Synthesis, biological evaluation, and in silico studies of new CDK2 inhibitors based on pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold with apoptotic activity

Figures & data

Figure 1. Structures of active drugs containing fused pyrimidine (I) Roscovitine, (II) Ibrutinib, and (III) Dinaciclib, Reported pyrazolo[3,4-d]pyrimidines derivatives (IV), (V), and (VI), and Reported pyrazolo[1,5-a]pyrimidines derivative (VII) as CDK2 inhibitors.

Figure 2. Binding mode schematic illustration of Roscovitine ligand (I) (A) and Newly synthesised pyrazolopyrimidine derivative compound 13 (B) with key amino acids at CDK2 binding site.

Figure 3. Features' similarities between Roscovitine ligand (I) and reported pyrazolo[1,5-a]pyrimidines derivative (VII) against the newly designed compounds as potent CDk2 inhibitors.

Scheme 1. Reagents and conditions; (a) 2-(ethoxymethylene)-malononitrile, EtOH, reflux, 6 h. (b) triethyl orthoformate, acetic anhydride, reflux, 6 h. (c) hydrazine hydrate, EtOH, reflux, 6 h. (d) D-Glucose, EtOH, glacial acetic acid, reflux, 3 h. (e) D-Xylose, EtOH, glacial acetic acid, reflux, 3 h.

Scheme 2. Reagents and condition; (a) CS₂, KOH, EtOH, reflux, 6 h. (b) Chloroacetonitrile, K₂CO₃, DMF, 25 °C, 8 h. (c) Chloroacetaldhyde dimethyl acetal, K₂CO₃, DMF, 25 °C, 8 h. (d) 2-chloro-1,2-propandiol, KOH, EtOH, reflux, 3 h. (e) Acetic anhydride, pyridine, reflux, 2 h. (f) 2-chloroethanol, KOH, EtOH, reflux, 3 h.

Scheme 3. Reagents and conditions; (a) 2,3,4,6-Tetra-O-acetyl-D-glucopyranosylbromide, KOH, acetone, 25 °C, 6 h. (b) 2,3,4,6-Tetra-O-acetyl-D-glactopyranosylbromide, KOH, acetone, 25 °C, 6 h.

Figure 4. The IC₅₀ of tested compounds against MCF-7, HepG-2, and HCT-116 cancer cell lines. Data expressed as mean ± SD from three independent repeats (n = 3). *Significant from sorafenib at p < 0.05.

Table 1. The In vitro anti-proliferative activity of the synthesised compounds against human cell lines.

Compound ID	In vitro IC50 (nM) Mean ± SD
	MCF-7	HepG-2	HCT-116
Sorafenib	144 ± 0.47	19 ± 0.29	176 ± 0.69
4	150 ± 1.5	603 ± 1.9	521 ± 0.67
5	663 ± 1.4	690 ± 0.48	503 ± 0.97
6	621 ± 2.7	98 ± 0.78	93 ± 0.35
7	937 ± 0.93	63 ± 0.11	926 ± 1.6
8	72 ± 0.53	8 ± 0.34	52 ± 0.44
9	60 ± 0.49	690 ± 1.6	71 ± 0.46
10	95 ± 1.9	987 ± 2.0	664 ± 0.93
11	63 ± 1.1	49 ± 0.17	7 ± 0.61
12	613 ± 0.33	928 ± 0.97	587 ± 0.88
13	55 ± 0.43	74 ± 1.1	9 ± 0.06
14	7 ± 0.17	7 ± 0.43	6 ± 0.17
15	8 ± 0.29	7 ± 0.3	10 ± 0.27

Figure 5. Inhibitory activity of most potent compounds on CDK/Cyclin A2. *Significant from sorafenib at P < 0.05, # Significant from dinaciclib at P < 0.05.

Table 2. CDK2/cyclin A2 inhibitory activity results of all synthesised compounds.

Compound ID	CDK2/cyclinA2 IC50 (µM) ± SD
Sorafenib	0.184 ± 0.01
Dinaciclib	0.029 ± 0.002
4	1.342 ± 0.021*^, #
5	2.050 ± 0.05*^, #
6	0.559 ± 0.013*^, #
7	0.274 ± 0.005*^, #
8	1.064 ± 0.059*^, #
9	0.881 ± 0.049*^, #
10	0.506 ± 0.011*^, #
11	0.089 ± 0.005*
12	0.961 ± 0.019*^, #
13	0.13 ± 0.007^#
14	0.118 ± 0.007*^, #
15	0.061 ± 0.003*

*Significant from sorafenib at p < 0.05.

^# Significant from dinaciclib at p < 0.05.

Table 3. The In vitro cytotoxicity of the most active compound 15 and Staurosporine against normal lung cells (WI-38).

Compound ID	In vitro IC50 (µM) as Mean ± SD
	Normal cell WI-38
15	69.60 ± 2.47
Staurosporine	57.57 ± 1.42

Figure 6. Flow cytometric analysis for cell cycle distribution. (A) Control HCT, (B) Compound 15, and (C) graphical representation for cell cycle distribution analysis among differently treated cells.

Table 4. Flow cytometric analysis for cell cycle distribution of compound 15 on HCT-116 cells.

	Results DNA content
	%G0-G1	%S	%G2/M	%Pre-G1	Comment
Control/HCT-116	49.51	38.11	12.38	1.85
Compound 15/HCT-116	43.79	47.41	8.8	32.73	Cell growth arrest at S phase

Figure 7. Flow cytometric analysis of apoptosis among treated cells. (A) Control HCT, (B) Compound 15, and (C) Graphical illustration of apoptosis % among differently treated cells.

Table 5. Effect of compound 15 on apoptosis in HCT-116 cells.

	Apoptosis			Necrosis
	Total	Early	Late
Control HCT-116	1.85	0.34	0.28	1.23
Compound 15/HCT-116	32.73	9.26	19.12	4.35

Figure 8. The superimposition 3D diagram of co-crystallised (green colour) and ligand after docking (purple colour) at the active site.

Table 6. In silico ADMET predictions of the newly synthesised compounds using Discovery Studio.

Name	BBB LEVEL	ADMET Absorption level	ADMET A log P98	ADMET PSA 2D	Cytochrome P450 2D6 (CYP2D6) (non-inhibitor)	Hepato-toxicity	ADMET Aq Solubility level	PPB-Level (poorly bounded)
2	3 (low)	0 (good)	1.051	96.195	−5.658	true	3 (good)	0.007
3	3 (low)	0 (good)	2.095	89.908	−3.936	true	3 (good)	2.494
4	4	3 (v. low)	0.143	168.67	−6.932	false(non-toxic)	3 (good)	−6.288
5	4	3 (v. low)	−0.648	189.486	−2.309	true	3 (good)	−11.215
6	3 (low)	0 (good)	0.913	111.023	−4.481	true	3 (good)	−2.893
7	3 (low)	0 (good)	2.291	85.529	−5.219	true	2 (low)	−1.158
8	4	0 (good)	2.275	108.786	−3.780	true	2 (low)	−2.374
9	3 (low)	0 (good)	2.482	103.711	−3.424	true	2 (low)	−2.444
10	4	0 (good)	1.494	127.482	−4.059	true	3 (good)	−7.124
11	4	1 (Moderate)	2.253	138.313	−5.385	true	3 (good)	−4.920
12	3 (low)	0 (good)	2.005	106.667	−3.563	true	3 (good)	−3.724
13	4	0 (good)	2.384	112.082	−3.468	true	3 (good))	−3.661
14	4	3 (v. low)	1.906	199.705	−6.713	true	3 (good)	−7.699
15	4	3 (v. low)	1.906	199.705	−6.713	true	3 (good)	−7.699

Table 7. Physicochemical properties predictions of the most potent promising compounds using ADMETlab.

Name	Mol wt	nHBAs	nHBDs	Log P	Log S	Log D	TPSA
8	382.08	9	1	2.278	−5.401	2.353	113.79
9	431.12	10	1	2.576	−4.731	2.65	108.46
11	501.12	12	1	2.984	−4.846	2.454	142.6
13	429.1	10	1	2.928	−5.276	2.61	116.3
14	637.16	17	1	2.66	−5.104	1.999	204.43
15	637.16	17	1	2.66	−5.104	1.999	204.43
Roscovitine	354.22	7	3	2.421	−2.807	−2.643	94.35

The bold values expresses values of 140 A or more showing poor absorption of compounds 11, 14 and 15.

Table 8. ADMETlab absorption, distribution, and excretion parameters of the most potent compounds with promising docking results.

Name	Absorption						Distribution			Excretion
	Caco-2	Pgp inh	Pgp sub	HIA	F20	F30	PPB	BBB	VD	T1/2	CL
8	−4.715	1	1	1	1	1	96.80	0	0.898	0.555	8.438 moderate
9	−4.647	1	1	1	1	1	97.15	0	0.977	0.218	9.419 moderate
11	−5.051	1	1	1	0	1	95.63	0	1.008	0.483	2.179 low
13	−4.77	1	1	1	1	1	96.21	0	1.372	0.365	5.718 moderate
14	−5.49	0	1	0	1	0	92.03	1	1.227	0.561	1.832 low
15	−5.49	0	1	0	1	0	92.03	1	1.227	0.561	1.832 low
Roscovitine	−4.343	1	0	0	1	1	30.14	0	1.143	0.844	9.767 moderate

Figure 10. The common physicochemical properties of compounds (A) Roscovitine, (B) Compound 15.

Figure 11. Structure–activity relationship for compounds (4–15).

Data availability statement

Supporting data is supplied with the manuscript.