Benzoxazole derivatives as new VEGFR-2 inhibitors and apoptosis inducers: design, synthesis, in silico studies, and antiproliferative evaluation

Figures & data

Figure 1. The four main pharmacophoric requirements of VEGFR-2 inhibitors.

Figure 2. Summary of the suggested rationale.

Scheme 1. Synthesis of the starting materials 3a-c.

Scheme 2. Synthesis of the intermediates 7a-d and 11.

Scheme 3. Synthesis of the final compounds 12a-l and 13a-c.

Table 1. In vitro anti-proliferative effects of the obtained compounds against HepG2 and MCF-7 cell lines.

Comp. No.	X	HBA-HBD	R	In vitro IC50 (µM) ^a
				HepG2	MCF-7
12a	H	-NH-CO-		38.83 ± 3.2	33.27 ± 2.9
12b	Cl	-NH-CO-		64.16 ± 6.1	77.03 ± 7.3
12c	CH3	-NH-CO-		74.30 ± 6.8	36.72 ± 3.3
12d	H	-NH-CO-		23.61 ± 2.1	44.09 ± 3.8
12e	Cl	-NH-CO-		71.59 ± 6.7	62.29 ± 5.8
12f	CH3	-NH-CO-		36.96 ± 3.4	22.54 ± 1.8
12g	H	-NH-CO-		36.67 ± 2.9	53.01 ± 5.1
12h	Cl	-NH-CO-		102.10 ± 8.5	85.62 ± 8.2
12i	CH3	-NH-CO-		27.30 ± 2.2	27.99 ± 2.1
12j	H	-NH-CO-		50.92 ± 4.6	33.61 ± 2.8
12k	Cl	-NH-CO-		28.36 ± 2.5	86.62 ± 7.8
12l	CH3	-NH-CO-		10.50 ± 0.8	15.21 ± 1.1
13a	H	-CO -NH-NH-CO-		25.47 ± 2.1	32.47 ± 2.9
13b	Cl	-CO -NH-NH-CO-		42.06 ± 3.8	26.31 ± 2.2
13c	CH3	-CO -NH-NH-CO-		24.25 ± 2.1	53.13 ± 3.7
Sorafenib	-	-	-	5.57 ± 0.4	6.46 ± 0.3

^aData are presented as mean of the IC₅₀ values from three different experiments.

Table 2. IC₅₀ values of the tested compounds on the inhibitory activities against VEGFR-2 Kinases Assay.

Comp. No.	X	HBA-HBD	R	VEGFR-2, IC50 (nM)
12d	H	-NH-CO-		194.60
12f	CH3	-NH-CO-		264.90
12i	CH3	-NH-CO-		155.00
12l	CH3	-NH-CO-		97.38
13a	H	-CO -NH-NH-CO-		267.80
Sorafenib	-	-	-	48.16

Figure 3. Correlation graph study.

Table 3. IC₅₀ results of 12d, 12i, and 12 l against WI-38 cell line.

Compound	WI-38, IC50 (µM)
12d	99.41
12i	76.78
12l	37.79
Sorafenib	22.10

Figure 4. Flow cytometry analysis of HepG2 cell cycle after the treatment of compound 12 l.

Table 4. Supressing potentialities of 12I on the cell cycle of HepG2 cells after 24 h treatment.

Sample	Cell cycle distribution (%)^a
	%Sub-G1	%G1	%S	% G2/M
HepG2	0.93 ± 0.02	51.07 ± 1.03	27.22 ± 1.24	20.78 ± 0.23
Compound 12 l /HepG2	0.79 ± 0.25	28.43 ± 0.37**	38.68 ± 1.81*	32.10 ± 181**

^aValues are given as mean ± SEM of two independent experiments and *p < 0.05; **p < 0.01.

Figure 5. Flow cytometry analysis of compound 12 l apoptotic induction against HepG2 cells.

Table 5. Apoptotic potentialities compound 12 l against HepG2 cells after 24 h treatment.

Sample	Viable a (Left Bottom)	Apoptosis a		Necrosis a (Left Top)
		Early (Right Bottom)	Late (Right Top)
HepG2	92.96 ± 0.55	5.34 ± 0.01	1.22 ± 0.77	0.48 ± 0.27
12l / HepG2	64.55 ± 3.43	32.45 ± 3.13*	2.86 ± 0.21	0.14 ± 0.06

^aValues are given as mean ± SEM of two independent experiments. *p < 0.05.

Figure 6. The immunoblotting of effect of compound 12 l against BAX, Bcl-2, and Caspase-3.

Table 6. Effect of compound 12 l on the levels of BAX, Bcl-2, and Caspase-3 proteins expression in HepG2 cells treated for 24 h.

Sample	Protein expression (normalized to β-actin) ^a
	BAX	Bcl-2	BAX/Bcl-2 ratio	Caspase-3
HepG2	1.00 ± 0.08	1.00 ± 0.32	1.00 ± 0.25	1.00 ± 0.06
12l	3.40 ± 0.15**	0.47 ± 0.05	6.83 ± 0.96*	2.98 ± 0.13**

^aValues are given as mean ± SEM of two independent experiments. *p < 0.05, **p < 0.01.

Figure 7. Results of the re-docking step into the VEGFR-2 catalytic site; native ligand (green) and the obtained pose (red).

Figure 8. Sorafenib binding interactions with VEGFR-2 catalytic site.

Figure 9. Binding pose of 12 l with the active site of VEGFR-2.

Figure 10. Superimposition of 12 l (red) and sorafenib (wheat) inside the VEGFR-2 catalytic site.

Table 7. Physicochemical properties of the tested compounds passed Lipinski and Veber Rules

Comp.	Lipinski Rules				Veber Rules
	Num HD	Num HA	M Wt	AlogP	Num Rotatable Bonds	TPSA
12a	2	4	395.475	3.685	6	109.53
12b	2	4	429.92	4.35	6	109.53
12c	2	4	409.501	4.171	6	109.53
12d	2	4	383.464	3.214	6	109.53
12e	2	4	417.909	3.879	6	109.53
12f	2	4	397.491	3.701	6	109.53
12g	2	5	433.48	3.843	7	118.76
12h	2	5	467.925	4.508	7	118.76
12i	2	5	447.506	4.33	7	118.76
12j	2	4	437.899	4.524	6	109.53
12k	2	4	472.344	5.189	6	109.53
12l	2	4	451.925	5.01	6	109.53
13a	3	5	446.478	3.117	7	138.63
13b	3	5	480.923	3.781	7	138.63
13c	3	5	460.505	3.603	7	138.63

Table 8. ADME profile of compounds 12d, 12i, and 12 l

Parameter	12d	12i	12l
Physicochemical properties
Molecular weight	383.46	447.51	451.93
Num. heavy atoms	27	32	31
Num. H-bond acceptors	4	5	4
Num. H-bond donors	2	2	2
Molar Refractivity	107.02	125.24	123.75
TPSA	109.53 Ų	118.76 Ų	109.53 Ų
Consensus Log Po/w	3.34	3.98	4.48
Log S (ESOL)	Moderately soluble	Moderately soluble	Moderately soluble
Drug likeness
Lipinski violations	Yes; 0 violation	Yes; 0 violation	Yes; 0 violation
Ghose violations	Yes	Yes	Yes
Veber violations	Yes	Yes	Yes
Bioavailability Score	0.55	0.55	0.55
Pharmacokinetics
GI absorption	High	Low	Low
BBB permeant	No	No	No
CYP1A2 inhibitor	Yes	Yes	Yes
CYP2C19 inhibitor	Yes	Yes	Yes
CYP2C9 inhibitor	Yes	Yes	Yes
CYP2D6 inhibitor	Yes	Yes	Yes
CYP3A4 inhibitor	Yes	Yes	Yes

Table 9. Radar charts for prediction of oral bioavailability profile of compounds 12d, 12i, and 12 l

Compounds	12d	12i	12l
Radar images

Table 10. Colours, yields, and meting points of the target compounds

Compounds	Color	Meting points (°C)
12a	White crystals	230–232
12b	White crystals	240–242
12c	White crystals	235–237
12d	White crystals	211–215
12e	White crystals	233–235
12f	White crystals	222–224
12g	White crystals	252–254
12h	White crystals	244–246
12i	White crystals	255–257
12j	White crystals	240–242
12k	White crystals	220–222
12l	White crystals	266–268
13a	White crystals	223–225
13b	White crystals	211–213
13c	White crystals	235–237