted from the in vitro data for 47 compared to 46. Compound 46 provided the best overall profile within all of the structural classes (indole, 4-F-6-azaindole, 4-MeO-6azaindole, 4-azaindole, and diazaindole core, with C-7 carbon linkage) examined during this round of compound optimization. However, 46 was not pursued further because it failed to offer sufficient differentiation from 2 in the preclinical profiling assays to predict for a clinical advantage. 4-Methoxy-6-azaindole Derivatives with N-Linked Heteroaryl Substituents at C-7. At this juncture of the program, C-7 substituents linked to the core azaindole via a nitrogen rather than a carbon atom were considered, with a view to modulating the electronic distribution within both the core heterocycle and the C-7 appendage. Target compounds for this phase of the study were selected for synthesis based on the SAR observations made with 16−33 and other series of attachment inhibitors that had been developed in parallel.13,14b Studies of C-7-substituted indoles had established that antiviral potency was optimal when the C-7 substituent adopted a topographically coplanar arrangement with the core heterocycle.13d,e,14b Analysis of the relative antiviral potency data for 6-azaindole analogs 16−33 revealed that the more potent antiviral activity in each subseries generally corresponded with C-7 heteroaryl rings that were predicted to be able to access a coplanar arrangement with the azaindole core. The optimal compounds were those that possessed arrangements on both flanks of the heteroaryl ring that could engage the pyrrolo N− H and the C-6 nitrogen atom of the azaindole, sites labeled as 6314



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The unsubstituted 1,2,3-triazoles 49 and 50 and the 1,2,4triazoles 51 and 52 demonstrated potent antiviral activity in cell culture, with EC50 values of less than 1 nM in the pseudotype assay while the prototypical pyrazole 53 was several-fold weaker, perhaps reflective of the reduced strength of the C−H H-bond donor properties.27,31 Gratifyingly, this series displayed promising profiles in the in vitro screens predictive of PK properties, with robust metabolic stability after 10 min of incubation in HLM and RLM predictive of low rates of metabolism in vivo. Compounds 49−52 also demonstrated good membrane permeability based on evaluation in the Caco-2 cell assay, representing an important step toward the sought-after compromise in properties, while the pyrazole 53 performed less impressively. In a preliminary evaluation of a small number of clinical isolates, 51 and 52 possessed a broader spectrum of inhibition than 2, data that foreshadowed the emergence of the C-7 nitrogen-linked heteroaryl series as the more promising class of AIs compared to the C-7 carbon-linked heteroaryl series. With these promising data in hand, elaboration of the C-7 heteroaryl moieties by the introduction of substituents was explored in the context of both the triazoles and pyrazoles, with the latter pursued because they offered greater potential for variation of the substitution pattern. This survey comprised compounds 3 and 54−88, with the profiling data compiled in Table 6. For the pyrazole series, the heterocyclic ring was decorated with a range of polar, nonpolar, and basic substituents with the result that in all but two of the examples compiled in Table 6, antiviral potency was enhanced compared to the progenitor 53. The 5-methyl derivative 57 and the 3dimethylaminomethyl analogue 62 were notable exceptions to this generalization, with the reduced potency associated with 57 attributed to a compromise of overall planarity based on the arguments developed above. This hypothesis is supported by the potent antiviral activity associated with the 5-amino homologue 60 which can engage the azaindole nitrogen atom in a H-bonding interaction that would reinforce the planar topography favored by a productive H-bond between the pyrazole nitrogen and pyrrole N−H of the core heterocycle. The 3-methyl derivatives 54 and 55 exhibited increased membrane permeability across a confluent Caco-2 cell layer compared to the poorly permeable 53, but both were only moderately stable in HLM, although 54 performed well in RLM. The physicochemical properties of HIV-1 AIs presented a significant challenge to development in general, and the solubility of 54 was not an exception, measured at 0.017 mg/ mL under equilibrium conditions. Methyl substitution proximal to the benzamide of the piperazine ring (compound 55) resulted in a modest 4-fold improvement in aqueous solubility to 0.069 mg/mL but, in this example, was associated with a 3-fold reduction in antiviral potency. In an effort to combine potent antiviral activity with improved solubility, the methyl substituent of 54 was either replaced or elaborated with polar elements that included basic amines, as exemplified by 58−62, the fluoride 63, hydroxymethyl derivative 64, and the carboxamide 65. However, while compounds 58−61 and 63− 65 expressed high antiviral potency, none provided the overall targeted profile in the context of the sought-after compromise between metabolic stability and membrane permeability. Excellent potency was observed for substituted 1,2,4triazoles with a range of substituents installed at the 3-position of the ring, providing several compounds for which the EC50 values in the pseudovirus infectivity assay were less than 0.10

Similar arguments related to intramolecular interactions stabilizing or destabilizing a planar conformation can be invoked to explain the differences in potency between the pair of pyridines 25 and 27 and the pyrimidine 31 and its less potent isomers 32 and 33 (Figure 1b). In the case of the latter series, the more potent 31 is stabilized in a planar topography by productive interactions between a pyrimidine C−H and the azaindole nitrogen atom and a pyrimidine nitrogen atom and the azaindole N−H, while 32 introduces an unfavorable nitrogen lone pair to nitrogen lone pair interaction and 33 incurs unfavorable allylic 1,3-strain (Figure 1b). These nonbonded interactions are presumed to underlie their reduced antiviral potency. In the cases of the furans 16 and 17, the thiazoles 21 and 22, the pyridines 25 and 26, and the pyrimidines 31−33, there appears to be a correlation between the presence of intramolecular H-bonding interactions and enhanced permeability across a confluent layer of Caco-2 cells.30 However, for the thiazole 20 and the pyridazine 29, this phenomenon does not appear to be operative since both exhibit low membrane permeability coefficient values, suggestive of the involvement of additional factors. The recognition of the importance of a coplanar relationship between the azaindole ring and the C-7 heteroaryl substituent in combination with trends that the properties facilitating permeability and enhancing metabolic stability were in opposition led to the proposal that C-7 substituents linked via a nitrogen atom may offer a compromise in properties. It was hypothesized that the electronic overlap between the nitrogen atom of a C-7 heteroaryl ring and the azaindole core would result in a shorter bond length between the two heterocycles and contribute to the preference for coplanarity. In the C-7 C-linked analogues, higher metabolic stability appeared to correlate with an increase in the polarity of the C7 heteroaryl moiety while excess polarity exposed at the periphery of the molecules resulted in reduced membrane permeability in the Caco-2 assay. This led to the concept that polar C-7 N-linked heteroaryl compounds might be associated with increased metabolic stability without incurring a significant penalty on membrane permeability since the polarity would be installed proximal to the azaindole core, an idea that converged with the topology concept depicted in Figure 2. An azole configured with a nitrogen atom at the α-

Figure 2. Structural concept for C-7 N-linked azole heterocycles that would favor a planar topography.

position and a C−H at the α′-site was targeted based on this premise, which necessitated that the C-7 heteroaryl ring incorporate at least two nitrogen atoms, with one being the site of attachment. This analysis led to a focus on the synthesis of C-7 N-linked pyrazoles, 1,2,3-triazoles, and 1,2,4-triazoles, with antiviral and developability data for compounds 49−53 prepared for this phase of the survey compiled in Table 5. 6315



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Table 5. Antiviral Activity, Cytotoxicity, Metabolic Stability, and Caco-2 Cell Permeability Data Associated with C-7 N-Linked Unsubstituted 1,2,3-Triazoles 49 and 50, the 1,2,4-Triazoles 51 and 52, and the Pyrazole 53

a

Antiviral data are the average of two separate test occasions with the individual data in parentheses except where the standard deviation is included.

nM. Exceptions were 68 and the two basic amine-containing analogues 76 and 80, compounds that recapitulated the SAR observations with 57 (methyl effect) and 62 (basic amine effect) in the pyrazole series. However, SARs in the triazole series exhibited some divergence from that associated with the pyrazoles since the introduction of a methyl group at the 3position of the triazole ring (compound 3, EC50 = 0.10 nM) did not result in a further boost in potency when compared to the parent molecule 51. Against this structural backdrop, installation of a methyl substituent on the piperazine ring proximal to the benzamide led to a 2- to 5-fold reduction in antiviral potency that showed some dependence on the absolute configuration, with the (R)-isomer 66 more potent than the (S)-isomer 67. An ethyl substituent at C-3 of the triazole ring, compound 69, was marginally more potent than 3, with the (R)-methylpiperazine homologue 70 comparably potent but of lower metabolic stability. The methyl ethers 71 and 72 exhibited targeted antiviral potency and metabolic stability in LM but were excluded from further consideration due to the poor membrane permeability values recorded in the Caco-2 cell assay. An amine substituent at the C-3 position of the 1,2,4-triazole ring was tolerated in the antiviral assay, but 73 exhibited poor membrane permeability. Elaboration of the amine of 73 was explored in the context of 74−76, but these analogues were not pursued further because they offered no potency advantage when compared to 3. For the other substituents explored in the context of 77−86 that extend the

boundaries of the pharmacophore and probe for the tolerance of polar functionality, deficiencies in either metabolic stability or membrane permeability or lack of an advantage in potency compared to the structurally more compact prototype 3, which had emerged as a promising compound, led to them not being evaluated in more advanced studies. The two tetrazoles 87 and 88, which significantly extend the silhouette at the C-7 position and present a polar azole element in this region of the pharmacophore, represent an interesting SAR point since they maintain potent antiviral activity in the pseudotype assay and toward another B clade isolate designated 93US143. Notably, both 87 and 88 exhibited 10-fold more potent antiviral activity than 3 toward two C clade isolates 97ZA009C and 98TZ017 and the D clade virus E94UG114 that were tested as part of an initiative to characterize potential microbicide candidates (data not shown).32 By use of the X-ray cocrystal structure of 2 bound to a construct of HIV-1 gp120 as the basis (Figure 3a), compounds 87 and 88 were modeled in the HIV-1 gp120 protein with the suggestion that these larger C-7 substituents are able to establish additional hydrophobic contacts.33 In addition, the modeling suggests that the tetrazole of 87 can access a conformation where it is poised to engage Lys117 (Figure 3b) while in 88, the tetrazole moiety has the potential to interact with Asp113 (Figure 3c). Several of the more promising compounds were advanced into rat PK studies administered by both the po and iv routes, with the results summarized in Table 7. Benchmark PK studies 6316



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Table 6. Antiviral Activity, Cytotoxicity Values, Metabolic Stability, and Caco-2 Cell Membrane Permeability Associated with a Series of 6-Azaindoles Substituted at C-7 with N-Linked Pyrazoles 54−65, 87, 88 and Triazoles 3, 66−86a

a

Antiviral data are the average of two separate test occasions with the individual data in parentheses except where the standard deviation is included. 6317



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with representatives of the three different heterocycle chemotypes compiled in Table 5 revealed that triazoles 49−51 and pyrazole 53 demonstrated good oral bioavailability, with 51 comparable to 2. The 1,2,3-triazole derivative 49 exhibited lower clearance and a lower volume of distribution than observed for 2, with an intrinsic half-life that was comparable to the prototype and an AUC measured over 24 h that was 3fold higher. However, the PK profile of the methylated piperazine homologue 50 was poorer than for 2, with similar clearance parameters, a lower volume of distribution and a shorter iv half-life. Both the 1,2,4-triazole 51 and pyrazole 53 offered lower iv clearance and higher po plasma exposure when compared to 2. Substitution of the pyrazole ring of 53 with a methyl substituent (compounds 54 and 55) preserved the PK profile of the prototype, but the more polar NH2 (58) and CH2OH (64) substituted analogues were poorly bioavailable and suffered from high iv clearance. However, the 3-methyl1,2,4-triazole derivative 3 exhibited improved PK properties compared to the unsubstituted progenitor 51, with 2-fold lower clearance and a 2-fold higher iv half-life that translated into a 2-fold higher AUC measured over 24 h. Moreover, 3 was a less potent inhibitor of CYP 3A4 (IC50 > 40 μM) than 51 (IC50 ∼ 1.0 μM), presumably a reflection of the steric masking of the triazole nitrogen atoms conferred by the CH 3 substituent.34,35 The PK profile of the methylpiperazine homologue 66 also reflected an improvement over 2. The ethyl-substituted compound 69 offered a further advance although in this case the methylpiperazine derivative was less impressive while the morpholine 74 was similar in profile to 2. Finally, the methylated 1,2,3-triazole 86 offered a PK profile that was comparable to the unsubstituted progenitor 49. Preclinical Profiling of 3 and the Discovery of Its Phosphonooxymethyl Prodrug 4. The overall profile of 3 that emerged from these studies identified it as a compound of sufficient interest to qualify for additional evaluation based on the 7-fold improved antiviral potency in the primary assay and 7-fold higher AUC in the rat following oral dosing, parameters that met the targeted criterion of an overall 10-fold enhancement of properties. Additional scrutiny of the antiviral effects of 3 toward representative HIV-1 clinical isolates expressing envelope subtypes A−G revealed improved inhibitory activity compared to 2, particularly toward subtype

Figure 3. (a) Cocrystal structure of 3 bound to HIV-1 gp120 taken from the published structure (PDB accession number 5U70) demonstrating key drug−target contacts with the two key H-bonding interactions to Glu113 and Trp427 highlighted. (b) Model of 87 bound to the gp120 construct that preserves the key drug−target interactions observed in the cocrystal structure of 3. (c) Model of 88 bound to the gp120 construct that preserves the key drug−target interactions observed in the cocrystal structure of 3.

Table 7. Rat PK Parameters for Selected C-7, N-Linked Heteroaryl 6-Azaindole HIV-1 AIsa compd

F (%)

po 24 h AUC (μM·h)

CL iv (mL min−1 kg−1)

Vss iv (L/kg)

t1/2 iv (h)

2 3 49 50 51 53 54 55 58 64 66 69 70 74 86

90 82 59 59 90 77.1 43.7 72.8 0.8 19 104 86 88.4 52 65.5

15 ± 6.3 111 ± 25 43.5 ± 10.0 8.6 ± 1.4 48.7 ± 15.7 25.6 ± 2.7 15.7 ± 7.2 13.2 ± 8.6 0.03 1.99 ± 0.50 88 ± 25 254 ± 87 82 ± 23 13 ± 5.0 34.8 ± 1.5

13 ± 4.0 1.3 ± 0.19 2.4 ± 0.08 12 ± 0.19 2.4 ± 0.5 5.6 ± 1.8 3.7 ± 1.0 11.2 ± 0.9 57 ± 21 29 ± 6.7 1.9 ± 0.26 0.7 ± 0.05 1.8 ± 0.4 6.1 ± 0.83 4.3 ± 0.5

1.1 ± 0.22 0.36 ± 0.098 0.23 ± 0.04 0.49 ± 0.14 0.4 ± 0.04 0.48 ± 0.06 0.23 ± 0.02 0.47 ± 0.02 0.4 ± 0.1 1.1 ± 0.3 0.34 ± 0.07 0.12 ± 0.007 0.2 ± 0.03 0.26 ± 0.02 0.29 ± 0.06

2.4 ± 0.33 4.3 ± 1.1 1.6 ± 0.3 0.70 ± 0.24 2.6 ± 0.5 1.8 ± 0.2 0.9 ± 0.2 0.56 ± 0.03 0.5 ± 0.3 2.9 ± 2.3 3 ± 0.9 3.3 ± 0.66 3.0 ± 1.4 1.5 ± 0.19 1.4 ± 0.5

a

Compounds were dosed at 1 mg/kg iv or 5 mg/kg po. 6318



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was not anticipated to be significantly affected because of the high intrinsic passive membrane permeability. The aqueous solubility of a crystalline sample of 3 was measured as 0.022 mg/mL at pH = 7.4, which is approximately 2-fold lower than the aqueous solubility of a crystalline form of 2. Modest increases in solubility were observed at pH = 1 (0.050 mg/mL) and pH = 10 (0.07 mg/mL), reflecting the amphoteric nature of the compound. Both 2 and 3 are defined as biopharmaceutics classification system (BSC) class II molecules based on their low aqueous solubility and high Caco-2 membrane permeability.37 Analogous to 2, the relative oral bioavailability of 3 when dosed as an unoptimized suspension formulation was lower (52%) than when dosed as a solution, indicative of a dissolution- or solubility-limited effect on drug absorption. Consistent with these data, plasma exposure increased in a less than dose-proportional fashion upon dose escalation. However, acceptable multiples of the predicted efficacious human exposure were achieved in preclinical toxicology studies using solution-based formulations and 3 was successfully advanced through IND-enabling studies and into phase 1 clinical trials. A dose escalation study in which doses of 25, 50, 100, 200, 400, 800, and 1200 mg were administered to normal healthy volunteers as a solid capsule formulation revealed a dose-related increase in exposure, although this was less than dose-proportional from 800 to 1200 mg. The mean plasma concentration 12 h following a 1200 mg was approximately 7-fold above the protein binding-adjusted EC50 value of 8.6 ng/mL.17,38 The successful development of a phosphonooxymethyl prodrug of 2 that provided a solution to the solubility- and dissolution-limited exposure observed with the parent compound led to an examination of the application of this prodrug moiety to 3 in an effort to maximize plasma exposure and promote coverage of a broader spectrum of viruses in clinical studies.39 The phosphonooxymethyl prodrug 4 was prepared as delineated in Scheme 3.17,40 The synthesis of 4 was accomplished by adding an excess of NaH (5 equiv) to a suspension of 3 in THF followed after 30 min by 1 equiv of iodine and a 10-fold excess of freshly prepared di-tert-butyl chloromethylphosphate which afforded 89.17,39,40 The tertbutyl ester moieties of 89 were unmasked under thermal hydrolytic conditions by heating the compound at 40 °C in a 1:1 mixture of acetone and H2O for 16 h which furnished the intermediate phosphoric acid 90.40 The monotromethamine salt 4 was formed in in situ and isolated after crystallization from the solution.39−41 Prodrug 4 possessed high aqueous solubility, measured as >11 mg/mL across the pH range of 1.5−8.2 at room temperature, and was stable in solution under acidic and neutral conditions and in its solid state for at least 24 h at 37 °C. Following oral administration, 4 was likely hydrolyzed by alkaline phosphatase(s) present at the brush border membranes of the intestinal lumen to form a hydroxymethyl intermediate which rapidly lost formaldehyde to produce 3. The high membrane permeability of 3 facilitated its rapid absorption, mitigating the potential for precipitation of the parent drug.42−44 This hypothesis was supported by the good to excellent oral bioavailability (80−122%) of 3 observed after administration of low doses of 4 to rats, dogs, and cynomolgus monkeys and the very low to absent systemic exposure of prodrug 4. Moreover, 4 was hydrolyzed to form 3 in the presence of human placental alkaline phosphatase and in

B virus where the median EC50 value of 3 toward 22 viruses from geographically diverse regions was 10-fold lower than for 2 and with no overt dependence on coreceptor tropism.36 In a PhenoSense virus entry assay that evaluated envelopes from 134 participants in Bristol-Myers Squibb-sponsored clinical trials and 108 samples from the Monogram collection, IC50 values ranged from 0.05 to >100 nM, reflecting results observed using a viral replication assay in peripheral blood mononuclear cells. Although the majority of these isolates represented subtype B viruses, envelopes from subtypes A, C, AE, F, F1, BF, and AG were also included. An analysis of these data indicated that 50% of the subtype B viruses would be inhibited by 50% at a concentration of 0.34 nM while concentrations of 2.26 and 1.30 nM would be required to capture 50% of the subtype A and C viruses in a similar fashion. The concentration of 3 that would exceed the IC50 values of 90% of the subtype B, A, and C viruses were calculated to be 4.59, 87.9, and 7.09 nM, respectively. In biochemical studies, the affinity of 3 for a soluble preparation of HIV-1 gp120 was high, with a Kd of 0.83 nM which compared to a Kd of 19 nM for 2 under comparable conditions. More importantly, the binding kinetics for 3 were enhanced in a favorable direction, with a 10-fold slower off rate (t1/2 = 458 min) compared to 2 (t1/2 = 43 min).36 With promising antiviral data, the PK profile of 3 was evaluated in the dog and cynomolgus monkey, with the results compiled in Table 8. In the rat and dog, the AUC of 3 Table 8. PK Parameters Associated with 3 Compared to 2 in the Rat, Dog, and Cynomolgus Monkey 3 Oral F (%) rat 82 dog 89 cynomolgus monkey 64 Oral AUC at 5 mg/kg (μM·h) rat 111 dog 61 cynomolgus monkey 14 Total CL (mL min−1 kg−1) rat 1.3 (low) dog 2.6 (low) cynomolgus monkey 7.5 (low)

2 90 57 60 15 43 28 13 (low) 2.4 (low) 4.3 (low)

following oral dosing was improved over 2 (111 μM·h compared to 15 μM·h in the rat for 2 and 3, respectively, and 61 μM·h compared to 43 μM·h in dog for 2 and 3, respectively) but not in the cynomolgus monkey, where higher clearance was observed, although the total body clearance was deemed to be low based on hepatic blood flow in this species. However, 3 showed improved stability in HLM, with very low turnover, and in human hepatocyte assays when compared to 2. Human plasma protein binding for compound 3 was 85%, offering a higher free fraction than 2 which bound more avidly (95%). However, the effects of the inclusion of 40% human serum on the antiviral activity were similarly modest for both compounds, with shifts in the EC50 values of 1.5- to 2.1-fold for 3 and 1.9- to 3.7-fold for 2. In liability assay screening, 3 did not significantly inhibit CYP 1A2, 2C19, 2C9, 2D6, and 3A4 (IC50 values of >40 μM) and was inactive in a PXR activation assay predictive of a low potential for CYP 3A4 induction. In Caco-2 cell studies, 3 had an efflux ratio of 3.1 but absorption 6319



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Scheme 3. Synthetic Approach to the Preparation of the Phosphonooxymethyl Prodrug 4 Derived from 3

Figure 4. Plasma exposure profiles of 3 administered as the parent of as prodrug 4 at escalating doses in the rat (a) and dog (b).

hepatocytes and thus was predicted to behave similarly in man to that observed in the in vivo studies in the preclinical species. A comparison of the AUC of 3 obtained in rats given either 3 or prodrug 4 is shown in Figure 4a. The AUC of 3 was similar after administration of either the parent 3 or prodrug 4 at lower dosages (≤25 mg/kg) because both can be formulated as a solution (PEG-400/EtOH/0.1 N NaOH for parent 3 and H2O for the prodrug 4), but at the higher dose of 200 mg/kg, the neutral parent could only be formulated as a suspension whereas the prodrug salt could be formulated as an aqueous solution and provided for superior plasma exposure of 3. Similar results were also observed in single-dose toxicokinetic and tolerability studies conducted in dogs, with data presented in Figure 4b. Prodrug 4 was administered either in dry-filled capsules or as an aqueous solution at daily doses of 25, 92, and 250 mg/kg, which were the molar equivalent to 20,

75, and 203 mg/kg of parent 3, respectively, or twice daily at 46 and 125 mg/kg, equivalent to a total daily dose of parent 3 of 75 and 203 mg/kg. Compound 3 was dosed either daily at doses of 15, 75, and 200 mg/kg or twice daily at 37.5 and 100 mg formulated as a solution in PEG400/EtOH/0.1 N NaOH. The higher single doses of 4 resulted in rapid emesis in some dogs which reduced exposure, so twice daily dosing (b.i.d.) of half doses was employed to avoid this problem. In these experiments, prodrug 4 was capable of delivering much higher systemic concentrations of 3 than dosing of the parent drug at higher doses. Prodrug 4 was administered at 5 equiv dosages of 100, 200, 400, 600, 800, and 1000 mg in a single ascending dose (SAD) study in a phase I clinical trial using a standard capsule formulation. The exposure of 3 increased more than proportionally with dose, as illustrated in Figure 5, and 6320



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Figure 5. Plasma exposure of 3 (BMS-626529) following administration of doses of 100, 200, 440, 600, 800, and 1000 mg of prodrug 4 to NHVs in a phase 1 clinical trial compared with the exposure of 3 following administration of the drug in a capsule formulation. Platform for LC operating in electrospray mode. The columns used were the following. Column A: YMC ODS-A S7 3.0 mm × 50 mm column. Column B: PHX-LUNA C18 4.6 mm × 30 mm column. Column C: Xterra 4.6 mm × 50 mm C18 5 μm column. Column D: XTERRA C18 S7 3.0 mm × 50 mm column. Column E: XTERRA C18 S5 4.6 mm × 50 mm column. Column F: Xterra MS C18 5 μm 4.6 mm × 30 mm column. Gradient was done from 100% solvent A to 100% solvent B. Solvent A is a mixture of 10% MeOH/90% H2O/ 0.1% CF3CO2H, while solvent B is a mixture of 10% H2O/90% MeOH/0.1% CF3CO2H. The flow rate was kept at 5 mL/min, and the gradient period was limited to 2 min, plus 1 min holding time. The detector wavelength was set at 220 nm. Preparation of 1-(4-Benzoylpiperazin-1-yl)-2-(4-methoxy-7(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (3). Step 1: Preparation of 3-Methyl-1,2,4triazole (ac-0). Solid formic hydrazide (68 g, 1.13 mol) was mixed with solid thioacetamide (85 g, 1.13 mol) in a 500 mL round-bottom flask and the mixture heated with stirring at 150 °C (oil bath temperature) for 1.5 h under a flow of N2 gas to remove the H2S and H2O formed from the reaction, with approximately 18 mL of liquid collected. Distillation of the reaction mixture under reduced pressure (102 °C/0.35−1 mmHg) provided 3-methyl-1,2,4-triazole (ac-0, 60.3 g, 63.3%) as a white solid after removing a liquid forerun. 1H NMR (500 MHz, CDCl3) δ ppm 9.5 (br, 1H), 8.03 (s, 1H), 2.51 (s, 3H). Step 2: Preparation of 4-Methoxy-7-(3-methyl-1H-1,2,4-triazol1-yl)-1H-pyrrolo[2,3-c]pyridine (13a). 4-Methoxy-7-chloro-6-azaindole (7, 9.1 g, 50 mmol), K2CO3 (13.8 g, 100 mmol), Cu powder (6.35 g, 100 mmol), and 3-methyl-1,2,4-triazole (83 g, 1.0 mol) were mixed in a 500 mL round-bottom flask. The flask was placed in an oil bath maintained at a temperature of 170−175 °C, and the melt heated under an atmosphere of N2 for 12 h. HPLC analysis indicated that the starting material, the desired product, and the isomeric byproduct were present in a ratio of ∼30% vs ∼45% vs ∼15%. After cooling the mixture to room temperature, MeOH (150 mL) was added and the insoluble material was removed by filtration through a Celite pad and washed with CH3OH. The filtrate was concentrated under vacuum to leave a residue that was partitioned between H2O (1 L) and EtOAc (150 mL). The aqueous phase was separated, extracted with EtOAc (2 × 150 mL) and the combined organic layer was dried over MgSO4, filtered and concentrated under vacuum to leave a residue (∼8 g) which was crystallized from a mixture of CH3CN and H2O (50 mL/ 100 mL) to furnish 1.45 g (12.7%) of 13a as white solid. The mother liquor was concentrated to leave a residue that was purified by C-18 reversed phase silica gel chromatography (YMC ODS-A 75 μm) using 15−30% CH3CN/H2O as the eluent to provide an additional 1.15 g of 13a. In addition, the aqueous layer was extracted multiple times with EtOAc and the combined extract dried over MgSO4, filtered, concentrated, and recrystallized from MeOH to produce an additional crop of 13a (200 mg). The total yield was 2.8 g (24%). 1H NMR (500 MHz, CDCl3) δ ppm 9.15 (s, 1H), 7.56 (s, 1H), 7.40 (s, 1H), 6.73 (s,

demonstrated the effectiveness of the prodrug at overcoming solubility- and/or dissolution-limited absorption at higher drug doses. However, prodrug 4 provided for a profile of rapid release and absorption of 3 that revealed a relatively short halflife of the parent molecule that had been masked by the phenomenon of flip-flop pharmacokinetics due to a prolonged absorption of 3 when the compound was dosed as a suspension.17,45,46 This observation necessitated the development of an extended-release formulation designed to minimize the peak to trough ratio drug in plasma and to achieve a twice daily dosing regimen.17,47 This provided a formulation suitable for phase 3 clinical trials of 4 which have been conducted in combination with other antiretroviral agents in heavily pretreated patients, and the conclusions were that the data supported continued development.48

■

CONCLUSION The SAR and properties of a series of 4-methoxy-6-azaindole HIV-1 attachment inhibitors with substitution at C-7 of the core heterocycle have been described. Incorporation of Nlinked heteroaryl substituents at C-7 of the azaindole heterocycle that reflected patterns of substitution designed to maximize coplanarity led to compounds with significantly improved potency, PK, and overall preclinical profiles. Compound 3 currently represents the most optimal molecule from this class of HIV-1 AIs and is being developed as the prodrug 4 in order to surmount drug delivery issues arising from dissolution- and/or solubility-limited absorption of the parent drug. Recently reported cocrystal structures of 3 with the target HIV-1 gp120 protein confirm the coplanarity model that was used to discover these agents and also provide an explanation for the SAR developed during our studies of the HIV-1 AI chemotype.11 Clinical studies with 4 as an extended release formulation in combination with other HIV-1 ARV agents are in progress, and the compound offers potential for use in salvage therapy for HIV-1 infected patients who have exhausted many of the other known drug classes of marketed antiretroviral agents.

■

EXPERIMENTAL SECTION

General Information. The purities of all compounds were were ≥95% and determined by analytical LC/MS methods. LC data were collected on a Shimadzu LC-10AS liquid chromotograph with a SPD10AV UV−vis detector. MS data were measured by a Micromass 6321



Article

1H), 4.05 (s, 3H) 2.54 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ ppm 161.8, 149.5, 141.2, 129.5, 127.5, 127.2, 123.5, 116.9, 100.5, 56.3, 14.2. Anal. Calcd for C11H11N5O: C 57.63, H 4.83, N 30.55. Found: C 57.37, H 4.64, N 30.68. MS m/z: 230 (MH+). HRMS (ESI) m/z: (M + H)+ calcd for C11H12N5O, 230.1042; found 230.1038. Step 3: Preparation of Methyl 2-(4-Methoxy-7-(3-methyl-1H1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoacetate (14a). AlCl3 (40 g, 0.3 mol) was dissolved in a mixture of CH2Cl2 and CH3NO2 (100 mL/20 mL) maintained under an atmosphere of N2, and 13a (4.58 g, 0.02 mol) and methyl chlorooxoacetate (9.8 g, 0.08 mol) were added. The mixture was stirred at room temperature for 1.5 h before being added dropwise to a cold, stirred solution of 20% aqueous NH4OAc solution (750 mL). The mixture was stirred for 20 min to afford a precipitate which was filtered, washed with H2O, and dried under vacuum to afford 14a (4.7 g, 75%) as a white solid. 1H NMR (500 MHz, CDCl3) δ ppm 11.0 (br, 1H), 9.15 (s, 1H), 8.34 (d, 1H, J = 3 Hz), 7.76 (s, 1H), 4.05 (s, 3H), 3.96 (s, 3H), 2.58 (s, 3H). MS m/z: 316 (MH+). HRMS (ESI) m/z: (M + H)+ calcd for C14H14N5O4, 316.1046; found 316.1041. Step 4: Preparation of 2-(4-Methoxy-7-(3-methyl-1H-1,2,4triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoacetic Acid (15a). Aqueous NaOH solution (0.25 M, 272 mL, 68 mmol) was added to a suspension of 14a (10.7 g, 34 mmol) in CH3OH (150 mL) at room temperature and the mixture stirred for 20 min. The CH3OH was removed under vacuum and the residual aqueous solution washed with EtOAc. 1 N HCl solution (68 mL, 68 mmol) was added to the aqueous phase to adjust the pH to 7. The resulting mixture was frozen and lyophilized to leave the Na salt of 15a (14.1 g, 99%) as an offwhite solid which was used without further purification. A sample of the Na salt of 15a was purified by C-18 reversed phase column chromatography after treatment with NaHCO3: HPLC >97% (AP, UV at 254 nm). 1H NMR (Na salt, 500 MHz, DMSO-d6) δ ppm 9.32 (s, 1H), 8.03 (s, 1H), 7.56 (s, 1H), 3.83 (s, 3H), 2.37 (s, 3H); 13C NMR (Na salt, 125 MHz, DMSO-d6) δ ppm 191.3, 171.7, 160.0, 149.8, 143.5, 125.1, 120.0, 114.8, 57.2, 13.8. MS m/z: 302 (MH+). HRMS (Na salt, ESI−) m/z: (M − H)+ calcd for C13H10N5O4, 300.0733; found 300.0724. Step 5: Preparation of 1-(4-Benzoylpiperazin-1-yl)-2-(4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin3-yl)ethane-1,2-dione (3). Et3N (10.1 g, 100 mmol) and EDC (5.75 g, 30 mmol) were added to a solution of 15a (3.01 g, 10 mmol) and benzoylpiperazine hydrochloride (3.39 g, 15 mmol) in DMF (50 mL) maintained under an atmosphere of N2. The mixture was stirred at room temperature for 22 h after sonication, then at 40 °C for 2 h. The DMF and Et3N were removed under vacuum to leave a residual solution which was diluted with H2O (200 mL) under stirring and sonication. The formed precipitates were collected, washed with H2O, and dried under vacuum to afford 3 (2.8 g, 59%) as an off-white solid. The filtrate was extracted with CH2Cl2 and the combined organic layer was dried over Na2SO4, filtered, and concentrated under vacuum to leave a residue that was triturated with Et2O to precipitate a solid which was further suspended and triturated with MeOH to afford an additional 400 mg of 3 as an off-white solid. The combined yield of 3 was 3.2 g (68%). 1H NMR (500 MHz, DMSO-d6) δ ppm 12.40 (s, 1H), 9.25 (s, 1H), 8.25 (s, 1H), 7.88 (s, 1H), 7.46 (br, 5H), 3.99 (s, 3H), 3.68 (br, 4H), 3.43 (br, 4H), 2.50 (s, 3H); 13C NMR (125 Hz, DMSO-d6) δ ppm 185.42, 169.22, 166.17, 161.29, 149.15, 142.10, 138.52, 135.43, 129.6, 128.34, 126.98, 123.60, 122.71, 120.95, 114.11, 56.78, 45.11, 40.58, 13.78. UV (MeOH) λmax 233.6 nm (ε 3.43 × 104), 314.9 nm (ε 1.73 × 104). Anal. Calcd for C24H24N7O4·0.2H2O: C 60.42, H 4.94, N 20.55. Found: C 60.42, H 5.03, N 20.65; KF (H2O) 0.75%. MS m/z: 474 (MH+). HRMS (ESI) m/z: (M + H)+ calcd for C24H24N7O4, 474.1890; found 474.1884. Preparation of 1-(4-Benzoylpiperazin-1-yl)-2-(4-methoxy-7(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (3), the Alternate Route. Compound 11a (150 mg, 0.35 mmol), 3-methyl-1,2,4-triazole (ac-0, 581 mg, 7 mmol), Cu powder (45 mg, 0.7 mmol), K2CO3 (97 mg, 0.7 mmol) were loaded into a sealed tube which was flushed several times with N2 before being sealed. The tube was heated at 160 °C for 11 h, the mixture

cooled, and CH3OH added, and the insoluble material was removed by filtration. The filtrate was concentrated under vacuum to leave a residue which was purified by C-18 reversed phase column chromatography eluting with MeOH−H2O (containing 0.1% CF3CO2H) to afford 3 as a CF3CO2H salt (19 mg, 11%). Preparation of 1,3-Dihydroxy-2-(hydroxymethyl)propan-2aminium (3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)methyl Hydrogen Phosphate (4). Step 1: Preparation of Di-tert-butyl Chloromethylphosphate. Tetrabutylammonium di-tert-butyl phosphate (57 g, 0.126 mol) and chloroiodomethane (221 g, 1.26 mol) were mixed in a 2 L round-bottom flask. The mixture was stirred at room temperature for 4 h, the volatiles were removed under vacuum, and Et2O (500 mL) was added to the residue. The insoluble material was removed by filtration and the filtrate concentrated under vacuum to afford 112 g of di-tert-butyl chloromethylphosphate as a light brown or yellow oil which was used without further purification. Step 2: Preparation of (3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3c]pyridin-1-yl)methyl Di-tert-butyl Phosphate (89). A mixture of 3 (200.00 g, 422.39 mmol), Cs2CO3 (344.06 g, 1.06 mol), KI (140.24 g, 844.81 mmol), and NMP (1.00 L) in a four-neck, 10 L reactor equipped with an overhead stirrer, a thermocouple, a distillation apparatus, and a N2 inlet was stirred at room temperature to provide a light brown suspension. Di-tert-butyl chloromethylphosphate (273.16 g, 1.06 mol) was added dropwise and the temperature of the mixture maintained at 30 °C for 16−24 h, before being cooled to 5 °C prior to the addition of CH2Cl2 (1.5 L). H2O (3.5 L) was added slowly with caution, maintaining the temperature of the mixture below 20 °C. The lower layer of the resulting biphasic mixture was collected, washed with H2O (3 × 3.5 L), and concentrated under vacuum to a volume of approximately 1 L while maintaining the temperature below 25 °C. Isopropanol (2 L) was added and the solution concentrated under vacuum to a volume of 2 L while maintaining the temperature below 25 °C. After 0.2 g of 89 was added as a seed, the solution was stirred for 16−24 h at room temperature to produce a suspension that was collected by filtration, washed with MTBE (1 L), and dried under vacuum at 50 °C for 16−24 h to afford 89 as a light yellow solid (207.1 g, 70%). 1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 8.18 (s, 1H), 7.91 (s, 1H), 7.42 (s, 5H), 5.95 (d, 2H, J = 14.2 Hz), 4.06 (s, 3H), 3.97−3.36 (m, 8H), 2.50 (s, 3H), 1.27 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 184.64, 170.65, 165.91, 161.60, 150.82, 145.38, 141.89, 134.96, 130.20, 129.59, 128.68, 127.58, 127.10, 124.77, 122.64, 115.22, 83.90, 83.83, 73.69, 73.63, 56.95, 46.04, 41.66, 29.61, 29.56, 13.90. MS m/z: 696 (MH+). Step 3: Preparation of 1,3-Dihydroxy-2-(hydroxymethyl)propan2-aminium (3-(2-(4-Benzoylpiperazin-1-yl)-2-oxoacetyl)-4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin1-yl)methyl Hydrogen Phosphate (4). A mixture of 89 (200.24 g, 287.82 mmol), acetone (800 mL), and H2O (800 mL) was charged in a four-neck 10 L reactor equipped with an overhead stirrer, a thermocouple, a condenser, and a N2 inlet. The solution was stirred at 40 °C for 18−24 h, cooled to 20 °C, tromethamine (33.62 g, 277.54 mmol) added, and the mixture stirred at 40 °C for 1 h until all of the solids had dissolved. The mixture was cooled to 20 °C, filtered through a 10 μm Cuno filter into a four-neck, 10 L reactor equipped with an overhead stirrer, a thermocouple, and a N2 inlet. Acetone (3 L) was added along with 0.5 g of 4 as a seed followed by an additional 3 L of acetone. The mixture was stirred at room temperature for 16− 24 h to provide a suspension that was collected by filtration, washed with acetone (800 mL), and dried under vacuum at 50 °C for 16−24 h to afford 4 a white powder (165.91 g, 82%), mp 203 °C. 1H NMR (500 MHz, CD3OD) δ 8.83 (s, 1H), 8.52 (s, 1H), 8.02 (s, 1H) 7.49 (br, 5H), 5.47 (d, 2H, J = 13 Hz), 4.11 (s, 3H), 4.00−3.40 (m, 8H), 3.66 (s, 6H), 2.50 (s, 3H); 13C NMR (125 MHz, CD3OD) δ 185.6, 171.9, 167.4, 161.4, 151.7, 146.9, 143.8, 135.4, 130.3, 129.7, 128.8, 127.2, 124.9, 122.6, 114.3, 73.5, 61.8, 59.9, 56,5, 46.0, 41.7, 12.6. HRMS (ESI) m/z: (M-trisamine + H)+ calcd for C25H27N7O8P, 584.1659, found 584.1664. Anal. Calcd for C29H37N8O11P: C 49.43, H 5.29, N 15.90, P 4.39. Found: C 49.18, H 5.38, N 15.59, P 4.26. 6322



Article

Antiviral Evaluation. Cells. The human embryonic kidney cell line 293T (HEK 293T, NIH AIDS Research and Reference Reagent Program) was propagated in DMEM (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated FBS (Sigma), 100 U/mL of penicillin, 100 μg/mL of streptomycin and subcultured twice a week. The HeLa cell line expressing CD4, CCR5, and CXCR4 (HeLa 67 cells) was propagated in DMEM containing 10% heat inactivated FBS with 400 μg/mL of zeocin, 100 μg/mL of hygromycin, and 200 μg/mL of geneticin (G418) added. The human T-cell leukemia cell line MT2 (NIH AIDS Research and Reference Reagent Program) was propagated in RPMI 1640 (Invitrogen) containing 10% FBS (Hyclone). Pseudotype Reporter Virus. Pseudotyped virus was generated by transfecting 293T cells with an envelope-deficient, proviral clone of HIV-1 (LAI-Δenv-luc, with the firefly luciferase in place of the envelope gene) along with a plasmid expressing the envelope of either JFRL (CCR5-tropic) virus or LAI (CXCR4-tropic) virus. Lipofectamine Plus reagent (Invitrogen) was used according to directions provided by the manufacturer. After a 3 day incubation, supernatant containing the pseudotyped viruses (JRFL-luc or LAI-luc) was harvested, aliquoted, and frozen at −80 °C. Viral titers were normalized based upon the luminescence signal in the absence of added compounds. Test Sample Preparation. Test compounds were initially dissolved in DMSO to create 30 mM stock solutions. 10-step sequential 3-fold dilutions were subsequently carried out in DMSO. Drug Anti-HIV Activity Assay and Data Analysis. A singlecycle viral infection system was utilized to determine drug susceptibility.49 Pseudotyped JRFL-luc or LAI-luc viruses were used to infect HeLa 67 cells in the presence of test compounds at a range of concentrations. Three days after infection, virus replication was monitored by measuring luciferase expression in the infected cells using a luciferase reporter gene assay kit (Steady-Glo), followed by quantification by measuring luminescence using an EnVision multilabel plate readers (PerkinElmer). The EC50 was calculated with the Microsoft Excel Xlfit curve fitting software, where percent inhibition = 1/[1 + (EC50/drug concn)m] and m is a parameter that reflects the slope of the concentration−response curve. Cytotoxicity Assay and Data Analysis. CC50 values (concentrations of drug required to reduce cell viability by 50%) were determined after a 3 day incubation in MT2 cells in the presence of serially diluted compounds. Cell viability was determined using a redox dye assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS, Promega) according to the manufacturer’s protocol.50 The 50% cytotoxic concentrations (CC50) were calculated by using the exponential form of the median effect equation, where percent inhibition = 1/[1 + (CC50/drug concn)m], where m is a parameter that reflects the slope of the concentration−response curve. Animal Studies. For iv and po PK studies in rats, test compounds were as a solution formulated in PEG-400/ethanol (90/10). For the iv and po PK studies in dogs and monkeys, test compounds were dissolved in PEG-400/ethanol (90/10) with pH adjustment using 0.1 N NaOH. Rat. Male Sprague-Dawley rats (300−350 g, Hilltop Lab Animals, Inc., Scottdale, PA) with cannulas implanted in the jugular vein and/ or bile duct were used for PK studies. For po PK evaluation, rats were fasted overnight prior to the studies. Blood samples of 0.3 mL were collected from the jugular vein in EDTA-containing microtainer tubes (Becton Dickinson, Franklin Lakes, NJ) and centrifuged to separate plasma. In the iv study, test compound was delivered at 1 mg/kg as a bolus over 0.5 min (n = 3). Serial blood samples were collected before dosing and at 2, 10, 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing. In the po study, rats (n = 3) received an oral dose of 5 mg/kg of compound. Serial blood samples were collected before dosing and at 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing. Dog. The iv and po studies of test compounds were conducted in a crossover fashion in three male beagle dogs (12 ± 0.4 kg, Marshall Farms USA Inc., North Rose, NY). There was a one-week washout

period between the iv and po studies. In the iv study, test compound was infused via the cephalic vein at 1 mg/kg over 5 min at a constant rate of 0.2 mL kg−1 min−1. Serial blood samples were collected from the femoral vein before dosing and at 5, 10, 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing. In the po study, the dogs were fasted overnight before dosing compound which was administered by oral gavage at 5 mg/kg. Serial blood samples were collected before dosing and at 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing. Monkey. The iv and po studies of test compounds were conducted in a crossover fashion in three male cynomolgus monkeys (10 ± 0.8 kg, Charles River Biomedical Research Foundation, Houston, TX). There was a one-week washout period between the iv and po studies. In the iv study, test compound was infused via the femoral vein at 1 mg/kg over 5 min at a constant rate of 0.2 mL kg−1 min−1. Serial blood samples were collected from the femoral artery before dosing and at 5, 10, 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing. In the po study, the monkeys were fasted overnight before dosing. Test compound was administered by oral gavage at 5 mg/kg, and serial blood samples were collected before dosing and at 15, 30, 45, 60, 120, 240, 360, 480, and 1440 min after dosing.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00759.

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Experimental details for synthetic procedures and associated analytical data (e.g., MS and NMR) for compounds 6−9, 11a−c, 11e, 13c−e, 14c−e, 15c−e, 16−47, 49−66, 68−73, 77−79, 81, 83−88 and intermediates ac-1 to ac-9 and a list of molecular formula strings (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*Phone: (203) 677-6584. E-mail: [email protected]. ORCID

Tao Wang: 0000-0002-5866-8148 Nicholas A. Meanwell: 0000-0002-8857-1515 Present Addresses ○

R.J.C.: Assembly Biosciences, 409 Illinois Street, San Francisco, CA 94158. ◆ J.F.K.: ViiV Healthcare, 36 East Industrial Road, Branford, CT 06405. Notes

The authors declare no competing financial interest. # Deceased, June 27, 2005. All studies were conducted in accordance with the BristolMyers Squibb Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee at Bristol-Myers Squibb or by the ethical review process at the institution where the work was performed.

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ACKNOWLEDGMENTS The authors express gratitude to Dr. Xiaohua Stella Huang and Elizabeth Bitel for their assistance in obtaining NMR spectra and HRMS and CHN data, respectively. The authors are grateful to the preclinical candidate optimization organization (PCO) at Bristol-Myers Squibb for generating in vitro and in 6323



Article

Antonio, C. A. T.; Faraon, E. J. A.; Anwari, P.; Arnlov, J.; Larsson, A.; Arsenijevic, V. S A.; Artaman, A.; Asghar, R. J.; Assadi, R.; Atkins, L. S.; Badawi, A.; Balakrishnan, K.; Banerjee, A.; Basu, S.; Beardsley, J.; Bekele, T.; Bell, M. L.; Huang, J. J.; Bernabe, E.; Beyene, T. J.; Deribe, K.; Bhala, N.; Bhalla, A.; Jha, V.; Bhutta, Z. A.; Abdulhak, A. B.; Binagwaho, A.; Kankindi, I.; Blore, J. D.; Balaji, A. L.; Lopez, A. D.; Basara, B. B.; Yentur, G. K.; Bose, D.; Brainin, M.; Breitborde, N.; Castaneda-Orjuela, C. A.; Catala-Lopez, F.; Chadha, V. K.; Chang, J.C.; Chiang, P. P.-C.; Chuang, T.-W.; Colomar, M.; Cooper, L. T.; Cooper, C.; Courville, K. J.; Cowie, B. C.; Maclachlan, J. H.; Criqui, M. H.; Dandona, R.; Kumar, G. A.; Murthy, K. S.; Dayama, A.; De Leo, D.; Degenhardt, L.; Del Pozo-Cruz, B.; Des Jarlais, D. C.; Dessalegn, M.; Dharmaratne, S. D.; Dilmen, U.; Uzun, S. B.; Ding, E. L.; Driscoll, T. R.; Durrani, A. M.; Ellenbogen, R. G.; Ermakov, S. P.; Esteghamati, A.; Hafezi-Nejad, N.; Sheikhbahaei, S.; Farzadfar, F.; Fereshtehnejad, S.-M.; Fijabi, D. O.; Gaffikin, L.; Gamkrelidze, A.; Kereselidze, M.; Khonelidze, I.; Gankpe, F. G.; Geleijnse, J. M.; Gessner, B. D.; Gibney, K. B.; Ginawi, I. A. M.; Glaser, E. L.; Gona, P.; Goto, A.; Gouda, H. N.; Knibbs, L.; Gugnani, H. C.; Gupta, R.; Gupta, R.l.; Hamadeh, R. R.; Hammami, M.; Hankey, G. J.; Haro, J. M.; Havmoeller, R.; Hedayati, M. T.; Pi, I. B. H.; Mejia-Rodriguez, F.; Quezada, A. D.; Servan-Mori, E. E.; Hoek, H. W.; Hornberger, J. C.; Hosgood, H. D.; Hotez, P. J.; Hoy, D. G.; Iburg, K. M.; Idrisov, B. T.; Innos, K.; Leinsalu, M.; Jacobsen, K. H.; Jeemon, P.; Jiang, G.; Jonas, J. B.; Juel, K.; Kan, H.; Karam, N. E.; Karch, A.; Karema, C. K.; Kaul, A.; Kawakami, N.; Kazi, D. S.; Kemp, A. H.; Lotufo, P. A.; Kengne, A. P.; Keren, A.; Khader, Y. S.; Khalifa, S. E. A. H.; Khan, E. A.; Khang, Y.-H.; Kinfu, Y.; Kinge, J. M.; Skirbekk, V.; Kokubo, Y.; Kosen, S.; Defo, B. K.; Kulkarni, V. S.; Kulkarni, C.; Kumar, K.; Kumar, R. B.; Kwan, G. F.; Lai, T.; Lam, H.; Lan, Q.; Lansingh, V. C.; Larson, H. J.; Pearce, N.; Lee, J.-T.; Yoon, S.-J.; Leigh, J.; Leung, R.; Li, Y.; Li, Y.; De Lima, G. M. F.; Lin, H.-H.; Lipshultz, S. E.; Liu, S.; Wang, L.; Zhou, M.; Liu, Y.; Narayan, K. M. V.; Omer, S. B.; Lloyd, B. K.; Machado, V. M. P.; Magis-Rodriguez, C.; Majdan, M.; Mapoma, C. C.; Marcenes, W.; Marzan, M. B.; Masci, J. R.; Mashal, M. T.; MasonJones, A. J.; Mayosi, B. M.; Mazorodze, T. T.; Mckay, A. C.; Meaney, P. A.; Mehndiratta, M. M.; Melaku, Y. A.; Memish, Z. A.; Saeedi, M. Y.; Mendoza, W.; Miller, T. R.; Mills, E. J.; Mohammad, K. A.; Mola, G. L.; Monasta, L.; Montico, M.; Ronfani, L.; Moore, A. R.; Mori, R.; Moturi, W. N.; Mukaigawara, M.; Naheed, A.; Naidoo, K. S.; Naldi, L.; Nangia, V.; Nash, D.; Nejjari, C.; Nelson, R. G.; Neupane, S. P.; Newton, C. R.; Nisar, M. I.; Norheim, O. F.; Nowaseb, V.; Oh, I.-H.; Ohkubo, T.; Olusanya, B. O.; Opio, J. N.; Orisakwe, O. E.; Pandian, J. D.; Patten, S. B.; Paul, V. K.; Pavlin, B. I.; Pereira, D. M.; Pervaiz, A.; Pesudovs, K.; Petzold, M.; Pourmalek, F.; Qato, D.; Rafay, A.; Rahimi-Movaghar, V.; Ur Rahman, S.; Raju, M.; Rana, S. M.; Razavi, H.; Reilly, R. Q.; Remuzzi, G.; Richardus, J. H.; Roy, N.; Sabin, N.; Sahraian, M. A.; Samonte, G. M. J.; Sawhney, M.; Schneider, I. J. C.; Schwebel, D. C.; Singh, J. A.; Seedat, S.; Sepanlou, S. G.; Shibuya, K.; Shin, H. H.; Weichenthal, S.; Shiue, I.; Shivakoti, R.; Sigfusdottir, I. D.; Silberberg, D. H.; Silva, A. P.; Simard, E. P.; Sliwa, K.; Soneji, S.; Soshnikov, S. S.; Sreeramareddy, C. T.; Stathopoulou, V. K.; Stroumpoulis, K.; Swaminathan, S.; Sykes, B. L.; Tabb, K. M.; Talongwa, R. T.; Tenkorang, E. Y.; Terkawi, A. S.n.; Thomson, A. J.; Thorne-Lyman, A. L.; Towbin, J. A.; Traebert, J.; Tran, B. X.; Dimbuene, Z. T.; Tsilimbaris, M.; Uchendu, U. S.; Ukwaja, K. N.; Vallely, A. J.; Vasankari, T. J.; Venketasubramanian, N.; Violante, F. S.; Vlassov, V. V.; Vollset, S. E.; Waller, S.; Wallin, M. T.; Wang, X. R.; Wang, Y.; Zhu, J.; Weiderpass, E.; Weintraub, R. G.; Westerman, R.; White, R. A.; Wilkinson, J. D.; Woldeyohannes, S. M.; Wong, J. Q.; Xu, G.; Yang, Y. C.; Yano, Y.; Yip, P.; Yonemoto, N.; Younis, M.; Yu, C.; Jin, K. Y.; El Sayed Zaki, M.; Zhao, Y.; Zheng, Y.-F.; Zou, X. N. Global, regional, and national incidence and mortality for HIV, tuberculosis, and malaria during 1990−2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2014, 384, 1005− 1070. (4) UNAIDS global HIV statistics fact sheet, July 2017. http://www. unaids.org/sites/default/files/media_asset/UNAIDS_FactSheet_en. pdf (accessed February 16, 2018).

vivo profiling data. The authors also thank Dr. Mark Krystal for helping in preparing and reviewing the manuscript.

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DEDICATION Dedicated to the memory of Professor Gilbert Stork, an inspiring philosopher and mentor. ∇

ABBREVIATIONS USED AIDS, acquired immunodeficiency syndrome; AlCl3, aluminum chloride; ART, antiretroviral therapy; AUC, area under the curve; Bz, benzoyl; Caco-2 cells, human colorectal adenocarcinoma cells; CC50, 50% cytotoxic concentration; CCR5, chemokine (C-C motif) receptor 5; CD4, cluster of differentiation 4; CXCR4, chemokine (C-X-C motif) receptor 4; CYP 450, cytochrome P450; DEBPT, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one; DMEM, Dulbecco’s modified Eagle medium; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EC50, 50% effective concentration; EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; FBS, fetal bovine serum; Luc, luciferase; gp120, glycoprotein 120; HAART, highly active antiretroviral therapy; hERG, human ether-à-go-go related gene product; HIV-1, human immunodeficiency virus type 1; HLM, human liver microsome; HPLC, high pressure liquid chromatography; HRMS, high resolution mass spectrometry; IPA, isopropyl alcohol; LC, liquid chromatography; LTR, long terminal repeat; mp, melting point; MS, mass spectrometry; MTBE, methyl tert-butyl ether; NMP, N-methyl-2-pyrrolidone; PBMC, peripheral blood mononuclear cell; PEG, poly(ethylene glycol); PK, pharmacokinetic; RLM, rat liver microsome; SAR, structure−activity relationship; XTT, 2,3bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt

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