Synthesis and In Vitro Cytotoxic Activity of Chromenopyridones

Novel substituted chromenopyridones (3a–j and 6a–d) were synthesized and evaluated in vitro for the cytotoxic activity against various human cancer cell lines such as prostate (PC-3), breast (MCF-7), CNS (IMR-32), cervix (Hela), and liver (Hep-G2). preliminary cytotoxic screening showed that all the compounds possess a good to moderate inhibitory activity against various cancer cell lines. Particularly, compound 6b bearing allyl moiety displayed a significant cytotoxic potential in comparison to standard drugs.


Introduction
Cancer is the most dreaded group of diseases in which abnormal cells divide aggressively without control invade and spread to other parts of the body through the blood and lymph systems [1]. Despite tremendous advancements both in early diagnosis and approaches to treatment, cancer still remains an unconquered problem [1]. e development of new chemotherapeutic agents for the treatment of cancer with fewer side effects is an important goal for medicinal chemists. Heterocyclic compounds display a high degree of structural diversity and constitute the largest, most varied family of organic compounds evaluated for anticancer potential in past decades [2]. A perusal of the literature revealed that chromone framework constitutes an integral part of several natural products and biologically active molecules. Chromone derivatives exhibit a remarkable spectrum of pharmacological activities including antitumor [3], anti-in�ammatory [4], antibacterial [5], antifungal [6], antioxidant [7], anti-HIV [8], vasodilator [9], antiviral [10], and antiallergenic [11]. Naturally occurring chromone based molecule, morusin, isolated from a Chinese herb, has been reported to be a potent antitumor agent [12]. Recently, we have developed some chromone based compounds such as substituted 3-(5-phenyl-3H- [1,2,4]dithiazol-3-yl)chromen-4-one and substituted 4-oxo-4H-chromene-3-carbothioic acid N-phenylthioamide, which display a promising cytotoxic activity against a number of human cancer cell lines [13]. Derivatives of 6-chloro-/�uorochromone have also been reported as potential topoisomerase inhibitor anticancer agents [14].
2-Pyridone moiety is another substructural unit of several natural and synthetic molecules exhibiting diverse biological activities such as antitumor [15,16], antimicrobial [17], anti-in�ammatory [18], antiviral [19], and so forth. For instance, camptothecin, isolated from Camptotheca acuminate [20], is a naturally occurring DNA topoisomerase I poison possessing a pyridone nucleus. Due to a remarkable antiproliferative activity of camptothecin, various synthetic derivatives have been synthesized and among them, two water-soluble derivatives have gained FDA approval, that is, irinotecan to treat colorectal cancers and topotecan for ovarian and smallcell lung cancers [21]. Additionally, some camptothecinoids such as lurtotecan and exatecan are currently under clinical trials [22]. Keeping in view the high anticancer activity of both chromone and pyridones, it was decided to synthesize chromenopyridones and evaluate their cytotoxic potential against various human cancer cell lines.
1 H NMR spectra of compound 3b showed two ole�nic doublets, one at 8.20 and another at 6.90 ppm attributed to C 4 -H and C 3 -H, respectively, with coupling constant value 9.0 Hz. 13 C NMR spectrum of 3b showed resonance at 175.3 and 164.6 ppm attributed to the (C=O) group of both chromone and pyridone moiety, respectively, which was further corroborated by IR spectrum which showed strong bands at 1693 and 1687 cm −1 . IR spectrum also showed C-N stretching at 1419 cm −1 of pyridone ring. e structure 3b is further con�rmed by mass spectrum which revealed a molecular ion peak at m/z 358 (M + ).
Yield (%) of products the concentration required to inhibit cancer cell proliferation by 50% aer the exposure of cells to test compounds have also been determined (Table 2). In the case of prostate cancer cell line ( [27]. Flavopiridol, derived from rohitukine, which is an alkaloidal-�avonoid present in the Indian plant Dysoxylum binectariferum, induces cell-cycle arrest at both G1 and G2 phases and is a potent inhibitor of CDK1, 2, 4, and 6 in a competitive manner with respect to ATP [28]. Quercetin, the natural �avonoid is reported to arrest the cell cycle and proliferation in prostate cancer cells by modulation of CDK1/Cyclin B; cell cycle analysis showed that it blocks G2-M transition with a signi�cant induction of apoptosis [29]. Psorospermin is a chromone-based molecule possessing an epoxide group and has been reported to display a signi�cant cytotoxic activity with topoisomerase II inhibitory potential. It is proposed that psorospermin's xanthone group intercalates with DNA and the epoxide ring undergoes alkylation at the N 7 -guanine base of DNA in the presence of topoisomerase II [30][31][32].
ough systemic establishment of the structure activity relationship has not been taken up, however, based on the presently observed cytotoxic activity of chromenopyridones against various human cancer cell lines, it emerges that compound bearing allyl moiety such as 6b showed better cytotoxic activity than simple chromenopyridones. Compounds bearing electron withdrawing groups such as chloro and bromo at C 6 , C 7 , and C 8 were found to be more active against cancer cells than compounds bearing an electron donating group.

Conclusion
e chromenopyridones 3a-j and 6a-d were synthesized and evaluated for their cytotoxic activity against various human cancer cell lines. All compounds were puri�ed with column chromatography and characterized by spectroscopic ( 1 H and 13 C NMR, IR, mass) and elemental analysis. e in vitro cytotoxic evaluation revealed that compounds bearing electron withdrawing groups on chromone moiety were more active against cancer cells than compounds bearing electron donating groups and allylated chromenopyridones (6a-d) displayed a higher activity. ese "lead" compounds can be used for further anticancer drug development and their mode of action studies.

Chemistry.
Starting materials and reagents were purchased from commercial suppliers and used aer further puri�cation (crystallisation/distillation). JE�L AL-300FT (300 MHz) NMR spectrometer was used to record the 1 H NMR and 13 C NMR (75 MHz). Chemical shi ( ) and coupling constant are reported in ppm and Hz, respectively. Tetramethylsilane is used as the internal standard. IR spectra of compounds were recorded with shimadzu FT-IR 8400S spectrophotometer by using KBr pellets and CHCl 3 as solvent. Mass spectra were recorded on Shimadzu GCMS-QP-2000A (EI method) and Bruker Daltonics Esquire 3000 (ESI-MS method) spectrometers.

Synthesis of Substituted 1-Phenyl
To solutions of substituted 2-anilino-3-formylchromone (1a-j, 0.53 g, 2.0 mmol) in dry benzene (100 mL), ethoxycarbonylmethylene-triphenylphosphorane (0.7 g, 2.0 mmol) was added and the reaction mixtures were re�uxed with stirring till the completion of reaction (12 h, TLC). e solvent was removed under vacuum so as to reduce the volume to about 10 mL and the solutions were kept under refrigeration until cream colored crystal separated. e crystals were �ltered out and recrystallized from benzene to obtain the titled compounds as off-white solids [24].

Biological
Activity. e sulforhodamine B (SRB) assay is a colorimetric assay used for cytotoxic screening to assess cell growth [26]. Cells are cultured in a 96-well tissue culture plates and the cell growth which depends upon the rate of multiplication is measured indirectly by the intensity of the color of the dye which is directly proportional to the number of cells present. e human cancer cell lines (procured from NCI, Frederick, USA) were grown in tissue culture �asks in complete growth medium (RPMI-1640 medium with 2 mM glutamine, pH 7.4, supplemented with 10% fetal calf serum, 100 g/mL streptomycin, and 100 units/ml penicillin) in a carbon dioxide incubator (37 ∘ C, 5% CO 2 , 90% RH). e cells at subcon�uent stage were harvested from the �ask by treatment with trypsin (0.05% in PBS (pH 7.4) containing 0.02% EDTA). Cells with viability of more than 98% as determined by trypan blue exclusion were used for the determination of cytotoxicity and the cell suspension of 1 × 10 5 cells/mL was prepared in a complete growth medium. Stock solutions (2 × 10 −2 M) of synthesized compounds 3a-j and 6a-d were prepared in DMSO. e stock solutions were serially diluted with a complete growth medium containing 50 g/mL of gentamycin to obtain working test solutions of required concentrations. In vitro cytotoxicity against four human cancer cell lines of different tissues was determined using 96-well tissue culture plates. e 100 L of cell suspension was added to each well of the 96-well tissue culture plate. e cells were allowed to grow in a carbon dioxide incubator (37 ∘ C, 5% CO 2 , 90% RH) for 24 hours. Test materials in complete growth medium (100 L) were added aer 24 hours of incubation to the wells containing cell suspension. e plates were further incubated for 48 hours in a carbon dioxide incubator. e cell growth was stopped by gently layering trichloroacetic acid (50%, 50 L) on top of the medium in all the wells. e plates were incubated at 4 ∘ C for one hour to �x the cells attached to the bottom of the wells. e liquid of all the wells was gently pipetted out and discarded. e plates were washed �ve times with distilled water to remove trichloroacetic acid, growth medium low molecular weight metabolites, serum proteins, and so forth, and were airdried. e plates were stained with sulforhodamine B dye (0.4% in 1% acetic acid, 100 L) for 30 minutes. e plates were washed �ve times with 1% acetic acid and then airdried. e adsorbed dye was dissolved in Tris-HCl buffer (100 L, 0.01 M, pH 10.4) and plates were gently stirred for 10 minutes on a mechanical stirrer. e optical density (OD) was recorded on ELISA reader at 540 nm. e cell growth was determined by subtracting the mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of the test material was calculated considering the growth in the absence of any test material as 100% and in turn percent growth inhibition in presence of the test material was calculated.