A survey of core replacements in indole-based HIV-1 attachment inhibitors.

Indole- and azaindole-based glyoxylyl amide derivatives have been described as HIV-1 attachment inhibitors (AIs) that act by blocking the interaction between the viral gp120 coat protein and the human host cell CD4 receptor. As part of an effort to more deeply understand the role of the indole/azaindole heterocycle in the expression of antiviral activity, a survey of potential replacements was conducted using parallel synthesis methodology. The design and optimization was guided by a simple 2-dimensional overlay based on an overall planar topography between the indole/azaindole and C-7 substituents that had been deduced from structure-activity studies leading to the discovery of temsavir (3). 2-Substituted naphthalene- and quinoline-derived chemotypes emerged as the most interesting prototypes, with C-5 and C-6 substituents enhancing antiviral potency. Despite the fact that neither of these chemotypes incorporated a H-bond donor that has been shown to engage the side chain carboxylate of Asp113 in gp120, the antiviral potency of several analogues met or exceeded that of 3, demonstrating that engaging Asp113 is not a prerequisite for potent antiviral activity.

Inhibitors of HIV-1 attachment. Part 10. The discovery and structure-activity relationships of 4-azaindole cores.

A series of 4-azaindole oxoacetic acid piperazine benzamides was synthesized and evaluated in an effort to identify an oral HIV-1 attachment inhibitor with the potential to improve upon the pre-clinical profile of BMS-378806 (7), an initial clinical compound. Modifications at the 7-position of the 4-azaindole core modulated potency significantly and SAR showed that certain compounds with a 5-membered ring heteroaryl group at that position were the most potent. Four of the compounds with the best profiles were evaluated in a rat pharmacokinetic model and all had superior oral bioavailability and lower clearance when compared with 7.

Inhibitors of HIV-1 attachment. Part 11: the discovery and structure-activity relationships associated with 4,6-diazaindole cores.

A series of HIV-1 attachment inhibitors containing a 4,6-diazaindole core were examined in an effort to identify a compound which improved upon the potency and oral exposure of BMS-488043 (2). BMS-488043 (2) is a 6-azaindole-based HIV-1 attachment inhibitor which established proof-of-concept for this mechanism in human clinical studies but required high doses and concomitant administration of a high fat meal to achieve efficacious exposures. Based on previous studies in indole and azaindole scaffolds, SAR investigation was concentrated around the key 7-position in the 4,6-diazaindole series and led to the discovery of molecules with 5- to 20-fold increases in potency and three- to seven-fold increases in exposure over 2 in a rat PK studies.

Inhibitors of HIV-1 attachment. Part 7: indole-7-carboxamides as potent and orally bioavailable antiviral agents.

A series of substituted carboxamides at the indole C7 position of the previously described 4-fluoro-substituted indole HIV-1 attachment inhibitor 1 was synthesized and the SAR delineated. Heteroaryl carboxamide inhibitors that exhibited pM potency in the primary cell-based assay against a pseudotype virus expressing a JRFL envelope were identified. The simple methyl amide analog 4 displayed a promising in vitro profile, with its favorable HLM stability and membrane permeability translating into favorable pharmacokinetic properties in preclinical species.

Inhibitors of HIV-1 attachment. Part 8: the effect of C7-heteroaryl substitution on the potency, and in vitro and in vivo profiles of indole-based inhibitors.

As part of the SAR profiling of the indole-oxoacetic piperazinyl benzamide class of HIV-1 attachment inhibitors, substitution at the C7 position of the lead 4-fluoroindole 2 with various 5- and 6-membered heteroaryl moieties was explored. Highly potent (picomolar) inhibitors of pseudotyped HIV-1 in a primary, cell-based assay were identified and select examples were shown to possess nanomolar inhibitory activity against M- and T-tropic viruses in cell culture. These C7-heteroaryl-indole analogs maintained the ligand efficiency (LE) of 2 and were also lipophilic efficient as measured by LLE and LELP. Pharmacokinetic studies of this class of inhibitor in rats showed that several possessed substantially improved IV clearance and half-lives compared to 2. Oral exposure in the rat correlated with membrane permeability as measured in a Caco-2 assay where the highly permeable 1,2,4-oxadiazole analog 13 exhibited the highest exposure.

In vitro antiviral characteristics of HIV-1 attachment inhibitor BMS-626529, the active component of the prodrug BMS-663068.

BMS-663068 is the phosphonooxymethyl prodrug of BMS-626529, a novel small-molecule attachment inhibitor that targets HIV-1 gp120 and prevents its binding to CD4(+) T cells. The activity of BMS-626529 is virus dependent, due to heterogeneity within gp120. In order to better understand the anti-HIV-1 spectrum of BMS-626529 against HIV-1, in vitro activities against a wide variety of laboratory strains and clinical isolates were determined. BMS-626529 had half-maximal effective concentration (EC(50)) values of <10 nM against the vast majority of viral isolates; however, susceptibility varied by >6 log(10), with half-maximal effective concentration values in the low pM range against the most susceptible viruses. The in vitro antiviral activity of BMS-626529 was generally not associated with either tropism or subtype, with few exceptions. Measurement of the binding affinity of BMS-626529 for purified gp120 suggests that a contributory factor to its inhibitory potency may be a relatively long dissociative half-life. Finally, in two-drug combination studies, BMS-626529 demonstrated additive or synergistic interactions with antiretroviral drugs of different mechanistic classes. These results suggest that BMS-626529 should be active against the majority of HIV-1 viruses and support the continued clinical development of the compound.

In vivo patterns of resistance to the HIV attachment inhibitor BMS-488043.

Attachment inhibitors (AI) are a novel class of HIV-1 antivirals, with little information available on clinical resistance. BMS-488043 is an orally bioavailable AI that binds to gp120 of HIV-1 and abrogates its binding to CD4(+) lymphocytes. A clinical proof-of-concept study of the AI BMS-488043, administered as monotherapy for 8 days, demonstrated significant viral load reductions. In order to examine the effects of AI monotherapy on HIV-1 sensitivity, phenotypic sensitivity assessment of baseline and postdosing (day 8) samples was performed. These analyses revealed that four subjects had emergent phenotypic resistance (a 50% effective concentration [EC(50)] >10-fold greater than the baseline value) and four had high baseline EC(50)s (>200 nM). Population sequencing and sequence determination of cloned envelope genes uncovered five gp120 mutations at four loci (V68A, L116I, S375I/N, and M426L) associated with BMS-488043 resistance. Substitution at the 375 locus, located near the CD4 binding pocket, was the most common (maintained in 5/8 subjects at day 8). The five substitutions were evaluated for their effects on AI sensitivity through reverse genetics in functional envelopes, confirming their role in decreasing sensitivity to the drug. Additional analyses revealed that these substitutions did not alter sensitivity to other HIV-1 entry inhibitors. Thus, our studies demonstrate that although the majority of the subjects' viruses maintained sensitivity to BMS-488043, substitutions can be selected that decrease HIV-1 susceptibility to the AI. Most importantly, the substitutions described here are not associated with resistance to other approved antiretrovirals, and therefore, attachment inhibitors could complement the current arsenal of anti-HIV agents.

Inhibitors of HIV-1 attachment. Part 4: A study of the effect of piperazine substitution patterns on antiviral potency in the context of indole-based derivatives.

4-Fluoro- and 4-methoxy-1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (2 and 3, respectively) have been characterized as potent inhibitors of HIV-1 attachment that interfere with the interaction of viral gp120 with the host cell receptor CD4. As part of an effort to understand fundamental aspects of this pharmacophore, discovered originally using a high throughput cell-based screen, modification and substitution of the piperazine ring was examined in the context of compounds 6a-ah. The piperazine ring was shown to be a critical element of the HIV-1 attachment inhibiting pharmacophore, acting as a scaffold to deploy the indole glyoxamide and benzamide in a topographical relationship that complements the binding site on gp120.

Inhibitors of HIV-1 attachment. Part 3: A preliminary survey of the effect of structural variation of the benzamide moiety on antiviral activity.

1-(4-Benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (1a) has been characterized as an inhibitor of HIV-1 attachment that interferes with the interaction of viral gp120 with the host cell receptor CD4. In previous studies, the effect of indole substitution pattern on antiviral activity was probed. In this Letter, the effect of structural variation of the benzamide moiety is described, a study that reveals the potential or the phenyl moiety to be replaced by five-membered heterocyclic rings and a restricted tolerance for the introduction of substituents to the phenyl ring.

Inhibitors of HIV-1 attachment. Part 2: An initial survey of indole substitution patterns.

The effects of introducing simple halogen, alkyl, and alkoxy substituents to the 4, 5, 6 and 7 positions of 1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione, an inhibitor of the interaction between HIV gp120 and host cell CD4 receptors, on activity in an HIV entry assay was examined. Small substituents at C-4 generally resulted in increased potency whilst substitution at C-7 was readily tolerated and uniformly produced more potent HIV entry inhibitors. Substituents deployed at C-6 and, particularly, C-5 generally produced a modest to marked weakening of potency compared to the prototype. Small alkyl substituents at N-1 exerted minimal effect on activity whilst increasing the size of the alkyl moiety led to progressively reduced inhibitory properties. These studies establish a basic understanding of the indole element of the HIV attachment inhibitor pharmacophore.

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

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.

Discovery of 4-benzoyl-1-[(4-methoxy-1H- pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2- (R)-methylpiperazine (BMS-378806): a novel HIV-1 attachment inhibitor that interferes with CD4-gp120 interactions.

Indole derivative 1 interferes with the interaction of the HIV surface protein gp120 with the host cell receptor CD4. The 4-fluoro derivative 2 exhibited markedly enhanced potency and was bioavailable in the rat, dog, and cynomolgus monkey when administered orally as a solution formulation. However, aqueous suspensions of 2 were poorly bioavailable, indicative of dissolution-limited absorption. The 7-azaindole derivative 3, BMS-378806, exhibited improved pharmaceutical properties while retaining the HIV-1 inhibitory profile of 2.

Inhibitors of human immunodeficiency virus type 1 (HIV-1) attachment. 12. Structure-activity relationships associated with 4-fluoro-6-azaindole derivatives leading to the identification of 1-(4-benzoylpiperazin-1-yl)-2-(4-fluoro-7-[1,2,3]triazol-1-yl-1h-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-585248).

A series of highly potent HIV-1 attachment inhibitors with 4-fluoro-6-azaindole core heterocycles that target the viral envelope protein gp120 has been prepared. Substitution in the 7-position of the azaindole core with amides (12a,b), C-linked heterocycles (12c-l), and N-linked heterocycles (12m-u) provided compounds with subnanomolar potency in a pseudotype infectivity assay and good pharmacokinetic profiles in vivo. A predictive model was developed from the initial SAR in which the potency of the analogues correlated with the ability of the substituent in the 7-position of the azaindole to adopt a coplanar conformation by either forming internal hydrogen bonds or avoiding repulsive substitution patterns. 1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-[1,2,3]triazol-1-yl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-585248, 12m) exhibited much improved in vitro potency and pharmacokinetic properties than the previous clinical candidate BMS-488043 (1). The predicted low clearance in humans, modest protein binding, and good potency in the presence of 40% human serum for 12m led to its selection for human clinical studies.

Inhibitors of human immunodeficiency virus type 1 (HIV-1) attachment. 5. An evolution from indole to azaindoles leading to the discovery of 1-(4-benzoylpiperazin-1-yl)-2-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-488043), a drug candidate that demonstrates antiviral activity in HIV-1-infected subjects.

Azaindole derivatives derived from the screening lead 1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (1) were prepared and characterized to assess their potential as inhibitors of HIV-1 attachment. Systematic replacement of each of the unfused carbon atoms in the phenyl ring of the indole moiety by a nitrogen atom provided four different azaindole derivatives that displayed a clear SAR for antiviral activity and all of which displayed marked improvements in pharmaceutical properties. Optimization of these azaindole leads resulted in the identification of two compounds that were advanced to clinical studies: (R)-1-(4-benzoyl-2-methylpiperazin-1-yl)-2-(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)ethane-1,2-dione (BMS-377806, 3) and 1-(4-benzoylpiperazin-1-yl)-2-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione (BMS-488043, 4). In a preliminary clinical study, 4 administered as monotherapy for 8 days, reduced viremia in HIV-1-infected subjects, providing proof of concept for this mechanistic class.

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding.

BMS-378806 is a recently discovered small molecule HIV-1 inhibitor that blocks viral entrance to cells. The compound exhibits potent inhibitory activity against a panel of R5-(virus using the CCR5 coreceptor), X4-(virus using the CXCR4 coreceptor), and R5/X4 HIV-1 laboratory and clinical isolates of the B subtype (median EC50 of 0.04 microM) in culture assays. BMS-378806 is selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses, and exhibits no significant cytotoxicity in the 14 cell types tested (concentration for 50% reduction of cell growth, >225 microM). Mechanism of action studies demonstrated that BMS-378806 binds to gp120 and inhibits the interactions of the HIV-1 envelope protein to cellular CD4 receptors. Further confirmation that BMS-378806 targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated in the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to compound inhibition. BMS-378806 displays many favorable pharmacological traits, such as low protein binding, minimal human serum effect on anti-HIV-1 potency, good oral bioavailability in animal species, and a clean safety profile in initial animal toxicology studies. Together, the data show that BMS-378806 is a representative of a new class of HIV inhibitors that has the potential to become a valued addition to our current armamentarium of antiretroviral drugs.