Synthesis and Hemostatic Activity of New Amide Derivatives

Abstract

Eight dipeptides containing antifibrinolytic agents (tranexamic acid, aminocaproic acid, 4-(aminomethyl)benzoic acid, and glycine-natural amino acids) were synthesized in a three-step process with good or very good yields. DMT/NMM/TsO^- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate) was used as a coupling reagent. Hemolysis tests were used to study the effects of the dipeptides on blood components. Blood plasma clotting tests were used to examine their effects on thrombin time (TT), prothrombin time (PT), and the activated partial thromboplastin time (aPTT). The level of hemolysis did not exceed 1%. In clotting tests, TT, PT, and aPTT did not differentiate any of the compounds. The prothrombin times for all amides 1-8 were similar. The obtained results in the presence of amides 1-4 and 8 were slightly lower than for the other compounds and the positive control, and they were similar to the results obtained for TA. In the case of amide 3, a significantly decreased aPTT was observed. The aPTTs observed for plasma treated with amide 3 and TA were comparable. In the case of amide 6 and 8, TT values significantly lower than for the other compounds were found. The clot formation and fibrinolysis (CFF) assay was used to assess the influence of the dipeptides on the blood plasma coagulation cascade and the fibrinolytic efficiency of the blood plasma. In the clot formation and fibrinolysis assay, amides 5 and 7 were among the most active compounds. The cytotoxicity and genotoxicity of the synthesized dipeptides were evaluated on the monocyte/macrophage peripheral blood cell line. The dipeptides did not cause hemolysis at any concentrations. They exhibited no significant cytotoxic effect on SC cells and did not induce significant DNA damage.

Keywords: 4-(aminomethyl)benzoic acid; aminocaproic acid; antifibrinolytic agents; hemostasis; lysine analogs; peptides; tranexamic acid.

Figure 1

Key steps in the blood plasma coagulation cascade. The extrinsic pathway is activated by the tissue factor, which is abundantly expressed in the subendothelial tissue but becomes available to blood plasma proteins after vascular injury. Induction of the intrinsic coagulation pathway requires activation of the coagulation factor XII and the presence of a high-molecular (HMW) kininogen and prekallikrein. Independently on the starting point, both pathways trigger the tenase and prothrombinase enzymatic complexes, leading to the generation of the thrombin enzyme. The thrombin-catalyzed removal of short peptides from the fibrinogen molecule activates fibrinogen polymerization and the formation of fibrin monomers and polymers. This complex cascade of reactions results in the conversion of soluble fibrinogen into an insoluble fibrin clot.

Figure 2

Main components of the fibrinolytic system (description in the text).

Figure 3

Structure of lysine and its synthetic analogs: aminocaproic acid (EACA), tranexamic acid (TA), and 4-(aminomethyl)benzoic acid (PAMBA).

Figure 4

Structures of dipeptides 1–8 with expected hemostatic activities.

Figure 5

Synthesis of N-Boc amides 18–25.

Figure 6

Synthesis of hydrochlorides of amides 1–8.

Figure 7

The influence of amides 1–8 on the stability of erythrocytes (hemolysis test); (a) c = 5 mg/L; (b) c = 50 mg/L; (c) c = 500 mg/L.

Figure 7

The influence of amides 1–8 on the stability of erythrocytes (hemolysis test); (a) c = 5 mg/L; (b) c = 50 mg/L; (c) c = 500 mg/L.

Figure 7

The influence of amides 1–8 on the stability of erythrocytes (hemolysis test); (a) c = 5 mg/L; (b) c = 50 mg/L; (c) c = 500 mg/L.

Figure 8

Prothrombin time (PT) for amides 1–8 and tranexamic acid (TA) at three different concentrations—10, 25, and 50 mg/L—and for the control sample of human blood plasma (untreated with the examined amides) (* p < 0.05).

Figure 9

Activated partial thromboplastin time (aPTT) for amides 1–8 and tranexamic acid (TA) at three different concentrations—10, 25, and 50 mg/L—and for the control sample of human blood plasma (untreated with the examined amides) (* p < 0.05).

Figure 10

Thrombin time (TT) for amides 1–8 and tranexamic acid (TA) at three different concentrations—10, 25 and 50 mg/L—and for the control sample of human blood plasma (untreated with the examined amides) (* p < 0.05).

Figure 11

An exemplary curve of blood plasma coagulation and clot lysis recorded during the clot formation and fibrinolysis (CFF) assay. The first step in the CFF test involves activation of the blood plasma coagulation cascade and fibrin clot formation (1). It is characterized by the maximal velocity of the fibrin polymerization parameter (V_maxC). The maximal absorbance (A_max) peak corresponds to the fibrin stabilization phase and is an indicator of fibrin clot thickness (2). The third step of the CFF assay covers the activation of fibrinolytic mechanisms and fibrin clot degradation (3) and is described by the maximal velocity of clot lysis (V_maxF).

Figure 12

Cytotoxicity of the examined amides on SC cells. The XTT assay was used to assess the cytotoxicity of the tested amides. All of the experiments were performed in triplicate. Data are expressed as mean ± SD (n = 3), *** p < 0.001 versus the negative control.

Figure 13

Genotoxicity of the tested compounds. Statistical analysis was based on the results of three independent tests. The differences were statistically significant as follows: *** p < 0.001 versus the negative control.