Novel Synthesis of Hydrazide-Hydrazone Derivatives and Their Utilization in the Synthesis of Coumarin, Pyridine, Thiazole and Thiophene Derivatives with Antitumor Activity

The reaction of cyanoacetyl hydrazine (1) with 3-acetylpyridine (2) gave 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), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268) was performed. Most of the synthesized compounds showed high inhibitory effects.


Introduction
Hydrazines and their derivatives constitute an important class of compounds that has found wide utility in organic synthesis [1,2]. While hydrazines have traditionally been employed as reagents for the derivatization and characterization of carbonyl compounds, in recent years the N-N linkage has been used as a key structural motif in various bioactive agents. In particular, an increasing number of N-N bond-containing heterocycles and peptidomimetics have made their way into commercial applications as pharmaceutical and agricultural agents [3,4]. Recently, hydrazide-hydrazones have gained great OPEN ACCESS importance due to their diverse biological properties including antibacterial, antifungal, anticonvulsant, anti-inflammatory, antimalarial and antituberculosis activities [5][6][7][8][9][10][11][12][13][14][15][16][17]. With the aim of obtaining novel hydrazide-hydrazones with a wide spectrum of pharmaceutical applications, we report herein the synthesis of a series of hydrazide-hydrazones together with their use in a series of heterocyclic transformations and their evaluation as anti-tumor agents [18][19][20][21].

Results and Discussion
Recently, our research group became involved with a comprehensive program involving the synthesis of a series of hydrazide-hydrazone derivatives and their utilization in the synthesis of heterocyclic compounds with potential pharmaceutical and biological activities [22,23]. In continuation to this program, we report herein the reaction of cyanoacetylhydrazine (1) with 3-acetylpyridine (2) in 1,4-dioxane to form the hydrazide-hydrazone derivative 3. The structure of compound 3 was confirmed based on its analytical and spectral data. Thus, the 1 H-NMR showed a singlet at δ 2.28 for the CH 3 group, a singlet at δ 4.26 for the CH 2 group, a multiplet at δ 7.43-8.99 for the pyridyl group and a singlet (D 2 O exchangeable) at δ 10.81 for the NH group. Moreover, the 13 C-NMR spectrum showed peaks at δ: 14.2 (CH 3 ), 28.9 (CH 2 ), 116.8 (CN), 122. 1, 123.4, 133.7, 150.2, 151.0 (pyridine C), 168.1 (C=N), 169.8 (C=O). Further structure elucidation of compound 3 was obtained through the study of its reactivity towards chemical reagents. Thus, the reaction of 3 with either benzaldehyde (4a), 4-chlorobenzaldehyde (4b) or 4-methoxybenzaldehyde (4c) gave the corresponding benzal derivatives 5a-c, respectively (Scheme 1).
On the other hand, the reaction of compound 3 with salicylaldehyde (6) gave the coumarin derivative 7 (Scheme 2). Analytical and spectral data of the product are in agreement with the proposed structure (see Experimental section). Next, we studied the reactivity of the active methylene group present in compound 3 towards diazonium salts. Thus, the reaction of 3 with either benzenediazonium chloride (8a), 4-chlorobenzenediazonium chloride (8b), 4-bromobenzenediazonium chloride (8c) or 4-nitrobenzenediazonium chloride (8d), gave the hydrazone derivatives 9a-d, respectively (Scheme 3). Analytical and spectral data of the latter reaction products are all consistent with the proposed structures. Scheme 3. Synthesis of phenylhydrazo derivatives 9a-d. R N NCl Moreover, the reaction of compound 3 with cyclohexanone (10) and elemental sulfur in the presence of triethylamine was studied as an application of Gewald's thiophene synthesis. The reaction led to the formation of 4,5,6,7-tetrahydrobenzo[b]thiophene derivative 11. On the other hand, the reaction of compound 3 with cyclopentanone (12) and sulfur gave the cyclopentene[b]thiophene derivative 13. The structures of compounds 11 and 13 were based on analytical and spectral data (see Experimental section). Further confirmation of structure 11 was obtained through its synthesis via another reaction route. Thus, the reaction of compound 3 with cyclohexanone in the presence of ammonium acetate in an oil bath at 140 °C gave the Knoevenagel condensation product 14. The latter reacted with elemental sulfur in the presence of triethylamine to produce the same tetrahydrobenzo[b]thiophene derivative 11. It is convenient to notice that the yield (70%) for formation of compound 11 using the latter procedure was higher than in the synthetic pathway described before (62%) (Scheme 4).  Next, we studied the reactivity of 3 towards phenyl isothiocyanate in basic dimethylformamide solution followed by heterocyclization with active methylene reagents like -haloketones with the aim of synthesizing thiazole derivatives with potential antitumor activity. Thus, compound 3 reacted with phenylisothiocyanate (15) in DMF/KOH solution at room temperature to give the intermediate potassium sulphide salt 16. Heterocyclization of 16 with α-haloketones like ethyl bromoacetate (17) gave the thiophene derivative 18. The structure of 18 was confirmed based on analytical and spectral data. The 1 H-NMR showed a singlet at δ 2.22 for the CH 3 group, a singlet at δ 7.46 for the thiazole hydrogen, a multiplet at δ 7.54-8.95 for the pyridine H and C 6 H 5 and two singlets at 8.80/10.13 for the NH and OH groups, respectively. Moreover, the 13  Ph N C S Next, we studied the reactivity of the hydrazide-hydrazone derivative 3 towards cinnamonitrile derivatives. Thus, the reaction of 3 with either 2-benzylidenemalononitrile (22a) or ethyl 2-cyano-3phenylacrylate (22b) gave the pyridine derivatives 23a and 23b, respectively (Scheme 6). Scheme 6. Synthesis of 23a,b. 3 CH Ph

Effect on the Growth of Human Tumor Cell Lines
The effect of compounds 3-25 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) was evaluated after a continuous 48 h exposure. The results are summarized in Table 1. All of the tested compounds were able to inhibit the growth of the tested human tumor cell lines in a dose-dependant manner (data not shown). The results indicated in Table 1 revealed that compound 3 showed the highest inhibitory effect against all the three tumor cell lines. In addition compound 5c showed the best inhibitory effect against CNS cancer (SF-268), while compounds 7 and 23a showed high inhibitory effects against non-small cell lung cancer (NCI-H460) and breast adenocarcinoma (MCF-7), respectively. Compounds 5b, 9a, 23b showed the lowest inhibitory effects. The rest of the compounds showed a moderate growth inhibitory effect. Comparing compound 5b with 5c, it is obvious thatthe presence of the CH 3 O group in 5c resulted in a higher inhibitory effect than 5b with the Cl group. Comparing the pyridine derivatives 23a (with the cyano group) and 23b (with the carboxyethyl group), the first has a greater inhibitory effect than the second towards the three cell lines.

General
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 and 13 C-NMR spectra were measured on a Varian EM-390-200 MHz in CD 3 SOCD 3 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. Antitumor evaluation for the newly synthesized products were performed by a research group at the National Research Center and the National Cancer Institute at Cairo University.
. To a solution of cyanoacetylhydrazine (2, 0.99 g, 0.01 mol) in 1,4-dioxane (20 mL), 3-acetylpyridine (1.21 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured onto a beaker containing an ice/water mixture. The formed solid product was collected by filtration and dried to give white crystals (from ethanol).

General Procedure for the Synthesis of 5a, 5b or 5c
To a solution of 3 (2.02 g, 0.01 mol) in 1,4-dioxane (20 mL), either benzaldehyde (1.06 g, 0.01 mol), p-chlorobenzaldehyde(1.12 g, 0.01 mol) or p-methoxybenzaldehyde (1.08 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 6 h then poured onto a beaker containing an ice/water mixture. The formed solid product was collected by filtration and dried.   (7). To a solution of 3 (2.02 g, 0.01 mol) in 1,4-dioxane (20 mL), salicylaldehyde (1.22 g,0.01 mol) was added. The reaction mixture was heated under reflux for 3 hours then poured onto a beaker containing an ice/water mixture. The formed solid product was collected by filtration and dried to give white crystals (from ethanol). Yield

General Procedure for the Synthesis of 9a, 9b, 9c or 9d
To a cold solution of 3 (2.02 g, 0.01 mol) in ethanol (20 mL) containing sodium acetate (3.0 g) was added with continuous stirring either of the appropriate substituted benzenediazonium salt (0.01 mol) [prepared by adding sodium nitrite (1.6 g, 0.02 mol) in water (8 mL) to a cold solution of either of the appropriate substitute aniline 8a-d in the appropriate amount of hydrochloric acid]. The reaction mixture was stirred for 2 h and the formed solid product, in each case, was collected by filtration.    CN), 116.3, 118.0, 118.6, 122.8, 124.5, 128.0, 138.9, 150.2
Method B: To a solution of compound 14 (2.82 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.5 mL), elemental sulfur (0.32 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water containing few drops of hydrochloric acid. The formed solid product was collected by filtration.
Yield: 1.95g, 62% (method A) and 2.  (13). To a solution of 3 (2.02 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (1.0 mL) and elemental sulfur (0.32 g, 0.01 mol), cyclopentanone 12 (0.98 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h then poured onto a beaker containing an ice/water mixture. The formed solid product was collected by filtration and dried obtaining pale yellow crystals (from ethanol).Yield  (14). Equimolar amounts of compound 3 (2.02 g, 0.01 mol) and cyclohexanone 10 (0.98 g, 0.01 mol) were heated in an oil bath at 140 °C for 1 h in presence of ammonium acetate. After cooling the reaction mixture, it was heated in ethanol, then poured into ice/water mixture and the formed solid product was collected by filtration and dried to give pale yellow crystals (from ethanol). Yield  (19) are stirred in the following day, and the solution is covered for another night, after which the reaction mixture is poured onto ice and the precipitated solid is filtered off.  (20 (21). To a solution of 3 (2.02 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (1.0 mL) and elemental sulfur (0.32 g, 0.01 mol), phenylisothiocyanate (

General Procedure for the Synthesis of 23a or 23b
To a solution of 3 (2.02 g, 0.01 mol) in 1,4-dioxane (20 mL) either 2-benzylidenemalononitrile (22a, 1.54 g, 0.01 mol) or ethyl 2-cyano-3-phenylacrylate (22b, 2.01 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h then poured onto a beaker containing an ice/water mixture. The formed solid product was collected by filtration and dried.

Conclusions
In this work, cyanoacetylhydrazine (1) reacted with 3-acetylpyridine (2) to afford the hydrazidehydrazone derivative 3. The latter was reacted with different reagents to give coumarin, pyridine, thiazole and thiophene derivatives. The antitumor evaluations of the newly synthesized products were carried out, showing that both the hydrazide-hydrazone derivative 3 and the benzylidene derivative 5c have the highest inhibitory effects.