Inhibitors of hepatitis C virus polymerase: Synthesis and characterization of novel 2-oxy-6-fluoro-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamides

SAR exploration from an initial hit, (S)-N-(2-cyclohexenylethyl)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)benzamide (1), identified using our proprietary automated ligand identification system (ALIS),¹ has led to a novel series of selective hepatitis C virus (HCV) NS5B polymerase inhibitors with improved in vitro potency as exemplified by (S)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)-N-isopentyl-N-methylbenzamidecarboxamide (41) (IC₅₀ = 0.5 μM). The crystal structure of an analogue (44) was solved and provided rationalization of the SAR of this series, which binds in a distinct manner in the palm domain of NS5B, consistent with biochemical analysis using enzyme mutant variants. These data warrant further lead optimization efforts on this novel series of non-nucleoside inhibitors targeting the HCV polymerase.     

N-Acetamideindolecarboxylic acid allosteric ‘finger-loop’ inhibitors of the hepatitis C virus NS5B polymerase: discovery and initial optimization studies

SAR studies at the N¹-position of allosteric indole-based HCV NS5B inhibitors has led to the discovery of acetamide derivatives with good cellular potency in subgenomic replicons (EC₅₀ <200 nM). This class of inhibitors displayed improved physicochemical properties and favorable ADME-PK profiles over previously described analogs in this class.     

Identification of novel inhibitors of HCV RNA-dependent RNA polymerase by pharmacophore-based virtual screening and in vitro evaluation

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B hepatitis where no effective treatment is available. The HCV NS5B with RNA-dependent RNA polymerase (RdRp) activity is a key target for the treatment of HCV infection. Here we report novel NS5B polymerase inhibitors identified by virtual screening and in vitro evaluation of their inhibitory activities. On the basis of a newly identified binding pocket of NS5B, distinct from the nucleotide binding site but highly conserved among various HCV isolates, we performed virtual screening of compounds that fit this binding pocket from the available chemical database of 3.5 million compounds. The inhibitory activities of the in silico selected 119 compounds were estimated with in vitro RdRp assay. Three compounds with IC50 values of about 20 μM were identified, and their kinetic analyses suggest that these compounds are noncompetitive inhibitors with respect to the ribonucleotide substrate. Furthermore, the single-point mutations of the conserved residues in the binding pocket of NS5B resulted in the significant decrease of the RdRp activity, indicating that the binding pocket presented here might be important for the therapeutic intervention of HCV. These novel inhibitors would be useful for the development of effective anti-HCV agents.     

Discovery of novel potent HCV NS5B polymerase non-nucleoside inhibitors bearing a fused benzofuran scaffold

This letter describes the discovery of a fused benzofuran scaffold viable for preparing a series of novel potent HCV NS5B polymerase non-nucleoside inhibitors. Designed on the basis of the functionalized benzofuran derivative nesbuvir (HCV-796), these compounds presumably bind similarly to the allosteric binding site in the “palm” domain of HCV NS5B protein. SAR of each potential hydrogen-bonding interaction site of this novel scaffold is discussed along with some preliminary genotypic profile and PK data of several advanced compounds.     

Inhibitors for the hepatitis C virus RNA polymerase explored by SAR with advanced machine learning methods

Hepatitis C virus (HCV) is a global health challenge, affecting approximately 200 million people worldwide. In this study we developed SAR models with advanced machine learning classifiers Random Forest and k Nearest Neighbor Simulated Annealing for 679 small molecules with measured inhibition activity for NS5B genotype 1b. The activity was expressed as a binary value (active/inactive), where actives were considered molecules with IC₅₀ ?0.95 μM. We applied our SAR models to various drug-like databases and identified novel chemical scaffolds for NS5B inhibitors. Subsequent in vitro antiviral assays suggested a new activity for an existing prodrug, Candesartan cilexetil, which is currently used to treat hypertension and heart failure but has not been previously tested for anti-HCV activity. We also identified NS5B inhibitors with two novel non-nucleoside chemical motifs.     

Design,synthesis, assessment,and molecular docking of novel pyrrolopyrimidine (7-deazapurine) derivatives as non-nucleoside hepatitis C virus NS5B polymerase inhibitors

Hepatitis C virus (HCV) infection is highly persistent and presents an unmet medical need requiring more effective treatment options. This has spurred intensive efforts to discover novel anti-HCV agents. The RNA-dependent RNA polymerase (RdRp), NS5B of HCV, constitutes a selective target for drug discovery due to its absence in human cells; also, it is the centerpiece for viral replication. Here, we synthesized novel pyrrole, pyrrolo[2,3-d]pyrimidine and pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives. The non-toxic doses of these compounds on Huh 7.5 cell line were determined and their antiviral activity against HCVcc genotype 4a was examined. Compounds 7j, 7f, 5c, 12i and 12f showed significant anti HCV activity. The percent of reduction for the non-toxic doses of 7j, 7f, 5c, 12i and 12f were 90%, 76.7 ± 5.8%, 73.3 ± 5.8%, 70% and 63.3 ± 5.8%, respectively. The activity of these compounds was interpreted by molecular docking against HCV NS5B polymerase enzyme.     

1,5-Benzodiazepine inhibitors of HCV NS5B polymerase

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC₅₀ = 400 nM and 270 nM, respectively) and selective (CC₅₀ > 20 μM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.     

5,6-Dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase

5,6-Dihydro-1H-pyridin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4ad displayed potent inhibitory activities in biochemical and replicon assays (IC₅₀ (1b) < 10 nM; IC₅₀ (1a) < 25 nM, EC₅₀ (1b) = 16 nM), good in vitro DMPK properties, as well as moderate oral bioavailability in monkeys (F = 24%).     

New hybrid molecules combining benzothiophene or benzofuran with rhodanine as dual COX-1/2 and 5-LOX inhibitors: Synthesis,biological evaluation and docking study

New molecular hybrids combining benzothiophene or its bioisostere benzofuran with rhodanine were synthesized as potential dual COX-2/5-LOX inhibitors. The benzothiophene or benzofuran scaffold was linked at position -2 with rhodanine which was further linked to various anti-inflammatory pharmacophores so as to investigate the effect of such molecular variation on the anti-inflammatory activity. The target compounds were evaluated for their in vitro COX/LOX inhibitory activity. The results revealed that, compound 5h exhibited significant COX-2 inhibition higher than celecoxib. Furthermore, compounds 5a, 5f and 5i showed COX-2 inhibitory activity comparable to celecoxib. Compound 5h showed selectivity index SI = 5.1 which was near to that of celecoxib (SI = 6.7). Compound 5h displayed LOX inhibitory activity twice than that of meclofenamate sodium. Moreover, compounds 5a, 5e and 5f showed significant LOX inhibitory activity higher than that of meclofenamate sodium. Compound 5h was screened for its in vivo anti-inflammatory activity using formalin-induced paw edema and gastric ulcerogenic activity tests. The results revealed that, it showed in vivo decrease in formalin-induced paw edema volume higher than celecoxib. It also displayed gastrointestinal safety profile as celecoxib. The biological results were also consistent with the docking studies at the active sites of the target enzymes COX-2 and 5-LOX. Also, compound 5h showed physicochemical, ADMET, and drug-like properties within those considered adequate for a drug candidate.     

Isosorbide-based peptidomimetics as inhibitors of hepatitis C virus serine protease

Hepatitis C infection is a cause of chronic liver diseases such as cirrhosis and carcinoma. The current therapy for hepatitis C has limited efficacy and low tolerance. The HCV encodes a serine protease which is critical for viral replication, and few protease inhibitors are currently on the market. In this paper, we describe the synthesis and screening of novel isosorbide-based peptidomimetic inhibitors, in which the compounds 1d, 1e, and 1i showed significant inhibition of the protease activity in vitro at 100 µM. The compound 1e also showed dose-response (IC₅₀ = 36 ± 3 µM) and inhibited the protease mutants D168A and V170A at 100 µM, indicating it as a promising inhibitor of the HCV NS3/4A protease. Our molecular modeling studies suggest that the activity of 1e is associated with a change in the interactions of S2 and S4 subsites, since that the increased flexibility favors a decrease in activity against D168A, whereas the appearance of a hydrophobic cavity in the S4 subsite increase the inhibition against V170A strain.     

The synthesis and SAR study of phenylalanine-derived (Z)-5-arylmethylidene rhodanines as anti-methicillin-resistant Staphylococcus aureus (MRSA) compounds

A focused library of rhodanine compounds containing novel substituents at the C5-position was synthesized and tested in vitro against a panel of clinically relevant MRSA strains. The present SAR study was based on our lead compound 1 (MIC = 1.95 μg/mL), with a focus on identifying optimal C5-arylidene substituents. In order to obtain this objective, we condensed several unique aromatic aldehydes with phenylalanine-derived rhodanine intermediates to obtain C5-substituted target rhodanine compounds for evaluation as anti-MRSA compounds. These efforts produced three compounds with significant efficacy: 23, 32 and 44, with MIC values ranging from 0.98 to 1.95 μg/mL against all tested MRSA strains as compared to the reference antibiotics penicillin G (MIC = 15.60–250.0 μg/mL) and ciprofloxacin (MIC = 7.80–62.50 μg/mL) and comparable to that of vancomycin (MIC = 0.48 μg/mL). In addition, compounds 24, 28, 37, 41, 46 and 48 (MIC = 1.95–3.90 μg/mL) were efficacious against all MRSA strains. The majority of the synthesized compounds had bactericidal activity at concentrations only two to fourfold higher than their MIC. Overall, the results suggest that compounds 23, 32 and 44 may be of potential use in the treatment of MRSA infections.     

Synthesis and biological evaluation of N9-cis-cyclobutylpurine derivatives for use as cyclin-dependent kinase (CDK) inhibitors

A novel 6-aminopurine scaffold bearing an N9-cis-cyclobutyl moiety was designed using structure-based molecular design based on two known CDK inhibitors, dinaciclib and Cmpd-27. A series of novel 6-aminopurine compounds was prepared for structure–activity relationship (SAR) studies of CDK2 and CDK5 inhibitors. Among the compounds synthesized, compound 8l displayed potent CDK2 and CDK5 inhibitory activities with low nanomolar ranges (IC₅₀ = 2.1 and 4.8 nM, respectively) and showed moderate cytotoxicity in HCT116 colon cancer and MCF7 breast cancer cell lines. Here, we report the synthesis and evaluation of novel 6-aminopurine derivatives and present molecular docking models of compound 81 with CDK2 and CDK5.     

Design and evaluation of novel tetracyclic benzofurans as palm site allosteric inhibitors of HCV NS5B polymerase

Hepatitis C virus (HCV) NS5B polymerase is a prime target for the development of direct-acting antiviral drugs for the treatment of chronic HCV infection. Several novel and potent HCV NS5B non-nucleoside inhibitors with unique tetracyclic bezonfuran-based structures were prepared and evaluated. Similar to clinical developmental compound MK-8876, N-linked (compounds 1 and 2) and C-linked (compounds 3 and 4) tetracyclic structures maintained broad spectrum anti-replicon potency profiles and demonstrated moderate to excellent oral bioavailability and pharmacokinetic parameters across the three preclinical animal species. To better understand the importance of tetracyclic structures related to pan genotypic potency profiles especially against clinically relevant GT1a variants, the teracycles with different ring size were prepared and in vitro evaluations suggested compounds with six number ring have better overall potency profiles.     

Analogs design,synthesis and biological evaluation of peptidomimetics with potential anti-HCV activity

Two series of peptidomimetics were designed, prepared and evaluated for their anti-HCV activity. One series possesses a C-terminal carboxylate functionality. In the other series, the electrophilic vinyl sulfonate moiety was introduced as a novel class of HCV NS3/4A protease inhibitors. In vitro based studies were then performed to evaluate the efficacies of the inhibitors using Human hepatoma cells, with the vinyl sulfonate ester (10) in particular, found to have highly potent anti-HCV activity with an EC₅₀ = 0.296 μM. Finally, molecular modeling studies were performed through docking of the synthesized compounds in the HCV NS3/4A protease active site to assess their binding modes with the enzyme and gain further insight into their structure–activity relationships.     

Anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors with sub-micromolar potency in the cell-based replicon assay

Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1 and 2 resulted in the identification of multiple structural elements that contributed to improved cell culture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic (IC₅₀) and cell culture (EC₅₀) potencies of less than 100 nanomolar.     

Discovery of novel HCV inhibitors: Synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4B

A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides was prepared and evaluated for their ability to inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series furnished compounds with low nanomolar potency against the HCV genotype 1b replicon. Among these, compound 8c has identified as a potent HCV replicon inhibitor (EC₅₀ = 4 nM) with a selectivity index with respect to cellular GAPDH of more than 2500. Further, compound 8c had a good pharmacokinetic profile in rats with an IV half-life of 6 h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance identified an amino acid substitution in HCV NS4B that confers resistance to these compounds. These compounds hold promise as a new chemotype with anti-HCV activity mediated through an underexploited viral target.     

Small molecule fusion inhibitors: Design,synthesis and biological evaluation of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives targeting HIV-1 gp41

By a scaffold elongation strategy, a series of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives with a linear multi-aromatic-ring skeleton were designed, synthesized, and evaluated in HIV-1 gp41 and cellular assays. Among them, the most active compounds, 12e, 12g, and 12k with a one-carbon linker (n = 1) between the rhodanine (C) and phenyl (D) rings, exhibited very promising inhibitory potency with IC₅₀ values of 1.8–2.6 μM and EC₅₀ values of 0.3–1.5 μM against gp41 6-HB formation and HIV-1 replication in MT-2 cells, respectively. Additionally, they were almost equally effective against both T20-sensitive and resistant strains. The related SAR studies and molecular modeling results provided potential for further developing a new class of non-peptide small molecular fusion inhibitors targeting the HIV-1 gp41.     

Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with dual NF-κB inhibitory and anti-proliferative activities

Twenty-five novel pregnenolone/2-cyanoacryloyl conjugates (6–30) were designed and prepared, with the aim of developing novel anticancer drugs with dual NF-κB inhibitory and anti-proliferative activities. Compounds 22 and 27–30 showed inhibition against TNF-α-induced NF-κB activation in luciferase assay, which was confirmed by Western blotting. Among them, compound 30 showed potent NF-κB inhibitory activity (IC₅₀ = 2.5 μM) and anti-proliferative against MCF-7, A549, H157, and HL-60 cell lines (IC₅₀ = 6.5–36.2 μM). The present study indicated that pregnenolone/2-cyanoacryloyl conjugate I can server as a novel scaffold for developing NF-κB inhibitors and anti-proliferative agents in cancer chemotherapy.     

A 2,6,9-hetero-trisubstituted purine inhibitor exhibits potent biological effects against multiple myeloma cells

A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was identified as being a promising inhibitor of MM tumour cells (IC₅₀’s <5 μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffold’s history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.     

Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit

A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC₅₀ < 0.1 μM. Cross-resistant analysis confirmed that the virus NS4B protein remained the target of the new oxopiperazine analogs obtained via scaffold morphing from the HTS hit 4.