Antiviral Properties of HIV-1 Capsid Inhibitor GSK878.

GSK878 is a newly described HIV-1 inhibitor that binds to the mature capsid (CA) hexamer in a pocket originally identified as the binding site of the well-studied CA inhibitor PF-74. Here, we show that GSK878 is highly potent, inhibiting an HIV-1 reporter virus in MT-2 cells with a mean 50% effective concentration (EC50) of 39 pM and inhibiting a panel of 48 chimeric viruses containing diverse CA sequences with a mean EC50 of 94 pM. CA mutations associated with reduced susceptibility to other inhibitors that bind to PF-74 binding site (L56I, M66I, Q67H, N74D, T107N, and Q67H/N74D) also reduced susceptibility to GSK878, with M66I, Q67H/N74D, and L56I having the greatest impact on antiviral activity. Amino acid substitutions in the CA cyclophilin A (CypA) binding loop (H87P and P90A), distal from the inhibitor binding site and associated with reduced CA-CypA binding, subtly, but reproducibly, also decreased GSK878 potency. Mechanism-of-action studies showed that GSK878 blocked both early (preintegration) and late (postintegration) steps in HIV-1 replication, with the early inhibition primarily determining the compound's antiviral activity. The early inhibition results from blocks to HIV-1 nuclear import and proviral integration and is associated with altered stability of the HIV-1 CA core.

Potent Long-Acting Inhibitors Targeting the HIV-1 Capsid Based on a Versatile Quinazolin-4-one Scaffold.

Long-acting (LA) human immunodeficiency virus-1 (HIV-1) antiretroviral therapy characterized by a ≥1 month dosing interval offers significant advantages over daily oral therapy. However, the criteria for compounds that enter clinical development are high. Exceptional potency and low plasma clearance are required to meet dose size requirements; excellent chemical stability and/or crystalline form stability is required to meet formulation requirements, and new antivirals in HIV-1 therapy need to be largely free of side effects and drug-drug interactions. In view of these challenges, the discovery that capsid inhibitors comprising a quinazolinone core tolerate a wide range of structural modifications while maintaining picomolar potency against HIV-1 infection in vitro, are assembled efficiently in a multi-component reaction, and can be isolated in a stereochemically pure form is reported herein. The detailed characterization of a prototypical compound, GSK878, is presented, including an X-ray co-crystal structure and subcutaneous and intramuscular pharmacokinetic data in rats and dogs.

Improving Drug Delivery While Tailoring Prodrug Activation to Modulate Cmax and Cmin by Optimization of (Carbonyl)oxyalkyl Linker-Based Prodrugs of Atazanavir.

Structure-property relationships associated with a series of (carbonyl)oxyalkyl amino acid ester prodrugs of the marketed HIV-1 protease inhibitor atazanavir (1), designed to enhance the systemic drug delivery, were examined. Compared to previously reported prodrugs, optimized candidates delivered significantly enhanced plasma exposure and trough concentration (Cmin at 24 h) of 1 in rats while revealing differentiated PK paradigms based on the kinetics of prodrug activation and drug release. Prodrugs incorporating primary amine-containing amino acid promoieties offered the benefit of rapid bioactivation that translated into low circulating levels of the prodrug while delivering a high Cmax value of 1. Interestingly, the kinetic profile of prodrug cleavage could be tailored for slower activation by structural modification of the amino terminus to either a tertiary amine or a dipeptide motif, which conferred a circulating depot of the prodrug that orchestrated a sustained release of 1 along with substantially reduced Cmax and a further enhanced Cmin.

Discovery and Preclinical Profiling of GSK3839919, a Potent HIV-1 Allosteric Integrase Inhibitor.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have been of interest recently because of their novel mechanism of action. Strategic modifications to the C5 moiety of a class of 4-(4,4-dimethylpiperidinyl)-2,6-dimethylpyridinyl ALLINIs led to the identification of a tetrahydroisoquinoline heterocycle as a suitable spacer element to project the distal hydrophobic aryl ring. Subsequent optimization of the aryl substitutions identified 12 as an ALLINI with single-digit nanomolar inhibitory potency and low clearance across preclinical species. In preclinical toxicology studies with 12 in rats, lipid hepatocellular vacuolation was observed. Removal of the C6 methyl group resulted in GSK3839919 (22), which exhibited a reduced incidence and severity of lipid vacuolation in both in vitro assays and in vivo studies while maintaining the potency and pharmacokinetic (PK) properties of the prototype. The virology, PK, and toxicology profiles of 22 are discussed.

Scaffold modifications to the 4-(4,4-dimethylpiperidinyl) 2,6-dimethylpyridinyl class of HIV-1 allosteric integrase inhibitors.

Allosteric integrase inhibitors (ALLINIs) of HIV-1 may hold promise as a novel mechanism for HIV therapeutics and cure. Scaffold modifications to the 4-(4,4-dimethylpiperidinyl) 2,6-dimethylpyridinyl class of ALLINIs provided a series of potent compounds with differentiated 5/6 fused ring systems. Notably, inhibitors containing the 1,2,4-triazolopyridine and imidazopyridine core exhibited single digit nM antiviral potency and low to moderate clearance after intravenous (IV) dosing in rat pharmacokinetic (PK) studies. The 1,2,4-triazolopyridines showed a higher oral exposure when compared to the imidazopyridines. Further modifications to the C5 substituent of the 1,2,4-triazolopyridines resulted in a new lead compound, which had improved rat IV/PO PK compared to the former lead compound GSK3739936, while maintaining antiviral potency. Structure-activity relationships (SAR) and rat pharmacokinetic profiles of this series are discussed.

The Genesis and Future Prospects of Small Molecule HIV-1 Attachment Inhibitors.

Gp120 is a critical viral proteins required for HIV-1 entry and infection. It facilitates HIV-1 binding to target cells, human-to-human transmission, relocation of virus from mucosa to lymph nodes, cell-cell infection and syncytium formation, and the bystander effect that kills uninfected CD4+ T-cells and other human cells. Molecules that bind to gp120 can inhibit its function by stabilizing conformations of the protein, leading to the inability to infect cells, and resulting in non-permissive. Small molecule-mediated stabilization of certain conformations of gp120 may also enhance recognition of HIV-1 infected cells by neutralizing antibodies and make the virus more susceptible to effector functions such as ADCC, which could potentially be part of future cure regimens. Additionally, HIV attachment inhibitors can complex with free gp120 and potentially repress both cytopathic effects from membrane-bound or soluble gp120. Fostemsavir (RukobiaTM), a phosphate prodrug of an HIV-1 attachment inhibitor that was recently approved for use in highly treatment experienced (HTE) patients with multidrug resistant HIV-1 is a first-in-class drug with a favorable safety profile that provides an additional treatment option for treatment in this population of patients with a high medical need.

Design, Synthesis, and Preclinical Profiling of GSK3739936 (BMS-986180), an Allosteric Inhibitor of HIV-1 Integrase with Broad-Spectrum Activity toward 124/125 Polymorphs.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have garnered special interest because of their novel mechanism of action: they inhibit HIV-1 replication by promoting aberrant integrase multimerization, leading to the production of replication-deficient viral particles. The binding site of ALLINIs is in a well-defined pocket formed at the interface of two integrase monomers that is characterized by conserved residues along with two polymorphic amino acids at residues 124 and 125. The design, synthesis, and optimization of pyridine-based allosteric integrase inhibitors are reported here. Optimization was conducted with a specific emphasis on the inhibition of the 124/125 polymorphs such that the designed compounds showed excellent potency in vitro against majority of the 124/125 variants. In vivo profiling of promising preclinical lead 29 showed that it exhibited a good pharmacokinetic (PK) profile in preclinical species, which resulted in a low predicted human efficacious dose. However, findings in rat toxicology studies precluded further development of 29.

Innovation in the discovery of the HIV-1 attachment inhibitor temsavir and its phosphonooxymethyl prodrug fostemsavir.

The discovery and development of fostemsavir (2), the tromethamine salt of the phosphonooxymethyl prodrug of temsavir (1), encountered significant challenges at many points in the preclinical and clinical development program that, in many cases, stimulated the implementation of innovative solutions in order to enable further progression. In the preclinical program, a range of novel chemistry methodologies were developed during the course of the discovery effort that enabled a thorough examination and definition of the HIV-1 attachment inhibitor (AI) pharmacophore. These discoveries helped to address the challenges associated with realizing a molecule with all of the properties necessary to successfully advance through development and this aspect of the program is the major focus of this retrospective. Although challenges and innovation are not unusual in drug discovery and development programs, the HIV-1 AI program is noteworthy not only because of the serial nature of the challenges encountered along the development path, but also because it resulted in a compound that remains the first and only example of a mechanistically novel class of HIV-1 inhibitor that is proving to be very beneficial for controlling virus levels in highly treatment-experienced HIV-1 infected patients.

(Carbonyl)oxyalkyl linker-based amino acid prodrugs of the HIV-1 protease inhibitor atazanavir that enhance oral bioavailability and plasma trough concentration.

We describe the design, synthesis and pharmacokinetic (PK) evaluation of a series of amino acid-based prodrugs of the HIV-1 protease inhibitor atazanavir (1) derivatized on the pharmacophoric secondary alcohol using a (carbonyl)oxyalkyl linker. Prodrugs of 1 incorporating simple (carbonyl)oxyalkyl-based linkers and a primary amine in the promoiety were found to exhibit low chemical stability. However, chemical stability was improved by modifying the primary amine moiety to a tertiary amine, resulting in a 2-fold enhancement of exposure in rats following oral dosing compared to dosing of the parent drug 1. Further refinement of the linker resulted in the discovery of 22 as a prodrug that delivered the parent 1 to rat plasma with a 5-fold higher AUC and 67-fold higher C24 when compared to oral administration of the parent drug. The PK profile of 22 indicated that plasma levels of this prodrug were higher than that of the parent, providing a more sustained release of 1 in vivo.

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.

Design, Synthesis, and Pharmacokinetic Evaluation of Phosphate and Amino Acid Ester Prodrugs for Improving the Oral Bioavailability of the HIV-1 Protease Inhibitor Atazanavir.

Phosphate and amino acid prodrugs of the HIV-1 protease inhibitor (PI) atazanavir (1) were prepared and evaluated to address solubility and absorption limitations. While the phosphate prodrug failed to release 1 in rats, the introduction of a methylene spacer facilitated prodrug activation, but parent exposure was lower than that following direct administration of 1. Val amino acid and Val-Val dipeptides imparted low plasma exposure of the parent, although the exposure of the prodrugs was high, reflecting good absorption. Screening of additional amino acids resulted in the identification of an l-Phe ester that offered an improved exposure of 1 and reduced levels of the circulating prodrug. Further molecular editing focusing on the linker design culminated in the discovery of the self-immolative l-Phe-Sar dipeptide derivative 74 that gave four-fold improved AUC and eight-fold higher Ctrough values of 1 compared with oral administration of the drug itself, demonstrating a successful prodrug approach to the oral delivery of 1.

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.

Coupling of an Acyl Migration Prodrug Strategy with Bio-activation To Improve Oral Delivery of the HIV-1 Protease Inhibitor Atazanavir.

HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, 1), remain important medicines to treat HIV-1 infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer, which results in additional drug-drug interactions that are sometimes difficult to manage. We investigated a chemo-activated, acyl migration-based prodrug design approach to improve the pharmacokinetic profile of 1 but failed to obtain improved oral bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1) yielded substantially better oral drug delivery of 1 when compared to the parent itself, the simple acyl migration-based prodrug, and the corresponding simple l-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo, and (3) offered the benefit of mitigating the pH-dependent absorption associated with 1, thereby potentially reducing the risk of decreased bioavailability with concurrent use of stomach-acid-reducing drugs.

Structure-Property Basis for Solving Transporter-Mediated Efflux and Pan-Genotypic Inhibition in HCV NS5B Inhibitors.

In solving the P-gp and BCRP transporter-mediated efflux issue in a series of benzofuran-derived pan-genotypic palm site inhibitors of the hepatitis C virus NS5B replicase, it was found that close attention to physicochemical properties was essential. In these compounds, where both molecular weight (MW >579) and TPSA (>110 Å2) were high, attenuation of polar surface area together with weakening of hydrogen bond acceptor strength of the molecule provided a higher intrinsic membrane permeability and more desirable Caco-2 parameters, as demonstrated by trifluoroacetamide 11 and the benchmark N-ethylamino analog 12. In addition, the tendency of these inhibitors to form intramolecular hydrogen bonds potentially contributes favorably to the improved membrane permeability and absorption. The functional group minimization that resolved the efflux problem simultaneously maintained potent inhibitory activity toward a gt-2 HCV replicon due to a switching of the role of substituents in interacting with the Gln414 binding pocket, as observed in gt-2a NS5B/inhibitor complex cocrystal structures, thus increasing the efficiency of the optimization. Noteworthy, a novel intermolecular S=O···C=O n → π* type interaction between the ligand sulfonamide oxygen atom and the carbonyl moiety of the side chain of Gln414 was observed. The insights from these structure-property studies and crystallography information provided a direction for optimization in a campaign to identify second generation pan-genotypic NS5B inhibitors.

Inhibitors of HIV-1 Attachment: The Discovery and Development of Temsavir and its Prodrug Fostemsavir.

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

Bioactivation of cyclopropyl rings by P450: an observation encountered during the optimisation of a series of hepatitis C virus NS5B inhibitors.

1. Due to its unique C-C and C-H bonding properties, conformational preferences and relative hydrophilicity, the cyclopropyl ring has been used as a synthetic building block in drug discovery to modulate potency and drug-like properties. During an effort to discover inhibitors of the hepatitis C virus non-structural protein 5B with improved potency and genotype-coverage profiles, the use of a pyrimidinylcyclopropylbenzamide moiety linked to a C6-substituted benzofuran or azabenzofuran core scaffold was explored in an effort to balance antiviral potency and metabolic stability. 2. In vitro metabolism studies of two compounds from this C6-substituted series revealed an NADPH-dependent bioactivation pathway leading to the formation of multiple glutathione (GSH) conjugates. Analysis of these conjugates by LC-MS and NMR demonstrated that the cyclopropyl group was the site of bioactivation. Based on the putative structures and molecular weights of the cyclopropyl-GSH conjugates, a multi-step mechanism was proposed to explain the formation of these metabolites by P450. This mechanism involves hydrogen atom abstraction to form a cyclopropyl radical, followed by a ring opening rearrangement and reaction with GSH. 3. These findings provided important information to the medicinal chemistry team which responded by replacing the cyclopropyl ring with a gem-dimethyl group. Subsequent compounds bearing this feature were shown to avert the bioactivation pathways in question.

Development of the Large-Scale Synthesis of Tetrahydropyran Glycine, a Precursor to the HCV NS5A Inhibitor BMS-986097.

An efficient large-scale synthesis of acid 1, a penultimate precursor to the HCV NS5A inhibitor BMS-986097, along with the final API step are described. Three routes were devised for the synthesis of 1 at the various stages of the program. The third generation route, the one that proved scalable and is the main subject of this paper, features a one-step Michael addition of t-butyl 2-((diphenylmethylene)amino)acetate (24) to (E)-benzyl 4-(1-hydroxycyclopropyl)but-2-enoate (28) followed by cyclization and chiral separation to form 27c, the core skeleton of cap piece 1. The epimerization and chiral resolution of 27c followed by further synthetic manipulations involving the carbamate formation, lactone reduction and cyclization, afforded cyclopropyl pyran 1. A detailed study of diphenylmethane deprotection via acid hydrolysis as well as a key lactone to tetrahydropyran conversion, in order to avoid a side reaction that afforded an alternative cyclization product, are discussed. This synthesis was applied to the preparation of more than 100 g of the final API BMS-986097 for toxicology studies.

Design strategies in the prodrugs of HIV-1 protease inhibitors to improve the pharmaceutical properties.

Combination antiretroviral therapy (cART) is currently the most effective treatment for HIV-1 infection. HIV-1 protease inhibitors (PIs) are an important component of some regimens of cART. However, PIs are known for sub-optimal ADME properties, resulting in poor oral bioavailability. This often necessitates high drug doses, combination with pharmacokinetic enhancers and/or special formulations in order to effectively deliver PIs, which may lead to a high pill burden and reduced patient compliance. As a remedy, improving the ADME properties of existing drugs via prodrug and other approaches has been pursued in addition to the development of next generation PIs with improved pharmacokinetic, resistance and side effect profiles. Phosphate prodrugs have been explored to address the solubility-limiting absorption and high excipient load. Prodrug design to target carrier-mediated drug delivery has also been explored. Amino acid prodrugs have been shown to improve permeability by engaging active transport mechanisms, reduce efflux and mitigate first pass metabolism while acyl migration prodrugs have been shown to improve solubility. Prodrug design efforts have led to the identification of one marketed agent, fosamprenavir, and clinical studies with two other prodrugs. Several of the reported approaches lack detailed in vivo characterization and hence the potential preclinical or clinical benefits of these approaches are yet to be fully determined.

Improving Metabolic Stability with Deuterium: The Discovery of BMT-052, a Pan-genotypic HCV NS5B Polymerase Inhibitor.

Iterative structure-activity analyses in a class of highly functionalized furo[2,3-b]pyridines led to the identification of the second generation pan-genotypic hepatitis C virus NS5B polymerase primer grip inhibitor BMT-052 (14), a potential clinical candidate. The key challenge of poor metabolic stability was overcome by strategic incorporation of deuterium at potential metabolic soft spots. The preclinical profile and status of BMT-052 (14) is described.

Discovery of BMS-961955, an allosteric inhibitor of the hepatitis C virus NS5B polymerase.

The synthesis, structure-activity relationship (SAR) data, and further optimization of the metabolic stability and pharmacokinetic (PK) properties for a previously disclosed class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors are described. These efforts led to the discovery of BMS-961955 as a viable contingency backup to beclabuvir which was recently approved in Japan for the treatment of HCV as part of a three drug, single pill combination marketed as XimencyTM.