ABSTRACT
Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).
Subject(s)
Antiviral Agents , SARS-CoV-2 , Animals , Mice , SARS-CoV-2/drug effects , Humans , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/genetics , COVID-19 Drug Treatment , Protease Inhibitors/pharmacology , COVID-19/virology , Coronavirus Infections/drug therapy , Coronavirus Infections/virologyABSTRACT
As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.
Subject(s)
COVID-19 , Glutamine , Humans , Glutamine/chemistry , SARS-CoV-2 , Cysteine Endopeptidases/chemistry , Inventions , Protease Inhibitors/pharmacology , Amides , Antiviral Agents/pharmacology , Antiviral Agents/chemistryABSTRACT
Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.
Subject(s)
HIV Infections , HIV-1 , Humans , HIV Infections/drug therapy , Antiviral Agents/therapeutic use , Apoptosis , Cell Death , CD4-Positive T-Lymphocytes , Virus ReplicationABSTRACT
Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.
Subject(s)
HIV Infections , HIV-1 , Alkynes , Benzoxazines , CARD Signaling Adaptor Proteins/metabolism , Cyclopropanes , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , HIV Infections/drug therapy , HIV-1/metabolism , Humans , Inflammasomes/metabolism , Leukocytes, Mononuclear , Neoplasm Proteins/metabolismABSTRACT
Using the HIV-1 protease binding mode of MK-8718 and PL-100 as inspiration, a novel aspartate binding bicyclic piperazine sulfonamide core was designed and synthesized. The resulting HIV-1 protease inhibitor containing this core showed an 60-fold increase in enzyme binding affinity and a 10-fold increase in antiviral activity relative to MK-8718.
ABSTRACT
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.
ABSTRACT
A series of 3-substituted aminocyclopentanes has been identified as highly potent and selective NR2B receptor antagonists. Incorporation of a 1,2,4-oxadiazole linker and substitution of the pendant phenyl ring led to the discovery of orally bioavailable analogues that showed efficient NR2B receptor occupancy in rats. Unlike nonselective NMDA antagonists, the NR2B-selective antagonist 22 showed no adverse affects on motor coordination in the rotarod assay at high dose. Compound 22 was efficacious following oral administration in a spinal nerve ligation model of neuropathic pain and in an acute model of Parkinson's disease in a dose dependent manner.
Subject(s)
Cyclopentanes/chemical synthesis , Cyclopentanes/pharmacology , Drug Discovery/methods , Excitatory Amino Acid Antagonists/chemical synthesis , Excitatory Amino Acid Antagonists/pharmacology , Oxadiazoles/chemical synthesis , Oxadiazoles/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Administration, Oral , Animals , Benzopyrans/metabolism , Biological Availability , Catalepsy/chemically induced , Catalepsy/drug therapy , Dogs , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/metabolism , Female , Half-Life , Indicators and Reagents , Isomerism , Ligation , Macaca mulatta , Male , Neuralgia/drug therapy , Parkinson Disease/drug therapy , Piperidines/metabolism , Rats , Rats, Sprague-Dawley , Spinal Nerves/pathologyABSTRACT
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.
Subject(s)
Amides/pharmacology , Calcitonin Gene-Related Peptide Receptor Antagonists , Amides/chemistry , StereoisomerismABSTRACT
A novel series of potent CGRP receptor antagonists containing a central quinoline ring constraint was identified. The combination of the quinoline constraint with a tricyclic benzimidazolinone left hand fragment produced an analog with picomolar potency (14, CGRP K(i)=23 pM). Further optimization of the tricycle produced a CGRP receptor antagonist that exhibited subnanomolar potency (19, CGRP K(i)=0.52 nM) and displayed a good pharmacokinetic profile in three preclinical species.
Subject(s)
Calcitonin Gene-Related Peptide Receptor Antagonists , Quinolines/pharmacology , Animals , Biological Availability , Dogs , Drug Evaluation, Preclinical , Macaca mulatta , Quinolines/chemistry , Quinolines/pharmacokinetics , RatsABSTRACT
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.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Calcitonin Gene-Related Peptide Receptor Antagonists , Spiro Compounds/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Autoradiography , Binding, Competitive , Biological Transport , Brain/metabolism , Bridged Bicyclo Compounds, Heterocyclic/blood , Bridged Bicyclo Compounds, Heterocyclic/cerebrospinal fluid , Cell Line , Chlorocebus aethiops , Female , Humans , Kinetics , Macaca mulatta , Male , Mice , Radioligand Assay , Receptors, Adrenomedullin , Receptors, Calcitonin/metabolism , Receptors, Calcitonin Gene-Related Peptide/physiology , Receptors, Islet Amyloid Polypeptide , Receptors, Peptide/metabolism , Spiro Compounds/blood , Spiro Compounds/cerebrospinal fluid , Vasodilation/drug effectsABSTRACT
Incorporation of polar functionality into a series of highly potent calcitonin gene-related peptide (CGRP) receptor antagonists was explored in an effort to improve pharmacokinetics. This strategy identified piperazinone analogues that possessed improved solubility at acidic pH and increased oral bioavailability in monkeys. Further optimization led to the discovery of the clinical candidate 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) (4), the most potent orally active CGRP receptor antagonist described to date.
ABSTRACT
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.
Subject(s)
Acetamides/chemistry , Calcitonin Gene-Related Peptide Receptor Antagonists , Spiro Compounds/chemistry , Acetamides/chemical synthesis , Acetamides/pharmacology , Animals , Cell Line , Drug Design , Humans , Rats , Receptors, Calcitonin Gene-Related Peptide/metabolism , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacology , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Calcitonin Gene-Related Peptide Receptor Antagonists , Heterocyclic Compounds, 3-Ring/pharmacokinetics , Spiro Compounds/pharmacokinetics , Administration, Oral , Animals , Biological Availability , Cell Line , Dogs , Haplorhini , Heterocyclic Compounds, 3-Ring/administration & dosage , Heterocyclic Compounds, 3-Ring/chemistry , Humans , Rats , Receptors, Calcitonin Gene-Related Peptide/metabolism , Spiro Compounds/administration & dosageABSTRACT
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 K(i)=40pM). The closely related compound 27 demonstrated good oral bioavailability in dog and rhesus.
Subject(s)
Calcitonin Gene-Related Peptide Receptor Antagonists , Indoles/chemical synthesis , Spiro Compounds/chemical synthesis , Administration, Oral , Animals , Biological Availability , Dogs , Drug Discovery , Humans , Indoles/pharmacology , Macaca mulatta , Oxindoles , Spiro Compounds/pharmacology , Structure-Activity RelationshipABSTRACT
The previously disclosed spirohydantoin-based CGRP receptor antagonists were optimized for potency through modification of the benzimidazolone substituents. Compounds were identified which had minimal shift in the cAMP functional assay containing 50% human serum. Blockade of CGRP-mediated vasodilation was observed with these compounds in a rhesus pharmacodynamic assay and the in vivo potency correlated with the in vitro activity in the serum-shifted functional assay.
Subject(s)
Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Calcitonin Gene-Related Peptide Receptor Antagonists , Indans/chemistry , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacology , Animals , Benzimidazoles/blood , Benzimidazoles/chemistry , Combinatorial Chemistry Techniques , Humans , Macaca mulatta , Molecular Structure , Spiro Compounds/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Azepines/administration & dosage , Azepines/chemistry , Calcitonin Gene-Related Peptide Receptor Antagonists , Imidazoles/administration & dosage , Imidazoles/chemistry , Migraine Disorders/drug therapy , Administration, Oral , Animals , Cell Line , Dogs , Dose-Response Relationship, Drug , Female , Humans , Macaca mulatta , Male , Migraine Disorders/metabolism , Protein Binding/drug effects , Protein Binding/physiology , Rats , Receptors, Calcitonin Gene-Related Peptide/metabolismABSTRACT
A rapid analogue approach to identification of spirohydantoin-based CGRP antagonists provided novel, low molecular weight leads. Modification of these leads afforded a series of nanomolar benzimidazolinone-based CGRP receptor antagonists. The oral bioavailability of these antagonists was inversely correlated with polar surface area, suggesting that membrane permeability was a key limitation to absorption. Optimization provided compound 12, a potent CGRP receptor antagonist (K(i)=21nM) with good oral bioavailability in three species.
Subject(s)
Calcitonin Gene-Related Peptide Receptor Antagonists , Hydantoins/pharmacology , Hydantoins/pharmacokinetics , Spiro Compounds/pharmacology , Spiro Compounds/pharmacokinetics , Administration, Oral , Benzimidazoles/chemistry , Benzimidazoles/pharmacokinetics , Benzimidazoles/pharmacology , Biological Availability , Cell Line , Humans , Hydantoins/chemistry , Kidney , Models, Molecular , Molecular Structure , Spiro Compounds/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Benzodiazepinones/pharmacology , Calcitonin Gene-Related Peptide Receptor Antagonists , Animals , Benzodiazepinones/pharmacokinetics , Biological Availability , Cell Line , Humans , Hydrogen Bonding , Rats , Structure-Activity RelationshipABSTRACT
Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/genetics , RNA, Viral/antagonists & inhibitors , Ribonucleosides/chemical synthesis , Adenosine Deaminase/chemistry , Administration, Oral , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Biological Availability , Cell Line , Drug Stability , Models, Molecular , Molecular Conformation , Molecular Structure , Phosphorylation , Purine-Nucleoside Phosphorylase/chemistry , RNA, Viral/biosynthesis , Rats , Ribonucleosides/chemistry , Ribonucleosides/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2'-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5'-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2'-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2'-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2'-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.