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

Abstract

Herein we report a microscale parallel synthetic approach allowing for rapid access to libraries of N‐acylated aminotriazoles and screening of their inhibitory activity against factor XIIa (FXIIa) and thrombin, which are targets for antithrombotic drugs. This approach, in combination with post‐screening structure optimization, yielded a potent 7 nM inhibitor of FXIIa and a 25 nM thrombin inhibitor; both compounds showed no inhibition of the other tested serine proteases. Selected N‐acylated aminotriazoles exhibited anticoagulant properties in vitro influencing the intrinsic blood coagulation pathway, but not extrinsic coagulation. Mechanistic studies of FXIIa inhibition suggested that synthesized N‐acylated aminotriazoles are covalent inhibitors of FXIIa. These synthesized compounds may serve as a promising starting point for the development of novel antithrombotic drugs.

Semisynthetic Approach toward Biologically Active Derivatives of Phenylspirodrimanes from S. chartarum

“…The reactions were performed at 25 °C. To derive IC 50 values, endpoint RFU (single fluorescence reading after 1 h) was used. , …”

“…All measurements were performed using citrated (3.8%) human pooled plasma (Dunn Labortechnik GmbH, Germany) on a semiautomated coagulometer (Thrombotimer-2, Behnk Elektronik, Germany) according to the manufacturer’s instructions, as previously reported. , For aPTT measurement, plasma (100 μL) was placed into the incubation cuvettes of the instrument and incubated for 2 min at 37 °C. Then, the test compound solution (10 μL) or solvent (DMSO, 10 μL) was added with a pipette.…”

“…After 3 min of incubation, the cuvettes were transferred to a measuring position, the coagulation was initiated by the addition of 100 μL of PT assay reagent already containing CaCl 2 (prewarmed at 37 °C, Convergent Technologies, Germany), and the clotting time was recorded. 42,47 Cytotoxicity Studies. The cytotoxic effects of the isolated and semisynthesized compounds were evaluated with the resazurin assay, performed analogous to previous studies.…”

New Blood Coagulation Factor XIIa Inhibitors: Molecular Modeling, Synthesis, and Experimental Confirmation

“…In a study using virtual highthroughput screening [20], 17 factor XIIa inhibitors with IC 50 values below 50 µM were identified. Figure 2 shows the structures of the reported coagulation factor XIIa inhibitors and their in vitro inhibitory activity values: inhibitors based on aminotriazole selective over thrombin, trypsin, and FXa [21,22]; inhibitors based on coumarin selective over thrombin, plasma kallikrein, TF/FXVIIa, and FXa [18]; boronic-acid-based inhibitor [11]. Nevertheless, the development of inhibitors of coagulation factor XIIa is at an early stage and the search for a lead compound to create a drug based on it with a low risk of bleeding is an urgent task today [1].…”

Amide-functionalized 1,2,4-Triazol-5-amines as Covalent Inhibitors of Blood Coagulation Factor XIIa and Thrombin

“…27,28 Despite promising enzyme inhibitory and anticoagulant properties, aminotriazoles (e.g., 4a,b, Figure 1) were less efficient than non-covalent anticoagulants dabigatran and rivaroxaban. 27,28 Therefore, further structure optimization of this series of compounds is required to access more potent and selective FXIIa and/or thrombin inhibitors. In this regard, 1,2,4-triazol-5-amine scaffold represents an excellent synthon, synthetic modifications of which allow for the preparation of compounds of different complexities.…”