Eco-friendly synthesis of novel pyrazole derivatives and their anticancer and CDK2 inhibitory activities

Abstract

A series of 1-(2-pyridinyl)-4-aryl-1H-pyrazole-3,5-diamine derivatives (2–12) were designed and synthesized using a one-pot multicomponent reaction via both microwave-assisted and conventional techniques. The structural properties of the new compounds were established using spectroscopic data. The new derivatives were biologically assessed as promising CDK2 enzyme inhibitors. Then, the target pyrazoles were screened against both HepG2 and MCF-7 malignant cell lines. Compounds 4, 7, and 10 revealed significant CDK2 inhibitory activities with comparable potencies (IC₅₀ = 0.75, 0.77 and 0.85 µM, respectively) to that of roscovitine (IC₅₀ = 0.99 µM). Additionally, compounds 5, 6, and 11 demonstrated the best inhibitory activities, which are twice the activity of roscovitine towards CDK2, with IC₅₀ values of 0.56, 0.46, and 0.45 µM, respectively. Concerning the cytotoxic activity, compound 5 displayed potent cytotoxicity (IC₅₀ = 13.14 and 8.03 µM) against the HepG2 and MCF-7 cell lines, respectively. Further investigation on the mechanism demonstrated that 5 induced apoptosis, increased the proapoptotic protein Bax level, and reduced the antiapoptotic Bcl-2 level in the cells of MCF-7. Finally, the molecular docking study showed bioactive analogues that fit well in the CDK2 active site via various interactions.

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1. Introduction

Interestingly, pyrazole derivatives are deemed as one of the most significant heterocyclic derivatives that have broad usage in nano-biotechnology,^1,2 DNA-encoded library technology,³ pigments, dyes,^4,5 and the petrochemical industry.^6,7 Also, recently, pyrazoles have shown widespread biological and pharmacological activities, such as anticancer,^8–10 antimicrobial,^11,12 anti-phlogistic,^13,14 analgesic,¹⁵ acaricidal,¹⁶ anthelmintic,¹⁷ antiviral,¹⁸ antagonistic,¹⁹ antioxidant,^20–22 herbicidal,²³ antifungal²⁴ and antibacterial activities.^25,26 Conversely, heterocyclics that contain more than one nitrogen atom have excellent and diverse therapeutic potential and are pivotal in many industrial applications.^27–30

Green chemistry is the science of designing chemical processes that reduce and or eliminate the hazardous products of reactions. It prevents pollution at a molecular level. Microwave techniques used in the synthesis of heterocyclic compounds are also significant branches of green chemistry methods. The one-pot multicomponent system^31–34 is considered one of the essential techniques for reaction preparation with more facile productivity performance, and it gives highly desired products from simple starting materials and a single run. So, these techniques attract more attention because they are environmentally safe, offer convenient and easy improvements to reaction time and yield, and provide extremely energy-efficient synthetic operations. Comparing both microwave irradiation techniques with the thermally conventional technique, it is clear that the microwave irradiation technique is environmentally friendly, easily controlled, and more ecologically tolerant. As advantages, many reactions of the heterocyclic derivatives are carried out under relatively clean and mild conditions with relatively high yields and short reaction times.^35–37 So, now, this type of green synthesis is considered a very important technique in heterocyclic derivative preparation because of its mild conditions and simplicity. Owing to the above advantages of heterocyclic derivatives and depending on their highly favoured site applications in the industry and biology, more efforts have been put into the preparation of many novel heterocyclic compounds.^38–40

On the other hand, cancer is a global health problem in which deviant cells in the body divide and proliferate uncontrollably, attacking nearby tissues and in extreme cases, complete biological systems.^41,42 While cell cycle control is mediated via governing proteins, which involve kinases that rely on cyclin for their functions (CDKs). CDKs belong to the threonine/serine kinases family that regulates the cell cycle phases, mostly division, gene transcription, and other post-translational events.⁴³ For instance, CDK2 has a crucial role in the cell cycle sequence from the G1 to S phase transition, in addition to encouraging the DNA repair system.⁴⁴ Therefore, CDK2 enzyme inhibition can lead to cell arrest at both the G2/M and G1/S phases and enhance apoptosis.^45,46 CDK2 is overexpressed in diverse kinds of cancer, such as breast cancer, liver cancer, colorectal carcinoma, pancreatic cancer, melanoma, ovarian carcinoma, lung carcinoma, and osteosarcoma. As a result, the inhibition of CDK2 is thought to be an attractive target for the evolution of novel cytotoxic agents.^47,48 The promising antitumor effect of several CDK inhibitors containing a pyrazole core has been reported in various literature (dinaciclib, roscovitine, olomoucine, purvalanol A and B, CVT313, CYC065, and BS-194), some of which have progressed into clinical investigation.^41,49–51

Investigating the complex crystal structure of CDK2-roscovitine shows an important interaction between the amino-pyrazolopyrimidine of roscovitine and Leu83 in the hinge region. Roscovitine also interacts with the active site through many hydrophobic interactions; the isopropyl group occupies a hydrophobic pocket. Lastly, the benzyl moiety is pointed out to the solvent-accessible area and has been found to interact with the Lys89 amino acid.^41,50

In light of published research studies on the design and synthesis of chemotherapeutic agents targeting CDKs, this work is dedicated to synthesizing a series of new pyrazole derivatives (2–12) via the reaction of 2-hydrazinylpyridine (1) with different 2-(aryl-diazenyl)malononitrile derivatives using conventional and microwave methods. The rational design of the new pyrazoles depends on the simplification of the pyrazolopyrimidine scaffold of dinaciclib and the purine ring of roscovitine (Fig. 1). The amino groups in all the prepared compounds are expected to interact with the crucial amino acid (Leu83) in the pivot region. Moreover, the roscovitine benzyl group and the pyridine oxide moiety of dinaciclib are replaced with a substituted phenyl moiety to maintain the compound's active-site interactions. Finally, the hydrophobic isopropyl group of roscovitine and the ethyl group of dinaciclib are replaced by a pyridine moiety in the new desired compounds. All the newly synthesized pyrazole derivatives are investigated as CDK2 inhibitors and antitumour versus both mammary breast cancer (MCF-7) and hepatic carcinoma (HCC; HepG2). Furthermore, a docking study of the promising compounds in CDK2 active sites is performed to visualize their relative binding interactions and positions compared with those of roscovitine.

Fig. 1 The rational design of the newly synthesized compounds.

2. Experimental

2.1. Synthesis

The chemicals, reagents, solvents, and starting materials were obtained from Sigma-Aldrich. All solvents used were dried following the chemical's purification handbook for laboratories. To track the reactions and development and control the newly synthesised heterocyclics' homogeneity, TLC was utilized via silica gel precoated plates “Merck Kieselgel 60F₂₅₄, BDH”. The melting temperatures of all the newly prepared compounds were determined on the digital Stuart electric equipment (SMP3) for melting points. Microwave irradiation reactions were carried out in an Anton Paar monowave microwave reactor using 10 mL borosilicate glass vials. Infrared absorption spectroscopy (IR, cm⁻¹) was conducted using a PerkinElmer spectrophotometer (model 293) with KBr disks. A Varian Mercury spectrometer (300 MHz) was used for measuring ¹H and ¹³C-NMR spectra in DMSO-d₆ as a solvent, with tetramethyl silane (TMS) as the reference standard. Multiplicity is symbolized as m for “multiple”, t for “triplet”, s for “singlet”, q for “quartet”, d for “doublet”, or combinations thereof. Coupling constants (J, Hz) and chemical shifts (δ, ppm) were measured. Mass spectroscopic analysis was carried out using the electron ionization technique on a Shimadzu Gas chromatograph (GC-2010, 70 eV) mass spectrometer. Elemental microanalysis was carried out on a PerkinElmer CHN analyzer (model 2400), and the excellent microanalysis results were within ±0.4% of the theoretical expectations. Different isolated pyrazoles were formed with very interesting structural features.

2.1.1. General preparation method of the 1-(2-pyridinyl)-4-(aryl)-1H-pyrazole-3,5-diamine derivatives (2–13). A solution of 2-hydrazinylpyridine (1) (0.01 mol, 1.09 mL) in ethanol (20 mL) was refluxed for 2–20 h with each of the following, separately: 2-(3-tolyldiazenyl)malononitrile (0.01 mol, 1.84 g), 2-((4-acetylphenyl)diazenyl)malononitrile (0.01 mol, 2.12 g), 4-((dicyanomethyl)diazenyl)benzenesulfonic acid (0.01 mol, 2.50 g), 4-((dicyanomethyl)diazenyl)-2-hydroxybenzoic acid (0.01 mol, 2.30 g), N-(diaminomethylene)-4-((dicyanomethyl)diazenyl)benzenesulfonamide (0.01 mol, 2.91 g), 2-((4-(phenyldiazenyl)phenyl)diazenyl)malononitrile (0.01 mol, 2.74 g), 2-((2,4,5-trichlorophenyl)diazenyl)malononitrile (0.01 mol, 2.71 g), 2-((3-nitrophenyl)diazenyl)malononitrile (0.01 mol, 2.15 g), 2-((4-methoxyphenyl)diazenyl)malononitrile (0.01 mol, 2.00 g), 2-((dicyanomethyl)diazenyl)benzoic acid (0.01 mol, 2.14 g), 2-((2-chlorophenyl)diazenyl)malononitrile (0.01 mol, 2.04 g) and 2-(thiazol-2-yldiazenyl)malononitrile (0.01 mol, 1.77 g). After the reaction mixture had cooled, the obtained solid substance was separated through filtration, washed four times using ethanol (25 mL), and crystallized using a suitable dissolving agent to give compounds 2–12, respectively.
2.1.1.1. 1-(2-Pyridinyl)-4-(3-tolyldiazenyl)-1H-pyrazole-3,5-diamine (2). Orange crystals from ethanol; yield 61%; mp 188–190 °C. IR (cm⁻¹) ν: 3419, 3390, 3313, 3167 (2NH₂), 1620 (C=N), 1595 (C=C), 1573 (N=N). ¹H NMR (DMSO-d₆, δ ppm): 2.37 (s, 3H, CH₃), 6.12 (br., 2H, NH₂, D₂O exchangeable), 7.10–7.67 (m, 7H, Ar–H), 7.93 (br., 2H, NH₂, D₂O exchangeable), 8.27–8.42 (s, 1H, Ar–H); ¹³C NMR (DMSO-d₆, δ ppm): 21.6, 112.3 (2C), 114.6, 118.7 (2C), 119.5, 121.6, 128.6, 129.1, 138.6, 139.7, 147.4, 153.7 and 154.2; anal. calcd for: C₁₅H₁₅N₇ (293): C, 61.43; H, 5.12; N, 33.45; found: C, 61.57; H, 5.09; N, 33.34.
2.1.1.2. 1-(4-((3,5-Diamino-1-(2-pyridinyl)-1H-pyrazol-4-yl)diazenyl)phenyl)ethan-1-one (3). Brown crystals from methanol; yield 65%; mp 264–266 °C. IR (cm⁻¹) ν: 3427, 3357, 3301 (2NH₂), 1650 (C=O), 1603 (C=N), 1595 (C=C), 1573 (N=N). ¹H NMR (DMSO-d₆, δ ppm): 2.33 (s, 3H, CH₃), 6.22 (br., 2H, 2NH₂, D₂O exchangeable), 7.25–8.43 (m, 8H, Ar–H), 9.74 (br., 2H, 2NH₂, D₂O exchangeable); anal. calcd for: C₁₆H₁₅N₇O (321): C, 59.81; H, 4.67; N, 30.53; found: C, 59.91; H, 4.59; N, 30.44.
2.1.1.3. 4-((3,5-Diamino-1-(2-pyridinyl)-1H-pyrazol-4-yl)diazenyl)benzenesulfonic acid (4). Black crystals from methanol; yield 62%; mp > 300 °C. IR (cm⁻¹) ν: 3421 (br., OH), 3340, 3313 (2NH₂), 3022 (CH aromatic), 1600 (C=N), 1572 (N=N), 1190 (S=O). ¹H NMR (DMSO-d₆, δ ppm): 6.22 (br., 2H, NH₂, D₂O exchangeable), 7.10–7.67 (m, 7H, Ar–H), 7.93 (br., 2H, NH₂, D₂O exchangeable), 8.27–8.42 (m, 1H, Ar–H), 11.25 (s, H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, δ ppm): 112.3 (2C), 115.0, 119.6 (2C), 120.5, 124.4, 126.8, 139.8, 147.5, 150.0, 153.7, 154.1 and 175.5; anal. calcd for: C₁₄H₁₃N₇O₃S (359): C, 46.80; H, 3.62; N, 27.30; S, 8.91; found: C, 46.68; H, 3.71; N, 27.34; S, 8.85.
2.1.1.4. 4-((3,5-Diamino-1-(2-pyridinyl)-1H-pyrazol-4-yl)diazenyl)-2-hydroxybenzoic acid (5). Brown crystals from ethanol; yield 61%; mp > 300 °C. IR (cm⁻¹) ν: 3600–2500 (br., OH & 2NH₂), 1653 (C=O), 1632 (C=N), 1593 (C=C), 1563 (N=N). ¹H NMR (DMSO-d₆, δ ppm): 6.13 (br., 4H, 2NH₂, D₂O exchangeable), 7.09–8.40 (m, 8H, Ar–H & COOH, D₂O exchangeable), 16.00 (br., H, OH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, δ ppm): 107.5, 110.8, 112.3, 114.8 (2C), 119.5 (2C), 119.8 (2C), 130.9, 139.7, 147.5, 156.4, 163.6 and 172.5. Anal. calcd for: C₁₅H₁₃N₇O₃ (339): C, 53.10; H, 3.83; N, 28.91; found: C, 53.02; H, 3.69; N, 29.01.
2.1.1.5. 4-((3,5-Diamino-1-(2-pyridinyl)-1H-pyrazol-4-yl)diazenyl)-N-(diaminomethylene)benzenesulfonamide (6). Reddish brown crystals from ethanol; yield 64%; mp 246–248 °C (MeOH); IR (cm⁻¹) ν: 3451, 3429, 3340, 3316, 3168, 3120 (4NH₂), 1627 (C=N), 1592, 1568 (C=C), 1551 (N=N), 1262 (SO₂); ¹H NMR (DMSO-d₆, δ ppm); 5.99 (br., 2H, NH₂, D₂O exchangeable), 6.41 (br., 2H, NH₂ exchangeable), 6.73 (br., 4H, 2NH₂, D₂O exchangeable), 7.22–8.43 (m, 8H, Ar–H); ¹³C NMR (DMSO-d₆, δ ppm): 112.3 (2C), 115.8, 119.7 (2C), 121.1 (2C), 127.0 (2C), 139.7, 142.7, 147.5, 154.0, 155.5 and 158.6; anal. calcd for: C₁₅H₁₆N₁₀O₂S (400): C, 45.00; H, 4.00; N, 35.00; S, 8.00. Found: C, 45.09; H, 3.95; N, 34.88; S, 8.07.
2.1.1.6. 4-((4-(Phenyldiazenyl)phenyl)diazenyl)-1-(2-pyridinyl)-1H-pyrazole-3,5-diamine (7). Orange crystals from ethanol; yield 62%; mp 242–245 °C. IR (cm⁻¹) ν: 3423, 3389, 3308, 3190 (2NH₂), 1615 (C=N), 1592 (C=C), 1565, 1511 (N=N); ¹H NMR (DMSO-d₆, δ ppm): 6.04 (br., 2H, NH₂, D₂O exchangeable), 6.50 (br., 2H, NH₂, D₂O exchangeable), 7.20–8.45 (m, 13H, Ar–H); ¹³C NMR (DMSO-d₆, δ ppm): 111.9, 115.9, 119.2 (2C), 121.6 (2C), 122.5 (2C), 123.7 (2C), 129.4 (2C), 131.2 (2C), 139.3, 147.0, 150.4, 152.2, 153.6, 155.5; anal. calcd for: C₂₀H₁₇N₉ (383): C, 62.66; H, 4.44; N, 32.90; found: C, 62.77; H, 4.38; N, 32.85.
2.1.1.7. 1-(2-Pyridinyl)-4-((2,4,5-trichlorophenyl)diazenyl)-1H-pyrazole-3,5-diamine (8). Orange crystals from methanol; yield 63%; mp 222–224 °C. IR (cm⁻¹) ν: 3443, 3408, 3267 (2NH₂), 1634, 1601 (C=N), 1592 (C=C), 1558 (N=N); ¹H NMR (DMSO-d₆, δ ppm): 6.21 (br., 2H, NH₂, D₂O exchangeable), 6.64 (br., 2H, NH₂, D₂O exchangeable), 7.24–8.55 (m, 6H, Ar–H); anal. calcd for: C₁₄H₁₀N₇Cl₃ (382.5): C, 43.92; H, 2.61; N, 25.62; Cl, 27.85. Found: C, 44.02; H, 2.55; N, 25.71; Cl, 27.72.
2.1.1.8. 4-((3-Nitrophenyl)diazenyl)-1-(2-pyridinyl)-1H-pyrazole-3,5-diamine (9). Brown crystals from acetic acid; yield 63%; mp 276–278 °C. IR (cm⁻¹) ν: 3423, 3374, 3308 (2NH₂), 1622 (C=N), 1589 (C=C), 1568 (N=N); ¹H NMR (DMSO-d₆, δ ppm): 6.04 (br., 2H, NH₂, D₂O exchangeable), 6.51 (br., 2H, NH₂, D₂O exchangeable), 7.18–8.71 (m, 8H, Ar–H); ¹³C NMR (DMSO-d₆, δ ppm): 112.0 (2C), 119.9, 121.0, 130.1 (2C), 137.3 (2C), 147.1 (2C), 148.9, 153.7, 154.4 (2C); anal. calcd for: C₁₄H₁₂N₈O₂ (324): C, 51.85; H, 3.70; N, 34.57; found: C, 51.91; H, 3.64; N, 34.51.
2.1.1.9. 4-((4-Methoxyphenyl)diazenyl)-1-(2-pyridinyl)-1H-pyrazole-3,5-diamine (10). Brown crystals from ethanol; yield 65%; mp 250–252 °C. IR (cm⁻¹) ν: 3388, 3280, 3207 (2NH₂), 1628, 1603 (C=N), 1592 (C=C), 1544 (N=N); ¹H NMR (DMSO-d₆, δ ppm): 3.84 (s, 3H, OCH₃), 6.90–8.84 (m, 8H, Ar–H), 9.46 (br., 2H, NH₂, D₂O exchangeable), 10.63 (br., 2H, NH₂, D₂O exchangeable), 14.49 (br., 1H, OH, D₂O exchangeable); anal. calcd for: C₁₅H₁₅N₇O (309): C, 58.25; H, 4.85; N, 31.72; found: C, 58.16; H, 5.01; N, 31.88.
2.1.1.10. 4-((2-Chlorophenyl)diazenyl)-1-(2-pyridinyl)-1H-pyrazole-3,5-diamine (11). Brown crystals from ethanol; yield 61%; mp 204–206 °C. IR (cm⁻¹) ν: 3444, 3413, 3300, 3212, 3154, 3112 (2NH₂), 1640 (C=N), 1606 (C=C), 1560 (N=N); ¹H NMR (DMSO-d₆, δ ppm): 5.99 (br., 2H, NH₂, D₂O exchangeable), 6.59 (br., 2H, NH₂, D₂O exchangeable), 7.20–8.43 (m, 8H, Ar–H); ¹³C NMR (DMSO-d₆, δ ppm): 111.8, 116.2, 116.8, 119.3, 120.5, 127.5, 128.1, 129.9, 130.5, 139.2, 139.9, 147.0, 148.6, and 153.5. Anal. calcd for: C₁₄H₁₂N₇Cl (313.5): C, 53.59; H, 3.83; N, 31.26; Cl, 11.32; found: C, 53.47; H, 3.91; N, 31.14; Cl, 11.48.
2.1.1.11. 1-(Pyridin-2-yl)-4-(thiazol-2-yldiazenyl)-1H-pyrazole-3,5-diamine (12). Black crystals from ethanol; yield 62%; mp 258–260 °C. IR (cm⁻¹) ν: 3301, 3180 (2NH₂), 1640 (C=N), 1599 (C=C), 1574 (N=N); ¹H NMR (DMSO-d₆, δ ppm): 6.55 (br., 2H, NH₂, D₂O exchangeable), 6.70–6.79 (m, 2H, Ar–H), 6.93 (br., 2H, NH₂, D₂O exchangeable), 7.10–8.56 (m, 4H, Ar–H); anal. calcd for: C₁₁H₁₀N₈S (286): C, 46.15; H, 3.49; N, 39.16; S, 11.20; found: C, 46.09; H, 3.60; N, 39.28; S, 11.03.

2.2. Microwave-promoted synthesis technique

Microwave-assisted synthesis was performed using the same stoichiometric ratios of reactants as in the conventional procedure. The reaction vial was irradiated at 120 °C under 2–5 bar pressure and 200–400 W power for 1–4 min (Table 1) with constant magnetic stirring. The completion of the reaction was confirmed by TLC. The resulting product was then purified by ethanol washing, followed by recrystallization using a suitable solvent.

Table 1 Physical data comparison between the desired derivatives (2–12) under microwave and conventional techniques

Cpd no.	Time “min”		Yield%		YE		OE		RME		AE
	MW	Conv.	MW	Conv.	MW	Conv.	MW	Conv.	MW	Conv.
2	1	120	92	61	92.00	0.5083	0.7551	0.5006	75.51	50.06	100
3	2	360	91	65	45.50	0.1806	0.7587	0.5419	75.87	54.19	100
4	4	780	95	62	23.75	0.0795	0.8063	0.5262	80.63	52.62	100
5	3	600	92	61	30.67	0.1017	0.7739	0.5131	77.39	51.31	100
6	3	900	92	64	30.67	0.0711	0.7931	0.5517	79.31	55.17	100
7	2	720	93	62	46.50	0.0861	0.7968	0.5312	79.68	53.12	100
8	4	1200	91	63	22.75	0.0525	0.7831	0.5421	78.31	54.21	100
9	3	840	92	63	30.67	0.0750	0.7684	0.5261	76.84	52.61	100
10	2	480	93	65	46.50	0.1354	0.7704	0.5385	77.04	53.85	100
11	3	960	95	61	31.67	0.0635	0.7696	0.4941	76.96	49.41	100
12	3	660	91	62	30.33	0.0939	0.7436	0.5066	74.36	50.66	100

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2.3. Biological assessment

2.3.1. In vitro cyclin-dependent kinase CDK2 inhibitory activity. The inhibitory efficiency of the CDK2 enzyme of our investigated pyrazoles was estimated using Bioscience CDK kits, CDK2 (Catalog #79599), following the manufacturer's instructions.

2.3.2. In vitro anticancer properties. The newly synthesized pyrazoles' anticancer effect was evaluated in vitro utilizing two different cancer cell lines, including MCF-7 and HepG2. The used cell lines were purchased from ATCC (Rockville, MD). The colorimetric assay aims to convert the yellow color of the tetrazolium bromide (MTT) into purple through formazan formation in viable cells via mitochondrial enzyme succinate dehydrogenase (SDH). The first step was culturing the cell lines using 10% FBS in RPMI-1640 medium. Streptomycin and penicillin were supplied as antibiotics, and the cell lines under 5% CO₂ at 37 °C were plated in a 96-well microplate. Following the treatment with changing concentrations of the prepared pyrazoles, cells were cultured at 37 °C for 48 h. Under dark conditions, the cells were maintained during incubation with an MTT reagent at 37 °C for 4 h. The obtained purple-colored products in each well were dissolved in DMSO. The colorimetric assay was conducted using a plate reader at 570 nm, and the result was recorded. The nonlinear regression fitting model was used to calculate the percentage of the relative cell viability (Prism version 8.0, GraphPad). The data were recorded from the means of three separate experiments.^52,53

2.3.3. Cell cycle analysis. Breast adenocarcinoma cells (MCF-7) were treated with derivative 5 for 48 h at its IC₅₀. Then, the cells furnished by trypsinization were washed twice in PBS and set at 4 °C in ice/ethanol (60%, w/w) for 12 h. After that, the cells were re-washed in PBS, and the ethanol was removed. Later, the cells were stained in a buffer solution containing RNase and propidium iodide (PI, 500 µL) and incubated for 30 min. Finally, a flow cytometer was utilized for cell cycle phase analysis.^54,55

2.3.4. Apoptotic assay. MCF-7 cell lines were treated using IC₅₀ of analogue 5 for 48 h and then trypsinized and subjected to PBS for washing. Thereafter, the cells were stained in a buffer containing 10 µL of V-fluorescein isothiocyanate and 5 µL of propidium iodide (staining solution) at r.t. in a dark place, based on the manufacturer's directions. After dyeing, the cell lines were analyzed using the FACScan analysis flow cytometer.⁵⁶
2.3.4.1. Apoptotic marker (Bcl-2) determination. The enzyme-linked immunosorbent Bcl-2 ELISA detecting kit from Zymed (Catalog #99-0042) was utilized for the quantitative assay of Bcl-2 in human cells following the manufacturer's instructions.
2.3.4.2. Apoptotic marker (Bax) determination. Human active Bax content was determined using the ELISA kit for detecting Human Bax (EIA-4487) based on the manufacturer's guidelines.

2.3.5. Molecular docking simulation study. The molecular docking investigation was achieved using Vina Autodock. The crystal structure information of CDK2 in complex with the ligand roscovitine was obtained from the protein databank (ID code 2A4L). The receptor and the ligand were adapted in the format of pdbqt to carry out the docking procedure using Vina Autodock. M.G.L. systems were utilized to measure the grid layout box features and binding site coordinates. The freely accessible BIOVIA Discovery Studio 2021 visualizer was employed to visualize the predicted 3D and 2D binding modes for the investigated compounds.

2.3.6. Computational study of ADME aspect prediction. ChemDraw 14.0 was used to transform the pyrazole structures into a SMILES dataset. After that, the obtained SMILES were inserted into the freely available SwissADME website to estimate the pharmacokinetic properties, physicochemical aspects, ADME parameters, and/or drug-likeness characteristics of the screened compounds.⁵⁷

3. Results, interpretation, and discussion

3.1. Chemistry and hetero synthesis

The novel pyrazole analogues were synthesized through the reaction sequences clarified in Schemes 1–3. The interesting pharmacological properties of the bifunctional starting 2-hydrazinopyridine (1), such as anticancer,^58,59 antibacterial,^60,61 antifungal,^62,63 and anti-inflammatory⁶⁴ activities, led us to synthesize a new series of pyrazole derivatives (2–13).

Scheme 1 Preparation of the targeted pyrazole derivatives 2–7.

Scheme 2 Preparation of the targeted pyrazole derivatives 8–12.

Scheme 3 Preparation mechanism of the pyrazole derivatives 2–12.

Therefore, the reaction of compound 1 with di-nitrogen electrophiles such as 2-(3-tolyldiazenyl)malononitrile, 2-((4-acetylphenyl)diazenyl)malononitrile, 4-((dicyanomethyl)diazenyl)benzenesulfonic acid, 4-((dicyanomethyl)diazenyl)-2-hydroxybenzoic acid, N-(diaminomethylene)-4-((dicyanomethyl)diazenyl)benzenesulfonamide, 2-((4-(phenyldiazenyl)phenyl)diazenyl)malononitrile, 2-((2,4,5-trichlorophenyl)diazenyl)malononitrile, 2-((3-nitrophenyl)diazenyl)malononitrile, 2-((4-methoxyphenyl)diazenyl)malononitrile, 2-((dicyanomethyl)diazenyl)benzoic acid, 2-((2-chlorophenyl)diazenyl)malononitrile and 2-(thiazol-2-yldiazenyl)malononitrile was investigated. The corresponding condensed derivatives (2–12) (Schemes 1 and 2) were produced via the hydrazine NH₂ group nucleophilic addition on the CN group of the malononitrile derivative, followed by the second addition of the NH group on the second cyano group, which was predicted to yield some interesting biological characteristics (Scheme 3).

3.2. A comparison between microwave and conventional procedures

The synthesis of the series of pyrazoles was performed under conventional and microwave-assisted methods.^65–71 TLC was employed to monitor the reaction process using the two techniques. Table 1 shows that despite the molar concentrations of all reactants in the three disciplines being equivalent, the product yields and reaction times were completely different. The most suitable method for a definite reaction was determined by calculating the yield economy (YE) according to the following equation.

Moreover, reaction mass efficiency (RME) is a beneficial parameter for the detection of the reaction efficiency, and it is calculated through the following equation.

The last parameter utilized in the differentiation between the green techniques (grinding and microwave) and the conventional method is the optimum efficiency (OE), which is determined via the following equation: (AE) refers to the atomic economy. Such parameters were applied to distinguish the efficacy of the three techniques in the preparation of an identical desired compound. It is to be mentioned that while the other parameters are greater in the microwave, followed by grinding technique and then conventional heating, the AE is the same in all methodologies utilized to synthesize the same compound.

In the microwave technique, the same reactant amount used in conventional reactions was used without a solvent. The TLC was used to monitor the completion of the reaction. The solids formed were washed with ethanol three times and recrystallized using the proper solvent. Microwave irradiation was carried out using an Anton Paar monowave 300 with 10 mL borosilicate glass vials for 1–4 min. The conventional thermal reactions were carried out in a porcelain mortar with a pestle for 2–20 h. The obtained products of the same reaction using the three techniques were identical in mp, mixed mp, and TLC. The comparison between the reaction time and yield of the prepared compounds by the two techniques was reported. The microwave reactions afforded higher yields with the lowest times than the conventional reactions. The microwave technique is considered a green method in heterocyclic synthesis. Thus, the microwave technique is better than the conventional techniques.

3.3. In vitro CDK2 inhibition

The investigated compounds were evaluated for their CDK2 inhibition activity to explore their action mechanism. A reference drug used for comparison was roscovitine, which is regarded as a highly competitive CDK2 ATP inhibitor. The results are reported in Table 2 as the 50% inhibition concentration value of CDK2 (IC₅₀). The enzyme assay results revealed that the tested compounds displayed moderate to potent inhibitory activities at sub-micromolar to low micromolar levels (IC₅₀ values of 0.45–1.5 µM).

Table 2 CDK2 inhibitory activities of our tested compounds versus roscovitine

Comp. ID	IC50 (µM) CDK-2
2	1.05 ± 0.2
3	1.2 ± 0.15
4	0.75 ± 0.05
5	0.56 ± 0.03
6	0.46 ± 0.02
7	0.77 ± 0.05
8	1.46 ± 0.5
9	1.5 ± 0.7
10	0.85 ± 0.05
11	0.45 ± 0.02
12	1.24 ± 0.85
Roscovitine	0.99 ± 0.08

---

Compounds 5, 6 and 11 demonstrated a significant inhibitory effect towards CDK2 and afforded IC₅₀ = 0.56, 0.46, and 0.45 µM, respectively. They revealed 2-fold the activity of roscovitine (IC₅₀ = 0.99 µM). However, compounds 4, 7, and 10 revealed comparable potency of roscovitine against CDK2 (IC₅₀ values of 0.75, 0.77 and 0.85 µM, respectively).

3.4. In vitro anticancer evaluation

The anticancer activities of the compounds (4, 5, 6, 7, 10, and 11) showing the most potent inhibitory effect on CDK2 were examined towards two human tumour cell lines, specifically hepatic (HepG2) and breast (MCF-7) carcinoma. The screened compounds' biological activities were measured by the MTT evaluation method. The IC₅₀ values were estimated compared with those of the standard drug, doxorubicin, which displayed IC₅₀ values of 4.50 and 4.17 µM for the two tested cell lines, respectively.

Derivative 5 displayed potent cytotoxicity (IC₅₀ = 13.14 and 8.03 µM), while derivative 6 showed good potency (IC₅₀ = 22.76 and 26.08 µM) against the HepG2 and MCF-7 cell lines, respectively. Moreover, pyrazole derivative 10 revealed the third-best activity, compared with doxorubicin, (IC₅₀ = 15.38 µM) against the MCF-7 cell line. Finally, the other synthesized pyrazoles possessed moderate or weak anticancer activities. The results are tabulated in Table 3.

Table 3 IC₅₀ (µM) of the tested compounds against human tumour cells (HepG2 and MCF-7) compared with that of doxorubicin

Comp. ID	IC50
	HepG2	MCF-7
4	53.84 ± 3.2	48.70 ± 2.8
5	13.14 ± 1.1	8.03 ± 0.6
6	22.76 ± 1.5	26.08 ± 1.7
7	67.12 ± 3.5	35.11 ± 2.2
10	41.59 ± 2.4	15.38 ± 1.2
11	74.18 ± 3.8	61.76 ± 3.4
DOX	4.50 ± 0.2	4.17 ± 0.2

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3.5. Cell cycle analysis

The analysis of the cell cycle was conducted to explore the possible underlying antiproliferative mechanism behind the most potent compound. Among the cancer cell lines under evaluation, MCF-7 exhibited the highest sensitivity towards our compounds. So, this work aims to evaluate the effect of pyrazole derivative 5 on the MCF-7 cell cycle. Concerning the compound 5 effect on the cell cycle, the data demonstrated a cell cycle arrest at the S phase from 35.52% to 41.02% and the G2 phase from 26.46% to 47% compared with the control. Further, a decrease in the cell population at the G1 phase, from 38.02% to 11.98%, was observed (Table 4 and Fig. 2). These results indicated that our tested compound showed a 1.8- and 1.2-fold increase in the G2 and S phases, respectively, in comparison to the control cancer cells.

Table 4 Results of the cell cycle analysis on the MCF-7 cells expressed by (%) of cells in each phase when treated with compound 5

Compound	% G1	% S	% G2
5/MCF-7	11.98	41.02	47.00
Control/MCF-7	38.02	35.52	26.46

---

Fig. 2 Cell cycle analysis of the control (on the left side) and compound 5 (on the right side) on the MCF-7 cells.

3.6. Apoptosis study

Apoptosis is a planned death process for damaged cells that occurs under physiological and pathological conditions. The disturbance or misguided monitoring of different apoptotic routes results in the out-of-control multiplication of malignant cells.^72,73

An annexin V binding assay was carried out to study the apoptosis-inducing potential of compound 5. The apoptotic nature of compound 5 against the MCF-7 cells was measured through flow cytometry detection with propidium iodide (PI) and annexin V-FITC double staining.

The results demonstrated that pyrazole derivative 5 elevated the early ratio of apoptosis from 0.43% to 71.1% and the late ratio of apoptosis from 0.04% to 8.10%. Compound 5 decreased the necrotic cell death from 0.18% to 0.02%, relative to untreated MCF-7 as a control. These data suggested that compound 5 induced programmed cell death via apoptosis instead of the necrotic pathway (Fig. 3 and Table 5).

Fig. 3 Effect of the control (the left side) and compound 5 (the right side) on the apoptosis of MCF-7.

Table 5 The effect of compound 5 on the apoptosis/necrosis of the MCF-7 cells

Compound	Total	Early apoptosis	Late apoptosis	Necrosis
5/MCF-7	79.22	71.1	8.10	0.02
Control/MCF-7	0.65	0.43	0.04	0.18

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The quadrants in the cytograms represent the following: necrotic cells (higher left quadrant of the cytogram); late apoptotic cells (higher right quadrant of the cytogram); non-apoptotic and non-necrotic cells (living cells) (lower left quadrant of the cytogram); early apoptotic cells (lower right quadrant of the cytogram).

3.7. The influence on mitochondrial apoptotic proteins (Bax & Bcl-2)

The family of the Bcl-2 protein shows a critical contribution in mitochondrial apoptosis control. Among them, Bcl-2 and Bax regulate this process properly; Bcl-2 suppresses it (anti-apoptotic), while Bax induces it (pro-apoptotic).⁷⁴ Therefore, achieving an optimal equilibrium between these two proteins is essential for determining the ability of the cells to pass through apoptosis.⁷⁵

During this study, the treatment of MCF-7 via the most active derivative (5) was performed, and its influence on the Bcl-2 and Bax levels is illustrated in Table 6.

Table 6 Effect of compound 5 on the Bax and Bcl-2 levels

Comp.	Bax		Bcl-2
	pg mL^−1	FLD	ng mL^−1	FLD
5	369.15 ± 0.7	6.45	1.72 ± 0.08	0.3
Control	57.22 ± 0.9	1	5.31 ± 0.5	1

---

The results revealed that compound 5 raised the Bax protein level by 6.4-fold and decreased the anti-apoptotic Bcl-2 level by 3-fold in comparison with the control. The important parameter is the two-protein ratio. Therefore, the Bax/Bcl-2 ratio of compound 5 was calculated to be approximately 20-fold in comparison with the control, indicating that this compound shifted the cells to undergo apoptosis.

3.8. Docking study

In this study, Autodock Vina software was used to carry out the reference docking simulation study, and the two promising derivatives (5 and 6) were docked into the CDK2 enzyme ATP binding site. The interaction and binding affinity of the desired pyrazoles were studied to clarify the biological outcomes, as well as to shed light on the binding poses and interactions. The CDK2 X-ray crystal structure in complex with roscovitine was downloaded from the protein data bank (code 2A4L).⁷⁶ The docking procedure was confirmed by re-docking roscovitine inside the CDK2 active site. After that, the RMS deviation was measured (0.99 Å), implying that the used docking procedure obtained an accurate pose orientation. The affinity mode of roscovitine showed that the amino pyrazolopyrimidine moiety was linked to the critical amino acid (Leu83) residue in the hinge region by two hydrogen bonds, in addition to its ability to form three hydrogen bonds, two of them with Asp86 and the last one with Gln131. The benzyl moiety formed a non-conventional hydrogen bond with Asp86. Moreover, the isopropyl group was able to interact with Glu81 and Ala31 through carbon–hydrogen bonds. Besides, the hydroxyl group formed an H-bond with Glu12. Furthermore, the interactions of different hydrophobic natures were recorded with Val18, Lys89, Leu134, Phe80, Phe82, and Ile10 (Fig. 4).

Fig. 4 The proposed 2D (left) and 3D (right) binding modes of roscovitine into the CDK2 active site.

The exploration of the compounds 5 and 6 binding modes exhibited good fitting in the CDK2 active site with energy scores of −7.40 and −6.88 kcal mol⁻¹, respectively. Both compounds established a complex with the ATP binding site that was stabilized by H bonds in the hinge region, together with many hydrophobic connections with the enzyme hydrophobic pocket (Table 7, Fig. 5 and 6).

Table 7 The proposed docking scores and interaction of roscovitine and the desired pyrazole derivatives (5 and 6)

Comp. no.	Energy score (kcal mol^−1)	Interaction types (amino-acid residues)	Bond length (Å)
Roscovitine	−7.94	H-bond (Leu83)	2.5
		H-bond (Leu83)	4.2
		H-bond (Asp86)	3.85
		H-bond (Asp86)	4.00
		H-bond (Glu12)	2.11
		H-bond (Gln131)	3.51
		C–H-bond (Leu83)	3.25
		C–H-bond (Asp86)	3.65
		C–H-bond (Glu81)	2.45
		C–H-bond (Glu81)	3.63
		C–H-bond (Gln131)	4.44
		C–H-bond (Ala31)	4.50
		Hydrophobic-π–alkyl (Phe80)	4.66
		Hydrophobic-π–alkyl (Ala31)	3.94
		Hydrophobic-π–alkyl (Ile10)	4.70
		Hydrophobic-π–alkyl (Ile10)	5.14
		Hydrophobic-π–alkyl (Val18)	5.29
		Hydrophobic-π–alkyl (Leu134)	4.29
		Hydrophobic-π–alkyl (Leu134)	4.53
		Hydrophobic-π–alkyl (Ala31)	3.89
		Hydrophobic-π–alkyl (Ile10)	4.23
		Hydrophobic-π–π (Phe82)	5.83
5	−7.40	H-bond (Leu83)	2.26
		H-bond (Leu83)	3.46
		H-bond (Leu83)	4.16
		H-bond (Lys20)	4.34
		H-bond (Asp86)	4.74
		H-bond (Asp86)	2.69
		H-bond (Asp86)	2.80
		H-bond (Gln131)	4.20
		H-bond (Glu81)	4.24
		H-bond (His84)	2.71
		H-bond (Glu8)	1.95
		C–H-bond (His84)	4.59
		C–H-bond (Ala31)	3.98
		C–H-bond (Glu81)	3.69
		Hydrophobic-π–alkyl (Ile10)	4.50
		Hydrophobic-π–alkyl (Ile10)	4.87
		Hydrophobic-π–alkyl (Leu134)	3.98
		π–anion (Glu8)	4.42
6	−6.88	H-bond (Leu83)	2.00
		H-bond (Leu83)	4.26
		H-bond (Lys20)	3.25
		H-bond (Lys20)	2.09
		H-bond (Lys89)	2.67
		H-bond (His84)	3.19
		H-bond (Ile10)	2.72
		H-bond (Leu298)	2.31
		H-bond (Glu8)	2.26
		C–H-bond (Lys89)	4.28
		C–H-bond (Lys20)	4.33
		C–H-bond (His84)	2.53
		C–H-bond (His84)	4.25
		C–H-bond (Glu12)	4.68
		π–alkyl (Ile10)	4.51
		π–anion (Asp86)	3.80
		π–anion (Asp86)	3.69
		π–π (His84)	5.98
		π–cation (Lys89)	2.49
		Hydrophobic amide–π (Gln85)	5.03

---

Fig. 5 The proposed 2D (left) and 3D (right) binding modes of 5 into the CDK2 active site.

Fig. 6 The proposed 2D (left) and 3D (right) binding modes of 6 into the CDK2 active site.

A similar direction was noticed for both derivatives into the enzyme active pocket, in which the diamino-4-diazenyl pyrazole moiety formed 2 H-bonds with the vital Leu83 amino acid. The NH₂ group at the 3-position formed three H-bonds with Asp86 and Gln131, while the amino group at the 5-position formed an H-bond with Glu81 in compound 5. The pyrazole ring was involved in a non-polar interaction with the Leu134 amino acid. The hydroxyl group interacted through three H-bonds with His84, Lys20, and Leu83. The carboxylic group formed an H-bond with Glu8. The phenyl ring could lie well in the hydrophobic pocket of ATP through nonpolar interactions with amino acid Ile10. However, a pi–cation connection was formed between the phenyl ring and Glu8. Concerning compound 6, the amino group at the 5-position was involved in an H-bond interaction with Ile10, and the pyrazole core formed hydrophobic interactions with Ile10 and Gln85. Diamino methylene-benzenesulfonamide formed five H-bonds with His84, Lys20, Leu298, and Glu8. The benzenesulfonamide ring could fit in the ATP nonpolar pocket, forming a pi–pi interaction with His84 amino acid. Finally, the pyridine moiety formed a carbon–hydrogen bond with Glu12 and a pi–anion interaction with Asp86. Thus, we can conclude that the simplification of the fused heterocyclic ring to pyrazole has a positive impact on the activity, as the diamino pyrazole ring could accommodate well and form connections with the same essential amino acids: Leu83, Asp86, Ile10, and Glu81. Moreover, the cyclization of the isopropyl group of roscovitine to pyridine in compound 5 could adapt the hydrophobic pocket and interact with similar amino acids (Ala31 and Glu81) as the isopropyl of the ligand. The overlay of the ligand, roscovitine, with our newly prepared derivatives (5 and 6) is presented in Fig. 7.

Fig. 7 Superimposition of roscovitine and active compounds 5 and 6.

3.9. In silico ADME prediction

According to Lipinski's rule for oral drugs, the permeation or absorption of a heterocyclic molecule is probable when the weight of the molecule is under 500 g mol⁻¹, the calculated value of the octanol–water partition coefficient is lower than 5, and the molecule possesses not more than five H-bond donors and not more than 10 H-bond acceptors.⁷⁷ However, drug-likeness constraints according to Veber's rule were defined as total rotatable bonds ≤ 10 and area occupied by polar surface (PSA) ≤ 140.⁷⁸

The pharmacokinetic properties of the promising derivatives (5 and 6) were predicted via the SwissADME online tool. The results revealed that compound 5 had zero Lipinski violations. Concerning Veber's rule, the tested compounds had rotatable bonds between 4 and 5, indicating molecular flexibility to their biotarget, and both compounds had one deviation from Veber rules (TPSA > 140) (Table 8).

Table 8 Physicochemical characteristics based on Veber's and Lipinski's rules of compounds 5 and 6

Cpd no.	HBD	HBA	M log P	MW	No. of rot. bonds	TPSA	Lipinski's violations	Veber's violations
5	4	7	0.28	339.31	4	165	0	1
								TPSA > 140
6	4	7	0.65	400.42	5	214.39	1	1
								TPSA > 140

---

Considering the medicinal chemistry and pharmacokinetic parameters of the desired compounds (Table 9), both tested pyrazole derivatives had low gastrointestinal tract absorption and were unable to penetrate the blood–brain barrier. This confirms that the targeted molecules work systemically with no side effects on the CNS.

Table 9 Pharmacokinetic properties and medicinal chemistry parameters of tested compounds 5 and 6

Cpd no.	GI absorption	BBB permeation	P-gp substrate	CYP2D6	CYP3A4	Synthetic accessibility
5	Low	No	Yes	No	No	3.03
6	Low	No	Yes	No	No	3.51

---

Furthermore, both studied derivatives have no activities on cytochrome P450 isomers “CYP3A2 and CYP2D6”, and consequently, they are expected to have no drug–drug interaction on administration (Table 9).

SwissADME chemical synthesis Accessibility Score (SA) assumed that the ease of the synthesis was correlated with the molecular fragment's frequency in the obtained molecules. The score ranged from very easy (1) to very difficult (10) to synthesize.⁷⁹ Both tested analogues' SA scores were 3.03 and 3.51, suggesting that both could be synthesized on a large scale.

4. Conclusion

In conclusion, novel azo-pyrazole moieties were designed, synthesized via two techniques, and evaluated for their potential CDK2-enzyme inhibition effects. The anticancer activity of the most active analogues in the enzyme inhibitory studies was assessed against two different human cancer cell lines, in comparison to doxorubicin. Compound 5 exhibited the best cytotoxic results relative to the standard. The CDK2 inhibition study revealed that compound 5 showed a prominent inhibitory effect (IC₅₀ = 0.56 µM), superior to that of the reference drug, roscovitine (IC₅₀ = 0.99 µM). Moreover, cell cycle analysis demonstrated that compound 5 induced cell arrest at both S phase and G2 phase compared with the control, in addition to its ability to trigger apoptosis, raise the Bax level, and reduce the Bcl-2 level in the MCF-7 cells. Finally, the process by which the synthesized pyrazole derivatives suppress the growth of cancer was examined by running CDK2 docking simulations.

Author contributions

Samar A. Abubshait: supervision, writing – original draft. Lamia H. T. Amin: investigation, methodology, writing – original draft. Abeer M. El-Naggar: data curation, supervision, writing – original draft. Mohamed G. Elbanna: methodology, writing – original draft. Kurls E. Anwer: investigation, methodology, data curation, formal analysis, writing, conceptualization, supervision, validation, writing – review & editing.

Conflicts of interest

All authors confirm that no competing personal, financial, and/or professional interests exist that may influence the work reported in this article.

Data availability

All data supporting the findings of this study are included in the article and its supplementary information (SI) files. Supplementary information: including FTIR, 1HNMR, 13CNMR, Rational, Cell cycle analysis, effects of compounds on apoptosis of MCF-7, and molecular docking studies. See DOI: https://doi.org/10.1039/d5ra05212b.

Acknowledgements

I would like to thank the Basic and Applied Scientific Research Centre at Imam Abdulrahman Bin Faisal University, KSA, for funding and supporting the analysis.

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