MECHANISM-BASED ISOCOUMARIN INHIBITORS FOR BLOOD-COAGULATION SERINE PROTEASES - EFFECT OF THE 7-SUBSTITUENT IN AMINO-4-CHLORO-3-(ISOTHIOUREIDOALKOXY)ISOCOUMARINS ON INHIBITORY AND ANTICOAGULANT POTENCY

A series of ino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-Ci TPrOIC) derivatives with various substituents at the 7- and 3-position s have been synthesized as inhibitors of several blood coagulation enz ymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irrevers ible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoet hoxy group at the 3-position and a large hydrophobic group at the 7-po sition are better inhibitors for thrombin, factor VIIa, factor Xa, fac tor XIa, factor XIIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-C iTEtOIC (14), (S)-Ph(CH3) CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHC ONH-CiTEtOIC (26) inhibit thrombin quite potently and have k(obs)/[I] values of (1-4) x 10(4) M(-1) s(-1). Modeled structures of several iso coumarins noncovalently complexed with human alpha-thrombin suggest th at H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubat ion for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 6 5 % of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine, With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 mu M. Thus, these compounds are effective anticoagulant s in vitro and may be useful in vivo.