Design and synthesis of novel phenylaminopyrimidines with antiproliferative activity against colorectal cancer

Abstract

New phenylaminopyrimidine (PAP) derivatives have been designed and synthesised as potential tyrosine kinase inhibitors for the treatment of cancer. The synthesized compounds share a general structure and vary in the substitution pattern at position-2 of the pyridine ring. Several derivatives have demonstrated potent anticancer activities against HCT-116, HT-29 and LS-174T colorectal cancer cells. Furthermore, a number of hits showed good selectivity to Src-kinase. The cytotoxic mechanisms of these compounds were also investigated by studying their effects on cell-cycle distribution. Among all the compounds examined, compound 8b (with a terminal pyridin-3-yl moiety at the pyridine ring) showed the highest inhibitory selectivity towards src-kinase, which was coupled with cell cycle arrest, and apoptotic and autophagic interference, in colorectal cancer cells. This report introduces a novel category of PAP derivatives with promising kinase inhibitory and anticancer effects against colon cancer.

Fig. 1. Structures of lead inhibitors, imatinib (1), CT5102 (2), and the PAP lead 3.

Fig. 2. General structure of the novel PAP inhibitors derived from the structures of the lead inhibitor 3.

Scheme 1. Synthesis of target PAP derivatives 8a–j. Reaction conditions and yields: (i) 2-chloro-4-methylpyridine, LHMDS, THF, N₂, rt, 18 h, 72%; (ii) DMF-DMA, reflux, 12 h; (iii) NaOEt, guanidine-HCl, abs. EtOH, reflux, 18 h, 58%; (iv) 3,5-bis(trifluoromethyl)phenyl bromide, Pd(PPh₃)₂Cl₂, xantphos, NaO^tBu, toluene, reflux, 12 h, 69%; (v) arylboronic acid, Pd(PPh₃)₂Cl₂, K₂CO₃, N₂, THF/H₂O; (4 : 1), 70 °C, 20 h.

Scheme 2. Synthesis of the hydroxyl analogues 9 and 10a–j. Reaction conditions and yields: (i) BF₃S(CH₃)₂, CHCl₃, N₂, rt, 48 h.

Fig. 3. The effect of test compounds at 10 μM concentration against the activity of total intracellular tyrosine kinase (A) and purified Src kinase (B). Data are expressed as mean ± SD; n = 3.

Fig. 4. Dose–response relationship of selected compounds against the activity of purified Src kinase enzyme. Data are expressed as mean ± SD; n = 3.

Fig. 5. Molecular docking simulation of compound 8b into src kinase co-crystal protein structure 4MXO. Upper panel: a 3D presentation of the most stable docking pose of 8b showing proposed HB interaction between the terminal pyridine nitrogen and glutamate 353. Lower panel: a 2D presentation for the docked structure.

Fig. 6. Effect of compounds under investigation on the viability of HCT-116 (A), HT-29 (B) and LS-174T colorectal cancer cells (C). Cells were treated with 10 μM and 100 μM of test compounds for 72 h and viability was determined using SRB assay. Data are expressed as mean ± SD; n = 6.

Fig. 7. Dose–response assessment for compounds 7 (A), 8b (B), 9 (C), 10a (D), 10b (E), 10f (F) and 10g (G) against HCT-116 (solid lines) and HT-29 cells (dotted lines). Cells were treated with test compounds for 72 h and viability was determined using SRB assay. Data are expressed as mean ± SD; n = 6.

Fig. 8. The effect of compounds 7, 8b, 9, 10a, 10b, and 10f on the cell cycle distribution of HCT-116 (A) and HT-29 (B) cells. Cells were exposed to 10 μM of test compounds for 48 h and cell cycle distribution was determined using DNA cytometry analysis and different cell phases were plotted as percentage of total events. Data is presented as mean ± SD; n = 3. (*): Significantly different from control group.

Fig. 9. Apoptosis/necrosis assessment in HCT-116 (A) and HT-29 (B) cells after exposure to test compounds. Cells were exposed to 10 μM of compounds 7, 8b, 9, 10a, 10b, and 10f for 48 h and were stained with annexin V-FITC/PI. Different cell populations were plotted as percentage of total events. Data is presented as mean ± SD; n = 3. (*): Significantly different from control group.

Fig. 10. Autophagic cell death assessment in HCT-116 (A) and HT-29 (B) cells after exposure to test compounds. Cells were exposed to 10 μM of compounds 7, 8b, 9, 10a, 10b, and 10f for 48 h. Cells were stained with Cyto-ID autophagosome tracker. Net fluorescent intensity (NFI) were plotted and compared to basal fluorescence of control group. Data is presented as mean ± SD; n = 3. (*): Significantly different from control.