Synthesis and characterisation of (Z)-styrylbenzene derivatives as potential selective anticancer agents

Figures & data

Figure 1. The structures of resveratrol, compound 3, CA-4, colchicine, and the design of target compounds 6a-h, 7a-c, 13a-d, and 14a-b.

Figure 2. NOE (nuclear overhauser effect) result of compound 6h.

Scheme 1. Reagents and conditions: (a) (CH₃)₂SO₄, (CH₃)₂CO, K₂CO₃, reflux; (b) LiAlH₄, THF, 0 °C-rt, 4–6 h; (c) CH₂Cl₂, PBr₃, 0 °C-rt, 3–6 h; (d) CH₃CN, TMSCN, TBAF, reflux, 4–6 h; (e) CH₃OH, CH₃ONa, aromatic aldehydes, 4–6 h.

Table 1. In vitro anti-proliferative activity of the synthesised compounds, CA-4, CA-4P, and taxol against eleven cell lines^a (IC₅₀ (µM^b))

Agent	R	IC50 (μM)
		MGC-803	A549	HepG-2	AGS	Bel-7402	HCT116	L-02	HeLa	SGC-7901	MCF-10A	MCF-7
6a	4-NO2	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
6b	4-OBn	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
6c	3-OCH3	22.6 ± 9.25	>100	>100	>100	>100	>100	>100	>100	>100	>100	65.13 ± 6.64
6d	4-OCH3	6.18 ± 0.36	>100	>100	0.05 ± 0.003	0.75 ± 0.21	1.51 ± 0.05	4.15 ± 4.16	>100	>100	3.07 ± 0.18	16.66 ± 4.77
6e	3.4.5-OCH3	>100	>100	>100	44.56 ± 9.11	31.24 ± 2.01	32.1 ± 5.25	70.6 ± 7.65	>100	>100	15.56 ± 2.45	32.88 ± 17.6
6f	4-CH2(CH3)2	7.21 ± 3.65	36.62 ± 0.134	>100	1.33 ± 0.15	0.72 ± 0.01	0.17 ± 0.1	>100	>100	>100	5.79 ± 0.48	4.37 ± 2.80
6g	4-CH2CH3	<0.01	>100	>100	4.38 ± 2.04	0.65 ± 0.05	0.34 ± 0.01	>100	>100	>100	1.19 ± 0.08	>100
6h	4-CH3	<0.01	0.53 ± 0.023	3.74 ± 1.73	0.51 ± 0.45	0.15 ± 0.03	0.61 ± 0.16	>100	>100	>100	9.45 ± 5.25	2.61 ± 0.98
7a	Thiophene-2-yl	0.67 ± 0.22	>100	>100	8.01 ± 1.65	1.82 ± 1.20	16.58 ± 10.26	21.2 ± 10.75	>100	>100	55.81 ± 10.4	15.02 ± 2.00
7b	Pyridine-2-yl	>100	>100	>100	>100	44.6 ± 32.7	>100	>100	>100	>100	33.44 ± 17.4	15.95 ± 5.67
7c	Naphthalene-1-yl	3.45 ± 2.11	>100	>100	3.51 ± 0.25	2.53 ± 0.12	4.58 ± 0.42	8.21 ± 0.051	>100	>100	1.03 ± 0.44	2.83 ± 0.35
13a	4-CH3	>100	>100	>100	27.86 ± 5.84	>100	>100	>100	>100	>100	>100	>100
13b	4-CH2CH3	>100	>100	>100	26.53 ± 3.89	>100	>100	>100	>100	>100	85.69 ± 3.12	35.03 ± 14.8
13c	3.4.5-OCH3	>100	>100	>100	66.20 ± 3.65	77.3 ± 0.26	79.2 ± 5.21	>100	>100	>100	>100	33.94 ± 7.30
13d	4-H	89.21 ± 3.20	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
14a	Thiophene-2-yl	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
14b	Pyridine-2-yl	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
CA-4		0.06 ± 0.02	>100	>100	3.42 ± 0.03	2.02 ± 0.03	0.16 ± 0.00	1.10 ± 0.03	>100	0.30 ± 0.00	3.23 ± 1.21	0.22 ± 0.09
CA-4P		0.03 ± 0.02	>100	>100	4.73 ± 0.22	5.45 ± 2.04	0.59 ± 0.22	2.67 ± 0.61	>100	0.12 ± 0.03	14.55 ± 1.36	0.48 ± 0.25
Taxol		0.06 ± 0.01	0.44 ± 0.02	0.94 ± 0.03	0.02 ± 0.01	0.09 ± 0.02	0.03 ± 0.01	>100	12.95 ± 0.47	>100	0.18 ± 0.02	0.08 ± 0.01

^a Cytotoxicity as IC₅₀ for each cell line, refers to the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using the MTT assay.

^b Data represent the mean values of three independent determinations.

Table 2. Selectivity index values of compounds relative to the effects on normal cell L-02 or MCF-10A cells.

Agent	R	SI
		IC50 (L-02/MGC-803)	IC50 (L-02/A549)	IC50 (L-02/HepG-2)	IC50 (L-02/AGS)	IC50 (L-02/BEL-7402)	IC50 (L-02/HCT116)	IC50 (MCF-10A/MCF-7)
6d	4-OCH3	–	–	–	>83.0	>5.5	>2.7	–
6f	4-CH2(CH3)2	>13.8	>2.7	–	>75.1	>138.8	>588.2	>1.3
6g	4-CH2CH3	>10000	–	–	>22.8	>153.8	>294.1	–
6h	4-CH3	>10000	>188.6	>26.7	>196.0	>666.6	>163.9	>3.6
7a	Thiophene-2-yl	>31.6	–	–	>2.6	>11.6	>1.2	>3.7
CA-4		>18.3	–	–	–	–	>6.8	>14.6
CA-4P		>89.0	–	–	–	–	>4.5	>30.3
Taxol		>1666	>227.2	>106.3	>5000	>1111	>3333	>2.2

–: not suitable for calculation; SI: selectivity index, IC₅₀ (L-02 or MCF-10A)/IC₅₀ (cancer cell).

Figure 3. Compound 6h decreases the expression of cyclin A and cyclin D1 and increases the expression of cyclin B1, thus reducing the cell cycle progression of BEL-7402 cells. (A) Cell numbers in the phases of the cell cycle were analyzed on 12 h after stimulation. Values were obtained from three separate experiments and represent the mean ± SD. *p < 0.05. **p < 0.01. ***p < 0.001 compared to respective control group. (B) Immunoblots were probed using antibodies directed against cyclin A, B1, and D1, and β-actin. Whole-cell protein lysates were extracted from BEL-7402 cells cultured after 12 h. Results are representative of three separate experiments.

Figure 4. Apoptosis induction in BEL-7402 cells after treatment for 12 h with (A) 0.1% DMSO (vehicle); (B) 0.01 μM 6h; (C) 0.1 μM 6h; (D) 0.1 μM taxol. (E) The results of the cell apoptosis.

Figure 5. Transwell assay of compound 6h showing inhibition of BEL-7402 cell migration. (A) The images of stained BEL-7402 cells adhering in the lower layer of insert of transwell with phase-contrast microscopy (200× magnification). (B) The relative migration ratio of BEL-7402 cells. Data expressed as mean ± SD (n ≥ 3). ***p < 0.001 compared to control group.

Figure 6. Colony formation ability was inhibited by 6h treatment. (A) The images of stained colonies under phase-contrast microscopy. (B) The curve was fitted as the inhibition ratio of colony formation against the concentration. Values are represented as mean ± SD from three independent colony formation experiments.

Figure 7. Molecular modelling of compounds in complex with tubulin (PDB: 1SA0). Shown is the proposed binding mode and interaction between tubulin and selected compounds. (A) 6h, (B) taxol, and (C) CA-4. The compounds and important amino acids in the binding pockets are shown in stick model. (D) Superposition of 6h, taxol, and CA-4 in the pocket of tubulin in surface representation. The compounds are shown in stick model with carbon atoms in grey (6h), yellow-green (taxol), or green (CA-4).