Thrombin is the most potent platelet aggregator. To discover the selective inhibitor of thrombin that is important to curing platelet aggregation-related diseases, docking experiments were performed to dock (1R,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1R,3S)-THCCA], and (1S,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1S,3S)-THCCA], into the p pocket of bovine thrombin. The ideal match supported that (1R,3S)-THCCA could be used as a potential lead compound. In this case 20 natural amino acids were theoretically introduced into the 3-carboxyl of (1R,3S)-THCCA and 20 derivatives, (1R,3S)-THCCA-amino acids, were docked into p pocket of bovine thrombin to perform virtual screening. The screening revealed that comparing to (1R,3S)-THCCA itself the DockScores of 16 derivatives were higher, and (1R,3S)-THCCA-Asn (4j) got the highest DockScore. Thus, 16 derivatives were synthesized for experimental study. The in vitro anti-platelet aggregation assay showed that at 100 μM of concentration the 16 derivatives failed to inhibit the platelet aggregation induced by both adenosine diphosphate and arachidonic acid. On the other hand, however, the IC₅₀ value of the 16 derivatives inhibiting the platelet aggregation induced by platelet activating factor and thrombin ranged from 9.44 μM to 194.64 μM and from 0.07 μM to 9.56 μM, respectively. The in vitro anti-platelet aggregation assay suggested that the 16 derivatives selectively inhibited the platelet aggregation induced by thrombin. In particular, the IC₅₀ of (1R,3S)-THCCA-Asn (4j) had the lowest value. On rat model at 1 nmol/kg of dosage the 16 derivatives effectively prevented thrombus formation. It is worth pointing out that even at 0.01 nmol/kg of dosage, 4j still effectively prevented thrombus formation. 4j hardly has effects on the proliferation of mammalian cells and rat tail bleeding time. In conclusion, the combination of virtual screening and biological assays successfully lead to the discovery of 4j as a promising candidate of selective inhibitor of thrombin.

凝血酶是最有效的血小板聚集剂。为发现对治疗血小板聚集相关疾病具有重要意义的凝血酶选择性抑制剂，对(1 R ,3 S )-2,3,4,9-四氢-β-咔啉-3-羧酸进行对接实验, [(1 R ,3 S )-THCCA], 和 (1 S ,3 S )-2,3,4,9-四氢-β-咔啉-3-羧酸, [(1 S ,3 S )- THCCA]，进入牛凝血酶的 p 口袋。理想匹配支持 (1 R ,3 S )-THCCA 可用作潜在的先导化合物。在这种情况下，理论上将 20 个天然氨基酸引入（1R ,3 S )-THCCA 和 20 种衍生物 (1 R ,3 S )-THCCA-氨基酸对接到牛凝血酶的 p 袋中以进行虚拟筛选。筛选结果显示，与(1 R ,3 S )-THCCA 本身相比，16 个衍生物的DockScore 更高，而(1 R ,3 S )-THCCA-Asn ( 4j ) 的DockScore 最高。因此，合成了 16 种衍生物用于实验研究。体外抗血小板聚集试验表明，在 100 μM 浓度下，16 种衍生物未能抑制由二磷酸腺苷和花生四烯酸诱导的血小板聚集。但另一方面，IC_{抑制血小板活化因子和凝血酶诱导的血小板聚集的16种衍生物的50}值分别为9.44 μM~194.64 μM和0.07 μM~9.56 μM。体外抗血小板聚集试验表明，16 种衍生物选择性地抑制凝血酶诱导的血小板聚集。特别地，(1 R ,3 S )-THCCA-Asn ( 4j ) 的IC ₅₀具有最低值。在大鼠模型中，1 nmol/kg 剂量的 16 种衍生物有效地防止了血栓形成。值得指出的是，即使在 0.01 nmol/kg 的剂量下，4j仍能有效防止血栓形成。4j对哺乳动物细胞的增殖和鼠尾出血时间几乎没有影响。总之，虚拟筛选和生物测定的结合成功地发现了4j作为凝血酶选择性抑制剂的有希望的候选者。