Heterocycle amide isosteres: An approach to overcoming resistance for HIV-1 integrase strand transfer inhibitors.

A series of heterocyclic pyrimidinedione-based HIV-1 integrase inhibitors was prepared and screened for activity against purified integrase enzyme and/or viruses modified with the following mutations within integrase: Q148R, Q148H/G140S and N155H. These are mutations that result in resistance to the first generation integrase inhibitors raltegravir and elvitegravir. Based on consideration of drug-target interactions, an approach was undertaken to replace the amide moiety of the first generation pyrimidinedione inhibitor with azole heterocycles that could retain potency against these key resistance mutations. An imidazole moiety was found to be the optimal amide substitute and the observed activity was rationalized with the use of calculated properties and modeling. Rat pharmacokinetic (PK) studies of the lead imidazole compounds demonstrated moderate clearance and moderate exposure.

Design, synthesis and SAR study of bridged tricyclic pyrimidinone carboxamides as HIV-1 integrase inhibitors.

The design, synthesis and structure-activity relationships associated with a series of bridged tricyclic pyrimidinone carboxamides as potent inhibitors of HIV-1 integrase strand transfer are described. Structural modifications to these molecules were made in order to examine the effect on potency towards wild-type and clinically-relevant resistant viruses. The [3.2.2]-bridged tricyclic system was identified as an advantageous chemotype, with representatives exhibiting excellent antiviral activity against both wild-type viruses and the G140S/Q148H resistant virus that arises in response to therapy with raltegravir and elvitegravir.

The discovery and preclinical evaluation of BMS-707035, a potent HIV-1 integrase strand transfer inhibitor.

BMS-707035 is an HIV-1 integrase strand transfer inhibitor (INSTI) discovered by systematic optimization of N-methylpyrimidinone carboxamides guided by structure-activity relationships (SARs) and the single crystal X-ray structure of compound 10. It was rationalized that the unexpectedly advantageous profiles of N-methylpyrimidinone carboxamides with a saturated C2-substitutent may be due, in part, to the geometric relationship between the C2-substituent and the pyrimidinone core. The single crystal X-ray structure of 10 provided support for this reasoning and guided the design of a spirocyclic series 12 which led to discovery of the morpholino-fused pyrimidinone series 13. Several carboxamides derived from this bicyclic scaffold displayed improved antiviral activity and pharmacokinetic profiles when compared with corresponding spirocyclic analogs. Based on the excellent antiviral activity, preclinical profiles and acceptable in vitro and in vivo toxicity profiles, 13a (BMS-707035) was selected for advancement into phase I clinical trials.

The design, synthesis and structure-activity relationships associated with C28 amine-based betulinic acid derivatives as inhibitors of HIV-1 maturation.

The design and synthesis of a series of C28 amine-based betulinic acid derivatives as HIV-1 maturation inhibitors is described. This series represents a continuation of efforts following on from previous studies of C-3 benzoic acid-substituted betulinic acid derivatives as HIV-1 maturation inhibitors (MIs) that were explored in the context of C-28 amide substituents. Compared to the C-28 amide series, the C-28 amine derivatives exhibited further improvements in HIV-1 inhibitory activity toward polymorphisms in the Gag polyprotein as well as improved activity in the presence of human serum. However, plasma exposure of basic amines following oral administration to rats was generally low, leading to a focus on moderating the basicity of the amine moiety distal from the triterpene core. The thiomorpholine dioxide (TMD) 20 emerged from this study as a compound with the optimal antiviral activity and an acceptable pharmacokinetic profile in the C-28 amine series. Compared to the C-28 amide 3, 20 offers a 2- to 4-fold improvement in potency towards the screening viruses, exhibits low shifts in the EC50 values toward the V370A and ΔV370 viruses in the presence of human serum or human serum albumin, and demonstrates improved potency towards the polymorphic T371A and V362I virus variants.

Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage.

BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat.

Inhibitors of HIV-1 maturation: Development of structure-activity relationship for C-28 amides based on C-3 benzoic acid-modified triterpenoids.

We have recently reported on the discovery of a C-3 benzoic acid (1) as a suitable replacement for the dimethyl succinate side chain of bevirimat (2), an HIV-1 maturation inhibitor that reached Phase II clinical trials before being discontinued. Recent SAR studies aimed at improving the antiviral properties of 2 have shown that the benzoic acid moiety conferred topographical constraint to the pharmacophore and was associated with a lower shift in potency in the presence of human serum albumin. In this manuscript, we describe efforts to improve the polymorphic coverage of the C-3 benzoic acid chemotype through modifications at the C-28 position of the triterpenoid core. The dimethylaminoethyl amides 17 and 23 delivered improved potency toward bevirimat-resistant viruses while increasing C24 in rat oral PK studies.

C-3 benzoic acid derivatives of C-3 deoxybetulinic acid and deoxybetulin as HIV-1 maturation inhibitors.

A series of C-3 phenyl- and heterocycle-substituted derivatives of C-3 deoxybetulinic acid and C-3 deoxybetulin was designed and synthesized as HIV-1 maturation inhibitors (MIs) and evaluated for their antiviral activity and cytotoxicity in cell culture. A 4-subsituted benzoic acid moiety was identified as an advantageous replacement for the 3'3'-dimethylsuccinate moiety present in previously disclosed MIs that illuminates new aspects of the topography of the pharmacophore. The new analogs exhibit excellent in vitro antiviral activity against wild-type (wt) virus and a lower serum shift when compared with the prototypical HIV-1 MI bevirimat (1, BVM), the first MI to be evaluated in clinical studies. Compound 9a exhibits comparable cell culture potency toward wt virus as 1 (WT EC50=16 nM for 9a compared to 10nM for 1). However, the potency of 9a is less affected by the presence of human serum, while the compound displays a similar pharmacokinetic profile in rats to 1. Hence 9a, the 4-benzoic acid derivative of deoxybetulinic acid, represents a new starting point from which to explore the design of a 2nd generation MI.

Synthesis and evaluation of C2-carbon-linked heterocyclic-5-hydroxy-6-oxo-dihydropyrimidine-4-carboxamides as HIV-1 integrase inhibitors.

Integration of viral DNA into the host cell genome is an obligatory process for successful replication of HIV-1. Integrase catalyzes the insertion of viral DNA into the target DNA and is a validated target for drug discovery. Herein, we report the synthesis, antiviral activity and pharmacokinetic profiles of several C2-carbon-linked heterocyclic pyrimidinone-4-carboxamides that inhibit the strand transfer step of the integration process.

In vitro cross-resistance profile of nucleoside reverse transcriptase inhibitor (NRTI) BMS-986001 against known NRTI resistance mutations.

BMS-986001 is a novel HIV nucleoside reverse transcriptase inhibitor (NRTI). To date, little is known about its resistance profile. In order to examine the cross-resistance profile of BMS-986001 to NRTI mutations, a replicating virus system was used to examine specific amino acid mutations known to confer resistance to various NRTIs. In addition, reverse transcriptases from 19 clinical isolates with various NRTI mutations were examined in the Monogram PhenoSense HIV assay. In the site-directed mutagenesis studies, a virus containing a K65R substitution exhibited a 0.4-fold change in 50% effective concentration (EC50) versus the wild type, while the majority of viruses with the Q151M constellation (without M184V) exhibited changes in EC50 versus wild type of 0.23- to 0.48-fold. Susceptibility to BMS-986001 was also maintained in an L74V-containing virus (0.7-fold change), while an M184V-only-containing virus induced a 2- to 3-fold decrease in susceptibility. Increasing numbers of thymidine analog mutation pattern 1 (TAM-1) pathway mutations correlated with decreases in susceptibility to BMS-986001, while viruses with TAM-2 pathway mutations exhibited a 5- to 8-fold decrease in susceptibility, regardless of the number of TAMs. A 22-fold decrease in susceptibility to BMS-986001 was observed in a site-directed mutant containing the T69 insertion complex. Common non-NRTI (NNRTI) mutations had little impact on susceptibility to BMS-986001. The results from the site-directed mutants correlated well with the more complicated genotypes found in NRTI-resistant clinical isolates. Data from clinical studies are needed to determine the clinically relevant resistance cutoff values for BMS-986001.

Entecavir exhibits inhibitory activity against human immunodeficiency virus under conditions of reduced viral challenge.

Entecavir (ETV) was developed for the treatment of chronic hepatitis B virus (HBV) infection and is globally approved for that indication. Initial preclinical studies indicated that ETV had no significant activity against human immunodeficiency virus type 1 (HIV-1) in cultured cell lines at physiologically relevant ETV concentrations, using traditional anti-HIV assays. In response to recent clinical observations of anti-HIV activity of ETV in HIV/HBV-coinfected patients not receiving highly active antiretroviral therapy (HAART), additional investigative studies were conducted to expand upon earlier results. An extended panel of HIV-1 laboratory and clinical strains and cell types was tested against ETV, along with a comparison of assay methodologies and resistance profiling. These latest studies confirmed that ETV has only weak activity against HIV, using established assay systems. However, a >100-fold enhancement of antiviral activity (equivalent to the antiviral activity of lamivudine) could be obtained when assay conditions were modified to reduce the initial viral challenge. Also, the selection of a M184I virus variant during the passage of HIV-1 at high concentrations of ETV confirmed that ETV can exert inhibitory pressure on the virus. These findings may have a significant impact on how future assays are performed with compounds to be used in patients infected with HIV. These results support the recommendation that ETV therapy should be administered in concert with HAART for HIV/HBV-coinfected patients.

Design, Synthesis, and SAR of C-3 Benzoic Acid, C-17 Triterpenoid Derivatives. Identification of the HIV-1 Maturation Inhibitor 4-((1 R,3a S,5a R,5b R,7a R,11a S,11b R,13a R,13b R)-3a-((2-(1,1-Dioxidothiomorpholino)ethyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1 H-cyclopenta[ a]chrysen-9-yl)benzoic Acid (GSK3532795, BMS-955176).

GSK3532795, formerly known as BMS-955176 (1), is a potent, orally active, second-generation HIV-1 maturation inhibitor (MI) that advanced through phase IIb clinical trials. The careful design, selection, and evaluation of substituents appended to the C-3 and C-17 positions of the natural product betulinic acid (3) was critical in attaining a molecule with the desired virological and pharmacokinetic profile. Herein, we highlight the key insights made in the discovery program and detail the evolution of the structure-activity relationships (SARs) that led to the design of the specific C-17 amine moiety in 1. These modifications ultimately enabled the discovery of 1 as a second-generation MI that combines broad coverage of polymorphic viruses (EC50 <15 nM toward a panel of common polymorphisms representative of 96.5% HIV-1 subtype B virus) with a favorable pharmacokinetic profile in preclinical species.

Hemorheologic abnormalities associated with HIV infection: altered erythrocyte aggregation and deformability.

PURPOSE: To investigate possible alterations of erythrocyte aggregation and deformability, which are factors that can influence blood flow, in human immunodeficiency virus (HIV)-infected individuals and to determine whether these factors are related to the severity of immunodeficiency. METHODS: Laboratory evaluations were performed on 46 HIV-infected individuals and 44 HIV-negative control subjects. Current and nadir (lowest previous) CD4+ T-lymphocyte counts were identified for each subject. Erythrocyte aggregation was measured using a fully automatic erythrocyte aggregometer. Factors related to erythrocyte aggregation were also determined: erythrocyte sedimentation rate (ESR), zeta sedimentation ratio (ZSR), and plasma fibrinogen levels. Erythrocyte deformability was observed at various fluid shear stress levels, with a laser diffraction ektacytometer. Correlations were sought between each of these measures and current or nadir CD4+ T-lymphocyte counts, and each measure was compared between three subgroups based on current and nadir CD4+ T-lymphocyte counts (severely immunosuppressed, immune reconstituted, never severely immunosuppressed). RESULTS: The following parameters were significantly different between HIV-infected subjects and controls: increased erythrocyte aggregation, at stasis (P < 0.001) and low shear stress (P < 0.001), increased ESR (P < 0.001), increased ZSR (P < 0.028), increased serum fibrinogen (P = 0.015), and decreased erythrocyte deformability (P < 0.001). Only erythrocyte aggregation at stasis correlated significantly with current CD4+ T-lymphocyte count (r = - 0.344, P = 0.022). None of the parameters was significantly different between HIV-infected subgroups. CONCLUSIONS: Increased aggregation and decreased deformability of erythrocytes are associated with HIV-infection regardless of the severity of immunodeficiency. HIV-infected individuals may be at risk for progressive retinal microvascular damage from persistent hemorheologic abnormalities, despite immune reconstitution associated with potent antiretroviral drug therapies.

Discovery of BMS-955176, a Second Generation HIV-1 Maturation Inhibitor with Broad Spectrum Antiviral Activity.

HIV-1 maturation inhibition (MI) has been clinically validated as an approach to the control of HIV-1 infection. However, identifying an MI with both broad polymorphic spectrum coverage and good oral exposure has been challenging. Herein, we describe the design, synthesis, and preclinical characterization of a potent, orally active, second generation HIV-1 MI, BMS-955176 (2), which is currently in Phase IIb clinical trials as part of a combination antiretroviral regimen.

An assessment of polymorphonuclear leukocyte rigidity in HIV-infected individuals after immune recovery.

PURPOSE: To determine whether polymorphonuclear leukocytes (PMNs) remain rigid after immune reconstitution in human immunodeficiency virus (HIV)-infected individuals with a history of severe immunosuppression. METHODS: PMN rigidity was measured in vitro in three groups: (1) HIV-infected individuals with a history of CD4+ T-lymphocyte counts of less than 50/microL, but with current counts of more than 200/microL attributable to potent antiretroviral therapy (group 1); (2) HIV-infected individuals whose CD4+ T-lymphocyte counts had always been more than 200/microL (group 2); and (3) HIV-negative control subjects. Rigidity was determined with a cell transit analyzer (containing a micropore filter with 30 identical, 8-microm diameter pores), representing a simple in vitro model of a capillary bed. A longer PMN pore transit time reflects increased PMN rigidity. RESULTS: PMN transit time (median) in group 1 (n = 11) was 3.34 ms, in group 2 (n = 9) was 3.19 ms, and in control subjects (n = 15) was 2.66 ms. PMN rigidity was significantly greater in groups 1 (P = 0.014) and 2 (P = 0.046) than in control subjects (Wilcoxon rank-sum test). A significant difference was not identified between groups 1 and 2 (P = 0.518). CONCLUSIONS: The increased PMN rigidity known to occur in severely immunosuppressed HIV-infected individuals persists after immune reconstitution. Furthermore, PMN rigidity is increased in those HIV-infected individuals who do not have a history of severe immunosuppression. Because PMN rigidity can alter microvascular blood flow, HIV-infected individuals may remain at risk for retinal vascular damage in the era of potent antiretroviral therapy.

Biochemical analysis of HIV-1 integrase variants resistant to strand transfer inhibitors.

In this study, eight different HIV-1 integrase proteins containing mutations observed in strand transfer inhibitor-resistant viruses were expressed, purified, and used for detailed enzymatic analyses. All the variants examined were impaired for strand transfer activity compared with the wild type enzyme, with relative catalytic efficiencies (k(p)/K(m)) ranging from 0.6 to 50% of wild type. The origin of the reduced strand transfer efficiencies of the variant enzymes was predominantly because of poorer catalytic turnover (k(p)) values. However, smaller second-order effects were caused by up to 4-fold increases in K(m) values for target DNA utilization in some of the variants. All the variants were less efficient than the wild type enzyme in assembling on the viral long terminal repeat, as each variant required more protein than wild type to attain maximal activity. In addition, the variant integrases displayed up to 8-fold reductions in their catalytic efficiencies for 3'-processing. The Q148R variant was the most defective enzyme. The molecular basis for resistance of these enzymes was shown to be due to lower affinity binding of the strand transfer inhibitor to the integrase complex, a consequence of faster dissociation rates. In the case of the Q148R variant, the origin of reduced compound affinity lies in alterations to the active site that reduce the binding of a catalytically essential magnesium ion. Finally, except for T66I, variant viruses harboring the resistance-inducing substitutions were defective for viral integration.

Exploration of the diketoacid integrase inhibitor chemotype leading to the discovery of the anilide-ketoacids chemotype.

Integrase is one of three enzymes expressed by HIV and represents a validated target for therapy. A previous study of the diketoacid-based chemotype suggested that there are two aryl-binding domains on integrase. In this study, modifications to the indole-based diketoacid chemotype are explored. It is demonstrated that the indole group can be replaced with secondary but not tertiary (e.g., N-methyl) aniline-based amides without sacrificing in vitro inhibitory activity. The difference in activity between the secondary and tertiary amides is most likely due to the opposite conformational preferences of the amide bonds, s-trans for the secondary-amide and s-cis for the tertiary-amide. However, it was found that the conformational preference of the tertiary amide can be reversed by incorporating the amide nitrogen atom into an indoline heterocycle, resulting in very potent integrase inhibitors.

Triketoacid inhibitors of HIV-integrase: a new chemotype useful for probing the integrase pharmacophore.

Integrase is one of three enzymes expressed by HIV and represents a validated target for therapy. This study reports on the discovery of a new triketoacid-based chemotype that selectively inhibits the strand transfer reaction of HIV-integrase. SAR studies showed that the template binds to integrase in a manner similar to the diketoacid-based inhibitors. Moreover, comparison of the new chemotype to two different diketoacid templates led us to propose two aryl-binding domains in the inhibitor binding site. This information was used to design a new diketoacid template with improved activity against the enzyme.