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Tumorigenesis is driven by the accumulation of adverse genetic changes resulting in dysregulated transcription promoting altered gene expression and the cancer cell state. Thus, tumors can develop dependencies on the transcriptional regulators that promote the reprogrammed gene expression landscape. One such regulator is cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation and promotes activation of transcription factors. We developed a potent, selective, and orally bioavailable CDK9 inhibitor, KB-0742. KB-0742 is highly selective for CDK9 as compared to other CDKs and has shown minimal off-target effects in kinase and receptor panel screens. Here we present the nonclinical pharmacologic characterization of KB-0742. The pharmacokinetics (PK) of KB-0742 in rats and dogs showed rapid absorption and high bioavailability with a %F of ≥84 after oral administration in rats and >100 in dogs. KB-0742 exhibited low turnover in human microsome and hepatocyte preparations and is projected to have low clearance and a high volume of distribution in humans based on allometric scaling from nonclinical species. These properties may facilitate achievement of sustained target engagement in patients using intermittent dosing which may mitigate the toxicity of CDK9 inhibition. To test this hypothesis, mice bearing MV4-11 (acute myeloid leukemia) xenografts were treated with KB-0742 at either 60 mg/kg for 3-days on/4-days off or continuous dosing at 25 mg/kg. Intermittent dosing showed similar tumor growth inhibition (81%) as seen with continuous dosing (74%). Target engagement was assessed in the tumors by measuring the inhibition of RNA polymerase II (pSER2). KB-0742 treatment resulted in an over 50% decrease in pSER2 compared with vehicle-treated controls. To support the phase I clinical trial, 2 pharmacodynamic assays were developed to measure drug target engagement in peripheral blood mononuclear cells (PBMCs). The first used RNA-expression profiling to measure alterations in gene expression with treatment, and the second used MSD protein analysis to measure changes in pSER2 levels. Ex vivo experiments using PBMCs from 3 healthy donors showed an over 80% reduction in pSER2 levels at 4 hours post-exposure to 1 µM of KB-0742. Similar results were observed using the RNA-expression profiling assay, in which decreased gene expression was observed in key genes at 1 µM exposures. KB-0742’s high specificity, oral bioavailability, and favorable PK properties distinguish it from other available CDK9 inhibitors. We are currently testing the hypothesis in the clinic with a phase I trial (NCT04718675) that the ability to dose intermittently will allow for favorable anti-tumor activity while minimizing adverse effects. Citation Format: Melinda A. L. Day, Douglas C. Saffran, Nathalie Rioux, Tom Chen, Christina Lee, David B. Freeman, Crystal MacKenzie, Joseph P. Vacca, Peter B. Rahl, Benjamin Wesley Trotter, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. Preclinical pharmacokinetics and pharmacodynamics of KB-0742, a selective, oral CDK9 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P228.

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.

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.

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 R155 K, A156T, A156 V, and D168 V 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.

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.

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.

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.

J. Neurochem. (2011) 118, 1016–1031. Abstract 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.

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 μM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.

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).

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.

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.

Activation Induced Cytidine Deaminase (AID) is a DNA directed cytidine deaminase that is normally only expressed in activated B-cells to promote somatic hypermutations and immunoglobulin class switching. Unlike site-specific recombinases (e.g. RAG1/2), AID lacks target site specificity and can generate DNA damage at widespread locations throughout the genome. In cancer cells, AID expression promotes high levels of DNA replication stress, and results in dependency upon the homologous recombination factor RAD51. We have shown previously that a novel RAD51 inhibitor, CYT01B, functions by disrupting RAD51 focus formation which reduces the nuclear concentration of RAD51 and promotes RAD51 protein degradation. Here we present the in vitro and in vivo pharmacological characterization of CYT01B. We first analyzed the permeability and stability of the compound using Caco-2 and liver microsome assays. CYT01B shows low efflux and is stable with low intrinsic clearance rates (< 30 ml/min/mg protein) in mouse, rat, dog, and human, but not in cynomolgus monkey liver microsomes. In vivo, CYT01B showed oral bioavailability that correlated well with Caco-2 permeability (36.9% in monkeys up to 86.5% in rats). Additionally, CYT01B exhibited a minimum half-life of 4 hours in all species tested (mouse, rat, dog, and monkey). We then examined off target liabilities by performing kinome inhibition and Panlabs safety panel screens. CYT01B showed negligible ( Disclosures Patrick: Cyteir Therapeutics: Consultancy. Bedard:Cyteir Therapeutics: Consultancy. Vacca:Cyteir Therapeutics: Consultancy. McComas:Cyteir Therapeutics: Consultancy. Castro:Cyteir Therapeutics: Consultancy. Day:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A previously utilized quinoline-for- N -phenylamide replacement strategy was employed against a central amide in a novel class of CGRP receptor antagonists. A unique and unexpected substitution pattern was ultimately required to maintain reasonable affinity for the CGRP receptor, while at the same time predicting acceptable heterocycle positioning for related analogs. Subsequently, specific quinoline and naphthyridine compounds were prepared which supported these structural predictions by displaying CGRP binding affinities in the 0.037–0.15 nM range.

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.

A new class of HCV NS3/4a protease inhibitors which contain a P2 to P4 macrocyclic constraint was designed using a molecular-modeling derived strategy. Exploration of the P2 heterocyclic region, the P2 to P4 linker, and the P1 side chain of this class of compounds via a modular synthetic strategy allowed for the optimization of enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 35b (vaniprevir, MK-7009), which is active against both the genotype 1 and genotype 2 NS3/4a protease enzymes and has good plasma exposure and excellent liver exposure in multiple species.

Calcitonin gene-related peptide (CGRP) has long been hypothesized to play a key role in migraine pathophysiology, and the advent of small-molecule antagonists has clearly demonstrated a clinical link between blocking the CGRP receptor and migraine efficacy. 2-[(8R)-8-(3,5-Difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(2R)-2'-oxo-1,1',2',3-tetrahydrospiro[indene-2,3'-pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207) represents the third CGRP receptor antagonist to display clinical efficacy in migraine trials. Here, we report the pharmacological characterization of MK-3207, a potent and orally bioavailable CGRP receptor antagonist. In vitro, MK-3207 is a potent antagonist of the human and rhesus monkey CGRP receptors (K(i) = 0.024 nM). In common with other CGRP receptor antagonists, MK-3207 displays lower affinity for CGRP receptors from other species, including canine and rodent. As a consequence of species selectivity, the in vivo potency was assessed in a rhesus monkey pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging. MK-3207 produced a concentration-dependent inhibition of dermal vasodilation, with plasma concentrations of 0.8 and 7 nM required to block 50 and 90% of the blood flow increase, respectively. The tritiated analog [3H]MK-3207 was used to study the binding characteristics on the human CGRP receptor. [3H]MK-3207 displayed reversible and saturable binding (K(D) = 0.06 nM), and the off-rate was determined to be 0.012 min(-1), with a t(1/2) value of 59 min. In vitro autoradiography studies on rhesus monkey brain slices identified the highest level of binding in the cerebellum, brainstem, and meninges. Finally, as an index of central nervous system penetrability, the in vivo cerebrospinal fluid/plasma ratio was determined to be 2 to 3% in cisterna magna-ported rhesus monkeys.

The administration of hepatitis C virus (HCV) NS3/4A protease inhibitors to patients with chronic HCV infections has demonstrated that they have dramatic antiviral effects and that compounds acting via this mechanism are likely to form a key component of future anti-HCV therapy. We report here on the preclinical profile of MK-7009, an inhibitor of genotype 1a and 1b proteases at subnanomolar concentrations with modestly shifted potency against genotype 2a and 2b proteases at low nanomolar concentrations. Potent activity was also observed in a cell-based HCV replicon assay in the presence of added human serum (50%). In multiple species evaluated in preclinical studies, the MK-7009 concentrations in the liver were maintained at a significant multiple of the cell-based replicon 50% effective concentration over 12 to 24 h following the administration of moderate oral doses (5 to 10 mg per kg of body weight). MK-7009 also had excellent selectivity against both a range of human proteases and a broad panel of pharmacologically relevant ion channels, receptors, and enzymes. On the basis of this favorable profile, MK-7009 was selected for clinical development and is currently being evaluated in controlled clinical trials with both healthy volunteers and HCV-infected patients.

A novel class of CGRP receptor antagonists was rationally designed by modifying a highly potent, but structurally complex, CGRP receptor antagonist. Initial modifications focused on simplified structures, with increased flexibility. Subsequent to the preparation of a less-potent but more flexible lead, classic medicinal chemistry methods were applied to restore high affinity (compound 22, CGRP Ki = 0.035 nM) while maintaining structural diversity relative to the lead. Good selectivity against the closely related adrenomedullin-2 receptor was also achieved.

We have developed a novel series of heteroaromatic BACE-1 inhibitors. These inhibitors interact with the enzyme in a unique fashion that allows for potent binding in a non-traditional paradigm. In addition to the elucidation of their binding profile, we have discovered a pH dependent effect on the binding affinity as a result of the intrinsic pK(a) of these inhibitors and the pH of the BACE-1 enzyme binding assay.

Rational modification of a previously identified spirohydantoin lead structure has identified a series of potent spiroazaoxindole CGRP receptor antagonists. The azaoxindole was found to be a general replacement for the hydantoin that consistently improved in vitro potency. The combination of the indanylspiroazaoxindole and optimized benzimidazolinones led to highly potent antagonists (e.g., 25, CGRP Ki = 40 pM). The closely related compound 27 demonstrated good oral bioavailability in dog and rhesus.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

Using a combination of traditional Medicinal Chemistry/SAR analysis, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad antiviral activity against a number of key clinical mutations.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide that plays a key role in the pathophysiology of migraine headache. CGRP levels in the cranial circulation are increased during a migraine attack, and CGRP itself has been shown to trigger migraine-like headache. The correlation between CGRP release and migraine headache points to the potential utility of CGRP receptor antagonists as novel therapeutics in the treatment of migraine. Indeed, clinical proof-of-concept in the acute treatment of migraine was demonstrated with an intravenous formulation of the CGRP receptor antagonist BIBN4096BS (olcegepant). Here we report on the pharmacological characterization of the first orally bioavailable CGRP receptor antagonist in clinical development, MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]. In vitro, MK-0974 is a potent antagonist of the human (K(i) = 0.77 nM) and rhesus (K(i) = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for the canine and rat receptors as determined via (125)I-human CGRP competition binding assays. A rhesus pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging was utilized to determine the in vivo activity of CGRP receptor antagonism. MK-0974 produced a concentration-dependent inhibition of dermal vasodilation, generated by capsaicin-induced release of endogenous CGRP, with plasma concentrations of 127 and 994 nM required to block 50 and 90% of the blood flow increase, respectively. In conclusion, MK-0974 is a highly potent, selective, and orally bioavailable CGRP receptor antagonist, which may be valuable in the acute treatment of migraine.

Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Herein we describe optimization of CGRP receptor antagonists based on an earlier lead structure containing a (3R)-amino-(6S)-phenylcaprolactam core. Replacement of the phenylimidazolinone with an azabenzimidazolone gave stable derivatives with lowered serum shifts. Extensive SAR studies of the C-6 aryl moiety revealed the potency-enhancing effect of the 2,3-difluorophenyl group, and trifluoroethylation of the N-1 amide position resulted in improved oral bioavailabilities, ultimately leading to clinical candidate 38 (MK-0974).

A series of potent novel dihydroxypyridopyrazine-1,6-dione HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 6 is active against replication of HIV with a CIC95 of 0.31 μM and exhibits no shift in potency in the presence of 50% normal human serum. It displays a good pharmacokinetic profile when dosed in rats and no covalent binding with microsomal proteins in both in vitro and in vivo models.

A series of low-molecular weight 2,6-diamino-isonicotinamide BACE-1 inhibitors containing an amine transition-state isostere were synthesized and shown to be highly potent in both enzymatic and cell-based assays. These inhibitors contain a trans-S,S-methyl cyclopropane P3 which bind BACE-1 in a 10s-loop down conformation giving rise to highly potent compounds with favorable molecular weight and moderate to high susceptibility to P-glycoprotein (P-gp) efflux.

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 μM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.

In our continuing effort to identify CGRP receptor antagonists for the acute treatment of migraine, we have undertaken a study to evaluate alternative 4-substituted piperidines to the lead dihydroquinazolinone 1. In this regard, we have identified the piperidinyl-azabenzimidazolone and phenylimidazolinone structures which, when incorporated into the benzodiazepine core, afford potent CGRP receptor antagonists (e.g., 18 and 29). These studies produced a potent analog (18) which overcomes the instability issues associated with the lead structure 1. A general pharmacophore for the 4-substituted piperidine component of these CGRP receptor antagonists is also presented.

Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4 R )- and (4 S )-4-fluoroproline was rationalized by analyzing inhibitor–enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4 R )-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41 , exhibits high potency in a coagulation assay in human plasma (2×APTT = 190 nM), excellent selectivity versus the digestive enzyme trypsin ( K i = 3300 nM), and excellent oral bioavailability in dogs with moderate clearance ( F = 100%, CL = 12 mL/min/kg).

High-throughput screening of the Merck sample collection identified benzodiazepinone tetralin-spirohydantoin 1 as a CGRP receptor antagonist with micromolar activity. Comparing the structure of 1 with those of earlier peptide-based antagonists such as BIBN 4096 BS, a key hydrogen bond donor-acceptor pharmacophore was hypothesized. Subsequent structure activity studies supported this hypothesis and led to benzodiazepinone piperidinyldihydroquinazolinone 7, CGRP receptor K(i)=44nM and IC(50)=38nM. Compound 7 was orally bioavailabile in rats and is a lead in the development of orally bioavailable CGRP antagonists for the treatment of migraine.

We have synthesized and evaluated a series of conformationally biased P3 amide replacements based on an isophthalamide lead structure. The studies resulted in the identification of the beta-secretase inhibitor 7m which has an in vitro IC(50)=35 nM. The synthesis and biological activities of these compounds are described.

Introduction of a 5,6-dihydrouracil functionality in the 5-position of N -(4-fluorobenzyl)-8-hydroxy-[1,6]naphthyridine-7-carboxamide 1 led to a series of highly active HIV-1 integrase inhibitors. These compounds displayed low nanomolar activity in inhibiting both the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 11 is a 150-fold more potent antiviral agent than 1 , with a CIC 95 of 40 nM in the presence of human serum. It displays good pharmacokinetics when dosed in rats and dogs.

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).

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.

Modification of lead compound 1 by reducing lipophilicity in the P3 group produced a series of low molecular weight thrombin inhibitors with excellent potency in functional assays, metabolic stability, and oral bioavailability. These modifications led to the identification of two optimized compounds, 14 and 16.

An effective approach for enhancing the selectivity of beta-site amyloid precursor protein cleaving enzyme (BACE 1) inhibitors is developed based on the unique features of the S1' pocket of the enzyme. A series of low molecular weight (

A series of potent and selective proline- and pyrazinone-based macrocyclic thrombin inhibitors is described. Detailed SAR studies led to the incorporation of specific functional groups in the tether that enhanced functional activity against thrombin and provided exquisite selectivity against trypsin and tPA. X-ray crystallography and molecular modeling studies revealed the inhibitor-enzyme interactions responsible for this selectivity.

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma half-life, and human liver microsome stability revealed 5 as the best candidate for advanced evaluation.

Naphthyridine 7 inhibits the strand transfer of the integration process catalyzed by integrase with an IC50 of 10 nM and inhibits 95% of the spread of HIV-1 infection in cell culture at 0.39 microM. It does not exhibit cytotoxicity in cell culture at < or =12.5 microM and shows a good pharmacokinetic profile when dosed orally to rats. The antiviral activity of 7 and its effect on integration were confirmed using viruses with specific integrase mutations.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

A new class of conformationally constrained thrombin inhibitors is described. These compounds contain a unique bicyclic pyridone scaffold which serves as a P3P2 dipeptide surrogate. The synthesis and antithrombotic activity of these inhibitors is reported.

A series of tricyclic CGRP receptor antagonists was optimized in order to improve oral bioavailability. Attenuation of polar surface area and incorporation of a weakly basic indoline nitrogen led to compound 5, a potent antagonist with good oral bioavailability in three species.

beta-Secretase inhibition offers an exciting opportunity for therapeutic intervention in the progression of Alzheimer's disease. A series of isonicotinamides derived from traditional aspartyl protease transition state isostere inhibitors has been optimized to yield low nanomolar inhibitors with sufficient penetration across the blood-brain barrier to demonstrate beta-amyloid lowering in a murine model.