Design, synthesis, molecular modeling and biological evaluation of novel Benzoxazole-Benzamide conjugates via a 2-Thioacetamido linker as potential anti-proliferative agents, VEGFR-2 inhibitors and apoptotic inducers

Figures & data

Figure 1. Design of the target benzoxazole-benzamide conjugates 1–15.

Figure 2. Target benzoxazoles fulfilled the pharmacophoric structural features of VEGFR-2 inhibitors.

Scheme 1 Synthesis of the compounds 1–12; Reagents/conditions: (i) CS₂/KOH/CH₃OH/reflux 6 h, (ii) KOH/C₂H₅OH/reflux 4 h, (iii) ClCH₂COCl, NaHCO₃/DMF/r.t./1h, (iv) SOCl₂/1,2-dichloroethane/reflux 4 h, (v) R-NH₂/acetonitrile/TEA/r.t. 8 h, (vi) DMF/KI/60 °C/6h.

Scheme 2. Synthesis of the compounds 13–15; Reagents/conditions: (i) CH₃OH/conc. H₂SO₄/reflux 2 h, (ii) NH₂-NH₂/C₂H₅OH/reflux 4 h, (iii) acetonitrile/TEA/r.t. 8 h, (vi) DMF/KI/60 °C/6h.

Figure 3. In vitro anti-proliferative activity of the target compounds 1–15.

Table 1. In vitro anti-proliferative activity of the compounds 1–15 against HCT-116, MCF-7 human cancer cell lines and W-180 normal cell line, and their corresponding selectivity indices.

Compounds	HCT-116		MCF-7		WI-38
	IC50 (µM)^a	SI^b	IC50 (µM)^a	SI^b	IC50 (µM)^a
1	7.8 ± 0.015	5.3	7.2 ± 0.010	5.8	41.9 ± 0.27
2	18.5 ± 0.014	3.0	11.7 ± 0.014	4.8	56.7 ± 0.36
3	24.2 ± 0.019	2.4	22.7 ± 0.008	2.6	59.3 ± 0.45
4	23.2 ± 0.012	2.2	23.6 ± 0.015	2.2	51.5 ± 0.40
5	20.9 ± 0.025	4.0	12.4 ± 0.007	6.9	85.4 ± 0.55
6	30.7 ± 0.011	4.0	19.1 ± 0.006	6.5	124.6 ± 0.70
7	32 ± 0.002	4.0	24 ± 0.011	5.3	128.2 ± 0.75
8	28.8 ± 0.010	4.7	22.3 ± 0.004	6.0	135.4 ± 0.85
9	17.1 ± 0.008	4.4	12.3 ± 0.008	6.0	74.6 ± 0.53
10	16.7 ± 0.012	7.5	16.1 ± 0.011	7.8	125.5 ± 0.72
11	12.2 ± 0.007	8.5	16.6 ± 0.013	6.2	103.5 ± 0.70
12	10.4 ± 0.010	12.1	9.4 ± 0.016	13.4	126.2 ± 0.75
13	9.1 ± 0.005	10.0	9.0 ± 0.005	10.1	91.3 ± 0.60
14	9.7 ± 0.013	9.9	9.5 ± 0.009	10.1	96.5 ± 0.65
15	12.9 ± 0.014	10.2	15.3 ± 0.01	8.6	131.5 ± 0.80
Sorafenib	11.6 ± 0.012	–	10.5 ± 0.014	–	–

^aIC₅₀ values are the mean ± SD of three separate experiments.

^bSelectivity index (SI) is the ratio of the IC₅₀ value for normal cells (WI-38) to the IC₅₀ values for HCT-116 and MCF-7 cells.

Figure 4. Inhibitory activity of 1, 9, 10, 11, 12 and 15 against VEGR-2 Protein Kinase.

Table 2. Inhibitory activity of 1, 9, 10, 11, 12 and 15 against VEGR-2 Protein Kinase.

No.	VEGFR-2 Protein Kinase IC50 (µM)
1	0.268 ± 0.005
9	0.649 ± 0.008
10	0.704 ± 0.009
11	0.361 ± 0.004
12	0.385 ± 0.005
15	0.597 ± 0.007
Sorafenib	0.352 ± 0.005

IC₅₀ values are the mean of three individual experiments.

Figure 5. Cell distribution in the subG1, G0/G1, S and G2/M phases for HCT116 cells (B) treated with vehicle control (A), compounds 1 (C) and 11 (D).

Figure 6. Cell distribution in the subG1, G0/G1, S and G2/M phases for MCF7 cells (B) treated with vehicle control (A), compounds 1 (C) and 11 (D).

Table 3. Effect of compounds 1, 11 and vehicle control on the cell cycle phases of HCT-116 and MCF-7 cells lines.

Compound / Cell line	%G0-G1	%S	%G2-M	%Pre-G1
1 / HCT116	42.34	33.25	24.41	15.36
11 / HCT116	45.37	26.19	28.44	17.41
Control / HCT116	49.51	31.56	18.93	4.52
1 / MCF7	40.66	29.72	29.62	19.78
11 / MCF7	43.84	30.92	25.24	16.93
Control / MCF7	55.14	28.33	16.53	3.41

Figure 7. Effect of compounds 1, 11 and vehicle control on the percentage of annexin V-FITC-positive staining in HCT-116 cell line. The experiments were done in triplicates. The four quadrants identified as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.

Figure 8. Effect of compounds 1, 11 and vehicle control on the percentage of annexin V-FITC-positive staining in MCF-7 cell lines. The experiments were done in triplicates.

Table 4. Percent of apoptosis and necrosis induced by compounds 1, 11 and vehicle control in HCT-116 and MCF-7 cell lines.

Compound / Cell line	%Apoptosis			%Necrosis
	%Total	%Early	%Late
1 / HCT-116	15.36	4.86	8.95	1.55
11 / HCT-116	17.41	6.01	9.62	1.78
Control / HCT-116	4.52	1.1	1.57	1.85
1 / MCF-7	19.78	6.02	12.17	1.59
11 / MCF-7	16.93	4.93	10.26	1.74
Control / MCF-7	3.41	0.47	1.34	1.6

Figure 9. Effect of compounds 1, 11 and vehicle control on anti-apoptotic proteins (Bcl-2 and Bcl-xL) in (A) HCT-116 cancer cells and (B) MCF-7 cancer cells.

Figure 10. Docking of compounds 1, 11 and sorafenib into the VEGFR active site. (A) Interaction of Sorafenib with amino-acids Leu840, Glu885, Lys920 and Asp1046. (B) Interaction of 1 with amino-acids Leu840, Lys868, Cys919, Asp1046 and Phe1047 and superimposition of 1 (shown as cyan sticks) with sorafenib (shown as green sticks). (C) Interaction of 11 with amino-acids Lys868 and Asp1046 and superimposition of 11 (shown as cyan sticks) with sorafenib (shown as green sticks).

Table 5. Docking energy scores (kcal/mol) obtained from the MOE software for compounds 1–15 and sorafenib.

Compound No.	Score	rmsd_refine	E_conf	E_place	E_score1	E_refine	E_score2
1	−8.45083	3.46348	7.13944	−61.18144	−10.73645	−15.61404	−8.450831
2	−8.07490	4.14820	24.33731	−65.02733	−11.56395	−16.29065	−8.074901
3	−7.18548	1.39598	29.55076	−82.09853	−11.23328	−8.114554	−7.185479
4	−8.41887	1.47159	27.91452	−89.37426	−11.65076	−19.99584	−8.418868
5	−7.63854	2.33253	2.78024	−88.87581	−12.35492	−4.167041	−7.638539
6	−7.88400	1.76917	20.26365	−77.02324	−11.26872	−15.04636	−7.884004
7	−9.36350	1.7113	17.05548	−84.28175	−11.35441	−22.20009	−9.363497
8	−9.38409	1.46039	21.96994	−79.49125	−12.83700	−22.23286	−9.384088
9	−7.93492	0.97358	−1.62790	−108.53313	−12.54650	−5.27634	−7.934922
10	−8.69645	2.34028	9.40272	−69.44931	−11.03539	−21.76292	−8.696453
11	−8.15044	3.47698	16.16184	−104.55356	−11.93168	−16.78374	−8.150444
12	−9.30653	2.37350	11.61155	−100.80922	−11.70867	−21.75587	−9.306530
13	−8.82812	1.14095	63.19030	−79.27531	−10.92340	−23.19646	−8.828117
14	−8.71854	2.22981	70.10210	−86.90809	−10.84697	−21.77860	−8.718533
15	−9.27842	1.94674	53.63672	−69.43458	−10.36066	−21.94845	−9.278418
Sorafenib	−6.98449	1.78143	−3.12607	−79.69516	−10.28793	−7.641235	−6.984490

Score: lower scores are more favourable; rmsd_refine: the root mean square deviation of the pose; E_conf: free binding energy (FBE) of the conformer; E_place: FBE from the placement stage; E_score 1: FBE from the first rescoring stage; E_refine: FBE from the refinement stage; E_score 2: FBE from the second rescoring stage.