Novel Potent and Selective Thrombin Inhibitors Based on a Central 1,4-Benzoxazin-3(4H)-one Scaffold 4 Janez Ilas ˇ, Tihomir Tomas ˇic ´, and Danijel Kikelj* UniVersity of Ljubljana, Faculty of Pharmacy, As ˇkerc ˇeVa 7, 1000 Ljubljana, SloVenia ReceiVed December 24, 2007 Novel thrombin inhibitors with the central 1,4-benzoxazine-3(4H)-one scaffold, benzamidine P 1 arginine side chain mimetic and various P 3 moieties are described. 3-(Benzyl(2-(4-carbamimidoylbenzyl)-4-methyl- 3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl)amino)-3-oxopropanoic acid (7b), the most potent compound in the series, exhibited a K i of 2.6 nM in vitro for thrombin and high selectivity against trypsin and factor Xa. Introduction Thromboembolic diseases, including stroke, deep vein throm- bosis, and myocardial infarction, are the leading causes of death in developed countries. 1 The search for low molecular weight inhibitors of thrombin, the key enzyme in the coagulation cascade, has been a major goal in the search for novel antithrombotic agents over the past decade. 2 Argatroban was the first small-molecule thrombin inhibitor introduced to the market, 3 but its widespread use has been hindered by the fact that it is not orally active. The withdrawal of ximelagatran, 4 the first orally available thrombin inhibitor, has shown that there is not yet a satisfactory thrombin inhibitor on the market. With ongoing progress in the development of efficient antithrombotic drugs, focus has also turned to other important enzymes in the coagulation cascade, mainly factor Xa 5 and factor VIIa, 6 as well as to dual inhibitors targeting two coagulation enzymes at the same time, 7 which have not yet resulted in marketable products. Therefore, thrombin remains an important target in the discovery of novel antithrombotic compounds. In our ongoing research directed toward a novel class of antithrombotic compounds with dual function, possessing in the same molecule both thrombin inhibitory and fibrinogen receptor antagonistic activity, 8 1,4-benzoxazine-3(4H)-one was found to be a suitable scaffold for their design. On the basis of its strong interaction with Asp189 in the thrombin S 1 pocket, the benza- midine group was chosen as arginine mimetic 9 for the first generation of antithrombotic compounds with dual action, although being aware of its strong basicity. The problem of poor bioavailability of compounds containing a benzamidine group, arising from its basicity, can be overcome by a prodrug approach, as shown in the case of ximelagatran 4 and dabigat- ran. 10 In the search for the optimal spacer between the 1,4- benzoxazin-3(4H)-one and benzamidine moieties to produce antithrombotic compounds with dual activity, we prepared a series of compounds incorporating the methylene group con- necting both moieties that, although devoid of fibrinogen receptor antagonistic activity, were found to be potent and highly selective thrombin inhibitors. Here we report on the design, synthesis, in vitro biological activity, and structure-activity relationship of this novel class of thrombin inhibitors possessing a central 1,4-benzoxazin-3(4H)-one core. Synthesis of the target 1,4-benzoxazine-3(4H)-one thrombin inhibitors 6a-d and 7a-d was achieved using the chemistry depicted in Scheme 1. The bromo derivative 1 11 was synthesized from 2-amino-5-nitrophenol in four steps involving N-acylation, cyclization, N-methylation, and bromination, with an overall yield of 89%. A Wittig reaction of 4-cyanobenzaldehyde and phosphorus ylide obtained from 1 with triphenylphosphine proceeded smoothly, yielding 3-oxo-3,4-dihydro-2H-1,4-ben- zoxazine-2-ylidene derivative 2, mainly as the Z-isomer (chemi- cal shift of olefinic proton: 7.05 ppm), although a small amount of E-isomer was also obtained (chemical shift of olefinic proton: 6.84 ppm). The two geometric isomers could be separated by recrystallization from petroleum ether/ethyl acetate (1:1). Com- pound 2 (mixture of Z- and E-isomers) was reduced catalytically at 6 bar to racemic 7-amino-3-oxo-3,4-dihydro-2H-1,4-benzox- azine-2-yl derivative 3, whereas under less vigorous catalytic hydrogenation conditions, only the nitro group of 2 was reduced to give amine 3a. N-Benzylation of 3, employing reductive amination of benzaldehyde, with sodium triacetoxyborohydride as reducing agent, afforded secondary amine 4, which was acylated with ethyl oxalyl chloride, methyl malonyl chloride, and ethyl succinyl chloride to introduce side chains of different lengths containing a carboxylic ester moiety, giving 5a-c. Alternatively, 3 was coupled to 2-benzyl-3-ethoxy-3-oxopro- panoic acid using EDC/HOBT a coupling strategy to give compound 5d. Nitriles 5a-d were transformed to the corre- sponding benzamidines 6a-d under Pinner reaction 12 condi- tions. The esters 6a-d were hydrolyzed to carboxylic acids 7a-d using 1.5 M sodium hydroxide in ethanol solution. Results and Discussion The in vitro thrombin inhibitory potencies of the synthesized compounds and their selectivities against factor Xa and trypsin are presented in Table 1. Our preliminary studies demonstrated (data not shown) that the best thrombin inhibition was obtained when the P 3 moiety was attached to a 2-substituted 1,4- benzoxazine-3(4H)-one scaffold at position 7. Introduction of a methyl group at position 4 additionally contributed to the hydrophobic interaction in the thrombin S 2 pocket. 8 Optimiza- tion of the P 3 moiety revealed the beneficial effect of benzylation of the amino group at position 7 which, to improve potency and increase solubility, was additionally substituted with acyl * To whom correspondence should be addressed. Tel.: +386 1 476 95 61. Fax: +386 1 425 80 31. E-mail: danijel.kikelj@ffa.uni-lj.si. 4 Dedicated to Professor Slavko Peèar on the occasion of his 60th birthday. a Abbreviations: ADT, Auto Dock Tools; DMF, N,N-dimethylformamide; EDC, N-ethyl N′-(3-dimethylaminopropyl)-carbodiimide; ESI, electrospray ionization; HOBT, 1-hydroxybenzotriazole; THF, tetrahydrofuran; PDB, Protein Data Bank. J. Med. Chem. 2008, 51, 2863–2867 2863 10.1021/jm701622y CCC: $40.75  2008 American Chemical Society Published on Web 04/16/2008