New benzothiazole hybrids as potential VEGFR-2 inhibitors: design, synthesis, anticancer evaluation, and in silico study

Figures & data

Figure 1. The necessary pharmacophoric properties of some FDA and clinically approved VEGFR-2 inhibitors.

Figure 2. Representative examples of some reported anticancer VEGFR-2 inhibitors carrying 2-aminobenzothiazole scaffold, thiazolidine-2,4-dione, cyanothiouracil, and thiadiazole-urea pharmacophores.

Figure 3. The fundamental structural requirements for SOR and rational of design of the novel postulated VEGFR-2 inhibitors.

Scheme 1. Preparation of benzothiazole/thiazolidine-2,4-dione hybrids 4a–e; reagents and conditions: (i) CH₂Cl₂/Et₃N 0 °C, rt; (ii) AcOH, NaOAc, reflux 14 h; (iii) EtOH, KOH, reflux 2 h; and (iv) DMF, K₂CO₃, reflux overnight.

Scheme 2. Synthesis of benzothiazole/1,3,4-thiadiazole-aryl urea hybrids 6a–d; reagents and conditions: (i) CS₂, EtOH, reflux overnight; (ii) CH₃CN, reflux overnight; and (iii) acetone, K₂CO₃, reflux overnight.

Scheme 3. Synthesis of benzothiazole/cyanothiouracil hybrids 8a–d; reagents and conditions: (i) EtOH, K₂CO₃, reflux 12 h and (ii) acetone, K₂CO₃, reflux overnight.

Table 1. Cytotoxicity (IC₅₀) of the target hybrids 4a–e, 6a–d, and 8a–d towards HCT-116, HepG-2, MCF-7, and WI-38 cell lines.

Comp.	R	IC50 (µM)^a
		HCT-116	HepG-2	MCF-7	WI-38
4a	4-F	5.61 ± 0.3	7.92 ± 0.6	3.84 ± 0.1	45.23 ± 2.6
4b	4-OCH3	29.92 ± 2.2	34.68 ± 2.4	22.27 ± 1.7	77.57 ± 4.3
4c	4-Br	47.94 ± 2.9	65.47 ± 3.6	50.05 ± 2.9	28.85 ± 2.2
4d	4-CH3	26.78 ± 2.1	21.13 ± 1.8	15.48 ± 1.2	74.01 ± 4.1
4e	3,4,5-(OCH3)3	8.24 ± 0.6	9.04 ± 0.8	6.11 ± 0.4	32.81 ± 2.3
6a	H	92.45 ± 4.9	83.20 ± 4.4	72.62 ± 3.9	57.84 ± 3.5
6b	4-CH3	41.68 ± 2.7	52.33 ± 3.1	39.79 ± 2.3	86.20 ± 4.8
6c	4-OCH3	80.82 ± 4.3	76.90 ± 4.0	67.38 ± 3.8	49.11 ± 2.8
6d	4-Cl	64.41 ± 3.6	69.26 ± 3.8	58.61 ± 3.2	85.66 ± 4.5
8a	4-CH3	13.89 ± 1.0	18.10 ± 1.4	10.86 ± 0.8	53.10 ± 3.1
8b	4-Cl	54.06 ± 3.2	36.98 ± 2.5	43.13 ± 2.5	>100
8c	4-Br	38.29 ± 2.5	27.94 ± 2.2	31.30 ± 2.1	>100
8d	4-OCH3	>100	87.52 ± 4.7	78.22 ± 4.1	63.24 ± 3.8
SOR	–	5.23 ± 0.3	4.50 ± 0.2	4.17 ± 0.2	6.72 ± 0.5

^aIC₅₀ is defined as the concentration needed to inhibit 50% of cancer cell proliferation. Data are represented as the mean ± SD from the dose-response curves as triplicate . IC₅₀, (µg/mL): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), 100–200 (very weak), above 200 (non-cytotoxic).

Figure 4. Cytotoxic effect of the most active compounds 4a, 4e, 8e, and SOR on human normal WI-38 cell line.

Table 2. Inhibitory effects of compounds 4a, 4e, and 8a against VEGFR-2.

Comp.	% Inhibition				IC50 (nM)
	10 nm	100 nm	1 μM	10 μM
4a	31.27	45.14	77.94	91.96	91
4e	24.09	42.84	70.45	88.69	161
8a	16.31	37.14	70.11	83.47	266
SOR	32.47	57.22	81.01	92.17	53

Figure 5. Effect of compounds 4a, 4e, and 8a on DNA-ploidy flow cytometric analysis of MCF-7 cells. The cells were treated with DMSO as control and 4a, 4e, and 8a for 24 h.

Table 3. Effect of compounds 4a, 4e, and 8a on the cell cycle progression in MCF-7 cells compared to SOR.

Comp.	Cell cycle distribution (%)
	G0-G1	S	G2-M
4a	46.87	49.85	3.28
4e	52.35	42.66	4.99
8a	61.36	36.11	2.53
Control (DMSO)	57.92	31.89	10.19

Figure 6. Effect of compounds 4a, 4e, and 8a on the percentage of annexin V-FITC-positive staining in MCF-7 cells. The cells were treated with DMSO as control and 4a, 4e, and 8a for 24 h. Q1 quadrant represents dead (necrotic) cells; Q2 quadrant represents late apoptosis; Q3 quadrant represent live cells; Q4 quadrant represents early apoptosis.

Figure 7. Compound 4a compound 4e compound 8a docked in the active site and aligned to the co-crystallized ligand SOR. Please refer to the online version for colored figure.

Table 4. Binding energy of the top three compounds docked in VEGFR-2 (PDB: 4ASD) in comparison to the co-crystallized ligand SOR.

Compound	FlexXscore
4a	−24.1
4e	−20
8a	−17.9
SOR	−35.6

Table 5. ADMET profile for active compounds 4a, 4e, 8a, and SOR.

Comp.	HIA	CaCo2	MDCK	BBB	CYP3A4 inhibition	PgP inhibition	Carcinogenicity
4a	91.96	0.67	0.13	0.11	Non	Inhibitor	Negative
4e	86.05	3.06	0.08	0.07	Non	Inhibitor	Negative
8a	83.30	0.44	0.13	0.07	Non	Inhibitor	Negative
SOR	93.50	22.55	0.08	1.00	inhibitor	Inhibitor	Negative

Notes: HIA: human intestinal absorption (%), CaCo2: permeability through cells derived from human colon adenocarcinoma (nm/s), MDCK: permeability through Maden Darby canine kidney cells (nm/s); tool for rapid permeability, BBB: blood brain barrier penetration, CYP3A4: cytochrome P450 3A4, PgP: P-glycoprotein.