The Reaction of Cyclopentanone with Cyanomethylene Reagents : Novel Synthesis of Pyrazole , Thiophene , and Pyridazine Derivatives

The reaction of cyclopentanone with either malononitrile or ethyl cyanoacetate gave the corresponding condensated products.The latter underwent some heterocyclic reactions to give new pyrazole, thiophene, and pyridazine derivatives.The antitumor evaluation of the newly synthesized products against the three cancer cells, namely, breast adenocarcinoma (MCF-7), nonsmall cell lung cancer (NCI-H460), andCNS cancer (SF-268) showed that some of themhave high inhibitory effect towards three cell lines which is higher than the standard.


Experimental
Melting points were determined on an Electrothermal melting point apparatus (Electrothermal 9100) and are uncorrected.IR spectra were recorded for KBr discs on a Pye Unicam SP-1000 spectrophotometer. 1 H NMR spectra were measured on a Varian EM-390 at 200 MHz in DMSO- 6 as solvent using TMS as internal standard.The mass spectra were recorded with Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu instruments.Analytical data were obtained from the Microanalytical Data Unit at Cairo University, Giza, Egypt.Antitumor evaluation for the newly synthesized products was performed by a research group at the National Research Center and the National Cancer Institute at Cairo University.Fetal bovine serum (FBS) and l-glutamine were from Gibco Invitrogen Co. (Scotland, UK).RPMI-1640 medium was from Cambrex (New Jersey, NJ, USA).Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin, and sulforhodamine B (SRB) were from Sigma Chemical Co.(Saint Louis, MO, USA).Stock solutions of all compounds were prepared in DMSO and kept at −20 ∘ C. Appropriate dilutions of the compounds were freshly prepared just prior to assays.Final concentrations of DMSO did not interfere with the cell growth.MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK), and NCI-H460 and SF-268 were kindly provided  1. Spectral data were inserted through Table 2.

2-Cyclopentylidenemalononitrile 3a and Ethyl 2-Cyano-2cyclopentylideneacetate 3b
General Procedure.To a dry solution of cyclopentanone 1 (0.84 g, 0.01 mol) either malononitrile 2a (0.66 g, 0.01 mol) or  ethyl cyanoacetate 2b (1.13 g, 0.01 mol) was added followed by the addition of ammonium acetate (0.50 g).The reaction mixture, in each case, was heated in an oil bath at 120 ∘ C then left to cool.The solidified product was triturated with ethanol, and the formed solid product was collected by filtration to give compounds 3a,b.

Results and Discussions
The reaction of cyclopentanone with either malononitrile or ethylcyanoacetate gave the Knoevenagel condensated products 3a and 3b, respectively.The structures of the latter products were based on analytical and spectral data.Thus, the 1 H NMR spectrum of 3a showed a multiplet at  1.34-2.44indicating the four CH 2 groups.Next, we studied the reaction of either 3a or 3b with either benzenediazonium chloride or p-chlorobenzenediazonium chloride.The reaction was carried out in ethanol 0-5 ∘ C and afforded the corresponding arylhydrazone derivatives 5a-d, respectively.Either compound 5a or 5c reacts with either hydrazine hydrate or phenylhydrazine to give the corresponding pyrazole derivatives 7a-d, respectively (Scheme 1).The analytical and spectral data of the latter products are in agreement with the proposed structures.Thus, the Scheme 2 The reaction of either (9a) or 9b with either malononitrile or ethyl cyanoacetate gave the benzocyclopentane derivatives 11a-d, respectively.Formation of the latter products is explained in terms of the first addition of the cyanomethylene reagent to the ylidene moiety followed by the Micheal addition of the CH group to the nitrile and subsequent elimination of HCN.Structures of 11a-d were confirmed on the basis of their analytical and spectral data, respectively.Thus, the 1 H NMR spectrum of 11a showed a multiplet at  1.30-2.36ppm, a singlet at  4.48 ppm for the NH 2 group, and a multiplet at  7.26-7.38ppm indicating the C 6 H 5 group.Further confirmations for the structures of such compounds were obtained through the reaction of either 3a or 3b with either -cyanocinnamonitrile 12a or ethyl -cyanocinnamate 12b to give the same products 11a-d, respectively (identical finger print IR, mixed m.p.).
Next, we moved towards studying reactivity of either 3a or 3b towards Gewald's thiophene synthesis.Thus, the reaction of either 3a or 3b with elemental sulphur in 1,4-dioxane and the presence of a catalytic amount of triethylamine gave the cyclopenta[]thiophene derivatives 13a and 13b, respectively (Scheme 2).The latter products were obtained previously by Mohareb and Al-Farouk [24] using another reaction route.Moreover, carrying the same reaction with the arylhydrazone derivatives 5a and 5b gave the thienocyclopentene derivatives 14a and 14b, respectively.Compounds 14a and 14b were reacted with acetic anhydride in dimethyl formamide to give the N-acetyl products 15a and 15b, respectively.Compounds 5a,b underwent ready cyclization in 1,4-dioxane solution containing triethylamine to give the cyclopenta[]pyridazine derivatives 16a and 16b, respectively (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), and NCI-H460 and SF-268 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 g/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.

4.3.
Tumor Cell Growth Assay.The effects of 3a,b-15a,b 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 [25].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 minimum concentration 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 [26].
For our newly synthesized products, we selected the three cancer cell lines the breast adenocarcinoma (MCF-7), nonsmall cell lung cancer (NCI-H460), and CNS cancer (SF-268) as our compounds are electron reach systems substituted with electronegative groups, and many reports from our previous work and others [25] used such cell lines together with the use of doxorubicin which was showed to be the best positive control against the three cell lines.not shown).The p-chlorophenylhydrazone derivative 15a, the indene derivatives 11d, and the pyridazin-6-one derivative 15b showed the best results among the tested compounds, and such reactivity is higher than the standard doxorubicin.On the other hand, compounds 3b, 7c, 9a, 9b, 12a, 12b, 13a, 14a, 15a, 16a, and 16b showed moderated growth inhibitory effect.

Materials
Comparing the activities of 15a and 15b, it is observed that the chloro group in 15b presents a stronger growth inhibitory effect than the amino substituent in 15a, although the results in NCI-H460 cell line are comparable.It is clear from Table 3 that some compounds like 7a, 7b, and 11a showed very low activity towards the three cancer cell lines.
Comparing the reactivities of compounds 11a-d, it is obvious that compound 11d with X = Y = COOEt showed the highest inhibitory effect among the four compounds.On the other hand, considering the cyclopentenopyridazine derivatives 15a and 15b, the presence of the electronegative C=N group is responsible for the higher cytotoxicity of 15b over 15a.It is clear from Table 3 that compounds 5b, 11d, and 15b showed the highest cytotoxicity among the newly synthesized products, and such activity is higher than that of the standard material doxorubicin.

Conclusions
In this work, we succeeded to synthesis a series of fused thiophene derivatives.The cytotoxicity of the newly synthesized products showed that compounds 5b, 11d, and 15b are the most active compounds towards the three cancer cell lines.

Table 1 :
Physical and microanalysis of the newly synthesized compounds 3a-16b.
by the National Cancer Institute (NCI, Cairo, Egypt).The physical properties, yield %, solvent of crystallization, and microanalytical data of the synthesized products were indicated through Table

Table 2 :
Spectral data of the newly synthesized products.

Table 3 .
All the compounds were able to inhibit the growth of the human tumor cell lines in a dose-dependent manner (data

Table 3 :
Effect of compounds 3a,b-15a,b on the growth of three human tumor cell lines.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.