Utility of Activated Nitriles in the Synthesis of Novel Heterocyclic Compounds with Antitumor Activity

Reaction of cyanoacetic acid hydrazide (1) with 4-methoxyacetophenone and 4-chlorobenzaldehyde (2a,b) afforded the corresponding 2-cyanoacetohydrazide derivatives (3a,b) respectively. The latter compounds were utilized as a key intermediate for the synthesis of new heterocyclic compounds. Newly synthesized compounds were characterized by elemental analyses and spectral data. The antitumor evaluation of some newly synthesized compounds was screened in vitro against human breast cancer cell line (MCF-7).


Introduction
In many reports, hydrazide-hydrazones are considered to be good candidates for different pharmaceutical applications, where such compounds were considered to exert anticonvulsant [1], analgesic [2], anti-inflammatory [3], antiplatelet [4], antimalarial [5], antimicrobial [6], antitumoral [7,8], vasodilator [9], and antiviral activity [10]. With the aim of obtaining new hydrazide-hydrazones with such wide spectrum of pharmaceutical applications, in this research, synthesis of a series of hydrazide-hydrazones and some of their heterocyclic transformations, followed by antitumor evaluations of newly synthesized products was done.

Experimental
All melting points were determined in open glass capillaries on a Gallenkamp apparatus and are uncorrected. IR spectra (cm −1 ) were recorded on a Pye-Unicam spectrophotometer type 1200 using KBr discs. 1 H-NMR spectra were recorded on a Varian EM-390 (90 MHz) spectrometer using TMS as an internal standard and DMSO-d6 as a solvent. Chemical shifts were expressed in (ppm) values and mass spectra were determined on Finnigan Incos 500 (70 ev). Elemental analyses were determined using a Parkin-Elmer 240C Microanalyzer. The microanalyses were performed at the Microanalytical Unit, Faculty of Science, Cairo University.

General Procedure for Synthesis of 3a,b.
To a solution of 2cyanoacetohydrazide (0. 99 g, 0.01 mol) in ethanol (20 mL), 4-methoxyacetophenone and/or p-chlorobenzaldehyde (0.01 mol) was added. The reaction mixture was heated under reflux for 2 h, then left to cool, and poured into ice/ water. The obtained product was filtered, washed with water, and recrystallized from the appropriate solvent to give 3a,b. [1-(4-methoxyphenyl)

General Procedure for Synthesis of Arylidene Derivatives (5a-d).
A solution of 3a,b (0.01 mol) in ethanol (30 mL) was treated with 0.01 mol of aromatic aldehydes such as 4-methoxybenzaldehyde and/or 4-chlorobenzaldehyde and few drops of TEA. The reaction mixture was heated under reflux for 6 h. The reaction mixture was left to cool at room temperature and poured into ice/water containing few drops of hydrochloric acid, and the formed solid was collected by filtration and recrystallized from the appropriate solvent to give 5a-d. [1-(4- 6a,b). A mixture of 5a,b (0.01) and hydrazine hydrate (0.50 g, 0.01 mol, 95%) was refluxed for 6 h. After cooling, the formed solid was filtered, washed with water, and recrystallized from the appropriate solvent to give 6a,b.   [1-(4-methoxyphenyl)ethylidene]amino}-6-oxo-1,6-dihydropyridine-3-carboxamide (8). To a solution of compound 3a (2.31 g, 0.01 mol) in 1,4-dioxane (30 mL) containing few drops of TEA, 3-(4-chlorophenyl)-2-cyanoacrylamide (2.06 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 4 h, then poured into ice/water, and the solid obtained was recrystallized from ethanol to give 8.  [2,3-d]pyrimidine-6-carbonitrile (9). A mixture of compound 8 (4.35 g, 0.01 mol) and 4-nitrobenzaldehyde (0.01 mol) in ethanol (20 mL) with few drops of TEA was refluxed in oil bath with stirring for 10-12 h. The reaction mixture was  (10). To a stirred solution of 8 (4.35 g, 0.01 mol) in DMF (10 mL), carbon disulfide (0.76 mL, 0.01 mol) and NaOH (0.2 g, 0.005 mol) were added. The reaction mixture was stirred at room temperature for 6 h, then diluted with an equal volume of water, and treated with dilute HCl (pH 4). The separated solid product was filtered, washed with water, and recrystallized from DMF to give 10.

General Procedure for Synthesis of 4,5,6,7-Tetrahydrobenzo[b]thiophene-2-carbohydrazide Derivatives (11a,b).
To a solution of 3a,b (0.01 mol) in ethanol (20 mL) containing TEA (1 mL) and elemental sulfur (0.01 mol), cyclohexanone (0.01 mol) was added. The reaction mixture was heated under reflux for 3 h, then poured into ice/water. The formed solid product was collected by filtration and recrystallized from the appropriate solvent to give 11a,b.  (14). To a stirred suspension of finely powdered potassium hydroxide (0.01 mole) in dry DMF (10 mL (15). Compound 15 was synthesized as mentioned for the synthesis of 14 but using dimethylsulfate instead of ethylchloroacetate; the resulting product was crystallized from ethanol to give 15.

General Procedure for Synthesis of Ethyl [2-Cyano-1mercapto-3-oxoprop-1-enyl)thio]acetate Derivatives (19a,b).
To a stirred suspension of finely powdered potassium hydroxide (0.02 mole) in dry DMF (10 mL), cyanoacetohydrazides, 3a,b, (0.01 mole) were added. The resulted mixture was cooled at 10 ∘ C in an ice bath; then (0.01 mol) carbon disulfide was added slowly over the course of 10 min. After complete addition, stirring of the reaction mixture was continued for 6 h Then ethyl chloroacetate (1.22, 0.01 mol) was added to the mixture and stirring continued for 3 h, then the mixture was poured into crushed ice and HCl; the resulting precipitate was filtrated off, dried, and recrystallized from the appropriate solvent to give 19a,b.   (20a,b). Compounds 20a,b were synthesized as mentioned for synthesis of 19, but using dimethyl sulfate instead of ethyl chloroacetate, the resulting product was recrystallized from the appropriate solvent to give 20a,b.   (21 and 22). A mixture of 20a (3.35 g, 0.01 mol), hydrazine hydrate, and/or 2-hydrazino-1,3-benzothiazole (1.89 g, 0.012 mol) in ethanol (30 mL) was heated under reflux for 3 hrs and allowed to cool. The solid product obtained was filtrate off and recrystallized from the appropriate solvent to give 21 and 22.    6a, 6b, 11b, 14, 15, 16, 18b, 19a, 19b, 21, and 22 were tested for their cytotoxicity in vitro, in comparison with doxorubicin (DXR) as a reference drug against human breast cancer cell line (MCF-7). MCF-7 cells (1 × 10 4 ) were incubated with synthesized compounds at various concentrations of 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 g/mL (incorporated with 10 L DMSO) at 37 ∘ C for 48 h, and viable cells yield was determined by colorimetric method [11]. Experiments were carried out in triplicate, and results are reported in Table 1.

Results and Discussion
3.1. Synthesis. The Schemes 1-4 describe the synthesis of the target molecules. Condensation of cyanoacetic acid hydrazide (1) with 4-methoxyacetophenone and 4-chlorobenzaldehyde(2a,b) in hot 1,4-dioxane afforded hydrazone derivatives (3a,b). The structures of compounds (3a,b) were established on the basis of analytical and spectral data. IR spectrum of 3a showed bands at 3204 cm −1 (NH), 2256 (CN), and 1675 (C=O). 1 H NMR spectrum of 3a showed the presence of a singlet at 2.23 ppm corresponding to CH 3 group, a singlet at 3.81 ppm for methoxy group, a singlet at 4.18 ppm for CH 2 group, and a singlet at 10.90 ppm for an NH group. Further evidence for the structures of 3a,b was obtained through studying their chemical reactivity through some chemical reagents. Thus, cyclocondensation of compounds 3a,b with salicylaldehyde in boiling ethanol and in the presence of a few drops of triethylamine (TEA) [12] afforded the corresponding 2H-chromene-3-carbohydrazide derivatives, 4a,b.
Condensed 3a,b with aryl aldehydes such as 4-methoxybenzaldehyde and/or 4-chlorobenzaldehyde [13] afforded the corresponding arylidene derivatives 5a-d. The structures of compounds 5a-d were based on analytical and spectral data (see Section 2). When compounds 5a,b reacted with hydrazine hydrate [14], they afforded the corresponding 1Hpyrazole-4-carbohydrazide derivatives, 6a,b. The structure of 6 was inferred from its 1 H NMR spectrum which appeared as new signals for NH 2 group (Scheme 1).
Reactivity of hydrazide-hydrazone derivative 3a toward cinnamonitrile derivative was studied [15]. Thus, the reaction of 3a with 3-(4-chlorophenyl)-2-cyanoacrylamide afforded the corresponding pyridinone derivative 8; the reaction took place through the intermediate 7. Treatment of pyridinone derivative 8 with 4-nitrobenzaldehyde in the presence of ethanol and catalytic amount of triethylamine (TEA) afforded the corresponding pyrido[2,3-d]pyrimidine-6-carbonitrile derivative 9. On the other hand, 2-thioxopyrido [2,3-d]pyrimidine-6-carbonitrile 10 was obtained by cyclizing 8 with carbon disulfide in the presence of sodium hydroxide at room temperature [16] followed by acidification with diluted hydrochloric acid. Structures of new compounds were based on analytical and spectral data. Thus, the 1 H NMR spectrum of 8 showed the presence of a singlet at 6.92 and 6.95 corresponding to the two NH 2 groups. The IR spectrum of 10 displayed absorption bands at 3332, 3188 cm −1 due to the 2NH function and 1243 cm −1 due to C=S group.
Reaction of compounds 3a,b with cyclohexanone and elemental sulfur in the presence of TEA afforded the corresponding 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives, 11a,b. Formation of 11 took place according to the similar reported reactions of cyclohexanone with methylene reagents and elemental sulfur [17]. Structure of compound 11 was based on analytical and spectral data (Scheme 2).
The active methylene moiety of 2-cyano-N - [1-(4- Scheme 3 [18] yielding the nonisolable intermediate potassium sulfide salt 12, and then ethylchloroacetate was added affording 1,3thiazolidinone derivative 14. Probably, the reaction mechanism is assumed to proceed via S-alkylation to give the intermediate 13 which was cyclized to the corresponding thiazolidinone derivatives 14. Elemental analyses and spectral data were in favor of these proposed 1,3-thiazolidinone structures. The IR spectrum of 14 showed absorption bands at 1742 cm −1 due to thiazolidinone CO. The 1 H NMR spectrum showed signals in the region at 4.02 ppm corresponding to C4 protons of the thiazolidinone ring. On the other hand, treatment of the nonisolable potassium sulfide salt 12 with dimethylsulfate afforded 3-anilino-2-cyano-3methylthio-acrylohydrazide 15. Cyclocondensation of the acrylohydrazide 15 with hydrazine hydrate in boiling ethanol afforded aminopyrazole derivative 16. The structure of 15 and 16 was identified as the reaction product on the basis of their elemental analysis and spectroscopic data. 1 H NMR spectrum of 15 displayed the following signals at 2.50 corresponding to the SCH 3 group, 10.10 corresponding to the NHPh group, and 11.60 corresponding to the NHCO group. IR spectrum of 16 showed the lacks of absorption band assignable to the CN group and the presence of a new absorption band at 3298, 3242 cm −1 assignable to NH 2 group. Its 1 H NMR spectrum showed signals at 6.13 ppm corresponding to the NH 2 protons, another three singlet signals at 8.00, 8.20, and 10.10 ppm assignable to three NH protons (Scheme 3).
Reaction of compounds 3a,b with carbon disulfide in boiling DMF containing catalytic amount of potassium hydroxide afforded nonisolable intermediate potassium sulfide salts 17a,b. Treatment of the nonisolable potassium salts 17a,b with dilute hydrochloric acid [19] afforded the corresponding dithiol derivatives 18a,b. The structures of compounds 18a,b were based on both elemental analyses and spectral data. The 1 H NMR spectrum of 18a revealed signal at 2.73 and 2.88 ppm for two SH protons and at 10.50 ppm for NHCO proton. Moreover, Alkylation of dithiol derivatives 17a,b with ethylchloroacetate yielded the corresponding ethyl [2-cyano-1-mercapto-3-oxoprop-1-enyl) thio]acetate derivatives 19a,b [20]. The structures of 19a,b were supported on the basis of elemental analyses and spectral data. Ketene S,S-dithio-acetals derivatives 20a,b can be prepared by alkylation of dithiol derivatives 17a,b with dimethylsulfate. The structure of 20 was elucidated on the basis of the elemental analyses and spectral data. The IR spectrums of 20a showed the appearance of absorption band at 3203 for NH and 2256 cm −1 for CN groups. 1 H NMR spectrum of 20b showed singlet signal at 2.50 ppm for 6 protons of two similar methyl protons, while a singlet signal for the methylene protons disappeared.
Cyclocondensation of bis-(methylthio)acrylohydrazide derivative 20a with hydrazine derivatives such as hydrazine hydrate and 2-hydrazino-1,3-benzothiazole affords the corresponding 5-(methylthio) 1H-pyrazole-4-carbohydrazide 21 and 22, respectively. The structures of compounds 21 and 22 were as established and confirmed as the reaction product on the basis of their elemental analyses and spectral data. The IR spectrum of 21 showed absorption band at 3300, 3243 cm −1 assignable for NH 2 , in addition to disappearance of nitrile function signal. Its 1 H NMR spectrum revealed the presence of singlet signals at 6.13 ppm assignable to the NH 2 protons (Scheme 4). 3a, 3b, 4a, 4b, 6a, 6b, 11b, 14, 15, 16, 18b, 19a, 19b, 21, and 22  Doxo. were compared to the antiproliferative effects of the reference control doxorubicin (Table 1, plotted in Figure 1). The results indicated that most compounds demonstrated substantial growth inhibitory effects against the human tumor cells at the tested concentrations. The antiproliferative activity of the test compounds against tumor cell lines may be arranged in a descending order due to measured concentration required to inhibit tumor cell proliferation by IC50 g/mL which corresponds to the concentration required for 50% inhibition of cell viability. In general, compounds 11b, 6a, 16, 19b, 21, and  22 with IC50 values 7.5, 12.2, 12.6, 15.3, 16.1, and 18 g/mL, respectively, showed significant activity on the tumor cell lines tested.