John F. Kadow

Envelope Conformational Changes Induced by Human Immunodeficiency Virus Type 1 Attachment Inhibitors Prevent CD4 Binding and Downstream Entry Events

ABSTRACT 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.

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.

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

ABSTRACT 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 (EC50) values of <10 nM against the vast majority of viral isolates; however, susceptibility varied by >6 log10, 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.

Diastereoselective addition of Grignard reagents to azetidine-2,3-dione: Synthesis of novel Taxol® analogues

Abstract Synthesis and cytotoxicity properties of novel C-2′ analogues of paclitaxel are described. The analogues were synthesized using Holtons β-lactam approach to append the side chain on baccatin III. The key intermediate to the synthesis of novel analogues was prepared employing an unprecedented stereocontrolled addition of Grignard reagent to a chiral azetidine-2,3-dione.

Structure-activity relationships of taxol®: synthesis and biological evaluation of C2 taxol analogs

Abstract Red-Al was used to selectively remove the C 2 benzoate of 7,13-bisTES baccatin ( 8 ). Derivatization of the resulting C 2 hydroxy compound provided the corresponding p-nitrophenyl carbamate, the p-methoxy and p-nitrobenzoates, and cyclohexyl ester. Attachment of the taxol side chain to the above four analogs via lactam ( 15 ) followed by desilylation provided five analogs of taxol modified at the C 2 position, including a C 1,2 cyclic carbonate. These five analogs were less potent than taxol in a tubulin polymerization assay and a cytotoxicity assay against human colon cancer cell line.

Discovery and Preclinical Characterization of the Cyclopropylindolobenzazepine BMS-791325, A Potent Allosteric Inhibitor of the Hepatitis C Virus NS5B Polymerase.

Described herein are structure-activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (2) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation.

Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase

ABSTRACT BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC50, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC50s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC50. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC50s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.

A chemoselective approach to functionalize the C-10 position of 10-deacetylbaccatin III. Synthesis and biological properties of novel C-10 Taxol® analogues

Abstract A chemoselective approach to functionalize the C-10 position of 10-deacetyl baccatin III, a key intermediate for the semi-synthesis of paclitaxel, is described. The chemistry provides an easy access to a variety of C-10 hydroxyl derivatives, such as, ethers, esters, carbonates, carbamates, and sulfonates under mild conditions. The C-10 modified baccatin derivatives were further employed in the synthesis of novel biologically active Taxol® analogues.

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 6. Preclinical and Human Pharmacokinetic Profiling of BMS-663749, a Phosphonooxymethyl Prodrug of the HIV-1 Attachment Inhibitor 2-(4-Benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanone (BMS-488043)

BMS-663749, a phosphonooxymethyl prodrug 4 of the HIV-1 attachment inhibitor 2-(4-benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanone (BMS-488043) (2) was prepared and profiled in a variety of preclinical in vitro and in vivo models designed to assess its ability to deliver parent drug following oral administration. The data showed that prodrug 4 had excellent potential to significantly reduce dissolution rate-limited absorption following oral dosing in humans. Clinical studies in normal healthy subjects confirmed the potential of 4, revealing that the prodrug significantly increased both the AUC and C(max) of 2 compared to a solid capsule formulation containing the parent drug upon dose escalation. These data provided guidance for further efforts to obtain an effective HIV-1 attachment inhibitor.