SYNTHESIS AND CYTOTOXICITY OF NOVEL THIOPHENE , PYRAN AND PYRIDINE DERIVATIVES

The reaction of 2-amino-3-cyano-tetrahydrobenzothiophene 3 with ethyl acetoacetate gave the amide derivatives 5. The reactivity of 5 toward a variety of chemical reagents was studied to give pyrans, pyridines, thiophenes and the thiazoles and their fused derivatives. The structures of the newly synthesized products were confirmed on the basis of their respective analytical and spectral data. The antitumor evaluations of the synthesized compounds against the three cancer cell lines MCF-7, NCI-H460 and SF-268 showed that compounds 9a, 9c, 12a and 14 were of the highest potencies against the three cancer cell lines among the tested compounds.


INTRODUCTION
Aromatic thiophenes play on part in animal metabolism; for examples, Biotin, one of the vitamins (Vitamin H) (Figure 1), is a tetrahydrothiophene, however aromatic thiophenes do occur in some plants, in association with polyacetylenes with which they are biogenetically linked and Banminth (pyrantel), available anthelmintic used in animal husbandry, is one of the thiophene compounds in chemotherapy.Thiophenes with a wide spectrum of biological activities are known, several of these derivatives possess potent analgesic [1,2], anticonvulsant, anti-inflammatory and antibacterial [3][4][5][6], antipyretics [7], antitumor [8,9], antiparasitic [10], antimicrobial [11], antihistaminic (H1) [12], antianexiety test in mice [13], antiarrhythmic [14] and serotonin antagonist [15].In previous work we have found that certain substituted thiophenes and their heterocyclic derivatives show antitumor activities [16][17][18].In the present work we were aiming the synthesis of new heterocyclic derivatives derived from the 2-amino-3cyano-4,5,6,7-tetrahydrobenzo[b]thiophene followed by studying the cytotoxicity of the newly synthesized compounds. .Moreover, the multi-component reaction of compound 5 with any of the aromatic aldehydes namely benzaldehyde (6a), 4chlorobenzaldehyde (6b) or 4-methoxybenzaldehyde (6c) in 1,4-dioxane containing ammonium acetate gave the pyridine derivatives 8a-c.Compounds 7a-c each with the acetyl group at the pyran ring found to be capable for the Gewald's thiophene synthesis.Thus, any of compounds 7a-c reacted with malononitrile and elemental sulfur to give the thiophene derivatives 9a-c, respectively.The analytical and spectral data of 9a-c were consistent with their respective structures as depicted in Scheme 2. Thus, the 1  Similarly, the pyridine derivatives 8a-c reacted with malononitrile and elemental sulfur to give the thiophene derivatives 10a-c, respectively.The reaction of compound 5 with either of malononitrile (2) or ethyl cyanoacetate (11) and elemental sulfur in 1,4-dioxane containing a catalytic amount of triethylamine gave the thiophene derivatives 12a and 12b, respectively.Analytical and spectral data were the basis of their respective structure elucidation.Finally, the reaction of compound 5 with thioglycollic acid (13) gave the thiazole derivative 14 (Scheme 3).
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 105 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 newly synthesized products 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 concentrations 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 relationship
It is clear from Table 1 that the 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (3) showed high cytotoxicity against MCF-7 with GI 50 0.30 mol L -1 but it showed low cytotoxicity against NCI-H460.The reaction of compound 3 with ethyl acetoacetate gave the amide derivative 5 which showed moderate cytotoxicity against NCI-H460 and SF-268 with GI 50 's 3.1 and 1.96 mol L -1 .For the multi-component products 7a-c it is obvious that compound 7c (X = OCH 3 ) showed the highest cytotoxicity among the three compounds.On the other hand, the pyridine derivatives 8a-c were of low potency towards the three cancer lines.The reaction of any of compounds 7a-c with elemental sulfur and malononitrile to yield the thiophene derivatives 9a-c where compounds 9a (X = H) and 9c (X = OCH 3 ) showed the highest cytotoxicity among the three compounds.On the other hand, compounds 10a-c showed low potencies.Considering the dithiophene derivatives 12a,b, it is clear that compound 12a with X = CN showed the higher potency than 12b, X = COOEt.It is clear that the presence of the CN group is responsible for its reactivity.Finally, the thiazole derivative 14 showed high potency against the three cancer cell lines.Table 1.Effect of newly synthesized compounds on the growth of three human tumor cell lines.

Chemistry
All melting points were determined on an Electrothermal digital melting point apparatus and are uncorrected.IR spectra (KBr discs) were recorded on a FTIR plus 460 or Pyeunicam SP-1000 spectrophotometer. 1 H NMR spectra were recorded with Mercury-300BB (300 MHz) (Cairo University) instrument in DMSO-d 6 as solvent using TMS as internal standard and chemical shifts are expressed as  ppm. 13C-NMR spectra were recorded in DMSO.Analytical data were obtained from the micro analytical data unit at Cairo University and were performed on Vario El III Elemental CHNS analyzer.
N- 5,6,thiophen-2-yl)-3-oxobutanamide (5) Ethyl acetoacetate (13.0 g, 0.1 mol) was heated till 140 o C then compound 3 (17.8g, 0.1 mol) was added with continuous heating till the temperature reach 125 o C at this moment the whole reaction mixture is heated under reflux for 20 min.The reaction mixture was allowed to cool overnight and the formed solid product was filtered, dried and crystallized from ethanol to give compound 5.