Discovery of KB-0742, a Potent, Selective, Orally Bioavailable Small Molecule Inhibitor of CDK9 for MYC-Dependent Cancers.

Transcriptional deregulation is a hallmark of many cancers and is exemplified by genomic amplifications of the MYC family of oncogenes, which occur in at least 20% of all solid tumors in adults. Targeting of transcriptional cofactors and the transcriptional cyclin-dependent kinase (CDK9) has emerged as a therapeutic strategy to interdict deregulated transcriptional activity including oncogenic MYC. Here, we report the structural optimization of a small molecule microarray hit, prioritizing maintenance of CDK9 selectivity while improving on-target potency and overall physicochemical and pharmacokinetic (PK) properties. This led to the discovery of the potent, selective, orally bioavailable CDK9 inhibitor 28 (KB-0742). Compound 28 exhibits in vivo antitumor activity in mouse xenograft models and a projected human PK profile anticipated to enable efficacious oral dosing. Notably, 28 is currently being investigated in a phase 1/2 dose escalation and expansion clinical trial in patients with relapsed or refractory solid tumors.

Identification of Highly Specific Diversity-Oriented Synthesis-Derived Inhibitors of Clostridium difficile.

In 2013, the Centers for Disease Control highlighted Clostridium difficile as an urgent threat for antibiotic-resistant infections, in part due to the emergence of highly virulent fluoroquinolone-resistant strains. Limited therapeutic options currently exist, many of which result in disease relapse. We sought to identify molecules specifically targeting C. difficile in high-throughput screens of our diversity-oriented synthesis compound collection. We identified two scaffolds with apparently novel mechanisms of action that selectively target C. difficile while having little to no activity against other intestinal anaerobes; preliminary evidence suggests that compounds from one of these scaffolds target the glutamate racemase. In vivo efficacy data suggest that both compound series may provide lead optimization candidates.

Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

Novel Quinoline-Based P2-P4 Macrocyclic Derivatives As Pan-Genotypic HCV NS3/4a Protease Inhibitors.

We have previously reported the discovery of our P2-P4 macrocyclic HCV NS3/4a protease inhibitor MK-5172, which in combination with the NS5a inhibitor MK-8742 recently received a breakthrough therapy designation from the US FDA for treatment of chronic HCV infection. Our goal for the next generation NS3/4a inhibitor was to achieve pan-genotypic activity while retaining the pharmacokinetic profile of MK-5172. One of the areas for follow-up investigation involved replacement of the quinoxaline moiety in MK-5172 with a quinoline and studying the effect of substitution at 4-position of the quinoline. The rationale for this effort was based on molecular modeling, which indicated that such modifications would improve interactions with the S2 subsite, in particular with D79. We wish to report herein the discovery of highly potent inhibitors with pan-genotypic activity and an improved profile over MK-5172, especially against gt-3a and A156 mutants.

Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity.

The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection.

Development of macrocyclic inhibitors of HCV NS3/4A protease with cyclic constrained P2-P4 linkers.

A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.

Development of potent macrocyclic inhibitors of genotype 3a HCV NS3/4A protease.

A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.

Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase.

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.

MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants.

HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.

MK-8825: a potent and selective CGRP receptor antagonist with good oral activity in rats.

Rational modification of the clinically tested CGRP receptor antagonist MK-3207 (3) afforded an analogue with increased unbound fraction in rat plasma and enhanced aqueous solubility, 2-[(8R)-8-(3,5-difluorophenyl)-8-methyl-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(6S)-2'-oxo-1',2',5,7-tetrahydrospiro[cyclopenta[b]pyridine-6,3'-pyrrolo[2,3-b]pyridin]-3-yl]acetamide (MK-8825) (6). Compound 6 maintained similar affinity to 3 at the human and rat CGRP receptors but possessed significantly improved in vivo potency in a rat pharmacodynamic model. The overall profile of 6 indicates it should find utility as a rat tool to investigate effects of CGRP receptor blockade in vivo.

Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor.

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

Discovery of pyrrolidine-based ß-secretase inhibitors: lead advancement through conformational design for maintenance of ligand binding efficiency.

We have developed a novel series of pyrrolidine derived BACE-1 inhibitors. The potency of the weak initial lead structure was enhanced using library-based SAR methods. The series was then further advanced by rational design while maintaining a minimal ligand binding efficiency threshold. Ultimately, the co-crystal structure was obtained revealing that these inhibitors interacted with the enzyme in a unique fashion. In all, the potency of the series was enhanced by 4 orders of magnitude from the HTS lead with concomitant increases in physical properties needed for series advancement. The progression of these developments in a systematic fashion is described.

Design and synthesis of conformationally constrained inhibitors of non-nucleoside reverse transcriptase.

Highly active antiretroviral therapy (HAART) significantly reduces human immunodeficiency virus (HIV) viral load and has led to a dramatic decrease in acquired immunodeficiency syndrome (AIDS) related mortality. Despite this success, there remains a critical need for new HIV therapies to address the emergence of drug resistant viral strains. Next generation NNRTIs are sought that are effective against these mutant forms of the HIV virus. The bound conformations of our lead inhibitors, MK-1107 (1) and MK-4965 (2), were divergent about the oxymethylene linker, and each of these conformations was rigidified using two isomeric cyclic constraints. The constraint derived from the bioactive conformation of 2provided novel, highly potent NNRTIs that possess broad spectrum antiviral activity and good pharmacokinetic profiles. Systematic SAR led to the identification of indazole as the optimal conformational constraint to provide MK-6186 (3) and MK-7445 (6). Despite their reduced flexibility, these compounds had potency comparable to that of the corresponding acyclic ethers in both recombinant enzyme and cell based assays against both the wild-type and the clinically relevant mutant strains.

Identification of a novel RAMP-independent CGRP receptor antagonist.

Identification of an HIV integrase inhibitor with micromolar affinity for the CGRP receptor led to the discovery of a series of structurally novel CGRP receptor antagonists. Optimization of this series produced compound 16, a low-molecular weight CGRP receptor antagonist with good pharmacokinetic properties in both rat and dog. In contrast to other nonpeptide antagonists, the activity of 16 was affected by the presence of divalent cations and showed evidence of an alternative, RAMP-independent CGRP receptor binding site.

Attenuation of scratch-induced reactive astrogliosis by novel EphA4 kinase inhibitors.

The EphA4 receptor and its ephrin ligands are involved in astrocytic gliosis following CNS injury. Therefore, a strategy aimed at the blockade of EphA4 signaling could have broad therapeutic interest in brain disorders. We have identified novel small molecule inhibitors of EphA4 kinase in specific enzymatic and cell-based assays. In addition, we have demonstrated in two in vitro models of scratch injury that EphA4 receptor kinase is activated through phosphorylation and is involved in the repopulation of the wound after the scratch. A potent EphA4 kinase inhibitor significantly inhibited wound closure and reduced the accumulation of the reactive astrocytes inside the scratch. We have also shown that after the transient focal cerebral ischemia in rats, a large glial scar is formed by the accumulation of astrocytes and chondroitin sulfate proteoglycan surrounding the infarcted tissue at 7 days and 14 days of reperfusion. EphA4 protein expression is highly up-regulated in the same areas at these time points, supporting its potential role in the glial scar formation and maintenance. Taken together, these results suggest that EphA4 kinase inhibitors might interfere with the astrogliosis reaction and thereby lead to improved neurological outcome after ischemic injury.

Raltegravir: the first HIV-1 integrase strand transfer inhibitor in the HIV armamentarium.

Raltegravir is the first integrase strand transfer inhibitor approved for the treatment of HIV-1 infection. As the first agent in this new class of antiretroviral therapies, raltegravir has demonstrated safety and efficacy in treatment-naive as well as heavily pretreated HIV-infected patients failing therapy with multidrug-resistant virus. Raltegravir has a favorable drug interaction profile that permits both administration to a wide, demographically diverse patient population and coadministration with many other therapeutic agents, including antiretroviral agents and supportive medications, without restrictions or dose adjustment. Data through 96 weeks of follow-up in three phase III studies, protocol 021 (STARTMRK) in treatment-naive patients, and protocols 018 (BENCHMRK-1) and 019 (BENCHMRK-2) in treatment-experienced patients, demonstrated the potent and durable antiretroviral and immunologic effects and the favorable long-term safety profile of raltegravir in both treatment-naive and treatment-experienced patients. Raltegravir represents an important addition to the current armamentarium for the treatment of HIV infection.

Orally bioavailable imidazoazepanes as calcitonin gene-related peptide (CGRP) receptor antagonists: discovery of MK-2918.

In our ongoing efforts to develop CGRP receptor antagonists for the treatment of migraine, we aimed to improve upon telecagepant by targeting a compound with a lower projected clinical dose. Imidazoazepanes were identified as potent caprolactam replacements and SAR of the imidazole yielded the tertiary methyl ether as an optimal substituent for potency and hERG selectivity. Combination with the azabenzoxazinone spiropiperidine ultimately led to preclinical candidate 30 (MK-2918).

Discovery of MK-1220: A Macrocyclic Inhibitor of Hepatitis C Virus NS3/4A Protease with Improved Preclinical Plasma Exposure.

The discovery of MK-1220 is reported along with the development of a series of HCV NS3/4A protease inhibitors containing a P2 to P4 macrocyclic constraint with improved preclinical pharmacokinetics. Optimization of the P2 heterocycle substitution pattern as well as the P3 amino acid led to compounds with greatly improved plasma exposure following oral dosing in both rats and dogs while maintaining excellent enzyme potency and cellular activity. These studies led to the identification of MK-1220.