USES OF ACETOACETANILIDE FOR THE SYNTHESIS OF THIOPHENE DERIVATIVES WITH CYTOTOXIC ACTIVITIES

The reaction of acetoacetanilide 1 with elemental sulfur and malononitrile 2 gave the thiophene derivative 3. The latter was the key starting material for the synthesis of different thiophene and fused derivatives through its reaction with aryldiazonium chloride, benzaldehyde and acetic anhydride. Moreover, the reaction with phenylisothiocyanate produced fused derivative. The reaction of compound 3 with elemental sulfur produced dithiophene derivative. In addition, the reaction of compound 3 with ethylcyanoacetate produced Ncyanoacetamide derivative that was capable for further heterocyclizations through its reaction with different reagents. The cytotoxicity of the newly synthesized products was evaluated against the three cancer cell lines namely MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer) where some compounds showed optimal cytotoxicity. The results showed that compounds 5a, 5b, 8, 22, and 23 exhibited optimal cytotoxic effect against cancer cell lines.


INTRODUCTION
Thiophene derivatives represent a class of important and well-studied heterocycles [1]. The interest in this kind of heterocycles has spread from early dye chemistry [2] to modern drug design [3], biodiagnostics [4], electronic and optoelectronic devices [5], conductivity-based sensors [6], and self-assembled superstructures [7]. The general synthetic approaches to such kind of compounds either involve the functionalization at the positions a and b to the sulfur atom of the pre-constructed thiophene nucleus [8], or the construction of thiophene ring from appropriately substituted open chain precursors [9]. The latter becomes much attractive for its general applicability to achieve more complicated substitution patterns [10]. Gewald and coworkers developed the synthesis of 2-aminothiophenes from the multicomponent condensation of ketones or aldehydes, cyanoacetate and elemental sulfur [11]. Later on, there are several reviews and papers reported on the variations and improvements on the originally published Gewald's synthesis of polysubstituted thiophenes [12]. Recently, we were involved through comprehensive program involving the synthesis of thiophene [13,14] derivatives together with their further reactions with chemical reagents to give heterocyclic and fused heterocyclic derivatives with antitumor activities. Moreover, we synthesized different thiophene derivatives that have been screened for antitumor activity against breast adenocarcinoma (MCF-7), nonsmall cell lung cancer (NCI-H460) and CNS cancer (SF-268); this is besides studying their cytotoxicity against the normal human cell line normal fibroblast cells WI 38 [15]. In this work we make extension for this program through the synthesis of thiophene derivatives using acetoacetanilide and maononitrile in the presence of elemental sulfur to produce potentially antitumor thiophene derivatives [16][17][18][19][20][21][22][23]. Thus, compound 3 reacted with ethyl cyanoacetate 21 in refluxing dimethylformamide to give the amide derivative 22. The analytical and spectral data of compound 22 are in agreement with its structure. The reaction of 22 with benzaldehyde 6 gave the benzylidene derivative 23. Moreover, the reaction of 22 with salicyladehyde 24 gave the coumarin derivative 25. The excellent yield of compound 22 encouraged us to study its reactivity towards aromatic diazonium salts in the aim of synthesizing new aryl hydrazine derivatives with different cytotoxic activities. Thus, the reaction of 22 with aromatic diazonium salts 4a-d gave the arylhydrazone derivatives 26a-d. The analytical and spectral data of the latter products were the

Structure activity relationship
From Table 1 it is clear that the benzimidazole moiety was found to be crucial for the cytotoxic effect of the cyclic compounds 3 to 27. Compounds 5a, 5b, 8, 22, and 23 exhibited optimal cytotoxic effect against cancer cell lines. On the other hand compound 9c, 26d and 27 showed very low activities toward the three cancer cell lines. Compounds 3, 5c, 5d, 7, 9d, 14, 20, 25 and 26c showed moderate activities. The thiophene derivative 3 showed low potency, however its reaction with difference aryl diazonium salts gave the arylhydrazone derivatives 5a-c. It is obvious that compounds 5a (R = H) and 5b (R = CH 3 ) showed the highest cytotoxicity among the three compounds. The reaction of compound 3 with benzaldehyde produced compound 7 with low potency. The thiophene derivative 7 showed low potency although its acetylation gave compound 8 with high potency against the three cancer cell lines. For the thiophenes derivatives 9a-d, it is clear that compound 9b with 6-the 4-methylphenyl group showed the highest activity among the four compounds which is considered as a moderate activity. Compound 12 which was a thieno[d]pyrimidine derivative showed highest activity among the tested compounds. Similarly, compound 17 which is a thieno[b]pyridine derivative showed high potency towards the three cancer cell lines. Compounds 22, 23, 26a and 26b showed relatively high potency and such activity is attributed to the presence of the N-acyl moieties. From the activity of the compounds towards the three cancer cell lines it is obvious that the presence of N-rich and Scontaining heterocyclic ring was the main effect through the high potency of the compound.  Tumor cell growth assay. The effects of 3-27 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 M. 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% (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.

Instruments
All melting points are uncorrected. IR spectra were recorded for KBr discs on a PyeUnicam SP-1000 spectrophotometer. 1 H NMR spectra were measured on a Varian EM-390-200 MHz in DMSO as solvent using TMS as internal standard and chemical shifts are expressed as δ. Analytical data were obtained from the Microanalytical Data Unit at Cairo University, Giza, Egypt.

5-Amino-4-cyano-N-phenyl-3-styrylthiophene-2-carboxamide (7)
To a solution of 3 (2.57 g, 0.01 mol) in 1,4-dioxane (40 mL) containing piperidine (1.0 mL), benzaldehyde (1.0 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 8 h then left to cool. The solid product formed upon evaporating the solution under vacuum followed by triturating the remaining product with ethanol, was collected by filtration and dried. The obtained product was crystallized from ethanol to give pale brown crystals.

5-Acetamido-4-cyano-3-methyl-N-phenylthiophene-2-carboxamide (8)
To a solution of 3 (2.57 g, 0.01 mol) in acetic acid (30 mL), acetic anhydride (1.02 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h then left to cool. The solid product formed upon pouring onto ice/water containing a few drops of hydrochloric acid was collected by filtration and dried. The obtained product was crystallized from ethanol to give pale yellow crystals.  The formed solid product, in each case, upon stirring at room temperature for 1 h was collected by filtration and dried. The obtained product was crystallized from ethanol to give yellowish brown crystals for 9a and 9b and reddish yellow crystals for 9c and 9d.

General procedure for the synthesis of 2-cyanoacetamido)-4-cyano-3-methyl-Nphenylthiophene-2-carboxamide derivatives 26a-d
To a cold solution of 22 (3.24 g, 0.01 mol) in 1,4-dioxane (40 mL) containing sodium hydroxide (2.5 g) a cold solution of the respective diazonium salt [prepared by the addition of sodium nitrite solution (0.70 g, 0.01 mol) to a cold solution of either aniline (0.94 g, 0.01 mol), ptoluidine (1.15 g, 0.01 mol), p-methoxy aniline (1.3 g, 0.01 mol) or p-chloroaniline (1.29 g, 0.01 mol) in concentrated hydrochloric acid (12 mL) with continuous stirring]was added while stirring. The formed solid product, in each case, upon stirring at room temperature for 1 h was collected by filtration and dried. The obtained product was crystallized from ethanol to give brown crystals for 26a and 26c and black crystals for 26b and 26d.