Synthesis and biological activity evaluation of 3-(hetero) arylideneindolin-2-ones as potential c-Src inhibitors

Figures & data

Figure 1. (a) Inactive conformation of c-Src shows phosphorylated Tyr530 on the SH2 domain; (b) Phosphorylation of Tyr419 on the kinase domain SH1 allows to activate of the enzyme.

Figure 2. Structures of the molecules approved by the FDA or in clinical trials as Src-family inhibitors.

Table 1. Evaluation of the inhibitory activity of c-Src by the in-house prepared collection of small molecules.

Dasatinib was used as reference compound.

Table 2. Inhibitory activity evaluation of indolinones 22–30.

	% Inhibition
Compound	100 µM	10 µM	ID50 (µM)	Ki (ATP competitive)
Dasatinib	100	100	1.60 nM ± 0.22 nM	0.80 nM ± 0.11 nM
	64.61 ± 3.80	32.06 ± 3.53
	57.44 ± 6.31	22.77 ± 2.94
	87.46 ± 7.41	34.18 ± 0.60
	35.79 ± 3.69	23.15 ± 5.55
	94.70 ± 0.30	67.03 ± 0.10	5.63 μM ± 2.46 μM	3.80 µM ± 0.63 μM
	45.58 ± 7.85	25.30 ± 9.71
	55.15 ± 0.91	30.69 ± 10.16
	78.56 ± 1.32	39.81 ± 3.16
	42.03 ± 8.91	9.39 ± 19.22

Dasatinib was used as reference compound.

Scheme 1. General synthesis by Knoevenagel condensation starting from 6-chloro oxindole and different aromatic and heteroaromatic compounds.

Table 3. Evaluation of the inhibitory activity of the novel indolinone derivatives.

	% Inhibition
Compound	100 µM	10 µM	ID50	Ki (ATP competitive)
Dasatinib	100	100	1.60 nM ± 0.22 nM	0.80 nM ± 0.11 nM
	88.96 ± 1.35	35.74 ± 9.66	>10 µM	–
	69.94 ± 0.19	53.03 ± 18.08	2.43 µM ± 1.03 µM	1.64 µM ± 0.69 µM
	75.72 ± 7.63	85.35 ± 6.98	0.71 µM ± 0.43 µM	0.48 µM ± 0.29 µM
	86.86 ± 2.50	37.90 ± 0.35	>10 µM	–
	47.19 ± 9.33	4.15 ± 1.28	–	–
	84.27 ± 0.82	16.86 ± 13.95	–	–
	53.62 ± 12.91	31.48 ± 3.16	–	–
	80.84 ± 6.47	71.67 ± 2.27	3.24 µM ± 0.43 µM	2.19 µM ± 1.03 µM
	92.13 ± 3.84	41.05 ± 10.30	–	–
	34.40 ± 13.26	14.44 ± 2.09	–	–
	70.56 ± 4.80	0	–	–
	93.88 ± 0.58	63.39 ± 1.06	5.31 µM ± 0.47 µM	3.58 µM
	87.71 ± 3.04	12.83 ± 1.32	–	–
	76.82 ± 9.22	53.85 ± 26.57	>10 µM	–
	96.08 ± 1.42	41.20 ± 1.12	9.14 µM ± 1.74 µM	6.17 µM

Dasatinib was used as reference compound.

Table 4. Cytotoxicity evaluation of selected compounds on human MCF-7 breast cancer cell line.

Compound	MCF-7 (IC50, μM)
Dasatinib	27 ± 1
33	>100
34	>100
43	85 ± 2
49	33 ± 2
50	68 ± 2

24 h after seeding, cells were exposed for 72 h to the compounds and cytotoxicity was measured using MTS assay. Experiments were performed in triplicate and data represent mean values ± SD. Dasatinib was used as reference compound.

Figure 3. Graphical representation of the best-scored binding pose of 33 (magenta) within the c-Src ATP binding site, in comparison to the co-crystallized inhibitor AP23464 (green). Yellow dashed lines represent hydrogen bonds between Met341, Glu310, Asp404, and both ligands.