Synthesis of new thienylpicolinamidine derivatives and possible mechanisms of antiproliferative activity

Abstract

Three thienylpicolinamidine derivatives 4a-c were prepared from their corresponding picolinonitriles 3a-c on treatment with lithium trimethylsilylamide, LiN(TMS)₂, followed by a de-protection step using ethanol/HCl (gas). DFT calculations were used to optimize the geometric structure of the newly synthesized picolinamidines. The comparison of DFT calculated spectral data with the experimental data (¹H-NMR and ¹³C-NMR) showed a good agreement. The in vitro antiproliferative activity of the cationic compounds 4a-c was determined against 60 cancer cell lines representing nine types of cancer. The tested picolinamidines were highly active with compounds 4a and 4b eliciting mainly cytotoxic activity with GI values ranging from -7.17 to -86.03. Leukemia (SR and K-562), colon (SW-620 and HT29), and non-small cell lung cancer (NCI-H460) cell lines were the most responsive to the investigated picolinamidines. In particular, 4-methoxyphenyl derivative 4a showed a profound growth deterring power with GI₅₀ of 0.34 μM against SR, 0.43 μM against SW-620, and 0.52 μM against NCI-H460. The three tested picolinamidines elicited potent GI₅₀ values against all tested cell lines at low micromolar to sub-micromolar level. The new picolinamidines were selective and did not affect normal human fibroblasts. The selectivity index ranged from 13-21 μM. The novel picolinamidines downregulated the expression of key genes in the cell cycle, cdk1 and topoII, but did not affect p53 or txnrd1. Compounds 4b and 4c caused a significant reduction in the concentrations of TopoII and MAPK proteins but were devoid of any effect on the activity of caspase 3. Taken together, these promising anticancer candidates are effective at very low concentrations and safe to normal cells, and most probably work through arresting the cell cycle, and therefore, they deserve further investigations.

Fig. 1. Synthesis scheme for the new thienylpicolinamidine derivatives. Reagents and conditions: (i) Pd(PPh₃)₄, anhydrous K₂CO₃, 1,4-dioxane; (ii) (a) LiN(TMS)₂, (b) ethanol/HCI (gas).

Fig. 2. The frontier molecular orbital of cationic picolinamidines 4a–c.

Fig. 3. The linear regression between experimental and DFT calculated ¹H NMR chemical shifts of the investigated cationic picolinamidines 4a–c.

Fig. 4. The linear regression between experimental and DFT calculated ¹³C NMR chemical shifts of the investigated cationic picolinamidines 4a–c.

Fig. 5. The effect of the picolinamidine derivatives 4a–c on (A) topoisomerase II (Topo II) concentration, (B) cleaved caspase 3 activity, and (C) MAPK concentration in HepG2 cells. Each compound has been assigned three wells and the experiment was performed twice. Data are expressed as mean ± SEM. *Significant change compared to untreated cells.