Synthesis, antitumour and antioxidant activities of novel α,β-unsaturated ketones and related heterocyclic analogues: EGFR inhibition and molecular modelling study.

New α,β-unsaturated ketones 4a,b; 5a-c; and 6a,b; as well as 4-H pyran 7; pyrazoline 8a,b; isoxazoline 9; pyridine 10-11; and quinoline-4-carboxylic acid 12a,b derivatives were synthesized and evaluated for in vitro antitumour activity against HepG2, MCF-7, HeLa, and PC-3 cancer cell lines. Antioxidant activity was investigated by the ability of these compounds to scavenge the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+). Compounds 6a, 6b, 7, and 8b exhibited potent antitumour activities against all tested cell lines with [IC50] ≅5.5-18.1 µΜ), in addition to significantly high ABTS•+ scavenging activities. In vitro EGFR kinase assay for 6a, 6b, 7, and 8b as the most potent antitumour compounds showed that; compounds 6b, and 7 exhibited worthy EGFR inhibition activity with IC50 values of 0.56 and 1.6 µM, respectively, while compounds 6a and 8b showed good inhibition activity with IC50 values of 4.66 and 2.16 µM, respectively, compared with sorafenib reference drug (IC50 = 1.28 µM). Molecular modelling studies for compounds 6b, 7, and 8b were conducted to exhibit the binding mode towards EGFR kinase, which showed similar interaction with erlotinib.

Introduction

Cancer is a group of diseases involving abnormal cell growth with the potential to spread into or invade nearby tissues. Although chemotherapy is the mainstay of cancer therapy, it produces substantial side effects that may be attributed to cytotoxic effects on normal cells. This clearly underlies the urgent need for developing novel chemotherapeutic agents that will be more selective for cancer cells, and thus produce fewer side effects. On the other hand, free radicals and the reactive oxygen species are constantly generated through many biological processes in the body. The capability of antioxidants to reduce the risk of certain cancer types is linked to their ability to scavenge free radicals, reduce oxidative stress, and decrease abnormal cell division. Administration of a single molecule acting through a different mechanism is a better drug candidate than drug combinations. Hence, several studies have investigated both anticancer and antioxidant activities of numerous newly synthesized molecules.

Furthermore, a high level of EGFR kinase enzyme is overexpressed in several tumours such as those in colon, prostate, breast, HeLa, HepG2, and non-small lung cancers. The inhibition of EGFR kinase enzyme is used in cancer treatment, and is effected by blocking this enzyme with small molecules such as erlotinib (A),, neratinib (B), sorafenib (C), and crizotinib (D) (Figure 1). Additionally, the α,β-unsaturated ketones, such as curcumin (E; Figure 1), are a major class of widespread natural products and constitute the core structure of many drugs covering a wide range of biological applications, including EGFR inhibition as well as antioxidant and antitumour activities,,. Moreover, heterocycles such as pyrazoline (F; Figure 1),, isoxazoline, pyran, pyridine, and quinoline derivatives possess potent antioxidant and antitumour activities as well as some of these compounds possessed EGFR inhibition activities,,.

Figure 1.

The reported antitumour (A–F) and the designed (G) compounds.

Taking all the aforementioned facts into account in our continuous efforts to develop new structures to serve as antitumour and antioxidant agents, we synthesized new α,β-unsaturated ketones, 4-H pyran, pyrazoline, isoxazoline, pyridine, and quinoline derivatives (G; Figure 1). The rationale for evaluating the antitumour, antioxidant, and EGFR kinase inhibition activities of the designed molecules (G; Figure 1) was as follows: (i) design the structure–activity relationship for compounds incorporating α,β-unsaturated ketones with diverse substituent groups; (ii) recognise the effectiveness of the cyclic α,β-unsaturated ketones versus the acyclic derivatives; (iii) thus, compare the cycloalkanones and their piperidinone analogues; (iv) heterocyclic compounds resulting from the addition reaction of α,β-unsaturated ketones such as pyrane, pyrazoline, oxazoline, and pyridine derivatives were also included in the study in order to cover the most relative analogues.

Furthermore, the most active antitumour compounds were subjected to EGFR kinase inhibition test and docked into the binding sites of EGFR kinase enzyme to explore their complementarity with the specified binding pockets.

Materials and methods

Chemistry

Melting points (°C, uncorrected) were measured using a Fisher-Johns apparatus. Elemental analyses were carried out at the microanalytical unit, Cairo University. IR spectra (potassium bromide [KBr]) were acquired using a Mattson 5000 FT-IR spectrometer (ν in cm−1). 1H NMR and 13C NMR spectra were obtained in deuterated dimethyl sulphoxide (DMSO-d6) or deuterated chloroform (CDCl3) on Bruker 400 and 100 MHz instruments, respectively, using tetramethyl silane (TMS) as an internal standard. Chemical shifts were reported downfield from TMS in ppm, δ units. Mass spectrometry (MS) measurements were performed on a JEOL JMS-600H spectrometer. The purities of the compounds were evaluated by thin layer chromatography (TLC), which was performed on silica gel G (Merck), and spots were visualised by irradiation with ultraviolet light (UV; 254 nm). Compound 3, 4-(cyclopentyloxy)benzaldehyde, was synthesized in accordance with the method described in the literature.

General method for the synthesis of α,β-unsaturated ketone derivatives (4a,b; 5a-c; and 6a,b)

A solution of 4-(cyclopentyloxy)benzaldehyde 3 (1.9 g, 0.01 mol) in ethanol (20 ml) was added to a stirred solution of the appropriate ketone (0.03 mol) in ethanol (20 ml) containing NaOH (0.8 g, 0.02 mol). The reaction mixture was refluxed for 8 h, cooled and the solvent was evaporated under reduced pressure. The resulting solid was triturated with diethyl ether, filtered, dried, and crystallised from the appropriate solvent.

4-(4-(Cyclopentyloxy)phenyl)but-3-en-2-one (4a)

Crystallisation solvent, ethanol; Yield, 40%; melting point (mp): 219–220 °C; IR (KBr) νmax/cm−1 1610 (C = O), 1530, 1525, 1510, 1470 (C = C). 1H NMR (DMSO-d6); δ: 7.95 (d, 2H, Ar-H, J = 8 Hz), 7.52 (d, 2H, Ar-H, J = 8 Hz), 7.28 (d, 1H, CH = CH, J = 8.4 Hz), 6.53 (d, 1H, CH = CH, J = 8.4 Hz), 4.80–4.70 (m, 1H, CH), 2.26 (s, 3H, CH3), 1.95–1.85 (m, 2H, CH2), 1.75–1.66 (m, 4H, 2CH2), 1.60–1.52 (m, 2H, CH2). MS m/z (%); 232.00 (6.39, M++2), 231.00 (20.31, M++1), 230.10 (21.46, M+), 224.00 (52.84), 147.00 (49.56), 142.10 (100.00), 121.00 (22.93), 100.00 (16.25). Anal. Calcd. for C15H18O2 (%): C, 78.23; H, 7.88. Found: C, 78.63; H, 8.18.

3-(4-(Cyclopentyloxy)phenyl)-1-(4-methylphenyl)prop-2-en-1-one (4b)

Crystallisation solvent, water; Yield, 85%; mp: 220–222 °C; IR (KBr) νmax/cm−1 1615 (C = O), 1540, 1535, 1525, 1480 (C = C). 1H NMR (CDCl3); δ: 7.85 (d, 4H, Ar-H, J = 8 Hz), 7.32 (d, 4H, Ar-H, J = 8 Hz), 7.18 (d, 1H, CH = CH, J = 8.4 Hz), 6.73 (d, 1H, CH = CH, J = 8.4 Hz), 4.80–4.72 (m, 1H, CH), 2.36 (s, 3H, CH3), 1.85–1.80 (m, 2H, CH2), 1.70–1.62 (m, 4H, 2CH2), 1.60–1.50 (m, 2H, CH2).13C NMR (DMSO-d6); δ: 198.2, 155.8, 143.3, 135.6, 134.3, 129.1, 128.4, 128.0, 114.8, 78.3, 32.2, 23.5, 21.0. MS m/z (%); 306.13 (17.66, M+), 238.10 (100.00), 237.08 (87.48), 210.08 (14.15), 209.07 (14.02), 195.06 (16.87), 144.03 (42.22). Anal. Calcd. for C21H22O2 (%): C, 82.32; H, 7.24. Found: C, 82.0 2; H, 7.05.

2-(4-(Cyclopentyloxy)benzylidene)cyclopentanone (5a)

Crystallisation solvent, ethanol; Yield, 55%; mp: 282–283 °C; IR (KBr) νmax/cm−1 1600 (C = O), 1535, 1520, 1510, 1500 (C = C). 1H NMR (DMSO-d6); δ: 7.55 (d, 2H, Ar-H, J = 8 Hz), 7.11 (brs, 1H, CH=), 6.90 (d, 2H, Ar-H, J = 8 Hz), 4.89–4.80 (m, 1H, CH), 3.20–3.10 (m, 2H, CH2), 2.11–1.88 (m, 4H, 2CH2), 1.72–1.61 (m, 4H, 2CH2), 1.60–1.45 (m, 4H, 2CH2). MS m/z (%); 257.08 (0.98, M++1), 256.09 (1.28, M+), 146.05 (54.79), 145.04 (35.70), 131.03 (100.00), 117.06 (25.47), 115.03 (38.21), 107.02 (73.33). Anal. Calcd. for C17H20O2 (%): C, 79.65; H, 7.86. Found: C, 80.05; H, 8.06.

2-(4-(Cyclopentyloxy)benzylidene)cyclohexanone (5b)

Crystallisation solvent, water; Yield, 50%; mp: 291–292 °C; IR (KBr) νmax/cm−1 1620 (C = O), 1550, 1642, 1530, 1470 (C = C). 1H NMR (DMSO-d6); δ: 7.25 (d, 2H, Ar-H, J = 8 Hz), 7.00 (brs, 1H, CH=), 6.85 (d, 2H, Ar-H, J = 8 Hz), 4.90–4.82 (m, 1H, CH), 2.80 (t, 2H, CH2, J = 4.5 Hz), 2.40 (t, 2H, CH2, J = 4.5 Hz), 2.00–1.90 (m, 2H, CH2), 1.85–1.60 (m, 6H, 3CH2), 1.50–1.30 (m, 2H, CH2), 1.20–1.00 (m, 2H, CH2). MS m/z (%); 272.15 (2.78, M++2), 271.13 (15.64, M++1), 270.11 (52.26, M+), 203.08 (26.47), 202.07 (74.25), 201.07 (25.10), 145.05 (21.80), 107.02 (100.00). Anal. Calcd. for C18H22O2 (%): C, 79.96; H, 8.20. Found: C, 80.01; H, 8.50.

2-(4-(Cyclopentyloxy)benzylidene)cycloheptanone (5c)

Crystallisation solvent, water; Yield, 60%; mp: 284–285 °C; IR (KBr) νmax/cm−1 1625 (C = O), 1555, 1540, 1534, 1490 (C = C). 1H NMR (DMSO-d6); δ: 7.21 (d, 2H, Ar-H, J = 8.2 Hz), 7.02 (brs, 1H, CH=), 6.80 (d, 2H, Ar-H, J = 8.2 Hz), 4.60–4.50 (m, 1H, CH), 3.20–3.10 (m, 2H, CH2), 2.11–1.75 (m, 10H, 5CH2), 1.60–1.47 (m, 6H, 3CH2). MS m/z (%); 286.30 (0.2, M++2), 285.20 (0.8, M++1), 284.10 (35.00, M+), 216.10 (58.00), 215.10 (100.00), 121.10 (34.00), 120.10 (46.08), 41.10 (37.07). Anal. Calcd. for C19H24O2 (%): C, 80.24; H, 8.51. Found: C, 80.70; H, 8.91.

3-(4-(Cyclopentyloxy)benzylidene)-1-methylpiperidin-4-one (6a)

Crystallisation solvent, water; Yield, 65%; mp: 248–250 °C. IR (KBr) νmax/cm−1 1600 (C = O), 1540, 1525, 1535, 1490 (C = C). 1H NMR (, DMSO-d6); δ: 7.00 (d, 2H, Ar-H, J = 8 Hz), 6.80–6.71 (m, 3H, Ar-H, CH=), 4.60–4.50 (m, 1H, CH), 3.15 (s, 2H, CH2), 2.85 (t, 2H, CH2,J = 4.5 Hz), 2.60 (t, 2H, CH2,J = 4.5 Hz), 2.30 (s, 3H, N–CH3), 2.10–1.90 (m, 2H, CH2), 1.80–1.71 (m, 4H, 2CH2), 1.55–1.45 (m, 2H, CH2). MS m/z (%); 287.16 (6.34, M++2), 286.15 (21.93, M++1), 285.15 (8.16, M+), 166.05 (17.92), 161.06 (4.83), 112.08 (34.02), 111.09 (42.67), 110.02 (100.00). Anal. Calcd. for C18H23NO2 (%): C, 75.76; H, 8.12; N, 4.91. Found: C, 75.86; H, 8.22; N, 5.01.

3-(4-(Cyclopentyloxy)benzylidene)-1-ethylpiperidin-4-one (6b)

Crystallisation solvent, water; Yield, 64%; mp: 240–241 °C. IR (KBr) νmax/cm−1 1635 (C = O), 1560, 1550, 1530, 1485 (C = C). 1H NMR (DMSO-d6); δ: 7.20 (d, 2H, Ar-H, J = 8 Hz), 6.90–6.80 (m, 3H, Ar-H, CH=), 4.90–4.80 (m, 1H, CH), 3.70 (q, 2H, CHCH3,J = 7 Hz), 3.20 (s, 2H, CH2), 2.85 (t, 2H, CH2, J = 4.5 Hz), 2.65 (m, 2H, CH2), 2.00–1.90 (m, 2H, CH2), 1.75–1.60 (m, 4H, 2CH2), 1.55–1.45 (m, 2H, CH2), 1.02 (t, 3H, CH2CHJ = 7 Hz). MS m/z (%); 301.30 (18.00, M++2), 300.20 (38.50, M++1), 299.20 (26.00, M+), 137.90 (36.09), 132.90 (42.35), 123.90 (100.00), 72.10 (58.50), 58.00 (51.77). Anal. Calcd. for C19H25NO2 (%): C, 76.22; H, 8.42; N, 4.68. Found: C, 76.62; H, 8.72; N, 5.00.

Synthesis of ethyl 6-amino-5-cyano-4-(4-(cyclopentyloxy)phenyl)-2-methyl-4H-pyran-3-carboxylate (7)

A mixture of 4-(cyclopentyloxy)benzaldehyde 3 (0.57 g, 0.003 mol), ethylacetoacetate (0.39 g, 0.003 mol), malononitrile (0.20 g, 0.003 mol), and sodium benzoate (15 mol%) in ethanol (20 ml) was stirred at room temperature for 24 h. The reaction mixture was filtered, and the solid product was washed with water and then with ethanol, dried and crystallised from dimethylformamide. Yield, 45%; mp >300 °C; IR (KBr) νmax/cm−1 3401 and 3326 (NH2), 2221 (C≡N), 1697 (C = O). 1H NMR (DMSO-d6); δ: 8.30 (brs, 2H, NH2, D2O exchangeable), 7.30 (d, 2H, Ar-H, J = 8 Hz), 7.90 (d, 2H, Ar-H, J = 8 Hz), 5.70 (s,1H, 4-H of pyran), 5.10–5.00 (m, 1H, CH), 4.10 (q, 2H, CH3CH2O, J = 7.5 Hz), 2.50 (s, 3H, CH3), 2.00–1.90 (m, 2H, CH2), 1.80–1.60 (m, 6H, 3CH2), 1.20 (t, 3H, CH3CH2O, J = 7.5 Hz). MS m/z (%); 368.25 (0.81, M+), 348.07 (18.46), 321.06 (28.72), 276.07(91.20), 275.06 (68.87), 274.06 (28.04), 248.05 (17.64), 107.06 (100.00). Anal. Calcd. for C21H24N2O4 (%): C, 68.46; H, 6.57; N, 7.60. Found: C, 68.66; H, 7.00; N, 8.00.

Synthesis of compounds 8a and 8b

A mixture of compound 4b (0.91 g, 0.003 mol) and hydrazine hydrate (0.15 g, 0.003 mol) in absolute ethanol (30 ml) or phenyl hydrazine (0.32 g, 0.003 mol) in glacial acetic acid (5 ml) was heated under reflux for 9–10 h. After cooling, the separated products were filtered, dried, and crystallised from ethanol to yield the title compounds.

5-(4-(Cyclopentyloxy)phenyl)-3-(4-methylphenyl)-4,5-dihydro-1H-pyrazole (8a)

Yield, 55%; mp: 145–146 °C. IR (KBr) νmax/cm−1 3450 (NH), 1547 (C = N). 1H NMR (DMSO-d6); δ: 7.91–7.10 (m, 8H, Ar-H), 6.75 (dd, J = 11.7, 4.5 Hz, 1H, 5-H of pyrazoline), 4.92–4.82 (m, 1H, CH), 3.75 (dd, J = 11.7, 18.0 Hz, 1H, 4-H of pyrazoline), 3.50 (dd, J = 4.5, 18.0 Hz, 1H, 4-H of pyrazoline), 2.38 (s, 3H, CH3), 2.10–1.93 (m, 2H, CH2), 1.90–1.49 (m, 6H, 3CH2), 9.20 (brs, 1H, NH, D2O exchangeable). MS m/z (%); 321.30 (2.00, M++1), 320.20 (6.50, M+), 318.90 (22.02), 261.00 (20.50), 145.10 (16.02), 143.90 (100.00), 120.10 (32.00), 90.90 (31.00). Anal. Calcd. for C21H24N2O (%): C, 78.71; H, 7.55; N, 8.74. Found: C, 79.01; H, 7.88; N, 8.95.

5-(4-(Cyclopentyloxy)phenyl)-1-phenyl-3-(4-methylphenyl)-4,5-dihydro-1H-pyrazole (8b)

Yield, 60%; mp: 139–141 °C. IR (KBr) νmax/cm−1 1547, 1560, 1550, 1545 (C = N, C = C). 1H NMR (DMSO-d6); δ: 7.00–7.90 (m, 13H, Ar-H), 6.85–6.75 (m, 1H, 5-H of pyrazoline), 4.80–4.70 (m, 1H, CH), 3.90–3.80 (m, 1H, 4-H of pyrazoline), 3.50–3.30 (m, 1H, 4-H of pyrazoline), 2.37 (s, 3H, CH3), 2.00–1.95 (m, 2H, CH2), 1.90–1.50 (m, 6H, 3CH2). MS m/z (%); 397.00 (10.81, M++1), 396.40 (13.81, M+), 281.05 (36.86), 233.06 (26.59), 220.05 (100.00), 180.04 (36.11), 151.10 (25.03), 117.00 (53.87). Anal. Calcd. for C27H28N2O (%): C, C, 81.78; H, 7.12; N, 7.06. Found: C, 82.08; H, 7.22; N, 7.16.

Synthesis of 5-(4-(cyclopentyloxy)phenyl)-3-(4-methylphenyl)-4,5-dihydroisoxazole (9)

A mixture of compound 4b (0.91 g, 0.003 mol), hydroxylamine hydrochloride (0.2 g, 0.003 mol), and potassium hydroxide (0.2 g, 0.003 mol) in ethanol (20 ml) was refluxed for 12 h. The solvent was evaporated under reduced pressure and the residue obtained was triturated with water, filtered, and dried to yield compound 9, which crystallised from ethanol. Yield, 56%; mp: 120–122 °C. IR (KBr) νmax/cm−1 1540, 1565, 1555, 1545 (C = N, C = C). 1H NMR (DMSO-d6); δ: 7.90–7.30 (m, 8H, Ar-H), 6.80–6.70 (m,1H, 5-H of isoxazole), 4.80–4.70 (m, 1H, CH), 3.90–3.80 (m, 2H, 4-H of isoxazoline), 2.36 (s, 3H, CH3), 2.00–1.85 (m, 2H, CH2), 1.80–1.50 (m, 6H, 3CH2). MS m/z (%); 321.09 (0.81, M+), 292.05 (21.82), 291.05 (46.86), 236.06 (36.59), 222.05 (100.00), 179.04 (26.11), 161.10 (25.93), 118.00 (63.87). Anal. Calcd. for C21H23NO2 (%): C, 78.47; H, 7.21; N, 4.36. Found: C, 78.90; H, 7.60; N, 4.96.

Synthesis of 4-(4-(cyclopentyloxy)phenyl)-2-oxo-6-(4-methylphenyl)-1,2-dihydropyridine-3-carbonitrile (10)

A mixture of compound 4b (1.53 g, 0.005 mol), ethylcyanoacetate (0.56 g, 0.005 mol), and ammonium acetate (3.1 g, 0.04 mol) in absolute ethanol (50 ml) was refluxed for 8 h. After cooling, the product was collected by filtration, washed with ethanol, dried, and crystallised from ethanol to yield the title compound. Yield, 40%; mp: 278–280 °C; IR (KBr) νmax/cm−1 3445 (NH), 2215 (C≡N), 1652 (C = O). 1H NMR (DMSO-d6); δ: 8.28–8.04 (m, 3H, Ar-H), 8.07 (br, s, 1H, NH, D2O exchangeable), 8.10–7.92 (m, 2H, Ar-H), 7.40–7.28 (m, 2H, Ar-H), 7.10–7.05 (m, 2H, Ar-H), 5.00–4.95 (m, 1H, CH), 2.39 (s, 3H, CH3), 2.10–1.95 (m, 2H, CH2), 1.85–1.61 (m, 6H, 3CH2). MS m/z (%); 370.00 (15.81, M+), 255.05 (21.12), 285.05 (38.06), 230.06 (29.49), 229.05 (100.00), 188.04 (66.11), 153.10 (15.13), 119.00 (50.7 7). Anal. Calcd. for C24H22N2O2 (%): C, 77.81; H, 5.99; N, 7.56. Found: C, 78.01; H, 6.10; N, 7.99.

Synthesis of 2-amino-4-(4-(cyclopentyloxy)phenyl)-6-(4-methylphenyl)nicotinonitrile (11)

A mixture of compound 4b (1.53 g, 0.005 mol), malononitrile (0.30 g, 0.005 mol), and ammonium acetate (3.1 g, 0.04 mol) in absolute ethanol (50 ml) was refluxed for 10 h. The reaction mixture was then cooled, poured into crushed ice, and the product separated out was filtered, washed with water, dried, and crystallised from water to yield compound 11. Yield, 45%; mp: 272–273 °C; IR (KBr) νmax/cm−1 3406 and 3336 (NH2), 2212 (C≡N), 1599(C = N). 1H NMR (DMSO-d6); δ: 8.29–8.19 (m, 3H, Ar-H), 8.10 (brs, 2H, NH2, D2O exchangeable), 7.90 (d, 2H, Ar-H, J = 4 Hz), 7.40 (d, 2H, Ar-H, J = 4 Hz), 7.10 (d, 2H, Ar-H, J = 4 Hz), 5.00–4.90 (m, 1H, CH), 2.40 (s, 3H, CH3), 2.10–2.00 (m, 2H, CH2), 1.90–1.75 (m, 4H, 2CH2), 1.70–1.60 (m, 2H, CH2). MS m/z (%); 369.14 (1.17, M+), 334.10 (20.39), 307.08 (19.44), 241.05 (20.53), 182.92 (13.80), 160.09 (35.90), 146.25 (100.00), 107.07 (24.46). Anal. Calcd. for C24H23N3O (%): C, 78.02; H, 6.27; N, 11.37. Found: C, 78.42; H, 6.66; N, 11.55.

General method for the synthesis of 2-(4-(cyclopentyloxy)styryl)-6-substituted quinoline-4-carboxylic acids (12a,b)

A mixture of compound 4a (1.15 g, 0.005 mol) and isatin derivatives (0.005 mol) in 50% aqueous ethanol (40 ml) containing potassium hydroxide (1.28 g, 0.023 mol) was refluxed for 24 h. The reaction mixture was filtered, and the filtrate was acidified with acetic acid and the solvent was evaporated under reduced pressure. The residue obtained was triturated with water, filtered, and dried to yield compounds 12a,b which crystallised from dimethylformamide.

6-Bromo-2-(4-(cyclopentyloxy)styryl)quinoline-4-carboxylicacid (12a)

Yield, 40%; mp >300 °C; IR (KBr) νmax/cm−1 3425 (OH), 1640 (C = O), 1565 (C = N). 1H NMR (CDCl3); δ 11.30 (brs, 1H, OH, D2O exchangeable), 7.78–6.88 (m, 10H, CH = CH, Ar-H), 4.80–4.70 (m, 1H, CH), 2.00–1.90 (m, 2H, CH2), 1.70–1.60 (m, 4H, 2CH2), 1.50–1.40 (m, 2H, CH2). MS m/z (%); 439.00 (12.41, M++2), 438.00 (16.05, M++1), 437.00 (13.00, M+), 239.20 (56.20), 145.10 (32.00), 97.10 (57.01), 94.90 (71.00), 71.10 (100.00), Anal. Calcd. for C23H20BrNO3 (%): C, 63.02; H, 4.60; Br, 18.23; N, 3.20. Found: C, 63.42; H, 4.70; Br, 18.00; N, 2.92.

2-(4-(Cyclopentyloxy)styryl)-6-fluoroquinoline-4-carboxylicacid (12b)

Yield, 50%; mp >300 °C; IR (KBr) νmax/cm−1 3421 (OH), 1690 (C = O), 1577 (-C = N-). 1H NMR (CDCl3); δ 11.40 (brs, 1H, OH, D2O exchangeable), 7.88–6.42 (m, 10H, CH = CH, Ar-H), 4.90–4.82 (m, 1H, CH), 2.10–1.90 (m, 2H, CH2), 1.80–1.70 (m, 4H, 2CH2), 1.55–1.45 (m, 2H, CH2). 13C NMR (DMSO-d6); δ: 183.2, 158.8, 149.6, 140.0, 135.1, 134.2, 128.1, 126.8, 144.3, 144.3, 82.1, 32.2, 24.1. MS m/z (%); 377.07 (0.86, M+), 373.99 (40.47), 283.00 (23.66), 270.01 (15.00), 240.07 (10.76), 187.07 (25.35), 151.04 (11.07), 142.12 (100.00). Anal. Calcd. for C23H20FNO3 (%): C, 73.20; H, 5.34; F, 5.03; N, 3.71. Found: C, 73.30; H, 5.34; F, 5.33; N, 4.01.

Biological testing

Antitumour evaluation

The evaluation of the antitumour activity was performed using tetrazolium salt MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay as reported,.

Antioxidant assay

The absorbance (Acontrol) of a green-blue solution (ABTS+ radical solution) resulted from a mixture of ABTS and manganese dioxide (MnO2) and was recorded at λmax 734 nm, according to the reported procedure,. The absorbance (Atest) was measured upon the addition of 20 µl of 1 mg/ml solution of the test sample in spectroscopic grade methanol/phosphate buffer (1:1 v/v) to the ABTS solution. The decrease in absorbance is expressed as % inhibition, which can be calculated from the following equation:

L-Ascorbic acid 20 µl (2 mM) solution was used as standard antioxidant (positive control). A blank sample was run using only methanol/phosphate buffer (1:1), while the negative control was run with ABTS and the methanol/phosphate buffer.

EGFR kinase inhibition assay

EGFR kinase activity was determined via EGFR Human In-Cell ELISA Kit in 96-well plates according to the manufacturer's instructions (EGFR Kinase Assay Kit Catalog # ab126419 of ABCAM, Cambridge, MA), as supplemental information. The EGFR kinase activities for each compound were expressed as IC50 values using seven concentrations (10.0, 5.0, 2.5, 1.25, 0.625, 0.31, and 0.15 µM).

Docking methodology

All modelling experiments were conducted with MOE programs running on PC computer (MOE 2008.10 of Chemical Computing Group. Inc, Montreal, QC, Canada). Starting coordinates of the X-ray crystal structure of EGFR enzyme in complex with eroltinib (PDB code 1M17) is obtained from the RCSB Protein Data Bank. All the hydrogen was added and enzyme structure was subjected to a refinement. The docking methodology was similar to that described in our previous reports,.

Results and discussion

Synthesis of compounds 4–7 (Scheme 1)

The compound 4-(cyclopentyloxy)benzaldehyde (3) was obtained as a key intermediate in a 75% yield by the reaction of 4-hydroxybenzaldehyde (1) with bromocyclopentane (2) in the presence of phase-transfer catalyst; t-butylammonium bromide (Bu4NBr). Condensation of 4-(cyclopentyloxy)benzaldehyde (3) with various aliphatic, aromatic, cyclic, and heterocyclic ketones in an ethanolic solution of sodium hydroxide afforded the corresponding compounds 4a,b; 5a–c; and 6a,b. The structures of the synthesized compounds were confirmed by their elemental and spectral analyses. Proton nuclear magnetic resonance (1H NMR) spectra of compounds 4a and 4b were confirmed by two doublets of vinylic protons at 7.28, 6.53, and 7.18, 6.73 ppm, respectively.

Synthesis of the designed α,β-unsaturated ketones and 4-H pyran derivatives.

Moreover, the 1H NMR spectrum of compound 4a showed a singlet signal at 2.26 ppm attributed to an acetyl group. The 1H NMR spectrum compound 4b was verified by the presence of new aromatic signals at 7.85–7.32 ppm in addition to a singlet signal at 2.36 ppm due to the presence of a 4-methyl group. The presence of a new peak at 198.2 ppm due to a carbonyl (CO) group was demonstrated in 13C NMR spectrum.

1H NMR spectra of compounds 5a–c were characterised by the presence of cycloalkane protons at 4.90–1.00 ppm. The 1H NMR spectrum of compound 6a is characterised by the presence of a singlet peak at 2.3 ppm corresponding to the methyl protons of the N-CH3 group, while a triplet–quartet pattern characteristic of an ethyl group (N-CH2CH3) was identified in the 1H NMR spectrum of compound 6b at 2.65 and 3.70 ppm, respectively. Synthesis of 4-H pyran derivative (7) was achieved by stirring 4-(cyclopentyloxy)benzaldehyde (3), malononitrile, and ethyl acetoacetate in ethanol in the presence of a catalytic amount of sodium benzoate at room temperature. The infra-red (IR) spectrum of compound 7 exhibited bands at 3401, 3326 (NH2), 2221 (C≡N), and 1697 (C = O) cm−1. Meanwhile, the 1H NMR spectrum showed a triplet and quartet at 1.20 and 4.10 ppm integrating for the COOCH2CH3 group, respectively. In addition, presence of two singlet peaks at 5.70 and 8.30 ppm for the methyl (CH3) and amine (NH2) groups, respectively.

Synthesis of compounds 8–12 (Scheme 2)

The compound 3-(4-(cyclopentyloxy)phenyl)-1-(4-methylphenyl)prop-2-en-1-one (4b) was heated under reflux with hydrazine hydrate or phenylhydrazine in ethanol or glacial acetic acid, resulting in the corresponding pyrazoline derivatives 8a and 8b. 1H NMR spectra of compounds 8a and 8b were characterised by the disappearance of the olefinic protons with the appearance of pyrazoline protons at 6.85–6.75, 3.90–3.75, and 3.50–3.30 ppm. Moreover, facile cyclocondensation of compound 4b with hydroxylamine hydrochloride in ethanolic potassium hydroxide gave the corresponding isoxazoline (9). The 1H NMR spectrum of compound 9 was characterised by the disappearance of the olefinic protons with the appearance of isoxazoline protons at 6.80–6.70 and 3.90–3.80 ppm. Reaction of the α,β-unsaturated ketone 4b with ethylcyanoacetate or malononitrile in ethanol in the presence of ammonium acetate yielded the cyanopyridine derivatives 10 and 11, respectively. IR spectra of compounds 10 and 11 were used to verify their structures through the appearance of characteristic absorption bands due to nitrile groups at 2215 and 2212 cm−1, respectively. In addition, a singlet peak at 8.07 ppm corresponding to the NH proton appeared in the 1H NMR spectrum of compound 10, while a singlet peak at 8.10 ppm was assignable to the NH2 group in compound 11, and both were deuterium oxide (D2O) exchangeable. Quinoline-4-carboxylic acid derivatives 12a,b were prepared by condensation of 4-(4-(cyclopentyloxy)phenyl)but-3-en-2-one (4a) and isatin derivatives in ethanolic potassium hydroxide. The IR spectrum of compound 12b was characterised by the presence of absorption bands at 3421 cm−1 and 1690 cm−1, representing hydroxy (OH) and carbonyl (C = O) groups, respectively. Moreover, a broad singlet at 11.40 ppm assignable to the exchangeable OH group was seen in the 1H NMR spectrum, and the 13C NMR spectrum showed the presence of a signal for the carbonyl group at 183.20 ppm.

Synthesis of the designed pyrazoline, isoxazoline, cyanopyridine, and quinoline-4-carboxylic acid derivatives.

Biological evaluation

Antitumour evaluation using MTT assay

The designed compounds were evaluated for their in vitro antitumour effects via the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method against a panel of four human tumour cell lines; namely, hepatocellular carcinoma cell line (HepG2), breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa), and prostate cancer cell line (PC-3),,. The antitumour activities of the designed compounds 4–12, along with that of the reference drugs 5-FU and afatinib are shown in Table 1. The α,β-unsaturated ketone 4a showed moderate antitumour activity against the investigated cell lines (IC50 ≅ 21.8–40.9 µM), while replacement of the methyl moiety in α,β-unsaturated ketone 4a with the 4-tolyl fragment in α,β-unsaturated ketone 4b resulted in slightly increase in antitumour activity against HepG2, MCF-7, HeLa, and PC-3 cell lines, with IC50 values at 20.0, 36.4, 18.8, and 17.1 µM, respectively. Weak antitumour activity was demonstrated by the 2-arylidene cyclic ketones 5a–c as shown by their IC50 values (30.1 to >100 µM). Interestingly, the 3-arylidene derivatives of piperidone 6a and 6b exhibited the greatest antitumour activities among the designed α,β-unsaturated ketone derivatives. For example, compound 6b displayed very strong antitumour effects against HeLa and PC-3 cell lines, as expressed by IC50 values of 6.7 and 9.1 µM, respectively. Moreover, compound 6b exhibited a strong inhibitory effect on the growth of HepG2 and MCF-7 cell lines, with IC50 values at 13.0 and 13.7 µM, respectively.

Table 1.

In vitro antitumour activity of 5-fluorouracil, afatinib, and the tested compounds.

	IC50 (μM)a
Compd no.	HepG2b	MCF-7c	HeLad	PC-3e
5-FU	7.9 ± 0.17	5.4 ± 0.20	4.8 ± 0.21	8.3 ± 0.35
Afatinib	5.4 ± 0.25	7.1 ± 0.49	6.2 ± 0.67	7.7 ± 0.57
4a	27.3 ± 1.96	40.9 ± 2.79	25.7 ± 1.97	21.8 ± 1.68
4b	20.0 ± 1.11	36.4 ± 2.60	18.8 ± 1.57	17.1 ± 1.58
5a	>100	>100	77.8 ± 4.41	94.1 ± 5.82
5b	55.4 ± 3.95	49.4 ± 3.16	30.1 ± 2.24	71.1 ± 4.93
5c	71.3 ± 4.53	64.7 ± 4.27	37.5 ± 2.81	26.9 ± 1.89
6a	15.9 ± 1.02	18.1 ± 1.58	9.4 ± 0.98	10.5 ± 0.97
6b	13.0 ± 0.87	13.7 ± 1.35	6.7 ± 0.67	9.1 ± 0.88
7	8.0 ± 0.38	7.5 ± 0.54	10.3 ± 1.13	13.3 ± 1.26
8a	18.9 ± 1.35	29.3 ± 1.97	16.2 ± 1.36	12.7 ± 1.13
8b	7.2 ± 0.24	5.6 ± 0.36	5.5 ± 0.45	7.8 ± 0.56
9	62.3 ± 4.10	58.4 ± 4.50	46.2 ± 3.30	50.1 ± 3.55
10	80.9 ± 5.34	70.9 ± 4.98	51.2 ± 3.82	41.9 ± 2.87
11	92.9 ± 5.82	97.3 ± 5.51	62.4 ± 3.80	87.7 ± 5.41
12a	85.4 ± 5.31	87.1 ± 5.24	89.4 ± 4.89	>100
12b	30.8 ± 2.07	48.1 ± 3.25	66.8 ± 4.07	69.4 ± 4.32

IC50, compound concentration required to inhibit tumour cell proliferation by 50% (mean ± SD, n = 3).

Human hepato-cellular carcinoma cell line (HepG2).

Human breast adenocarcinoma cell line (MCF-7).

Human cervical epithelioid carcinoma cell line (HeLa).

Human prostate cancer cell line (PC-3).

IC50, (μM): 1–10 (very strong), 11–25 (strong), 26–50 (moderate), 51–100 (weak), above 100 (non-cytotoxic).

More interestingly, compound 7, which contained a 4-H pyran core, exerted good activities against HepG2 (IC50 = 8.0 µM), MCF-7 (IC50 = 7.5 µM), HeLa (IC50 = 10.3 µM), and PC-3 (IC50 = 13.3 µM) cancer cell lines. Moreover, N-phenylpyrazoline 8b showed a sharp increase in antitumour activity when compared with α,β-unsaturated ketone analogue 4b. IC50 values of compound 8b against HepG2, MCF-7, HeLa, and PC-3 cell lines were 7.2, 5.6, 5.5, and 7.8 µM, respectively, in comparison with IC50 values of the reference drugs 5-FU (7.9, 5.4, 4.8, and 8.3 µM, respectively) and afatinib (5.4, 7.1, 6.2, and 7.6 µM, respectively). In addition, replacement of the phenyl ring in compound 8b with the hydrogen atom in pyrazoline 8a led to a decrease in antitumour activity against the MCF-7 cell line (IC50 = 29.3 µM), HepG2 (IC50 = 18.9 µM), HeLa (IC50 = 16.2 µM), and PC-3 (IC50 = 12.7 µM) cell lines. However, cyclisation of compounds 4a,b to isoxazoline 9; pyridines 10–11; and quinolines 12a,b analogues produced moderate to weak antitumour activity with IC50 values in the range of 30.8–97.3 µM.

Antioxidant activity using ABTS•+radical-scavenging assay

The assay is based on measuring the ability of the tested compounds to scavenge the long-life radical cation of ABTS,,,. In this study, all the newly synthesized compounds 4–12 and L-ascorbic acid, as a positive control, were evaluated and showed considerable free radical-scavenging activities. The reduction in colour intensity was expressed as inhibition percentage of the ABTS+ as shown in Table 2. From the listed results, we concluded that all tested compounds exhibited more than 50% inhibition of the ABTS radical cation except derivatives 5a, 10, 11, and 12a. It is clear that the conversion of α,β-unsaturated ketones 4a,b (% inhibition =52%) to the corresponding heterocyclic molecules generally led to sharp increases in antioxidant effects. Among them, the N-phenylpyrazoline derivative 8b displayed the highest free radical-trapping properties, with 88.5% inhibition, which was comparable to L-ascorbic acid at 90.0%. Moreover, 4-H pyran 7 and isoxazoline 9 derivatives showed inhibition of 75.8% and 60.0%, respectively. Conversion of the acyclic α,β-unsaturated ketones 4a,b (52% inhibition) into their corresponding cyclic α,β-unsaturated ketones 5a–c (51% inhibition) and 6a,b (54–55% inhibition) showed no change in activity. However, we concluded that compounds characterised by having pyrane 7, pyrazoline 8b, and isoxazoline 9 ring systems were among the most active compounds (60–88.5% inhibition), indicating that these core structures may play a role in trapping ABTS free radicals.

Table 2.

The percentage inhibition of the ABTS radical cation by L-ascorbic acid and the tested compounds.

Compound no	Absorbance	%Inhibition
Control of ABTS	0.512	0
Ascorbic acid	0.051	90.0
4a	0.245	52.0
4b	0.243	52.6
5a	0.281	45.0
5b	0.249	51.4
5c	0.251	51.0
6a	0.234	54.3
6b	0.229	55.1
7	0.124	75.8
8a	0.240	53.0
8b	0.058	88.5
9	0.204	60.0
10	0.270	47.3
11	0.279	45.5
12a	0.275	46.3
12b	0.256	50.0

Correlations between antioxidant and antitumour activities

The correlation between the antioxidant and the antitumour activities was investigated using SigmaPlot software (London, UK). The overall correlation between the antioxidant and antitumour activities of the synthesized compounds against individual cancer cell lines is shown in Figure 2. Most of the synthesized compounds showed moderate correlation (a moderate uphill relationship) between antioxidant and antitumour activities, as indicated by their coefficients of determination (R2). These R2 values were 0.573 (HepG2 cancer cell), 0.653 (MCF-7 cancer cell), 0.547 (HeLa cancer cell), and 0.480 (PC-3 cancer cell). The results indicate only a moderate linear relationship between the antioxidant and antitumour activities, which lead to the conclusion that antioxidant activity is not the only mechanism responsible for antitumour activity.

Figure 2.

The overall correlation between the antioxidant activity (%Inhibition) and the antitumour activity of the synthesized compounds against cancer cell lines (HepG2, MCF-7, HeLa, and PC-3 cells).

EGFR inhibitory activity

The antitumour activity results of compounds 6a, 6b, 7, and 8b encourage us to study the mechanism of antitumour activity using ELISA-based EGFR-TK assay with sorafenib as the reference drug. The % inhibition and IC50 values of the tested compounds were calculated and are listed in Table 3. Compound 6b and 7 revealed worthy EGFR inhibition activity with IC50 value of 0.56 and 1.6 µM, respectively, while compound 8b showed good inhibitory activity against EGFR with IC50 value of 2.16 µM, compared to sorafenib reference drug (IC50 = 1.28 µM). On the other hand, compounds 6a showed moderate inhibitory activity against EGFR with IC50 value of 4.66 µM, comparable to those of sorafenib (IC50 = 1.28 µM). We concluded, based on these results, that the designed compounds such as 6a, 6b, 7, and 8b are EGFR inhibitors which could be a new scaffold for the design of future analogues.

Table 3.

In vitro IC50 values of the designed compounds towards EGFR kinase enzyme.

	% Inhibition
Compd no.	10.0a	5.0a	2.5a	1.25a	0.625a	0.31a	0.15a	EGFR IC50 (μM)
6a	57.65	50.34	44.35	34.55	26.84	18.16	6.67	4.66
6b	83.66	76.71	64.44	61.23	55.33	48.95	26.52	0.56
7	72.78	65.98	52.86	49.65	43.27	31.78	8.22	1.6
8b	64.88	60.72	50.51	47.28	43.85	26.25	6.96	2.16
Sorafenib	80.88	71.63	56.72	49.48	43.27	33.92	10.82	1.28

Concentarion in μM.

Molecular docking results

The preceding results encouraged us to study the molecular docking of the most active compounds 6b, 7, and 8b using EGFR, which are overexpressed in numerous tumours such as prostate (PC-3), breast (MCF-7), hepatocellular carcinoma (HepG2), and human cervical (HeLa) cancer cell lines. All docking calculations were performed using MOE 2008.10 software.

The docked compounds 6b, 7, 8b, and the reference inhibitor erlotinib (Protein Data Bank [PDB] code 1M17) into the putative active site of EGFR are shown in Figure 3. The molecular modelling results of the compound, 6b, demonstrated an approximate orientation of the molecule in comparison with erlotinib inside the putative binding site of receptor pocket with some additional hydrogen bond interactions with surrounding amino acids. These docking results showed three classical and five non-classical hydrogen bonds, where the distinctive residue Thr766 formed bifurcated hydrogen bonds with oxygen and carbon atoms of the piperidin-4-one ring system (Figure 3, middle left panel). In addition, the amino acid residue Thr830 formed bifurcated hydrogen bonds through NH–aliphatic-CH and NH–N interactions of N-ethylpiperidin-4-one, while the amino acid Asp831 showed another hydrogen bond with N-ethyl group of piperidin-4-one core through the C = O–aliphatic–CH interaction. Additionally, the surrounding amino acids Met769, and Gly772 showed another three interactions with aromatic ring and pentyloxy moiety through C = O–Aromatic-CH, O–aliphatic-CH and O–NH bonds (Figure 3, middle left panel).

Figure 3.

Three-dimensional (3D) interactions of erlotinib (upper panel), compounds 6b (middle left panel), 7 (middle right panel), and 8b (lower panel) with the receptor pocket of EGFR kinase. Hydrogen bonds are shown as green lines and CH–π interactions as dotted lines.

Similarly, compound 7 binds into the putative active site of EGFR with three classical and one non-classical hydrogen bond. It was found that the amino acid Thr766 formed bifurcated classical hydrogen bonds with the 2-amino moiety and the oxygen atom of the 4-H pyran ring system (Figure 3, middle right panel). Moreover, the distinctive amino acid residue Met769 was involved in two hydrogen bonds: with the oxygen atom and with alkyl moieties of the ester group.

Moreover, compound, 8b, demonstrated similar results as compounds 6b and 7 inside the putative binding site of receptor pocket. These docking results showed two classical hydrogen bonds, where the distinctive residue Thr766 formed bifurcated classical hydrogen bonds with nitrogen atoms of the pyrazoline ring system (Figure 3, lower panel). In addition, three non-classical hydrogen bonds formed with surrounding amino acids, as shown in Figure 3 (lower panel). The amino acid residue Leu768 formed bifurcated hydrogen bonds through NH—Ar-CH interaction and one with the methyl group of the 4-tolyl moiety (NH—aliphatic-CH), while the third non-classical hydrogen bond was observed between the amino acid Thr830 and an aromatic ring through the OH—Ar-CH interaction. Additionally, the surrounding amino acids Leu768, Leu820, and Thr766 showed hydrophobic interactions with aromatic rings through CH—π and OH—π (Figure 3, lower panel).

Conclusions

Novel α,β-unsaturated ketone 4–6a,b, 4-H pyran 7, pyrazoline 8a,b, isoxazoline 9, pyridine 10–11, and quinoline-4-carboxylic acid 12a,b derivatives have been synthesized, and the antitumour, antioxidant, and EGFR kinase inhibition activities have been evaluated. It is clear that most of the synthesized compounds exert significant antitumour activities. Among the tested derivatives, 6a, 6b, 7, and 8b showed potent IC50 values ≅ 5.5–18.1 µM, which were comparable to that of 5-FU (IC50 ≅ 4.8–8.3 µM) and afatinib (IC50 ≅ 5.4–7.6 µM). Moreover, compound 8b has been shown promising, broad spectrum antitumour activity against the tested cell lines with an IC50 range of 5.5–7.8 µM. Additionally, compounds 6a, 6b, 7, 8b, and 9 exhibited the highest antioxidant effects using the ABTS radical-scavenging assay. Moreover, we observed a moderate relationship between the antitumour activity and the antioxidant effects of the tested compounds, which suggested that antioxidant effect is not the major role in the antitumour activity. Additionally, compounds 6b, and 7 exhibited excellent inhibition towards EGFR kinase enzyme with IC50 values range of 0.56–1.6 µM, respectively, while compounds 6a and 8b have good activity with IC50 = 4.66 and 2.16 µM, respectively, compared with the reference drug sorafenib (IC50 = 1.28 µM). Molecular docking studies were conducted for compounds 6b, 7, and 8b into putative binding sites of EGFR kinase enzyme, which showed similar binding modes to erlotinib (EGFR kinase inhibitor).