THE USES OF ETHYL 2-(1H-BENZO[D]IMIDAZOL-2-YL)ACETATE TO SYNTHESIS PYRAZOLE, THIOPHENE, PYRIDINE AND COUMARIN DERIVATIVES WITH ANTITUMOR ACTIVITIES

In the present work, the ethyl 2-(1H-benzo[d]imidazol-2-yl)acetate (3) was subjected to a series of heterocyclization reactions through its reaction with different chemical reagents. The resulting molecules were thiophene, pyrazole, coumarin derivatives incorporated benzo[d]imidazole moiety. All the synthesized compounds were determined by elemental analysis, H NMR, C NMR, and MS. The antitumor evaluations of the newly synthesized products toward the three cancer cell lines MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer) showed that compounds 5a, 9b, 9c, 17, 23b and 38 were of the highest potencies among the synthesized compounds.


INTRODUCTION
In recent years, benzimidazole derivatives have provided a large number of biologically active compounds that have been intensively used in medicinal chemistry as drugs. They are structural isosteres of naturally occurring nucleotides, which allow them to interact easily with the biopolymers of the living system and different kinds of biological activity have been obtained. Some 2-aminobenzimidazoles displayed an appreciable antimicrobial effect. Their corresponding carbamate derivatives have been synthesized for their significant in vivo antifilarial activity [1]. Concerning the high affinity that they display towards a variety of enzymes and protein receptors, they could be considered as pivotal structures in drug design [2]. Optimization of benzimidazole-based structures has resulted in marketed drugs, e.g. Omeprazole [3] and Pimobendan [4] that are therapeutically useful in the management of peptic ulcer and congestive heart failure respectively. Many derivatives of benzimidazoles are well known for their antimicrobial [5,6] anthelmintic [7] antiviral [8,9] and antifungal [10,11] activities. Since 1985, benzimidazole containing compounds have been reported as well known anticancer agents [12,13]. The role of mammalian DNA topoisomerases as molecular targets showed the presence of a singlet at  5.90 ppm indicating the presence of the pyrimidine CH 2 group and a multiplet at  7.26-7.38 ppm for the C 6   The reactivity of compound 3 towards thiophene synthesis using the Gewald's thiophene synthesis was studied. Thus, the reaction of compound 3 with elemental sulfur and either of malononitrile (10) or ethyl cyanoacetate (22) gave the thiophene derivatives 23a and 23b, respectively. Moreover, the reaction of compound 3 with ethylorthoformate (24) gave the ethoxymethylyeno derivatives 25. On the other hand, the multi-component reaction of compound 3 with ethyl orthoformate (24) and aniline (4a) gave the phenylaminomethyleno derivative 26. The analytical and spectral data of compounds 25 and 26 were consistent with their respective structures (see experimental section). The multi-component reaction (MCRs) of compound 3 with ethyl orthoformate (24) and malononitrile (10) gave the ethyl 1-amino-2cyano-3-ethoxybenzo [4,5] Further confirmations for the structure of compound 30 were obtained through studying its reactivity towards some chemical reagents. Thus, compound 30 reacted with either chloroacetone (31a) or ethyl -cholroacetate (31b) to give the 1H-benzo [4,5]imidazo [1,2a]chromeno [3,4-c]pyridin-1-one derivatives 32a and 32b, respectively. Formation of the latter product was explained in terms of the first reaction of the NH of compound 30 with the -chloro group of 32a or 32b followed by either elimination of water or ethanol, respectively. Moreover, the reaction of compound 30 with chloroacetylchloride (33) gave the 8-hydroxy-1Hbenzo [4,5]imidazo [1,2-a]chromeno [3,4-c]pyridin-1-one 35 its analytical and spectral data confirm the proposed structure. The reaction of compound 30 with acetic anhydride (36) gave the N-acetyl derivative 37. Our trials to make cyclization of 37 under different conditions were failed. Finally, the reaction of compound 30 with phenylisothiocyanate (20) gave the Nphenylthiourea derivative 38 (Scheme 4).
Cell cultures. Three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (nonsmall cell lung cancer), and SF-268 (CNS cancer) were used. MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK), NCI-H460, SF-268 and normal fibroblast cells (WI 38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 M glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL), at 37 o C in a humidified atmosphere containing 5% CO 2 . Exponentially growing cells were obtained by plating 1.5 x 10 5 cells/mL for MCF-7 and SF-268 and 0.75 x 104 cells/mL for NCI-H460, followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.
Tumor cell growth assay. The effects of the synthesized compounds on the in vitro growth of human tumor cell lines were evaluated according to the procedure adopted by the National Cancer Institute (NCI, USA) in the 'In vitro Anticancer Drug Discovery Screen' that uses the protein-binding dye sulforhodamine B to assess cell growth. Briefly, exponentially, cells growing in 96-well plates were then exposed for 48 h to five serial concentrations of each compound, starting from a maximum concentration of 150 µM. Following this exposure period adherent cells were fixed, washed, and stained. The bound stain was solubilized and the absorbance was measured at 492 nm in a plate reader (Bio-Tek Instruments Inc., Power wave XS, Wincoski, USA). For each test compound and cell line, a dose-response curve was obtained and the growth inhibition of 50% (GI 50 ), corresponding to the concentration of the compounds that inhibited 50% of the net cell growth was calculated as described elsewhere. Doxorubicin was used as a positive control and tested in the same manner.
Results are given in cconcentrations that were able to cause 50% of cell growth inhibition (GI 50 ) after a continuous exposure of 48 h and show means ± SEM of three-independent experiments performed in duplicate.

Structure activity relation ship
It is clear from Table 1 that many of the synthesized compounds showed high activity against the three cancer cell lines. It is obvious that compound 5a showed higher potency than compound 5b as it seemed that the presence of the 4-methylphenyl group responsible for the decreasing of compound 5b. On the other hand the compounds 7a-c showed low potencies against the three cancer cell lines. Considering the pyrazole derivatives 9a-c, it is obvious that compounds 9b (R = H, X = Cl) and compound 9c (R = Ph, X = H) revealed high potencies against the three cancer cell lines whereas compound 11 showed low inhibitions. Considering compounds 13, 5a,b and 17 it is clear that the ethyl 1,3-dimethylbenzo [4,5]imidazo[1,2a]pyridine-4-carboxylate (17) showed the highest inhibitions among the four compounds. The same also for compounds 18, 19, 21, 23a and 23b, it is clear that compound 23b showed the highest inhibitions and its reactivity was due to the presence the COOEt group attached to the thiophene ring. Considering compounds 25, 26, 28 and 30, it is clear that compound 30 was the only one with high potencies. For compounds 32a, 32b, 37 and 38 it is obvious from Table 1 that the 2-(2-oxo-2H-chromen-3-yl)-N-phenyl-1H-benzo[d]imidazole-1-carbothioamide 38 was of the highest potency among these four compounds. Its reactivity is attributed to the presence of the coumarin and the N-phenylthiourea moieties. From Table 1 it is clear that the cytotoxic effect of the cyclic compounds. Compounds 5a, 9a, 9b, 17, 23a, 23b, 30, 38 exhibited optimal cytotoxic effect against cancer cell lines, with GI 50 ,s in the M range. On the other hand, compounds 11, 13, 15a, 15b, 18, 19, 21, 25, 27, 29, 32a, 32b, 35 and 37 showed low cytotoxicity effect toward the three cancer lines. It is clear from the demostracted "SAR" that the presence of electronegative groups together with the nature of the heteroatom enhances the antitumor effect of the target molecules.

EXPERIMENTAL General
All melting points were determined on an electrothermal apparatus (Büchi 535, Switzerland) in an open capillary tube and are uncorrected. 13 C-NMR and 1 H-NMR spectra were recorded on Bruker DPX200 instrument in DMSO with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values are mentioned in δ (ppm). Mass spectra were recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument. Elemental analyses were carried out by the Microanalytical Data Unit Ludwig-Maximilians-Universitat-Munchen, Germany. The progress of all reactions was monitored by TLC on 2 x 5 cm pre-coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck).

Ethyl 2-(1H-benzo[d]imidazol-2-yl)acetate (3)
To the dry solid of o-phenylene diamine (1.08 g, 0.01 mol) diethylmalonate (1.60 g, 0.01 mol) was added. The reaction mixture was heated in an oil bath at 120 o C for 1 h then left to cool. The remaining product was triturated with ethanol and the formed solid product was collected by filtration. Brown crystals from ethanol; yield: 1.73 g (85%); m.p. General procedure for the synthesis of the anilide derivatives 5a,b To a solution of compound 3 (2.04 g, 0.01 mol) in dimethylformamide (30 mL) either aniline (0.93 g, 0.01 mol) or 4-methylaniline (1.08 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 4 h then poured onto ice/water mixture containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

General procedure for the synthesis of the pyrazole derivatives 9a-c
To a solution of either compound 7a (3.22 g, 0.01 mol) with either of hydrazine hydrate (0.50 g, 0.01 mol) or phenylhydrazine (1.08 g, 0.01 mol) or compound 7c (3.26 g, 0.01 mol) with hydrazine hydrate (0.50 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 6 h then poured onto ice/water mixture containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

General procedure for the synthesis of the ylidene derivatives 15a, b
To the dry solid of compound 3 (2.04 g, 0.01 mol) ammonium acetate (0.50 g) either cyclophentanone (0.78 g, 0.01 mol) or cyclohexanone (0.92 g, 0.01 mol) were added. The reaction mixture was heated in an oil bath at 120 o C for 1 h then left to cool. The product was triturated with ethanol and the formed solid product was collected by filtration.

General procedure for the synthesis of the thiophene derivatives 23a,b
To a solution of compound 3 (2.04 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (0.50 mL) elemental sulfur or either of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 4 h then poured onto ice/water mixture containing few drops of hydrochloric acid and the formed solid product was collected by filtration.

CONCLUSION
Ethyl 2-(1H-benzo[d]imidazol-2-yl)acetate (3) was used for several heterocyclic transformation reactions. The antitumor evaluations of the newly synthesized products toward the three cancer cell lines MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer) showed that compounds 5a, 9b, 9c, 17, 23b and 38 were of the highest potencies among the synthesized compounds. The work showed that benzimidazole derivatives were excellent compounds that can be used as anticancer agents.