To cite this article: Wong PC, Crain EJ, Xin B, Wexler RR, Lam PYS, Pinto DJ, Luettgen JM, Knabb RM. Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies. J Thromb Haemost 2008; 6: 820-9.Summary. Background: Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. Objective: We evaluated the in vitro properties of apixaban and its in vivo activities in rabbit models of thrombosis and hemostasis. Methods: Studies were conducted in arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), electrically mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. Results: In vitro, apixaban is potent and selective, with a K i of 0.08 nM for human FXa. It exhibited species difference in FXa inhibition [FXa K i (nM): 0.16, rabbit; 1.3, rat; 1.7, dog] and anticoagulation [EC 2· (lM, concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 lM did not alter human and rabbit platelet aggregation to ADP, cthrombin, and collagen. In vivo, the values for antithrombotic ED 50 (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT and the values for BT ED 3· (dose that increased BT by 3-fold) were 0.27 ± 0.03, 0.11 ± 0.03, 0.07 ± 0.02 and > 3 mg kg warfarin, respectively. Conclusions: In summary, apixaban was effective in the prevention of experimental thrombosis at doses that preserve hemostasis in rabbits.

Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P1 moieties that resulted in the identification of the p-methoxyphenyl P1, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P4 ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4.

Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.