Design, Synthesis and Anticancer Evaluation of Substituted Cinnamic Acid Bearing 2-Quinolone Hybrid Derivatives

Abstract

A new series of hybrid molecules containing cinnamic acid and 2-quinolinone derivatives were designed and synthesized. Their structures were confirmed by ¹H-NMR, ¹³C-NMR and mass analyses. All the synthesized hybrid molecules were assessed for their in vitro antiproliferative activity against more than one cancer cell lines. Compound 3-(3,5-dibromo-7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-phenylacrylic acid (5a) with IC₅₀ = 1.89 μM against HCT-116 was proved to the most potent compound in this study, as compared to standard drug staurosporin. DNA flow cytometry assay of compound 5a revealed G2/M phase arrest and pre-G1 apoptosis. Annexin V-FITC showed that the percentage of early and late apoptosis was increased. The results of topoisomerase enzyme inhibition activity showed that the hybrid molecule 5a displays potent inhibitory activity compared with control.

1. Introduction

Molecular hybridization is a commonly molecular modification approach to obtain multiple compounds with therapeutic advantages over the two different drugs [1,2,3,4,5,6,7,8]. The novel hybrid molecules have the potential to enhance efficacy, improve safety, be cost-effective and reduce the propensity to elicit resistance relative to the parent drugs [9,10]. It is therefore understandable that the investigation of new hybrid anticancer drugs has recently become of great therapeutic interest.

2-quinolones (1-azacoumarins) play an important role in anticancer drug design since their derivatives have shown excellent anticancer activity through different mechanisms such as growth inhibition via apoptosis or inhibition of angiogenesis [11,12,13,14]. Additionally, the antineoplastic activity of these quinolones was attributed to intercalating binding to DNA [15]. Moreover, 6-substituted-1-azacoumarins are under human clinical experiments as an effective oral antitumor medicine [16]. Furthermore, subsequent, introduction of hydrophilic side chain to 2-quinolone scaffold led to the discovery of topotecan and irinotecan which currently used as anticancer drugs [17].

Cinnamic acid and its natural analogues are known for the treatment of cancer for over centuries [18,19]. The hydroxy cinnamic acids are natural products arisen from the deamination of the phenyl alanine. Natural hydroxy cinnamates are extremely potent antitumor agents [20,21,22]. Chemically, it is an aromatic fatty acid composed of a phenyl ring substituted with an acrylic acid group, commonly in the trans-geometry, and has low toxicity in human exposure. Cinnamic acid derivatives have been evaluated as pharmacologically active compounds [23]. They show a remarkable variety of biological activities and are often used as promising starting compounds for the development of new, highly effective drugs [24,25,26]. Cinnamic acid possesses α,β unsaturated carbonyl moiety, which can be considered as a Michael acceptor, an active moiety often employed in the design of anticancer drugs [27].

Based on the potent anticancer activity displayed by known quinolone derivatives, a new series of hybrid molecules were designed and synthesized. The hybrid structure contains cinnamic acids bearing 7,8-dihydroxy-4-methyl-1-amino-2-quinolinone as shown in Figure 1.

2. Results and Discussion

2.1. Chemistry

The synthetic pathway leading to the 7,8-dihydroxy-4-methyl-1-amino-quinoline-2-one (3) and 3-(2-oxoquinolin-1(2H)-ylamino)-3-arylacrylic acid derivatives 4 and 5 is outlined in Scheme 1.

7,8-dihydroxy-4-methylcoumarin (2) was obtained via the condensation of pyrogallol with ethyl acetoacetate in the presence of acid catalyst according to literature procedure [28]. Reaction of 7,8-dihydroxy-4-methyl-coumarin (2) with hydrazine hydrate in pyridine led to the formation of 1-amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (3). FT-IR spectrum of compound 3 showed the absence absorption band of carbonyl function for the coumarin ring, in addition to new absorption band at 1688 cm⁻¹ due to carbonyl function of the amide group, in addition two new bands at 3225, and 3178 cm⁻¹ related to NH₂ group. ¹H-NMR spectrum of compound 3 showed characteristic two singlet signals at δ 2.35 and 6.12 ppm due to the protons of methyl group (CH₃) and H-3 of quinolinone ring. Protons of the aromatic ring were appeared at δ 6.82 and 7.10 as doublet signal of H-5 and H-6 for the quinolinone ring. The ¹³C-NMR spectrum of compound 3 showed four carbon signals at δ 160.73, 154.44, 150.01 and 143.75 ppm assigned to carbonyl function (C=O), two carbons (C-O) and one carbon (C-N) groups.

Treatment of 1-amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (3) with substituted cinnamic acids in reflexing ethanol in the presence of fused sodium acetate provided the corresponding 3-(7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-arylacrylic acid 4a–c.

The structure of compounds 4a–c has been described by IR, ¹H-NMR and ¹³C-NMR. The IR spectrum of compound 4a as representative example, revealed the presence of two stretching vibration bands for NH and carbonyl of acid at 3232 and 1695 cm⁻¹, respectively. The ¹H-NMR spectrum of compound 4a showed additional signals in the region δ 6.52–7.69 ppm with the presence of aromatic, olefinic and NH protons. In addition, the ¹³C-NMR spectrum of compound 4a showed three signals at δ 168.31, 160.74 and 18.73 ppm assigned to the carbons of two carbonyl and methyl groups, respectively. In addition, the ¹³C-NMR spectrum of compound 4a displayed an additional carbon signals in the region of δ 134.77–110.62 ppm attributed to the carbons of quinoline, aromatic and olefinic carbons.

The halogenation of compounds 4a,b with bromine in glacial acetic with stirring at 60 °C led to the formation of 3-(3,5-dibromo-7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-arylacrylic acid 5a,b. In the ¹H-NMR spectrum of compound 5a Protons of the aromatic and H-6 of quinolinone ring were observed as multiplet signals within the expected chemical shift region at δ 7.35–7.69 ppm. The ¹³C-NMR spectrum of 5a showed four carbons appeared as four signals at δ 152.09, 147.53, 144.41 and 141.54 ppm due to two carbons of C-O and C-N groups. In addition, carbons of the aromatic and quinolinone rings were observed a characteristic carbon signals in the region δ 138.68–107.12 ppm.

2.2. Evaluation of Biological Activity

2.2.1. In Vitro Cytotoxic Activity against Three Cancer Cell Line

The effect of quinolone derivatives 3, 4a,b and 5a,b on the viability of three cancer cell lines were studied using MTT assay (Table 1). The cytotoxicity was assessed using Staurosporin (STU) as positive control. The three cancer cell lines are MCF-7 (breast cancer cell line), HepG2 (hepatocellular carcinoma cell line) and colon carcinoma (HCT-116). The obtained results indicated that compound 5b exhibited the most potent cytotoxic activity against MCF-7 cells with IC₅₀ value 8.48 μM. In addition, compound 5a exhibited potential cytotoxic activity against HepG2 and HCT-116 cells with IC₅₀ values 4.05 and 1.89 μM, respectively. From the obtained results it can be concluded that, the presence of bromine at C3 and C5 of quinolone ring has better anticancer activity against three cancer cell lines.

2.2.2. Cell Cycle Analysis

Targeting the cell cycle of tumor cells has been recognized as a promising strategy for cancer therapy [29,30]. In this study, cell cycle analysis was followed via DNA flow cytometric analysis using propidium iodide (PI) as staining agent following hybrid molecule 5a treatment at its IC₅₀ concentration for 48 h. The results showed that the tested compound increase the percentage of cell population at G2/M phase by 2.83-fold compared to untreated control group. While the number of cells in the G1 and S phases were reduced. Additionally, the result showed that compound 5a increased the percentage of cells at pre-G1 phase by 8.29-fold compared to the control (Figure 2).

2.2.3. Apoptosis Detection Assay

To characterize the mode of cell death caused by compound 5a, a biparametric cytofluorimetric analysis using PI and Annexin-V-FITC was performed in HCT-116 cells after treatment with compound 5a at its IC₅₀ concentration dose value for 48 h (Figure 3). The results revealed that compound 5a increases in the percentage of total apoptosis compared with untreated control group. The percentage of early apoptosis was increased from 0.71% to 6.65% compared with the untreated control group. Additionally, compound 5a can increase the percentage of late apoptosis from 0.52% to 7.87% compared with untreated control.

2.2.4. Topoisomerase II Inhibitory Activity

Compound 5a was evaluated for inhibitory activity on Topoisomerase II (topo II). Five dose concentrations were used and IC₅₀ concentration was determined. Podophyllotoxin (podo) was employed as the standard. It is observed that, compound 5a revealed strong inhibitory activity against topo II with IC₅₀ value of 75.82 ng/mL, compared to the reference compound podo (IC₅₀ = 31.24 ng/mL) (Figure 4).

2.3. Molecular Docking Study

Molecular docking is a vital tool in drug design [31,32,33,34]. Molecular docking achieves a confirmation for the protein and ligand interactions. In this work, molecular docking was conducted using the synthesized compounds (3, 4a,b, and 5a,b) against breast cancer protein (PDB: 3HB5). The docking results showed a potential structure-activity relationship between the target compounds (3, 4a,b, and 5a,b) against 3HB5 protein as shown in (Table 2, Figure 5 and Figure 6). Compounds 4a and 5a showed the highest binding interaction against the key amino acids of the 3HB5 with docking scores −9.8 and −9.56 kcal.mol⁻¹ respectively. The binding affinity and interaction bonds between docked compounds and active site of target protein 3HB5 were hydrogen bond, polar and hydrophobic interaction as shown in (Table 2). Compounds 5a exhibited a potential interaction toward (3HB5) receptor which is compatible with cytotoxicity and biological results.

3. Material and Methods

3.1. Chemistry

The melting point was measured on electro thermal 200 digital melting point device and was uncorrected. The ¹H and ¹³C-NMR spectra were measured with a Bruker Avance 400 MHz spectrometer (Chichago, Elk Grove Village, USA) using the DMSO-d₆ as solvent. The IR data were obtained with a shimadzu 470 spectrometer (Kyoto, Japan). The molecular weight of the synthesized compounds was determined by electron ionization (EI) mass spectrometer performed using a probe Agilent MSD 5975 spectrometer (Agilent Technologies, Inc. 5301 Stevens Creek Boulevard Santa Clara, CA, USA) operating at 70 eV. The elemental analysis was performed on a Perkin-Elmer 2400 series CHN elemental analyzer (Haan, Germany) chemical and reagents were purchased from either Aldrich or Sigma and all reagents were analytical grade.

3.1.1. Synthesis of 7,8-Dihydroxy-4-methyl Coumarin (2)

A mixture of pyrogallol (0.01 mol) and ethyl acetoacetate (0.01 mol) in the presence of concentrated sulfuric acid (2 mL) as acid catalyzed was heated under reflux on a water bath for 2 h. The reaction mixture was cooled and poured into water, the solid formed was filtered off, washed with water and dried. Finally, the product was crystallized from hot water.

7,8-Dihydroxy-4-methyl coumarin (2, known). IR (KBr) ν_max: 3458 (br. OH), 1725 (C=O), 1605, 1583 (C=C), 1170, 1065, 1035 (C-O) cm⁻¹. ¹H-NMR (DMSO-d₆) δ: 2.34 (s, 3H, CH₃), 6.21 (s, 1H, H-3 of coumarin ring), 6.91 (d, 1H, H-6 of coumarin ring), 7.21 (d, 1H, H-5 of coumarin ring), 9.68 (br. s, 1H, OH), 9.14 (br. s, 1H, OH) ppm. MS (m/z, %) = 192 (M⁺, 76.30). Anal. Calcd. for C₈H₈O₄ (192): C, 62.50; H, 4.17. Found: C, 62.33; H, 4.03.

3.1.2. Synthesis of 1-Amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (3)

A solution of 7,8-dihydroxy-4-methyl coumarin (0.01 mol) in pyridine (30 mL) was added hydrazine hydrate (0.02 mol), then heating under reflux for 6 h. The reaction mixture was cooled, poured into ice-water and neutralized with dilute hydrochloric acid (1N). The solid obtained was filtered off, washed with water, dried and purified by crystallization from absolute ethanol.

1-Amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (3) as yellow crystals, yield 71%, m.p. 265–267 °C. IR (K Br) ν_max: 3418 (br OH), 3225, 3178 (NH₂), 1648 (C=O), 1619, 1587 (C=C), 1061, 1006 (C-O) cm⁻¹. ¹H-NMR (DMSO-d₆) δ: 2.35 (s, 3H, CH₃), 4.12 (br.s, 2H, NH₂), 6.12 (s, 1H, H-3 of quinolinone), 6.82 (d, 1H, H-6 of quinolinone), 7.09 (d, 1H, H-5 of quinolinone) ppm. ¹³C-NMR (DMSO-d₆) δ: 160.73 (C-2), 154.44 (C-7), 150.01 (C-8), 143.75 (C-9), 132.69 (C-4), 115.92 (C-10), 113.17 (C-5), 112.60 (C-6), 110.59 (C-3), 18.73 (C-11) ppm. MS (m/z, %) = 206 (M⁺, 17.30). Anal. Calcd. for C₁₀H₁₀N₂O₂ (206): C, 58.25; H, 4.85; N, 13.59. Found: C, 58.09; H, 4.66; N, 13.39.

3.1.3. Synthesis of 3-(7,8-Dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-arylacrylic Acid (4a–c)

A suspension of a suitable cinnamic acid derivative (0.01 mol) and 1-amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (3, 0.01 mol) in ethanol (30 mL), in the presence of fused sodium acetate (0.03 mol) was refluxed for 4 h. The reaction mixture was cooled down, poured into water, and the solid formed was filtered. The product was crystallized from a suitable solvent to give compound 4a–c.

3-(7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-phenylacrylic acid (4a) Pale yellow crystals, yield 76%, m.p. 185–187 °C. Crystallized from ethanol/H₂O (3:1). IR (KBr) ν_max: 3550–3350 (br. OH), 3232 (NH), 1705–1675 (br. C=O), 1629, 1584 (C=C), 1229, 1061, 1006 (C-O) cm⁻¹. ¹H-NMR (DMSO-d₆) δ: 2.36 (s, 3H, CH₃), 6.13 (s, 1H, H-3 of quinolinone), 6.56 (s, 1H, H-olefinic of cinnamic acid), 6.38 (d, 1H, H-6 of quinoline ring), 7.10 (d, 1H, H-5 of quinolinone ring), 7.41–7.69 (m, 6H, Ar-H and NH) ppm. ¹³C-NMR (DMSO-d₆) δ: 168.31 (C-21), 160.74 (C-7),150.00 (C-8), 144.11 (C-9), 143.82 (C-13), 134.77 (C-17), 132.72 (C-4), 130.62 (C-14), 129.37 (C-15,9), 128.63 (C-16,18), 120.09 (C-20), 115.92 (C-10), 113.21 (C-5), 112.67 (C-6), 110.62 (C-3), 18.73 (C-H) ppm. MS (m/z, %) = 352 (M⁺, 13.51), Anal. Calcd. for C₁₉H₁₆N₃O₅ (352), C, 64.77; H, 4.54; N, 7.95. Found: C, 64.58; H, 4.33; N, 7.77.

3-(7,8-Dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-(4-hydroxyphenyl)acrylic acid (4b) Pale yellow crystals, yield 69%, m.p. 181–183 °C. Crystallized from ethanol/H₂O (3:1). IR (KBr) ν_max: 3560–3100 (br. OH), 3232 (NH), 1701–1660 (C=O), 1626, 1593 (C=C), 1170, 1062, 1007 (C-O) cm⁻¹. ¹H-NMR (DMSO-d₆) δ: 2.35 (s, 3H, CH₃), 6.12 (s, 1H, H-3 of quinolinone ring), 6.28–7.53 (m, 8H, Ar-H, olefinic and NH) ppm. ¹³C-NMR (DMSO-d₆) δ: 168.45 (C-21), 160.70 (C-2), 160.70 (C-17), 154.39 (C-7), 149.87 (C-8), 144.65 (C-9), 143.79 (C-13), 132.63 (C-4), 130.56 (C-15,19), 125.74 (C-14), 116.22 (C-16,18), 115.95 (C-10), 115.81 (C-20), 113.24 (C-5), 112.60 (C-6), 110.66 (C-3), 18.71 (C-11) ppm. MS (m/z, %) = 368 (M⁺, 11.51). Anal. Calcd. for C₁₉H₁₆N₂O₆ (368): C, 61.96; H, 3.35; N, 7.61. Found: C, 61.73; H, 3.11; N, 7.51.

3-(7,8-Dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid (4c). Pale yellow crystals, yield 67%, m.p. 175–177 °C. Crystallized from DMF/H₂O (1:1). IR (KBr) ν_max: 3550–2984 (br. OH), 3232 (NH), 1700–1652 (C=O), 1620, 1595 (C=C), 1172, 1141, 1062 (C-O) cm⁻¹. ¹H-NMR (DMSO-d₆) δ: 2.35 (s, 3H, CH₃), 3.82 (s, 3H, OCH₃), 6.12 (s, 1H, H-3 of quinolinone ring), 6.36–7.52 (m, 7H, Ar-H, H-olefinic and NH), 9.35 (br.s, 1H, OH), 10.02 (br.s, 2H, 2xOH) ppm. ¹³C-NMR (DMSO-d₆) δ: 168.51 (C-21), 160.70(C-2), 154.41 (C-7), 149.87 (C-8), 149.53 (C-17), 148.37 (C-18), 144.99 (C-9), 143.78 (C-13), 132.26 (C-4), 126.23 (C-15), 123.21 (C-19), 116.10 (C-10), 115.95 (C-14,16), 113.23 (C-5), 112.58 (C-6), 111.56 (C-20), 110.65 (C-3), 56.13 (C-22), 18.72 (C-11) ppm. MS (m/z, %) = 398 (M⁺, 8.60). Anal. Calcd. for C₂₀H₁₈N₂O₇ (398): C, 60.30; H, 4.52; N, 7.03. Found: C, 60.11; H, 4.29; N, 6.89.

3.1.4. Synthesis of 3-(3,5-Dibromo-7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-arylacrylic Acid (5a,b)

In 15 mL of glacial acetic acid, compound 4 (0.01 mol) was dissolved. Then, 10 mL of bromine (0.02 mol) in glacial acetic acid was added dropwise to compound 4 solution with stirring at 60 °C. After 5–10 min. the bromine color was discharged and a yellow solution remained. At this point, an additional 0.5–1 mL of the bromine-AcOH solution was added with stirring at room temperature for 30–45 min. The reaction mixture was poured into water and the resulting product was filtered off, washed with water and dried. Finally, The product was crystallized from DMF/H₂O (1:1) to give 5a,b.

3-(3,5-Dibromo-7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-phenylacrylic acid (5a) Yellow crystals, yield 71%, m.p. 225–227 °C. IR(KBr) ν_max: 3452 (br. OH), 3254 (NH),1710–1705 (C=O), 1627, 1604, 1558 (C=C), 1223, 1072, 1026 (C-O) cm⁻¹. ¹H-NMR (DMSO-d₆) δ: 2.55 (s, 3H, CH₃), 5.32 (d, 1H, H-olefinic) 5.54 (d, 1H, H-olefinic of two isomer), 6.54 (d, 1H, NH), 7.38–7.69 (m, 6H, Ar-H for two isomer) 10.35 (br.s, 1H, OH), 10.53 (br.s, 1H, OH) ppm. ¹³C-NMR (DMSO-d₆) δ: 169.56, 168.05 (C=O of acid for two isomer), 156.46 (C-2), 152.09, 147.5, (C-O), 144.41, 141.54 (C-N), 138.68, 134.67, 133.30,130.68, 129.54, 129.35, 129.15, 128.65, 128.65, 119.66, 119.38, 113.62, 109.24, 107.12 (C-aromatic acid Quinolinone ring), 52.38, 47.69 (C-olefinic), 20.01 (CH₃) ppm. MS (m/z, %) = 508(M⁺, unstable). Anal. Calcd. for C₁₉H₁₄N₂Br₂O₅ (508): C, 44,88, H, 2.75; N, 5.51. Found: C, 44.64; H, 2.52; N, 5.33.

3-(3,5-dibromo-7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-(4-hydroxyphenyl)acrylic acid (5b). Pale yellow crystals, yield 69%, m.p. 233–235 °C. IR (KBr) ν_max: 3580–2955 (br. OH and NH), 1717–1705 (br. C=O), 1607, 1558 (C=C), 1239, 1160, 1101, 1028 (C-O) cm⁻¹. ¹H-NMR (DMSO-d₆) δ: 2.53 (s, 3H, CH₃), 7.01–8.16 (m, 6H, Ar-H and H-olefinic) ppm. ¹³C-NMR (DMSO-d₆) δ: 166.21, 156.46 (C=O), 152.08, 151.84, 151.04 (C-O), 147.52, 141.53 (C-N), 134.49, 134.31, 133.31, 132.87, 132.46, 130.87, 130.77, 130.23, 119.36, 116.83, 113.61, 112.63, 112.34, 111.94, 109.24, 108.50, 107.78, 106.22 (C-Quinolinone, aromatic and olefinic carbons), 20.16, 20.01 (CH₃,two isomer). MS: (m/z, %) = 524(M⁺, unstable). Anal. Calcd. for C₁₉H₁₄N₂Br₂O₆ (524): C, 43.51; H, 2.67; N, 5.34. Found: C, 43.34; H, 2.42; N, 5.51. ¹H and ¹³C NMR of compound 3, 4a–c and 5a–b are available in Supplementary Materials.

3.2. Biological Evaluation

3.2.1. Anti-Tumor Activity against Three Cancer Cell Lines

The cytotoxic activity was measured in vitro using the MTT assay. Cells were plated in 96-multiwall plate (10⁵ cells/well) for 24 h before treatment with the compounds. Test compounds were dissolved in dimethyl sulfoxide (DMSO) which did not exceed 1% final concentration. Different concentrations of the compound under test (0.39, 1.56, 6.25, 25, and 100 μg/mL) were added to the cell’s monolayer. Triplicate wells were prepared for each individual concentration. Monolayer cells were incubated with the compound(s) for 48 h at 37 °C and in atmosphere of 5% CO₂. After 48 h, cells were fixed, washed with phosphate buffer saline (PBS, pH = 7.4) and stained with 40 μL of MTT solution (5 mg/mL of MTT in 0.9% NaCl) in each well was added and incubated for an additional 4 h. MTT crystals were solubilized by adding 180 μL of acidified isopropanol/well and the plate was shaken at room temperature, followed by photometric determination of the absorbance at 570 nm using ELISA reader. The molar concentration required to inhibit 50% of cell viability (IC₅₀) was calculated and compared with the reference drug STU. The surviving fractions were expressed as means ± SD.

3.2.2. Cell Cycle Analysis of Compound 5a

HCT-116 cells, (3.0 × 10⁵ cells/well) and incubated at 37 °C for 12 h. The target cells were then treated with the compound 5a at its IC₅₀ concentration dose value for 48 h. After treatment, cells were collected and fixed with 75% ethanol at 20 °C overnight, then, cells were washed with PBS followed by centrifugation and incubated with (10 mg/mL) Rnase (Sigma, St. Louis, MO, USA) and (5 mg/mL) propidium iodide (PI, Sigma) before flow cytometry analysis (FACSCalibur cytometer using Cellquest software, BD Bioscience, San Jose, CA, USA).

3.2.3. Apoptosis Determination by Annexin-V Assay

The HCT-116 cells (2 × 10⁵/well) were treated with compound 5a at its IC₅₀ concentration value for 48 h. After treatment, cells were harvested and washed twice (180 g, 10 min, 4 °C) with PBS. Each cell well was resuspended in 100 μL of binding buffer, and 5 µL Annexin V-FITC were added. After an incubation time of 10 min at room temperature, additional 400 µL of binding buffer were added for a final volume of 500 µL. Cells were stained with PI immediately before measurement. Cells were the analyzed by using FACS Calibur Flow cytometer (Becton and Dickinson, Heidelberg, Germany). Data thus obtained were analyzed with Cell-Quest software (Becton and Dickinson, Heidelberg, Germany).

3.2.4. In Vitro Topoismerase Inhibitory Assay

Compound 5a was selected to be evaluated against topoisomerase II [MBS#942146] using human DNA topoisomerase 2-β (TOP2β) ELISA kit according to manufacturer’s instructions. Prepare all reagents, working standards, and samples. Add 100 μL of standard and sample per well and incubate for 2 h at 37 °C. Remove the liquid of each well, do not wash. Add 100 μL of biotin-antibody to each well and incubate for 1 h at 37 °C. Aspirate each well and wash three times. Add 100 μL of horseradish peroxidase (HRP-avidin) to each well and incubate for 1 h at 37 °C. Repeat the aspiration/wash process for five times. Add 90 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to each well and incubate for 15–30 min at 37 °C, protect from light. Add 50 μL of stop solution to each well and determine the optical density of each well within 5 min, using a microplate reader set to 450 nm. The values of % activity versus a series of compound concentrations (2.5, 5, 10, and 15 µM) were then plotted using non-linear regression analysis of sigmoidal dose-response curve. The IC₅₀ values for compound 5a against topoisomerase II was determined by the concentration causing a half-maximal percent activity and the data were compared with Dox as standard.

3.3. Molecular Docking Study

Docking calculations were estimated using a Docking Server [35]. Docking scores were examined on the 3HB5 protein model. Crucial hydrogen atoms, Kollman united atom type charges, and solvation parameters were inserted with the support of AutoDock tools. Affinity (grid) maps of 61 × 61 × 61 points and 0.375 Å spacing were created using the Autogrid program [36]. AutoDock parameter set and distance-dependent dielectric purposes were utilized in the calculation of the van der Waals and the electrostatic terms, correspondingly. Docking simulations were done via the Lamarckian genetic algorithm (LGA) and the Solis and Wets local search approach [37]. The initial position, orientation, and torsions of the ligand molecules were fixed arbitrarily. Wholly rotatable torsions were discharged throughout docking. Each docking experimentation was originated from 2 different runs that were established to end afterward a maximum of 250,000 energy evaluations. The population size was set to 150. Throughout the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 were applied.

4. Conclusions

In conclusion, hybrid molecules containing cinnamic acid and 2-quinolinone derivatives (4a–c) were synthesized via the reaction of cinnamic acid derivatives with 1-amino-7,8-dihydroxy-4-methylquinolin-2(1H)-one (3), which was obtainable in the reaction of 7,8-hydroxy-4-methylcoumarin (2) with hydrazine hydrate in pyridine. Brominated derivatives 5a,b were prepared via the halogenation of compounds 4a,b with bromine in glacial acetic acid. Cinnamic acids bearing 2-quinolone 4a,b and their brominated derivatives 5a,b were assessed for their in vitro antiproliferative activity against three cancer cell lines and the brominated derivatives were found to be more active. DNA flow cytometry assay of compound 5a revealed G2/M phase arrest and pre-G1 apoptosis. Annexin V-FITC showed the percentage of early and late apoptosis was increased. The results of topoisomerase enzyme inhibition activity showed that the hybrid molecule 5a display potent inhibitory activity compared with control. In conclusion, the preliminary study of the anticancer activity of the prepared compounds represents a novel strategy for the discovery of anticancer compounds which needs further investigation.

Supplementary Materials

The following are available online: ¹H and ¹³C NMR of compound 3, 4a–c and 5a,b.

Author Contributions

All authors participated in the production of the article. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Taif University Researchers Supporting project number (TURSP-2020/220), Taif University, Taif, Saudi Arabia.

Institutional Review Board Statement

Not applicable. Studies not involving humans or animals. Biological and cytotoxicity experiments were conducted on in vitro cancer cell lines.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

Taif University Researchers Supporting Project number (TURSP-2020/220), Taif University, Taif, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of compounds are not available from authors.

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Figures, Scheme and Tables

Figure 1. Molecular strategy related to cinnamic acid-2-quinolone hybrid III obtained from hybridization of cinnamic acid (I) and 1-amino-4-methyl-2-quinolinone (II).

Scheme 1. Synthesis of 3-(2-oxoquinolin-1(2H)-ylamino)-3-arylacrylic acid (4 and 5). Reagents and reaction condition: (i) CH3COCH2COOEt, conc H2SO4, (ii) N2H4, pyridine; (iii) Ar-CH=CH-COOH, NaOAc, (iv) Br2, AcOH.

Figure 2. (A) Graphical representation of cell cycle distribution of HCT-116 cell line induced by compound 5a. (B) Cell cycle distribution of HCT-116 cell line induced by compound 5a.

Figure 3. (A) Graphical representation of apoptosis effect of compound 5a on HCT-116 cell line. (B) Apoptosis effect of compound 5a on HCT-116 cell line.

Figure 4. Graphical presentation for comparison of topo II IC50 (ng/mL) of the compound 5a and podo.

Figure 5. Interaction of docked compounds 3, 4a and 4b inside the binding site of (3HB5): (A) 2D interaction; (B) 3D interaction.

Figure 6. Interaction of docked compounds 5a and 5b inside the binding site of (3HB5). (A) 2D interaction; (B) 3D interaction.

Table 1

In vitro antitumor activity of quinolone derivatives 3, 4a,b and 5a,b over MCF-7, HepG2 and HCT-116, cell lines. Data are expressed as the mean ± SD.

Comp No.	IC50 (μM)
	MCF-7	HepG2	HCT-116
3	32.9 ± 1.81	8.23 ± 0.42	15.0 ± 0.79
4a	18.7 ± 1.03	19.4 ± 0.99	21.3 ± 1.12
4b	11.8 ± 0.65	15.5 ± 0.79	7.56 ± 0.4
5a	19.3 ± 1.06	4.05 ± 0.21	1.89 ± 0.10
5b	8.48 ± 0.47	30.9 ± 1.57	10.7 ± 0.57
STU	6.91 ± 0.38	5.23 ± 0.27	4.25 ± 0.22

Table 2

Docking scores of synthesized compounds (3, 4a,b, and 5a,b) against 3HB5 (PDB: 3HB5).

Comp No.	Docking Scores (kcal.mol−1)	Hydrogen Bonds	Polar Interaction	Hydrophobic Interactions
3	−6.42	ASN90	LYS159SER12	PHE192ILE14
4a	−9.81	LYS195SER142		PHE192ILE14PHE226
4b	−7.56	PHE192	SER12ASN90LYS195THR190	ILE14
5a	−9.56	THR190	GLY141VAL188SER142TYR155	PHE192ILE14
5b	−6.06	GLY92SER12ASN90SER11	ARG37THR190	ALA191