Mechanism-Based Isocoumarin Inhibitors for Blood Coagulation Serine Proteases. Effect of the 7-Substituent in 7-Amino-4-chloro-3-(isothioureidoalkoxy)isocoumarins on Inhibitory and Anticoagulant Potency

• Published in: Journal of medicinal chemistry 1994-04, Vol.37 (9), p.1298-1306
• Main Authors: Kam, Chih-Min, Kerrigan, John E, Plaskon, R. Richard, Duffy, Edward J, Lollar, Pete, Suddath, F. L, Powers, James C
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Anticoagulants - chemistry; Anticoagulants - pharmacology; Binding Sites; Blood Coagulation - drug effects; Cattle; Chemistry; Coumarins - chemistry; Coumarins - metabolism; Coumarins - pharmacology; Exact sciences and technology; Factor IXa - antagonists & inhibitors; Factor VIIIa - pharmacology; Heterocyclic compounds; Heterocyclic compounds with o, s, se, te hetero atom and condensed derivatives; Humans; Hydrolysis; Isocoumarins; Liposomes - metabolism; Models, Molecular; Molecular Sequence Data; Molecular Structure; Organic chemistry; Preparations and properties; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - pharmacology; Swine; Thrombin - antagonists & inhibitors; Thrombin - chemistry
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/jm00035a009

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that 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 incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% 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 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.