Innovative Three-Step Microwave-Promoted Synthesis of N-Propargyltetrahydroquinoline and 1,2,3-Triazole Derivatives as a Potential Factor Xa (FXa) Inhibitors: Drug Design, Synthesis, and Biological Evaluation

Abstract

The coagulation cascade is the process of the conversion of soluble fibrinogen to insoluble fibrin that terminates in production of a clot. Factor Xa (FXa) is a serine protease involved in the blood coagulation cascade. Moreover, FXa plays a vital role in the enzymatic sequence which ends with the thrombus production. Thrombosis is a common causal pathology for three widespread cardiovascular syndromes: acute coronary syndrome (ACS), venous thromboembolism (VTE), and strokes. In this research a series of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXa) inhibitor were designed, synthesized, and evaluated for their FXa inhibitor activity, cytotoxicity activity and coagulation parameters. Rational design for the desired novel molecules was performed through protein-ligand complexes selection and ligand clustering. The microwave-assisted synthetic strategy of selected compounds was carried out by using Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar cycloaddition type reactions under microwave irradiation. The microwave methodology proved to be an efficient way to obtain all novel compounds in high yields (73-93%). Furthermore, a thermochemical analysis, optimization and reactivity indexes such as electronic chemical potential (µ), chemical hardness (η), and electrophilicity (ω) were performed to understand the relationship between the structure and the energetic behavior of all the series. Then, in vitro analysis showed that compounds 27, 29-31, and 34 exhibited inhibitory activity against FXa and the corresponding half maximal inhibitory concentration (IC₅₀) values were calculated. Next, a cell viability assay in HEK293 and HepG2 cell lines, and coagulation parameters (anti FXa, Prothrombin time (PT), activated Partial Thromboplastin Time (aPTT)) of the most active novel molecules were performed to determine the corresponding cytotoxicity and possible action on clotting pathways. The obtained results suggest that compounds 27 and 29 inhibited FXa targeting through coagulation factors in the intrinsic and extrinsic pathways. However, compound 34 may target coagulation FXa mainly by the extrinsic and common pathway. Interestingly, the most active compounds in relation to the inhibition activity against FXa and coagulation parameters did not show toxicity at the performed coagulation assay concentrations. Finally, docking studies confirmed the preferential binding mode of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives inside the active site of FXa.

Keywords: 1,2,3-triazole; N-propargyltetrahydroquinoline; cell viability assay; coagulation parameters; factor Xa inhibitors; microwave-assisted synthesis.

Figure 1

Blood coagulation cascade: extrinsic, intrinsic, and common pathway.

Figure 2

Currently clinically used FXa Inhibitors and the Food and Drug Administration (FDA) year of approval.

Figure 3

(a) Apixaban and (b) Rivaroxaban as gold standard reference compounds. The S₄ binding scaffolds phenyloxopiperidine or phenyloxomorphonilo are highlighted in blue.

Figure 4

(a) 5-heptyl-1,2,3,4-tetrahydroquinoline [44]. (b) Aflaquinolone A [46]. (c) 1,4-disubstituted 1,2,3,4-tetrahydroquinoline [50] (d) Tetrahydroquinoline chemical structure. (e) 1,4-disubstituted-1,2,3-triazole chemical structure.

Figure 5

Virtual screening of interest fragment in FXa PDB crystallography data base.

Figure 6

Shape-based query for phenyloxomorphonilo or phenyloxopiperidine containing FXa inhibitors. (a) Three-dimensional (3D) structural alignment of ligands after protein complexes superposition. (b) Shape-based query derived with color representing the combined shape with chemical features represented as spheres (hydrophobic = brown; rings = green; HB acceptor = red; HB donor = blue). (c) ROC area under the curve. HB: hydrogen bonding

Scheme 1

General synthetic pathway for selected compounds. (a) N,N′-dimethylethylenediamine (DMEDA), CuI, K₃PO₄, PhMe, microwave irradiation (MW), 160 °C. (b) propargylbromide, K₂CO₃, KI, CH₃CN, MW, 160 °C. (c) formaldehyde, 1-vinyl-2-pyrrolidinone, InCl₃, CH₃CN, MW, 160 °C. (d) HCl, NaNO₂, NaNO₃, H₂O, 40 °C. (e) (11–14), (24–26), copper nanoparticles supported on ZnO (CuNPs/ZnO), Et₃N, tetrahydrofuran (THF), 160 °C.

Scheme 2

Synthesis of aniline derivative 6 [80].

Scheme 3

Synthesis of Anilines Derivatives (7–9) [80].

Scheme 4

Synthesis of N-allyl/propargyl aniline derivatives (11–14) [80].

Scheme 5

Synthesis of N-propargyl tetrahydroquinoline derivatives (17–20).

Scheme 6

Synthesis of aryl azides derivatives (24–26).

Scheme 7

Synthesis of 1H-1,2,3-triazole derivatives (27–38). Compounds 27–29 (X = CH₂; R = H, Cl, F, respectively); 30–32 (X = N-Boc; R = H, Cl, F, respectively); 33–35 (X = S; R = H, Cl, F, respectively); 36–38 (X = O; R = H, Cl, F, respectively). Novel 1H-1,2,3-triazole derivatives (27–38) were obtained in good to excellent yield (73–93%) using a lower loading of catalyst and in shorter reaction time (15 min) than previously reported by assisted microwave methodologies.

Figure 7

(a) PT and (b) aPTT coagulation parameters for compounds 27 and 34.

Figure 8

Effect of selected compounds on cell viability using the MTS assay in (a) HEK293 and (b) HepG2 cell lines. Concentrations used were 100 mM (a), 10 mM (b), 100 nM (c), 50 nM (d), 10 nM (e), and 1 nM (f). Blue and red lines defines ± 2 SD limits from the average value of control wells treated only with vehicle (green line). All values are means ± S.D (n = 6) vs. control. Api: Apixaban and Riv: Rivaroxaban. SD: standard deviation.

Figure 9

(a) Overlay of Apixaban (green) and compound 19 over active site of FXa. (b) Aniline derivative 8 on FXa.

Figure 10

(a) Interaction diagram of Compound 19 in the FXa active site. (b) Apixaban interactions with different residues in the FXa active site.

Figure 11

Molecular docking of structures 31, 30, 34, and 27 ((a), (b), (c), and (d), respectively) into the active site of FXa enzyme (PDB: 2HYY). The dotted lines show the most common ligand-protein interactions in all ligand binding poses, HB in yellow, π-π stacking in orange, halogen bond in magenta and CH···O HB in green.