SYNTHESIS AND ANTICANCER ACTIVITY OF IMIDAZOLE DERIVATIVES INCORPORATING CHROMENE MOIETY

The oxo-chromene-6-carbaldehyde derivative 1 was used as a starting material for the synthesis of some novel pyrano[3,2-b] xanthene-4,7-dione 2 and chromen-oxazol-5(4H)-one derivatives 4,5. Furthermore, the oxazolone derivative 4 was used as key intermediates to synthesize some novel imidazol derivatives 6-14. The structures of target compounds were confirmed by elemental analyses and spectral data. All the target compounds were subjected to in-vitro antitumor activity against breast and colon human tumor cell lines (MCF7 and HCT). Most of the screened compounds showed interesting cytotoxic activities compared to a reference drug. Kerwords: Chromene, Oxazolone, Imidazole, Antitumor activity.

The C 3, 8 -H of coumarin, CH= and CH-aromatic were resonated in the range of ä 6.13-8.19ppm.OH proton was observed as singlet at 11.34 ppm.Its mass spectrum which revealed a molecular ion peak at m/z 411 (M-1, 5.16 %) corresponding to the molecular formula C 21 H 14 ClNO 6 . 1 H NMR (DMSO-d 6 , 300MHz) of 5 raveled a singlet was observed at 2.23 ppm due to C 2 -CH 3 protons.The CH 3 protons of oxazolone were observed a singlet at 3.41 ppm.The C 5 -OCH 3 protons were observed downfield as singlet at ä 3.89 ppm.The C 3, 8 -H of coumarin and CH= were resonated in the range of ä 6.53-8.21ppm.The OH proton was observed as singlet at 11.26 ppm.Its mass spectrum which revealed a molecular ion peak at m/z 315 (6.04 %) corresponding to the molecular formula C 16 H 13 NO 6 .Oxazolone derivative 4 was used as intermediate for the synthesis of the some imidazole derivatives via its reaction with some aromatic and hetero aromatic amines.Thus, Oxazolone derivative 4 reacted with 4-aminoacetophenone, 7-aminothiophenol, sulfaquionoxaline , 2-amino-4hydroxypyrimidine and 4-aminoantipyrine in glacial acetic acid in presence of fused sodium acetate to yield yield 2-(2-chlorophenyl)-4-(7-hydroxy-5-methoxy-2-methyl-4-oxo-4Hchromen-6-yl)methylene)-1-(4-aryl)-1H-imidazol-5(4H)-one 6-10, respectively, (Scheme 3).The structure of compounds 6-10 were confirmed by their IR, 1 HNMR and Mass spectra.The IR spectra revealed the imidazol carbonyl stretching frequency and the coumarin carbonyl stretching with addition special group for each compounds, its mass spectrum and 1 H NMR spectra respectively in experimental section.Finally, treatment of oxazoles 4,5 with thiosemicarbazide afford imidazotriazole derivatives 13, 14, respectively.Compounds 13, 14 confirmed by IR spectra which revealed disappear the imidazol carbonyl stretching frequency and appear the coumarin carbonyl stretching frequency at 1632, 1650 and (C=S) at 1568, 1567, respectively and presence of stretching broad band for OH group at 3432, 3426 cm -1 .Also, structures 13, 14 were supported by its mass spectrum and 1 H NMR spectra showed in experimental section.The formation of imidazotriazole derivatives 13, 14 is assumed to proceed through the in situ intramolecular cyclization of the non isolable intermediate via nucleophilic addition of the amino group to the carbonyl group followed by elimination of water, (Scheme 5).

In virto anticancer screening
Chemotherapy is a major therapeutic approach for the two human cultured cell lines which breast cancer cell and colon cancer cell lines (MCF7, HCT) in comparison to the known anti cancer drugs; doxorubicin and vinblastine as reference drugs, is one of the most effective antitumor agents used to produce regression in acute leukemias, Hodgkin's disease, and other lymphomas.The cytotoxic effects of the newly synthesized compounds were evaluated in the MCF7 breast cancer cell line with doxorubicin was used as a reference drug in six different concentrations.Also, HCT colon cancer cell line with vinblastine was used as a reference drug in three different concentrations.The primary anticancer assay was performed in accordance with the protocol of the Drug Evaluation Branch (Wasylyk, et al., 2008;Wilson and Bottiglier, 1962).From the results obtained in table1, the relationship between surviving fraction and drug concentration was plotted to obtain the survival curve of the MCF7 breast cancer cell line and another of the HCT colon cancer cell line.The response parameter calculated was the IC 50 value which corresponds to the compound concentration causing 50% mortality in net cells as depicted in Table 1.In the new work, seven of the newly synthesized compounds 2, 6, 7, 8, 9, 10, 13, 14 were selected.The most of the screened compounds showed interesting cytotoxic activities compared to a reference drug, according to IC 50 (which concentration causing 50% inhibition of cell growth.Chemotherapy is a major therapeutic for the both localized and metastasized cancers.Covering all structural variations in these analogs, being of tetrahydropyrano [3,2b]xanthene-4,7-dione 2 on colon cell line HCT was more active compound (IC 50 : 3.1 µg/ml) and was found to be near to a reference drug (Doxorubicin, IC 50 : 3.7µg/ml).However the activity of compound 2 against MCF7 was (IC 50 : 4.2 µg/ml) has near to a reference drug (vinblastine IC 50 : 2.97µg/ml).Imidazole moiety attached to mercaptophenyl derivative 7 (IC 50 : 6.63 µg/ml) or to imidazotriazole derivatives 13 (IC 50 : 7.85 µg/ml) and 14 (IC 50 : 6.9 µg/ml) have moderate activity which have one half that of doxorubicin (IC 50 : 3.743 µg/ml) against HCT.For MCF7, compound 7 (IC 50 : 8.5 µg/ml), compound 13 (IC 50 : 9.2 µg/ml) and compound 14 (IC 50 : 8.8 µg/ml) which have one third that of vinblastine (IC 50 : 2.97 µg/ml) according to the presence of mercapto group.The antitumor activity of another compounds containing hetero atom as compound 8 (IC 50 : 10.5 µg/ml for MCF7, (C 50 : 11.2 µg/ml for HCT), compound 9 (IC 50 : 10.9 µg/ml for MCF7, IC 50 : 12.3 µg/ml for HCT) and compound 10 (IC 50 : 12 µg/ml for MCF7, IC 50 : 12.8 µg/ml for HCT) have one forth activity against MCF7 which that of vinblastine (IC 50 : 2.97µg/ml) and against HCT which have one third that of doxorubicin (IC 50 : 3.743 µg/ml) respectively.All have signification value according to presence of quinoxaline, pyrimidine and pyrazole moiety.Also compound 6 (IC 50 : 14 µg/ml for MCF7, IC 50 : 13.3 µg/ml for HCT) containing acetylphenyl group has less activity compare to the six compounds but has considering value against to antitumor activity.Furthermore, all testing compounds gave antitumor activity against colon cancer cell HCT more than the activity against breast cancer cell MCF7.Finally , the decreasing order of growth inhibitory activities against breast cancer cell MCF7 and colon cancer cell HCT in comparison to the known anticancer drugs are 2>14 >13 >7 >8 >9 >10>6.Table (1).

CONCLUSIONS
In this study, seven of the newly synthesized compounds 2, 6, 7, 8, 9, 10, 13, 14 were selected to evaluation their in vitro growth inhibitory activities against breast cancer cell MCF7 and colon cancer cell HCT in comparison to the known anticancer drugs: doxorubicin and vinblastine as reference drugs.In virto growth inhibitory activities of imidazole derivatives against MHF7 and HCT cell lines revealed significant potential antitumor activity.Best results were gained by compound 2 since it showed approximately similar potency against MCF7 and HCT respectively.Chromone derivatives containing Imidazole moiety attached to mercaptophenyl 7 or to imidazotriazole derivatives 13, 14 is moderate activity according to the presence of mercapto group.Hetero compounds as 7, 8 and 9 have signification value according to presence of quinoxaline, pyrimidine and pyrazole groups.Also, compound 6 containing acetylphenyl group has considerting value to antitumor activity.There is provided chromone derivatives of the general formula which an antitumor pharmaceutical composition comprising as an effective ingredient the novel chromone derivatives.

General methods
Melting points were determined on a Stuart melting point apparatus.IR spectra were recorded on a Shimadzu-440 IR spectrophotometer using the KBr technique (Shimadzu, Japan).NMR spectra were measured on a Bruker Avance 300 (300Hz) in DMSO-d 6 as a solvent, using tetramethylsilane (TMS) as an internal standard.The mass spectra were recorded on Shimadzu GCMS-QP-1000EX mass spectrometers at 70 eV.Elemental analyses were carried out by the Micro-analytical Research Centre, Cairo University.Analytical results for C, H and N were within ±0.4% of the calculated values.All reagents were of commercial quality and used without purification, follow up of the reaction and checking the purity of the compounds.

Materials and methods
The human tumor cell lines (MCF7 and HCT) were available at the regional center for mycology & biotechnology at Al-Azhar University, Cairo, Egypt.Mammalian cell lines: MCF7 and HCT cells (human cell line of a well differentiated breast and colon carcinoma isolated from a breast or colon biopsy of a male Caucasian aged 15 years) were obtained from the American Type Culture Collection (ATCC).Chemicals Used: Dimethyl sulfoxide (DMSO), crystal violet and trypan blue dye were purchased from Sigma (St. Louis, Mo., USA).Fetal Bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from Lonza.Crystal violet stain (1%): It composed of 0.5% (w/v) crystal violet and 50% methanol then made up to volume with dH 2 O and filtered through a Whatmann No.1 filter paper.

Procedure
The cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and 50 µg/ml genta-mycin.All cells were maintained at 37 º C in a humidified atmosphere with 5% CO 2 and were subcultured two times a week.Cell toxicity was monitored by determining the effect of the test samples on cell morphology and cell viability.
Cytotoxicity evaluation using viability assay (Mosmann, 1983 and Vijayan, 2004): For cytotoxicity assay, the cells were seeded in 96-well plate at a cell concentration of 1×10 4 cells per well in 100µl of growth medium.Fresh medium containing different concentrations of the test sample was added after 24 h of seeding.Serial two-fold dilutions of the tested chemical compound were added to confluent cell monolayers dispensed into 96-well, flatbottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette.The microtiter plates were incubated at 37 º C in a humidified incubator with 5% CO 2 for a period of 48 h.Three wells were used for each concentration of the test sample.Control cells were incubated without test sample and with or without DMSO.The little percentage of DMSO present in the wells (maximal 0.1%) has no effect on the cell line.After incubation of the cells for 24 h at 37°C, various concentrations of sample (50, 25, 12.5, 6.25, 3.125 & 1.56 µg) were added, and the incubation was continued for 48 h and viable cells yield was determined by a colorimetric method.In brief, after the end of the incubation period, media were aspirated and the crystal violet solution (1%) was added to each well for at least 30 minutes.The stain was removed and the plates were rinsed using tap water until all excess stain is removed.Glacial acetic acid (30%) was then added to all wells and mixed thoroughly, and then the absorbance of the plates were measured on Microplate reader (TECAN, Inc.), using a test wavelength of 490 nm.All results were corrected for background absorbance detected in wells without added stain.Treated samples were compared with the cell control in the absence of the tested compounds.All experiments were carried out in triplicate.The relation between surviving fraction and drug concentration was plotted to get the survival curve of each tumor cell line.The concentration required for 50% inhibition of cell viability (IC 50 ) was calculated and compared with the reference drug vinblastine and the results are given in table 1.

Table 1 .
In-vitro anticancer evaluation against human tumor breast and colon cell lines (MCF7 and HCT).
bValues are means of three experiments.