Novel pyrimidine Schiff bases and their selenium-containing nanoparticles as dual inhibitors of CDK1 and tubulin polymerase: design, synthesis, anti-proliferative evaluation, and molecular modelling

Figures & data

Figure 1. (a) Cyclin-dependent kinase 1 (CDK1) inhibitors. (b) Anti-microtubule and CBSI drugs.

Figure 2. Design of novel pyrimidine Schiff bases and their SeNPs as dual CDK1 and tubulin polymerase inhibitors.

Scheme 1. Formation of Schiff′s bases 4–9. Reagents and conditions: (i) NaNO₂/AcOH/H₂O, r.t, 30 min; (ii) (NH₄)₂S, 75 °C, 15 min and (iii) aromatic aldehydes/gl. AcOH/heated under fusion, 20 min.

Figure 3. Chemical structures of synthesised Het-SeNPs 4NPs–9NPs.

Figure 4. (a) A schematic diagram of the synthesis of selenium nanoparticles; (b) UV-Vis spectrum of selenium nanoparticles.

Figure 5. TEM of SeNPs, (a), (b), (c), (d), (e), and (f) TEM of SeNPs loaded onto 4–9, respectively.

Table 1. Particle size and zeta potential of Het-SeNPs 4NPs–9NPs.

Compounds no.	MPS (nm)	PDI	SD	Zeta potential
4NPs	80.9	0.336	±5.876	7.70
5NPs	58.55	0.666	±8.414	8.15
6NPs	75.96	0.202	±7.324	0.229
7NPs	50.43	0.013	±5.143	−1.51
8NPs	78.22	0.033	±6.653	−17.30
9NPs	60.47	0.626	±7.987	−0.948

Table 2. The anti-proliferative activity of pyrimidine Schiff bases (4–9) and their nano-sized forms (4NPs–9NPs) against human cancer cell lines (IC₅₀ µM).

Ser. no.	Compounds no.	IC50 VALUE (µM ± SD)^a
		R	R^1	R^2	MCF-7	HepG-2	A549
1	4	Me	N(CH3)2	H	3.17 ± 0.04	1.07 ± 0.03	1.53 ± 0.01
2	5	Me	OH	H	3.59 ± 0.08	4.07 ± 0.08	4.27 ± 0.08
3	6	Et	OH	H	1.15 ± 0.03	1.97 ± 0.05	1.14 ± 0.04
4	7	Et	NO2	H	3.37 ± 0.04	3.38 ± 0.11	4.16 ± 0.07
5	8	Et	H	NO2	2.03 ± 0.07	2.1 ± 0.09	2.3 ± 0.05
6	9	Et	Br	H	3.16 ± 0.13	3.47 ± 0.09	3.43 ± 0.05
7	4NPs	Me	N(CH3)2	H	0.04 ± 0	0.03 ± 0.01	0.04 ± 0
8	5NPs	Me	OH	H	0.06 ± 0	0.09 ± 0	0.07 ± 0
9	6NPs	Et	OH	H	0.04 ± 0	0.07 ± 0	0.07 ± 0
10	7NPs	Et	NO2	H	0.07 ± 0	0.03 ± 0	0.04 ± 0
11	8NPs	Et	H	NO2	0.04 ± 0	0.05 ± 0	0.07 ± 0
12	9NPs	Et	Br	H	0.07 ± 0	0.05 ± 0	0.1 ± 0.01
13	SeNPs	–	–	–	0.03 ± 0	0.04 ± 0	0.04 ± 0
14	5-FU				1.82 ± 0.03	1.36 ± 0.01	0.76 ± 0.04

^aIC₅₀ value is the concentration of the test compound required to inhibit the growth of 50% of cancer cell population. Values represent the mean for three experiments performed in triplicate.

Table 3. The selectivity of the most potent candidates 4 and 6 in both normal and nanoformulations (4NPs and 6NPs).

Ser. no.	Compounds no.	Cytotoxicity (IC50 µM ± SD)
		HepG-2	Vero	SI^a
1	4	1.07 ± 0.03	4 ± 0.05	3.74
2	6	1.97 ± 0.05	3.34 ± 0.06	1.69
3	4NPs	0.03 ± 0.01	0.59 ± 0.01	19.67
4	6NPs	0.07 ± 0	0.35 ± 0.01	5.00
5	5-FU	1.36 ± 0.01	3.04 ± 0.06	2.24

^aSelectivity index = IC₅₀ normal cell/IC₅₀ cancer cell.

Figure 6. The inhibitory effect of the compounds 4 and 4NPs on (a) cyclin-dependent kinase 1, (b) Tubulin polymerisation through colchicine binding site. (*) indicate to the significant differences between compound 4NPs-treated and compound 4-treated cells, where (***) indicates to p < 0.001. All experiments were performed in triplicates.

Table 4. The inhibitory activity of the most potent candidates 4 and 4NPs against both CDK1 and tubulin polymerase (CBS) compared to two reference drugs.

Ser. no.	Compounds no.	CDK1 (IC50 µM ± SD)	Tubulin polymerase (CBS) (IC50 µM ± SD)
1	4	2.183 ± 0.06	1.926 ± 0.119
2	4NPs	0.475 ± 0.31	0.614 ± 0.038
3	Roscovitine	0.273 ± 0.03	–
4	Combretastatin-A4	–	0.25 ± 0.015

Figure 7. (a) The effect of the most potent compounds 4 and 4NPs on cell cycle phases of HepG-2. Flow cytometric histograms of HepG-2 cell cycle phases; (b) untreated cells, (c) treated with compound 4 alone, and (d) treated with compound 4NPs. All experiments were performed in triplicate.

Table 5. The effect of the most potent candidates 4 and 4NPs on cell cycle phases of HepG-2.

Ser no.	Compounds no.	%G0–G1	%S	%G2/M
1	4 /HepG-2	49.22 ± 2.61	36.55 ± 1.93	14.23 ± 0.81
2	4NPs/HepG-2	52.61 ± 2.72	39.57 ± 4.01	7.82 ± 1.02
3	Cont.HepG-2	43.59 ± 2.66	41.03 ± 3.42	15.38 ± 0.77

Figure 8. (a) The apoptosis-inducing effect of the most potent candidates 4 and 4NPs on HepG-2. (*) indicate to the significant differences between compound 4NPs-treated and compound 4-treated cells, where (*) indicates to p < 0.05, (**) p < 0.01. All experiments were performed in triplicates. (b–d). Flow cytometric dot plot of PI/annexin V screening of HepG-2; (b) untreated cells, (c) cells treated with compound 4, and (d) cells treated with compound 4NPs. All experiments were performed in triplicate.

Table 6. The apoptotic effect of the most potent candidates 4 and 4NPs on HepG-2.

Ser. no.	Compound no.	Apoptosis			Necrosis
		Total	Early	Late
1	4 /HepG-2	41.04 ± 2.39	26.42 ± 1.47	7.46 ± 0.54	7.16 ± 0.44
2	4NPs/ HepG-2	53.62 ± 2.42	31.29 ± 1.72	12.82 ± 0.44	9.51 ± 0.27
3	Cont.HepG-2	2.47 ± 0.07	0.55 ± 0.04	0.19 ± 0.02	1.73 ± 0.14

Figure 9. (a) Compound 4 at the active site of CDK1 (PDB ID: 6GU6), and (b) alignment of compound 4 and dinaciclib at the active site of CDK1 (PDB ID: 6GU6).

Figure 10. (a) Nano compound 4NPs at the active site of CDK1 (PDB ID: 6GU6). (b) The alignment of 4NPs and dinaciclib at the active site of CDK1 (PDB ID: 6GU6).

Figure 11. (a) Compound 4 at the CBS of microtubules (PDB ID: 5LYJ). (b) The alignment of compound 4 and combretastatin-A4 at the CBS of microtubules (PDB ID: 5LYJ).

Figure 12. (a) Nano compound 4NPs at the CBS of microtubules (PDB ID: 5LYJ). (b) The alignment of nano compound 4NPs and combretastatin-A4 at the CBS of microtubules (PDB ID: 5LYJ).