Design, synthesis and biological activity of amidinobicyclic compounds (derivatives of DX-9065a) as factor Xa inhibitors: SAR study of S1 and aryl binding sites

Since factor Xa (fXa) plays a pivotal role in the blood coagulation cascade, inhibition of fXa is thought to be an effective treatment for a variety of thrombotic events. (2S)-2-[4-[[(3S)-1-Acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-amidino-2-naphthyl)propanoic acid hydrochloride pentahydrate (DX-9065a) was previously found in our laboratory as a novel orally active factor Xa inhibitor. DX-9065a exhibits a strong inhibitory activity toward fXa by occupying the substrate recognition (called S1) sites and aryl binding sites of fXa. Herein we describe conversions of the amidinonaphthalene and the acetimidoylpyrrolidine moieties of DX-9065a. Some compounds showed remarkably increased in vitro anti-factor Xa and PRCT activities compared with those of DX-9065a. The most promising compound 38 showed four times the prolongation of APTT against DX-9065a after oral administration to rats.     

1-(2-Naphthyl)-1H-pyrazole-5-carboxylamides as potent factor Xa inhibitors. Part 3: Design, synthesis and SAR of orally bioavailable benzamidine-P4 inhibitors

Using N,N-dialkylated benzamidines as the novel P4 motifs, we have designed and synthesized a class of 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides as highly potent and selective fXa inhibitors with significantly improved hydrophilicity and in vitro anticoagulant activity. These benzamidine-P4 fXa inhibitors have displayed excellent oral bioavailability and long half-life.     

Studies on the different modes of action of the anticoagulant protease inhibitors DX-9065a and Argatroban. I. Effects on thrombin generation

The present study began with mathematical modeling of how inhibitors of both factor Xa (fXa) and thrombin affect extrinsic pathway-triggered blood coagulation. Numerical simulation demonstrated a stronger inhibition of thrombin generation by a thrombin inhibitor than a fXa inhibitor, but both prolonged clot time to a similar extent when they were given an equal dissociation constant (30 nm) for interaction with their respective target enzymes. These differences were then tested by comparison with the real inhibitors DX-9065a and argatroban, specific competitive inhibitors of fXa and thrombin, respectively, with similar K(i) values. Comparisons were made in extrinsically triggered human citrated plasma, for which endogenous thrombin potential and clot formation were simultaneously measured with a Wallac multilabel counter equipped with both fluorometric and photometric detectors and a fluorogenic reporter substrate. The results demonstrated stronger inhibition of endogenous thrombin potential by argatroban than by DX-9065a, especially when coagulation was initiated at higher tissue factor concentrations, while argatroban appeared to be slightly less potent in its ability to prolong clot time. This study demonstrates differential inhibition of thrombin generation by fXa and thrombin inhibitors and has implications for the pharmacological regulation of blood coagulation by the anticoagulant protease inhibitors.     

A novel,potent, and orally active VLA-4 antagonist with good aqueous solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.     

Novel strategy to boost oral anticoagulant activity of blood coagulation enzyme inhibitors based on biotransformation into hydrophilic conjugates

The blood coagulation cascade represents an attractive target for antithrombotic drug development, and recent studies have attempted to identify oral anticoagulants with inhibitory activity for enzymes in this cascade, with particular attention focused on thrombin and factor Xa (fXa) as typical targets. We previously described the discovery of the orally active fXa inhibitor darexaban (1) and reported a unique profile that compound 1 rapidly transformed into glucuronide YM-222714 (2) after oral administration. Here, we propose a novel strategy towards the discovery of an orally active anticoagulant that is based on the bioconversion of a non-amidine inhibitor into the corresponding conjugate to boost ex vivo anticoagulant activity via an increase in hydrophilicity. Computational molecular modeling was utilized to select a template scaffold and design a substitution point to install a potential functional group for conjugation. This strategy led to the identification of the phenol-derived fXa inhibitor ASP8102 (14), which demonstrated highly potent anticoagulant activity after biotransformation into the corresponding glucuronide (16) via oral dosing.     

Design,synthesis and biological evaluation of anthranilamide derivatives as potential factor Xa (fXa) inhibitors

Factor Xa (fXa) is a crucial player in various thromboembolic disorders. Inhibition of fXa can provide safe and effective antithrombotic effects. In this study, a series of anthranilamide compounds were designed by utilizing structure-based design strategies. Optimization at P1 and P4 groups led to the discovery of compound 16g: a highly potent, selective fXa inhibitor with pronounced in vitro anticoagulant activity. Moreover, 16g also displayed excellent in vivo antithrombotic activity in the rat venous thrombosis (VT) and arteriovenous shunt (AV-SHUNT) models. The bleeding risk evaluation showed that 16g had a safer profile than that of betrixaban at 1?mg/kg and 5?mg/kg dose. Additionally, 16g also exhibited satisfactory PK profiles. Eventually, 16g was selected to investigate its effect on hypoxia-reoxygenation- induced H9C2 cell viability. MTT results showed that H9C2 cell viability can be remarkably alleviated by 16g.     

1-(2-Naphthyl)-1H-pyrazole-5-carboxylamides as potent factor Xa inhibitors. Part 2: A survey of P4 motifs

A variety of P4 motifs have been examined to increase the binding affinity and in vitro anticoagulant potency of our biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamide-based fXa inhibitors. Highly potent 2-naphthyl-P1 fXa inhibitors (K(i)< or =2 nM) with improved in vitro anticoagulant activity (2xTG< or =1 microM) and respectable pharmacokinetic properties have been discovered.     

(3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone: A potent,selective, orally active dipeptidyl peptidase IV inhibitor

A series of 4-substituted proline amides was synthesized and evaluated as inhibitors of dipeptidyl pepdidase IV for the treatment of type 2 diabetes. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone (5) emerged as a potent (IC₅₀ = 13 nM) and selective compound, with high oral bioavailability in preclinical species and low plasma protein binding. Compound 5, PF-00734200, was selected for development as a potential new treatment for type 2 diabetes.     

Discovery of orally bioavailable imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors

We report a series of potent imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors. Optimization of the solvent accessible 8-position led to improvements in both oral bioavailability and off-target kinase inhibition. Compound 25 demonstrates anti-tumor activity in an A2780 ovarian tumor xenograft model.     

Design and synthesis of positional isomers of 5 and 6-bromo-1-[(phenyl)sulfonyl]-2-[(4-nitrophenoxy)methyl]-1H-benzimidazoles as possible antimicrobial and antitubercular agents

In this Letter, we report the structure–activity relationship (SAR) studies on series of positional isomers of 5(6)-bromo-1-[(phenyl)sulfonyl]-2-[(4-nitrophenoxy)methyl]-1H-benzimidazoles derivatives 7(a–j) and 8(a–j) synthesized in good yields and characterized by ¹H NMR, ¹³C NMR and mass spectral analyses. The crystal structure of 7a was evidenced by X-ray diffraction study. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus, (Gram-positive), Escherichia coli and Klebsiella pneumoniae (Gram-negative), antifungal activity against Candida albicans, Aspergillus flavus and Rhizopus sp. and antitubercular activity against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis, Mycobacterium fortuitum and MDR-TB strains. The synthesized compounds displayed interesting antimicrobial activity. The compounds 7b, 7e and 7h displayed significant activity against Mycobacterium tuberculosis H37Rv strain.     

(2S,4S)-1-[2-(1,1-Dimethyl-3-oxo-3-pyrrolidin-1-yl-propylamino)acetyl]-4-fluoro-pyrrolidine-2-carbonitrile: A potent,selective, and orally bioavailable dipeptide-derived inhibitor of dipeptidyl peptidase IV

A series of 2-[3-[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino]-3-methyl-1-oxobutyl]-based DPP-IV inhibitors with various monocyclic amines were synthesized. The structure–activity relationships (SAR) led to the discovery of potent DPP-IV inhibitors, having IC₅₀ values of <100 nM with excellent selectivity over the closely related enzymes, DPP-II, DPP8, DPP9 and FAP (IC₅₀ > 20 μM). Of these compounds, the analogues 12a, 12h and 12i exhibited a long-lasting ex vivo DPP-IV inhibition in rats.     

The design,synthesis and evaluation of 2-aminobenzoxazole analogues as potent and orally efficacious ChemR23 inhibitors

We previously reported 2-aminobenzoxazole analogue 1 as a potent ChemR23 inhibitor. The compound showed inhibitory activity against chemerin-induced calcium signaling through ChemR23 internalization in CAL-1 cells, which are cell lines of plasmacytoid dendric cells (pDCs). Furthermore, compound 2 inhibited chemotaxis of CAL-1 triggered by chemerin in vitro. However, we noted a difference in the ChemR23 response to our inhibitor between rodents and non-rodents in a previous study. To address this issue, we performed optimization of ChemR23 inhibitors using CAL-1 cells endogenously expressing human ChemR23 and conducted a pharmacokinetics study in cynomolgus monkeys. Various substituents at the 4-position of the benzoxazole ring exhibited potent in vitro bioactivity, while those at the 6-position were not tolerated. Among substituents, a carboxyl group was identified as key for improving the oral bioavailability in cynomolgus monkeys. Compound 38a with the acidic part changed from a tetrazole group to a 1,2,4-oxadiazol-5-one group to improve bioactivity and pharmacokinetic parameters exhibited inhibitory activity against chemerin-induced chemotaxis in vitro. In addition, we confirmed the ChemR23 internalization of pDCs by compound 38a orally administered to cynomolgus monkeys. These 2-aminobenzoxazole-based ChemR23 inhibitors may be useful as novel immunotherapeutic agents capable of suppressing the migration of pDCs, which are known to be major producers of type I interferons in the lesion area of certain autoimmune diseases, such as systemic lupus erythematosus and psoriasis.     

Discovery of 9-(1-anilinoethyl)-2-morpholino-4-oxo-pyrido[1,2-a]pyrimidine-7-carboxamides as PI3Kβ/δ inhibitors for the treatment of PTEN-deficient tumours

Starting from TGX-221, we designed a series of 9-(1-anilinoethyl)-2-morpholino-4-oxo-pyrido[1,2-a]pyrimidine-7-carboxamides as potent and selective PI3Kβ/δ inhibitors. Structure–activity relationships and structure–property relationships around the aniline and the amide substituents are discussed. We identified compounds 17 and 18, which showed profound pharmacodynamic modulation of phosphorylated Akt in the PC3 prostate tumour xenograft, after a single oral dose. Compound 17 also gave significant inhibition of tumour growth in the PC3 prostate tumour xenograft model after chronic oral dosing.     

Discovery of 5-substituent-N-arylbenzamide derivatives as potent,selective and orally bioavailable LRRK2 inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson’s disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.     

Prodrug-based design, synthesis, and biological evaluation of N-benzenesulfonylpiperidine derivatives as novel, orally active factor Xa inhibitors

We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding element. This series resulted in compound 39, which exhibited potent inhibitory activity against fXa (IC50 = 13 nM) and excellent selectivity over thrombin (>7000-fold). The masking of its highly hydrophilic groups led to the creation of related prodrug 28, which demonstrated an anticoagulant effect after oral dosing.     

Design,synthesis, and pharmacological evaluation of 2-(4-sulfonylphenyl)-2-[(E)-pyrrolidin-1-ylimino]-N-thiazoleacetamides as glucokinase activators

This paper presents the synthesis and glucokinase activity of novel hydrazone derivatives. The 2-(4-cyclopropylsulfonylphenyl)-2-[(E)-pyrrolidin-1-ylimino]-acetamide derivatives 5a–5h presented the in vitro glucokinase activities and in vivo blood glucose-lowering effects in mice. Particularly, 5h showed an oral hypoglycemic effect in rats at 1 mg/kg. These hydrazone derivatives are a potential new class of glucokinase activators for the treatment of type 2 diabetes.     

Structure activity relationship studies of 3-arylsulfonyl-pyrido[1,2-a]pyrimidin-4-imines as potent 5-HT6 antagonists

Comprehensive structure activity relationship (SAR) studies were conducted on a focused screening hit, 2-(methylthio)-3-(phenylsulfonyl)-4H-pyrido[1,2-a]pyrimidin-4-imine (1, IC₅₀: 4.0 nM), as 5-HT₆ selective antagonists. Activity was improved some 2–4 fold when small, electron-donating groups were added to the central core as observed in 19, 20 and 26. Molecular docking of key compounds in a homology model of the human 5-HT₆ receptor was used to rationalize our structure–activity relationship (SAR) findings. In pharmacokinetic experiments, compound 1 displayed good brain uptake in rats following intra-peritoneal administration, but limited oral bioavailability.     

2-Benzimidazolyl-9-(chroman-4-yl)-purinone derivatives as JAK3 inhibitors

A novel class of Janus tyrosine kinase 3 (JAK3) inhibitors based on a 2-benzimidazoylpurinone core structure is described. Through substitution of the benzimidazoyl moiety and optimization of the N-9 substituent of the purinone, compound 24 was identified incorporating a chroman-based functional group. Compound 24 shows excellent kinase activity, good oral bioavailability and demonstrates efficacy in an acute mechanistic mouse model through inhibition of interleukin-2 (IL-2) induced interferon-γ (INF-γ) production.     

Design,synthesis and SAR investigation of thienosultam derivatives as ADAMTS-5 (aggrecanase-2) inhibitors

A series of 1,1-dioxothieno[2,3-d]isothiazole (thienosultam) derivatives were designed and synthesized as novel ADAMTS-5 inhibitors for an investigation into a side chain of thienosultam for the S1′ pocket. The resulting compounds (19 and 24) show high ADAMTS-5 inhibition and other MMP selectivity, and these compounds show good oral bioavailability.     

Discovery of inhibitors of plasminogen activator inhibitor-1: structure-activity study of 5-nitro-2-phenoxybenzoic acid derivatives

Two novel series of 5-nitro-2-phenoxybenzoic acid derivatives are designed as potent PAI-1 inhibitors using hybridization and conformational restriction strategy in the tiplaxtinin and piperazine chemo types. The lead compounds 5a, 6c, and 6e exhibited potent PAI-1 inhibitory activity and favorable oral bioavailability in the rodents.