New 1,2,4-Triazole Derivatives with a N-Mannich Base Structure Based on a 4,6-Dimethylpyridine Scaffold as Anticancer Agents: Design, Synthesis, Biological Evaluation, and Molecular Modeling

Abstract

A series of novel N-Mannich bases derived from a dimethylpyridine-1,2,4-triazole hybrid was synthesized and evaluated in vitro for cytotoxic activity on several human gastrointestinal cancer cells (EPG, Caco-2, LoVo, LoVo/Dx, and HT-29). Compound 6 bearing a phenyl group at the N-4 position and a 4-methylphenyl piperazine moiety at the N-2 position of the 1,2,4-triazole-3-thione scaffold exerted good cytotoxic activities on EPG and Caco-2 cell lines, along with pronounced selectivity, showing lower cytotoxicity against normal colonic epithelial cells (CCD 841 CoTr). Further evaluation revealed the good ability of compound 6 to inhibit the efflux function of P-glycoprotein in P-gp-expressing cell lines (HT-29, LoVo, and LoVo/Dx). Moreover, compound 6 induced apoptotic cell death through a significant increase in the caspase-3 and p53 protein levels in HT-29 cells. Finally, the molecular docking method was applied to explain our experimental findings. The molecular modeling study based on Density Functional Theory (DFT) and the Quantum Theory of Atoms in Molecules (QTAIM) analysis provided insight into the geometric and electronic structure properties of the compounds.

Keywords: 1,2,4-triazole; DFT; N-Mannich base; QTAIM; anticancer activity; cytotoxicity; dimethylpyridine; molecular docking.

Figure 1

Market-available anticancer drugs containing 1,2,4-triazole and cytotoxic compounds comprising pyridine, 1,2,4-triazole, and/or N-Mannich base cores.

Figure 2

Target 1,2,4-triazole-derived N-Mannich bases.

Scheme 1

Scheme for synthesis of N-Mannich bases derivatives 4–13 (a—appropriate isothiocyanate/EtOH/reflux/12–14 h, b—NaOH/reflux/2 h/HCl, c—EtOH, HCHO, appropriate phenylpiperazine/r.t./4 h).

Figure 3

Influence of compounds 5 and 6 on rhodamine 123 accumulation in HT-29, LoVo, and LoVo/Dx cancer cells. The mean fluorescence intensity of accumulated Rh-123 dye, which means the results were normalized to control untreated cells (E is the result for the measured sample, and E₀ is the result for the control). Verapamil was used as a positive control.

Figure 4

The effect of compounds 5 and 6 on the level of apoptotic and necrotic HT-29 cells after 24 h of incubation. Doxorubicin was used as a reference drug.

Figure 5

Molecular structures of N-Mannich 6, 8, and 11 obtained as a result of DFT/ωB97-XD/def2-TZVP (selected metric parameters of all compounds were gathered in Table S3) simulations in vacuo (left) and with solvent reaction field with water as a solvent (right).

Figure 6

Selected QTAIM molecular graphs obtained based on results from the DFT/ωB97-XD/def2-TZVP level of theory in vacuo for compounds 6, 8, and 11. The dotted line indicates the presence of a non-covalent interaction. The green and red spheres are Bond and Ring Critical Points (BCPs and RCPs), respectively. The complete set of graphs is presented in the SI.

Figure 7

Visualization of the studied enzymes (A)—caspase-3, (B1)—MDM-2 (model 1), and (B2) MDM-2 (model 2) with their native ligand structures as well as their binding pockets (transparent surfaces) calculated with PyVol.

Figure 8

The electrostatic potential (ESP) visualization of the studied enzyme models with their native ligands. Enzymes (A)—caspase-3, (B1)—MDM-2 (model 1), and (B2)—MDM-2 (model 2). The red color indicates regions with an excess of negative charge, while blue indicates those with a positive charge.

Figure 9

Molecular docking results obtained for the set of studied N-Mannich bases. The host macromolecules are MDM-2 (models 1 and 2) and caspase-3, respectively.

Figure 10

Visualization of selected positions of ligand–protein complexes, (A)—caspase-3 with compounds denoted as 8, 10, and 13, (B1)—MDM-2 (model 1) with 8, and (B2) MDM-2 (model 2) with 4, 5, 6, 7, and 8 positions of ligands that obtained the most favorable binding affinities in the docking experiment. Coloration of the carbon atoms of the ligands: (in order of increasing number of ligands) tan, light blue, pink, light green, light brown.

Figure 11

Diagrams of the interactions of selected ligand–protein complexes for the model enzymes (A)—caspase-3 and (B1)—MDM-2 (model 1). Hydrophobic interactions were indicated by the presence of red combs, while hydrogen bonds were indicated by green pathways between the H-bond donor and acceptor atoms.

Figure 12

Diagrams of interactions of selected ligand–protein complexes for model enzyme (B2)—MDM-2 (model 2). Hydrophobic interactions are indicated by the presence of red combs, while hydrogen bonds are indicated by green paths between the H-bond donor and acceptor atoms.