The Reaction of Cyanoacetylhydrazine with ω-Bromo(4-methyl)acetophenone: Synthesis of Heterocyclic Derivatives with Antitumor Activity

New approaches for the synthesis of hydrazide-hydrazone derivatives were demonstrated as well as some heterocyclizations of such derivatives to afford 1,3,4-triazine, pyridine and 1,3,4-oxadiazine derivatives. 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) were recorded. Most of the synthesized compounds showed high inhibitory effects.


Results and Discussion
In this work we report the reaction of cyanoacetylhydrazine (1) with ω-bromo-(4-methylacetophenone) (2) in 1,4-dioxane which gave the condensed product 3. The structure of compound 3 was confirmed based on analytical and spectral data. Thus, the 1 H-NMR showed a singlet at δ 2.51 for the CH 3 , two singlets at δ 4.31, 4.72 for the two CH 2 groups, a multiplet at δ 6.50-7.76 for the C 6 H 4 group and a singlet at δ 11.46 (D 2 O exchangeable) for the NH group. The reactivity of compound 3 towards different chemical reagents was studied. The reaction of 3 with either potassium cyanide or potassium thiocyanate gave the corresponding cyanide or the thiocyanate derivatives 4a and 4b, respectively (Scheme 1).

Scheme 1.
Synthesis of the hydrazide-hydrazone 3 and 4a, b. The reaction of compound 3 with either hydrazine hydrate (5a) or phenylhydrazine (5b) gave the hydrazine derivative 6a or 6b, respectively. Analytical and spectral data of the reaction products are in agreement with the proposed structures (see Experimental section). The reaction of either 6a or 6b with benzaldehyde (7) gave the benzal derivative 8a or 8b, respectively (Scheme 2).

12a-d
On the other hand, the reaction of either 6a or 6b with either acetylacetone (13a) or ethyl acetoacetate (13b) gave the pyridine derivatives 14a-d, respectively (Scheme 5). The structures of the latter products were confirmed by their analytical and spectral data (see Experimental section). Compound 3 underwent ready cyclization when heated in sodium ethoxide solution to give the 1,3,4-oxadiazine derivative 15, whose structure was established from its analytical and spectral data (see Experimental section). The oxadiazine derivatives 15 seemed to be an intermediate for many reactions between 3 and many chemical reagents. Thus, the reaction of 3 with benzaldehyde (7) gave the 2-(α-benzalacetonitrilo)-1,3,4-oxadiazine derivative 16. The analytical and spectral data of 16 were in agreement with the proposed structure. Thus, the 1 H-NMR spectrum showed a singlet at δ 2.51 for the CH 3 group, a singlet at δ 4.22 for the CH 2 group, a singlet at δ 5.16 for the (=CH) group and a multiplet at δ 7.35-8.02 for the C 6 H 5 and C 6 H 4 groups. The same product 16 was obtained through the reaction of compound 15 with benzaldehyde (7) (confirmed by m.p., mixed m.p. and fingerprint IR spectrum). On the other hand, the reaction of 15 with benzenediazonium chloride (9) gave the 2-(αphenylhydrazo)-1,3,4-oxadiazine derivative 17 (Scheme 6).

Effect on the Growth of Human Tumor Cell Lines
The effect of compounds 4-17 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 results are summarized in Table 1. All the compounds were able to inhibit the growth of the tested human tumor cell lines in a dose-dependent manner (data not shown). The results showed that compound 14b, with its 2-hydroxypyridine group, showed the highest inhibitory effect against all the three tumor cell lines. In addition compounds 10a, 10f, 12a, 12b and 14c showed high inhibitory effects. On the other hand, compounds 4b, 6a, 8b, 10b, 10d, 12c, 12d, 14a, 14c, 16 and 17 showed the lowest inhibitory effect towards adenocarcinoma (MCF-7). The rest of compounds showed the moderate growth inhibitory effects. Comparing compound 4a with 4b, it is obvious that the presence of the α-SCN present in 4b showed lower inhibitory effect than 4a with it's α-CN group. Comparing 1,2,4-triazine derivatives 10a (with the 4-phenylamino group) and 10b (with the 4-thiophenoamino group), the first has a greater inhibitory effect than the second towards the three cell lines. 60.4 ± 6.0 77.8 ± 3.1 47.0 ± 6.4 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. * Results from two-independent experiments performed in duplicate. Doxorubicin was used as positive control, GI 50 : MCF-7 = 42.8 ± 8.2 nM, NCI-H460 = 94.0 ± 8.7 nM, and SF-268 = 94.0 ± 7.0 nM.

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 & 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 & the National Cancer Institute at Cairo University. (3). To a solution of cyanoacetylhydrazine (1, 2.44 g, 0.02 mol) in 1,4-dioxane (20 mL), ω-bromo-(4-methylacetophenone) (5.24 g, 0.02 mol) was added. The reaction mixture was stirred at room temperature for 1 hr then poured onto a beaker containing an ice/water mixture. The solid product formed was collected by filtration and dried obtaining pale yellow crystals (from ethanol).Yield

General Procedure for the Synthesis of 4a or 4b
To a solution of 3 (0.54 g, 1.83 × 10 −3 mol) in ethanol (25 mL) in a water bath at 60 °C, either potassium cyanide (0.11 g, 1.83 × 10 −3 mol) or potassium thiocyanate (0.17 g, 1.83 × 10 −3 mol) was added with continuous stirring. The reaction mixture was left in the water bath for 30 min at 60 °C then poured onto a beaker containing ice/water mixture and few drops of hydrochloric acid. The formed solid product was collected by filtration and dried.

General Procedure for the Synthesis of 12a and 12b
To a solution of compound 6a (0.47 g, 1.91 × 10 −3 mol) in ethanol (20 mL) containing triethylamine (0.5 mL), either malononitrile (0.12 g, 1.91 × 10 −3 mol) or ethyl cyanoacetate (0.21 g, 1.91 × 10 −3 mol) was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a beaker containing an ice/water mixture and a few drops of hydrochloric acid. The solid product formed was collected by filtration and dried.

General Procedure for the Synthesis of 12c and 12d
To a solution of compound 6b (0.60 g, 1.86 × 10 −3 mol) in ethanol (20 mL) containing triethylamine (0.5 mL), either malononitrile (0.12 g, 1.86 × 10 −3 mol) or ethyl cyanoacetate (0.21 g, 1.86 × 10 −3 mol) was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a beaker containing an ice/water mixture and a few drops of hydrochloric acid. The solid product formed was collected by filtration and dried.

General Procedure for the Synthesis of 14a and 14b
To a solution of compound 6a (0.52 g, 2.12 × 10 −3 mol) in ethanol (20 mL) containing piperidine (0.5 mL), either acetylacetone (0.21 g, 2.21 × 10 −3 mol) or ethyl acetoacetate (0.27 g, 2.21 × 10 −3 mol) was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a beaker containing an ice/water mixture and a few drops of hydrochloric acid. The solid product formed was collected by filtration and dried.

General Procedure for the Synthesis of 14c and 14d
To a solution of compound 6b (0.60 g, 1.86 × 10 −3 mol) in ethanol (20 mL) containing piperidine (0.5 mL), either acetylacetone (0.18 g, 1.86 × 10 −3 mol) or ethyl acetoacetate (0.24 g, 1.86 × 10 −3 mol) was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a beaker containing an ice/water mixture and a few drops of hydrochloric acid. The solid product formed was collected by filtration and dried.  (15). A solution of compound 3 (1.00 g, 3.39 × 10 −3 mol) in sodium ethoxide (50 mL) was heated under reflux for 3 hrs then poured onto a beaker containing an ice/water mixture and a few drops of hydrochloric acid. The formed solid product was collected by filtration and dried to give pale brown crystals (from ethanol).  (16). To a solution of compound 3 (2.00 g, 6.79 × 10 −3 mol) in ethanol (30 mL) containing piperidine (0.5 mL), benzaldehyde (0.72 g, 6.79 × 10 −3 mol) was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a beaker containing an ice/water mixture with a few drops of hydrochloric acid. The formed solid product was collected by filtration and dried to afford brown crystals (from ethanol). Yield: 1.35 g (58%), m.p. 100 °C; IR (KBr) υ/cm −1 : 3050 (CH aromatic), 3029 (CH 3 ), 2921 (CH 2 ),