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. 2019 Feb;14(1):74–83. doi: 10.4103/1735-5362.251855

Searching for new cytotoxic agents based on chromen-4-one and chromane-2,4-dione scaffolds

Alexandra Gaspar 1, Maryam Mohabbati 2, Fernando Cagide 1, Nima Razzaghi-Asl 3,4, Ramin Miri 2, Omidreza Firuzi 2,*, Fernanda Borges 1,*
PMCID: PMC6407335  PMID: 30936935

Abstract

Cancer is a major cause of death worldwide and novel anticancer agents for its better management are much needed. Benzopyrone-based compounds, such as chromones, possess several distinctive chemical and biological properties, of which the cytotoxicity against cancer cells seems to be prominent. In this study, two series of compounds based on chromen-4-one (3-10) and chromane-2,4-dione (11-18) scaffolds were synthesized in moderate/high yields and evaluated for cytotoxicity against HL-60, MOLT-4, and MCF-7 cancer cells using MTT assay. In general, the compounds exhibited moderate cytotoxic effects against the cancer cell lines, among which, a superior potency could be observed against MOLT-4 cells. Chroman-2,4-dione (11-18) derivatives had overall higher potencies compared to their chromen-4-one (3-10) counterparts. Compound 13 displayed the lowest IC50 values against HL-60 (IC50, 42.0 ± 2.7 μM) and MOLT-4 cell lines (IC50, 24.4 ± 2.6 μM), while derivative 11 showed the highest activity against MCF-7 cells (IC50, 68.4 ± 3.9 μM). In conclusion, this study provides important information on the cytotoxic effects of chromone derivatives. Benzochroman-2,4-dione has been identified as a promising scaffold, which its potency can be modulated by tailored synthesis with the aim of finding novel and dissimilar anticancer compounds.

Keywords: Antineoplastic agents, Cancer, Chromones, Drug screening

INTRODUCTION

Benzopyrone-based compounds, such as chromone, coumarin, and flavonoids have come to the attention of many investigators in recent years, due to their distinctive chemical and biological properties (1,2,3,4). In this context, benzopyran and its derivatives constitute an important class of natural compounds representing a broad range of biological activities such as antibacterial and antiviral (5,6), anticancer (7,8), antioxidant (5,9), anti-inflammatory (10), monoamine oxidase B inhibition (11,12,13), interaction with A3 adenosine receptor (14,15), and anticoagulant effects (16). Moreover, they are important intermediates in the synthesis of several natural products and medicinal agents (1,3,17).

Cancer continues to be among one of the most important causes of death worldwide (18). Chemotherapeutic agents that are available today invariably suffer from poor toxicity profile and adverse effects that cause dose reduction and therefore lack of efficacy (19). Therefore, discovery of novel anticancer agents is one of the top priorities of drug discovery programs. In this regard, benzopyran based compounds, like chromones, may be regarded as privileged scaffolds for rational design of anti-cancer agents (20). Cytotoxic activity has been reported for several chromone derivatives such as 3-formylchromones (21), 2,3-diarylchromanones (22), 3-hydroxychromones (23), 4H-chromen-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylates (24), 3-(3-oxobenzofuran-2(3H)-ylidene)methyl)-4H-chromones (25) and also for organometallic complexes of chromone derivatives (26).

The anticancer effect of these compounds has been reported against leukemia (22,25,27) and breast cancer cells (20,28,29). Furthermore, chromones seem also to exert antiproliferative effects against lung and colon cancer cells (8). Several derivatives of chromones seem to induce apoptosis in different cancer cells (28,29).

In view of the literature evidence on the potential anticancer effect of chromone derivatives and also in continuation of our work on the development of novel heterocyclic based cytotoxic agents (30,31,32), herein we report on the synthesis, characterization, and in vitro cell based cytotoxic activities of a series of chromen-4-one and chromane-2,4-dione derivatives.

MATERIALS AND METHODS

Reagents, general procedures, and apparatus

3- (4,5-Dimethylthiazol-2-yl)- 2, 5- diphenyltetrazolium bromide (MTT), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N-diisopropylethylamine (DIPEA), chromone-2-carboxylic (1), and chromone-3-carboxylic (2) acids as well as primary amines were obtained from Sigma-Aldrich (USA).

Fetal bovine serum (FBS), RPMI1640, phenol red free RPMI1640, phosphate buffered saline (PBS), trypsin, and trypan blue were purchased from Biosera (France). Penicillin/streptomycin was purchased from Invitrogen (USA). Dimethyl sulfoxide (DMSO) and cisplatin were from Merck (Germany) and EBEWE Pharma (Austria), respectively. All other reagents and solvents were pro analysis grade and acquired from Merck, Sigma-Aldrich (USA) and PanReac AppliChem (Germany) and used without further purification.

Thin-layer chromatography (TLC) was carried out on pre-coated silica gel 60 F254 (Merck, Portugal). The thickness of TLC layer was 0.2 mm. The spots were visualized under UV detection at 254 and 366 nm. Flash column chromatography was performed using silica gel 60 (0.2-0.5 or 0.040-0.063 mm; Carlo Erba, Portugal). The organic phases were dried over anhydrous Na2SO4 after workup and extraction. Whenever needed, the solutions were decolorized using activated charcoal. A Buchi Rotavapor® (Switzerland) was used to evaporate the solvents.

Proton nuclear magnetic resonance (1H NMR) and carbon-13 NMR (13C NMR) data were acquired, at room temperature, on a Brüker AMX 400 spectrometer (Spain) operating at 400.15 and 100.63 MHz, respectively. Chemical shifts were expressed in δ (ppm) values relative to tetramethylsilane (TMS) as internal reference; coupling constants (J) were reported in Hz. Electron impact mass spectra (EI-MS) were carried out on a VG AutoSpec instrument (Spain); the data were reported as m/z (% of relative intensity of the most important fragments).

General synthesis procedure

A solution of PyBOP (1 mmol) in dichloromethane (2.5 mL) was added to a solution of chromone carboxylic acid (1 mmol) in dimethylformamide (2.5 mL) and DIPEA (1 mmol) at 4 °C. The mixture was stirred on ice for 30 min. Afterwards the (hetero) aromatic amine was added to the reaction that was then warmed up to the ambient temperature. Then, the reaction was stirred for 4 h. The crude product was extracted (CH2Cl2) and purified by flash chromatography (CH2Cl2/MeOH or EtOAc/nhexane). Final purification was performed by recrystallization (EtOAc/n-hexane).

N- Cyclohexyl - 4- oxo - 4 H –chromene - 2 -carboxamide (3)

Yield: 60%. 1H NMR (CDCl3): δ = 1.73 – 1.18 (6H, m, 2 x H(3’), 2 x H(4’), 2 x H(5’)), 2.11 – 1.74 (4H, m, 2 x H(2’), 2 x H(6’)), 4.06-3.91 (1H, m, H(1’)), 6.70 (1H, d, J = 7.2, CONH), 7.17 (1H, s, H(3)), 7.45 (1H, ddd, J = 8.1, 7.2, 1.0, H(6)), 7.53 (1H, dd, J = 8.5, 0.6, H(8)), 7.74 (1H, ddd, J = 8.7, 7.2, 1.7, H(7)), 8.22 (1H, dd, J = 8.0, 1.5 Hz, H(5)). 13C NMR (DMSO): 24.9 (C3’, C5’), 25.4 (C4’), 32.9 (C2’, C6’), 49.1 (C1’), 112.1 (C3), 118.0 (C8), 124.4 (C4a), 125.9 (C6), 126.2 (C5), 134.4 (C7), 155.0 (C8a), 155.3 (C2), 158.2 (CONH), 178.2 (C4). EI-MS m/z: 271 (M+•, 38), 228 (12), 191 (19), 190 (100), 173 (16), 145 (12), 89 (39).

N- Phenyl - 4 -oxo - 4H – chromene – 2 -carboxamide (4)

Yield: 81 %. 1H NMR (DMSO): δ = 6.99 (1H, s, H(3)), 7.21 (1H, ddd, J = 7.6, 7.5, 1.1 H(4’)) 7.44 (2H, ddd, J = 7.0, 6.9, 1.8, H(3’), H(5’)), 7.58 (1H, ddd, J = 8.0, 6.9, 1.1, H(6)), 7.81 (2H, dd, J = 8.1, 1.1, H(2’), H(6’)), 7.86 (1H, dd, J = 8.3, 0.8, H(8)), 7.95 (1H, ddd, J = 8.5, 6.8,1.6, H(7)), 8.10 (1H, dd, J = 7.9, 1.6, H(5)), 10.77 (1H, s, CONH). 13C NMR (DMSO): δ = 111.1 (C3), 119.1 (C8), 121.1 (C2’, C6’), 123.7 (C4a), 124.9 (C6), 125.0 (C5), 126.2 (C4’), 128.9 (C3’, C5’), 135.1 (C7), 137.5 (C1’), 155.2 (C8a), 155.7 (C2), 157.8 (CONH), 177.4 (C4). EI-MS m/z: 265 (M+•, 18), 264 (83), 236 (32), 145 (18), 117 (19), 92 (1689 (100), 69 (15), 65 (16), 63 (19).

N-(4-Chlorophenyl)-4-oxo-4H- chromene- 2 -carboxamide (5)

Yield: 50%.1H NMR (DMSO): δ = 6.97 (1H, s, H(3)), 7.48 (2H, d, J = 8.8, H(3’), H(5’)), 7.56 (1H, m, H(6)), 7.83-7.96 (4H, m, H(7), H(8), H(2’), H(6’)), 8.08 (1H, dd, J = 7.9, 1.6, H(5)), 10.87 (1H, s, CONH).13C NMR (DMSO): δ = 111.2 (C3), 119.0 (C8), 122.7 (C2’, C6’), 123.7 (C4a), 125.0 (C5), 126.2 (C6), 128.7 (C4’), 128.8 (C3’, C5’), 135.2 (C7), 136.6 (C1’), 155.2 (C8a), 155.5 (CONH), 157.9 (C2), 177.3 (C4). EI-MS m/z: 301 (34), 300 (33), 299 (M+•, 100), 298 (50), 282 (15), 270 (24), 173 (14), 145 (28), 101 (18), 90 (10), 89 (89), 69 (16), 63 (14).

N-(4- (Methylthio) phenyl)-4 -oxo- 4H -chromene-2-carboxamide (6)

Yield: 60%.1H NMR (CDCl3): δ = 2.51(3H, s, SCH3 ), 7.28 (1H, s, H(3)), 7.31 (2H, d, J = 8.6, H(3’), H(5’)), 7.50 (1H, ddd, J = 8.0, 7.2, 1.0, H(6)), 7.60 (1H, d, J = 8.5, H(8)), 7.66 (2H, d, J = 8.6 H(2’), H(6’)), 7.78 (1H, ddd, J = 8.0, 7.1, 1.0, H(7)), 8.26 (1H, dd, J = 8.0, 1.5, H(5)), 8.51 (1H, s, CONH)).13C NMR (CDCl3): δ = 16.2 (SCH3), 112.7 (C3), 118.0 (C8), 121.0 (C3’, C5’), 124.4 (C4a), 126.2 (C5), 126.3 (C6), 127.6 (C2’, C6’), 133.7 (C1’), 134.7 (C7), 135.74 (C4’), 154.4 (C8a), 155.1 (C2), 156.8 (CONH), 178.7 (C4). EI-MS m/z: 312 (M+1, 21), 311 (M+•, 100), 278 (16), 140 (11), 138 (65), 89 (23).

N-(4- (Methylsulfonyl)phenyl ) -4 -oxo- 4H -chromene -2- carboxamide (7)

Yield: 54%.1H NMR (DMSO): δ = 3.23 (3H, s, CH3), 7.03 (1H, s, H(3)), 7.59 (1H, ddd, J = 8.1, 7.1, 1.1, H(6)), 7.86 (1H, dd, J = 8.5, 0.7, H(8)), 7.98 – 7.92 (1H, m, H(7)), 8.01-7.98 (2H, m, H(3’), H(5’)), 8.12-8.07 (3H, m, H(2’), H(5), H(6’)), 11.10 (1H, s, NH).13C NMR (DMSO): δ = 43.7 (CH3), 111.5 (C3), 119.1 (C8), 121.0 (C2’, C6’), 123.8 (C8a), 125.0 (C6), 126.3 (C5), 128.2 (C3’, C5’), 135.2 (C7), 136.4 (C4’), 142.2 (C1’), 155.1 (C8a), 155.2 (C2), 158.4 (CONH), 177.3 (C4). EI-MS m/z: 343 (M+•, 19), 343 (100), 214 (9).

N- (2-Thiazolyl) -4-oxo- 4H-chromene- 2 -carboxamide (8)

Yield: 55%.1H NMR (CDCl3): δ= 7.05 (1H, s, H(3)), 7.38 (1H , d, J = 3.7, H(3’), 7.55 (1H, ddd, J = 8.1, 7.0, 1.2, H(6)), 7.64 (1H, d, J = 3.7, H(4’)), 7.84 (1H, dd, J = 8.5, 0.8, H(8)), 7.90 (1H, ddd, J = 8.6, 7.0, 1.7, H(7)), 8.06 (1H, dd, J = 8.0, 1.3 Hz, H(5)), 13.32 (1H, bs, NH).13C NMR (CDCl3): δ = 111.8 (C3), 114.7 (C3’), 119.1 (C8), 124.9 (C4a, C6), 126.2 (C5), 135.1 (C7, C4’), 155.3 (C1’, C2, C8a, CONH), 159. 5 (C1’), 177.4 (C4). EI-MS m/z: 272.1 (M+•, 58), 244 (18), 216 (13), 173 (49), 145 (30), 89 (100).

N- (5-Methyl -2 -thiazolyl) – 4 - oxo- 4H -chromene-2-carboxamide (9)

Yield: 48%. 1H NMR (CDCl3): δ = 2.39 (3H, d, J = 1.1 Hz, CH3), 7.04 (1H, s, H(3)), 7.32 (1H, q, J = 1.1 Hz, H(4’)), 7.56 (1H, ddd, J = 8.1, 7.2, 1.1 Hz, H(6)), 7.83 (1H, d, J = 7.9 Hz, H(8)), 7.91 (1H, ddd, J = 8.6, 7.1, 1.7 Hz, H(7)), 8.07 (1H, dd, J = 8.0, 1.4 Hz, H(5)).13C NMR (CDCl3): δ = 11.4 (CH3), 111.7 (C3), 119.1 (C8), 123.8 (C3’), 124.9 (C4a, C6), 126.1 (C5), 135.1 (C7, C4’), 155.4 (C1’, C2, C8a, CONH), 177.4 (C4). EI-MS m/z: 288 (M+•, 53), 286 (86), 258 (72), 230 (62), 230 (62), 173 (95), 145 (82) 68 (100).

N- (Pyridin-2-yl )- 4-oxo - 4H-chromene – 2 -carboxamide (10)

Yield: 22%.1H NMR (CDCl3): δ = 7.19 (1H, ddd, J = 7.4, 5.0, 0.9, H(4’)), 7.30 (1H, s, H(3)), 7.50 (1H, ddd, J = 8.1, 7.2, 1.0, H(6)), 7.63 (1H, dd, J = 8.5, 0.6, H(6’)), 7.89-7.72 (2H, m, H(5’),H(8)), 8.25 (1H, dd, J = 8.0, 1.4, H(3’)), 8.45-8.32 (2H, m, H(5), H(7)), 9.31 (1H, s, NH).13C NMR (CDCl3): δ = 113.0 (C3), 114.8 (C6’), 118.2 (C8), 121.1 (C4’), 124.4 (C4a), 126.2 (C6), 126.3 (C5), 134.9 (C7), 139.0 (C5’), 148.1 (C3’), 150.1 (C1’), 154.0 (C8a), 155.2 (C2), 157.3 (CONH), 177.9 (C4). EI-MS m/z: 267 (22), 266 (M+•, 99), 238 (58), 237 (66), 210 (83), 89 (100).

(E/Z) - 3 - ((Cyclohexylamino) methylene) chromane-2,4-dione (11)

Yield: 30%.1HNMR (DMSO): δ = 1.32-1.94 (10H, m, 2 x H(2’), 2 x H(3’), 2 x H(4’), 2 x H(5’), 2 x H(6’)), 3.70 (1H, m, H(1’)), 7.32 (2H, m, H(6), H(8)), 7.66 (1H, dd, J = 8.0, 7.3, H(7)), 8.02 (0.7H, d, J = 7.6, H(5)), 8.11 (0.3H, d, J = 7.6, H(5)), 8.44 (0.7H, d, J = 14.8, H(CHNH)), 8.60 (0.3H, d, J = 14.8, H(CHNH)), 10.31 (0.3H, brs, NH), 11.85 (0.7H, brs, NH).13C NMR (DMSO): δ: 23.89 (C), 24.41 (C), 30.74 (C,), 32.18 (C), 35.75 (C), 58.56 (C1’), 95.6 (C3), 116.9 (C8), 120.2 (C4a), 123.9 (C6), 125.2 (C5), 134.3 (C7), 154.2 (C8a), 160.3 (CHNH), 162.4 (C2), 179.6 (C4). EI-MS m/z: 271 (M+1, 100), 228 (18), 188 (23), 175 (23), 173 (18), 121 (31), 97 (22), 57 (17).

(E/Z)-3- ((Phenylamino)methylene)chromane -2,4-dione (12)

Yield 60%.1H NMR (CDCl3) δ: 7.40-7.26 (5H, m, H(2’), H(3’), H(4’), H(5’), H(6’)), 7.52 - 7.45 (2H, m, H(6), H(8)), 7.64-7.57 (1H, m, H(7)), 8.14 (0.7H, dd, J = 7.8, 1.6, H(5)), 8.08 (0.3H, dd, J = 7.8, 1.7, H(5)), 8.91 (0.7H, d, J = 13.6, H(CHNH), 9.05 (0.3H, d, J = 14.5, H(CHNH), 11,94 (0.3H, d, J = 13.8, NH), 13.70 (0.7H, d, J = 12.2, NH).13C NMR (CDCl3) δ: 99.0/98.9 (C3), 117.6/117.5 (C8), 118.6/118.7 (C2’, C6’), 120.9/120.5 (C4a), 124.6/124.4 (C6), 126.7/126.0 (C5), 127.7/127.5 (C4’), 130.4/130.3 (C3’, C5’), 135.0/134.9 (C7), 137.9 (C1’), 154.9/153.6 (C8a), 155.2/155.1 (CHNH), 165.4/163.8 (C2), 182.0/178.8 (C4). EI-MS m/z: 266 (6), 265(M+•, 61), 173 (100), 144 (16), 121 (35), 117 (36).

(E/Z)-3- ((4-Chlorophenyl)amino)methylene) chromane-2,4-dione (13)

Yield: 47%.1H NMR (DMSO) δ: 7.34 (1H, d, J = 7.8, H(8)), 7.38 (1H, dd, J = 7.6, 1.3, H(6)), 7.54 (2H, d, J = 8.8, (H3’), H(5’)), 7.70-7.73 (3H, m, H(2’), H(6’), H(7)), 7.99 (1H, dd, J = 7.8, 1.5 Hz , H(5)), 8.85 (0.7H, d, J = 13.8, CHNH), 8.88 (0.3H, d, J = 14.8, CHNH), 11.84 (0.3H, d, J = 14.8, NH), 11.84 (0.3H, d, J = 14.8, NH), 13.39 (0.7H, d, J = 13.8, NH).13C NMR (DMSO) δ: 98.3 (C3), 117.2 (C8), 119.9 (C4a), 121.6/121.3 (C2’, C6’), 124.3 (C5), 125.5 (C6), 129.6/129.1 (C3’, C5’), 131.2 (C4’), 135.1 (C7), 137.1 (C1’), 154.5 (C8a) 155.8/154.5 (CHNH), 162.2 (C2), 180.3 (C4). EI-MS m/z: 301 (37), 300 (21), 299 (M+•, 88), 174 (16), 173(100), 151 (18), 121 (37), 92 (11), 89 (10).

(E/Z) – 3 - ((4-(Methylthio)phenyl)amino) methylene)chromane-2,4-dione (14)

Yield: 51%.1H NMR (CDCl3) δ: 2.52 (3H, s, CH3), 7.35-7.25 (5H, m, H(6), H(8), H(2’), H(3’), H(5’), H(6’)), 7.64-7.57 (1H, m, H7), 8.07 (0.7H, dd, J = 7.8, 1.7, H(5)), 8.14 (1H, dd, J = 7.8, 1.7, H(5)), 8.85 (1H, d, J = 13.6 Hz, CHNH), 8.99 (0.3H, d, J = 14.5, CHNH), 11.93 (0.3H, d, J = 14.5, NH), 13.73 (0.7 H, d, J = 13.2, NH).13C NMR (DMSO) δ: 15.9/15.8 (CH3), 98.8/98.7 (C3), 117.5/117.4 (C8), 119.0/118.9 (C2’, C6’), 120.4 (C4a), 124.5/124.2 (C5), 126.6/125.8 (C6), 127.9/127.8 (C3’, C5’), 134.82/134.7 (C7), 134.83 (C4’), 138.6/138.4 (C1’), 154.3/152.9 (CHNH), 155.0 (C8a), 163.6 (C2), 181.8 (C4). EI-MS m/z: 313 (27), 312 (76), 311 (M+•, 100), 296 (25), 174(29), 173(90), 148 (24), 121 (70).

(E/Z) - 3 - ((4-(Methylsulfonyl)phenyl)amino) methylene)chromane-2,4-dione (15)

Yield: 47%.1H NMR (DMSO) δ: 2.78 (3H, s, CH3), 7.40-7.33 (2H, m, H(8), H(6)), 7.75-7.69 (1H, m, H(7)), 7.81-7.76 (2H, m, H(3’), H(5’)), 7.91-7.86 (2H, m, H(2’), H(6’)), 8.00 (1H, dd, J = 7.8, 1.5, H(5)), 8.94 (0.7H, d, J = 13.7, CHNH), 8.97 (0.3H, d, J = 14.7, CHNH), 11.91 (0.3H, d, J = 14.7 Hz, NH), 13.46 (0.7, d, J = 13.8, NH).13C NMR (DMSO) δ: 43.1 (CH3), 98.6 (C3), 117.3/117.2 (C8), 119.9 (C4a), 120.4/120.1 (C2’, C6’), 124.5/124.4 (C5), 125.3/125.2 (C3’, C5’), 125.5 (C6), 135.2/135.1 (C7), 140.0 (C4’), 144.5/1144.4 (C1’), 154.4/154.2 (C8a), 155.8 (C2), 163.2/162.2 (CHNH), 180.5/177.5 (C4). EI-MS m/z: 343 (M+•, 17), 183(20), 173(21), 155 (100).

(E/Z)- 3 - ((Thiazol-2-ylamino) methylene) chromane-2,4-dione (16)

Yield 15%.1H NMR (CDCl3) δ: 7.17-7.13 (1H, m, H(4’)) 7.36 – 7.25 (2H, m, H(8), H(7)), 7.59-7.55 (1H, m, H(3’)), 7.67-7.60 (1H, m, H(6)), 8.07 (0.7H, dd, J = 7.8, 1.5, H(5)), 8.14 (0.3H, dd, J = 7.8, 1.5, H(5)), 9.18 (0.7H, d, J = 11.9, CHNH), 9.25 (0.3H d, J = 13.1 Hz, CHNH), 12.20 (0.3H, d, J = 12.4 Hz, NH), 13.97 (0.7H, d, J = 9.2 Hz, NH).13C NMR (CDCl3) δ: 100.7/100.6 (C3), 115.9/116.1 (C3’), 117.8/117.1 (C8), 120.6/120.1 (C4a), 124.9/124.6 (C5), 127.0/126.4 (C6), 135.8/135.5 (C7), 141.3/141.1 (C4’), 153.2 (C8a), 154.8/153.3 (CHNH), 159.8/159.7 (C2), 165.1/162.7 (C1’), 182.8/178.7 (C4). EI-MS m/z: 272 (M+•, 58), 173 (49), 145 (30), 89 (100).

(E/Z) – 3 - ((5- Methylthiazol - 2 - yl)amino )methylene)chromane-2,4-dione (17)

Yield 10%.1H NMR (CDCl3) δ: 2.46 (5H, d, J = 1.3 Hz, CH3), 7.34-7.19 (3H m, H(3’), H(8), H(7)), 7.64-7.55 (1H, m, H(6)), 8.02 (0.7H, dd, J = 7.8, 1.6, H(5)), 8.10 (0.3H, dd, J = 7.8, 1.6, H(5)), 9.04 (0.7H,), 9.12 (0.3H, s, CHNH), 12.08 (0.3H, brs, NH), 13.82 (0.7H, brs, NH).13C NMR (CDCl3) δ: 12.2 (CH3), 100.2/100.1 (C3), 117.6/117.5 (C8), 120.5/120.0 (C4a), 124.7/124.4 (C5), 126.8/126.2 (C6), 131.5/131.5 (C7), 135.5/135.2 (C3’), 138.3/138.2 (C4’), 154.3/152.7 (CHNH), 155.1/154.7 (C8a), 157.5/157.3 (C2), 164.9/162.7 (C1’), 182.4/178.5 (C4). EI-MS m/z: 286 (M+•, 100), 258 (27), 188 (39), 173 (50), 121 (56), 90 (74).

(E/Z)-3-((Pyridin-2-ylamino)methylene)chromane-2,4-dione (18)

Yield 61%.1H NMR (DMSO) δ: 7.42 – 7.28 (3H, m, H(4’), H(5’), H(6’)), 7.77-7.64 (2H, m, H(6), H(8)), 8.05-7.90 (2H, m, H(3’), H(7’)), 8.50 (1H, d, J = 4.7, CHNH), 9.39 (0.7H, d, J = 13.2, CHNH), 9.58 (0.3H, d, J = 14.1, CHNH), 11.84 (0.3H, d, J = 14.6 Hz, NH), 13.15 (0.7H, d, J = 13.1 Hz, NH),13C NMR (DMSO) δ: 99.0/98.9 (C3), 114.9/114.6 (C6’), 117.3/117.2 (C8), 120.0/120.2 (C4a), 122.4/122.2 (C4’), 124.5/124.4 (C6), 126.1/125.6 (C5), 135.4/135.3 (C7), 139.6/139.5 (C5’), 148.8/148.7 (C3’), 149.5/149.5 (C1’), 153.0/151.7 (C8a), 154.5/154.3 (CHNH), 162.9/162.6 (C2), 180.5/178 (C4). EI-MS m/z: 266 (M+•, 19), 265 (55), 210 (30), 209 (100), 79 (35).

Biological activity

Cell lines and culture

HL-60 (human promyelocytic leukemia), MOLT-4 cells (human acute lymphoblastic leukemia), and MCF-7 (human breast adenocarcinoma) were provided by Pasteur Institute of Iran (Tehran, I.R. Iran). The growth medium was prepared adding 10% FBS and antibiotics (penicillin-G and streptomycin) to RPMI1640 medium.

MTT assay

In order to evaluate the cytotoxic effect of synthesized compounds, MTT reduction assay was performed as described previously (33,34). Stock solutions of synthesized derivatives were prepared by their dissolving in DMSO. The stock solutions were diluted several times in growth medium in order to keep the final DMSO concentration below 0.25%. Three cell lines including HL-60, MCF-7, and MOLT-4 at densities of 4 × 104, 3 × 104 and 5×104 cells/mL, respectively, were plated in 96-well microplates (100 μL per well). The cells were cultured at 37 °C in humidified air (containing 5% CO2). Six wells were used as controls, which did not contain any synthetic compound. Blank wells were also used which contained only growth medium for background correction. After 24 h of incubation at 37 °C, 50 μL of the growth medium was removed and 50 μL media containing 3-4 different concentrations of synthesized derivative in the range 10-100 μM, depending on the potency of the compound, were added in duplicate. In experiments with HL-60 and MOLT-4 cells the plates were centrifuged before media removal. After 72 h, the medium was removed and replaced with 80 μL of MTT solution dissolved in RPMI without phenol red at a final concentration of 0.5 mg/mL and the plates were incubated for additional 4 h at 37 °C. In order to solubilize the formazan crystals, 200 μL DMSO was added to each well. The optical density was measured at 570 nm by a microplate reader. For each concentration of the test compounds, the percent viability was calculated compared to untreated cells and IC50 values were calculated with Curve Expert version 1.34 for Windows. Each test was repeated between 3-5 times.

RESULTS

Chemistry

Two series of compounds based on chromen-4-one and chromane-2,4-dione scaffolds were synthesized by a straightforward one-pot synthetic strategy in moderate/high yields (Scheme 1). The chromone carboxamide derivatives from series I (3-10, Table 1) were obtained by the condensation of chromone-2-carboxylic acid (1) with the appropriate amine (Scheme 1) using the coupling reagent PyBOP in the presence of DIPEA (35,36,37). Chromane-2,4-dione derivatives from series II (11-18, Table 1) were obtained with similar reaction conditions by using chromone-3-carboxylic acid (2) as starting material (Scheme 1). As it was previously reported (13), the high reactivity at C-2 position of the ester intermediate formed in situ between the carboxylic acid and PyBOP, changed the course of the reaction resulting in a nucleophilic attack by the (hetero) aromatic amine at C-2 position of the benzopyran ring. As stated before (13) after the ring-opening and the ring closing assisted-processes the formation of the chromane-2,4-dione derivatives took place in the reaction.

Scheme 1.

Scheme 1

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Strategy followed to synthetize chromen-4-one and chromane-2,4-dione derivatives. a, Reagents: PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; DIPEA, N,N-diisopropylethylamine; RNH2.

Table 1.

Structure and reaction yields of synthesized chromone-2-carboxamide (series I) and chromane-2,4-dione (series II) derivatives.

R graphic file with name RPS-14-74-g002.jpg graphic file with name RPS-14-74-g003.jpg
Series I Series II
Compounds Yield (%) Compounds Yield (%)
Cyclohexyl 3 60 11 30
Phenyl 4 81 12 60
4-Chlorophenyl 5 50 13 47
4-(Methylthio)phenyl) 6 60 14 51
4-(Methylsulfonyl)phenyl 7 54 15 47
2-Thiazolyl 8 55 16 15
5-Methyl-2-thiazolyl 9 48 17 10
Pyridin-2-yl 10 22 18 61
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Cytotoxic activity

Chromone-3-carboxamide (3-10) and chromane-2,4-dione (11-18) derivatives were assayed for their cytotoxic activity on HL-60, MOLT-4, and MCF-7 cancer cells and IC50 values are reported in Table 2 as mean ± SEM.

Table 2.

Cell growth inhibitory activities of chromone-2-carboxamide (series I) and chromane-2,4-dione (series II) derivatives were assessed by the MTT reduction assay (IC50 values, μM; mean ± SEM).

R graphic file with name RPS-14-74-g004.jpg graphic file with name RPS-14-74-g005.jpg
Series I Series II
Compound HL-60 MCF-7 MOLT-4 Compound HL-60 MCF-7 MOLT-4
Cyclohexyl 3 > 100 > 100 90.8 ± 1.2 11 64.6 ± 7.1 68.4 ± 3.9 33.2 ± 2.1
Phenyl 4 > 100 > 100 > 100  12 > 50 > 50 47.1 ± 3.6
4-Chlorophenyl 5 > 50 > 50 > 50  13 42.0 ± 2.7 > 50 24.4 ± 2.6
4-(Methylthio)phenyl) 6 > 100 > 100 > 100  14 > 50 > 50 41.6 ± 4.1
4(Methylsulfonyl)phenyl 7 > 50 > 50 > 50  15 71.8 ± 6.1 86.1 ± 19.0 48.6 ± 8.2
2-Thiazolyl 8 > 100 > 100 > 100  16 95.2 ± 7.0 85.8 ± 9.7 48.5 ± 8.3
5-Methyl-2-thiazolyl 9 > 100 > 100 > 100  17 91.5 ± 3.7 > 100 55.6 ± 3.6
Pyridin-2-yl 10 > 100 > 100 > 100  18 > 50 > 50 > 50
Cisplatin 2.7 ± 0.2 9.3 ± 2.4 2.9 ± 0.1
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DISCUSSION

In this study, two series of compounds with chromen-4-one and chromane-2,4-dione scaffolds were synthesized and their cytotoxicity was assessed against 3 human cancer cell lines using MTT assay. Chroman-2,4-dione (11-18) derivatives possessed higher cytotoxic capacities compared to their chromen-4-one (3-10) counterparts.

Several benzopyran derivatives have been reported to hold considerable cytotoxic effects against cancer cells. A series of synthesized chromone derivatives based on lavendustin structure showed anticancer effects against A-549 (lung adenocarcinoma) and HCT-15 (colon adenocarcinoma) cells (8). Also 2,3-diarylchromanones have shown potential cytotoxic effect against HL-60 promyelocytic leukemia cells (22). In another report, a series of 3-benzylidene-4-chromanones were synthesized and tested against MDA-MB-231 (triple negative breast adenocarcinoma), SK-N-MC (neuroblastoma), and KB (nasopharyngeal carcinoma) cells. Some of the synthesized derivatives were more effective than etoposide, a standard chemotherapeutic agent, against these cancer cells (20).

Different mechanisms of action have been proposed for the anticancer effect of chromone derivatives. They have been suggested to exert their effect via inhibition of topoisomerase enzymes (7,8,20). An in silico study has shown that 4H-chromone-1,2,3,4-tetrahydropyrimidine -5-carboxylates derivatives could be inhibitors of Bcr-Abl tyrosine kinase, which has important oncogenic functions in certain types of leukemia (27). Another investigation on breast and lung cancer cells has demonstrated that sulfonamide containing chromone derivatives possess inhibitory activity against carbonic anhydrase IX and XII, two important targets in certain types of cancer (29).

Our findings showed that chroman-2,4-dione derivatives possessed cytotoxic activity specially against leukemia cell lines and to a less extent against breast cancer cells. Although, the synthesized set of compounds in this study is not a numerous, in comparison with their anticancer activities (Table 2), the following structure activity relationships could be proposed: a) Chromane-2,4-dione derivatives (11-18) displayed a superior cytotoxicity profile when compared to the chromene-2-carboxamide compounds (3-10); b) Chromane derivatives (11-18) exhibited higher cytotoxic effects against MOLT-4 cell line compared to HL-60 or MCF-7 cell lines; 3) The observed order of cytotoxic activity in MOLT-4 cell line was as follows: e13 > 11 > 14 > 12 > 16 > 15 > 17 > 18; 4) In our synthesized series of compounds, it seems that the chromane-2,4-dione derivative bearing halogen in the exocyclic phenyl ring (13) shows the highest potency against MOLT-4 and HL-60 cell lines; 5) Aromatization of the N-substituted ring reduced the cytotoxic activity against MOLT-4 cell line (12, IC50 = 47.1 ± 3.6 and 11, IC50 = 33.2 ± 2.1 μM).

CONCLUSION

Our in vitro cell based cytotoxic evaluation showed that chromane-2,4-dione derivatives were better cytotoxic agents compared to chromone-2-carboxamide compounds against HL-60, MCF-7, and MOLT-4 human cancer lines. In particular, superior potencies have been detected in the case of acute lymphoblastic leukemia (MOLT-4) cells. Chromone-2-carboxamide compounds exhibited no cytotoxic activity against tested cancer cells; this activity loss may be related to the orientation of 2-carboxamide substituents in the site of action. The outcomes of this study may provide some information for rational discovery of chromane-2,4-dione based derivatives with the aim of finding more potent cytotoxic agents.

ACKNOWLEDGEMENTS

This project was financially supported by Science and Technology (FCT, Grant No. UID/QUI/00081/2013) and FEDER/COMPETE (Grant No. NORTE-01-0145-FEDER-000028). The authors thank FCT, POPH, and FEDER/COMPETE for postdoctoral grants awarded to A. Gaspar (SFRH/BPD/93331/2013) and F. Cagide (NORTE-01-0145-FEDER-000028). We are also grateful for the financial support of the National Institute for Medical Research Development (NIMAD), Tehran, I.R. Iran (Grant No.: 957652). The support of the Vice-Chancellor for Research, Shiraz University of Medical Sciences, Shiraz, I.R. Iran is much appreciated.

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