Acylated 1H-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action

Abstract

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.

Microscale Parallel Synthesis of Acylated Aminotriazoles Enabling the Development of Factor XIIa and Thrombin Inhibitors

“…The unbound fragments FR1 and FR2 (shown as orange and green figures) and the coupling reagents (shown as X‐ and square‐shaped figures) are low active or inactive inhibitors of FXIIa and thrombin and do not interfere with the assay at low concentrations (Figure 4, A). We previously showed that the unbound fragments FR1 and FR2 cannot efficiently inhibit the enzymes as only connected fragments (FR1‐FR2) are capable of covalent interaction with the enzymes’ catalytic Ser195 (also see the “Mechanism of Inhibition” section) [3f] . Accordingly, when FR1 and FR2 are bound to each other forming N‐acylated aminotriazole, a significant shift in inhibitory activity is observed (toward lower doses, Figure 4, B).…”

“…The resultant library of compounds was screened against FXIIa and thrombin revealing potent dual and selective inhibitors of FXIIa and/or thrombin. The results of this screening have been disclosed by us earlier and will not be repeated here [3f] . The screening was limited only to the variations on the inhibitors’ acyl fragment (FR2), leaving the aminotriazole core (FR1) unchanged and therefore unexplored.…”

“…Aminotriazoles 15 a – k were synthesized in a fusion reaction between aminoguanidine hydrochloride and carboxylic acids 14 a – k at 200 °C (Scheme 1). The reaction proceeds via intermediate amidoguanidines, which subsequently, without isolation undergo a cyclocondensation reaction affording desired products 15 a – k [3f] . Similarly, aminotriazoles 15 l – s were accessed reacting corresponding acid chlorides with aminoguanidine salts under basic conditions (Scheme 1).…”

“…To access 1 H ‐1,2,4‐triazol‐5‐amines 24 a – d exhibiting a quinoxalin‐2‐yl moiety as an alternative substituent in 3‐position of the triazole core, synthesis shown in Scheme 2 was performed. This extension of the pyridyl moiety might lead to additional interactions with S1’ and S2‐S3 binding sites of FXIIa or thrombin and improve the compounds’ affinity or selectivity e. g., toward FXIIa over thrombin, as thrombin exhibits a more narrow S2 binding site [3f,14] . For this, commercially available β‐ketoesters 20 a – c were brominated and then condensed with the symmetric o ‐phenylenediamines to afford fused heterocyclic esters 22 a – d [15] .…”

“…Significantly fewer compounds were found to inhibit thrombin (Figure 8). This was, however, expected as the employed acyl building blocks (FR2) are known to be more selective toward FXIIa [3f] . Nevertheless, the screening allowed to identify four potentially interesting thrombin inhibitors such as compounds comprising isoquinolin‐1‐yl (A6), pyrazin‐2‐yl (C11), quinoline‐2‐yl (A5), and benzo[ b ]thiophen‐2‐yl (D5) fragments.…”