Antitumor properties of certain spirooxindoles towards hepatocellular carcinoma endowed with antioxidant activity

Figures & data

Figure 1. Chemical structure of some naturally isolated anticancer spirooxindoles, and the synthetic spirooxindoles 6a–p.

Scheme 1. Synthesis of target compounds 6a–p; Reagents and conditions: (i) CH₃CN, DMF, NaH, benzene, reflux 4 h; (ii) Ethanol, phenylhydrazine, reflux 1 h; (iii) HOAc/H₂O (1:1 v/v), heating at 120 °C, 8–11 h.

Table 1. Anti-proliferative activities of spirooxindoles 6a–p against HepG2 hepatocellular carcinoma and PC-3 prostate cancer cell lines.

Compound	R	Ar	IC50 (µM)
			HepG2	PC-3
6a	H	C6H5––	6.9	11.8
6b	H	4–CH3–C6H4–	21.0	34.2
6c	H	4–OCH3–C6H4–	19.1	43.7
6d	H	4–Cl–C6H4–	49.1	>50.0
6e	Cl	C6H5–	8.4	13.5
6f	Cl	4–CH3–C6H4–	32.7	29.3
6g	Cl	4–OCH3–C6H4–	>50.0	>50.0
6h	Cl	4–Cl–C6H4–	19.9	>50.0
6i	Br	C6H5–	6.3	17.9
6j	Br	4–CH3–C6H4–	9.9	26.6
6k	Br	4–OCH3–C6H4–	13.2	39.1
6l	Br	4–Cl–C6H4–	12.7	>50.0
6m	OCH3	C6H5–	30.6	24.3
6n	OCH3	4–CH3–C6H4–	>50.0	42.8
6o	OCH3	4–OCH3–C6H4–	>50.0	>50.0
6p	OCH3	4–Cl–C6H4–	>50.0	>50.0
Adriamycin			0.12	0.62

Figure 2. Morphological changes following 48 h exposure of HepG2 cells to indicated concentrations of 6a, 6e and 6i. Signs of toxicity indicated with arrows represent cell rounding, shrinkage and/or loss of monolayer integrity. Total magnification = 300.

Table 2. Cytotoxic action of spirooxindoles 6a–c, 6e, 6f, 6i–k and 6 m towards non-tumorigenic human MCF-10A cell line, and selectivity index (S. I.) for tumour cells (MCF-10A/HepG2).

Compound	IC50 (µM)		Tumour S. I.
	MCF-10A	HepG2
6a	94.56	6.9	13.7
6b	121.61	21.0	5.8
6c	141.30	19.1	7.4
6e	69.74	8.4	8.3
6f	158.79	32.7	4.9
6i	76.94	6.3	12.2
6j	146.41	9.9	14.8
6k	136.27	13.2	10.3
6m	185.96	30.6	6.1

Figure 3. Effect of spirooxindole 6a on the phases of cell cycle of HepG2 cells.

Table 3. Effect of compound 6a on the expression levels of Bax and Bcl-2 in HepG2 cells treated with the compound at its IC₅₀.

Comp.	Bax ng/mL	Bcl-2 ng/mL	Bax/Bcl-2 ratio
Control	40.63	4.34	9.4
6a	232.2 (5.7)*	2.51 (0.57)*	92.5

*Numbers given between parentheses are the numbers of folds of control.

Table 4. Effect of compound 6a on the expression levels of active caspases-3 and -9, and p53 in HepG2 cells treated with the compound at its IC₅₀.

Comp.	Caspase-3 pg/mL	Caspase-9 ng/mL	p53 pg/mL
Control	53.57	2.19	77.15
6a	475.9 (8.88)*	26.92 (12.27)*	774.5 (10.03)*

*Numbers given between parentheses are the numbers of folds of control.

Figure 4. Effect of spirooxindole 6a on the percentage of annexin V-FITC-positive staining in HepG2 cells. The experiments were done in triplicates. The four quadrants identified as: LL: viable; LR: early apoptotic; UR: late apoptotic; UL: necrotic.

Table 5. The in vitro antioxidant activity of spirooxindoles (6a–p) in DPPH˙ scavenging assay.

Compound	R	Ar	IC50 (µg/mL)^a
6a	H	C6H5–	29.76 ± 1.82
6b	H	4–CH3–C6H4–	113.45 ± 5.59
6c	H	4–OCH3–C6H4–	43.04 ± 2.39
6d	H	4–Cl–C6H4–	33.43 ± 1.24
6e	Cl	C6H5–	36.03 ± 1.37
6f	Cl	4–CH3–C6H4–	40.31 ± 2.61
6g	Cl	4–OCH3–C6H4–	97.47 ± 5.62
6h	Cl	4–Cl–C6H4–	46.29 ± 2.18
6i	Br	C6H5–	36.38 ± 2.71
6j	Br	4–CH3–C6H4–	43.42 ± 3.04
6k	Br	4–OCH3–C6H4–	69.25 ± 4.25
6l	Br	4–Cl–C6H4–	41.36 ± 2.17
6m	OCH3	C6H5–	28.07 ± 1.63
6n	OCH3	4–CH3–C6H4–	46.29 ± 2.52
6o	OCH3	4–OCH3–C6H4–	58.82 ± 3.14
6p	OCH3	4–Cl–C6H4–	55.05 ± 3.41
Ascorbic acid	–	–	45.89 ± 2.39

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

Table 6. The in vitro antioxidant activity of spirooxindoles (6a–p) in FRAP assay.

Compound	µM (Fe^2+ Eq.)
6a	11.98
6b	6.933
6c	10.32
6d	10.99
6e	10.12
6f	11.36
6g	7.79
6h	11.35
6i	11.88
6j	10.34
6k	8.499
6l	11.59
6m	12.13
6n	11.58
6o	11.90
6p	11.18
Ascorbic acid	13.42

Figure 5. (A) Predicted Boiled-Egg plot from swissADME online web tool for spirooxindole 6a; (B) Bioavailability radar chart for spirooxindole 6a; The pink area represents the range of the optimal property values for oral bioavailability and the red line is spirooxindole 6a predicted properties.

Table 7. In silico predictions of the pharmacokinetics properties for spirooxindole 6a.

Cpd.	GIA	BBB	P-gp substrate	CYP1A2 inhibitor	CYP2C9 inhibitor	CYP2C19 inhibitor	CYP3A4 inhibitor	CYP2D6 inhibitor
6a	High	No	No	Yes	Yes	Yes	No	No

GIA: gastrointestinal absorption; BBB: blood-brain barrier permeation; P-gp: permeability glycoprotein; CYP1A2, CYP2C9, CYP2C19, CYP3A4 and CYP2D6 are the five major isoforms of cytochromes P450.

Table 8. In silico predictions of the drug-likeness properties for spirooxindole 6a.

Cpd.	Lipinski #violations	Ghose #violations	Veber #violations	Egan #violations	PAINS #alerts	Brenk #alerts	Bioavailability Score
6a	0	2	0	0	0	0	0.55

All calculations were performed using SwissADME [36].

Swiss Institute of Bioinformatics. http://www.swissadme.ch/index.php.

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