ABSTRACT
The discovery of potent, bioavailable small molecule inhibitors of p53-HDM2 PPI led us to investigate subsequent modifications to address a CYP3A4 time-dependent inhibition liability. On the basis of the crystal structure of HDM2 in complex with 2, further functionalization of the solvent exposed area of the molecule that binds to Phe19 pocket were investigated as a strategy to modulate the molecule liphophilicity. Introduction of 2-oxo-nicotinic amide at Phe19 proved a viable strategy in obtaining inhibitors exempt from CYP3A4 time-dependent inhibition liability.
Subject(s)
Cytochrome P-450 CYP3A/metabolism , Phenylalanine/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Tumor Suppressor Protein p53/antagonists & inhibitors , Dose-Response Relationship, Drug , Humans , Molecular Structure , Phenylalanine/chemistry , Protein Binding/drug effects , Proto-Oncogene Proteins c-mdm2/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Tumor Suppressor Protein p53/metabolismABSTRACT
(2'R)-Ethynyl uridine 3, and its (2'S)-diastereomer 10, are synthesised in a divergent fashion from the inexpensive parent nucleoside. Both nucleoside analogues are obtained from a total of 5 simple synthetic steps and 3 trivial column chromatography purifications. To evaluate their effectiveness against HCV NS5B polymerase, the nucleosides were converted to their respective 5'-O-triphosphates. Subsequently, this lead to the discovery of the 2'-ß-ethynyl 18 and -propynyl 20 nucleotides having significantly improved potency over Sofosbuvir triphosphate 24.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Nucleosides/pharmacology , Uridine/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Conformation , Nucleosides/chemical synthesis , Nucleosides/chemistry , Structure-Activity Relationship , Uridine/analogs & derivatives , Uridine/chemistryABSTRACT
Led by the structural information of the screening hit with mDM2 protein, a structure modification of Leu26 moiety of the novel p53-hDM2 inhibitors was conducted. A structure-activity relationship study of 4-substituted piperidines revealed compound 20t with good potencies and excellent CYP450 profiles.
Subject(s)
Leucine/chemistry , Piperidines/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Tumor Suppressor Protein p53/antagonists & inhibitors , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Structure-Activity Relationship , Tumor Suppressor Protein p53/metabolismABSTRACT
A new subseries of substituted piperidines as p53-HDM2 inhibitors exemplified by 21 has been developed from the initial lead 1. Research focused on optimization of a crucial HDM2 Trp23-ligand interaction led to the identification of 2-(trifluoromethyl)thiophene as the preferred moiety. Further investigation of the Leu26 pocket resulted in potent, novel substituted piperidine inhibitors of the HDM2-p53 interaction that demonstrated tumor regression in several human cancer xenograft models in mice. The structure of HDM2 in complex with inhibitors 3, 10, and 21 is described.
ABSTRACT
Introduction of an aliphatic side chain to a key position of a novel piperidine-based HDM2 inhibitor scaffold resulted in significant potency gains, enabling further series progression.
ABSTRACT
The discovery of 3,3-disubstituted piperidine 1 as novel p53-HDM2 inhibitors prompted us to implement subsequent SAR follow up directed towards piperidine core modifications. Conformational restrictions and further functionalization of the piperidine core were investigated as a strategy to gain additional interactions with HDM2. Substitutions at positions 4, 5 and 6 of the piperidine ring were explored. Although some substitutions were tolerated, no significant improvement in potency was observed compared to 1. Incorporation of an allyl side chain at position 2 provided a drastic improvement in binding potency.
Subject(s)
Piperidines/chemical synthesis , Piperidines/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Tumor Suppressor Protein p53/antagonists & inhibitors , Biological Assay , Cell Proliferation/drug effects , Humans , Inhibitory Concentration 50 , Molecular Structure , Piperidines/chemistry , Protein Binding/drug effects , Proto-Oncogene Proteins c-mdm2/metabolism , Structure-Activity Relationship , Tumor Suppressor Protein p53/metabolismABSTRACT
In the search for a second generation HCV protease inhibitor, molecular modeling studies of the X-ray crystal structure of Boceprevir1 bound to the NS3 protein suggest that expansion into the S4 pocket could provide additional hydrophobic Van der Waals interactions. Effective replacement of the P4 tert-butyl with a cyclohexylmethyl ligand led to inhibitor 2 with improved enzyme and replicon activities. Subsequent modeling and SAR studies led to the pyridine 38 and sulfone analogues 52 and 53 with vastly improved PK parameters in monkeys, forming a new foundation for further exploration.
Subject(s)
Antiviral Agents/chemistry , Proline/analogs & derivatives , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Area Under Curve , Biological Availability , Crystallography, X-Ray , Haplorhini , Models, Molecular , Proline/chemistry , Proline/pharmacokinetics , Proline/pharmacology , Protease Inhibitors/pharmacokinetics , Protease Inhibitors/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
Hepatitis is a disease characterized by inflammation of the liver, usually producing swelling and, in many cases, permanent damage to liver tissues. Viral hepatitis C (HCV), a small (+)-RNA virus, infects chronically 3% of the world's population. Boceprevir, SCH 503034, (1) our first generation HCV inhibitor, has already established proof-of- concept and is currently in late stage (phase III) clinical trials. In view of the positive data from our first generation compound, further work aimed at optimizing its overall profile was undertaken. Herein, we report that extension of our earlier inhibitor to the P(4) pocket by introducing a new sulfonamide moiety and optimization of the P1/P(1)' capping led to the discovery of a novel series of inhibitors of the HCV NS3 serine protease. Optimization of the P(1) residue significantly improved potency and selectivity. The combination of optimal moieties led to the discovery of compound 47 which, in addition to being a potent inhibitor of HCV subgenomic RNA replication, was also found to have good PK profile in rat, dog and monkey.
Subject(s)
Amides/chemistry , Antiviral Agents/chemistry , Serine Proteinase Inhibitors/chemistry , Sulfonamides/chemistry , Urea/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacokinetics , Binding Sites , Computer Simulation , Dogs , Drug Evaluation, Preclinical , Escherichia coli Proteins , Haplorhini , Humans , Membrane Proteins , Models, Molecular , Rats , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacokinetics , Sulfonamides/chemical synthesis , Sulfonamides/pharmacokinetics , Urea/chemical synthesis , Urea/chemistry , Urea/pharmacokinetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effectsABSTRACT
Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (â¼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.
ABSTRACT
Hepatitis C is the most prevalent liver disease. Viral hepatitis C (HCV), a small (+)-RNA virus, infects chronically an estimated 300 million people worldwide. Results of Phase I clinical studies with our first generation HCV inhibitor Boceprevir, SCH 503034 (1), presented at the 56th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD) were encouraging, and thus, additional human clinical studies are underway. In view of the positive data from our first generation compound, further work aimed at optimizing its overall profile was undertaken. Herein, we report that extension of our earlier inhibitor to the P(4) pocket and optimization of the P(1)' capping led to the discovery of new ketoamide inhibitors of the HCV NS3 serine protease with improved in vitro potency. In addition to being potent inhibitors of HCV subgenomic RNA replication, some of the new P(4)-capped inhibitors were also found to have improved PK profile.
Subject(s)
Amides/pharmacology , Drug Discovery , Proline/analogs & derivatives , Serine Proteinase Inhibitors/pharmacology , Serine Proteinase Inhibitors/pharmacokinetics , Viral Nonstructural Proteins/antagonists & inhibitors , Amides/chemical synthesis , Amides/chemistry , Animals , Binding Sites , Genome, Viral/drug effects , Hepacivirus/drug effects , Hepacivirus/enzymology , Hepacivirus/genetics , Models, Molecular , Molecular Conformation , Proline/chemistry , Proline/pharmacology , RNA, Viral/drug effects , Rats , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/chemistry , Stereoisomerism , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
Hepatitis C virus (HCV) infection is a global health crisis leading to liver cirrhosis, hepatocellular carcinoma, and liver failure in humans. Recently, we disclosed the discovery of Boceprevir, SCH 503034 (1), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that is currently undergoing phase III clinical trials. Our efforts toward a second generation HCV NS3 serine protease inhibitor were directed at improving the overall profile of the inhibitor. This article will elaborate on our studies leading to the discovery of new P4 modified inhibitors with enhanced potency and improved oral bioavailability. Thus, introduction of ether and carbamate-derived P4 moieties resulted in improving the replicon potency significantly. Incorporation of the P' secondary amide residue afforded significant improvement in pharmacokinetic properties. Combining the preferred moieties, identified from comprehensive SAR studies, resulted in inhibitors that displayed superior potency and very good oral as well as target organ exposure in rats.
Subject(s)
Drug Discovery , Hepacivirus/enzymology , Protease Inhibitors/pharmacology , Protease Inhibitors/pharmacokinetics , Viral Nonstructural Proteins/antagonists & inhibitors , Amides/chemistry , Animals , Models, Molecular , Molecular Conformation , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Rats , Structure-Activity RelationshipABSTRACT
The hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. The moderate efficacy along with side effects of the current pegylated interferon and ribavirin combination therapy underscores the need for more effective and safer new treatment. In an effort to improve upon our current clinical candidate, Boceprevir (SCH 503034), extensive SAR studies were performed on the P3 capping moieties. This led to the discovery of tert-leucinol derived cyclic imides as a potent series of novel P3 capping groups. Thus, the introduction of these imide caps improved the cell-based replicon EC(90) by more than 10-fold. A number of imides with various substitutions, ring sizes, bicyclic systems, and heterocyclic rings were explored. The 4,4-dimethyl substituted glutarimide emerged as the best cap as exemplified in compound 21 (K(i)* = 4 nM, EC(90) = 40 nM). Systematic optimization of different positions (P', P3, and P1) of the inhibitor resulted in the identification of the lead compound 46, which had an excellent potency (K(i)* = 4 nM, EC(90) = 30 nM) and good pharmacokinetic profile (22% and 35% bioavailability in rats and dogs, respectively). X-ray structure of inhibitor 46 bound to the enzyme revealed that there was an additional hydrogen bonding interaction between one of the imide carbonyls and Cys159.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/enzymology , Piperidones/chemical synthesis , Serine Proteinase Inhibitors/chemical synthesis , Urea/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Biological Availability , Crystallography, X-Ray , Dogs , Haplorhini , Hepacivirus/genetics , Hydrogen Bonding , Imides/chemical synthesis , Imides/chemistry , Leucine/analogs & derivatives , Leucine/chemical synthesis , Leucine/chemistry , Models, Molecular , Piperidones/pharmacokinetics , Piperidones/pharmacology , Rats , Serine Proteinase Inhibitors/pharmacokinetics , Serine Proteinase Inhibitors/pharmacology , Stereoisomerism , Structure-Activity Relationship , Urea/chemical synthesis , Urea/pharmacokinetics , Urea/pharmacologyABSTRACT
The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials.
Subject(s)
Hepacivirus/enzymology , Proline/analogs & derivatives , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Models, Molecular , Molecular Structure , Proline/chemistryABSTRACT
The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.
Subject(s)
Antiviral Agents/chemical synthesis , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/chemistry , Hepacivirus/enzymology , Proline/analogs & derivatives , Serine Proteinase Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , Antiviral Agents/chemistry , Binding Sites , Crystallography, X-Ray , Intracellular Signaling Peptides and Proteins , Models, Molecular , Proline/chemical synthesis , Proline/chemistry , Protein Conformation , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or polyethylene glycol (PEG)-interferon-alpha alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virological response. Herein, the SAR leading to the discovery of 70 (SCH 503034), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been advanced to clinical trials in human beings for the treatment of hepatitis C viral infections is described. X-ray structure of inhibitor 70 complexed with the NS3 protease and biological data are also discussed.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/enzymology , Proline/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Administration, Oral , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Area Under Curve , Binding Sites , Biological Availability , Crystallography, X-Ray , Dogs , Haplorhini , Molecular Structure , Proline/chemical synthesis , Proline/chemistry , Proline/pharmacokinetics , Rats , Structure-Activity Relationship , Tissue Distribution , Viral Nonstructural Proteins/chemistryABSTRACT
Introduction of various modified prolines at P(2) and optimization of the P(1) side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K(i)*= 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC(50) and IC(90) of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.
Subject(s)
Amides/chemistry , Amides/pharmacology , Genome, Viral/genetics , Hepacivirus/drug effects , Oligopeptides/chemistry , Oligopeptides/pharmacology , Serine Endopeptidases/metabolism , Animals , Haplorhini , Hepacivirus/enzymology , Hepacivirus/genetics , Models, Molecular , Molecular Structure , RNA, Viral/genetics , Rats , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolismABSTRACT
Depeptidization efforts of the P(3)-P(2) region of P(3) capped alpha-ketoamide inhibitor of HCV NS3 serine protease 1 are reported. We clearly established that N-methylation of the P(2) nitrogen and modification of the P(2)' carboxylic acid terminus were essential for activity in the replicon assay.
Subject(s)
Amides , Hepacivirus/drug effects , Nitrogen/chemistry , Replicon/drug effects , Serine Proteinase Inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Amides/chemical synthesis , Amides/chemistry , Amides/pharmacology , Binding Sites , Hepacivirus/chemistry , Hepacivirus/enzymology , Methylation , Molecular Structure , Protein Binding , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistry , X-Ray DiffractionABSTRACT
Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P'-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.
Subject(s)
Carrier Proteins/antagonists & inhibitors , Drug Resistance/genetics , Enzyme Inhibitors/pharmacology , Mutation , Oligopeptides/pharmacology , RNA, Viral/genetics , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Binding Sites , Blotting, Western , Cell Line, Tumor , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Genetic Vectors , Genotype , Humans , Inhibitory Concentration 50 , Intracellular Signaling Peptides and Proteins , Kinetics , Models, Chemical , Models, Molecular , Oligopeptides/chemistry , Polyproteins/chemistry , Protein Binding , Protein Conformation , Protein Structure, Tertiary , RNA/chemistry , Recombinant Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Structure-Activity Relationship , Time Factors , TransfectionABSTRACT
Photochemically generated acyl nitrenes undergo facile addition to 4,5-dihydrofurans 20 and 24b to yield the novel 2-ethoxyoxazolines 21 and 25. The regiocontrolled C=C insertion has provided for introduction of the sterically hindered C-3 amido appendage of the lankacidins 1-4 with high stereoselectivity. High chemoselectivity for the C=C insertion pathway was demonstrated upon production of the acyl nitrene intermediate from azide 33b. Intramolecular competition for allylic C(3)-H insertion versus C=C addition yielded exclusive formation of seven-membered N-acyl aziridines 34a,b. The latent aldehydic functionality of oxazolines such as 21 and 25 is exposed upon a brief hydrolysis, permitting further chemical elaboration. Wittig condensation of the lactol from 25 has led to the synthesis of the lactone fragment 5, containing all of the necessary stereochemistry and functionality for incorporation into the lankacidin antibiotics. The acyl nitrene insertion into 4,5-dihydrofurans affords a route toward unusual beta-amido acids and amino sugar derivatives as shown via stereocontrolled formation of the amidofuranose derivatives 31 and 32. The three-step process of acyl nitrene addition, hydrolysis of the resulting 2,5-dialkoxy oxazoline intermediates, and Wittig carbon chain elongation provides the stereocontrolled formation of novel beta-amido esters.