Synthesis of quercetin derivatives as cytotoxic against breast cancer MCF-7 cell line in vitro and in silico studies

Abstract

Background: Quercetin being antioxidant and antiproliferative agent acts by inhibiting CDK2, with an increase in cancer prevalence there is a need to profile quercetin derivatives as CDK2 inhibitors.

Materials & method: Schiff bases of quercetin were synthesized as cytotoxic agents against the MCF7 cell line. FTIR, ¹H-NMR and ¹³C-NMR, CHNS/O analysis were employed along with in vivo and in silico activities.

Results & conclusion: 2q, 4q, 8q and 9q derivatives have maximum cytotoxic effect with IC₅₀ values 39.7 ± 0.7, 36.65 ± 0.25, 35.49 ± 0.21 and 36.99 ± 0.45, respectively. Molecular docking also confirmed these results 8q has the highest binding potential of -9.165 KJ/mole making it a potent inhibitor of CDK2. These derivatives can be used as lead compounds as potent CDK2 inhibitors.

Graphical Abstract

Plain language summary

Articles highlights.

Derivatization

• Quercetin derived Schiff bases were synthesized having better cytotoxic profile against breast cancer cell line.

Characterization

• Spectral analysis confirms the new synthesized compounds.

In vitro studies

• The new derivatives were evaluated CDK-2 inhibitors as potential cytotoxic agent against MCF-7 cell line, 2q, 4q, 8q and 9q derivatives have maximum cytotoxic effect with IC₅₀ values 39.7 ± 0.7, 36.65 ± 0.25, 35.49 ± 0.21 and 36.99 ± 0.45 respectively.

In silico studies

• Molecular docking also confirmed these results 8q has the highest binding potential of -9.165 KJ/mole making it a potent inhibitor of cyclin-dependent kinase 2.

Conclusion

• These derivatives can be used as lead compounds as potent cyclin-dependent kinase 2 inhibitors.

1. Background

Carcinogenesis has six main characteristics and can occur in any cell, tissue or organ, leading to various types of cancer. The main processes that allow it to proceed include evading apoptosis, having an infinite proliferative potential, increased angiogenesis, resistance to antigrowth signals and activation of its growth signals and the ability to metastasize. Breast cancer accounts for about 29% of newly diagnosed cancer cases in women [1]. In 2020 2.26 million [95% uncertainty interval (UI), 2.24–2.79 million] new cases of breast cancer were reported, making it the most prevalent disease among women diagnosed worldwide [2].

Certain biomolecules found in the human diet, such as flavonoids, can act as ‘chemo-preventers’ by halting the growth of cancer cells. Apart from their antioxidant qualities, these macromolecules also induce cell-cycle arrest and apoptosis, contributing to their potential to prevent cancer [3,4]. The antioxidant properties of chemo-preventers have been getting a lot of attention due to the role of oxidative stress in the onset and progression of many diseases including cancer [5].

Quercetin derived from quercetum or oak forest has been used since 1857. It is named after Quercus. It is present in many natural plants, such as seeds, nuts, flowers, bark, leaves, apples, berries, brassica vegetables, capers, grapes, onions, spring onions, tea and tomatoes [6]. Quercetin (3,3′,4′,5,7-pentahydroxyflavone) belongs to the class of bioflavonoids and is known for shielding the tissues from damage caused by certain drug toxicities [7,8]. Quercetin is classified as a flavanol and a flavonoid has a chemical structure consisting of three rings: ring C, which joins the two rings (A and B), is a heterocyclic pyran ring – the addition of hydroxyl (-OH) group that enhances its antioxidant properties [9]. Among the several functional groups found in there quercetin are double bonds (C=C) within the rings, hydroxyl (-OH) groups and carbonyl (C=O) groups, the chemical reactivity and wide range of its biological activities are attributed to these functional groups [8].

It is used as a dietary supplement because it protects against various ailments, such as inflammatory and metabolic problems. Additionally, it has antiviral, anti-inflammatory, immunomodulatory, anticancer, antitumor and anti-allergy activities [10]. Quercetin is one of the most significant endogenous antioxidants and is commonly used in pharmaceutical, cosmetic and nutraceutical products because it shields against oxidants and inflammation [11].

Recent studies have shown that quercetin can inhibit the development, invasion and metastasis of tumor cells, as well as promote apoptosis in cancer cells, aiding in the prevention of breast cancer [12].

Cyclin-dependent kinases (CDKs) are a class of serine/threonine kinases, essential in controlling the advancement of the cell cycle. Cyclins stimulate the activity of these kinases. In reality, the orderly progression of the cell cycle is regulated by CDK/cyclin complexes [13]. Cyclin-dependent kinase 2 (CDK2) is known to be crucial for the emergence and metastasis of several cancer forms, including ovarian, lung, cholangiocarcinoma, oral squamous cell carcinoma, breast, liver, colon and prostate cancers [14]. Studies revealed that CDK2 is a potential target in cancer treatment [14].

Due to the potential anticancer activity of quercetin, there is a need for more active derivatives with promising anticancer activity. Quercetin is a polyfunctional group compound and has the potential to be a target for drug modification. The Carbonyl group of quercetin is considered an active target for Schiff Bases derivatization [15].

Mutation in genes and proteins is considered a major cause of drug resistance in anticancer therapy [16]. Due to the increasing incidence of drug-resistant malignancies, more research and treatment development are needed [16]. Quercetin has been approved by the FDA as a GRAS (Generally Recognized As Safe) dietary supplement because of its well-established safety and tolerability profile in humans [17]. Extensive research is being conducted on quercetin as a potential cancer treatment due to its remarkable qualities, which include the ability to regulate significant biochemical processes linked to apoptosis and the power to successfully prevent drug efflux in tumors resistant to numerous treatments [18]. Several studies have demonstrated that quercetin possesses anticancer effects. The regulation of these characteristics is mediated by many signaling pathways inside the cancer cell. Although few Schiff bases of quercetin were synthesized as cytotoxic agents against the MCF-7 cell line, there is a need to synthesize more biologically active compounds to profile better anticancer agents against breast cancer with better therapeutic efficacy and less adverse effects [15,19]. Our study aims to synthesize the new Schiff base derivatives of quercetin, with improved anticancer activity against the MCF-7 cell line. This novel approach also aims to significant improvements in the field of anticancer drug development by minimizing anticancer drug resistance and developing better candidates for the treatment of breast cancer having better efficacy. Moreover, in silico studies were conducted to validate the newly derived compounds from parent molecules.

2. Materials

Quercetin was purchased from Sigma Aldrich (Beijing, China), while other chemicals e.g., p-amino benzoic acid, p-amino phenol, aniline, phenylhydrazine, aspartic acid, glycine, glutamic acid, phenylalanine, tyrosine and ethanol. All the chemicals used in the recent investigation were of pure and analytical grade. TLC examination of chemical purification and reaction progress was carried out on silica gel-coated aluminum plates at F254 nm (Merck KGaA, Beijing, China). The solubility of novel compounds was evaluated in a variety of solvents, including DMSO-d6, dichloromethane, chloroform, distilled water, methanol and ethanol. A Gallen Kamp melting point apparatus (Cambridge, UK) was used to determine the melting point. Thermo Scientific’s Flash 2000 analyzer (MA, USA) analyzed carbon, hydrogen, nitrogen and sulphur levels. A high-performance digital Fourier transform from Bruker (UK) was used for the spectroscopic study.

3. Methodology

3.1. Preparation of derivatives

Quercetin (0.302 g, 1 mmol) was diluted in 15 ml of ethanol. The solution was treated with a few drops of mineral acid. After 30 min, add (1 mmol) aliphatic and aromatic amine 1q-11q solution in ethanol dropwise to the round bottom flask. The reaction mixture was refluxed and stirred at 70°C for 4 h (Figure 1). The resulting dark red solution was concentrated with a rotary evaporator, providing an orange crystalline precipitate after recrystallizing from a hot ethanol solution and drying under a vacuum. The Schiff base had a faint orange color [20].

Figure 1.

Synthesis of Schiff base derivatives of quercetin.

3.2. Cell viability assay

The synthesized compounds were assessed for cytotoxicity using an MTT test (Promega, Madison, USA). The percentage of cell viability in the MCF-7 cell line after drug-loaded therapy was compared with the control, parent drug and derivatives. Cells (1 × 10⁵/well) were seeded in 96-well plates and treated with medication, derivatives and blank solution in three different concentrations (20, 40 and 80 μM) for 6 and 24 h, respectively. An automated microplate reader measured absorbance at 570 nm after MTT treatment. The ratio of cells treated against controls was estimated using the formula below [21,22]. The inhibitory concentration (IC50) was determined by using Graph Pad prism software 9.02 (CA, USA) [21,23].

$$\text{Cel} \text{viability} (\%)=\frac{A570\left(\mathrm{sample}\right)- A570\left(\mathrm{blank}\right)}{\text{A}570\left(\text{control}\right)- \text{A}570\left(\text{blank}\right)}\times 100$$

3.3. Molecular docking

The binding characteristic of derived compounds’ the drug at the active site of human CDK2 was determined from Grid-based Ligand Docking with Energetics (Glide, Schrodinger, LLC, NY, USA) ¨ in silico “ protocol (Glide). For molecular docking studies, the protein HUMAN CDK-2 Protein Data Bank PDB id 1HCK was derived from www.rcsb.org. The ligand structures were drawn in ChemDraw 19.1 and imported into Schrodinger software for ligand preparation. Every ligand molecule is optimized by the ligand preparation process by use of the LigPrep module (LigPrep, Schrodinger). The x-ray structure of the protein CDK2 was imported in the wizard of protein preparation by using Maestro 12.8 (Schrodinger, 2021) [24].

• 1.The protein’s crystal structure (1HCK) was modified by adding missing side chains using Schrodinger’s prime 3.0
• 2.All water molecules were eliminated from the protein and polar hydrogen bonds were added.
• 3.Protonated states were preserved at pH 7.0 ± 2.0 using the Epik instrument.
• 4.Enhanced networks of hydrogen bonds and flip-off orientations/tautomeric states for residues of His, Asn and Gln.
• 5.The Optimised Potentials for Liquid Simulations force field was used to optimize the geometry [25].

The protein grid was generated by the receptor grid generation module of the Glide program. A cocrystal ligand was used to characterize the binding position. Using the extra precision technique [26,27], which offers suitable ligand postures for discovering additional places for ligand binding, Glide was employed in molecular docking studies. The docking investigations indicate the best postures with the Glide and Dock score [28].

3.4. Pharmacokinetic analysis

The pharmacokinetic properties of newly synthesized derivatives were assessed by using the SWISS absorption, distribution, metabolism and excretion (ADME) webserver. It predicts ADME. The data obtained included GI absorption number of rotatable bonds, H-bond acceptors and donors and log values Po/w.

4. Results & discussion

4.1. Results

The compound structures obtained are outlined in Figure 2. The physical and chemical characteristics of each derivative are detailed below in discussion. In addition, in vitro study data is provided in Supplementary Table S1, while molecular docking and Swiss ADME studies are presented in Table 1, Supplementary Table S2 & Table 2, respectively

Figure 2.

Structures of analogs of newly synthesized derivatives of quercetin.

Table 1.

Molecular docking results of derivatives 1q-11q.

Sr No	Code	Binding energy
1	1q	-7.93
2	2q	-9.04
3	3q	-7.06
4	4q	-8.41
5	5q	-7.44
6	6q	-7.25
7	7q	-7.53
8	8q	-9.16
9	9q	-8.28
10	10q	-7.41
11	11q	-6.93
12	q	-7.85

Table 2.

Pharmacokinetic properties of derivatives 1q-11q.

Code	No. of rotatable bonds	No. of H-bond acceptors	No. of H-bond donors	LogP o/w	GI absorption	Lipinski #violations	Bioavailability score	Synthetic accessibility
1q	3	9	6	2	Low	1	0.11	3.77
2q	2	7	5	2.14	High	0	0.55	3.68
3q	2	7	5	2.45	High	0	0.55	3.75
4q	3	6	5	2.39	High	0	0.55	3.64
5q	5	10	6	0.9	Low	1	0.11	4.2
6q	3	8	5	1.23	Low	0	0.56	3.5
7q	6	10	6	1.13	Low	1	0.11	4.17
8q	5	8	5	1.87	Low	0	0.56	4.26
9q	5	9	6	1.79	Low	1	0.11	4.29
10q	5	9	6	1.01	Low	1	0.55	4.19
11q	1	7	5	1.54	High	0	0.55	3.25

4.2. Discussion

4.2.1. 1q

IUPAC Name: 4-((2-(3,4-dihydroxy phenyl)-3,5,7-trihydroxychroman-4-ylidene)amino)benzoic acid. Yield (66%): M. P. 160°C. Molecular formula: C₂₂H₁₅NO₈ and Molecular weight: 423.38 gm/mol. Elemental analysis (calculated) for C₂₂H₁₅NO₈: C, 62.71; H, 3.59; N, 3.32; O, 30.38, FT-IR ν(cm^-1), 3309 (C-OH), 1719 (C=O), 1660 (C=N), 1450 (C=C), 1086 (C–O–C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09s (1H-CH), 5.88–5.89s (1H-CH), 6.52–6.53s (1H-CH), 7.04–7.05d (1H-CH), 6.82–6.83d (1H-CH), 7.43–7.44d (2H-CH), 8.18–8.19d(2H-CH), 10.27(1H-OH), 10.18 (1H-OH), 10.68 (1H-OH), 9.48 (2H-OH),12.71 (1H-OH). ¹³C NMR (DMSO, ppm) δ: 153.2(C1), 128.7 (C2), 147.1(C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 99.5 (C8), 159.3 (C9), 122.8(C10), 115.3 (C11), 145.9 (C12),146.2 (C13), 117.2 (C14), 121.8 (C15), 158.4 (C16), 122.2 (C17), 131.6 (C18), 128.7 (C19), 131.6 (C20), 122.2 (C21), 169.3(C22).

4.2.2. 2q

IUPAC Name: 2-(3,4-dihydroxyphenyl)-4-((4-hydroxyphenyl)imino)chromane-3,5,7-triol. Yield (67%); M. P. 198–200°C. Molecular formula: C₂₁H₁₅NO₇ and Molecular weight: 395.10 gm/mol. Elemental analysis (calculated) for C₂₁H₁₅NO₇: C, 64.12; H, 3.84; N, 3.56; O, 28.47, FT-IR ν(cm^-1), 3309 (C-OH), 1660 (C=N), 1450 (C=C) 1086 (C–O–C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09 s (1H-CH), 5.88–5.89s (1H-CH), 6.52–6.53s (1H-CH), 7.04–7.05d (1H-CH), 6.82–6.83d (1H-CH), 7.22–7.23d (2H-CH), 6.83–6.84d(2H-CH), 10.27(1H-OH), 10.18 (1H-OH), 10.68 (1H-OH), 9.48 (2H-OH),9.41 (1H-OH)¹³C NMR (DMSO, ppm) δ:153.2(C1), 128.7 (C2), 147.1(C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 99.5 (C8), 159.3 (C9), 122.8(C10), 115.3 (C11), 145.9 (C12),146.2 (C13), 117.2 (C14), 121.8 (C15), 158.4 (C16), 122.2 (C17), 131.6 (C18), 157.7 (C19), 131.6 (C20), 122.2 (C21).

4.2.3. 3q

IUPAC Name: 2-(3,4-dihydroxyphenyl)-4-(phenylimino)chromane-3,5,7-triol. Yield (67%): M. P. 170°C. Molecular formula: C₂₁H₁₅NO₆ and Molecular weight: 377.09 gm/mol. Elemental analysis (calculated) for C₂₁H₁₅NO₆: C, 66.84; H, 4.01; N, 3.71; O, 25.44. FT-IR ν(cm^-1), 3371 (C-OH), 1719 (C=O), 1666 (C=N), 1510 (C=C), 1060 (C–O–C) ¹H NMR (DMSO, ppm)δ: 7.08–7.09s (1H-CH), 5.88–5.89s (1H-CH), 6.52–6.53s (1H-CH), 7.04–7.05d (1H-CH), 6.82–6.83d (1H-CH), 6.93–6.94d (2H-CH), 7.34–7.35t (2H-C₆H₅), 7.07–7.08t (1H-C₆H₅) 10.27(1H-OH), 10.18 (1H-OH), 10.68 (1H-OH), 9.48 (2H-OH). ¹³C NMR (DMSO, ppm) δ: : 153.2(C1), 128.7 (C2), 147.1(C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 99.5 (C8), 159.3 (C9), 122.8(C10), 115.3 (C11), 145.9 (C12),146.2 (C13), 117.2 (C14), 121.8 (C15), 158.4 (C16), 118.8 (C17), 130 (C18), 127.2 (C19), 130.0 (C20), 118.8 (C21).

4.2.4. 4q

IUPAC Name: 2-(3,4-dihydroxyphenyl)-4-(2-phenylhydrazineylidene)-4H-chromene-3,5,7-triol. Yield (67%); M. P 142–143°C. Molecular formula: C₂₁H₁₈N₂O₆ and Molecular weight: 392.37 gm/mol. Elemental analysis (calculated) for C₂₁H₁₆N₂O₆: : C, 64.28; H, 4.11; N, 7.14; O, 24.47, FT-IR ν(cm^-1), 3371 (C-OH), 1719 (C=O), 1666 (C=N), 1510 (C=C), 1060 (C–O–C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09 s (1H-CH), 5.88–5.89 d (1H,CH), 7.04–7.05d (1H-CH) 6.53–6.54s (1H-CH), 6.82–6.83d (1H-CH), 7.34–7.35d (2H-C₆H₅), 7.35–7.36t (2H-C₆H₅), 7.06–7.07t (1H-C₆H₅), 10.68 s (1H-OH), 9.48 s (2H-OH) 10.27 s (1H-OH), 10.18 s (1H-OH) ¹³C NMR (DMSO, ppm) δ: 154.0 (C1), 143.0 (C2), 155.6 (C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 99.5 (C8), 159.1 (C9), 122.8 (C10), 115.2 (C11), 145.9 (C12),146.2 (C13), 117.2 (C14), 121.8 (C15), 143.0 (C16), 113.9 (C17), 129.5 (C18), 122.4 (C19), 129.5 (C20), 113.9 (C21).

4.2.5. 5q

IUPAC Name: 2-((2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-ylidene)amino)succinic acid. Yield (67%): M. P. 161–162°C. Molecular formula: C₁₉H₁₅NO₁₀ and Molecular weight: 417.07 gm/mol. Elemental analysis (calculated) for C₁₉H₁₅NO₁₀:: C, 54.68; H, 3.62; N, 3.62; O, 38.34, FT-IR ν(cm^-1), 3371 (C-OH), 1719 (C=O), 1666 (C=N), 1510 (C=C), 1060 (C–O–C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09 s (1H-CH), 5.88–5.89 d (1H-CH), 7.04–7.05d (1H-CH) 6.52–6.53s (1H-CH), 6.82–6.83d (1H-CH),4.33–4.34 (1H-CH), 2.94 (2H -CH₂) 10.68 s (1H-OH), 9.48 s (2H-OH) 10.27 s (1H-OH), 10.18 s (1H-OH) 13.72 s (2H-OH). ¹³C NMR (DMSO, ppm) δ: 153.2 (C1), 128.7 (C2), 164.6 (C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 105.6 (C8), 159.3 (C9), 122.9 (C10), 115.3 (C11), 145.9 (C12),146.2 (C13), 117.2 (C14), 121.8 (C15), 177.5 (C16), 66.1 (C17), 39.7(C18), 177.3(C19).

4.2.6. 6q

IUPAC Name: 2-((2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-ylidene)amino)acetic acid. Yield (78%); M. P. 141–144°C. Molecular formula: C₁₇H₁₃NO₈ and Molecular weight: 359.29 gm/mol. Elemental analysis (calculated) for C₁₇H₁₃NO₈:: C, 56.83; H, 3.65; N, 3.90; O, 35.62, FT-IR ν(cm^-1), 3370 (C-OH), 1683 (C=O), 1630 (C=N), 1531 (C=C) 1069 (C–O–C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09s (1H-CH), 5.88–5.87 d (1H-CH), 7.04–7.05d (1H-CH) 6.52–6.53s (1H-CH), 6.82–6.83d (1H-CH),4.49 (1H-CH), 10.68 s (1H-OH), 9.48 s (2H-OH) 10.27 s (1H-OH), 10.18 s (1H-OH) 12.22s (1H-OH). ¹³C NMR (DMSO, ppm) δ: 153.2 (C1), 128.7 (C2), 164.6 (C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 105.6 (C8), 159.3 (C9), 122.9 (C10), 115.3 (C11), 145.9 (C12),146.5 (C13), 117.2 (C14), 121.8 (C15), 51.7 (C16), 171.3 (C17).

4.2.7. 7q

IUPAC Name: 2-((2-(3,4-dihydroxy phenyl)-3,5,7-trihydroxy-4H-chromen-4-ylidene)amino)pentanedioic acid. Yield (75%); M. P. 145–147°C. Molecular formula: C₂₀H₁₇NO₁₀ and Molecular weight: 431.09 gm/mol. Elemental analysis (calculated) for C₂₀H₁₇NO₁₀:: C, 55.69; H, 3.97; N, 3.25; O, 37.09, FT-IR ν(cm-1), 3213 (C-OH), 1665 (C=N), 1505 (C=C) aromatic, 1090 (C–O–C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09 s (1H-CH), 5.88–5.89 d (1H-CH), 7.04–7.05d (1H-CH) 6.52–6.54 s (1H-CH), 6.82–6.83 d (1H-CH),3.91–3.92 t (1H-CH), 2.15 m(2H-CH₂), 2.33 t (2H-CH) 10.68 s (1H-OH), 9.48 s (2H-OH) 10.27 s (1H-OH), 10.18 s (1H-OH) 12.01 s (2H-OH). ¹³C NMR (DMSO, ppm) δ: 153.2 (C1), 128.7 (C2), 164.6 (C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 105.6 (C8), 159.3 (C9), 122.9 (C10), 115.3 (C11), 145.9 (C12),146.2 (C13), 117.2 (C14), 121.8 (C15), 177.5 (C16), 69.3 (C17), 29.8(C18), 30.9 (C19) 178.4 (C20).

4.2.8. 8q

IUPAC Name: 2-((2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-ylidene)amino)-3-phenylpropanoic acid. Yield (65%); M. P.145–146°C. Molecular formula: C₂₄H₁₉ NO₈ and Molecular weight: 449.11 gm/mol. Elemental analysis (calculated) for C₂₄H₁₉ NO₈ C, 64.14; H, 4.26; N, 3.12; O, 28.48 FT-IR ν(cm-1), 3313 (C-OH), 1634 (C=N), 1506 (C=C), 1091 (C–O–C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09 s (1H-CH), 5.88–5.89 d (1H-CH), 7.04–7.05 d (1H-CH) 6.52–6.53 s (1H-CH), 6.82–6.83d (1H-CH),4.39–4.40 t (1H-CH), 3.27–3.28 m(2H-CH₂) 7.21–7.22 d (2H -C₆H₅), 7.23–7.24t (2H -C₆H₅), 7.19–7.20t (1H -C₆H₅) 10.68s (1H-OH), 9.48 s (2H-OH) 10.27s (1H-OH), 10.18s (1H-OH) 12.72 (1H-OH). ¹³C NMR (DMSO, ppm) δ: 153.2 (C1), 128.7 (C2), 164.6 (C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 105.6 (C8), 159.3 (C9), 122.9 (C10), 115.3 (C11), 145.9 (C12),146.2 (C13), 117.2 (C14), 121.8 (C15), 71.0 (C16), 38.5 (C17), 137.5(C18), 127.7 (C19) 128.6 (C20), 125.9 (C21). 128.6 (C22), 127.7 (C23), 177.5 (C24).

4.2.9. 9q

IUPAC Name: 2-((2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-ylidene)amino)-3-(4-hydroxyphenyl)propanoic acid. Yield (65%); M. P. 144–146°C. Molecular formula: and Molecular weight: 465.41 gm/mol. Elemental analysis (calculated) for C₂₄H₂₀NO₉ C, 61.94; H, 4.12; N, 3.01; O, 30.94 FT-IR ν(cm-1), 3313 (C-OH), 1634 (C=N), 1506 (C=C) aromatic, 1091 (C–O–C), 1H NMR (DMSO, ppm)δ: 7.08–7.09 s (1H-CH), 5.88–5.89 d (1H-CH), 7.04–7.05d (1H) 6.52–6.53s (1H-CH), 6.82–6.83d (1H-CH),4.39–4.40 t (1H-CH), 3.27–3.28 m(2H CH₂) 6.96–6.97d (2H -CH), 6.67–6.68d (2H -CH), 10.68 s(1H-OH), 9.48 s (2H-OH) 10.27 s (1H-OH), 10.18s (1H-OH), 9.06s (1H-OH), 12.72 (1H-OH). ¹³C NMR (DMSO, ppm) δ: 153.2 (C1), 128.7 (C2), 164.6 (C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 105.6 (C8), 159.3 (C9), 122.9 (C10), 115.3 (C11), 145.9 (C12),146.2 (C13), 117.2 (C14), 121.8 (C15), 71.0 (C16), 38.5 (C17), 130.5(C18), 130.2 (C19) 115.8 (C20), 155.7 (C21). 115.8(C22), 130.2 (C23), 177.5 (C24).

4.2.10. 10q

IUPAC Name: 2-((2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-ylidene)amino)-3-(1H-imidazol-5-yl)propanoic acid. Yield (75%); M. P. 178–179°C. Molecular formula: C₂₁H₁₇N₃O₈ and Molecular weight: 439.38 gm/mol. Elemental analysis (calculated) for C₂₁H₁₇N₃O₇:, 57.41; H, 3.90; N, 9.56; O, 29.13 FT-IR ν(cm^-1), 3350 (C-OH), 1794 (C=O), 1630 (C=N), 1533 (C=C), 1013 (C-C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09, s (1H-CH), 5.88–5.89, s (1H-CH) 6.53–6.54, s (1H-CH), 6.82–6.83, d (1H-CH) 7.04–7.05, d (1H-CH), 4.39 t (1H-CH), 2.96 d, (2H-CH₂), 2.13–2.15, d (2H-CH₂) 7.33, s (1H-CH), 8.47, s (1H-CH) 9.48, s (1H-OH), 10.17, s (1H-OH), 10.1, s (1H-OH), 9.07, s (1H, OH), 12.90, s (1H-NH), 12.72, s (1H-OH), ¹³C NMR (DMSO, ppm) δ: 157.3 (C1), 128.7(C2), 164.6(C3), 163.2(C4), 97.9 (C5), 162.6 (C6), 93.6 (C7), 159.3 (C8), 105.6(C9), 122.9 (C10), 115.3 (C11), 145.9 (C12),146.5 (C13), 117.2 (C14), 121.8 (C15), 70.4 (C16), 34.5 (C17), 145.5 (C18), 135.9 (C19), 118.6 (C20), 177.5 (C21).

4.2.11. 11q

IUPAC Name: (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one oxime. Yield (78%): M.P.190–191°C. Molecular formula: C₁₅H₁₁NO₇ and Molecular weight: 317.05 gm/mol. Elemental analysis (calculated) for C₁₅H₁₁NO₇: C, 56.79; H, 3.50; N, 4.42; O, 35.30 -FTIR ν(cm^-1), 3228 (C-OH), 1656 (C=N), 1533 (C=C), 1092 (C–O–C), ¹H NMR (DMSO, ppm)δ: 7.08–7.09, s (1H-CH) 5.88–5.89, s (1H-CH), 7.04–7.05, d (1H-CH) 6.52–6.53, s (1H-CH) 6.82–6.83,d (1H-CH), 9.48, s (2H-OH) 10.18, s (1H-OH) 10.27, s (1H-OH), 10.68,s (1H-OH) 13.82, s (1H-OH) ¹³C NMR (DMSO, ppm) δ: 157.2 (C1), 144.7 (C2), 164.6 (C3), 163.2 (C4), 97.9 (C5), 162.9 (C6), 93.6 (C7), 99.8 (C8), 159.3 (C9), 122.9 (C10), 115.3 (C11), 145.9 (C12),146.5 (C13), 117.2 (C14), 121.8 (C15).

5. Discussion

5.1. Cell cytotoxicity studies

MCF-7 cells were treated with different concentrations (25, 50, 100 μg/ml) of quercetin derivatives 1q and 11q. Cell viability was assessed using the MTT assay after 6 and 24 h of treatment. It was observed that cell growth was inhibited in a dose-dependent manner after 24 h of treatment with the derived ligands. Figure 3A shows % cell viability at the dose of 20 μM after 24h compounds 2q and 8q significantly decrease in cell viability and has exhibited more cytotoxicity effects as compared with the parent drug molecule. Similarly in Figure 3B at the dose of 40 μM and in Fig 3C 100 μM dose of all under study compounds have shown cytotoxicity effect. 1q, 3q, 5q, 6q, 7q, 9q, 10q and 11q exhibited comparable cytotoxic effect. But 2q, 4q and 8q showed a significant decrease in cell viability i.e., 50 ± 2, 52 ± 2,46 ± 2 after treatment with 80 μM concentration. All data is mentioned in Supplementary Table S1. The observed IC50 in μM for compound 1q-11 43.07 ± 1, 37.95 ± 0.5,39.7 ± 0.02, 36.65 ± 0.3, 38.41 ± 0.3, 39.1033 ± 0.25, 38.23 ± 0.3, 35.49 ± 0.56, 36.99 ± 0.35, 39.1269 ± 0.12, 41.71 ± 0.43 the compound q stands for quercetin having IC50 value is 37.06 ± 0.08 against the MCF7 cells line. The IC50 concentration of derived compounds 4q, 8q and 9q in 24 h was significantly comparable to quercetin. The most significant one is that 8q exhibits toxic effects and as well as significantly reduces cell viability.

Figure 3.

(A–D) MCF-7 cell line treated against quercetin and its derived 11 compounds the % cell viability was determined at different concentrations. (A) 20 μM, (B) 40 μM and (C) 80 μM have shown %cell viability as compared parent compound q. 2q, 4q and 8q exhibit significantly comparable toxic effects than q. (D) shows IC50 values newly of synthesized derivatives which is comparable to the parent drug molecule.

5.2. Molecular docking

All derived compounds of quercetin 1q–11q were assessed against human CDK2, to evaluate their bioactive and specificity for the binding site of docked protein PDB id 1HCK. Quercetin belongs to a group of compounds that are potent inhibitors of CDK2 [23]. The CDK2 X-ray structure coupled with ATP and Mg⁺⁺ was used for validation of the above-mentioned derivatives. After analyzing the docking scores of every component under investigation, the best-conforming structures were selected the results are displayed in Table 1. Quercetin being the parent compound has shown H-bond interaction with two amino acid residues Leu 83 and Asp 143 with a hydroxyl group attached with six-membered ring and fused flavanone ring of quercetin the H-bond length is 4.89 Å and 4.38 Å the docking pose is represented in Supplementary Figure S12. There are some pi-alkyl interactions but ketonic functional moiety exhibited some unfavorable interactions with Asp 86 due to interaction between two donor atoms of residue and ligand, 2D binding interaction is represented in Supplementary Figure S12. Overall, it exhibits the most favorable binding energy -7.856 kj/mol making it a good inhibitor of CDK-2. The docking studies showed that most of our synthesized compounds made contact with the binding pocket of the target protein, with the change in the functional group of derivatives binding changes, the binding energy of all the derived compounds ranged from -9.165 to -6.93. The molecule has the lowest binding energy active inhibitor of protein. Table 1 describes the binding affinity and nonbonding interaction i.e., H-bonding with protein residues unit along with the interaction length between ligand and amino acids of protein. All the derived compounds show significant antiproliferative activities against the CDK2 PDB id HCK1. The potential inhibition of CDK2 sequence on the base of docking score is given as 8q >2q >4q >9q >1q >7q >5q >10q >6q >3q >11q. Compound 8q has binding affinity -9.165; it shows more significant activity due to the aromatic side chain substituted with the aliphatic -COOH group of this side chain having H-bond interaction Lys33 5.77Å, making it more potent (Figure 4A & B). Other H-bond interactions are with the parent structure of quercetin, the docking pose and the 2D interaction of 8q docked protein given in the Figure 4 A & B, another most significant active compound is 2q have a more binding affinity -9.045 kj/mol the hydroxyl group of substituted aromatic side chain, helps to bind more actively. The -OH substitution at the para position of phenyl ring helps to make H-bond interaction with Glu12 side chain residue, the distance of this interaction is 5.26Å demonstrated in Supplementary Figure S14A & B. As compared with 2q and 8q non-substituted aromatic side chains such as 3q have binding affinity of -7.06. 4q is an aromatic side chain hydrazide derivative this substitution helps the parent drug molecule to fit in the binding pocket of the HCK1 figure and helps each hydroxyl group of the parent drug molecule to form a favorable H-bond interaction. The derived compounds having an aliphatic side chain have less significant activity as compared with the substituted aromatic side chain.

Figure 4.

Docking Results of 8q. (A–B) (A) represents the 2D docking interaction 8q with CDK2 residue, (B) 3D docking pose of 8q with CDK2 molecule.

CDK2: Cyclin-dependent kinase 2.

5.3. In silico ADME studies

Using the Swiss ADME server, the pharmacokinetic characteristics of all the synthesized compounds discovered through molecular docking were examined. The ADME server predicts the physicochemical parameters of compounds, which are listed in Table 2. These values include Number of Rotatable bonds, Number of H-bond acceptors, Number of H-bond donors logP_o/w, GI absorption, Lipinski no violations, bioavailability score and synthetic accessibility. A good drug candidate should have a molecular weight of 500 Dalton or fewer, rotatable bonds of 10 or less, hydrogen bond acceptors of 10 or less, hydrogen bond donors of 5 or less and a log value (P o/w) of 5 or less. These are Lipinski’s five rules (RO5) for determining the likelihood of a medication for preclinical trials [29,30]. In our synthesized derivatives all have rotatable bond numbers less than 10 and H-bond acceptors are also less than 10 except a few molecules 5q and 7q have 10 H-bond acceptors. LogPo/w of all molecules is less than 5. Some molecules have high GI absorption e.g., 2q, 3q, 4q and 11q. Maximum Lipinski rule violation is one only due to a slight increase in molecular mass from 500 Dalton. A bioavailability score of ≥0.55 indicates optimal absorption by the body [31]. Most of the derivatives i.e. 2q, 3q,4q, 6q, 8q, 10q and 11q has optimal bioavailability score. Synthetic accessibility score has ranged from 1–10. SA score of 1 means the compound is easy to synthesize 10 means it is hard to synthesize [32]. All the derived compounds have scored less than demonstrates the synthesis of these compounds is not too hard.

6. Conclusion

All the newly synthesized derivatives are biologically active and some of them are more potent than the parent drug molecule. The derivative 8q has an IC₅₀ 35.49 ± 0.56 μM is cytotoxic against MCF7 cell lines. Similarly, three other derivatives i.e. 2q, 4q and 9q. These three derivatives also have more prominent IC₅₀ as well as molecular docking results. Those confirmed their inhibitory activity against CDK2 protein PDB id 1HCK, that has a major role in cell proliferation in tumor cells. The above-mentioned activities lead us to synthesize more potent anticancer drugs from quercetin. Quercetin itself is used as an antioxidant in food supplements. These derivatives may lead us to reevaluate the flavonoids for their drug delivery and efficacy with better sustainability. In future for enhancement of safety efficacy and target specificity, modified-release tablets, injectables, microencapsulation and nanoparticles may be synthesized.

7. Future perspective

We synthesized the biologically active compounds with better cytotoxic in vitro activity. In the future in vivo activities will be performed for further evaluation.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2379241

Author contributions

All authors contributed to the study’s conception and design. MR Khan wrote the initial text, reviewed it, carried out the tests, collected the results and handled all other aspects of the study. MA Khan overall supervise all the research process and discussed the concepts, study targets, methods and models. I Ahmad managed the activities. J Ahmed was in charge of maintaining research data as well as coordinating the design and implementation of research activities. H Ahmed verified and reproduced the results and provided study materials and reagents. I Mubeen used statistical tools to analyse the research data. B Awana helped compile the results. F Ullah contributed to programming and software development.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Data availability statement

Data can be provided on demand from corresponding author.