Investigation of the mechanism of anticoagulant action of a new triazolopyrimidine derivative

BACKGROUND: The development of new domestic oral anticoagulants is an urgent task. During a search for anticoagulant compounds among heterocyclic derivatives, a compound designated NAR0273b was identified, exhibiting antithrombin activity.

AIM: To study the mechanism of anticoagulant action of compound NAR0273b.

METHODS: The antithrombin activity of the 5,7di(thiophen2yl)4,5dihydro[1,2,4]triazolo[1,5a]pyrimidine derivative NAR0273b and the comparator drug dabigatran etexilate was evaluated using the Ecarin time assay in vitro on rabbit blood. The study included three groups: control, NAR0273b, and comparator, with five replicates per group. Experiments were performed on six male Chinchilla rabbits weighing 3–3.5 kg. The binding of NAR0273b to factor IIa was assessed using chromogenic substrate S2238 in vivo. The effect of NAR0273b on factor IIa levels was studied in male outbred white rats by enzymelinked immunosorbent assay. Experiments were performed on 35 animals weighing 250–270 g. The study included a control group, NAR0273b groups at 2.5, 5.5, and 11.0 mg/kg, and dabigatran etexilate groups at 3, 6, and 12.0 mg/kg, with five replicates per group. Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software Inc., USA). EC₅₀ and ED₅₀ values were calculated by regression analysis.

RESULTS: In the Ecarin time assay, NAR0273b showed direct antithrombin activity comparable to dabigatran in terms of EC₅₀. In vivo, after intragastric administration, NAR0273b bound factor IIa on a chromogenic substrate and was comparable to dabigatran in ED₅₀. ELISA confirmed the ability of NAR0273b to bind thrombin: bound thrombin in controls was 187.2 ng/mL, whereas in the NAR0273b group (5.5 mg/kg) it reached 401.4 ng/mL, a 2.2fold increase over control. Thus, the ability of NAR0273b to block thrombin (factor IIa) was confirmed quantitatively by ELISA.

CONCLUSION: The anticoagulant mechanism of NAR0273b is associated with thrombin (factor IIa) blockade.