Development of 3-methyl/3-(morpholinomethyl)benzofuran derivatives as novel antitumor agents towards non-small cell lung cancer cells

Figures & data

Figure 1. Chemical structures of benzofurans Fruquintinib and PF-00337210, as well as structures of target 2-methylbenzofurans (4a–d, 6a–c and 8a–c) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b and 17a–b).

Scheme 1. Synthesis of target 2-methylbenzofurans 4a–d and 6a–c; reagents and conditions: (i) NH₂NH₂.H₂O/isopropyl alcohol/reflux 2 h and (ii) dry toluene/reflux 7 h.

Scheme 2. Synthesis of target 2-methylbenzofurans 8a–c and 11; reagents and conditions: (i) Ethanol/Cat. Acetic acid/reflux 3 h, (ii) ethanol/cat. Acetic acid/reflux 7 h.

Scheme 3. Synthesis of key intermediate 3-(morpholinomethyl)benzofuran-2-carbohydrazide 14; Reagents and conditions: (i) NBS/carbon tetrachloride/dibenzoyl peroxide/reflux 16 h, (ii) Morpholine/Acetonitrile/K₂CO₃/KI/reflux 8 h, and (iii) NH₂NH₂.H₂O/isopropyl alcohol/reflux 2 h.

Scheme 4. Synthesis of target 3-(morpholinomethyl)benzofurans 15–18; reagents and conditions: (i) dry toluene/reflux 7 h and (ii) ethanol/cat. Acetic acid/reflux 3 h.

Figure 2. Effect of 3-methylbenzofuran derivative 4b on the phases of cell cycle of A549 cells, and effect of 3-(morpholinomethyl)benzofuran derivatives 15a and 16a on the phases of cell cycle of NCI-H23 cells.

Figure 3. Effect of 3-methylbenzofuran derivative 4b on the percentage of annexin V-FITC-positive staining in Non-small cell lung cancer A549 cells.

Figure 4. Effect of 3-(morpholinomethyl)benzofuran derivatives 15a and 16a on the percentage of annexin V-FITC-positive staining in Non-small cell lung cancer NCI-H23 cells.

Table 1. Cytotoxic impact against non-tumorigenic human lung WI-38 cell line, as well as mean tumour selectivity index (S.I.) (WI-38/A549 and NCI-H23).

Compounds	IC50 (µM)			Mean tumour selectivity
	WI-38	A549	NCI-H23
4b	19.51 ± 1.18	1.48 ± 0.08	5.90 ± 0.29	5.3
15a	32.81 ± 1.98	5.27 ± 0.29	2.52 ± 0.13	8.4
16a	12.86 ± 0.78	1.50 ± 0.08	0.49 ± 0.02	12.9

Table 2. In vitro anti-proliferative activity of target 3-methylbenzofurans (4a–d, 6a–c, 8a–c and 11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b and 18) against lung A549 and NCI-H23 cancer cell lines.

Compounds	X	R	IC50 (µM)^a
			A549	NCI-H23
4a	S	3-OCH3	10.09 ± 0.55	34.18 ± 1.71
4b	S	4-OCH3	1.48 ± 0.08	5.90 ± 0.29
4c	S	3-CF3	36.49 ± 0.2	24.36 ± 0.12
4d	S	4-F	12.79 ± 0.7	67.22 ± 3.36
6a	O	3-OCH3	15.11 ± 0.83	56.66 ± 2.83
6b	O	4-OCH3	12.5 ± 0.68	9.24 ± 0.46
6c	O	4-Cl-3-CF3	47.02 ± 2.57	24.54 ± 1.23
8a	H	3-OCH3	44.1 ± 2.41	14.89 ± 0.74
8b	H	4-OCH3	9.204 ± 0.5	6.148 ± 0.31
8c	CH3	4-OCH3	16.35 ± 0.89	13.95 ± 0.7
11	–	–	30.19 ± 1.65	12.05 ± 0.6
15a	S	3-OCH3	5.273 ± 0.29	2.525 ± 0.13
15b	S	4-OCH3	5.7 ± 0.31	68.9 ± 3.44
15c	S	3-CF3	6.303 ± 0.34	2.218 ± 0.11
16a	O	3-OCH3	1.50 ± 0.08	0.49 ± 0.02
16b	O	4-OCH3	18.89 ± 1.03	5.089 ± 0.25
17a	H	H	6.01 ± 0.03	22.97 ± 1.15
17b	H	4-OCH3	10.83 ± 0.59	3.043 ± 0.15
18	–	–	3.697 ± 0.2	29.75 ± 1.49
Staurosporine	–	–	1.52 ± 0.05	1.24 ± 0.02

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

Table 3. Inhibitory activity of target benzofurans 4b, 15a and 16a against VEGFR-2.

Compounds	IC50 (nM)
	VEGFR-2
4b	77.97 ± 4.6
15a	132.5 ± 7.8
16a	45.4 ± 2.7
Sorafenib	34.68 ± 2.6

Table 4. Anti-tubercular activity of the target compounds 4b, 15a and 16a.

Compounds	MIC (µg/ml)
4b	500
15a	62.5
16a	125
INH	0.24

Figure 5. 2D (A) and 3D (B) interactions of 3-methylbenzofuran derivative 4b within VEGFR-2 binding site (PDB: 4ASD).

Figure 6. 2D (A) and 3D (B) interactions of 3-(morpholinomethyl)benzofuran derivative 15a within VEGFR-2 binding site (PDB: 4ASD).

Figure 7. 2D (A) and 3D (B) interactions of 3-(morpholinomethyl)benzofuran derivative 16a within VEGFR-2 binding site (PDB: 4ASD).

Table 5. The detailed bonding interactions and binding scores for the benzofurans 4b, 15a and 16a.

Compounds	Score kcal\mole	Bonding interaction	Distance (Å)
4b	−9.9	Hydrogen bond with GLU885	2.66
		Hydrogen bond with CYS1045	2.38
		Hydrogen bond with ASP1046	3.05
		Pi-alkyl with LEU889	5.37
		Pi-anion with ASP1046	4.88
		Pi-donor with ASP1046	2.67
		Pi-alkyl with LYS868	5.41
		Pi-alkyl with CYS1045	4.17
		Pi–Pi interaction with PHE1047	4.91
		Pi-lone pair with PHE1047	2.68
15a	−10.4	Hydrogen bond with GLU885	2.88
		Hydrogen bond with CYS1045	2.47
		Hydrogen bond with ASP1046	2.58
		Pi-alkyl with LEU889	5.33
		Alkyl–alkyl with VAL898	5.13
		Alkyl–alkyl with LEU1019	4.57
		Alkyl–alkyl with ILE1044	5.04
		Pi-alkyl with HIS1026	5.46
		Pi-alkyl with LYS868	5.34
		Pi-alkyl with CYS1045	4.33
		Pi–Pi interaction with PHE1047	4.66
		Pi-lone pair with PHE1047	2.29
16a	−10.5	Hydrogen bond with GLU885	2.87
		Hydrogen bond with CYS1045	2.47
		Hydrogen bond with ASP1046	2.93
		Pi-alkyl with LEU889	5.33
		Pi-alkyl with ILE888	5.25
		Alkyl–alkyl with VAL898	5.18
		Alkyl–alkyl with LEU1019	4.54
		Alkyl–alkyl with ILE1044	5.04
		Pi-alkyl with HIS1026	5.40
		Pi-alkyl with LYS868	5.48
		Pi-alkyl with CYS1045	4.07
		Pi–Pi interaction with PHE1047	4.79
		Pi-lone pair with PHE1047	2.29