1.                   Introduction

Coumarin agents (known as 1,2-benzopyrone), consisting of fused benzene and α-pyrone rings are present in significant amounts in plants and more than 1300 coumarins were identified from natural sources [1]. The synthesis of Coumarins and their derivatives has attracted considerable attention from organic and medicinal chemists for many years as a large number of natural products contain this heterocyclic nucleus [2]. Coumarins comprise a vast array of biologically active compounds ubiquitous in plants, many of which have been used in traditional medicine since ancient times. The antitumor activity of coumarin against human tumor cell lines was first noted by Weber et al. [3]. The selective tumor cell specific cytotoxicity of coumarins has been well documented by Riveiro et al. [4]. However, the use of coumarin in cancer chemotherapy was first established by the successful application of Warfarin sodium on V2 cancer cell, granulocytes, lymphocytes and macrophages in different animal models. Later, clinical trials demonstrated activity of coumarins in many different cancers, including prostate cancer, malignant melanoma and metastatic renal malignant melanoma and metastatic renal cell carcinoma [5].

 2.                   Results and Discussion

 The synthesis of the intermediate and target compounds was performed according to the reactions outlined in (Schemes 1-8) and the establishment of the structure of these compounds has been confirmed by spectroscopic data. The starting compound 1 was prepared following the reported literature procedure [6]. Compound 1 was reacted with acetophenone derivatives namely, 4-chloroacetophenone and 4-aminoacetophenone under microwave irradiation and solvent free conditions to afford 2a,b in excellent yield (Scheme 1) [7 ]. The structure of 2a was supported by IR spectrum which showed characteristic absorption band at 1723cm^-1 attributable to δ-Lactone whereas, the IR spectrum of 2b showed absorption bands at 1727,3334 and 3299cm^-1 attributable to δ-Lactone and NH₂ groups. The mass spectrum of 2a showed molecular ion peak at m/z 354.6(20% M⁺). Similarly, compound 1 was reacted with active methylene compounds namely, malononitrile under solvent free and microwave irradiation conditions to yield the corresponding [(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl) methylidene] propanedinitrile 3 (Scheme1) [8,9]. The structure of compound 3 was elucidated on the basis of elemental analysis, spectral data and chemical transformation. The IR spectrum of 3 showed an absorption band at 2220cm^-1 attributable to C≡N whereas the 1H-NMR spectrum revealed the presence of singlet signals of three protons at δ 1.2 for CH₃, three protons at δ 3.8 for OCH₃ and one proton at δ 8.4 for C(CN)₂=CH.

 2.1 Scheme: 1

Treatment of compound 1 with benzenesulfonohydrazide in ethanolic solution and few drops of acetic acid afforded the corresponding N'-[(E)-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl) methylidene] benzene sulfonohydrazide 4 which was reacted with acetic anhydride to afford N'-[(E)-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl) methylidene]-N(phenylsulfonyl)acetohydrazide 5. The IR spectrum of 4 showed absorption bands at , 3349 , 1727, 1640 and 1088cm^-1 attributable to NH, δ-Lactone, C=N and SO_2, functions. Its 1H NMR spectrum revealed singlet signals of one proton at δ 8.35 for NH and one proton at δ 7.59 for CH=N . The IR spectrum of 5 showed absorption band at 1763cm^-1 for COCH₃ group and devoid the presence of NH. Its 1H-NMR revealed singlet signal of three protons at δ 2.53 for N-COCH₃ and disappearance of one Proton for NH group.

 On the other hand, compound 1 was reacted with hydrazine derivatives namely; hydrazine hydrate and cyanoacetic hydrazide to afford the corresponding 6-[(E)-hydrazinylidenemethyl]-8-methoxy-4-methyl-2H-coumarin-2-one 6 and 2-cyano-N'-[(E)-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene] acetohydrazide 7, consequently. The IR spectrum of 6 showed absorption bands at 3396.3, 3428, 1707, 1575 cm^-1 indicated the presence of  NH₂, δ-Lactone and C=N functions. Its ¹H-NMR revealed signals at δ 8.34, 8.887 (1H, s, CH=N-), (1H, s, NH) .The IR spectrum of 7 showed absorption bands at 1607.60cm^-1C=N, 1682.38 cm^-1 CO-NH amide, 2183.33cm^-1CN and 3152.33 cm^-1 NH . Its ¹H-NMR (DMSO-d6) spectrum showed signals at 1.410(3H,s,CH₃), 3.332, 3.524(4H, s, 2CH₂), 3.854(3H, s ,OCH₃), 6.251(1H, s, CH-coumarin), 7.919-7.550(2H, m, Ar-H), 8.362(1H, s, CH=N) and 8.889(1H, s, NH).

 Also, 8-methoxy-4-methyl-2-oxo-2H-coumarin-6-carbaldehyde 1 was reacted with [(phenylcarbonyl) amino]acetic acid in the presence of acetic anhydride and sodium acetate under refluxing to give (5Z)-5-[(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methyldene]-2-phenyl-1,3-oxazol-4(5H)-one 8 [10]. The IR spectrum of compound 8 indicated the presence of characteristic absorption bands at 1605.56 cm^-1C=N,1712.06 cm^-1 δ-Lactone and 1759.44cm^-1 C=O(Oxazole ring) .

 Compound 3 was reacted with ethyl cyanoacetate in the presence of catalytic amount of piperidine under refluxing to afford 2-amino-6-ethoxy-4-(8-methoxy-4-methyl-2-oxo-2H-coumain-6-yl)-4H-pyran-3,5-dicarbonitrile 9 (Scheme 2) [11]. The IR spectrum of 9 showed absorption bands at 1646.7cm^-1 C=C, 1720.6 cm^-1 δ-Lactone, 2196.6 cm^-1CN and 3213.8 , 3336.9 cm^-1NH₂ . The ¹H-NMR (DMSO-d6) spectrum showed signals at 1.224 (3H, s, CH₃), 1.646 (3H, t, CH₃CH₂) 4.250(2H, q, CH_2,CH₃), 3.770 (3H, s, OCH₃), 6.409 (1H, s, CH-coumarin), 7.272-6.932 (3H, m, Ar-H) and 9.024(2H, s, NH₂) .

 Similarly, Compound 3 was reacted with thiourea in the presence of of catalytic amount of piperidine under refluxing to gave (4E)-6-amino-4-[(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene]-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 10 [12]. The IR spectrum of 10 showed absorption bands at 1203.3 cm^-1C=S, 1651.4 cm^-1 C=N 1703.4cm^-1 δ-Lactone, 2213.8 cm^-1CN, 3178.3cm^-1NH and 3243.8,3332.8cm^-1NH₂ .

 2.2                Scheme: 2a

 In addition, compound 1 was reacted with hydrazine carbothioamide in glacial acetic acid under refluxing to afford (2E)-2-[(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene] hydrazinecarbothioamide that formulated as 11 in excellent yield (Scheme 2) [13]. The IR spectrum of 11 showed absorption bands at 1285.35cm^-1C=S, 1645.2cm^-1 C=N(imine),1696.92 cm^-1 δ-Lactone, 2696.97cm^-1 SH and 3371.0,3266.5cm^-1 NH₂. ¹H-NMR (DMSO-d6) spectrum of 11 showed signals at 1.821(3H,s,CH₃), 3.796(3H,s,OCH₃), 6.497(1H,s,CH-coumarin), 7.400-7.230(2H,m,Ar-H), 8.243(1H,s,CH=N), 9.070(1H, s, C-SH), 4.479(2H,s,NH₂) and 9.640(1H, s ,NH) . Its mass spectrum showed ion peak at m/z 291(0.01%).

 Reaction of compound 11 with acetic anhydride under refluxing yielded N-[4-acetyl-5-(8-methoxy-4 methyl-2-oxo-2H-coumarin-6-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl]acetamide 12 [13]. The IR spectrum of 12 showed absorption bands at 1614.4 cm-¹C=N, 1717.4 cm-¹ δ-Lactone, 1763.0 cm-¹ C=O (thiadiazole ring), and 3208.6cm-¹NH. Its 1H-NMR (DMSO-d6) spectrum showed signals at 1.245 (3H,s,CH₃), 1.348 (3H,s,CO-CH₃),1.909(3H, s, CO-CH₃), 3.757(3H, s ,OCH₃), 6.295(1H,s,CH-thiadiazol ring), 6.911(1H, s, CH-coumarin), 7.934-7.464(2H, m, Ar-H) and 8.372(1H, s ,NH) . Its mass spectrum showed ion peak at m/z m/z 375(0.01%) .

 Also, treatment of compound 11 with 2-bromo-1-phenylethanone under refluxing lead to the formation of (2Z)-2-[(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene]-N-(2-oxo-2-phenylethyl) hydrazine carbothioamide 13 [14]. The IR spectrum of 13 showed absorption bands at 1549.01 cm^-1C=N and1710.23 cm^-1 δ-Lactone. Its ¹H-NMR

 (DMSO-d6) spectrum showed signals at 1.055(3H, s ,CH₃), 2.498(2H, s, CH₂), 3.661(3H, s ,OCH₃), 6.355(1H, s, CH-coumarin), 7.945-6.678(7H, m, Ar-H) and 8.406 (1H, s, CH=N).

 2.3                Scheme: 2b

A mild and solvent-free conditions was used for synthesis of 1,4-dihydropyridine derivatives from ethyl acetoacetate, 8-methoxy-4-methyl-2-oxo-2H-coumarin-6-carbaldehyde 1 and ammonium acetate in the presence of zinc chloride as a catalyst under microwave irradiation conditions to give diethyl-4-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 14 [15] . The IR spectrum of 14 showed absorption bands at 1647.7 cm^-1C=N, 1725.3 cm^-1 δ-Lactone and 3269.5cm^-1 NH . Its ¹H-NMR (DMSO-d6) spectrum showed signals at 1.060(3H, s ,CH₃), 1.318-1.209 (6H, m, 2XCH₃-pyridine ring), 4.258-3.980 (4H, m, CH₂) ,4.575(1H,S,CH-Pyridine) ,3.843(3H, s ,OCH₃), 5.908(1H, s, CH-coumarin), 7.386-6.540(2H, m, Ar-H) and 8.234 (1H, s, NH).

 As consequently, The structure of 14 was confirmed by interaction with hydrazine hydrate in ethanolic solution under refluxing afforded the corresponding diethyl-4-(1-amino-8-methoxy-4-methyl-2-oxo-1,2-dihydroquinolin-6-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 15. The IR spectrum of 15 showed absorption bands at 2260.8cm^-1CN, 2966.6-2936.6cm^-1 CH₃ and 3299.8,3319.6cm^-1 NH, NH₂ . Its mass spectrum showed ion peak at m/z 427.0(0.01%).

2.4    Scheme: 3

On the other hand, reaction of 8-methoxy-4-methyl-2-oxo-2H-coumarin-6-carbaldehyde 1 with cyclohexanone and ammonium acetate in 2:1:1M ratio in the presence of anhydrous zinc chloride under microwave irradiation conditions yielded 6-(1,2,3,4,5,6,7,8,9,10-decahydroacridin-9-yl)-8-methoxy-4-methyl-2H-coumarin-2-one 16 (Scheme 4) [15]. The IR spectrum of 16 showed absorption bands at 1702.4cm^-1δ-Lactone and 3265.1cm^-1 NH. Its mass spectrum showed ion peak at m/z 377(0.41%).

 2.5                Scheme: 4

In the same manner, 8-methoxy-4-methyl-2-oxo-2H-coumarin-6-carbaldehyde 1 was reacted with thiourea and cyclohexanone in 2:1:1 M ratio under microwave irradiation conditions to give (E)-8-methoxy-6-(5-((8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylene)-2-thioxo-decahydroquinazolin-4-yl)-4-methyl coumarin-2-one17 (Scheme 5) [7]. The IR spectrum of 17 showed absorption bands at 1272.20cm-1C=S, 1597.94cm-1C=N, 1701.81cm-1 δ-Lactone and 3193.02cm-1NH. Its mass spectrum showed ion peak at m/z 554 which fragmented to give the base peak 202 (100%).

2.6                Scheme:5

In the same manner, reaction of 8-methoxy-4-methyl-2-oxo-2H-coumarin-6-carbaldehyde 1 with 3,4-dimethoxyacetophenone and thiourea in the presence of sodium ethoxide under microwave irradiated gave 6-(6-(3,4-dimethoxyphenyl)-2-thioxo-2,3-dihydropyrimidin-4-yl)-8-methoxy-4-methyl-2H-coumarin-2-one 18 (Scheme 6) [16]. The infrared spectrum of 18 showed absorption bands at at 1199.22cm^-1C=S, 1617.60 cm^-1C=N, 1722.71cm^-1 δ-Lactone and 3169.36 cm^-1NH . Its mass spectrum showed ion peak at m/z 496 which fragmented to give the base peak 75 (M⁺³100%).

2.7                Scheme: 6

 In addition, compound 1 was reacted with phenacyl bromide, cyclohexanone in acetonitrile and catalytic amount of pyridine to give 6-(3-benzoyl-2,3,4,5,6,7-hexahydrobenzofuran-2-yl)-8-methoxy-4-2H-coumarin-2-one 19 (Scheme 7) [17]. The infrared spectrum of 19 showed absorption bands at 1633.6 cm^-1C=O and 1704.0 cm^-1 δ-Lactone. Its ¹H-NMR (DMSO-d6) spectrum showed signals at 1.18(3H, s ,CH₃), 2.499-2.444(2H, m, CH₂), 2.509-2.503 (2H, m, CH₂) ,3.707(3H, s ,OCH₃), 6.434(1H, s, CH-coumarin) 6.46(OCH-Furan), 3.32(COCH-Furan), 7.976-7.469 (7H, m, Ar-H).

Compound 1 was reacted with malononitrile and hydrazine hydrate under microwave irradiation in the presence of sodium bisulphate to give 6-amino-4-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)-3-methyl-1,4-dihydropyrano [2,3-c] pyrazole-5-carbonitrile 20 (Scheme 8) [18]. The infrared spectrum of 20 showed absorption bands at 1634.2 cm^-1C=N, 1715.5 cm^-1 δ-Lactone, 2207.3 cm^-1CN and 3329.5,3204.9cm^-1 NH₂. ¹H-NMR (DMSO-d6) spectrum which showed signals at 1.202(3H, s,CH₃), 2.074( 3H, s, CH₃-pyrazol ring), 3.846(3H, s ,OCH₃), 5.494(1H,s,CH-Pyran), 6.365(1H, s, CH-coumarin), 7.951-6.509(2H, m, Ar-H), 4.180(2H, s,br, NH₂) and 12.953(1H,S,NH-Pyrazol).

 2.8                Scheme: 8

2.8.1           Cytotoxicity Study

 The anti-tumor activity of some newly synthesized coumarins against four cell lines: Human breast adenocarcinoma (MCF-7), colorectal carcinoma cells (HCT-116), hepatocellular carcinoma (Hep-G2) and prostate carcinoma (PC-3) has been evaluated by using MTT method [19, 20]. The cytotoxicity of the tested compounds (1, 5 and 15) at different concentrations against the four human cell lines. Compound (15) was the most potent against MCF-7 and HEP-G2 (IC₅₀=12.9 and 11.6µg/ml, respectively), while compound (1) was most potent against HCT-116 (IC50= 15.9 µg/ml) and compound (5) was most potent against PC-3 with IC₅₀= 26.7 µg/ml (Table 1).

The tested compounds down regulate significantly (p<0001) the expression of EGFR in both the MCF-7 and HEP-G2 cell lines, compared to their respective control cells (Figure 1).

As regard to CDKN 1, all tested compounds up regulate significantly (p<001) the expression of CDKN 1 in MCF-7 and HEP-G2, compared to their respective control cells (Figure 2).

Multiple comparison analysis showed that compounds 5 and 15 increased significantly (p<0001) the expression of CDKN 1 in MCF-7 cell line, compared to compound 1 while in HEP-G2 cell line, compound 15 increased significantly (p<004 and 003) the expression of CDKN 1 as compared to compounds 1 and 5, respectively.

 3.                   Experimental

 All melting points are not corrected. The infrared spectra on KBr disks were carried on a Pye Unicam SP³- 200 spectrophotometer. The NMR Spectra were measured on a varian EM 60 and JE OL-90 MHz Spectrometers with TMS as internal reference, chemical shifts were expressed in δ ppm. The mass spectra were determined on a FINNI-Gas 3300 mass spectrometer by direct inlet (Source temperature 90-300ºC, beam energy 70 ev).

 3.1                General procedure

 3.1.1                                                                                      Synthesis of 8-methoxy-4-methyl-2-oxo-2H-coumarin-6-carbaldehyde (1)

 A mixture of 4-hydroxy-3-methoxybenzaldehyde (0.01 mole; 1.09 gm), (0.01 mol) ethylaceto acetate in concentrated H₂SO₄ (25 ml) was heated with stirring on water bath for half an hours. The reaction product was poured onto ice/cold water, the solid that separated was crystallized from ethanol to give 1.

 1: C₁₂H₁₀O₄ (M W218): dark green powder; 92% yield; m.p 168ºC. Anal calcd. C, 66.06;H, 4.59 . Found; C, 66.04;H, 4.57. FT-IR (cm-¹): 1616 cm^-1 C=C, 1696 cm^-1 C=O, 1703cm^-1 δ-Lactone. δH=1.35 (3H,s,CH₃), 3.81(3H, s, OCH₃), 6.87(1H, s, CH-coumarin), 7.60-7.33 (3H, m, Ar-H) and 8.36 (1H, s, CHO).

 3.1.2           Synthesis of 6-[(1E)-3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl]-4,8-dimethyl-2H-coumarin-2-one (2a) and 6-[(1E)-3-(4-aminophenyl)-3-oxoprop-1-en-1-yl]-4,8-dimethyl-2H-coumarin-2-one (2b)

 To (0.01 mole; 2.18 gm) of compound 1 , (0.01mol; 1.54gm) 4-chloroacetophenone and /or (0.01mol; 1.54gm) 4-aminoacetophenone were heated under microwave irradiation for 4 minutes. The solid that separated was dried and crystallized from ethanol to give 2a,b.

 2a: C₂₀H₁₅ClO₄ (MW 354.5); Dark brown crystals 80%yield; m.p.375⁰C. Anal. Calcd for: C, 67.71; H, 4.23; Cl, 10.01: Found; C, 67.70; H, 4.21; Cl, 10.00. FT-IR (cm^-1): 669(C-Cl), 1723( δ-Lactone), 2983(CH-Coumarin). MS (m/z,%), 354.6(20%) [M⁺].

 2b: C₂₀H₁₇NO₄( M W335): blue powder with m.p. 325C⁰ and yield 75% . Analy calc. for C, 71.64;H, 5.07; N, 4.18. Found; C, 71.62;H, 5.05;N, 4.16. IR (cm^-1): 1727 δ-Lactone, 2900CH-Coumarin and 3334-3299 NH₂. 

5.1.3       3.1.3  Synthesis [(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl) methylidene] propanedinitrile 3

A mixture of 1 (0.01mol; 2.18gm) and (0.01mol; 0.66gm) propanedinitrile was heated under microwave irradiation and solvent free conditions for 6 minutes. The reaction product was recrystallized ethanol/petroleum ether (b.p.60-80˚C) (1:1) to give [(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl) methylidene] propanedinitrile 3.

 3 as brown powder with m.p.260˚C and yield 70%. Analy. Calc. for C₁₅H₁₀N₂O₃ (266)C 67.67 ; H 3.67; N 10.53. Found: C 67.65; H₃.65; N 10.51. MS(m/z,%)391(5.90%)M⁺. IR (cm^-1): at1605 cm^-1 C=C, 1723 cm^-1 δ-Lactone and 2220cm^-1CN. δH=1.23 (3H, s, CH₃) , 3.75 (3H,s,OCH₃) , 6.11(1H, s, CH-coumarin) , 8.39(1H, s, C(CN)₂=CH) ,7.96-7.11 (2H, m, Ar-H) .

5.1.4           3.1.4   Synthesis of N'-[(E)-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl) methylidene] benzene sulfonohydrazide 4.

A mixture of  1 (0.01mol; 2.18gm) and (0.01mol;1.72gm) benzene sulfonohydrazide in ethanolic solution (25ml) and drops of acetic acid was refluxed for 6 hrs. The result solution was poured into crushed ice/water . The solid that separated was filtered off, dried and recrystallized from ethanol/water to give N'-[(E)-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl) methylidene] benzene sulfonohydrazide 4.

4 as gray powder with m.p 195ºC and yield 70% . Anal. Calc. for C₁₈H₁₆N₂O₅S (372): C 58.06; H 4.30; N 7.53 ; S 8.61. Found: C 58.04; H 4.28; N 7.51 ; S 8.60. IR(cm^-1) 1088.82cm^-1SO₂, 1570 cm^-1C=C,1640 cm^-1C=N, 1727 cm^-1 δ-Lactone and 2920-3054 cm^-1 CH(aliphatic-aromatic), 3349 cm^-1 NH .δ H=1.11(3H, s ,CH₃), 8.35(1H, s ,NH), 3.66(3H, s ,OCH₃), 6.21(1H, s, CH-coumarin), 7.59-6.65 (7H, m, Ar-H) and 7.59(1H,s,CH=N).

 3.1.5                         Synthesis of (5Z)-5-[(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene]-2-phenyl-1,3-oxazol-4(5H)-one 5

A mixture of  1 (0.01 mol; 2.18gm) and [(phenylcarbonyl)amino]acetic acid (0.01mol; 1.79gm) in the presence of acetic anhydride and sodium acetate was heated under refluxing for 2 hours. The product obtained was poured into crushed ice, filtered off, dried then, recrystallized from ethanol/petroleum ether (b.p.60-80˚C) (1:1) to give 5

  5 as brown powder with m.p. ˃ 300ºC(420ºC) and yield 60%. Anal. Calc. for C₂₁H₁₇NO₅ (363): C 69.41; H 4.72 ; N 3.85 .Found: C 69.40; H 4.70;N 3.82. MS m/z 361(2.08%) and base peak at m/z 64(100%) . IR(cm^-1): 1605 cm^-1C=N, 1712 cm^-1 δ-Lactone and1759 cm^-1 C=O(oxazole ring) .

 3.1.6           Synthesis of 6-[(E)-hydrazinylidenemethyl]-8-methoxy-4-methyl-2H-coumarin-2-one 6

(0.01mol; 2.18gm) of  1 (0.01mol) of hydrazine hydrate in ethanolic solution (25ml) were heated under refluxing and stirring for 4 hrs. The precipitate was washed well, dried and recrystallised from ethanol to give 6.

6 as Pale yellow crystals with m.p.235⁰C and yield 85%. Anal. Calc. for C₁₂H₁₂N₂O₃ (232): C 62.07; H 5.17; N 12.07. Found: C 62.05; H 5.15; N 12.05. IR(cm^-1): 1574 cm^-1 C=N,1707 cm^-1 δ-Lactone and 3396 ,3428 cm^-1 NH₂ . δH=1.18 (3H, s ,CH₃), 3.79 (3H, s ,OCH₃), 8.34(1H, s, CH=N-), 6.29(1H, s, CH-coumarin), 7.40-6.78(7H, m, Ar-H) and 8.89(1H, s, NH).

 3.1.7           Synthesis of 2-cyano-N'-[(E)-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene]aceto hydrazide 7

 (0.01mol; 2.18gm) of 1, (0.02mol) 2-cyanoacetohydrazide in ethanolic solution (25ml) and in the presence of drops of acetic acid were heated under refluxing and stirring for 4 hrs. The precipitate was washed well, dried and recrystallised from ethanol to give 7 .

7 as Dark brown crystals with m.p. 218⁰C and 70% yield, anal calc for C₁₅H₁₃N₃O₄ (299): C 60.20; H 4.35 ; N 14.05 . Found: C 60.20; H 4.32; N 14.03 . IR(cm^-1): 1607cm^-1C=N, 1682 cm^-1CO-NHamide, 2183cm^-1CN and 3152 cm^-1 NH. δH=1.41(3H, s ,CH₃), 3.33, 3.52(4H, s, 2CH₂), 3.85(3H, s ,OCH₃), 6.25(1H, s, CH-coumarin), 7.92-7.55(2H, m, Ar-H), 8.36(1H, s, CH=N) and 8.89(1H, s, NH).

 3.1.8           Synthesis of N'-[(E)-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene]-N-( phenylsulfonyl) aceto-hydrazide 8

 A mixture of 4 (0.01mol; 3.72gm), (0.01mol) acetic anhydride was heated under refluxing for 2 hour. After cooling, the reaction mixture poured slowly onto crushed ice/H₂O. The solid that separated was filtered off, washed well with water, dried and recrystallized from ethanol to give 8.

 8 as dark brown powder in 65 % yield and m.p. 170ºC. anal calc for C₂₀H₁₈N₂O₆S (414): C57.97 ;H 4.35; N 6.76 ; S7.73; Found: C 57.95; H 4.33; N 6.75; S 7.71. IR (cm^-1): 1074cm^-1 SO₂,1610 cm^-1C=N, 1725cm^-1 δ-Lactone, 1763cm^-1 CH₃C=O and devoid of NH. δ H= 1.19(3H, s ,CH₃), 2.53(3H, s ,N-C=O(CH₃) , 3.68(3H, s, OCH₃), 6.14(1H, s, CH-coumarin), 7.64-6.84(7H, m, Ar-H) and 7.89(1H, s, -N=CH-aldimine).

 3.1.9           Synthesis of 2-amino-6-ethoxy-4-(8-methoxy-4-methyl-2-oxo-2H-coumain-6-yl)-4H-pyran-3,5-dicarbonitrile 9

 A mixture of 3 (0.01 mol; 2.66 gm), ethyl cyanoacetate (1.13 mol) and catalytic amount of piperidine in ethanolic solution (50ml) was heated under refluxing for 6 hrs. The excess solvent was then removed by distillation and the reaction residue poured onto crushed ice/water. The separated solid was filtered off, dried and recrystallized from acetone/water (1:1) to give 9

 9 as a violet powder with m.p.˃ 380˚C and yield 90%. anal. Calc. for C₂₀H₁₇N₃O₅ (379): C 63.32; H 4.52; N 11.08; Found: C 63.30; H 4.50; N 11.06. IR (cm^-1): at 1720 cm^-1 δ-Lactone, 2196 cm^-1 CN, 2851-2955 cm^-1, 3213 cm^-1 and 3336 cm^-1 NH₂. δH=1.22(3H, s, CH₃), 1.65(3H, t, CH₃CH₂) 4.25(2H, q, CH₂ CH₃), 3.77(3H, s, OCH₃), 6.41(1H, s, CH-coumarin), 7.27-6.93 (3H, m, Ar-H) and 9.02(2H, s, NH₂) .

 3.1.10        Synthesis of (4E)-6-amino-4-[(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene]-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile 10

 (0.01 mol; 2.66 gm) of 3 reacted with (0.01mol; 0.76gm) of thiourea in ethanolic solution (25ml) was refluxed for 6 hrs. The solid that separated after concentration was filtered off, dried and recrystallized from ethanol/water to give 10 .

 10 as a brown powder with m.p 350ºC and yield 75 %. Anal. Calc for C₁₆H₁₄N₄O₃S (342): C, 56.13; H, 4.12; N, 16.36; S, 9.37; Found: C, 56.11; H, 4.10; N, 16.34; S, 9.35. IR(cm^-1): 1203 cm^-1C=S, 1651 cm^-1C=N, 1703cm^-1 δ-Lactone, 2213 cm^-1CN, 3178cm^-1 NH and 3243, 3332cm^-1 NH₂. δH=1.15(3H, s ,CH₃), 3.72(3H, s,OCH₃), 4.60(2H, s ,NH₂), 6.24(1H, s, CH-coumarin), 7.83 - 6.82 (2H, m, Ar-H), 8.30, 7.95(2H, s,2NH).

 3.1.11        Synthesis of (2E)-2-[(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylidene] hydrazine carbothioamide 11.

 A mixture of 1 (0.01 mol; 2.18gm) and hydrazine carbothioamide (0.01mol; 0.91gm) in glacial acetic acid was refluxed for 3 hours. After cooling, the solid that separated by filteration, washed well with water, dried and recrystallized from acetone/ethanol (1:1) to give 11.

 11 as a yellow crystals with m.p. 202ºC and yield 90 %. anal calc for C₁₃H₁₃N₃O₃S (291):C, 53.61; H, 4.47; N, 14.43; S, 11.01; Found: C, 53.60; H, 4.45; N, 14.41; S, 11.00, MS m/z 291(0.01%) and base peak at m/z 91.06 (100%). IR (cm^-1): 1285cm^-1C=S, 1619cm^-1C=C, 1645cm^-1 C=N (imine),1696 cm^-1 δ-Lactone, 2696cm^-1 SH,3025-2970 cm^-1 CH (aliphatic-aromatic), 3371-3266cm-1 NH₂. δH=1.82(3H, s ,CH₃), 3.80(3H, s ,OCH₃), 6.50(1H, s, CH-coumarin), 7.40-7.23(2H, m, Ar-H), 8.24(1H, s, CH=N), 9.10(1H, s, C-SH), 4.48(2H,s,NH₂) and 9.64(1H, s ,NH) .

 3.1.12        Synthesis of N-[4-acetyl-5-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl]acetamide 12

 Acetic anhydride (40ml) was added to (0.01 mol; 2.91gm) of 10 and heated under refluxing for 4 hours. After cooling, the solid product was filtered off, washed well with water and recrystallized from ethanol/water (1:1) to give 12.

 12 as violet crystals with m.p. 320ºC and yield 70 % ; anal calc for C₁₇H₁₇N₃O₅S (375): C, 54.39; H, 4.53 ; N, 11.19; S, 8.53; Found: C, 54.37; H, 4.51; N, 11.17; S, 8.51. MS m/z 375(0.01%) and its base peak at m/z 44(100%). IR(cm^-1) 1614 cm^-1C=N, 1717 cm^-1 δ-Lactone_,, 1763cm^-1 C=O (thiadiazole ring) and 3208 cm^-1NH. δH=1.25(3H, s ,CH₃), 1.35 (3H, s, CO-CH₃),1.91(3H, s, CO-CH₃), 3.76(3H, s ,OCH₃), 6.30(1H, s ,CH-thiadiazol ring), 6.91(1H, s, CH-coumarin), 7.93-7.46(2H, m, Ar-H) and 8.37(1H, s ,NH) .

 3.1.13        Synthesis of (2Z)-2-[(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)-methylidene]-N-(2-oxo-2-phenylethyl) hydrazine carbothioamide 13

 To ethanolic solution of 10, (0.01mol;1.99 gm) 2-bromo-1-phenylethanone was added. The reaction mixture was heated under refluxing for 8 hrs. The solid that separated after concentration and cooling was filtered off and recrystallized from ethanol to give 13 .

 13 as brown crystals with m.p. 298ºC and yield 80%, anal. calc. C₂₁H₁₇N₃O₃S (391); C, 64.43; H, 4.38; N, 10.73; S, 8.19; Found: C, 64.41; H, 4.36; N, 10.71; S, 8.17. IR(cm-¹) 1549 cm^-1C=N, 1710 cm^-1 δ-Lactone_,. δH=1.06(3H, s ,CH₃), 2.50(2H, s, CH₂), 3.66(3H, s ,OCH₃), 6.36(1H, s, CH-coumarin), 7.95-6.68(7H, m, Ar-H) and 8.41 (1H, s, CH=N) .

 3.1.14        Synthesis of diethyl4-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 14

 (0.01mol; 2.18) gm1 reacted with (0.02mol; 2.66gm) ethyl acetoacetate and (0.01mol; 0.77gm) ammonium acetate under microwave irradiation for 7 minutes in the presence of anhydrous zinc chloride as a catalyst. The solid that separated was washed well with water, filtered off, dried and recrystallized from ethanol/acetone/water (1:1:1) to give 14

 14 as reddish brown powder with m.p ˃300ºC and yield 80% .Anal. Calc.: C₂₄H₂₇NO₇ (414); C 65.29; H 6.16; N 3.17. Found: C 65.27; H 6.14; N 3.15.IR (cm^-1); 1647 cm^-1C=N, 1725 cm^-1 δ-Lactone and 3269cm^-1 NH. δH=1.06(3H, s ,CH₃), 1.32-1.21( 6H, m, 2XCH₃-pyridine ring), 4.26-3.98 (4H, m, CH₂) ,4.58(1H,S,CH-Pyridine) ,3.85(3H, s ,OCH₃), 5.91(1H, s, CH-coumarin), 7.39-6.54(2H, m, Ar-H) and 8.23 (1H, s, NH).

 3.1.15        Synthaesis of diethyl 4-(1-amino-8-methoxy-4-methyl-2-oxo-1,2-dihydroquinolin-6-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 15.

 A mixture of (0.01 mol; 4.41 gm) of 13 and (0.01mol) hydrazine hydrate in ethanolic solution was reacted under refluxing for 4 hours . The reaction mixture was allowed to cool .The solid that separated was filtered off, dried and recrystallized from ethanol/water (1:1) to give 15.

 15 as pale brown crystals with m.p. 270ºC and yield 85 %. Anal. Calc.: C₂₀H₂₅N₇O₄(427); C 56.20; H 5.89; N 22.94. Found: C 56.18 ; H 5.87; N 22.92. MS m/z 427.0(0.01%) and base peak at m/z 44(100%). IR (cm^-1); 2260cm^-1CN, and 3299, 3319cm^-1 NH, NH₂.

 3.1.16        Synthesis of (E)-8-methoxy-6-(5-((8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)methylene)-2-thioxo-2,4a,5,6,7,8-hexahydroquinazolin-4-yl)-4-methyl-2H-coumarin-2-one 16.

 A mixture of (0.02mol; 4.36gm) of 1 , (0.01mol; 0.98gm) cyclohexanone and (0.01mol; 0.76gm) thiourea was reacted under microwave irradiation for 7minutes. The solid that separated was washed well with water, filtered off, dried and recrystallized from ethanol/water (1:1) to give 16.

 16 as dark brown powder with m.p. ˃ 300ºC and yield 80% . Anal. Calc.: C₃₁H₃₀N₂O₆ (558); C 66.65; H 5.41 ; N 5.01; S 5.74 . Found: C 66.63; H 5.40; N 5.00; S 5.72. MS m/z 554 which fragmented to give the base peak 202 (M⁺²100%). IR (cm^-1); at 1272cm^-1 C=S, 1597cm^-1C=N,1701cm^-1 δ-Lactone and 3193cm^-1NH .

 3.1.17        Synthesis of 6-(1,2,3,4,5,6,7,8,9,10-decahydroacridin-9-yl)-8-methoxy-4-methyl-2H-coumarin-2-one 17

 (0.01mol; 2.18gm) of 1 reacted with (0.02mol; 2.66gm) cyclohexanone and (0.01mol; 0.77gm) ammonium acetate under microwave irradiation for 7 minutes in the presence of anhydrous zinc chloride as a catalyst. The solid that separated was washed well with water, filtered off, dried and recrystallized from ethanol/acetone/water (1:1:1) to give 17

 17 as dark gray powder with m.p. ˃ 300ºC and yield 80% . Anal. Calc. : C₂₄H₂₇NO₃(377); C76.36; H 7.21; N 3.71. Found: C76.35; H 7.20; N 3.70.MS m/z 377(0.41%) and its base peak at m/z 80(100%). IR (cm^-1);1702cm^-1δ-Lactone, and 3265cm^-1 NH.

 3.1.18        Synthesis of 6-(6-(3,4-dimethoxyphenyl)-2-thioxo-2,3,4,5-tetrahydropyrimidin-4-yl)-8-methoxy-4-methyl-2-oxoquinoline-1(2H)-carbothioamide 18.

 A mixture of (0.01mol; 2.18gm) of 1,(0.01mol; 1.80gm) 3,4-dimethoxyacetophenone and (0.01mol; 0.76gm) thiourea was reacted in the presence of sodium ethoxide solution under microwave irradiation for 7minutes. The solid that separated was washed well with water, filtered off, dried and recrystallized from ethanol/petroleum ether(b.p.60-80˚C) (1:1) to give 18

 18 as brown powder with m.p. ˃ 300ºC and yield 80%. Anal. Calc.: C₂₃H₂₀N₂O₅S (436); C63.29; H 4.62; N 6.40; S 7.35. Found: C63.27; H 4.60; N 6.40; S 7.33 MS m/z 496 which fragmented to give the base peak 75 (M⁺³100%). IR (cm^-1);1702cm^-1δ-Lactone, and 3265 cm^-1 NH .

 3.1.19        Synthesis of 6-(3-benzoyl-2,3,4,5,6,7-hexahydrobenzofuran-2-yl)-8-methoxy-4-2H-coumarin-2-one 19.

 A mixture of (0.01mol; 2.18gm) of 1, (0.01mol; 0.98gm) cyclohexanone and (0.01mol; 1.99gm) 2-bromo-1-phenylethanone in the presence of acetonitrile solution and catalytic amount of pyridine was reacted under stirring for 2 hours at rt. Triethylamine (1ml) was added with continuous stirring. The product obtained was poured into crushed ice. The solid that separated was filtered off, dried and recrystallized from ethanol /water (1:1) to give 19.

 19 as reddish brown powder with m.p. 320ºC and yield 80%. Anal. Calc.; C₂₆H₂₄O₅ (416); C 74.98; H 5.81. Found C74.96; H 5.80 . IR (cm-¹) 1633 cm^-1C=O and 1704 cm^-1 δ-Lactone. δ H= 1.18(3H, s ,CH₃), 2.50-2.44(2H, m, CH₂), 2.51-2.50 (2H, m, CH₂) ,3.71(3H, s ,OCH₃), 6.43(1H, s, CH-coumarin) 6.46(OCH-Furan),3.32(COCH-Furan),7.98-7.47 (7H, m, Ar-H) .

 3.1.20        Synthesis of 6-amino-4-(8-methoxy-4-methyl-2-oxo-2H-coumarin-6-yl)-3-methyl-1,4-dihydro- pyrano[2,3-c]pyrazole-5-carbonitrile 20

 A mixture of (0.01mol; 2.18gm) of 1 (0.01mol; 1.33gm) ethyl acetoacetate, (0.01mol; 0.66 gm) malononitrile and (0.01mol) hydrazine hydrate was reacted under microwave irradiation for 7 minutes in the presence of sodium bisulphite. The solid that separated was washed well with water, filtered off, dried and recrystallized from ethanol to give 20.

 20 as reddish brown powder with m.p 380ºC and yield 85%. Anal. Calc.  C₁₉H₁₆N₄O₄ (364); C62.63; H 4.43; N 15.38. Found; C 62.61; H 4.41; N 15.36. IR (cm^-1) ; 1634 cm^-1C=N, 1715 cm^-1 δ-Lactone, 2207 cm^-1 CN and 3329,3204cm^-1 NH₂. ¹H-NMR (DMSO-d6) spectrum which showed signals at 1.20(3H, s ,CH₃), 2.07( 3H, s, CH₃-pyrazol ring), 3.85(3H, s ,OCH₃), 5.49(1H,s,CH-Pyran), 6.37(1H, s, CH-coumarin), 7.95-6.51(2H, m, Ar-H) and 4.18(2H, s,br, NH₂) and 12.95(1H,S,NH-Pyrazol). 21

 3.2. Cytotoxicity Assay

 The cytotoxicity was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay . Briefly, the cells were seeded in 96-well microtitre plate (Falcon, NJ, USA) at a cell concentration of 1x 10⁴ cells per well in 100μ l of growth medium. Fresh medium containing different concentrations of the tested compounds was added after 24 hours of seeding. Serial two fold dilutions of the metabolites were added confluent cell monolayer. The micro titer plates (polystyrene sterile tissue culture plates) were incubated at 37°C in a humidified incubator with 5 % CO₂ for a period of 48 hours. Three wells were used for each concentration of the tested compounds. Control cells were incubated without the tested compounds and with or without DMSO. The little percentage of DMSO present in the wells (maximal 0.1%) was found not to affect the experiment. After incubation of the cells for 24 hours at 37°C, various concentrations of the tested compounds were added, and the incubation was continued for 48 hours. After the end of the incubation period, crystal violet solution (1%) was added to each well for 30 min. The stain was removed and the plates were rinsed using tap water until all excess stain is removed. Glacial acetic acid was then added to all wells and mixed thoroughly; the plates were read on ELISA reader, using a test wavelength of 490 nm. Treated samples were compared with the control. All experiments were carried out in triplicate. The percent cytotoxicity was calculated by the formula: percent cytotoxicity (cell death) = [1-(absorbance of experimental wells/absorbance of control wells)] × 100%.

Figure-1

Figure-2

Figure-3

Figure-4

Figure-5

Figure-6

Figure-7

Figure-8

Figure-9

Figure 1: The mRNA level of EGFR and CDKN-1 in MCF-7 cell line.

Figure 2: The mRNA level of EGFR and CDKN-1 in HEP-G2 cell line.

Table 1: The half maximal inhibitory concentration (IC₅₀) of the tested compounds against the different studied human cell lines.

1.       Hoult JR, Paya M (1996) Pharmacological and biochemical action of simple coumarins: Natural product with therapeutic potential. Gen Pharmacol 27: 713-722.

2.       Rajitha B, Kumar VN, Someshwar P, Madhav J, Venu, et al. (2006) Dipyridine copper chloride catalyzed coumarin synthesis via pechmann-condensation under conventional heating and microwave irradiation. ARKIVOC 12: 23-27.

3.       Weber US, Steffen B, Siegers CP (1998) Res Commun Mol Pathol Pharmacol. 99: 193-206.

4.       Riveiro ME, De Kimpe N, Moglioni A, Vázquez R, Monczor F (2010) Coumarins: old compounds with novel promising therapeutic perspectives. Curr Med Chem 17: 1325-1338.

5.       Wu L, Wang X, Xu W, Farzaneh F, Xu R (2009) The structure and pharmacological functions of coumarins and their derivatives. Curr Med Chem 16: 4236-4260.

6.       Swayam SS, Smita S, Subhangankar N, Himanshu BS (2012) Synthesis of novel coumarin derivatives and its biological evaluations. Eur J Exp Bio 2: 899-908.

7.       Dawoud NTA (2013) An Efficient and Environ- mentally Friendly Procedure for Synthesis of Quinazolinone Derivatives by use of a Biginelli-Type Reaction. Chem Sci Trans 2: 129-134.

8.       Nirmal JP, Patel MP, Patel RJ (2009) Microwave assisted synthesis of some new bi quinoline compounds catalyzed by DMAP and their biological activities. Ind J Chem 48B: 712-717.

9.       Kumar SD, Sandhu JS (2012) Uncatalysed Knoevenagel condensation of 3-formylchromones in green media: polyethylene glycol-400(PEG-400). Ind J Chem 51B: 1743-1748.

10.    Parab RH, Dixit BC (2012) Synthesis, characterization and antimicrobial activity of imidazole derivatives based on 2-chloro-7-methyl-3-formylquinoline. E J Chem 9: 1188-1195.

11.    Mashkouri S, Naiwi R (2004) Mechanochemical solvent free and catalysis free one pot synthesis of pyrano 2,3-d pyrimidino -2,4-(1H, 3H)-Diones with quantitative yields. Molecules 14: 414-479.

12.    Hassani Z (2016) Synthesis of Pyrimidine Derivatives from Three-component Reaction of Malononitrile, Aldehydes and Thiourea/Urea in the Presence of High Surface Area and Nanosized MgO as a Highly Effective Heterogeneous Base Catalyst. Lett Org Chem 11: 546-549.

13.    Kaur H, Kumar S, Singh I, Kumar A. (2010) Synthesis, characterization and antibacterial activity of various substituted oxadiazolylpyrazolinyl/ isoxazolinylcoumarin derivatives. Inter J Pharm Sci Res 1: 58-65.

14.    Desai NC, Harsora JP (2012) Synthesis, characterization and antimicrobial screening of some quinoline based dihydropyridine and 2-oxo-azetidine derivatives. Ind J Chem 51(B): 1011-1019.

15.    Syamala M (2009) Recent progress in three component reactions An Update. Org Prep Proced Inter J 41: 1-68.

16.    Ghashang M, Mansoor SS, Aswin K (2013) An Efficient and Environmentally Friendly Procedure for the Synthesis of Some Novel 8-Benzylidene-4-phenyl-3,4,5,6,7,8-hexahydro-1H-quinazolin-2-ones/thiones using Tetrabutylammonium Hexatungstate as a Reusable Heterogeneous Catalyst under Solvent-Free Conditions. Bull Kor Chem Soc 34: 3289-3294.

17.    Chunduru VSR, Vedula RR (2012) One pot synthesis of coumarin substituted dihydrofurans. Ind J Chem 51B: 1417-1420.

18.    Ameta KL, Penoni A (2015) Heterogeneous catalysis: a versatile tool for the synthesis of bioactive heterocycles. CRC press,Taylor and Francis groups 345.

19.    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.

20.    Brȕnner TN, Frandson LC, Holst-Hansen C, Bei EM, Thompson WA et al. (1993) MCF7/LCC2: a 4-hydroxy tomoxifen resistant human breast cancer variant that retains sensitivity to the steroidal anti estrogen ICI 182, 780. Cancer Res 53: 3229-3232.