The Synthesis and Cytotoxicity of Novel Thiophene Derivatives Derived from 2-( 4-Oxo-4 , 4-Dihydrothiazol-2-yl ) Acetonitrile

The reaction of the 2-(4-oxo-4,4-dihydrothiazol-2-yl)acetonitrile 1 with cyaclopentanone (2) afforded the condensed product 3. The latter underwent a series of heterocyclizations through its reaction with different reagents. Moreover, compound 1 underwent the Gewald’s thiophene to afford compounds 15 and 17. The reaction of either hydrazine hydrate or phenylhydrazine with compound 17 gave the hydrazide derivatives 19a and 19b, respectively. The cytotoxicity of the newly synthesized products was measured towards the three cancer cell lines MCF-7, NCI-H460 and SF-268. The study showed that compounds 3, 5, 9c, 11, 13a, 13c, 17 and 19b were the most active compounds towards the three cancer cell lines.


Introduction
Although the number of drugs is available in the market, the need of discovering the new anti-tumor drugs with better pharmacokinetic profile and lesser toxicity has become the main objective in the field of medicinal chemistry, and it is also due to the fast microbial resistance to the existing molecules [1]- [3].A large number of compounds containing thiophene system have been investigated because of their broad spectrum of biological activities which include analgesic [4], antibacterial [5], antifungal [6], antiparasitic [7], antiviral [8], anti-inflammatory [9], anticonvulsant [10], anti-nociceptive [11], DNA cleavage [12], herbicidal [13], antitubercular [14], protein kinase inhibition [15], respiratory syndrome protease inactivation [16], an active ester in the peptide synthesis and agonists of peroxisome proliferator activated receptors [17].In the present work, we study the reactivity of compound 3 resulting from reaction of the 2-(4-oxo-4,4-dihydrothiazol-2-yl)acetonitrile (1) with cyclopentanone to produce novel thiophene derivatives together with cytotoxic evaluations of the newly synthesized products towards different cell lines.

Chemistry
The reaction of the 2-(4-oxo-4,4-dihydrothiazol-2-yl)acetonitrile (1) with cyaclopentanone (2) in the presence of ammonium acetate at 120˚C gave the Knoevenagel condensation compound 3.The structure of the compound 3 was confirmed on the basis of analytical and spectral data.The reaction of compound 3 with elemental sulphur in the presence of ethanol and triethylamine gave the 4,5,6,7-tetrahydrobenzo[b]thiophene derivative 4. The 2-amino group present in compound 4 showed interesting reactivity as primary aromatic amine.Thus, compound 4 reacted with acetic anhydride in presence of acetic acid gave the N-acetyl derivative 5. On the other hand the reaction of compound 4 with ethyl cyanoacetate 6 gave the N-cyanomethylacetamide derivatives 7. The analytical and spectral data are the tools of the structure elucidation of compound 7. Thus, the 1 H NMR spectrum showed a multiplet at δ1.18 -1.69 ppm indicating the cyclopentene three CH 2 , a singlet at δ2.50 ppm corresponding to the CH 2 group, a singlet at δ4.29 ppm for the thiazol CH 2 , and a singlet at δ8.27 ppm for the NH group (Figure 1).
The high yield of compound 4 encouraged us to study its further reactivity towards some chemical reagents.Thus, the reaction of 4 with any of benzene diazoniumchloride 8a, 4-chlorobenzene-diazonium chloride 8b or 4-methoxybenzene-diazonium chloride 8c in the presence of ethanol and sodiumhydroxide gave the arylhydrazonederivatives 9a-c, respectively.The analytical and spectral data of the latter products are consistent with their respective structures.On the other hand, compound 4 is capable for diazotization and coupling.Thus, compound 4 reacted with sodium nitrite in the presence of sodium nitrite and acetic acid at 0˚C -5˚C gave the non isolablediazonium salt 10.The latter coupled with acetylacetone to give the hydrazoderivative 11.

Antitumor and Normal Cell Line Activity Tests
The cytotoxicity of the synthesized compounds was tested for Three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small 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 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL), at 37˚C in a humidified atmosphere containing 5% CO 2 .Exponentially growing cells were obtained by plating 1.5 × 10 5 cells/mL for MCF-7 and SF-268 and 0.75 × 10 4 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.
The effects of 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 (12).Briefly, exponentially, cells growing in 96-wellplates 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., Powerwave XS, Wincoski, USA).For each test compound and cell line, a dose-response curve was obtained and the growth inhibition of 50% (IC 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.

Structure Activity Relationship
It is clear from Table 1 that compounds 3, 5, 9c, 11, 13a, 13c, 17 and 19b were the most active compounds towards the three cancer cell lines with IC50's against MCF-7 cell line (as an example) 0.02, 0.2, 0.01 1.6, 0.6, 0.4, 0.01 and 0.20, respectively.On the other hand, compounds 12, 13b and 19a were of moderate activities with IC50's against MCF-7 6.1, 11.1 and 10.6, respectively.The rest of compounds showed low activity.Consider compounds 9a-c it is clear that compound 9c showed the highest activity among the three compounds which is attributed to the presence of the OCH 3 group.Considering compounds 13a, b it is clear that compound 13a with the 4-CH 3 group showed higher activity than 13b with the 4-Cl group.For the hydrazide derivatives 19a, b it is obvious that compound 19b with the phenyl moiety is more potent than compound 19a.

Conclusion
We have reported a convenient synthesis of variety compounds from compound 1 to 19b derivatives.The cyto- toxicity of some derivatives towards three types of cancer cell lines were studied most of the synthesized compounds were found to be cytotoxic and hence deserve further pharmacological investigation.The results of these investigation will be published in due time.

Experimental
All melting points determined on an electrothermal digital melting point apparatus and are uncorrected.

Conclusion
We have reported a convenient synthesis of variety compounds from compound 1 to 19b derivatives.The cytotoxicity of some derivatives towards three types of cancer cell lines was studied.Most of the synthesized compounds were found to be cytotoxic and hence deserve further pharmacological investigation.Compounds 3, 5, 9c, 11, 13a, 13c, 17 and 19b were the most active compounds towards the three cancer cell lines.The results of these investigations will be published in due time.

Table 1 .
Effect of the synthesized compounds on the growth of three human tumor cell lines.