J. zyxwvutsrq Med. Chem. zyxwvut 1996,38, zyxwvu 1511-1522 1511 Characterization of a Class of Peptide Boronates with Neutral P1 Side Chains as Highly Selective Inhibitors of Thrombin John J. Deadman,* Said Elgendy, Christopher A. Goodwin, Donovan Green, Jehan A. Baban, Geeta Patel, Emmanuel Skordalakes, Naoyoshi Chino,? Goran Claeson, Vijay V. Kakkar, and Michael F. Scully Thrombosis Research Institute, No. 1B Manresa Road, London zyxwvuts SM2 5TF, United Kingdom Received August 23, 1994@ Z-D-Phe-Pro-boroMpg-OPin (9a)lP2 has been shown previously to be a highly specific inhibitor of thrombin in spite of lacking an arginine-like guanidino group at the P1 site.' A range of compounds have been synthesized based upon this lead compound, varying the neutral side chain at the P1 site. Of the 20 examples based upon the structures at P2 and P3 Of Z-D-X-PI-0 (X being Phe or B,B-diphenylalanine), all were found to be effective inhibitors of thrombin (K;s between 10 and 100 nM). Furthermore all exhibited a high specificity toward thrombin having values for a zyxwvut Ki(trypsin)/Ki(thrombin) ratio of between 10- and 100-fold. High ratio values were found for a number of the compounds tested against a range of serine proteinases (plasmin, factor Xa, kallikrein, urokinase, protein Ca, chymotrypsin, elastase, and cathepsin Gh2 As far as potency toward thrombin, compounds containing the methoxypropyl group at P1 were favored over those with a methoxy grouping on a shorter alkyl chain zyxw (8) or without the methoxy group (1-5). The compounds display potent anticoagulant activity with values for 18 in thrombin time of 0.63 pM and in activated partial thromboplastin time of 2.0 pM. llB NMR has been used to confirm interaction of the boron atom with the active site. From the high specificity shown with all the compounds we propose that the compounds, constitute a new class of thrombin inhibitors. Introduction The impetus for the development of specific inhibitors of thrombin has increased over recent years with the realization that although thrombo occlusive events in the venous circulation are amenable to prophylaxis or treatment by the established pharmaceuticals warfarin and heparin, in the arterial circulation thrombotic processes are less responsive to these drugs. A resistant thrombi3 may be caused at sites of atherosclerotic stenosis and plaque rupture leading to heart attack and stroke or as a result of treatment for symptomatic atherosclerotic disease, for example, coronary angio- plasty (PTCAh4 More recently such thrombi have been found to be more easily prevented by hirudin,5 the specific, tight binding inhibitor of thrombin, a 65-amino acid polypeptide isolated from the saliva of leeches, which has become available through recombinant tech- nology. The success in preclinical development of hiru- din and its shorter synthetic analogue, h i r ~ l o g , ~ ? ~ has increased interest in the development of a low molecular weight inhibitor of thrombin, with properties akin to hirudin but administerable orally. In the development of such inhibitors, we and previ- ous workers have considered the properties of thrombin as a trypsin-like proteinase which preferably cleaves polypeptide substrates at ArgLys-Xaa bonds.s-10 Un- like trypsin and helpful to the development, a-thrombin displays highly restricted specificity at adjacent sites, exhibiting particular preference for hydrophobic P2- P4 residues. The requirements have been used in the design of a number of potent inhibitors of thrombin built around a P1 arginine or arginine analogue, considered to be obligatory for binding to the primary specificity + Present address: Peptide Institute, Inc., Minoh-shi, Osaka 562, @ Abstract published in Advance ACS Abstracts, April 1, 1995. Japan. 0022-2623/95/1838-1511$09.00/0 pocket of the enzyme. Indeed a number of agmatine- based inhibitors have been made that retain the posi- tively charged group binding to the specificity pocket, yet, like the so-called tripod-like inhibitors, they do not interact covalently with th active site serine and still retain submicromolar K/s.ll However, the inclusion of such a group immediately increases affinity of the inhibitor to other trypsin-like proteinases, trypsin itself with a wide specificity being particularly succeptible to inhibition. This is an important consideration for drugs of potential oral administration, since trypsin inhibition leads to gastric toxicity.12 The P1 arginal inhibitor ~-Phe-Pro-Arg-Hl~ has Ki(Try)/Ki(Thr) of only 4.14 Re- placements at P3 had deleterious effects on selectivity against trypsin with Boc-D-Phe having a Ki(Try)/Ki(Thr) ratio of 0.3 and H-D-MePhe with Ki(Try)/Ki(Thr)de- creased to 2-fold.15 Unnatural amino acids at P3 showed higher binding affinity for trypsin than throm- bin16 and lost 10-20-fold activity against thrombin (IC50 > M) on P3 Phe substitution by desamino-Phe14 and desaminopheny1glycine.l6 In selection of candidiate compounds from in vitro data that are likely to possess optimum in vivo antithrombotic efficacy, potential an- tifibrinolytic activity is critical. We have demonstrated previously that lack of selectivity to the target protein- ase, manifested in inhibition of plasmin and urokinase, has a disastrous effect on the in vitro activity, leading to prolongation of fibrinolysis. Recently17 this has been shown to extend to in vivo situations, in a rabbit model of thrombolysis, where arginine-based inhibitors such a s Ac-D-Phe-Pro-boroArg-OH (DuP 714)18lead to sig- nificant fibrinolytic compromise. Since this experiment is representative of potential clinical use of high con- centrations of thrombin inhibitors, say during FYTCA, selectivity for thrombin is clearly essential in any potential clinical candidate. In the arginal series H-D- Phe14 and ~ - M e P h e l ~ have similar Ki(Pla)/Ki(Thr) of 0 1995 American Chemical Society