1,2,3-Triazole Derivatives as Novel Antifibrinolytic Drugs

Abstract

Fibrinolysis is a natural process that ensures blood fluidity through the removal of fibrin deposits. However, excessive fibrinolytic activity can lead to complications in different circumstances, such as general surgery or severe trauma. The current antifibrinolytic drugs in the market, aminocaproic acid (EACA) and tranexamic acid (TXA), require high doses repetitively to maintain their therapeutic effect. These high doses are related to a number of side effects such as headaches, nasal symptoms, or gastrointestinal discomfort and severely limit their use in patients with renal impairment. Therefore, the discovery of novel antifibrinolytics with a higher specificity and lower dosage could vastly improve the applicability of these drugs. Herein, we synthesized a total of ten compounds consisting of a combination of three key moieties: an oxadiazolone, a triazole, and a terminal amine. The IC₅₀ of each compound was calculated in our clot lysis assays, and the best candidate (1) provided approximately a 2.5-fold improvement over the current gold standard, TXA. Molecular docking and molecular dynamics were used to perform a structure-activity relationship (SAR) analysis with the lysine binding site in the Kringle 1 domain of plasminogen. This analysis revealed that 1,2,3-triazole was crucial for the activity, enhancing the binding affinity through pi-pi stacking and polar interactions with Tyr72. The results presented in this work open the door to further investigate this new family as potential antifibrinolytic drugs.

Keywords: antifibrinolytic; fibrinolysis; oxadiazolone; plasmin; plasminogen; tPa; triazole.

Figure 1

Previously studied small molecules with antifibrinolytic activity by inhibition of the lysine binding sites of plasminogen.

Figure 2

General structure for the hypothesized components, which include a piperidine ring, a triazole ring, and an oxadiazolone ring.

Scheme 1

Studied compounds for their antifibrinolytic activity.

Scheme 2

1,2,3-triazole formation. (a) TEA, MsCl, DCM, 0–5 °C to 23 °C, 2 h. (b) NaN₃, DMF, 80 °C, 8 h. (c) Ethyl propiolate, CuI, ACN, 23 °C, 12 h.

Scheme 3

Oxadiazolone formation for 1,2,3-triazole derivatives. (a) Hydrazine hydrate, n-butanol, reflux, 3 h. (b) CDI, DBU, ACN, reflux, 15 h. (c) NH₃, MeOH, 23 °C, 12 h. (d) TEA, TFAA, DCM, 0–5 °C to 23 °C, 5 h. (e) NH₂OH·HCl, NaHCO₃, MeOH, reflux, 14 h. (f) CDI, DBU, ACN, reflux, 15 h.

Scheme 4

1,2,4-triazole derivatives complete synthetic pathway. (a) methyl 1H,1,2,4-triazole-3-carboxylate, NaH, DMF, 70 °C, 48 h. (b) Hydrazine hydrate, n-butanol, reflux, 3 h. (c) CDI, DBU, ACN, reflux, 15 h. (d) NH3, MeOH, 23 °C, 12 h. (e) TEA, TFAA, DCM, 0–5 °C to 23 °C, 5 h. (f) NH₂OH·HCl, NaHCO₃, MeOH, reflux, 14 h. (g) CDI, DBU, ACN, reflux, 15 h.

Scheme 5

Tranexamic acid derivative complete synthetic pathway. (a) TsCl, EtOH, reflux, 2 h. (b) Boc₂O, TEA, MeOH, 23 °C, 12 h. (c) NH₃, MeOH, 85 °C (sealed vial), 5 days. (d) TEA, TFAA, DCM, 0–5 °C to 23 °C, 5 h. (e) NH₂OH·HCl, NaHCO₃, MeOH, reflux, 14 h. (f) CDI, DBU, ACN, reflux, 15 h.

Figure 3

Dose–response curve for each molecule represented as a percentage of fibrinolysis vs. concentration (µM) of inhibitor measured in plasma clot lysis assays. Compounds not included in this graph showed undetectable activity below 1000 µM.

Figure 4

Docking of most representative poses for lysine analogues (a) ε-aminocaproic acid (EACA) and (b) tranexamic acid (TXA). Docking studies were performed with Kringle 1 lysine binding site (pdb code 1cea) using AutoDock 4.2.

Figure 5

Docking of most representative poses for 1,2,3-triazole derivatives combined with oxadiazolone rings. (a) 1,2,4-oxadiazolone compounds: 1 (grey), 2 (pink), 3 (cyan), 4 (orange). (b) 1,3,4-oxadiazolone compounds: 5 (grey), 6 (cyan), 7 (pink). Docking studies were performed with Kringle 1 lysine binding site (pdb code 1cea) using AutoDock 4.2. H-bond interactions with the oxadiazolone ring are indicated as green dashed lines.

Figure 6

Docking of most representative poses for compounds 11 (a) and 8 (b). Docking studies were performed with Kringle 1 lysine binding site (pdb code 1cea) using AutoDock 4.2.

Figure 7

Docking of most representative poses for 1,2,4-triazole derivatives (compounds 9 and 10). Docking studies were performed with Kringle 1 lysine binding site (pdb code 1cea) using AutoDock 4.2.

Figure 8

Representation of the most important distances for molecular dynamics simulations of compounds 1 and 9. Distances for compound 1: (a) piperidine and Asp57, (b) triazole (nitrogen N2) and Tyr72, (c) triazole (nitrogen N3) and Tyr72. Distances for compound 9: (d) piperidine and Asp57, (e) triazole (nitrogen N2) and Tyr72, (f) triazole (nitrogen N4) and Tyr72.

Figure 9

Binding poses with GABA binding pocket of GABA_A receptor for tranexamic acid (left) and 4-PIOL (right). GABA_A receptor file obtained from PDB code 6d6u.