The Reaction of Cyanoacetylhdrazine with Furan-2-Aldehyde : Novel Synthesis of Thiophene , Azole , Azine and Coumarin Derivatives and Their Antitumor Evaluation

The reaction of cyanoacetylhydrazine 1 with furan-2-aldehyde 2 gives the hydrazide-hydrazone derivative 3. The latter compound undergoes a series of heterocyclization reactions to give new heterocyclic compounds. The antitumor evaluation of the newly synthesized products against three cancer cell lines, namely breast adenocarcinoma (MCF-7), nonsmall cell lung cancer (NCI-H460) and CNS cancer (SF-268) were recorded. Some of the synthesized compounds show high inhibitory effects.


Introduction
Substituted aminothioxomethyl hydrazides are important building blocks in synthetic heterocyclic chemistry.The S/N regioselective nucleophilic competition in the synthesis of heterocyclic systems by intermolecular and intramolecular cyclization, as well as the change in reaction conditions which might favor N-attack or S-attack or even attack on the substituted terminals are important factors for the diversity of the produced heterocyclic systems from the title reaction precursor.On the other hand, the synthetic potential and biological activity of several heterocyclic related to the named hydrazides have been explored to the maximum extent.Among the pharmacological profiles are their antimicrobial [1,2], antitubercular [3,4], anticonvulsant [5,6] anti-inflammatory [7,8], antidepressant [9], antitumor [10], and analgesic activities [11].Continuing our interest in developing new heterocyclic systems based on hydrazide-hydrazones [12,13] as well as design and synthesis of heterocyclic with promising biological activities [14][15][16][17], we focused our work on developing novel polyfunctionalized heterocyclic compounds with potential bioactivity.

Results and Discussion
In this work we report the reaction of cyanoacetylhydra-zine (1) with furan-2-aldehyde (2) to give the hydrazidehydrazone derivative 3, the structure of which was confirmed on the basis of analytical and spectral data.Thus, the 1 H NMR spectrum showed a singlet at δ 6.60 (2H) ppm corresponding to CH 2 group, a multiplet at δ 6.52 -7.88 (3H) ppm corresponding to furan protons, a singlet at δ 7.54 (1H) ppm corresponding to CH group, and a singlet (D 2 O-exchangeable) at δ 11.62 (1H) ppm corresponding to NH group.
The formed hydrazide-hydrazone derivative 3 was subjected through a series of heterocyclization reactions to form products with expected pharmaceutical applications.Thus, condensation of 3 with benzaldehyde (4) in ethanolic/piperidene solution gave the benzylidene derivative 5.The reaction of compound 5 with either hydrazine hydrate (6a) or phenylhydrazine (6b) gave the pyrazole derivatives 7a and 7b, respectively.
On the other hand, the reaction of compound 5 with either malononitrile (8a) or ethyl cyanoacetate (8b) gave the pyridone derivatives 10a and 10b, respectively.The reaction took place via 1,6-intramolecular dipolar cyclization of the intermediate 9a,b (cf.Scheme 1), analyticcal and spectral data of the latter compounds were consistent with the assigned structures.Thus, the 1 H NMR spectrum of 10b showed, a triplet at δ 1.29 (3H) ppm indicating the presence of the CH 3 group, a quartet at δ 4.24 (2H) ppm corresponding to CH 2 group, a multiplet at δ 6.52 -7.88 (3H) ppm for furan protons, a singlet (D 2 O-exchangeable) at δ 7.05 (2H) ppm equivalent to the NH 2 group, a multiplet at δ 7.17 other hand, compound 3 condensed with salicylaldehyde (13) to give the coumarin derivative 14.The analytical and spectral data of compound 14 are in agreement with the proposed structure.Thus, the 1 H NMR spectrum showed, a singlet at δ 6.95 (1H) ppm corresponding to coumarin H-4, a singlet at δ 7.52 (1H) ppm corresponding to CH group, a multiplet at δ 6.52 -7.88 (3H) ppm corresponding to furan protons, a multiplet at δ 7.33 -7.55 (4H) ppm corresponding to aromatic protons and a singlet (D 2 O-exchangeable) at δ 11.61 (1H) ppm corresponding to NH group.The reactivity of compound 3 towards 1,3-dicarbonyl compounds was studied to afford pyridine derivatives with wide range of biological activities thus, the reaction of 3 with either acetylacetone (15a) or ethyl acetoacetate (15b) gave the pyridine derivatives 16a and 16b, respectively (cf.Scheme 2).The structures of the latter products were established on the basis of analytical and spectral data.Thus, 1 H NMR spectrum of 16b showed a triplet at δ 1.35 (3H) ppm corresponding to CH 3 group, a singlet at δ 2.34 (3H) ppm corresponding to CH 3 group, a quartet at δ 4.22 (2H) ppm corresponding to CH 2 group, a singlet at δ 5.79 (1H) ppm corresponding to pyridine proton, a singlet at δ 7.51 (1H) ppm corresponding to CH group and a multiplet at δ 6.52 -7.88 (3H) ppm corresponding to furan protons.On the other hand, we studied the reactivity of compound 3 with cyanomethylene reagents thus, the reaction of 3 with either malononitrile (8a) or ethyl cyanoacetate (8b) gave the 2-pyridone derivatives 19a,b.The formation of the latter products took place via the intermediate formation of 17a,b (cf.Scheme 3).The 1 H NMR spectrum of 18b as an example showed a singlet at δ 5.80 (1H) ppm corresponding to the pyridine proton, a multiplet at δ 6.52 -7.88 (3H) ppm corresponding to furan protons, a singlet (D 2 O-exchangeable) at δ 7.00 (2H) ppm corresponding to NH 2 group, a singlet at δ 7.52 (1H) ppm corresponding to CH group, and a singlet (D 2 O-exchangeable) at δ 12.56 (1H) ppm corresponding to OH group.
Next, we studied the reactivity of the active methylene group present in compound 3 towards diazonium salts.Thus, the reaction of compound 3 with aryldiazonium chlorides 23a-23d gave the phenylhydrazone derivatives 24a-24d.Analytical and spectral data of the latter products are all consistent with the proposed structures (see experimental section).Compound 24a reacted with ethyl cyanoacetate (8b) to give the pyridazine derivative 26.The reaction took place via the intermediate formation of 25 (cf.Scheme 3) On the other hand, the reaction of 24a with phenyl isothiocynate (27) gave the triazine derivative 29 via the intermediate formation of 28 (cf.Scheme 3).Analytical and spectral data of the latter product are in agreement with the proposed structure.
Recently our research group was involved through a comprehensive program dealing with studying the reaction of phenylisothiocyanate with active methylene reagents followed by heterocylization with α-haloketones.The reactions lead to the formation of either thiazole or thiophene derivatives depending on the reaction conditions and the nature of the α-halocarbonyl compounds.
In continuation of this program we report here the reaction of compound 3 with phenylisothiocyanate (27) in DMF solution containing KOH to form the intermediate potassium sulphide salt 30.The latter intermediate under went heterocyclization upon reaction with ethyl chloroacetate (31) to give the thiazole derivative 33.Formation of the latter product took place via the intermediate formation of 32.Similarly the reaction of 30 with αchloroacetone (34a) or phenacyl bromide (34b) gave the thiazole derivatives 35a and 35b, respectively (Scheme 4).Analytical and spectral data are consistent with the proposed structures.Thus, 1 H NMR spectrum of 35a showed a singlet at δ 2.35 (3H) ppm corresponding to CH 3 group, a singlet at δ 5.81 (1H) ppm corresponding to thiazole proton, a multiplet at δ 6.25 -7.20 (5H) ppm corresponding to aromatic protons, a multiplet at δ 6.52 -7.88 (3H) ppm corresponding to furan protons, a singlet at δ 7.52 (1H) ppm corresponding to CH group and a singlet (D 2 O-exchangeable) at δ 11.62 (1H) ppm corresponding to NH group.

Material, Methods and Reagents
Fetal bovine serum (FBS) and L-glutamine, were from Gibco Invitrogen Co. (Scotland, UK).RPMI-1640 medium was from Cambrex (New Jersey, USA).Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, streptomycin and sulforhodamine B (SRB) were from Sigma Chemical Co.(Saint Louis, USA).Samples Stock solutions of selected compounds from 3-34a,b were prepared in DMSO and kept at −20˚C.Appropriate dilutions of the compounds were freshly prepared just prior the assays.Final concentrations of DMSO did not interfere with the cell growth.

Cell Cultures
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) 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 μ/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.

Effect on the Growth of Human Tumor Cell
Lines The effect of selected compounds from the newly synthesized products 3-35a,b was evaluated on the in vitro growth of three human tumor cell lines representing different tumor types, namely, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268), after a continuous exposure of 48 h. the evaluations of the newly synthesized products were measured using doxorubicin as the positive control and the results are summarized in Table 1.
All the compounds were able to inhibit the growth of the human tumor cell lines in a dose-dependent manner (data not shown).The pyrazole derivative 7b and the coumarine derivative 14 showed the best results, exhibiting an equivalent potency in all the three tumor cell lines which is still much lower than the gram positive control doxorubicin.On the other hand, compounds 3, 7a, 10a, 18a, 18b, 21, 22, 24c, 26 and 33 showed moderated growth inhibitory effect.Comparing the activities of 18a and 18b it is observed that the hydroxyl group in 18b presents a stronger growth inhibitory effect than the amino substituent in 18a although the results in NCI-H460 cell line are comparable.It is clear from Table 1 that some compounds like 10b, 16a,b and 35b showed very low activity towards certain cell line, MCF-7, and moderate activity towards other 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.

Conclusion
In conclusion, we have developed efficient methods for the synthesis of new hydrazide-hydrazone derivatives via the reaction of cyanoacetylhydrazine with furan-2-aldehyde.The produced product underwent a series of heterocyclizations.Their antitumor evaluations through the three cancer cell lines showed that compounds 7b and 14 showed the maximum inhibitory effect among the tested compounds.

Experimental
All melting points were determined in open capillaries and are uncorrected.Elemental analyses were performed on a Yanaco CHNS Corder elemental analyzer (Japan).IR spectra were measured using KBr discs on a Pye Unicam SP-1000 spectrophotometer. 1 H NMR spectra were measured on a Varian EM 390 -200 MHz instrument in CD 3 SOCD 3 as solvent using TMS as internal standard and chemical shifts are expressed as δ ppm.Mass spectra were recorded on Kratos (75 eV) MS equipment (Germany).Elemental analyses were carried out by the Micro-analytical Data Center at Cairo University and were performed on Vario EL III Elemental CHNS analyzer.

Synthesis of pyrazole derivatives 7a,b.
General procedure: To a solution of compound 5 (0.265 g, 1.0 mmol) in 1,4-dioxane (25 mL) and dimethylformamide (10 mL), either hydrazine hydrate (6a) (0.05 g, 1.0 mmol) or phenylhydrazine (6b) (0.10 g, 1.0 mmol) was added.The reaction mixture in each case was heated under reflux for 3 h, and then poured on an ice/ water mixture containing a few drops of hydrochloric acid.The formed solid product in each case was collected by filtration.