HETEROCYCLES

Short Paper | Regular issue | Vol. 91, No. 6, 2015, pp. 1212-1226
Received, 11th March, 2015, Accepted, 20th April, 2015, Published online, 11th May, 2015.
DOI: 10.3987/COM-15-13209

■ Synthesis and Cytotoxicity Activity of Some Novel Hydrazide, Pyrazole, Isoxazole, Pyrimidine and Fused Pyran-2-one Derivatives

Hala M. Refat* and Ahmed A. Fadda

Faculty of Science, Department of Chemistry, Suez Canal University, Al-Arish, 45511, Egypt

Abstract

The reaction of 2-cyano-3-(dimethylamino)-N-((2-methoxynaphthalen-1-yl)methylene)acrylohydrazide (2) with some nitrogen nucleophiles, phenols and compounds, having an active methylene group to synthesis polyfunctionally substituted azoles (3-5), azine (6), fused azines (7-10) and fused pyran-2-one (11-17) derivatives. All the synthesized products were confirmed by elemental analysis, IR, 1H-NMR, 13C-NMR and MS data. The antitumor evaluation of the newly products against the four human tumor cells lines namely heptacelluar carcinoma (liver) HepG-2, Colorectal carcinoma (colon) HCT-116, mammary gland (breast) MCF-7 and Epdermoid carcinoma (larynx) Hep-2 was investigated. Compound 4 exhibited superior in vitro antitumor activity in the 3-cell lines assay.

Enaminonitriles are important intermediates for the synthesis of heterocyclic compounds having various biological activities.1-7 It's investigated and exhibited a wide range of bioactivities including antitumor,8 antimicrobial,9 antiviral,10 analgesic, and anti-inflammatory drugs11 have been reported. On the other hand, hydrazones possessing an azomethine group (-CONH-N=CH-) constituted an important class of compounds for new drug development12 and showed a lot of bioactivity such as antimicrobial,13 antitubercular,14,15 anticonvulsant,16 analgesic,17 anti-inflammatory,18,19 antiplatelet aggregation,20 anticancer,21,22 antifungal,23 antiviral,24 antibacterial25 and antimalarial26 activities. These facts have prompted us for the development of new hydrazones and evaluated for their antitumor activities. In continuation of our interest an ongoing program aiming at finding new structural leads with potential chemotherapeutic activities;27-30 it was an excuse to synthesize some novel polysubstituted heterocycles linked to bicyclic naphthalene skeleton through a hydrazone linkage of potential interest. We report herein a facial synthesis of the highly versatile, hitherto unreported 2-cyano-3-(dimethylamino)-N-((2-methoxynaphthalen-1-yl)methylene)acrylohydrazide (2) and the results of its utility as a building block for the synthesis of the title compounds that would produce anticancer activity.
The target compounds are depicted in Schemes 1-4. The starting compound, 2-cyano-3-(dimethylamino)-N-((2-methoxynaphthalen-1-yl)methylene)acrylohydrazide (2) was prepared by refluxing an equimolar amounts of 2-cyano-N-((2-methoxynaphthalen-1-yl)methylene)acetohydrazide (1)31 and dimethylformamide dimethyl acetal (DMF-DMA) in dry xylene. The assignment of structure 2 was supported by elemental analysis and spectral data. For example, its 1H-NMR spectrum revealed two singlet signals at δ 3.27 and 3.33 ppm due to NMe2 group, also, a singlet signals at δ 4.07 and 8.17 ppm due to OMe and vinylic protons, respectively. Further, the mass spectrum of 2 showed the molecular ion peak at m/z = 322 (M+, 86.17), which matches with its molecular formula C18H18N4O2.
The reactivity of enaminonitrile 2 towards some nitrogen nucleophiles was investigated. Thus, when enaminonitrile 2 was reacted with hydrazine hydrate and phenylhydrazine in refluxing ethanol furnished the pyrazole derivatives 3 and 4, respectively. Structures of compounds 3 and 4 were established on the basis of elemental analysis and spectral data. The IR spectra of pyrazoles 3 and 4 were free of nitrile function and showed absorption bands for NH2 at 3450-3320 cm-1. The 1H-NMR spectra of 3 and 4 displayed a singlet signal at δ 8.35 ppm, which represents the C3 proton of the pyrazole ring. Also, the mass spectra showed the molecular ion peaks at m/z = 309 (M+, 5.25%), and 385 (M+, 7.49%), respectively, which are in agreement with their molecular formulas.
Similarly, the enaminonitrile 2 reacts with hydroxylamine hydrochloride in refluxing ethanol containing anhydrous potassium carbonate afforded isoxazole derivative 5. The assignment of structure 5 was supported by elemental analysis and spectral data. The IR spectrum displayed stretching vibration bands at 3410, 3379, 3201 and 1635 cm-1 corresponding to NH2, NH and CO groups, respectively. Its 1H-NMR spectrum revealed the presence of signals at δ 4.05, 6.13, 9.04 and 11.74 ppm assignable for OCH3, CH=N and NH protons, respectively. The mass spectrum showed the molecular ion peak at m/z = 310 (M+, 36.58%) corresponding to the molecular formula (C16H14N4O3).

We have also investigated the reactivity of enaminonitrile 2 towards guanidine. Thus, when 2 was treated with guanidine hydrochloride in refluxing ethanol containing anhydrous potassium carbonate afforded 2,4-diamino-N-((2-methoxynaphthalen-1-yl)methylene)pyrimidine-5-carbohydrazide (6). The assignment of structure 6 was supported by elemental analysis and spectral data. The mass spectrum of 6 showed the molecular ion peak at m/z = 336 (M+, 10.37) corresponding to a molecular formula C17H16N6O2.
Compounds 3-6 are assumed to be formed via addition of N-nucleophiles to the activated ethylenic double bond of enaminonitrile 2, followed by intramolecular cyclization and elimination of a dimethylamine molecule to give target compounds 3-6, is in line with the reported results of the reaction of enaminonitrile with nucleophiles under basic conditions.32
On the other hand, we investigated the reactivity of enaminonitrile 2 towards some N,N-binucleophiles to give benzothiazolo[3,2-a]pyrimidine (7), 2,3-dimethylpyrimido[1,2-b][1,2,4]triazine (8), pyrimido[1,2-b][1,2,4]triazine (9) and pyrido[1,2-a]pyrimidine (10), which expected pharmaceutical interest. Thus, reaction of enaminonitrile 2 with equimolar amounts of heterocyclic amines namely 2-aminobenzothiazole, 3-amino-5,6-dimethyl triazole, 3-aminotriazole and 2-amino-6-methylpyridine in refluxing acetic acid afforded the corresponding fused pyrimidine derivatives 7-10 (Scheme 2).
The structures 7-10 were established by the correct elemental analysis and compatible spectroscopic data. These products were confirmed by the disappearance of a cyano absorption band around 2186 cm-1 in their IR spectra, and the presence of a characteristic singlet signal was due to the C4 proton of pyrimidine ring at δ 8.96 ppm in their 1H-NMR spectra.

Next, we studied the reactivity of the enaminonitrile 2 towards some phenols, having an active methylene group to obtain fused pyran-2-one linked to indanone and pyrimidine moieties through a carbohydrazide linkage has been investigated. Thus, treatment of enaminonitrile 2 with phenolic group such as α-naphthol, β-naphthol and resorcinol under acidic medium gave the corresponding chromene derivatives 11-13. Analytical and spectral data for the later compounds were in agreement with the proposed structures. Generally, the IR spectra showed the absence of absorption band of cyano group and presence of the absorption band at 1713-1724 cm-1due to lactone carbonyl group, (see experimental).

Moreover, refluxing enaminonitrile 2 with active methylene compound such as dimedone in acetic acid afforded N-((2-methoxynaphthalen-1-yl)methylene)-7,7-dimethyl-2,5-dioxo-5,6,7,8-tetrahydro-2H- chromene-3-carbohydrazide (14). The assignment of structure 14 was supported by elemental analysis and spectral data. Its IR spectrum displayed stretching vibration bands at 3437 cm-1 corresponding to NH group, in addition to, the stretching vibration of three carbonyl groups at 1719-1642 cm-1. Its 1H-NMR spectrum revealed the presence of singlet signals at δ 1.09, 1.91, 2.33 ppm assignable for two CH2 and CH3 protons. Also, the mass spectrum showed the molecular ion peak at m/z = 418 corresponding to the molecular formula C24H22N2O5.
Furthermore, N-((2-methoxynaphthalen-1-yl)methylene)-2,5-dioxo-2,5-dihydroindeno[1,2-b]pyran-3- carbohydrazide (15) could be achieved by reaction of enaminonitrile 2 with 1,3-indandione, in acetic acid under reflux. The assignment of structure 15 was based on analytical and spectral data. The IR spectrum displayed stretching vibration bands at 3435, 1705 and 1637 cm-1 corresponding to NH and CO groups, respectively. Its 1H-NMR spectrum revealed the presence of signals at δ 3.84, 8.54, 9.06 and 11.07 ppm assignable for OCH3, C4 of coumarin ring, CH=N and NH protons, respectively. The mass spectrum showed the molecular ion peak at m/z = 424 (M+, 20.68%) corresponding to the molecular formula C25H16N2O5.

Finally, the reaction of enaminonitrile 2 with either barbituric acid or thiobarbituric acid in acetic acid afforded the corresponding pyrano[2,3-d]pyrimidine derivatives 16, 17, respectively. The structures 16, 17 were established by the correct elemental analysis and compatible spectroscopic data (see experimental).
Similar to the proposed reaction mechanism in scheme 3, compounds 11-17 were formed.

CYTOTOXICITY ACTIVITY
All the newly synthesized compounds, ten analogs were selected to be evaluated for their in vitro anticancer effect via the standard MTT method,33-35 against a panel of four human tumor cell lines namely; hepatocellular carcinoma (liver) HepG-2, colorectal carcinoma (colon) HCT-116, mammary gland (breast) MCF-7 and epidermoid carcinoma (larynx) Hep-2. The cell lines were obtained from ATCC via the Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. 5-Fluorouracil (5-Fu) was used as a standard anticancer drug for comparison. The results of cytotoxic activity are reported in Table 1.

The obtained results revealed that compound 4 are more potent and efficacious than 5-fluorouracil as reference drug towards the three tested human tumor cell lines. As for activity against hepatocellular carcinoma HepG-2, the highest cytotoxic activity was displayed by compounds 2, 4, 13 and 14 which showed the percentage viability IC50 at 2.60, 2.16, 2.41 and 2.51 µg/mL, respectively.
Colorectal carcinoma (colon) HCT-116 cell line showed the highest sensitivity towards the tested compounds, as its growth was found to be initiated by five compounds. The best activity was demonstrated by compounds 2, 4, 5, 13 and 14 which have IC50 at 5.22, 4.86, 5.47, 5.02 and 5.02 µg/mL, respectively. On the other hand, mammary gland (breast) MCF-7 cell line showed highest sensitivity towards the tested compounds, as its growth was found to be initiated by five compounds. The best activity was demonstrated by compounds 2, 4, 5, 13 and 14 which have IC50 at 6.87, 5.90, 7.10, 6.24 and 6.82 µg/mL, respectively.
Further interpretation of the results revealed that compounds 2, 4, 5, 13 and 14 showed high anticancer activity against breast cancer MCF-7 with percentage inhibition range of 3.56-4.24 µg/mL.

STRUCTURE ACTIVITY RELATIONSHIP
By comparing the experimental cytotoxicity of the compounds reported in this study to their structures, the following structure activity relationships (SAR) were postulated.
- The presence of a basic skeleton carbohydrazide is necessary for the broad spectrum of cytotoxic activity towards different cell lines (HepG-2, HCT-116, MCF-7 and Hep-2).
- Introducing of N-phenyl pyrazole ring to carbohydrazide derivative increases the cytotoxic activity.
- Transformation of carbohydrazide derivative to pyrimido[1,2-b][1,2,4]triazine reduces the antitumor activity, may be due its bulky size and fused heterocyclic system.
- The pyran ring containing hydroxyl group increases the cytotoxic activity.
- Introducing of isoxazole ring to carbohydrazide derivative increases the activity towards HCT-116 and MCF-7.
In conclusion, the objective of the present study was to synthesize and investigate the anticancer activity of some novel carbohydrazide with the hope of discovering new structure leads to serving as anticancer agents. The results of the anticancer screening revealed that compounds 2, 4, 5, 13 and 14 exhibited the highest in vitro cytotoxic activity when compared with the other tested compounds and 5-fluorouracil as a reference drug. In particular compound 4 proved to the most active member in this study with special effective against the human HepG-2, HCT-116 and MCF-7.

EXPERIMENTAL
Melting points Melting points were measured with a Gallenkamp apparatus are uncorrected. IR spectra were recorded KBr discs on a Mattson 5000 FTIR spectrophotometer at Microanalytical Unit, Faculty of Science, Mansoura University. The 1H NMR and 13C NMR spectra were measured on Bruker WP AC 300 (75 MHz) in DMSO-d6 as solvent, using tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed as δppm. Mass spectra were determined on Finnigan Incos 500 (70 eV). Elemental analyses were carried out at the Microanalytical Centre, Faculty of Science, Cairo University, Egypt. The results were found to be in good agreement with the calculated values.
Synthesis of 2-cyano-3-(dimethylamino)-N-(2-methoxynaphthalen-1-yl)methylene)acrylohydrazide (2). A mixture of compound 1 (2.67 g, 0.01 mol) and dimethylformamide dimethyl acetal (1.32 mL, 0.01 mol) in dry xylene (25 mL) was refluxed for 4 h, then left to cool at room temperature. The orange precipitate product was filtered off and recrystallized from toluene to give compound 2; Orange crystals; yield (82%); mp 178-180 oC (toluene); IR (KBr): ν/cm-1= 3245 (NH), 2186 (CN), 1666 (C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.27 (s, 3H, NCH3), 3.33 (s, 3H, NCH3), 4.07 (s, 3H, OCH3), 7.44-8.14 (m, 6H, Ar-H), 8.17 (s, 1H, CH=C), 9.05 (s, 1H, CH=N), 11.74 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 43.5 (2C), 55.5, 96.0, 104.3, 115.5, 119.5 (2C), 123.0, 126.2, 128.0, 129.4, 132.2, 133.2, 143.0, 153, 155.5, 172.0; MS (EI, 70 eV) m/z (%) = 322 (M+, 86.17), 292 (57.45), 251 (72.21), 221 (67.02), 199 (58.51), 184 (55.32), 169 (100.00). Anal. Calcd for C18H18N4O2 (322.37): C, 67.07; H, 5.63; N, 17.38%. Found: C, 67.02; H, 5.57; N, 17.29%.
General procedure for the reaction of enaminonitrile 2 with N-nucleophiles. To a solution of enaminonitrile 2 (0.64 g, 2 mmol) in EtOH (20 mL), hydrazine hydrate (80%, 0.2 mL) or phenylhydrazine (0.2 mL, 2 mmol) was added. The reaction mixture was refluxed for 8 h, and then cooled. The solid product so formed was filtered off, washed with EtOH, dried and recrystallised from a mixture of DMF/EtOH (1:2) to give compounds 3 and 4.
5-Amino-N-((2-methoxynaphthalen-1-yl)methylene)-1H-pyrazole-4-carbohydrazide (3). Yellow powder; yield (76%); mp 265-266 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3450, 3369 (NH2), 3275, 3125 (2NH), 1661 (C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.06 (s, 3H, OCH3), 6.81 (s, 2H, NH2), 7.44-8.16 (m, 6H, Ar-H), 8.35 (s, 1H, pyrazole H3), 9.02 (s, 1H, CH=N), 9.50, 11.74 (s, 2H, 2NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.5, 104.5, 114.0, 119.1 (2C), 123.4, 126.3, 128.2, 129.5, 130.6, 132.4, 133.5, 134.6, 143.0, 153, 168.0; MS (EI, 70 eV) m/z (%) = 309 (M+, 5.25), 265 (4.01), 225 (4.01), 184 (7.43), 169 (14.27), 139 (3.24). Anal. Calcd for C16H15N5O2 (309.33): C, 62.13; H, 4.89; N, 22.64%. Found: C, 62.02; H, 4.83; N, 22.54%.
5-Amino-N-((2-methoxynaphthalen-1-yl)methylene)-1-phenyl-1H-pyrazole-4-carbohydrazide (4). Yellow powder; yield (65%); mp 198-200 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3428, 3320 (NH2), 3228 (NH), 1662 (C=O), 1624 (C=N); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.06 (s, 3H, OCH3), 6.74 (s, 2H, NH2), 7.52-8.21 (m, 6H, Ar-H), 8.34 (s, 1H, pyrazole H3), 9.02 (s, 1H, CH=N), 11.72 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.6, 104.5, 119.2 (2C), 123.4 (3C), 126.4 (2C), 128.0, 129.5 (3C), 132.0, 133.0, 139.0, 140.0, 143.3, 150, 153.5 (2C), 168.0; MS (EI, 70 eV) m/z (%) = 385 (M+, 7.49), 351 (5.95), 276 (10.56), 228 (5.85), 213 (8.10), 185 (29.95). Anal. Calcd for C22H19N5O2 (385.43): C, 68.56; H, 4.97; N, 18.17%. Found: C, 68.52; H, 4.93; N, 18.12%.
Synthesis of 5-amino-N-((2-methoxynaphthalen-1-yl)methylene)isoxazole-4-carbohydrazide (5). A mixture of enaminonitrile 2 (0.64 g, 2 mmol) and hydroxylamine hydrochloride (0.18 g, 2.3 mmol) in EtOH (20 mL) containing anhydrous potassium carbonate (0.55 g, 4 mmol) was heated under refluxed for 6 h, then allowed to cool at room temperature and diluted with ice cold water (30 mL). The solid product so formed was filtered off, washed with water and recrystallised from EtOH to afford compound 5; Yellow crystals; yield (74%); mp 198-200 oC (EtOH); IR (KBr): ν/cm-1= 3410, 3379 (NH2), 3201 (NH), 1661 (C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.05 (s, 3H, OCH3), 6.13 (s, 2H, NH2), 7.49-8.15 (m, 6H, Ar-H), 8.31 (s, 1H, isoxazole H3), 9.04 (s, 1H, CH=N), 11.74 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.5, 104.5, 108.4, 119.0 (2C), 124, 126.6, 128.0, 129.0, 132.0, 133.0, 143.0, 153.3, 160.0, 168.0, 171.0; MS (EI, 70 eV) m/z (%) = 310 (M+, 36.58), 296 (22.18), 280 (28.79), 227 (22.57), 186 (28.40), 153 (35.80). Anal. Calcd for C16H14N4O3 (310.31): C, 61.93; H, 4.55; N, 16.06%. Found: C, 61.89; H, 4.47; N, 16.09%.
Synthesis of 2,4-diamino-N-((2-methoxynaphthalen-1-yl)methylene)pyrimidine-5-carbohydrazide (6). A mixture of enaminonitrile 2 (0.64 g, 2 mmol) and guanidine hydrochloride (0.22 g, 2.3 mmol) in EtOH (20 mL) containing anhydrous potassium carbonate (0.55 g, 4 mmol) was heated under refluxed for 8 h. The reaction mixture was allowed to cool to room temperature and diluted with ice-cold water (30 mL) containing few drops with HCl. The solid product so formed was filtered off, washed with water and recrystallised from EtOH to afford compound 6; Yellow crystals; yield (86%); mp 190-192 oC (EtOH); IR (KBr): ν/cm-1= 3396, 3340 (2NH2), 3197 (NH), 1660 (C=O); 1H-NMR (300 MHz, DMSO- d6) δ (ppm): 3.98 (s, 3H, OCH3), 6.61, 6.92 (s, 4H, 2NH2), 7.39-8.25 (m, 6H, Ar-H), 8.56 (s, 1H, pyrimidine H6), 9.05 (s, 1H, CH=N), 11.53 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO- d6) δ (ppm): 55.5, 104.2, 108.2, 119.0 (2C), 124.3, 126.6, 128.0, 129.5, 132.4, 133.0, 143.2, 153.4, 155.0, 162.0, 163.0, 168.0; MS (EI, 70 eV) m/z (%) = 336 (M+, 10.37), 287 (11.81), 214 (11.29), 183 (54.72), 153 (35.43), 137 (100.00). Anal. Calcd for C17H16N6O2 (336.35): C, 60.71; H, 4.79; N, 24.99%. Found: C, 60.63; H, 4.70; N, 24.93%.
General procedure for the reaction of enaminonitrile 2 with N,N-binucleophiles. To a solution of enaminonitrile 2 (0.64 g, 2 mmol) in acetic acid (10 mL), an equimolar amount of the appropriate heterocyclic amines (2-aminobenzothiazole, 5,6-dimethyl-3-amino-1,2,4-triazine , 3-amino-1,2,4-triazine and 2-amino-6-methylpyridine) was added and the mixture was heated under reflux for 10-14 h, then evaporated in vacuo. The residue was triturated with EtOH and the was collected by filtration, dried well, and recrystallized from a mixture of EtOH/DMF (1:1) to give compounds 7-10.
N-((2-Methoxynaphthalen-1-yl)methylene)-4-oxo-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carbo-hydrazide (7). Brown powder; yield (65%); mp 286-288 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3437 (NH), 1666 (2C=O), 1627 (C=N); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.98 (s, 3H, OCH3), 7.60-8.68 (m, 10H, Ar-H), 8.96 (s, 1H, pyrimidine-H4), 9.09 (s, 1H, CH=N), 11.06 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.5, 104.3, 119.2 (2C), 122.4, 124.2 (2C), 125.5, 126.4 (2C), 128.3, 129.4 (2C), 130.5, 132.0, 133.0, 136.0, 143.3, 153.2, 155.0, 158.0, 163.5, 168.0; MS (EI, 70 eV) m/z (%) = 428 (M+, 35.68), 307 (24.07), 281 (21.58), 230 (26.14), 184 (24.90), 153 (26.97), 126 (34.85). Anal. Calcd for C23H16N4O3S (428.47): C, 64.47; H, 3.76; N, 13.08%. Found: C, 64.40; H, 3.68; N, 13.12%.
N-((2-Methoxynaphthalen-1-yl)methylene)-2,3-dimethyl-8-oxo-8H-pyrimido[1,2-b][1,2,4]triazine-7-carbohydrazide (8). Brown powder; yield (68%); mp > 300 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3403 (NH), 1662 (2C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 2.49 (s, 6H, CH3), 3.97 (s, 3H, OCH3), 7.09-8.22 (m, 6H, Ar-H), 8.97 (s, 1H, pyrimidine-H4), 9.08 (s, 1H, CH=N), 11.07 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 18.5, 23.2, 55.5, 104.5, 119.5 (2C), 124.4, 126.2, 128.0, 129.4 (2C), 132.2, 133.3, 143.5, 145.3, 153.4 (2C), 155.0, 160.0, 166.3, 168.2; MS (EI, 70 eV) m/z (%) = 402 (M+, 11.20), 386 (10.16), 296 (11.33), 199 (7.55), 180 (11.33), 153 (6.77). Anal. Calcd for C21H18N6O3 (402.41): C, 62.68; H, 4.51; N, 20.88%. Found: C, 64.31; H, 4.46; N, 20.82%.
N-((2-Methoxynaphthalen-1-yl)methylene)-8-oxo-8H-pyrimido[1,2-b][1,2,4]triazine-7-carbohydraz-ide (9). Brown powder; yield (74%); mp > 300 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3432 (NH), 1663 (2C=O), 1621 (C=N); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.05 (s, 3H, OCH3), 7.61-8.65 (m, 8H, Ar-H), 8.96 (s, 1H, pyrimidine-H4), 9.09 (s, 1H, CH=N), 11.08 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.3, 104.2, 119.0 (2C), 124.3, 126.5, 128.0, 129.2 (2C), 132.3, 133.1, 136.0, 143.3, 150.1, 153.4 (2C), 155.0, 166.0, 168.0; MS (EI, 70 eV) m/z (%) = 374 (M+, 26.62), 327 (23.05), 280 (20.13), 225 (26.30), 186 (34.42), 173 (27.27). Anal. Calcd for C19H14N6O3 (374.36): C, 60.96; H, 3.77; N, 22.45%. Found: C, 60.86; H, 3.70; N, 22.39%.
N-((2-Methoxynaphthalen-1-yl)methylene)-6-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbohy-drazide (10). Reddish brown powder; yield (77%); mp > 300 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3430 (NH), 1663 (2C=O), 1627(C=N); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 2.45 (s, 3H, CH3), 4.06 (s, 3H, OCH3), 6.65-8.35 (m, 9H, Ar-H), 8.97 (s, 1H, pyrimidine-H4), 9.06 (s, 1H, CH=N), 11.07 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 23.5, 55.5, 104.4, 118.2, 119.2 (2C), 124.1, 125.0, 126.2 , 128.0, 129.3 (2C), 132.1, 133.3, 134.0, 143.2 (2C), 150.0, 153.1, 155.0, 160.0, 168.0; MS (EI, 70 eV) m/z (%) = 386 (M+, 42.92), 340 (24.66), 290 (36.99), 265 (47.03), 215 (33.79), 180 (38.36), 155 (39.73). Anal. Calcd for C22H18N4O3 (386.41): C, 68.38; H, 4.70; N, 14.50%. Found: C, 68.33; H, 3.62; N, 14.55%.
General procedure for the reaction of enaminonitrile 2 with phenols and compounds, have an active methylene group. To a solution of enaminonitrile 2 (0.64 g, 2 mmol) in acetic acid (10 mL), and α-naphthol or β-naphthol (0.28 g, 2 mmol) or resorcinol (0.22 g, 2 mmol) or dimedone (0.28 g, 2 mmol) or 1,3-indandione (0.29 g, 2 mmol) or barbituric acid (0.25 g, 2 mmol) or thiobarbituric acid (0.28 g, 2 mmol) was refluxed for 10 h. The obtained solid product was collected while it hot by filtration and recrystallized from a mixture of EtOH/DMF (1:1) to give compounds 11-17.
N-((2-Methoxynaphthalen-1-yl)methylene)-2-oxo-2H-benzo[h]chromene-3-carbohydrazide (11). Brown powder; yield (73%); mp > 300 oC (EtOH-DMF) ; IR (KBr): ν/cm-1= 3423 (NH), 1716, 1662 (2C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.04 (s, 3H, OCH3), 7.20-8.14 (m, 12H, Ar-H), 8.54 (s, 1H, coumarin-H4), 9.09 (s, 1H, CH=N), 11.07 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.4, 104.5, 114.2, 117.5, 119.6 (3C), 120.5 (3C), 122.0, 124.3, 126.4 (3C), 127.5 , 128.2, 129.4, 132.2, 133.1, 134.3, 143.2, 153.3, 155.5, 160.0, 168.0; MS (EI, 70 eV) m/z (%) = 422 (M+, 54.30), 350 (40.00), 285 (22.90), 214 (28.60), 185 (25.70), 167 (31.40), 126 (34.30). Anal. Calcd for C26H18N2O4 (422.44): C, 73.93; H, 4.30; N, 6.63%. Found: C, 73.87; H, 4.27; N, 6.55%.
N-((2-Methoxynaphthalen-1-yl)methylene)-3-oxo-3H-benzo[f]chromene-2-carbohydrazide (12). Red powder; yield (78%); mp > 300 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3418 (NH), 1713, 1660 (2C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.06 (s, 3H, OCH3), 7.29-8.16 (m, 12H, Ar-H), 8.54 (s, 1H, coumarin-H4), 9.09 (s, 1H, CH=N),11.07 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.6, 104.2, 114.1, 115.5, 117.2, 119.3 (2C), 122.2, 123.5, 124.2, 126.6 (2C), 128.5 (3C), 129.4, 130.2 (2C), 132.1, 133.1, 143.3, 144.2, 150.3, 153.5, 160.2, 168.4; MS (EI, 70 eV) m/z (%) = 422 (M+, 23.14), 391 (18.57), 346 (22.57), 320 (24.00), 213 (18.86), 199 (18.57), 186 (15.71). Anal. Calcd for C26H18N2O4 (422.44): C, 73.92; H, 4.30; N, 6.63%. Found: C, 73.83; H, 4.22; N, 6.65%.
7-Hydroxy-N'-((2-methoxynaphthalen-1-yl)methylene)-2-oxo-2H-chromene-3-carbohydrazide (13). Orange powder; yield (81%); mp > 300 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3453 (OH), 3204 (NH), 1724, 1663 (2C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.06 (s, 3H, OCH3), 7.12-8.18 (m, 9H, Ar-H), 8.54 (s, 1H, coumarin-H4), 9.09 (s, 1H, CH=N), 10.23 (s, 1H, OH), 11.07 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.5, 104.0 (2C), 112.3, 114.1, 117.2, 119.4 (3C), 124.2, 126.5, 128.5, 129.2, 130.1, 132.2, 133.1, 143.2, 153.3, 156.0, 158.5, 160.0, 168.2; MS (EI, 70 eV) m/z (%) = 388 (M+, 33.50), 370 (26.60), 353 (36.45), 313 (42.86), 226 (28.08), 199 (38.42), 181 (32.02). Anal. Calcd for C22H16N2O5 (388.38): C, 68.04; H, 4.15; N, 7.21%. Found: C, 67.97; H, 4.09; N, 7.18%.
N-((2-Methoxynaphthalen-1-yl)methylene)-7,7-dimethyl-2,5-dioxo-5,6,7,8-tetrahydro-2H-chromene3-carbohydrazide (14). Buff powder; yield (85%); mp 165-166 C (EtOH-DMF); IR (KBr): ν/cm-1= 3437 (NH), 1719-1642 (3C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 1.09 (s, 6H, 2CH3), 1.91 (s, 2H, CH2), 2.33 (s, 2H, CH2), 4.07 (s, 3H, OCH3), 7.26-7.95 (m, 6H, Ar-H), 8.54 (s, 1H, coumarin-H4), 9.02 (s, 1H, CH=N), 11.07 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 28.1 (2C), 33.2, 42.5, 43.6, 55.6, 104.5, 114.2, 119.3 (2C), 123.1, 124.6, 126.7, 128.3, 129.5, 132.2, 133.3, 143.2, 153.4, 154.5, 156.4, 160.3, 168.5, 186.2; MS (EI, 70 eV) m/z (%) = 418 (M+, 17.93), 387 (13.64), 374 (14.39), 357 (16.41), 266 (14.65), 252 (23.23), 185 (15.66), 173 (17.93), 126 (25.25). Anal. Calcd for C24H22N2O5 (418.45): C, 68.89; H, 5.30; N, 6.69%. Found: C, 68.79; H, 5.21; N, 6.63%.
N-((2-Methoxynaphthalen-1-yl)methylene)-2,5-dioxo-2,5-dihydroindeno[1,2-b]pyran-3- carbohydrazide (15). Red powder; yield (82%); mp > 300 C (EtOH-DMF); IR (KBr): ν/cm-1= 3435 (NH), 1705, 1637 (3C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.84 (s, 3H, OCH3), 7.28-8.30 (m, 10H, Ar-H), 8.54 (s, 1H, coumarin-H4), 9.06 (s, 1H, CH=N), 11.07 (s, 1H, NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.4, 104.0, 116.4, 119.6 (2C), 123.2 (2C), 124.4, 126.2 (2C), 128.4 (2C), 129.0, 132.1, 133.2, 134.5, 136.3, 138.5, 143.1, 153.5, 154.6, 156.3, 160.0, 168.5, 186.2; MS (EI, 70 eV) m/z (%) = 424 (M+, 20.68), 349 (53.93), 306 (24.61), 276 (17.28), 228 (30.89), 213 (21.20), 182 (19.90), 152 (18.59), 126 (43.46). Anal. Calcd for C25H16N2O5 (424.41): C, 70.75; H, 3.80; N, 6.60%. Found: C, 70.71; H, 3.75; N, 6.52%.
N-((2-Methoxynaphthalen-1-yl)methylene)-2,4,7-trioxo-1,3,4,7-tetrahydro-2H-pyrano[2,3-d]pyrimi-dine-6-carbohydrazide (16). Orange powder; yield (76%); mp 270-272 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3453-3206 (3NH), 1709-1642 (4C=O); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.97 (s, 3H, OCH3), 7.29-8.34 (m, 6H, Ar-H), 8.54 (s, 1H, coumarin-H4), 9.02 (s, 1H, CH=N), 10.32, 11.18, 11.74 (s, 3H, 3NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.6, 88.4, 104.2, 119.5 (2C), 123.1, 124.3, 126.5, 128.2, 129.4, 132.0, 133.0, 143.2, 153.5 (2C), 155.0, 156.8, 160.5 (2C), 168.5; MS (EI, 70 eV) m/z (%) = 406 (M+, 10.51), 375 (11.54), 276 (9.08), 241 (8.17), 182 (6.74), 126 (8.17). Anal. Calcd for C20H14N4O6 (406.35): C, 59.12; H, 3.47; N, 13.79%. Found: C, 59.03; H, 3.41; N, 13.71%.
N-((2-Methoxynaphthalen-1-yl)methylene)-4,7-dioxo-2-thioxo-1,3,4,7-tetrahydro-2H-pyrano[2,3-d]-pyrimidine-6-carbohydrazide (17). Brown powder; yield (78%); mp > 300 oC (EtOH-DMF); IR (KBr): ν/cm-1= 3139 (3NH), 1709-1641 (3C=O), 1140 (C=S); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.97 (s, 3H, OCH3), 7.78-8.35 (m, 6H, Ar-H), 8.56 (s, 1H, coumarin-H4), 8.92 (s, 1H, CH=N), 10.32, 11.97, 12.53 (s, 3H, 3NH, D2O exchangeable); 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 55.6, 88.5, 104.1, 119.2 (2C), 123.2, 124.3, 126.6, 128.2, 129.5, 132.2, 133.0, 143.2, 153.5, 157.1, 159.1, 160.5, 165.0, 168.5, 170.4; MS (EI, 70 eV) m/z (%) = 422 (M+, 7.31), 391 (6.81), 348 (7.31), 276 (10.84), 227 (23.71), 184 (7.19), 126 (9.58). Anal. Calcd for C20H14N4O5S (422.41): C, 56.87; H, 3.34; N, 13.26%. Found: C, 56.83; H, 3.28; N, 13.21%.

ANTITUMOR EVALUATION
The stock samples were diluted with RPMI-1640 medium to desired concentrations ranging from 1 to 50 µg/mL. The final concentration of dimethyl sulfoxide (DMSO) in each sample did not exceed 1% v/v. The cytotoxic activity of the compounds was tested against human hepatocellular carcinoma (liver) cell line (HepG-2), human colorectal carcinoma (colon) cell line (HCT-116), human mammary gland (breast) cell line (MCF-7), and human epidermoid carcinoma (larynx) cell line (Hep-2). The % viability of cell was examined visually. 5-Fluorouracil was used as a standard anticancer drug for comparison.
Briefly, cell were batch cultured for 10 d, then seeded in 96- well plates of 10 x 103 cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37 ºC for 24 h under 5% CO2 using a water jacketed carbon dioxide incubator (Shedon.TC2323.Cornelius, OR, USA). The medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations ranging from 1 to 50 µg/mL. Cells were suspended in RPMI-1640 medium, 1% antibiotic-antimycotic mixture (104 µg/mL potassium penicillin, 104 µg/mL streptomycin sulfate and 25 µg/mL Amphotericin B) and 1% L-glutamin in 96-well flat bottom microplates at 37 ºC under 5% CO2. After 96 h of incubation, the medium was again aspirated, trays were inverted onto a pad of paper towels, the remaining cells rinsed carefully with medium, and fixed with 3.7% (v/v) formaldehyde in saline for at least 20 min. The fixed cells were rinsed with water, and examined. The cytotoxic activity was identified as confluent, relatively unaltered mono-layers of stained cells treated with compounds.

ACKNOWLEDGEMENTS
The authors are thankful to Department of Pharmacology, Faculty of Pharmacy, Mansoura University, Egypt for performing the antitumor evaluation

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