Synthesis and in vitro anti-proliferative activity of some novel isatins conjugated with quinazoline/phthalazine hydrazines against triple-negative breast cancer MDA-MB-231 cells as apoptosis-inducing agents

Figures & data

Figure 1. Structures of some isatin-, quinazoline- and phthalazine-based I–VI approved anticancer drugs.

Figure 2. Structures of some reported isatins, quinazolines and phthalazines VII–XVII with anti-proliferative activity against the triple-negative breast cancer MDA-MB-231 cells, and structures of the target hybrids 5a–h, 10a–h and 13a–c.

Table 1. In vitro anti-proliferative activity of the newly synthesized hybrids against MDA-MB-231 cell line.

Compound	R	R1	IC50 (μM)^aMDA-MB-231
			5a	4-Cl	H	13.18 ± 0.36
5b	4-Cl	F	15.15 ± 0.15
5c	4-Cl	Cl	17.93 ± 0.11
5d	4-Cl	Br	19.68 ± 0.26
5e	2,6-Cl2	H	12.35 ± 0.12
5f	2,6-Cl2	F	12.95 ± 0.31
5g	2,6-Cl2	Cl	14.59 ± 0.23
5h	2,6-Cl2	Br	17.31 ± 0.55
10a	H	H	13.96 ± 0.10
10b	H	F	15.62 ± 0.14
10c	H	Cl	17.63 ± 0.37
10d	H	Br	21.39 ± 0.13
10e	Cl	H	12.86 ± 0.12
10f	Cl	F	12.94 ± 0.23
10g	Cl	Cl	12.00 ± 0.13
10h	Cl	Br	15.21 ± 0.15
13a	4-Cl	H	26.21 ± 2.07
13b	2,6-Cl2	H	48.72 ± 2.59
13c	4-Cl	F	34.17 ± 2.42
5-FU			29.38 ± 1.24

a IC₅₀ values are the mean ± SD of three separate experiments.

Scheme 1. Reagents and conditions: i, POCl₃/N,N-dimethylaniline/reflux 6 h; ii, NH₂NH₂.H₂O/EtOH/reflux 4 h; iii, EtOH/AcOH (catalytic)/reflux 0.5 h.

Scheme 2. Reagents and conditions: i, NH₂NH₂.H₂SO₄/NaOH/reflux 1 h; ii, POCl₃/N,N-dimethylaniline/reflux 6 h; iii, NH₂NH₂.H₂O/EtOH/reflux 7 h. iv, EtOH/AcOH (catalytic)/reflux 1 h.

Scheme 3. Reagents and conditions: i, DMF/K₂CO₃/reflux 3 h; ii, Compounds 4a,b/EtOH/AcOH (catalytic)/reflux 0.5 h.

Figure 3. Effect of compounds 5e and 10g on the protein levels of A) Bax; B) Bcl-2 in MDA-MB-231 cells treated with the compounds at their IC₅₀ concentrations against control (1% DMSO). Data are mean ± SD (n = 3). The experiment was done in triplicates. *Significantly different from control at p < 0.05. **Significantly different from control at p < 0.01. ***Significantly different from control at p < 0.001.

Table 2. Effect of compounds 5e and 10g on active caspase-9 and -3 levels, and the expression levels of Bcl-2 and Bax in MDA-MB-231 cancer cells treated with the compounds at their IC₅₀ concentrations.

Compound	Bcl-2 ng/mg protein	Bax ng/mg protein	Bax/Bcl-2 ratio	Caspase-9 nmol/mg protein	Caspase-3 nmol/mg protein
Control (1% DMSO)	12.31 ± 0.97	6.92 ± 0.51	0.56	3.07 ± 0.22	2.56 ± 0.17
5e	8.76 ± 0.42^b	9.05 ± 0.77^a	1.033	6.98 ± 0.73^c	6.12 ± 0.54^c
10g	5.90 ± 0.53^c	12.47 ± 1.08^c	2.11	8.27 ± 0.139^c	9.48 ± 0.73^c

Data are mean ± SD of three separate experiments.

^aSignificantly different from control (1% DMSO) at p < 0.05.

^bSignificantly different from control (1% DMSO) at p < 0.01.

^cSignificantly different from control (1% DMSO) at p < 0.001.

Figure 4. Effect of compounds 5e and 10g on the protein levels of A) active caspase-9; B) active caspase-3 in MDA-MB-231 cells treated with the compounds at their IC₅₀ concentrations against control (1% DMSO). Data are mean ± SD (n = 3). The experiment was done in triplicates. ***Significantly different from control at p < 0.001.

Figure 5. Effect of compounds 10g on the percentage of Annexin V-FITC-positive staining in MDA-MB-231 cells versus control (1% DMSO). Data are mean ± SD (n = 3). The experiments were done in triplicates. The four quadrants identified as: Normal, viable; Apo, early apoptotic; LATE APO, late apoptotic; NEC, necrotic. ***Significantly different from control at p < 0.001 (student’s t-test).

Table 3. In vitro anti-proliferative activities of the newly synthesized hybrids against A549, Caco-2, LoVo and HepG2 cell lines.

Compound	IC50 (μM)^a
	A549	Caco-2	LoVo	HepG2
	5a	97.04 ± 4.31	NA^b	15.71 ± 1.52	NA^b
5b	NA^b	NA^b	17.58 ± 2.18	NA^b
5c	NA^b	NA^b	36.59 ± 3.05	NA^b
5d	28.73 ± 1.59	NA^b	NA^b	NA^b
5e	NA^b	NA^b	14.48 ± 0.78	NA^b
5f	37.51 ± 2.08	NA^b	23.77 ± 1.75	NA^b
5g	NA^b	NA^b	13.86 ± 0.96	NA^b
5h	NA^b	NA^b	39.00 ± 2.13	NA^b
10a	NA^b	NA^b	13.83 ± 0.66	NA^b
10b	NA^b	NA^b	38.92 ± 2.73	NA^b
10c	NA^b	NA^b	15.38 ± 0.83	NA^b
10d	18.42 ± 1.52	NA^b	25.46 ± 2.07	NA^b
10e	91.57 ± 5.36	NA^b	14.29 ± 1.32	NA^b
10f	56.71 ± 2.93	NA^b	13.09 ± 0.33	NA^b
10g	NA^b	NA^b	14.40 ± 1.92	NA^b
10h	NA^b	NA^b	16.91 ± 1.36	NA^b
13a	NA^b	NA^b	48.62 ± 2.21	NA^b
13b	NA^b	NA^b	NA^b	NA^b
13c	NA^b	NA^b	NA^b	NA^b
Dox.	0.82 ± 0.05	3.01 ± 0.17	5.23 ± 0.49	2.74 ± 0.11

a IC₅₀ values are the mean ± SD of three separate experiments.

b NA: Compounds having IC₅₀ value >100 μM.

Table 4. Computer aided ADME study for the prepared hybrids.

Compound	AlogP98^a	PSA_2D^b	Solubility^c	Solubility level^d	Absorption level^e	CYP2D6^f	CYP2D6 probability
5a	4.911	76.766	−6.987	1	0	1	0.534
5b	5.117	76.766	−7.346	1	0	1	0.544
5c	5.576	76.766	−7.757	1	1	1	0.613
5d	5.660	76.766	−7.834	1	1	1	0.574
5e	5.576	76.766	−7.784	1	1	1	0.524
5f	5.781	76.766	−8.138	0	1	1	0.683
5g	6.240	76.766	−8.549	0	2	1	0.673
5h	6.324	76.766	−8.626	0	2	1	0.584
10a	4.306	76.766	−6.276	1	0	0	0.455
10b	4.511	76.766	−6.638	1	0	1	0.613
10c	4.970	76.766	−7.050	1	0	1	0.514
10d	5.054	76.766	−7.127	1	0	1	0.514
10e	4.970	76.766	−7.035	1	0	0	0.356
10f	5.176	76.766	−7.390	1	0	0	0.316
10g	5.635	76.766	−7.802	1	1	0	0.386
10h	5.719	76.766	−7.879	1	1	0	0.316
13a	6.701	67.308	−8.103	0	2	1	0.544
13b	7.365	67.308	−8.777	0	3	0	0.366
13c	6.906	67.308	−8.316	0	2	1	0.544

a Lipophilicity descriptor.

b Polar surface area.

c Solubility parameter. (0:−2 = optimal, −2:−4 = good, −4:−6 = low, −6:−8 = very low).

d Solubility level (0 = extremely low, 1 = very low but possible, 2 = low, 3 = good, 4 = optimal).

e Absorption level (0 = good, 1 = moderate, 2 = low, 3 = very low).

f CYP2D inhibition (0 = non inhibitor, 1 = inhibitor).