Since activated factor X (FXa) is a coagulant enzyme that generates thrombin and participates in both intrinsic and extrinsic coagulation pathways, inhibition of FXa may be more effective than inactivation of thrombin for interrupting blood coagulation. To assess the possible effectiveness of FXa inhibition as an anticoagulant, we designed and synthesized 3-(amidinoaryl)-2-[4-[(3S)-3-pyrrolidinyloxy]phenyl]propanoi c acid derivatives as low molecular weight, nonpeptidic, orally active FXa inhibitors. These derivatives exhibited potent and highly selective anti-FXa activity in vitro and anticoagulant activity on oral administration. The most promising compound, (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]- 3-(7-amidino-2-naphthyl)propanoic acid hydrochloride pentahydrate (4,DX-9065a), inhibited 50% of FXa activity (IC50) at 0.07 microM, doubled plasma recalcification time (PRCT) at 0.5 microM, and significantly prolonged activated partial thromboplastin time (APTT) at a dose of 100 mg/kg on oral administration. In contrast with FXa inhibition, 4 showed no activity against thrombin (IC50 > 2000 microM).

BACKGROUND AND PURPOSENuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of the Nrf2 pathway seems protective for many organs, and although a well-known Nrf2 activator, bardoxolone methyl, was evaluated clinically for treating chronic kidney disease, it was found to induce adverse events. Many bardoxolone methyl derivatives, mostly derived by chemical modifications, have already been studied. However, we adopted a biotransformation technique to obtain a novel Nrf2 activator. EXPERIMENTAL APPROACHThe potent novel Nrf2 activator, RS9, was obtained from microbial transformation products. Its Nrf2 activity was evaluated by determining NADPH:quinone oxidoreductase-1 induction activity in Hepa1c1c7 cells. We also investigated the effects of RS9 on oxygen-induced retinopathy in rats and glycated albumin-induced blood-retinal barrier permeability in rabbits because many ocular diseases are associated with oxidative stress and inflammation. KEY RESULTSBardoxolone methyl doubled the specific activity of Nrf2 in Hepa1c1c7 cells at a much higher concentration than RS9. Moreover, the induction of Nrf2-targeted genes was observed at a one-tenth lower concentration of RS9. Interestingly, the cytotoxicity of RS9 was substantially reduced compared with bardoxolone methyl. Oral and intravitreal administration of RS9 ameliorated the pathological scores and leakage in the models of retinopathy in rats and ocular inflammation in rabbits respectively. CONCLUSION AND IMPLICATIONSNrf2 activators are applicable for treating ocular diseases and novel Nrf2 activators have potential as a unique method for prevention and treatment of retinovascular disease. AbbreviationsAU, arbitrary unit; BEACON study, bardoxolone methyl evaluation in patients with chronic kidney disease and type 2 diabetes: the occurrence of renal events; BEAM study, bardoxolone methyl treatment: renal function in chronic kidney disease/type 2 diabetes; CD, concentration to double NQO1 activity; CDDO, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid; Ct, threshold cycle; Keap1, Kelch-like ECH-associated protein 1; NQO1, NADPH:quinone oxidoreductase-1; Nrf2, nuclear factor erythroid 2-related factor 2; P, post-natal day; PLA, poly lactic acid; tBHP, tert-butyl hydroperoxide

Our exploratory study was based on the concept that a non-amidine factor Xa (fXa) inhibitor is suitable for an orally available anticoagulant. We synthesized and evaluated a series of N-(6-chloronaphthalen-2-yl)sulfonylpiperazine derivatives incorporating various fused-bicyclic rings containing an aliphatic amine expected to be S4 binding element. Among this series, 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine type 61 displayed orally potent anti-fXa activity and evident prolongation of prothrombin time (PT) with the moderate bioavailability in rats. The X-ray crystal analysis afforded an obvious binding mode that 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine and 6-chloronaphthalene respectively bound to S4 and S1 subsites. In this X-ray study, we discovered a novel intramolecular S-O close contact. Ab initio energy calculations of model compounds deduced that conformers with the most close S-O proximity were most stable. The Mulliken population analysis proposed that this energy profile was caused by both of electrostatic S-O affinity and N-O repulsion. The results of these calculations and X-ray analysis suggested a possibility that the restricted conformation effected the affinity to S4 subsite of fXa.

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