HETEROCYCLES

Short Paper | Regular issue | Vol. 91, No. 1, 2015, pp. 134-148
Received, 17th November, 2014, Accepted, 19th December, 2014, Published online, 7th January, 2015.
DOI: 10.3987/COM-14-13129

■ Synthesis, Characterization and Biological Activity of Some Novel Coumarin Derivatives Containing Pyridine Moiety

Ahmed A. Fadda,* Khaled S. Mohamed, Hala M. Refat, and Engy E. El-Bialy

Department of Chemistry, Faculty of Science, University of Mansoura, Mansoura 35516, Egypt

Abstract

The 2-acetyl-3H-benzo[f]chromen-3-one (1) was used as a key intermediate for the synthesis of 3-(1-amino-3-oxo-3H-benzo[f]chromen-2-yl) - but-2-enenitrile derivatives 3a-d via condensation reactions with activated nitrile derivatives in the presence of ammonium acetate. Moreover, the 3a-d underwent interamolecular cyclization to form 3-alkyl-2-amino-4-methyl-5-oxo-5H-benzo [5, 6]chromeno[4,3-b]pyridine 4a-d. Compound 1 reacts with acetophenone and cyclohexanone in the presence of cyanoacetamide to afford the benzo[5,6]chromeno[3,4-c]pyridin-5-one derivatives 5 and 6, respectively. Also, 4-aryl-6-[benzo[h]coumarin-3-yl]-3-cyano-2-pyridone derivatives 8a-d were synthesized by an efficient and convenient method by the one-pot reaction of 1 with aromatic aldehydes 7a-d and malononitrile in the presence of sodium hydroxide under a solvent free condition. This method has the advantages of mild reaction conditions, easy workup and inexpensive reagents. Moreover, 2-(4,6-diphenylpyridin-2-yl)-3H-benzo[f]chromen-3-one (16) was prepared via reaction of α-pyrdinium salt of methyl ketone of 1 with benzalacetophenone in the presence of ammonium acetate. The structures of the new synthesized compounds were confirmed by spectral data and elemental analyses. Newly compounds were tested for in vitro cytotoxicity against heptacelluar carcinoma (HepG2) and breast cancer (MCF-7) in addition to their antibacterial evaluation.

Coumarins or benzo-2-pyrone derivatives are one of the most significant families of natural product compounds and are important in synthetic organic chemistry. They have been widely used as starting materials or intermediates in the pharmaceutical, perfumery and agrochemical industries.
Coumarins are also used as fluorescent brighteners, efficient laser dyes and additives in food and cosmetics.1 The coumarins represent a large group of compounds that have been reported to possess a wide range of biological activities,2-4 including anticoagulant and antithrombotic properties.5-7
Recently, coumarins have attracted considerable attention for electronic and photonic applications8,9 due to their inherent photochemical characteristics, reasonable stability and solubility in various organic solvents. Many coumarin derivatives have been commercialized as blue-green lasers for fluorescent labels, fluorescent probes10-12 and enzymatic measurements.13 They exhibit intense fluorescence upon substitution with various functional groups at different positions.14, 15
Fused heterocyclic scaffolds with nitrogen and oxygen atoms are fundamental to the medicinal chemistry for the development of several new drugs. To date, few reports are available in the literature regarding the synthesis of fused chromeno-pyridine or chromene ring bearing pyridine moiety. Owing to the necessity for an improved methodology for the synthesis of these fused scaffolds and prevalence of impressive biological properties of both pyridine and coumarins, we were interested in developing a mild synthetic protocol for the synthesis of the fused and isolated chromeno-pyridine.
In the present work, we have developed the synthesis of new 2-amino-4-methyl-5- oxo-5H-benzo[5,6]chromeno[4,3-b]pyridine derivatives 4a-d via interamolecular cyclization of 3-(1-amino-3-oxo-3H-benzo[f]chromen-2-yl)- but-2-enenitrile derivatives 3a-d.

The expected crotononitrile derivatives 3a-d, presumably formed by condensation reaction, Michael addition of ammonia to α, β-unsaturated lactone followed by autooxidation (Scheme 1). The structures of the products 3a-d were indicated by IR, 1H-NMR, 13C-NMR, mass spectroscopy and elemental analyses. The IR spectra of compounds 3a-d showed a characteristic absorption band in the region 2000-2205 cm-1 corresponding to the stretching vibration of the cyano group. The high frequency region of the spectra showed two strong absorption bands at 3300-3400 cm-1 due to the stretching vibrations of the NH2 group, in addition to a strong absorption band in the region 1710-1722 cm-1 corresponding to the stretching vibration of the α, β-unsaturated lactone in the coumarin ring.
The 1H-NMR spectra of 3a-d showed the presence of a singlet signal within the region δ 2.15-2.30 ppm due to the methyl protons and a multiplet signal within the region δ 7.28-8.20 ppm due to aromatic protons in addition to a broad singlet signal (D2O exchangeable) in the region 6.00-6.20 ppm due to an amino group.
In addition, the structure of compounds 3a-d was confirmed by its mass spectroscopic measurement.
When compounds 3a-d were refluxed in glacial acetic acid, they afforded 3-alkyl-2-amino-4-methyl-5-oxo-5H-benzo[5,6]chromeno[4,3-b]pyridin (4a-d) (Scheme 2).

The structure of compounds 4b and 4c was established on the basis of their IR spectra which showed the absence of any peak around 2100-2250 cm-1 due to a cyano group, which confirms that cyano group was involved in the reaction, while structure of compounds 4a and 4d was established on the basis of their IR spectra which showed the presence of one absorption band in the region 2100-2250 cm-1 due to a cyano group instead of two absorption bands in the region 2100-2250 cm-1 due to cyano group in 3a and 3d. Also, structure of compounds 4a-d was established on the basis of their 1 H-NMR, 13C-NMR and mass spectroscopy. When 1 was treated with acetophenone and cyanoacetamide for furnishing ammonia,16 it afforded 2-phenyl-4-methyl-5H-benzo[5,6]chromeno[3,4-c]pyridin-5-one (5) (Scheme 3).

The structure of the product 5 was inferred from its analytical and spectral data. Thus, its IR spectrum showed characteristic absorption band at 1718 cm-1 due to a carbonyl group. The 1H-NMR spectra of 5 exhibited a singlet signal at δ 2.43 ppm due to methyl protons in addition to a multiplet signal in the region 7.10-8.40 ppm due to aromatic protons.
Typically, on treating 1 with cyclohexanone and cyanoacetamide as ammonia source, it gave 2-methyl-11,12,13,14-tetrahydro-3H-benzo[5,6]chromeno[3,4-c]quinolin-3-one (6) (Scheme 4).

Newly synthesized compound 6 was characterized based on an elemental analysis, IR, 1H-NMR and mass spectral data. The IR spectrum of compound 6 showed absorption bands in the range from 3090–2890 cm−1 due to aliphatic –CH2- and CH aromatic. The strong band at 1725 cm−1 is attributed to the C=O stretching vibration. The 1H-NMR spectrum of 6 showed a multiplet signal at δ 1.60-1.70 ppm due to CH2-12 and CH2-13, a singlet signal at δ 2.50 ppm due to a methyl group, a triplet signal at δ 2.60 ppm due to CH2-11 and a triplet signal at δ 3.10 ppm due to CH2-14, in addition to multiplet signals in region 7.20-8.40 ppm. The mass spectrum gave a molecular ion peak at m/z = 315 which confirm with the proposed structure. The combined spectral data gave strong support for the proposed structure.
Coumarins and 2-pyridones are classic heterocyclic scaffolds which constitute vital substructures of several natural products and received enormous admiration for their wide range of applications. Secondary metabolites and synthetic intermediates of 2- pyridone scaffolds demonstrate broad spectrum of synthetic, material and biological applications.17 Ricinine18 with its remarkable CNS stimulant activity was the first isolated 2-pyridone natural product followed by the discovery of analogous antibiotic natural products such as elfamycin, 19 ilicolicin20 and efratomycin, 21 with excellent vasodilating properties.22
Numerous methods23 have been reported for the synthesis of 2-pyridone derivatives because of the biological importance associated with these compounds. However, these methods suffer from several drawbacks such as a long reaction time, an excess of a volatile organic solvent, lower product yields, and harsh refluxing conditions. Therefore, the development of a simple and efficient method for the preparation of 2- pyridone derivatives is an active area of research and there is scope for further improvement involving mild reaction conditions and higher product yields.
In recent years, solvent-free organic reactions24 have caused great interests which have many advantages such as high efficiency and selectivity, easy separation and purification, mild reaction conditions and a benefit to industry as well as environment. Some solvent-free reactions can be carried out with just heating.25 We herein describe a practical and simple method to prepare 4-aryl-6-[benzo[h]coumarin-3-yl]-3-cyano-2-pyridone 8a-d with heating raw material under dry conditions. The synthesis of 4-aryl-6-[benzo[f]coumarin-3-yl]-3-cyano-2-pyridone 8a-d is illustrated in the Scheme 5.

In the presence of sodium hydroxide, the reactions of various aromatic aldehydes 7a-d and 1 with malononitrile were carried out respectively to afford the corresponding products 8a-d. All reactions were completed within 30-45 min (TLC controlled) with high yields of products.
At first, the aldol condensation takes place from aromatic aldehydes 7a-d and methyl ketone 1 to give intermediate 9, or alternatively, the Knoevenagel condensation takes place by aldehydes 7a-d and malononitrile to give intermediate 10. Then, the intermediate 9 undergoes the Michael reaction with malononitrile or the intermediate 10 undergoes the Michael reaction with 1 to afford the intermediate 11. Partial hydrolysis of the intermediate 11 gives the intermediate 12, which undergoes intramoleculer nucleophilic addition and dehydration. The formation product 8a-d from 13 could be due to dehydrogenation by the atmospheric oxygen.
Because the reaction worked under a solvent-free condition, the handling procedure of reaction was very simple. The structure of compounds 8a-d was established based on elemental and spectroscopic data. IR spectra of compounds 8a-d showed a strong absorption peak in the region 3180-3300 cm-1 due to NH group, 2100-2250 cm -1 attributable to cyano group, 1625-1640 cm-1 due to amidic carbonyl. 1H NMR showed a singlet signal 11.88-12.60 ppm due to NH, 6.70-6.90 ppm due to C5-H pyridine, in addition to a multiplet in the region of 7.20-8.30 ppm due to aromatic protons and C4-H in benzo[f]coumarin ring.
2-(4,6-Diphenylpyridin-2-yl)-3H-benzo[f]chromen-3-one (16) was synthesized by Kröhnke pyridine synthesis via the formation of α-pyridinium salt,26 then reaction of the product 15 with chalcone in the presence of ammonium acetate as shown in Scheme 6.

BIOLOGICAL ACTIVITY
ANTIBACTERIAL STUDIES
The newly synthesized compounds were checked for their in vitro against Grampositive bacteria such as Bacillus sunbtills and Enterococcus faecalis E61 and Gram-negative bacteria such as Salmonella typhimurium and Escherichia coli in order to establish their bioactivities. In these tests, Ampicillin and Chloramphenicol were used as the standard drugs. Disk diffusion technique was used for the determination of the antibacterial. The results obtained against these microorganisms are given in Table 1

The results obtained clearly show the efficiency of some of the new compounds. The results indicated that some of the synthesized compounds have higher activity than the standard such as 3a, 4a and 4b against E. coli.
We found that the activity of the synthesized compounds depends on their concentration and the strain of tested bacteria. Grampositive bacteria were more susceptible to the synthesized compounds than Gram negative ones.
This effect can be attributed in part to the great complexity of the double membrane containing cell envelope in Gram-negative bacteria, compared to the single membrane structure of positive ones.
The results depicted in Table 1 revealed that most of tested compounds displayed variable moderately inhibitory effects on the growth of the tested Grampositive and low effect against Gramnegative bacterial strains. The compounds 3a, 3b, 3c, 8a, 8b and 8c showed relative activity towards Gram-positive bacteria but less than the reference drugs. Regarding the structure-activity relationship, it was revealed that all compounds have higher activity than the others that contain either a polar group as NH2 (3a, 3b, 3c) or NH in other compounds.
Compounds 3a, 4a and 4b revealed mean diameters of the clear inhibition zones 38, 29 and 34 mm against E. Coli Gram negative bacteria, respectively, i.e. greater clear inhibition zones than obtained by two reference drugs Ampicilin and Chloroamphincol 19 and 21 mm, respectively. The activity of 3a may be due presence of ethylidenemalononitrile moiety, while 4b may be attributed to presence of sulphonyl group in its structure.

CYTOTOXIC SCREENING
The in vitro cytotoxicity IC50 (µmol/L) of the new synthesized compounds was studied using the 5-fluorouracil as reference drug, including MCF-7 (breast) and HePG2 (liver). The results are listed in Table 2

All compounds showed cytotoxicity against MCF-7 (breast) and HePG2 (liver). Compound 3a showed a very strong cytotoxicity against HePG2 (liver) even more strong than 5-FU, while compound 3a showed very weak cytotoxicity against MCF-7 (breast). Compounds 3c and 3d showed strong cytotoxicity against HePG2 (liver). Also compound 3d showed strong cytotoxicity against MCF-7 (breast). Compound 3c showed moderate cytotoxicity against MCF-7 and 4a showed moderate cytotoxicity against MCF-7 (breast) and HePG2 (liver). The other compounds showed a weak cytotoxicity against MCF-7 (breast) and HePG2 (liver). From above data, compounds, which contain crotonitrile moiety, have the strong cytotoxicity effect.
In conclusion, we have developed a simple and novel method for the synthesis of 4,6-diaryl-2-pyridone under a solvent-free condition by one-pot reactions of 1, aromatic aldehydes 7a-d, and malononitrile. Because of avoiding the use of a toxic organic solvent, this protocol has advantages of cheap starting materials, an excellent yield, mild reaction conditions, a simple experimental procedure and a friendly environment. We believe that the present methodology addresses the current devise toward green chemistry.

EXPERIMENTAL
Melting points were recorded on Gallenkamp electric melting point apparatus (Electronic Melting Point Apparatus, Great Britain, London) and are uncorrected. Precoated Merck silica gel 60F-254 plates were used for thin-layer chromatography (TLC) and the spots were detected under UV light (254 nm). The infrared spectra were obtained from potassium bromide triturate containing 0.5% of the product on Pye Unicam SP 1000 IR spectrophotometer (Thermoelectron Co. Egelsbach, Germany. The 1H-NMR spectra were determined on Varian Gemini 400 MHz (Varian Co., Cairo university, Egypt), 13C-NMR = 100 MHz. Deuterated DMSO-d6 and CDCl3 was used as a solvents, tetramethylsilane (TMS) was used as an internal standard and chemical shifts were measured in δ ppm. Mass spectra were determined on a GC-MS.QP-100 EX Shimadzu (Japan). Elemental analyses were recorded on Perkin-Elmer 2400 Elemental analyzer at the Micro-analytical Center at Cairo University, Cairo, Egypt.
Synthesis of 3-(1-amino-3-oxo-3H-benzo[f]chromen-2-yl)-but-2-enenitrile derivatives 3a-d. A mixture of each of 1 (2.38 g, 0.01 mol) and appropriate nitriles 2a-d (0.01 mol) in the presence of ammonium acetate (1.54 g, 0.02 mol) was heated in an oil-bath at 150 oC for 45 min. The reaction mixture was poured onto ice/HCl. The solid that separated out was filtered, dried and recrystallized from EtOH-DMF to give compounds 3a-d.
2-(1-(1-Amino-3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)malononitrile (3a). Dark brown solid; yield (85%); mp 200 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 3358, 3312,( NH2), 2203, 2189 ( two CN ), 1720 ( C=O); 1H-NMR (400 MHz , DMSO-d6) δ (ppm): 2.30 (s, 3H, CH3), 6.10 (s, 2H, NH2), 7.28-8.20 (m, 6H, Ar-H); 13 C NMR (100 MHz , DMSO-d6) δ (ppm): 11.9, 84.6, 104.4, 113.9, 115.0, 117.8, 121.5, 123.1, 124.4, 127.5, 128.9, 130.2, 132.1, 153.6, 154.00, 159.2, 161.00, 175.1; MS (EI, 70 eV) m/z = 301 (M+). Anal. Calcd for C18H11N3O2 (301.31): C, 71.75; H, 3.68; N, 13.95. Found: C, 71.72; H, 3.70; N, 13.97.
3-(1-Amino-3-oxo-3H-benzo[f]chromen-2-yl)-2-(phenylsulfonyl)but-2-enenitrile (3b). Dark brown solid; yield (88%); mp 104 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 3368, 3315 ( NH2), 2187 (CN ),1718 (C=O); 1H-NMR (400 MHz , DMSO-d6) δ (ppm): δ 2.23 (s, 3H, CH3), 6.14 (s, 2H, NH2), 7.28-8.20 (m, 11H, Ar-H); 13C-NMR (100 MHz , DMSO-d6) δ (ppm): 12.2 , 104.5, 114.9, 115.3, 117.1, 122.0, 122.6, 123.9, 127.2, 128.5 (2C), 128.9, 129.1, 129.9 (2C), 130.7, 132.1, 135.2, 144.1, 150.5, 158.7, 159.5, 161.5; MS (EI, 70 eV) m/z = 390 (M+- CN). Anal. Calcd for C23H16N2O4S (416.45): C, 66.34; H, 3.87; N, 6.73; S, 7.70. Found: C, 66.35; H, 3.84; N, 6.70; S, 7.70.
3-(1-Amino-3-oxo-3H-benzo[f]chromen-2-yl)-2-(benzo[d]thiazol-2-yl)but-2-enenitrile (3c) Yellow solid; yield (70 %); mp 233 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 3397, 3323 (NH2), 2200 (CN), 1721 (C=O); 1H-NMR (400 MHz, DMSO-d6,) δ (ppm): 2.30 (s, 3H, CH3), 6.20 (s, 2H, NH2), 7.28-8.20 (m, 10H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 12.5, 103.1, 106.6, 115.0, 117.2, 118.4, 121.7, 122.6, 123.4, 124.5, 124.7, 125.8, 126.9, 128.2, 128.6, 130.7, 133.2, 137.8, 151.5, 153.8, 156.1, 159.2, 159.7, 162.7; MS (EI, 70 eV) m/z = 393 (M+- NH2). Anal. Calcd for C24H15N3O2S (409.46): C, 70.40; H, 3.69; N, 10.26; S, 7.83. Found: C, 70.42; H, 3.70; N, 10.26; S, 7.84.
2-(2-(1-Amino-3-oxo-3H-benzo[f]chromen-2-yl)-1-cyanoprop-1-en-1-yl)benzonitrile (3d). Brown solid; yield (81 %); mp 118 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 3377, 3342 (NH2), 2203, 2199 (two CN), 1720 (C=O); 1H-NMR (400 MHz, DMSO-d6,) δ (ppm): 2.30 (s, 3H, CH3), 6.20 (s, 2H, NH2), 7.28-8.20 (m, 10H, Ar-H); 13C-NMR (100 MHz , DMSO-d6) δ (ppm): 12.8, 100.9, 104.1, 108.9, 114.5, 115.9, 117.2, 118.8, 122.4, 125.0, 126.2, 127.7, 128.3, 128.6, 128.9, 130.4, 131.5, 132.4, 132.9, 139.9, 147.2, 152.8, 159.4, 161.6; MS (EI, 70 eV) m/z = 377 (M+). Anal. Calcd for C24H15N3O2 (377.40): C, 76.38; H, 4.01; N, 11.13. Found: C, 76.30; H, 3.99; N, 11.10.
Synthesis of 3-alkyl-2-amino-4-methyl-5-oxo-5H-benzo[5,6]chromeno[4,3-b]pyridine 4a-d. A solution of 3a-d (0.01 mol) in glacial acetic acid (30 mL) was refluxed for 3 h. The solids that separated on concentration the solvent and cooling the solution were filtered off and recrystallized from EtOH-DMF as compounds 4a-d.
2-Amino-4-methyl-5-oxo-5H-benzo[5,6]chromeno[4,3-b]pyridine-3-carbonitrile (4a). Dark brown solid; yield (30%); mp > 300 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 3398, 3315 (NH2), 2202 (CN ),1722 (C=O); 1H-NMR (400 MHz , DMSO-d6,) δ (ppm): 2.50 (s, 3H, CH3), 7.10 (s, 2H, NH2), 7.28-8.20 (m, 6H, Ar-H); 13C-NMR (100 MHz , DMSO-d6) δ (ppm): 16.8, 84. 6, 104.4, 113.9, 115.0, 117.8, 121.5, 123.1, 124.4, 127. 5, 128.9, 130.2, 132.1, 153.6, 154.00, 159.2, 175.1, 161.00; MS (EI, 70 eV) m/z = 301 (M+). Anal. Calcd for C18H11N3O2 (301.31): C, 71.75; H, 3.68; N, 13.95. Found: C, 71.74; H, 3.67; N, 13.97.
2-Amino-4-methyl-3-(phenylsulfonyl)-5H-benzo[5,6]chromeno[4,3-b]pyridin-5-one (4b). Dark brown solid; yield (55%); mp >300 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 3364, 3349 (NH2), 1725 (C=O); 1H-NMR (400 MHz , DMSO-d6,) δ (ppm): 2.50 (s, 3H, CH3), 7.04 (s, 2H, NH2), 7.28-8.20 (m, 11H, Ar-H); 13C-NMR (100 MHz , DMSO-d6) δ (ppm): 17.1, 116.6, 116.9, 118.8, 123.9, 124.8, 126.0, 127.2, 128.6 (2C), 128.9, 129.4, 129.9 (2C), 133.7, 135.1, 137.3, 141.4, 148.8¸ 153.4, 155.8, 158.9, 163.1; MS (EI, 70 eV) m/z = 416 (M+). Anal. Calcd for C23H16N2O4S (416.45): C, 66.34; H, 3.87; N, 6.73; S, 7.70. Found: C, 66.35; H, 3.84; N, 6.70; S, 7.70.
2-Amino-3-(benzo[d]thiazol-2-yl)-4-methyl-5H-benzo[5,6]chromeno[4,3-b]pyridin-5-one (4c). Red solid; yield (32%); mp 280 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 3412, 3356 (NH2), 1725 (C=O); 1H-NMR (400 MHz , DMSO-d6) δ (ppm): 2.51 (s, 3H, CH3), 6.98 (s, 2H, NH2), 7.28-8.20 (m, 10H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm):18.9, 115.4, 116.3, 116.9, 119.2, 121.0, 121.8, 123.7, 124.1, 124.9, 125.5, 127.4, 128.2, 128.8, 129.6, 133.1, 133.8, 142.5, 148.7, 151.6, 153.3, 153.9, 158.5, 162.7; MS (EI, 70 eV) m/z = 409 (M+). Anal. Calcd for C24H15N3O2S (409.46): C, 70.40; H, 3.69; N, 10.26; S, 7.83. Found: C, 70.42; H, 3.70; N, 10.26; S, 7.84.
2-(2-Amino-4-methyl-5-oxo-5H-benzo[5,6]chromeno[4,3-b]pyridin-3-yl)benzonitrile (4d). Brown solid; yield (35%); mp >300 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 3403, 3353 (NH2), 2208 (CN), 1724 (C=O); 1H-NMR (400 MHz , DMSO-d6) δ (ppm): 2.51 (s, 3H, CH3),7.10 (s, 2H, NH2), 7.28-8.20 (m, 10H, Ar-H); 13C-NMR (100 MHz , DMSO-d6) δ (ppm): 18.9, 105.3, 107.4, 114.8, 116.0, 116.9, 117.5, 118.8, 124.2, 126.9, 127.8, 128.3, 158.7, 128.9, 129.2, 130.5, 132.9, 133.3, 134.9, 141.6, 142.8, 149.4, 153.5, 162.7; MS (EI, 70 eV) m/z = 377 (M+). Anal. Calcd for C24H15N3O2 (377.40): C, 76.38; H, 4.01; N, 11.13. Found: C, 76.36; H, 4.04; N, 11.14.
Synthesis of 2-phenyl-4-methyl-5H-benzo[5,6]chromeno[3,4-c]pyridin-5-one (5). A solution of compound 1 (1.19 g, 5 mmol ) and 2-cyanoacetamide ( 0.42 g, 5 mmol ) was heated to reflux in 20 mL acetophenone for 1 h on an oil bath in 170 °C (monitored by TLC). The solid product was filtered off and recrystallized from EtOH-DMF to give compound 5; dark yellow solid; yield (34%); mp 257 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 1718 ( C=O); 1H-NMR (400 MHz , DMSO-d6) δ (ppm): 2.43 (s, 3H, CH3), 7.10-8.40 (m, 12H, Ar-H); 13C-NMR (100 MHz , DMSO-d6) δ (ppm): 23.7, 119.1, 121.9, 123.3, 124.8, 126.0, 126.9, 127.1, 127.4 (2C), 129.8 (2C), 130.5, 132.6, 136.7, 141.7, 152.2, 154.0, 157.9, 159.2, 164.2; MS (EI, 70 eV) m/z = 337 (M+)Anal. Calcd for C23H15NO2 (337.38): C, 81.88; H, 4.48; N, 4.15. Found: C, 81.79; H, 4.45; N, 4.13.
Synthesis of 2-methyl-11,12,13,14-tetrahydro-3H-benzo[5,6]chromeno[3,4-c]quinolin-3-one (6). A solution of compound 1 (1.19 g, 5 mmol) and 2-cyanoacetamide ( 0.42 g, 5 mmol ) was heated to reflux in 20 mL cyclohexanone for 8 h on an oil bath in 170 °C (monitored by TLC). The solid product was filtered off and recrystallized from EtOH-DMF to give compound 6; Dark yellow solid; yield (41%); mp 216 °C (EtOH-DMF); IR (KBr): ν/cm-1 = 2890-3000 (CH2 aliphatic ), 3000-3090 (CH aromatic ), 1725 ( C=O ); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 1.60-1.70 (m, 4H, CH2 -12,CH2-13), 2.50 (s, 3H, CH3), 2.60 (t, J = 7.80 Hz, 2H, CH2-11), 3.10 (t, J = 7.50 Hz, 2H, CH2-14) , 7.20-8.40 (m, 12H, Ar-H); 13C-NMR (100 MHz , DMSO-d6) δ (ppm): 22.4, 22.9, 23.8, 25.1, 30.4, 119.6, 122.4, 124.3, 125.8, 126.2, 127.1, 128.9, 129.3, 131.2, 132.8, 135.3, 151.9, 154.7, 158.2, 159.7, 163.4; MS (EI, 70 eV) m/z = 315 (M+). Anal. Calcd for C21H17NO2 (315.37): C, 79.98; H, 5.43; N, 4.44. Found: C, 79.96; H, 5.46; N, 4.43.
Synthesis of 4-aryl-6-[benzo[f]coumarin-3-yl]-3-cyano-2-pyridone derivatives 8a-d. A mixture of aromatic aldehydes 7a-d (1 mmol), 1 (1 mmol), malononitrile 3 (1.5 mmol) and NaOH (1.5 mmol) was put in a reaction flask and heated to a temperature of 75 °C for about 45 min. After completing the reaction, the reaction mixture was poured into water, and then washed with water thoroughly. The product was collected by filtration, dried and recrystallized from 95% EtOH.
2-Oxo-6-(3-oxo-3H-benzo[f]chromen-2-yl)-4-phenyl-1,2-dihydropyridine-3-carbonitrile (8a). Dark brown solid; yield (87%); mp 80 °C (EtOH); IR (KBr): ν/cm-1 =3288 (NH), 2200 (CN), 1724, 1633 ( two C=O); 1H-NMR (400 MHz, CDCl3) δ (ppm): 6.80 (s, 1H, C5-H pyridine), 7.20-8.30 (m, 12H, Ar-H), 12.55 (s, 1H, NH) ; 13C-NMR (100 MHz, CDCl3) δ (ppm): 101.1, 114.7, 115.9, 118.6, 120.7, 121.6, 122.1, 123.9, 125.0, 126.8, 127.4, 128.2, 128.8 (2C), 129.1, 129.8(2C), 132.3, 134.9, 136.6, 139.4, 155.3, 159.6, 161.4, 169.5; MS (EI, 70 eV) m/z = 390 (M+). Anal. Calcd for C25H14N2O3 (390.40): C, 76.92; H, 3.61; N, 7.18. Found: C, 76.95; H, 3.58; N, 7.15.
4-(4-Hydroxyphenyl)-2-oxo-6-(3-oxo-3H-benzo[f]chromen-2-yl)-1,2-dihydropyridine-3-carbonitrile (8b). Brown solid; yield (85%), mp 143 °C (EtOH); IR (KBr): ν/cm-1 = br 3405 (OH), 3219 (NH), 2196 (CN), 1725, 1636 ( two C=O); 1H-NMR (400 MHz, CDCl3) δ (ppm): 6.80 (s, 1H, C5-H pyridine), 7.20-8.30 (m, 11H, Ar-H), 10.20 (s, 1H, OH), 12.24 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ (ppm): 101.3, 114.8, 115.9, 117.3 (2C), 118.6, 120.7, 121.6, 122.2, 123.9, 125.4, 126.8, 127.4, 128.5, 129.6, 131.1(2C), 132.6, 134.9, 136.6, 139.9, 157.8, 159.6, 161.4, 169.6; MS (EI, 70 eV) m/z = 406 (M+). Anal. Calcd for C25H14N2O4 (406.40): C, 73.89; H, 3.47; N, 6.89. Found: C, 73.90; H, 3.44; N, 6.86.
4-(4-Methoxyphenyl)-2-oxo-6-(3-oxo-3H-benzo[f]chromen-2-yl)-1,2-dihydropyridine-3-carbonitrile (8c). Dark brown solid; yield (79%); mp 110 °C (EtOH); IR (KBr): ν/cm-1 = 3198 (NH), 2204 (CN), 1725, 1635 ( two C=O); 1H-NMR (400 MHz, CDCl3) δ (ppm): 3.90 (s, 3H, OCH3), 6.80 (s, 1H, C5-H pyridine), 7.20-8.30 (m, 11H, Ar-H), 11.98 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ (ppm): 59.2, 101.3, 114.8, 115.9, 116.4 (2C), 118.5, 120.5, 121.8, 122.2, 123.9, 125.6, 126.8, 127.4, 128.5, 129.6, 130.4 (2C), 132.6, 134.9, 136.8, 140.2, 157.8, 159.6, 161.4, 168.8; MS (EI, 70 eV) m/z = 420 (M+). Anal. Calcd for C26H16N2O4 (420.42) : C, 74.28; H, 3.84; N, 6.66. Found: C, 74.26; H, 3.85; N, 6.64.

4-(4-(Dimethylamino)phenyl)-2-oxo-6-(3-oxo-3H-benzo[f]chromen-2-yl)-1,2-dihydropyridine-3- carbonitrile (8d). Red solid; yield (71%); mp 136 °C (EtOH); IR (KBr): ν/cm-1 = 3188 (NH), 2194 (CN), 1725, 1640 (two C=O); 1H-NMR (400 MHz , CDCl3) δ (ppm): 3.10 (s, 6H, 2CH3 ), 6.80 (s, 1H, C5-H pyridine), 7.20-8.30 (m, 12H, Ar-H), 11.90 (s, 1H, NH); 13C-NMR (100 MHz, CDCl3) δ (ppm): 42.6 (2C), 101.3, 113.5 (2C), 114.4, 116.0, 118.5, 120.7, 121.8, 122.2, 124.0, 125.6, 126.9, 127.4, 128.7, 129.8, 130.1 (2C), 132.9, 134.4, 136.8, 140.5, 157.9, 159.8, 161.7 , 168.9; MS (EI, 70 eV) m/z = 433 (M+). Anal. Calcd for C27H19N3O3 (433.47): C, 74.81; H, 4.42; N, 9.69. Found: C, 74.80; H, 4.44; N, 9.70.
Synthesis of 2-(2-bromoacetyl)-3H-benzo[f]chromen-3-one (14)
The compound 14 was prepared according to the reported work26
Synthesis of 1-(2-oxo-2-(3-oxo-3H-benzo[f]chromen-2-yl)ethyl)pyridin-1-ium bromide (15)
The compound 15 was prepared according to the reported work26
Synthesis of 2-(4,6-diphenylpyridin-2-yl)-3H-benzo[f]chromen-3-one (16). A solution of 15 (0.395g, 1 mmol), benzalacetophenone (0.208 g, 1 mmol) and ammonium acetate (0.77 g, 1 mmol) in 10 mL glacial acetic acid was refluxed for about 6 h. The solid product was isolated by filtration. The solid product was washed with EtOH. The crude product was dried and crystallized from DMF-EtOH to furnish pure solid product; Dark brown solid; yield (42%); mp 255 °C (DMF-EtOH); IR (KBr): ν/cm-1 =3000-3090 (CH aromatic), 1725 (C=O), 1623 (C=N); 1H-NMR (400 MHz , DMSO-d6) δ (ppm): 7.20-8.40 (m, 19H, Ar-H); 13C-NMR (100 MHz , DMSO-d6) δ (ppm):112.3, 115.9, 118.3, 119.7, 121.9, 123.8, 127.1 (2C), 127.3, 127.8 (2C), 128.7, 128.9, 129.2 (2C), 129.3, 129.4, 129.8 (2C), 130.7, 131.4, 133.3, 136.8, 144.1, 145.4, 150.9, 152.6, 154.9, 157.2 162.5; MS (EI, 70 eV) m/z = 425 (M+). Anal. Calcd for C30H19NO2 (425.49): C, 84.69; H, 4.50; N, 3.29. Found: C, 84.68; H, 4.52; N, 3.31.

ANTIMICROBIAL SCREENING
ANTIBACTERIAL ASSAY
The newly synthesized compounds obtained were preliminary evaluated for their in vitro antibacterial activity against a narrow spectrum of bacterial species procured from the Laboratory of Microbial Biochemistry (Faculty of Pharmacy, Mansoura Univ.). The paper disc assay described by Cooper27 using nutrient agar medium was applied. Suspensions of each microorganism were prepared from their 24h-cultures to obtain approximately 106 colony forming units (cfu) per mL for plating. Paper discs (Whatman No.1) of 8 mm diameter were loaded individually with a constant amount (100 μg/disc) of the compounds to be tested. Discs were aseptically transferred and applied onto the dry surface of the inoculated plates and then incubated at 37 °C for overnight (~18-20 h). This assay was performed in duplicates and the mean diameters of the clear inhibition zones (mm) were recorded disregard a single colony or a faint haze caused by the inoculums.
CYTOTOXICITY ACTIVITY
The synthesized compounds were evaluated for their in-vitro anticancer effect via the standard MTT method,28 against a panel of two human tumor cell lines namely; heptacelluar carcinoma (liver) HepG and, mammary gland (breast) MCF-7.
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.

ACKNOWLEDGEMENTS
The authors owe to Department of Pharmacology, Faculty of Pharmacy, Mansoura University, Egypt for performing the antimicrobial and cytotoxicity evaluation.

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