Republic of Iraq Kurdistan Regional Government Ministry of Higher Education and Scientific Research University of Sulaimani College of Pharmacy Department of Pharmacognosy and Pharmaceutical Chemistry.

“SYNTHESIS, CHARACTERIZATION AND ANTIBACTERIAL EVALUATION” OF SOME COUMARIN DERIVATIVES AT C-4 OF 7-HYDROXY-4METHYL COUMARIN. A THESIS SUBMITTED TO THE COUNCIL OF PHARMACY COLLEGE AT THE UNIVERSITY OF SULAIMANI IN PARTIAL FULFILMENT OF THE REQUIERMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACEUTICAL CHEMISTRY By Dana Mohammed Hussein B.Sc. Pharmacy 2011 Supervised by Assist. Prof. Dr. Ammar A. Mahmood Kubba PhD Pharmaceutical Chemistry 2017

2717 KURDISH

1438 HIJRI

Dedicated To my

Parents, Beloved wife, Brothers and sister.

Acknowledgements Thanks to God Almighty for the completion of this master’s thesis. Only due to his blessings I could finish my thesis. I would like to express my deepest gratitude to my supervisor Assist prof.Dr. Ammar A. Kubba for his patience, invaluable advices and suggestions throughout the study. I would like to express my great thanks and appreciation to Dr. Emad Manhal AlKhafaji in the Faculty of Science/ School of Chemistry for his unlimited help, guidance and suggestions during the study. Big thanks and appreciation to Dr. Hiwa K. Saaed, Dean of the Pharmacy College/ University of Sulaimany for his great help and support. Great appreciation to Dr. Ban M. Rasheed, Vice dean and Head of Higher Education department /Pharmacy College/ University of Sulaimany for her great help and support. Special thanks to Dr. Nigar Najim, former head of Pharmacognosy and Pharmaceutical Chemistry department Pharmacy College/ University of Sulaimany for her great support and cooperation throughout the study. Special thanks for Dr. Shokhan Jamal, Pharmacy College/ University of Sulaimany, for her knowledge and support throughout my study. All the appreciation and gratitude for Pioneer Pharmaceutical Company/ Sulaimany/ Iraq, for their invaluable and countless help, support and cooperation in using their laboratories throughout my whole study and special thanks to Mrs. Salar Kolak, Mr. Mohammed Nawras, Mr. Ayman Al-faris, Mrs. Shayda Omer and Mr. Arsalan Othman . I would like to thank my whole family specially my parents, brothers and sister for their help and support from the first day of primary school, without them I would not have reached this point. Last but not least I would like to thank my best friend Hazha for her support, encouragement, quiet patience, devotion and love.

Dana M. Hussein

I

Abstract In the present study some new derivatives of coumarin had been synthesized by oxidation of the methyl group at C-4 of 7-hydroxy-4-methyl coumarin to formyl group using selenium dioxide, as an oxidizing agent. The reactions series for the synthesis of the new derivatives were as follow: Compound [D1], 7-hydroxy-4-formyl coumarin, was synthesized by the reaction of 7-hydroxy-4-methyl coumarin with selenium dioxide in hot xylene under continuous reflux for the period of 12 hours. Schiff bases [D2-D6], were prepared by the reaction of compound [D1] with different aromatic amines in absolute ethanol , using few drops of glacial acetic acid. Amino acid Schiff bases [D7-D8], were synthesized by the reaction of compound [D1] with different amino acids in a basic medium using absolute ethanol as a solvent. Chalcones, [D9-D11] were prepared by the reaction of compound [D1] with different aromatic ketones in absolute ethanol,with maintaining the temperature of the reaction below 25 0C by using 10% NaOH solution. Hydrazone derivative of thiosemicarbazide, compound [D12], was prepared by refluxing compound [D1] with thiosemicarbazide in absolute ethanol. Hydrazone derivative, compound [D13], was prepared by refluxing compound [D1] with 2,4-dinitrophenyl hydrazine in absolute ethanol using few drops of glacial acetic acid . Thiourea derivative, compound [D14], was prepared by refluxing compound [D1] with thiourea in glacial acetic acid ,using a few drops of concentrated hydrochloric acid. Compound [D15], which is a hydrazinyl thiazole derivative of coumarin, was prepared by refluxing compound [D12] with chloroacetic acid in glacial acetic acid as a solvent , containing anhydrous sodium acetate. II

The progress of the reactions and purity of the synthesized compounds were followed by using thin layer chromatography. Spectroscopic analyses such as FTIR, 13C-NMR, Mass spectroscopy and physical properties were carried out for structural elucidation. The preliminary antibacterial activity of the newly synthesized coumarin derivatives was screened using serial broth dilution method, and determining the (MIC), against two-gram positive bacteria Staphylococcus aureus and Micrococcus luteus and two-gram negative bacteria Escherichia coli and Pseudomonas aeruginosa that found to be active in vitro. Compound [D7] showed the lowest MIC (more potent) against Escherichia coli, while the compound [D8] and [D11] exhibited the highest MIC (less potent) against Staphylococcus aureus and Pseudomonas aeruginosa respectively.

III

Table of contents Subjects

Page

Acknowledgments

I

Abstract

II

Table of contents

IV

List of tables

VIII

List of schemes

X

List of figures

XII

List of abbreviations

XV

Chapter One: Literature Review 1.1: Heterocyclic Compounds

1

1.2: Coumarin

2

1.3: Coumarins structure and classification

3

1.4: Occurrence of coumarin

5

1.5: Synthesis of coumarin

6

1.5.1: Knoevenagel condensation

6

1.5.2: Kostanecki-Robinson reaction

7

1.5.3: Pechmann reaction

7

1.5.4: Perkin’s reaction

7

1.5.5: Reformatsky reaction

8

1.5.6: Wittig reaction

9

1.6: Reactions of coumarin derivatives

9

1.6.1: Reaction with nucleophilic reagents

9

1.6.2: Reaction with electrophilic reagents

10

IV

1.6.3: Claisen rearrangement

11

1.6.4: Halogenation of coumarin

11

1.6.5: Nitration

12

1.6.6: Oxidation of alkyl-coumarins

12

1.7: Synthesis and biological activity of amines and amino acids coumarin Schiff bases 1.8: Synthesis and biological activity of coumarin chalcones

15

1.9: Synthesis and biological activity of thiosemicarbazone coumarin derivatives and their corresponding cyclized forms 1.10: Synthesis and biological activity of thiourea and hydrazine derivatives of coumarin 1.11: Biological activities of coumarin

19

1.11.1: Antibacterial activity

23

1.11.2: Antiviral activity

24

1.11.3: Antioxidant activity

25

1.11.4: Anti-Inflammatory activity

26

1.11.5: Anti-cancer activity

26

1.11.6: Anticoagulant activity

27

1.11.7: Enzyme inhibition activity

28

1.12: Toxicity of coumarin

28

1.13: Aim of the study

29

17

22 23

Chapter Two: Experimental 2.1: Chemicals

32

2.2: Instruments and analytical techniques

33

2.2.1: Melting point apparatus

33

2.2.2: Thin layer chromatography (TLC)

33

2.2.3: Infrared spectrometry

33

V

2.2.4: 13C-NMR spectrometry

33

2.2.5: Mass spectrometry

34

2.3: Procedures

34

2.3.1: Synthesis of 7-Hydroxy-4-formyl coumarin [D1]

34

2.3.2: Synthesis of aromatic amine Schiff bases [D2-D6]

35

2.3.3: Synthesis of amino acid Schiff bases [D7-D8]

37

2.3.4: Synthesis of Chalcones [D9-D11]

38

2.3.5: Synthesis of 2-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methylene) hydrazine carbothioamide [D12]

39

2.3.6: Synthesis of 4-((2-(2,4-Dinitrophenyl)hydrazono)methyl)-7-hydroxy-2Hchromen-2-one [D13]

40

2.3.7: Synthesis of 1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methylene) thiourea [D14]

41

2.3.8: Synthesis of 2-(2-((7-Hydroxy-2-oxo-2H-chromen-4yl)methylene)hydrazinyl)thiazol-4(5H)-one [D15]

41

2.4: Anti-microbial activity

42

2.4.1: Antibacterial susceptibility tests

42

2.4.1.1: Dilution methods

43

2.4.1.2: Disk diffusion method

43

Chapter Three: Results and Discussion Results and Discussion

45

3.1: Synthesis of 7-Hydroxy-4-formyl coumarin [D1]

48

3.2: Synthesis of aromatic amine Schiff bases [D2-D6]

51

3.3: Synthesis of amino acid Schiff bases [D7-D8]

64

3.4: Synthesis of Chalcones [D9-D11]

69

3.5: Synthesis of 2-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methylene) hydrazine carbothioamide [D12]

75

VI

3.6: Synthesis of 4-((2-(2,4-Dinitrophenyl)hydrazono)methyl)-7-hydroxy-2Hchromen-2-one [D13]

79

3.7: Synthesis of 1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methylene) thiourea [D14]

83

3.8: Synthesis of 2-(2-((7-Hydroxy-2-oxo-2H-chromen-4 yl)methylene)hydrazinyl)thiazol-4(5H)-one [D15]

87

3.9: Antibacterial activity test results

91

Chapter Four: Conclusions and Recommendations 4.1: Conclusions

115

4.2: Recommendations

115

References References

116

VII

List of Tables Table

Title

Page

1-1

Examples of main subtypes of coumarin

4

1-2

Chemical structure and names of the synthesized compounds

30

2-1

The chemicals, reagents and their suppliers

32

2-2

The physical properties of compound [D1]

35

2-3

The physical properties of compounds [D2-D6]

36

2-4

The physical properties of compound [D7-D8]

37

2-5

The physical properties of compound [D9-D11]

38

2-6

The physical properties of compound [D12]

39

2-7

The physical properties of compound [D13]

40

2-8

The physical properties of compound [D14]

41

2-9

The physical properties of compound [D15]

42

3-1

FT-IR spectral data of compound [D1]

50

3-2

13

51

3-3

FR-IR spectral data of compounds [D2-D6]

62

3-4

13

63

3-5

FR-IR spectral data of compounds [D7-D8]

68

3-6

13

69

3-7

FR-IR spectral data of compounds [D9-D11]

74

3-8

MS spectral data of compounds [D9-D11]

75

3-9

FR-IR spectral data of compound [D12]

78

3-10

13

79

3-11

FR-IR spectral data of compound [D13]

82

C-NMR and MS spectral data of compound [D1]

C-NMR and MS spectral data of compounds [D2-D6]

C-NMR and MS spectral data of compounds [D7-D8]

C-NMR and MS spectral data of compound [D12]

VIII

3-12

13

83

3-13

FR-IR spectral data of compound [D14]

86

3-14

13

87

3-15

FR-IR spectral data of compound [D15]

90

3-16

13

91

3-17

Antibacterial activity of title coumarin derivatives[D2-D15]

91

C-NMR and MS spectral data of compound [D13]

C-NMR and MS spectral data of compound [D14]

C-NMR and MS spectral data of compound [D15]

IX

List of Schemes Scheme

Title

Page

1-1

Synthesis of 3-(methoxycarbonyl) coumarin

6

1-2

Kostanecki-Robinson synthesis of coumarin

7

1-3

Pechman reaction for synthesis of 7-hydroxy-4-methyl coumarin

7

1-4

Perkin synthesis of coumarin

8

1-5

Reformatsky synthesis of coumarin

8

1-6

Synthesis of 7-methoxy coumarin

9

1-7

Reaction of coumarin with nucleophilic reagent

10

1-8

Reaction of 3-Acetyl benzo coumarin with ethylenediamine

10

1-9

Reaction of coumarin with electrophilic reagent

11

1-10

Claisen rearrangement of coumarin

11

1-11

Halogenation of coumarin

12

1-12

Nitration of coumarin

12

1-13

Oxidation of methyl coumarin to formyl coumarin

13

1-14

Lewis structure of selenium dioxide

13

1-15

General mechanism of allylic methyl group oxidation by selenium dioxide

14

1-16

Stereo selectivity of oxidation by selenium dioxide

14

1-17

Synthesis of some aromatic amine Schiff bases of coumarin

15

1-18

Synthesis of some Schiff bases from 7-amino-4-methyl coumarin

16

1-19

Synthesis of some aromatic amine Schiff bases from 4-methyl benzocoumarin

16

1-20

Synthesis of coumarin-chalcone hybrid

18

1-21

Synthesis of coumarinyl chalcones.

19

1-22

Synthesis of thiosemicarbazone and di-hydro thiazole derivatives

20

X

1-23

Synthesis of thiosemicarbazone and hydrazonothiazolidin-4-one derivatives of acetyl coumarins

21

1-24

Synthesis of thiourea derivatives of coumarin

22

1-25

Synthesis of thiazole derivatives of coumarin and thiourea

22

1-26

Synthesis of phenylhydrazine derivatives of coumarin

23

3-1

Synthesis of coumarin derivatives [D1-D8]

46

3-2

Synthesis of coumarin derivatives [D9-D15]

47

3-3

Synthesis of compound [D1]

48

3-4

Mechanism of synthesis of compound [D1]

49

3-5

Synthesis of compounds [D2, D3 and D5]

52

3-6

Synthesis of compounds [D4 and D6]

52

3-7

The mechanism of synthesis of aromatic amine Schiff bases [D2-D6]

53

3-8

Synthesis of compounds [D7-D8]

64

3-9

Mechanism of synthesis of amino acid Schiff bases [D7-D8]

65

3-10

Synthesis of compound [D9-D11]

70

3-11

The mechanism of synthesis of compound [D9-D11]

71

3-12

Synthesis of compound [D12]

76

3-13

The mechanism of synthesis of compound [D12]

76

3-14

Synthesis of compound [D13]

79

3-15

The mechanism of synthesis of compound [D13]

80

3-16

Synthesis of compound [D14]

83

3-17

The mechanism of synthesis of compound [D14]

84

3-18

Synthesis of compound [D15]

87

3-19

The mechanism of synthesis of compound [D15]

88

XI

List of Figures Figure

Title

Page

1-1

Examples of heterocyclic compounds

2

1-2

Chemical structures of benzopyrone subclasses

3

1-3

Examples of biologically active coumarin based chalcones

18

1-4

3-acetyl coumarin and 3-acetyl thiosemicarbazone derivative

21

1-5

Example of coumarins with high antibacterial activities

24

1-6

Example of coumarins with antiviral activities

25

1-7

Example of coumarins with antioxidant activities

26

1-8

Example of coumarins with anti-inflammatory activities

26

1-9

Example of coumarins with anti-cancer activities

27

1-10

Example of coumarins with anticoagulant activities

28

1-11

Example of coumarins with enzyme inhibitory activities

28

3-1

FT-IR spectrum of compound [D1]

93

3-2

FT-IR spectrum of compound [D2]

93

3-3

FT-IR spectrum of compound [D3]

94

3-4

FT-IR spectrum of compound [D4]

94

3-5

FT-IR spectrum of compound [D5]

95

3-6

FT-IR spectrum of compound [D6]

95

3-7

FT-IR spectrum of compound [D7]

96

3-8

FT-IR spectrum of compound [D8]

96

3-9

FT-IR spectrum of compound [D9]

97

3-10

FT-IR spectrum of compound [D10]

97

3-11

FT-IR spectrum of compound [D11]

98

XII

3-12

FT-IR spectrum of compound [D12]

98

3-13

FT-IR spectrum of compound [D13]

99

3-14

FT-IR spectrum of compound [D14]

99

3-15

FT-IR spectrum of compound [D15]

100

3-16

13

101

3-17

13

101

3-18

13

102

3-19

13

102

3-20

13

103

3-21

13

103

3-22

13

104

3-23

13

104

3-24

13

105

3-25

13

105

3-26

13

106

3-27

13

106

3-28

Mass spectrum of compound [D1]

107

3-29

Mass spectrum of compound [D2]

107

3-30

Mass spectrum of compound [D3]

108

3-31

Mass spectrum of compound [D4]

108

3-32

Mass spectrum of compound [D5]

109

3-33

Mass spectrum of compound [D6]

109

3-34

Mass spectrum of compound [D7]

110

3-35

Mass spectrum of compound [D8]

110

C-NMR spectrum of compound [D1] C-NMR spectrum of compound [D2] C-NMR spectrum of compound [D3] C-NMR spectrum of compound [D4] C-NMR spectrum of compound [D5] C-NMR spectrum of compound [D6] C-NMR spectrum of compound [D7] C-NMR spectrum of compound [D8] C-NMR spectrum of compound [D12] C-NMR spectrum of compound [D13] C-NMR spectrum of compound [D14] C-NMR spectrum of compound [D15]

XIII

3-36

Mass spectrum of compound [D9]

111

3-37

Mass spectrum of compound [D10]

111

3-38

Mass spectrum of compound [D11]

112

3-39

Mass spectrum of compound [D12]

112

3-40

Mass spectrum of compound [D13]

113

3-41

Mass spectrum of compound [D14]

113

3-42

Mass spectrum of compound [D15]

114

XIV

List of Abbreviations Abbreviations

Meaning

13

Carbon 13 Nuclear Magnetic Resonance

AIDS

Acquired Immune Deficiency Syndrome

bend.

Bending

conc.

Concentrated

DMSO

Dimethyl Sulfoxide

DNA

DeoxyriboNucleic Acid

FT-IR

Fourier Transform Infrared spectroscopy

G

Gram

G+ve

Gram-positive

G-ve

Gram-negative

h

Hour

HCV

Hepatitis C Virus

HIV

Human Immunodeficiency Virus

HOMO

Highest Occupied Molecular Orbital

IUPAC

International Union of Pure and Applied Chemistry

LUMO

Lowest Unoccupied Molecular Orbital

m.p

Melting Point

m/z

Mass-to-Charge ratio

MIC

Minimum Inhibitory Concentration

ml

Milliliter

MS

Mass Spectrometry

ppm

Parts Per Million

C-NMR

XV

SAR

Structure Activity Relationship

str.

Stretching

TLC

Thin Layer Chromatography

XVI

Chapter One Literature Review

Chapter One

Chapter One

Literature Review

Literature Review 1.1: Heterocyclic Compounds: Heterocyclic compounds are defined by the IUPAC Gold Book as: “Cyclic compounds having as ring members atoms of at least two different elements, e.g. quinoline, 1,2-thiazole, bicyclo[3.3.1]tetrasiloxane” (1). They are known to be the counterparts of the carbocyclic compounds, which have only ring atoms from the same element. The Encyclopaedia Britannica describes the heterocyclic compounds as “Any of a class of organic compounds whose molecules contain one or more rings of atoms with at least one atom (the heteroatom) being an element other than carbon, most frequently oxygen, nitrogen, or sulfur” (2). Classification by ring size is convenient because heterocyclic rings of a given size have many common features. Three- and four-membered rings, because of their small size, are geometrically strained and thus readily opened; they are also readily formed. Such heterocycles are well-known reactive intermediates (2, 3). Five- and six-membered rings are readily formed and are very stable; their sizes also allow the development of aromatic character, whereas seven-membered rings and larger are stable but less readily formed and relatively less well investigated (4). υ Pyrrole, a five membered heterocycle, is among that are found in the alkaloids, a large class of alkaline organic nitrogen compounds produced primarily by plants. Nicotine is the best-known pyrrole-containing alkaloid. The heme group of the oxygen-carrying protein hemoglobin is formed from four pyrrole units joined in a larger ring system known as a porphyrin (5). Pharmaceutically important pyridines include the tuberculostat isoniazid (isonicotinic acid hydrazide), the anti-AIDS-virus drug nevirapine, the vasodilator nicorandil, used for treating angina and the urinary-tract analgesic phenazopyridine. Also heterocycles may contain more than one heteroatom which may be similar or dissimilar to each other such as imidazole, isoxazole and isothiazole (6). Figure (1-1) shows some examples of heterocyclic compounds. Though pyridine is overwhelmingly the most important of the six-membered aromatic heterocycles, there are oxygen heterocycles, pyrones, which contains one oxygen atom and a ketone functional group, that resemble the pyridones. The pharmaceutically important compound coumarin is composed of a benzene ring fused with an α-pyrone ring (7). 1

Chapter One

Literature Review

Figure (1-1): Examples of heterocyclic compounds

1.2: Coumarin: The coumarin (benzopyran-2-one, or chromene-2-one) ring system, which is one of six-membered oxygen containing heterocyclic compounds, figure (1-2), is present in natural products (such as the anticoagulant warfarin) that display interesting pharmacological properties. Coumarin and its derivatives (coumarins) are widely distributed throughout nature and many exhibit useful and diverse biological activities. Coumarins occur as secondary metabolites in the seeds, roots 2

Chapter One

Literature Review

and leaves of many plant species, notably in high concentration in the tonka. It is suggested that their function may include plant growth regulations, fungistasis, bacteriostasis and, even, waste products (8-10). 1.3: Coumarins structure and classification: Coumarin class of organic compounds consists of 1,2-benzopyrone ring system as a basic parent scaffold. These benzopyrones are subdivided into: 1) Alpha-benzopyrones, which are consisted of fused benzene and α-pyrone rings, figure (1-2)-compound [A]. 2) Gamma-benzopyrones; which are consisted of fused benzene and γ-pyrone rings, figure (1-2)-compound [B]. The coumarin class of compounds belongs to α -benzopyrones(9, 11, 12). Figure (1-2), shows the basic structure of α & γ-benzopyrones.

Figure (1-2): Chemical structures of benzopyrone subclasses

Coumarins are classified based on substitution in benzene and pyrone rings into: Simple coumarins with 5,6-benzene-2-pyrone skeleton with hydroxyl, alkoxy, alkyl substituents in benzene ring and pyrone ring, Furanocoumarins containing linear or angular type with substituents on benzene nucleus or pyrone ring including dihydrofuranocoumarines, Pyranocoumarins containing linear or angular type with substituents on benzene and pyrone rings and Bis- and Tri-coumarins. table (1-1) (13, 14).

3

Chapter One

Literature Review

Table (1-1): Examples of main subtypes of coumarin Classification Examples

Simple coumarins

Furanocoumarins

O

O

Xylotenin

Dihydrofurano coumarins

Rutaretin Pyranocoumarins

Grandivittin

4

H3C

O

CH3

CH2

Chapter One

Literature Review

Bis-coumarins

Tri-coumarins

1.4: Occurrence of coumarin: Coumarin (2H-1-benzopyran-2-one) is a plant-derived natural product that is an old class of compounds, which is a naturally occurring benzopyrene derivative. A lot of coumarins have been identified from natural sources, especially green plants, including Tonka bean, licorice, lavender and sweet clover grass. In addition, coumarin is also found naturally in certain food plants, for instance, apricots, strawberries, cinnamon, cherries and dong quai. These compounds are found in vegetables, fruits, seeds, nuts, coffee, tea, and wine. The very long association of plant coumarins with various animal species and other organisms throughout evolution may account for the extraordinary range of biochemical and pharmacological activities of these chemicals in mammalian and other biological systems. Coumarins have important effects in plant biochemistry and physiology, acting as antioxidants, enzyme inhibitors and precursors of toxic substances. In addition, these compounds are involved in the actions of plant growth hormones and growth regulators, the control of respiration, photosynthesis, as well as defense 5

Chapter One

Literature Review

against infection. The hydroxy coumarins are typical phenolic compounds and, therefore, act as potent metal chelators and free radical scavengers. They are powerful chain-breaking antioxidants. In view of the established low toxicity, relative cheapness, presence in the diet, and occurrence in various herbal remedies of coumarins, it appears prudent to evaluate their properties and applications further(15, 16). 1.5: Synthesis of coumarin: Coumarins are ubiquitously found in higher plants where they originate from the phenylpropanoid pathway. Their biosynthesis in plants is mainly done via hydroxylation, glycosis and cyclization of o-hydroxy cinnamic acid (17). Coumarin is synthesized via Perkin reaction by refluxing a mixture of salicylaldehyde and acetic acid in the presence of anhydrous sodium acetate and cobaltous chloride hexahydrate and by the condensation of salicylaldehyde with acetic anhydride in the presence of a base catalyst trimethylamine. Knoevenagel, Witting, Reformatsky, Claisen rearrangement, Michael condensation, and Pechmann reaction are also employed in the synthesis of coumarins (18). 1.5.1: Knoevenagel condensation: The Knoevenagel which is a very useful reaction and been employed widely for carbon-carbon bond formation in organic chemistry. Condensation is accomplished by treating a carbonyl compound such as an ortho-hydroxyaryl aldehyde with an active methylene in the presence of a catalyst in an organic solvent. In this reaction, various homogeneous catalysts such as ammonia or ammonium salts, pyridine and piperidine are typically used. It is one of the simplest and most important methods of synthesis of coumarin derivatives in relatively high yields especially for 3-substituted coumarin derivatives such as 3-carboxylic acid and 3acetylcoumarins, as shown in scheme (1-1) (19-22).

Scheme (1-1): Synthesis of 3-(methoxycarbonyl) coumarin

6

Chapter One

Literature Review

1.5.2: Kostanecki-Robinson reaction:

This reaction was first reported by Kostanecki and Rozycki in 1901, by which formation of coumarins, usually 3- and 4- substituted coumarins, is achieved by acylation of ortho-hydroxyaryl ketones with aliphatic acid anhydrides, followed by cyclization, scheme (1-2) (10).

Scheme (1-2): Kostanecki-Robinson synthesis of coumarin

1.5.3: Pechmann reaction: The Pechmann reaction is an acid-catalyzed reaction that proceeds through three main steps. The first step is transesterification, which involved an exchange between phenol and β-ketoester followed by intramolecular hydroxyl alkylation in the second step and elimination of a water molecule in the third step. Pechmann reaction is the most widely used method for the preparation of substituted coumarins such as 7-hydroxy-4-methyl coumarin since it proceeds from very simple starting materials and gives good yields of various substituted coumarins. Substituted coumarins can be prepared by using various reagents such as H2SO4, POCl3, AlCl3, cation exchange resins, trifluoroacetic acid, Montmorillonite clay and solid acid catalysts. As shown in scheme (1-3), (23, 24).

Scheme (1-3): Pechman reaction for synthesis of 7-hydroxy-4-methyl coumarin

1.5.4: Perkin’s reaction: Perkin reaction is developed by William Henry Perkin, in which coumarin is synthesized from the aldol condensation reaction of salicylaldehyde and acid 7

Chapter One

Literature Review

anhydrides in the presence of anhydrous sodium acetate acting as an alkali salt of the acid, the main disadvantage of this reaction is poor yield as shown in scheme (1-4) (18, 23).

Scheme (1-4): Perkin synthesis of coumarin

1.5.5: Reformatsky reaction: Aldehydes or ketones condensation with organozinc derivatives of α-halo esters to obtain β-hydroxy esters is known as the Reformatsky reaction. In appropriate reaction conditions, lactonisation could occur with the formation of coumarins, scheme (1-5) (25). R COR

O Br

C(OZnBr)CH2CO2CH2CH3 H3O+

O

Zn OH

R

OH

C(OH)CH2CO2CH2CH3

OH

Base

R

O

Scheme (1-5): Reformatsky synthesis of coumarin

8

O

Chapter One

Literature Review

1.5.6: Wittig reaction:

It is the reaction in which an aldehyde or ketone is converted into an alkene by condensation with a phosphonium ylide. A method developed for the preparation of coumarins via Wittig olefination-cyclisation of 3-(2-hydroxyaryl)propenoic esters. Cyclization takes places in consistently high yield when the isolated 3-(2hydroxyaryl)propenoic esters are subjected to flash vacuum pyrolysis (FVP)(26). The reaction of 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-4methylbenzaldehyde and 1-hydroxynaphthaldehyde with pyridine, chloroacetyl chloride, and triphenyl phosphine, followed by reaction with trimethylamine gave the corresponding 7-methoxycoumarin, scheme (1-6) (27).

Scheme (1-6): Synthesis of 7-methoxy coumarin

1.6: Reactions of coumarin derivatives: 1.6.1: Reaction with nucleophilic reagents: All coumarins, being lactones, react with alkalies. Their reactivity towards the base and the nature of products formed depends on the structure of the particular coumarin and the rigor of reaction conditions. The initial action of the alkali is always opening of the lactone ring and the formation of a salt of coumarinic acid. 9

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Acidification of this salt regenerates original coumarin. The opening of lactone ring by alkali is affected by substitution. 7-Methoxy coumarin is much harder to hydrolyze than coumarin; 7-hydroxy coumarin is still harder than 7-methoxy coumarin as it at once forms the negatively charged phenolic ion, scheme (1-7) (28).

Scheme (1-7): Reaction of coumarin with nucleophilic reagent

The reaction of coumarin derivatives such as 3-Acetyl benzo coumarin with ethylenediamine in ethanol results in ring opening and production of 1,2-bis[(ohydroxy-1-naphthaldiene)amino]-ethane, scheme (1-8) (29).

Scheme (1-8): Reaction of 3-Acetyl benzo coumarin with ethylenediamine

1.6.2: Reaction with electrophilic reagents: Reaction of electrophilic reagents with coumarin derivatives will be occurred either at the C=C of the lactone ring or at positions C8 and C6 of the benzene ring, such as chlorosulphonation of 7-hydroxy-4-methyl coumarin with chlorosulphonic acid to give both 6 & 8-sulphonyl chlorides or sometimes substitution occurs on the hydroxyl group such as the reaction of 7-hydroxycoumarins with acetylenic diesters and aryl aldehydes in the presence of triethyl amine in tetrahydrofuran at ambient temperature, scheme (1-9) (30).

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Scheme (1-9): Reaction of coumarin with electrophilic reagent

1.6.3: Claisen rearrangement: Coumarin with hydroxyl group in the benzene ring behaves as phenol and undergoes all the typical reactions of phenols, including Claisen rearrangement. 4Methyl-7 allyloxy coumarin rearranges to 8-position, scheme (1-10) (31).

Scheme (1-10): Claisen rearrangement of coumarin

1.6.4: Halogenation of coumarin: The addition of bromine to the 3,4-double bond of coumarin, followed by facile loss of hydrogen bromide gives 3-bromo coumarin. The presence of 4-alkyl group decreases the stability of dibromide. When coumarin is treated with bromine in carbon disulfide at 140°C or in the presence of iodine at 170°C it gives 3,6dibromo coumarin and 3,6,8tribromo coumarin respectively, scheme (1-11). 4Halogeno coumarins are made by treating 4-hydroxy coumarin with phosphorus pentahalide and this becomes the only method for introducing halogen at 4position unless negative groups are present in the benzene ring, (31).

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Scheme (1-11): Halogenation of coumarin

1.6.5: Nitration: Coumarin on nitration gives mainly 6-nitro coumarin derivative along with the formation of 8-nitro coumarin. On further nitration at 100°C, 6-nitro coumarin gives 3,6-dinitro coumarin and 8-nitro coumarin gives 6,8dinitro coumarin. The ease of nitration increases with the introduction of alkyl groups, especially, in 4position. To obtain mono-nitro coumarin, the theoretical amount of nitric acid must be employed. The presence of hydroxyl groups in the aromatic part makes this ring more like phenol and more susceptible to nitration. The nitration of coumarin with benzoyl nitrates is reported to give 5-nitro coumarin, scheme (1-12) (32).

Scheme (1-12): Nitration of coumarin

1.6.6: Oxidation of alkyl-coumarins: Selenium dioxide has been recognized as a selective reagent for incorporation of oxygen functionality at the allylic positions, and the reagent had been applied for the preparation of 4-formyl derivatives of coumarins from the corresponding 4methyl derivatives, scheme (1-13) (33).

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Scheme (1-13): Oxidation of methyl coumarin to formyl coumarin

The lewis structure of selenium dioxide is shown in scheme (1-14), illustrates that the electron density around the more electronegative oxygen atom is higher than that of selenium, so selenium dioxide could act as an electron acceptor or lewis acid and selenium is the center of this lewis acid, and when it is acting as an oxidant it would undergo lewis acid-base reaction, which results in Se-O being broken followed by production of elemental selenium (33).

Scheme (1-14): Lewis structure of selenium dioxide

Selenium dioxide will react with alkenes in a [3,2] group transfer reaction. The initial product will be allylic seleninic acid which undergoes allylic rearrangement to give an unstable compound that rapidly decomposes to an allylic alcohol. In some cases, particularly this most useful oxidation of methyl groups, the oxidation continues to give an aldehyde or ketone, scheme (1-15). Overall, methyl group has been replaced by a C=O group (34).

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Scheme (1-15): General mechanism of allylic methyl group oxidation by selenium dioxide

There is some interesting selectivity in this sequence. Only one of the three groups next to the alkene is oxidized and only one (E-) isomer of the enal is formed. All these decisions are taken in the initial cycloaddition step. The most nucleophilic double bond uses its more nucleophilic end to attack selenium dioxide at selenium. HOMO (π) of the alkene to attack the LUMO (π*) of (Se=O). Meanwhile, the HOMO (π) of (Se=O) attacks the LUMO (C–H σ*) of the allylic system (34). The stereoselectivity also appears to be determined in this step and it is reasonable to assume that the methyl group trans to the main chain will react rather than the other for simple steric reasons. Though this is true, the stereochemistry actually disappears in the intermediate and is finally fixed only in the [2,3]-sigmatropic rearrangement step. Both [2,3]- and [3,3]-sigmatropic rearrangements are usually E-selective, scheme (1-16) (34). O

Se

HO

O H

HO

O

Se O

Se

E

E R

R

[2,3]

R

Z E-methyl group reactsselectively, but... no alkene stereochemistryin intermediate

[2,3] sigmatropic rearrangement is E-selective

product is E-enal

Scheme (1-16): Stereo selectivity of oxidation by selenium dioxide

14

CHO

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1.7: Synthesis and biological activity of amines and amino acids coumarin Schiff bases: Schiff bases are the most significant class of compounds that has gained much importance in recent years due to their wide range of biological activities and industrial application. Compounds containing the >C=N- (azomethine group) structure are known as Schiff bases, usually synthesized from the condensation of primary amines with active carbonyl groups. These compounds are also known as imines or azomethines, and found to possess many pharmacological activities such as antimalarial, anticancer, antibacterial, antifungal, antitubercular, antiinflammatory, antioxidant, antiviral, anticonvulsant, antitumor, anti-HIV, cytotoxic, antidiabetic, antihypertensive and analgesic activities (35). Several studies showed that the presence of a lone pair of electrons in sp2 hybridized orbital of the nitrogen atom of the azomethine group plays an important role in chemical and biological activity (36). Many coumarin carboxaldehydes have the ability to be condensed with aromatic amines and amino acids to produce Schiff bases (37). According to some literature many Schiff bases had been synthesized from the reaction of 8-acetyl-7-hydroxy-4-methyl coumarin with substituted anilines and evaluation of their antibacterial and antifungal activities revealed that they have moderate to significant properties compared to the standards against Streptococci, Staphylococcus aureus, Pseudomonas aeruginisa, Escherichia coli, Candida albicans and Aspergillus Niger, scheme (1-17) (38).

Scheme (1-17): Synthesis of some aromatic amine Schiff bases of coumarin

Also, the condensation reaction of 7-amino-4-methyl-coumarin had been done with a number of substituted salicylaldehydes yielded a series of Schiff bases and a number of them showed anti-Candida activities comparable to that of the commercially available antifungal drugs such as ketoconazole and amphotericin B, scheme (1-18) (39).

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Scheme (1-18): Synthesis of some Schiff bases from 7-amino-4-methyl coumarin

Some literatures show that the reaction of 4-methyl benzo coumarin with selenium dioxide reveals the corresponding 4-formyl derivative, which eventually has been reacted with some primary aromatic amines to get Schiff bases with significant antibacterial activities, scheme (1-19) (40).

Scheme (1-19): Synthesis of some aromatic amine Schiff bases from 4-methyl benzocoumarin

Many Schiff bases of coumarins can be prepared from the reaction of formyl coumarins with aliphatic and aromatic amino acids such as the reaction of 8-formyl coumarin derivatives with glycine, alanine and phenylalanine, also they 16

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demonstrate very well antibacterial activities against both Gram-positive and Gram-negative bacteria (41). 1.8: Synthesis and biological activity of coumarin chalcones: Chalcones (α,β-unsaturated ketones) are an important group of natural or synthetic compounds that are known to exhibit an impressive array of biological properties. Owing to their pharmacological properties such as antimicrobial, antifungal, antimalarial and cytotoxic activity against various cancer cells by prevention of tubulin polymerization by binding to the colchicine binding site. Naturally occurring chalcones and their synthetic analogs had been significantly studied. Structure-activity relationship (SAR) elucidation of antimalarial chalcones demonstrated that the presence of the α,β -unsaturated ketone linker and E configuration are critical for their activities in which the alkoxylated chalcones displayed superior antimalarial activity than those of the hydroxylated analogs. The design and development of new bioactive agents based on the molecular hybridization strategy, involving the integration of two or more pharmacophoric units having different mechanisms of action in the same molecule, is a rationally attractive approach.These combined pharmacophores probably offer some advantages such as in overcoming drug resistance as well as improving their biological potency. Such hybrid approaches had previously reported the coupling of chalcones with various bioactive compounds including nucleosides, quinolones, ciprofloxacin, β-lactam antibiotics and coumarins (42, 43). A number of new chalcone-coumarin hybrids affording anticancer, antimalarial, vasorelaxant, anti-inflammatory, antioxidant and trypanocidal activities have been documented (42). For instance, hybrid (1) (Figure 1-3) has been found to exert significant c ytotoxic activity against the paclitaxel-resistant cancer cells. Derivative (2) has shown cytotoxic activity against cervical carcinoma cells without affecting normal fibroblast cells. Additionally, conjugate (3) exhibited antimalarial activity against both chloroquine sensitive and chloroquine-resistant strains. Such chalcone analogs (1-3) constitute coumaryl group, but the different substituents are pyridyl and phenyl groups on the 2-propen-1-one core structure (44).

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Figure (1-3): Examples of biologically active coumarin based chalcones

A series of coumarin–chalcone hybrids synthesized shows significant tumor inhibition, in taxol resistant cancers. Also it was reported that a coumarin containing compound neo-tanshinlactone, showed significant inhibition against two ER+ human breast cancer cell lines and was 10-fold more potent and 20-fold more selective than tamoxifen (45). Many coumarin-chalcones have been prepared, including the reaction of 3acetylcoumarin with substituted benzaldehydes, these coumarin-chalcones showed very promising trypanocidal activities, scheme (1-20) (42, 46).

Scheme (1-20): Synthesis of coumarin-chalcone hybrid

Coumarinyl chalcones can also be prepared under solvent-free condition and the catalyst will be Silica-Supported perchloric acid (HClO4-SiO2), such as in case of the reaction between 4-chloro-3-formyl coumarin and 4-hydroxy-3-acetyl coumarin, in this reaction when the optimum amount of the catalyst used which is about 100 mg, the best results in terms of yield and time is obtained. This method is convenient, efficient, and environmentally benign for the synthesis of coumarin 18

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based chalcones under thermal solvent-free conditions using HClO4–SiO2 as heterogeneous catalyst. The catalyst is easily preparable, stable (up to 500 0C) and can be recycled for four runs without any loss of its catalytic activity. The significant advantages of this clean methodology are excellent yield of the products, shorter reaction time, simple work-up procedure and mild reactions conditions, scheme (1-21) (47).

Scheme (1-21): Synthesis of coumarinyl chalcones.

1.9: Synthesis and biological activity of thiosemicarbazone coumarin derivatives and their corresponding cyclized forms: Thiosemicarbazone derivatives of formyl and acetyl coumarins are known to be biologically active at the same time these derivatives when undergo cyclization to form heterocycles are also showing very interesting pharmacological activities (48, 49) . The reaction 3-formyl-4-hydroxy coumarin with thiosemicarbazide resulted in production of the corresponding thiosemicarbazone derivative, which on further treatment with chloroacetic acid and sodium acetate revealed production of dihydro thiazole-one derivative, scheme (1-22) (49-51).

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Scheme (1-22): Synthesis of thiosemicarbazone and di-hydro thiazole derivatives

On the other hand condensation reaction of acetyl coumarin derivatives with thiosemicarbazide affords the corresponding thiosemicarbazone derivatives, which is on reaction with chloroacetic acid or ethyl chloroacetate affords the corresponding hydrazonothiazolidin-4-one derivative. In vitro antibacterial activity of these synthesized compounds have been done against Gram-negative Bordetella bronchiseptica and Escherichia coli and Gram-positive Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis pathogenic bacteria and two fungi Candida albicans and Saccharomyces cervesia, and showed very promising and significant antimicrobial activities, scheme(1-23) (48, 52, 53).

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Scheme (1-23): Synthesis of thiosemicarbazone and hydrazonothiazolidin-4-one derivatives of acetyl coumarins

Aggregation of amyloid ß peptide (Aß) is an important event in the progression of Alzheimer’s disease. Therefore, among the available therapeutic approaches to fight with disease, inhibition of Aß aggregation is widely studied and one of the promising approach for the development of treatments for Alzheimer’s disease. The efficacy of 3-acetyl coumarin and its corresponding thiosemicarbazone derivative is investigated for inhibition of Aß peptide aggregation and it was found that the thiosemicarbazone derivative is more potent regarding this issue, also 3acetyl thiosemicarbazone provides neuroprotection against Aß-induced cytotoxicity in SHS5Y5 cell line, which indicates that thiosemicarbazone modification renders 3-acetyl coumarin neuroprotective properties, figure (1-4) (52, 54) .

Figure (1-4): 3-acetyl coumarin and 3-acetyl thiosemicarbazone derivative

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1.10: Synthesis and biological activity of thiourea and hydrazine derivatives of coumarin: Thiourea derivatives of coumarin are known to be biologically active and attempts had been made for synthesizing these kinds of derivatives, these compounds are known to have their own biological importance by exerting analgesic and antiinflammatory activities In-vivo also they have good antibacterial activities against Gram-positive bacterial species like: Bacillus subtilis and Staphylococcus aureus and Gram-negative strains like Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Scheme (1-24) (49, 55).

Scheme (1-24): Synthesis of thiourea derivatives of coumarin

Reaction of 3-acetyl coumarin and its analogues in refluxing ethanol with thiourea in the presence of Iodine gives the corresponding 2-amino-4-(3coumarinyl)thiazoles, these newly synthesized heterocycles showed significant anti-oxidant activities when compared to ascorbic acid and manifested the best protective effects against DNA damages induced by bleomycin and also showed good antibacterial activities against Escherichia coli and Staphyllococcus aureus, scheme (1-25) (48, 56).

Scheme (1-25): Synthesis of thiazole derivatives of coumarin and thiourea

It has been reported that the azomethine functional group of the Schiff bases including that of the hydrazones contribute to the bioactivity of the hydrazones, the azomethine nitrogen C=N may interact and form intramolecular hydrogen bonding with some responding sites within the cell structure, which thus affects the regular 22

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cell processes; hence, their biological significance. The reaction of 8-acetyl-7hydroxy-4methyl coumarin with a series of phenyl hydrazines produces the corresponding hydrazone derivatives. These derivatives are tested for their antibacterial and antifungal activities and in was observed that the presence of chloro-substitution increases the inhibition extent of the microorganisms’ growth, such as gram-negative bacteria species Escherichia coli, Pseudomonas aeruginosa, gram-positive bacteria species Staphylococcus aureus, Streptococcus pyogenes and fungal strains like Cryptococcus neoformans, Aspergillus nigar, Aspergillus flavus, Candila albicans, scheme (1-26) (57, 58).

Scheme (1-26): Synthesis of phenylhydrazine derivatives of coumarin

1.11: Biological activities of coumarin: The plant kingdom constitutes a source of new chemicals, which may be important for their potential use in medicine as antimicrobial agents. Coumarins are plant secondary metabolite compounds whose biological activity varies according to their substitution patterns. Substituted 4-(1-piperazinyl) coumarins exhibit antiplatelet aggregation activity ,8-substituted 7-geranyloxycoumarin derivatives (specially the 8-methoxy and 8-acetoxy derivative) have anti-inflammatory activity and 8-substituted 7-methoxycoumarins show potent anti-tumor promoting effects (59) . 1.11.1: Antibacterial activity: The antibacterial activity of parent coumarin and many of its analogues have been extensively studied against many gram-positive and gram-negative bacteria. Some researchers tested their antibacterial activities against Escherichia coli Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus. Coumarin per se showed broad antibacterial activity against all the tested strains, this fairly 23

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high antibacterial activity of coumarin per se is mostly due to its lipophilic character and planar molecular structure, which contribute in penetration through bacterial cell membrane or cell walls. Within the group of mono-oxygenated coumarins, the addition of a methyl or O-methyl group at position C6 or C7 to the aromatic nucleus of the coumarin per se core structure maintained the antibacterial activity against Gram-negative bacteria, but diminished against Gram-positive strains when compared to the parent coumarin. On the other hand, substitution of the less polar functions (OMe, Me) at C6 by an OH function reduces the antibacterial activity against both Gram-positive and Gram-negative bacteria. The addition of an OH group at C7 of the coumarin per se significantly reduced the antibacterial activity against all the tested bacteria. These findings suggested that the antibacterial activity of oxygenated coumarins apparently depended on the position of polar (OH) and less polar (OMe, Me) functions at the aromatic nucleus of the coumarin structure. Osthenol is a compound with prenylation at C8 and an OH group at C7, suggesting that those groups are required for good antibacterial activity mostly against Gram-positive bacteria (59, 60). Compounds having long chain hydrocarbon substitutions such as ammoresinol and ostruthin show activity against a wide spectrum of gram-positive bacteria such as Bacillus megaterium, Micrococcus luteus, Micrococcus lysodeikticus and Staphylococcus aureus, figure (1-5) (15).

Figure (1-5): Example of coumarins with high antibacterial activities

1.11.2: Antiviral activity: A large variety of natural products have been described as anti-HIV agents, and compounds having coumarin nucleus are among them. Diverse coumarins analogues were found to display remarkable array of affinity with the different molecular targets for antiviral agents and slight modifications around the central motif result in pronounced changes in its antiviral spectrum. The inophyllums and calanolides represent novel HIV inhibitory coumarin derivatives. Inophyllum A, inophyllum B, inophyllum C, inophyllum E, inophyllum P, inophyllum G1 and inophyllum G2 are known to be effective inhibitors of HIV reverse transcriptase 24

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Calanolides A and B were completely protective against HIV-1 replication. (+)Calanolide A is a nonnucleoside RT inhibitor with potent activity against HIV-1. ()-Calanolide B and (-)-dihydrocalanolide B possess antiviral properties similar to those of (+)-calanolide A, figure(1-6) (15). Viral hepatitis is a potentially serious infectious disease mainly affecting liver causing hepatocellular carcinoma and is a major cause of morbidity and mortality worldwide. Several coumarins analogues also proved to have a substantial antihepatitis virus activity. Recently a new compound library of hybrid coumarinbenzimidazole and imidazopyridine-coumarine derivatives connected through methylenethio linker (-SCH) as anti-hepatitis C virus (HCV) agents. These conjugates exhibited very appealing anti-HCV activity and become promising leads in the further development of assortment of hybrid molecules, figure (1-6) (61) .

Figure (1-6): Example of coumarins with antiviral activities

1.11.3: Antioxidant activity: Fraxin showed free radical scavenging effect at high concentration and cell protective effect against H2O2-mediated oxidative stress. Esculetin exhibited antioxidant property. The antioxidant activity of the coumarins grandivittin , agasyllin, aegelinol benzoate and osthol was evaluated by their effects on human whole blood leukocytes and on isolated polymorphonucleated chemiluminescence which also showed promising results, figure (1-7) (62, 63).

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Figure (1-7): Example of coumarins with antioxidant activities

1.11.4: Anti-Inflammatory activity: Coumarin per se exhibits anti-inflammatory property and is used in the treatment of edema. This removes protein and edema fluid from injured tissue by stimulating phagocytosis, enzyme production, and thus proteolysis. Another compound imperatorin also exhibit anti-inflammatory and showed this activity in lipopolysaccharide-stimulated mouse macrophage in vitro and a carrageenaninduced mouse paw edema model in vivo. Imperatorin blocks the protein expression of inducible nitric oxide synthase and cyclooxygenase-2. Esculetin exhibited anti-inflammatory activity in rat colitis induced by trinitrobenzene sulfonic acid also inhibits the cyclooxygenase and lipoxygenase enzymes, also of the neutrophil dependent superoxide anion generation, figure (1-8) (64-67).

Figure (1-8): Example of coumarins with anti-inflammatory activities

1.11.5: Anti-cancer activity: Due to the potential applications of coumarins in the cancer chemotherapy, extensive efforts have been made on the design and synthesis of coumarin derivatives with improved anticancer activity (68). Many coumarins showed anticancer properties. Imperatorin exhibited anticancer effects. Osthole is effective in inhibiting the migration and invasion of breast cancer cells by wound healing and trans well assays. Many studies revealed that 26

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osthole effectively inhibits matrix metalloproteinase-s promoter and enzyme activity, which might be one of the causes that lead to the inhibition of migration and invasion by osthole (69). Esculetin exhibited antitumor activities. Protective effects of fraxin against cytotoxicity induced by hydrogen peroxide were examined in human umbilical vein endothelial cells. Another coumarins, such as grandivittin, agasyllin and aegelinol benzoate, exhibits marginally cytotoxic activity against lung cancercell lines. Chartreusin was shown to exhibit antitumor properties against murine leukemias, and B16 melanoma, figure (1-9) (70).

Figure (1-9): Example of coumarins with anti-cancer activities

1.11.6: Anticoagulant activity: Coumarins are vitamin K antagonists that produce their anticoagulant effect by interfering with the cyclic interconversion of vitamin K and its 2,3 epoxide (vitamin K epoxide). Thereby inhibition the biological activities of the coagulation factors (II, VII, IX, and X) require -carboxylation for their biological activity (71). The most well-known coumarin with anticoagulant activity is dicoumarol which was found in sweet clover. The isolated coumarins from Ferulago carduchorum (suberosin and suberenol) showed anticoagulant activity which could be more effective as a new industrial formulation with anticoagulant agent or a toxic compound in excess uses (72). Currently, most clinical anticoagulant agents are coumarins, such as warfarin and acenocoumarol. Also new coumarins were isolated from Ainsliaea fragrans proved to be less toxic than warfarin and showed no significant liver or kidney toxicity, figure (1-10) (73).

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Figure (1-10): Example of coumarins with anticoagulant activities

1.11.7: Enzyme inhibition activity: Methoxsalen (8-methoxypsoralen) is found in the seeds of the Ammi majus (Umbelliferae) and exhibited potent mechanism-based microsomal P 450 inhibitor in vitro and single dose methoxsalen effects on human cytochrome P450-2A6 activity, figure (1-11) (74).

O

O

O

OCH3

Methoxsalen Figure (1-11): Example of coumarins with enzyme inhibitory activities

1.12: Toxicity of coumarin: Coumarin is a secondary phytochemical with hepatotoxic and carcinogenic properties. However, clinical data on hepatotoxicity from patients treated with coumarin as medicinal drug is also available. This data revealed a subgroup of the human population being more susceptible for the hepatotoxic effect than the animal species investigated. After chemical synthesis in 1868, coumarin was marketed and used as a food flavoring for a long time. In the middle of the last century, coumarin was discovered to cause hepatic damage in laboratory animals, and the addition of synthetic coumarin to foods was banned, first in the USA in 1954 (9, 75). 28

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Tumor formation was observed in long term animal experiments. Extensive data is available on the toxicity of coumarin in laboratory animals, and these have been evaluated in various overview articles and expert opinions of scientific bodies. Of the effects observed in vivo, the carcinogenic and hepatotoxic properties are of major importance. Hepatotoxicity was observed not only in rodents, but also in many other mammal species. Tumor formation was observed in long term experiments with rodents: adenomas and carcinomas of the liver and bile ducts and adenomas of the kidney in rats, as well as adenomas and carcinomas of the lung and liver adenomas in mice (76). Coumarin induces tumors by a mechanism, which is preceded by toxicity in the same target organ, and this allows a threshold-based approach and the establishment of no observed adverse effect level. As to the mechanism of hepatotoxicity, many in vivo and in vitro studies have been performed in laboratory animals to elucidate the metabolism of coumarin. Briefly, the two most important pathways of coumarin metabolism are 7-hydroxylation leading to detoxification, which is predominant in primates, and metabolism of the lactone ring to form a coumarin 3,4-epoxide intermediate which can be conjugated with glutathione or may spontaneously degrade with the loss of carbon dioxide to form ohydroxyphenylacetaldehyde (o-HPA). The latter compound was found to be a hepatotoxic metabolite and is detoxified by oxidation to o-hydroxyphenylacetic acid (o-HPAA). Much less o-HPAA is formed in rats than mice, explaining the higher susceptibility of rats to coumarin-induced hepatotoxicity. Therefore, differences in detoxification of o-HPA are assumed to be a determining factor for species differences in sensitivity to coumarin hepatotoxicity (77). 1.13: Aim of the study: Coumarin itself and its derivatives own significant biological activities and they are important organic compounds (35). Depending on the previous reviews that proved these facts, we are interesting to synthesize some new derivatives which are: Schiff bases, chalcones, hydrazones and cyclized thiosemicarbazone, by oxidation of the methyl group at C4 of the parent coumarin nucleus, using selenium dioxide (40), then the new derivatives will be evaluated for their preliminary antibacterial activities, using serial broth dilution method.

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Table (1-2): Chemical structure and names of the synthesized compounds Compound Chemical structure Chemical name number D1 7-hydroxy-4-formyl coumarin

D2

7-hydroxy-4-(((4nitrophenyl)imino)methyl)-2Hchromen-2-one

D3

4-(((4acetylphenyl)imino)methyl)-7hydroxy-2H-chromen-2-one

D4

HO

O

O H3C

4-(((7-hydroxy-2-oxo-2Hchromen-4yl)methylene)amino)-1,5dimethyl-2-phenyl-1H-pyrazol3(2H)-one

CH3 N

HC N

N O

D5

D6

ethyl 4-(((7-hydroxy-2-oxo-2Hchromen-4yl)methylene)amino)benzoate

HO

O

O

7-hydroxy-4-(((5-mercapto1,3,4-thiadiazol-2yl)imino)methyl)-2H-chromen2-one

S HC

SH

N N

N

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D7

2-(((7-hydroxy-2-oxo-2Hchromen-4yl)methylene)amino)acetic acid

D8

2-(((7-hydroxy-2-oxo-2Hchromen-4yl)methylene)amino)propanoic acid

D9

7-hydroxy-4-(3-oxo-3phenylprop-1-en-1-yl)-2Hchromen-2-one

D10

7-hydroxy-4-(3-(2hydroxyphenyl)-3-oxoprop-1-en1-yl)-2H-chromen-2-one

D11

4-(3-(4-aminophenyl)-3oxoprop-1-en-1-yl)-7-hydroxy2H-chromen-2-one

D12

2-((7-hydroxy-2-oxo-2Hchromen-4yl)methylene)hydrazinecarbothio amide

D13

4-((2-(2,4dinitrophenyl)hydrazono)methyl) -7-hydroxy-2H-chromen-2-one

D14

1-((7-hydroxy-2-oxo-2Hchromen-4yl)methylene)thiourea

D15

2-(2-((7-hydroxy-2-oxo-2Hchromen-4yl)methylene)hydrazinyl)thiazol4(5H)-one

31

Chapter Two Experimental

Chapter Two

Chapter Two

Experimental

Experimental 2.1: Chemicals: The specific chemicals used in this work are listed in table (2-1) with their chemical structures and suppliers. Table (2-1): The chemicals, reagents and their suppliers Chemicals

Supplier

2,4-dinitro phenylhydrazine

GCC

2-amino-5-mercapto thiadiazole

Merck

2-hydroxy acetophenone

Sigma-Aldrich

4-amino acetophenone

Roth

4-amino antipyrine

Sigma-Aldrich

4-amino ethylbenzoate

Roth

4-nitro aniline

Merck

7-hydroxy-4methyl coumarin

Roth and Sigma-Aldrich

Absolute ethanol

Scharlau

Acetophenone

Sigma-Aldrich

Anhydrous sodium acetate

Scharlau

Chloro aceticacid

Sigma-Aldrich

Chloroform

Prolabo

Ethyl acetate

Alpha chemica

Glacial acetic acid

GCC

Glacial acetic acid

Avonchem

Glycine

Fluca 32

Chapter Two

Hydrochloric acid 99%

Alpha chemical

L-Alanine

Panreac

Methanol

Scharlau

P-Xylene

Roth

Selenium dioxide

Roth

Sodium hydroxide

Alpha chemical

Thiosemicarbazide

Fluca

Thiourea

Panreac

Experimental

Note: The solvents used were purified by distillation. 2.2: Instruments and analytical techniques: 2.2.1: Melting point apparatus: Melting points were determined by using Stuart/SMP3 melting point apparatus version 5.0 in open capillary tubes, and are uncorrected. 2.2.2: Thin layer chromatography (TLC): The purity of the synthesized compounds and progress of the reactions were determined using thin Layer chromatography on aluminum silica gel 60 F254 nm (Merck), detected by UV light (320 nm). 2.2.3: Infrared spectrophotometry: Infrared spectra were recorded using Thermo Scientific™ Nicolet™ iS™10 FT-IR Spectrophotometer in Pioneer company for pharmaceutical industry-SulaimaniKurdistan-Iraq. 2.2.4: 13C-NMR spectrometry: The 13C-NMR spectra were recorded on Bruker FT –NMR spectrometer 100 MHz, in College of Pharmacy-Hamedan University of Medical Sciences- Hamedan-Iran, 33

Chapter Two

Experimental

using deuterated dimethyl sulfoxide (DMSO-d6) and deuterated Chloroform (CDCl3) as a solvent. Chemical shifts were related to the solvent, (δ = ppm). 2.2.5: Mass spectrometry: Mass spectra were done at College of Pharmacy-Hamedan University of Medical Sciences- Hamedan-Iran, using Agilent Technology (HP), GC/MS/MS model 5973 network mass selective detector. 2.3: Procedures: 2.3.1: Synthesis of 7-Hydroxy-4-formyl coumarin [D1](37, 40, 78-80):

[D1] 7-Hydroxy-4-methyl coumarin (0.005 mol, 1 g) was dissolved in hot xylene (50 ml), the solution was cooled and selenium dioxide (0.009 mol, 1 g) was added. The solution was then refluxed for a period of 12 h, and then filtered while hot to remove the insoluble selenium. The solvent was removed by rotary evaporator to get the desired product. The yellow colored powder was obtained and recrystallized from ethanol. The physical properties of compound [D1] are listed in table (2-2).

34

Chapter Two

Table (2-2): The physical properties of compound [D1]

Comp. No.

m.p/ 0C Yield %

Molecular Formula

TLC Solvent system

D1

222224

C10H6O4

Ethyl acetate: chloroform 2:8

35%

Rf value 0.272

Experimental

Tollens reagent test

2,4-Dinitrophenyl hydrazine test

+ve mirror image

+ve

2.3.2: Synthesis of Aromatic Amine Schiff Bases [D2-D6] (35-37, 39, 81-83):

[D2]

[D4]

[D3]

[D5]

[D6]

Appropriate aromatic amine [(0.005) mol of [4-Nitro aniline0.69 g , 4-amino acetophenone, 0.67 g, 4-amino antipyrine, 1.01g ,4-amino ethyl benzoate0.82 g and 2-amino-5-mercapto-1,3,4-thiadiazole 0.66 g,] was added with continuous stirring to a solution of compound [D1] (0.005 mole, 0.95g) and two drops of glacial acetic acid in absolute ethanol (30 ml). The mixture was refluxed for 7 h, 35

Chapter Two

Experimental

and the reaction was monitored by TLC, the yellow colored precipitate was formed and collected by filtration, dried and recrystallized from ethanol. The physical properties of compounds (D2-D6) are listed in table (2-3). Table (2-3): The physical properties of compounds [D2-D6] Comp. No.

m.p/ o C

Yield %

Molecular Formula

TLC

Color

D2

201204

65

C16H10N2O5

Ethylacetate: chloroform 2:8

D3

187189

53

C18H13NO4

Ethylacetate: chloroform 1:9

0.455

Yellow

D4

192195

71

C21H17N3O4

Ethylacetate: chloroform 3:7

0.245

Intense yellow

D5

211213

44

C19H15NO5

Ethylacetate: chloroform 1:1

0.873

Pale yellow

D6

251254

61

C12H7N3O3S2

Ethylacetate: chloroform 4:5

0.524

Light yellow

Solvent system

36

Rf value 0.673

Bright yellow

R-group

Chapter Two

2.3.3: Synthesis of amino acid Schiff bases [D7-D8] (84-86):

Experimental

A solution of compound [D1] (0.01 mol, 1.9 g) in absolute ethanol(20 ml) was added dropwise to a homogenous solution of amino acid (0.01mol) ;glycine (0.75 g or alanine 0.89 g) and sodium hydroxide (0.01mole, 0.4 g) in absolute ethanol (20 ml) with continuous stirring. The solution’s color changed immediately and after 2-3 minutes (1 ml) acetic acid was added. Stirring continued for 24 h and 12 h for glycine and alanine respectively, the obtained precipitate was collected by filtration, washed with cold ethanol, dried and recrystallized from methanol. The physical properties of compound (D7-D8) are listed in table (2-4). Table (2-4): The physical properties of compound [D7-D8] Comp. m.p/ 0C No.

Yield%

D7

61

D8

198-200

173-176 73 (Dec.) *Dec.: Decomposition

Molecular formula

TLC Solvent system

C12H9NO5

Ethylacetate:Chl oroform 3.5:6.5

0.295

Light yellow

C13H11NO5

Ethylacetate:Chl oroform 4:6

0.349

Milky

37

Color Rf value

Rgrou p

Chapter Two

Experimental

2.3.4: Synthesis of Chalcones [D9-D11] (87-90):

Compound [D1] (0.01 mol, 1.9 g)was mixed with appropriate aromatic ketones (0.02 mol) of (acetophenone 2.4 g, 2-hydroxy acetophenone 2.7 g or 0.01 mol- 4amino acetophenone, 1.35 g) and dissolved in absolute ethanol (30 ml) in a 100 ml round-bottom flask equipped with a magnetic stirrer. To the reaction mixture sodium hydroxide (10 ml, 10%) was added with vigorous stirring for about 30 minutes until the solution became turbid. By the use of cold water bath on the magnetic stirrer temperature of the reaction was maintained at 20-25 0C. After vigorous stirring for 8-9 h, the reaction mixture was left to stand 24 h in the refrigerator. The precipitate was obtained, collected by filtration, washed with cold ice water and recrystallized from ethylacetate. The physical properties of compounds (D9-D11) are listed in table (2-5). Table (2-5): The physical properties of compound [D9-D11] Comp. No.

m.p/ 0C

Yield%

Molecular Formula

Color

TLC Solvent system Ethylacetate: chloroform 3:7

Rf value 0.765

Chartreuse

D9

178-181

34

C18H12O4

D10

163-167

41

C18H12O5

Ethylacetate: chloroform 2.5:7.5

0.67

Chartreuse

D11

192-195

29

C24H18NO4

Ethylacetate: chloroform 2.3:7.7

0.715

Red

38

R-group

Chapter Two 2.3.5:

Synthesis

of

Experimental

2-((7-hydroxy-2-oxo-2H-chromen-4-yl)methylene)

hydrazine carbothioamide [D12] (91):

[D12]

Equimolar amounts (0.005 mol, 0.95 g) of compound [D1] and (0.005 mol,0.45 g) thiosemicarbazide were refluxed in absolute ethanol (100 ml) in a round-bottom flask for 2 h, then the reaction mixture was cooled down. The obtained faint yellow-colored powder were collected by filtration and recrystallized from methanol. The physical properties of compound [D12] are listed in table (2-6).

Table (2-6): The physical properties of compound [D12] Comp. No. D12

m.p/ 0C

Yield%

203-207

67

Molecular Formula C11H9N3O3S

TLC Solvent system Ethylacetate:chloroform 3:7

39

Rf value 0.45

Chapter Two

Experimental

2.3.6: Synthesis of 4-((2-(2,4-dinitrophenyl)hydrazono)methyl)-7-hydroxy-2Hchromen-2-one [D13] (57):

[D13]

Compound [D1] ,(0.005 mol, 0.95 g) of and) 2,4-dinitro phenylhydrazine(0.005 mol, 1 g) was mixed in a round bottom flask using absolute ethanol (30 ml) as a solvent, (3-4) drops of glacial acetic acid was added and the mixture was refluxed for 7 h. The reaction monitored by TLC, an intense orange-colored precipitate was formed, collected by filtration and recrystallized from ethanol. Physical properties of compound [D13] are listed in table (2-7). Table (2-7): The physical properties of compound [D13] Comp. No. D13

m.p/ 0C 237-241

Yield % 72

Molecular Formula C16H10N4O7

TLC Solvent system Ethylacetate:chloroform 1.5:8.5

40

Rf value 0.82

Chapter Two

Experimental

2.3.7: Synthesis of 1-((7-hydroxy-2-oxo-2H-chromen-4-yl)methylene) thiourea [D14] (91):

[D14]

A solution of equimolar amounts of compound [D1] (0.005 mole, 0.95 g) and thiourea (0.005 mole, 0.38 g) is prepared in glacial acetic acid(35 ml), (2-3) drops of conc. HCl acid was added to the solution. The reaction mixture was refluxed for 2 h. Yellow colored powder was formed, collected by filtration and recrystallized from glacial acetic acid. Physical properties of compound [D14] are listed in table (2-8). Table (2-8): The physical properties of compound [D14] Comp. No. D14

m.p/ 0C

Yield%

247-250

49

Molecular Formula C11H8N2O3S

TLC Solvent system Ethylacetate:chloroform

Rf value 0.342

2.3.8: Synthesis of 2-(2-((7-hydroxy-2-oxo-2H-chromen-4yl)methylene)hydrazinyl)thiazol-4(5H)-one [D15] (91-93): A mixture of chloroacetic acid (0.01 mol,0.94 g) and compound [D12] (0.01 mol, 2.63 g) in glacial acetic acid (40 ml) containing anhydrous sodium acetate (0.04 mol, 3.28 g) was refluxed for 12h, and monitored by TLC. The reaction mixture was cooled; a pale yellow colored precipitate was formed, collected by filtration and recrystallized from ethanol. The physical properties of compound [D15] are listed in table (2-9).

41

Chapter Two

Experimental

[D15] Table (2-9): The physical properties of compound [D15] Comp. No. D15

m.p/ 0C 310-314

Yield % 63

Molecular Formula C13H9N3O4S

TLC Solvent system Ethylacetate:chloroform 1:9

Rf value 0.525

2.4: Anti-microbial activity: An antimicrobial is any substance of natural, semisynthetic or synthetic origin that kills or inhibits the growth of microorganisms but causes little or no damage to the host. The term antimicrobial includes antibiotics, antifungals, antivirals and other natural bioactive compounds (94).

2.4.1: Antibacterial susceptibility tests: Antibacterial susceptibility tests can be performed by either diffusion or dilution methods. The relative ease of performance, flexibility, use of automated or semiautomated devices are the main factors that affect the choice of the method. Conventional methods for testing the antimicrobial activities are broth dilution, agar dilution and disc diffusion methods (95).

42

Chapter Two

Experimental

2.4.1.1: Dilution methods:

The minimal concentration, which is usually expressed in microgram per milliliter of the antimicrobial agent required to inhibit the growth of a microorganism is determined by the dilution susceptibility testing methods. The procedures are carried out by either agar or broth based methods. The agents are usually tested as (twofold) serial dilutions, also any other concentrations can be set in between. After overnight incubation the lowest concentration that inhibits visible growth of an organism in vitro is recorded as the minimum inhibitory concentration (MIC) (96, 97) . A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism. Because a lower MIC value indicates that less of the drug is required in order to inhibit growth of the organism, drugs with lower MIC scores are more effective antimicrobial agents. MIC scores, which provide quantitative results, are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents (98). The broth methods include macro-broth dilution, in which the broth volume of each antimicrobial concentration is equal to or greater than 1.0 ml contained in test tubes and micro-broth dilution, in which antimicrobial dilutions are in 0.05 to 0.1 ml volumes contained in wells of micro-titer trays. The first method is a well standardized and reliable reference method that is useful for research purposes, but because of the availability of more convenient dilution systems(micro dilution), the macro dilution method is not useful for routine susceptibility testing in clinical laboratories (99). 2.4.1.2: Disk diffusion method: This method of susceptibility testing was developed in 1940, it is the official method used in most of the clinical microbiology laboratories for routine antimicrobial susceptibility testing including antibiotics. This method allows categorization of the bacterial isolates as resistant, intermediate or susceptible to a variety of antimicrobial agents. It is technically simple and very easily interpreted by clinicians; its primary disadvantage is that it provides only qualitative results without quantitative results (100, 101). In the present work the antibacterial activity of the newly synthesized compounds screened using macro-broth dilution method against two Gram-positive (G+ve) bacteria Staphylococcus aureus and Micrococcus luteus, and two Gram-negative 43

Chapter Two

Experimental

(G-ve) bacteria Escherichia coli and Pseudomonas aeruginosa to determine the MIC in vitro. A series of 13 tubes containing 1 ml of Muller Hinton media autoclaved and cooled then inoculated with 50 µl bacterial inoculum of bacterial suspension at McFarland turbidity of 0.5. Then defined concentrations of coumarin derivatives are added to each tube except the negative control in which the solvent (DMSO) was added and a positive control with bacterial inoculum without adding any derivatives, all are incubated aerobically at 37 0C for 24 hour, then MIC is determined by visual observation as the lowest concentration that inhibits bacterial growth appears clear (59).

44

Chapter Three Results and Discussion

Chapter Three

Chapter Three

Results and Discussion

Results and Discussion Coumarin and its derivatives are very well known and important organic compounds. They possess many biological activities such as: antibacterial activity, antifungal activity, antimalarial activity, antiviral activity, anticoagulant activity, cytotoxic activity against cancer cell lines, vasorelaxant activity, antioxidant activity, anti-Alzheimer’s disease activity alongside with analgesic and anti-inflammatory activity. In the present work 7-Hydroxy-4-methyl coumarin is used as a starting material, which is oxidized to 7-Hydroxy-4-formyl coumarin using selenium dioxide. Reaction of 7-Hydroxy-4-formyl coumarin with different aromatic amines, amino acids, aromatic ketones and hydrazine group containing compounds yielded different Schiff bases, chalcones and hydrazone derivatives. One hydrazone derivative undergoes cyclization to form thiazol-4(5H)-one derivative using chloroacetic acid in the presence of anhydrous sodium acetate in glacial acetic acid as a solvent. The reaction sequence of synthesized coumarin derivatives are outlined in schemes (3-1 and 3-2) respectively.

45

Chapter Three

Results and Discussion

Scheme (3-1): Synthesis of coumarin derivatives [D1-D8]

46

Chapter Three

Results and Discussion

Scheme (3-2): Synthesis of coumarin derivatives [D9-D15]

47

Chapter Three

Results and Discussion

3.1: Synthesis of 7-Hydroxy-4-formyl coumarin [D1]:

Synthesis of compound [D1], scheme (3-3), was achieved by the reaction of 7hydroxy-4-methyl coumarin with the oxidizing agent selenium dioxide by refluxing for continuous 12 hr. using xylene as a solvent. The reaction process was monitored by TLC using ethylacetate and chloroform (2:8).

Scheme (3-3): Synthesis of compound [D1]

The first step of the reaction is a group transfer. The allylic seleninic acid produced in the first step undergoes a [2,3]-sigmatropic rearrangement to reinstate the double bond position. Rapid decomposition of the selenium (II) intermediate leads to an allylic alcohol. Oxidation continues to give the α,β-unsaturated carbonyl product as outlined in the scheme (3-4) (34, 78).

48

Chapter Three

Results and Discussion

Scheme (3-4): Mechanism of synthesis of compound [D1]

The physical properties of compound [D1] are listed in table (2-2). The FT-IR spectrum of compound [D1], figure (3-1), shows the O-H stretching frequency at (3233) cm-1, C-H stretching of aldehyde at (2840) cm-1, C=O stretching of lactone at (1723) cm-1, C=O stretching of aldehyde at (1698) cm-1, C=C stretching of aromatic ring at (1599, 1516) cm-1, O-H bending of phenol at (1410) cm-1 , C-O stretching of lactone at (1068) cm-1. The FT-IR data of compound [D1] are listed in table (3-1).

49

Chapter Three Comp. No. D1

υ O-H 3233

Results and Discussion

Table (3-1): FT-IR spectral data of compound [D1]

Characteristic bands of FT-IR (cm-1) υ C-H υ C=O υ C=C Others stretching stretching stretching 3050 1723 (1599 and C-O str.,at 1068 (aromatic) (lactone) 1516) Aromatic O-H bend. of phenol at 2840 1698 1410 cm-1 (aldehydic (aldehyde) hydrogen)

The 13C-NMR spectrum of compound [D1], which is recorded in DMSO-d6, figure (3-16), shows the following bands of carbon: 194.31 (C=O aldehyde), 161.54 (C=O lactone), 160.78, 155.26, 127.08, 112.49 and 102.57 (C aromatic and alkene). The 13 C-NMR peaks of compound [D1] are listed in table (3-2).

The mass spectrum of compound [D1], figure (3-28), shows a molecular ion peak at m/z 190 [M]+. corresponding to the molecular formula C10H6O4. The other bands at m/z 162, 161, 148 and 104 are listed in table (3-2).

Sigma bond cleavage involved mechanism

50

Chapter Three

Results and Discussion

Table (3-2): 13C-NMR and MS spectral data of compound [D1] 13

Comp. No. D1

C=O 194.31 (aldehyde)

C-NMR, (δ)= ppm C aromatic and alkene 160.78, 155.26, 127.08, 112.49, 102.57

Mass spectroscopy data m/z +. 190[M] (27.6%), 162 [M-CO] (10%), 161[M-COH] (14.9%), 148[M-ketene] (100%), 104 (17%)

161.54 (lactone)

3.2: Synthesis of Aromatic Amine Schiff Bases [D2-D6]: Aromatic amine Schiff bases [D2-D6], are illustrated in schemes (3-5) and (3-6) , have been synthesized by condensation of compound [D1] with appropriate aromatic amines with the addition of two drops glacial acetic acid to activate the C=O of aldehyde and facilitate dehydration. The reactions’ processes were monitored by TLC.

51

Chapter Three

Results and Discussion

Scheme (3-5): Synthesis of compounds [D2, D3 and D5]

Scheme (3-6): Synthesis of compounds [D4 and D6]

As outlined in the scheme (3-7), the mechanism of the reaction includes protonation of the aldehyde carbonyl to increase the intensity of carbon positive charge followed by the nucleophilic attack of the amine group then the loss of water molecule by the action of heat(34, 38).

52

Chapter Three

Results and Discussion

Scheme (3-7): The mechanism of synthesis of aromatic amine Schiff bases [D2-D6]

The physical properties of the compounds [D2-D6] are listed in table (2-3). The FT-IR spectrum of compound [D2], figure (3-2), shows O-H stretching frequency at (3481) cm-1, C=O stretching of lactone at (1720) cm-1, C=C stretching 53

Chapter Three

Results and Discussion

at (1682) cm-1, C=N stretching at (1624) cm-1, (1592) cm-1 C=C stretching of aromatic ring, O-H bending ( rocking) at (1389) cm-1, (1500) cm-1 asymmetric N-O stretching, (1389) cm-1 symmetric N-O stretching and C-O stretching of lactone at (1068) cm-1. The characteristic FT-IR bands of compound [D2] are listed in table (3-3). The 13CNMR (ppm) spectrum of compound [D2], which is recorded in CDCl3, figure (3-17) shows the following bands: 162.61 (C=O lactone), 160.46 (C=N), 155.02, 154.02, 154.00, 152.78, 138.92, 128.52, 126.41, 125.99, 113.26, 111.07, and 102.96 (C aromatic and alkene). 13CNMR peaks of compound [D2] are listed in table (3-4).

Mass spectrum of compound [D2], figure (3-29), shows a molecular ion peak at 310[M]+. , corresponding to the molecular formula (C16H10N2O5) and other bands at m/z (293, 264, 188 and 161), results are listed in table (3-4).

Non-bonding electron involved fragmentation mechanism

54

Chapter Three

Results and Discussion

Pi bond cleavage involved fragmentation mechanism

The FT-IR spectrum of compound [D3], figure (3-3), shows O-H stretching frequency at (3359) cm-1, aromatic C-H stretching at (3080) cm-1, C-H stretching of CH3 at 2920 cm-1,(1704) cm-1 C=O stretching of ketone or lactone, (1670) cm-1 55

Chapter Three

Results and Discussion

C=C stretching, C=N stretching at (1610) cm-1, aromatic C=C stretching at (1589) cm-1, O-H bending at (1388) cm-1 and C-O stretching of lactone at 1068 cm-1. The characteristic FT-IR bands of compound [D3] are listed in table (3-3). 13

The C -NMR (ppm) spectrum of compound [D3], which is recorded in CDCl3, figure (3-18) shows the following bands: 197.51 (C=O ketone), 162.52 (C=O lactone), 160.90 (C=N), 155.09, 153.86, 151.66, 133.5, 131.05, 129.93, 125.90, 113.52, 112.53 and 103.31 (C aromatic and alkene) and 26.07 (CH3-C=O). 13C-NMR peaks of compound [D3] are listed in table (3-4).

The mass spectrum of compound [D3], figure (3-30), shows a molecular ion peak at 307 [M]+., corresponding to the molecular formula (C18H13NO4) and other bands at m/z (292, 264, 188 and 161), results are listed in table (3-4).

56

Chapter Three

Results and Discussion

The FT-IR spectrum of compound [D4], figure (3-4), shows O-H stretching frequency at (3300) cm-1, C-H stretching of aromatic at (3065) cm-1, C=O stretching of lactone at (1718)cm-1,C=C stretching at (1652)cm-1, 1137 C-N stretching of diazole C=N stretching at (1607) cm-1, aromatic C=C stretching at (1507) cm-1 , O-H bending at (1403) cm-1, C-H bending of CH3 at (1368) cm-1 and C-O stretching of lactone at (1070) cm-1. The characteristic FT-IR bands of compound [D4] are listed in table (3-3). The 13C-NMR (ppm) spectrum of compound [D4], which is recorded in CDCl3, figure (3-19), shows the following bands of carbon: 166.28 (C=O lactone), 161.8 (C=O amide), 160.80 (C=N), 158.1, 151.10, 129.50, 125.73, 113.28, 111.12, 103.25 (C aromatic and alkene), 34.7 (CH3-N), 18.73 (CH3). 13C-NMR peaks of compound [D4] are listed in table (3-4).

57

Chapter Three

Results and Discussion

The mass spectrum of compound [D4], figure (3-31), shows a molecular ion peak at 375[M]+. corresponding to the molecular formula (C21H18N3O4) and other bands at m/z (374, 188 and 161), results are listed in table (3-4).

58

Chapter Three

Results and Discussion

The FT-IR spectrum of compound [D5], figure (3-5), shows O-H stretching frequency at (3350) cm-1, C-H stretching of aromatic at (3080) cm-1, C-H stretching of aliphatic CH3 at (2978) cm-1, C=C stretching at (1682) cm-1 ,C=N stretching at (1640) cm-1, aromatic C=C stretching at (1597 and 1517) cm-1, C-H bending of CH2 at (1474) cm-1, O-H bending at (1389) cm-1 and C-O stretching of lactone at (1069) cm-1, and C-O stretching of ester at (1044) cm-1.The characteristic FT-IR bands of compound [D5] are listed in table (3-3). The 13C-NMR (ppm) spectrum of compound [D5], which is recorded in CDCl3, figure (3-20) shows the following bands 166.97 (C=O ester), 162.58 (C=O lactone), 160.74 (C=N), 158.63, 155.03, 150.76, 131.60, 130.77, 125.90, 112.48, 111.29, 103.26 (C aromatic and alkene), 60.56 (CH2-O), 14.42(CH3). 13C-NMR peaks of compound [D5] are listed in table (3-4). HO

O

O

162.58

14.42

O HC N

C

160.74

166.97

O

CH2CH3

60.56

The mass spectrum of compound [D5], figure (3-32), shows a molecular ion peak at 337[M]+. ,corresponding to the molecular formula (C19H15NO5) and other bands at m/z (320, 308, 292 and 264), results are listed in table (3-4).

59

Chapter Three

CH H2 C -

Results and Discussion

3

The FT-IR spectrum of compound [D6], figure (3-6), shows O-H stretching frequency at (3380) cm-1,(3074) C-H cm-1 aromatic stretching, S-H stretching frequency at (2550) cm-1, C=O stretching of lactone at (1760) cm-1 , C=C stretching at (1676) cm-1, C=N stretching at (1639) cm-1, aromatic C=C stretching at (1598 and 1505) cm-1, and C-O stretching of lactone at (1066) cm-1. The characteristic FT-IR bands of compound [D6] are listed in table (3-3). The 13C-NMR (ppm) spectrum of compound [D6], which is recorded in CDCl3, figure (3-21) shows the following bands: 181.11 (C-SH), 162.17 (C=O lactone), 159.88 (C=N), 155.09, 153.46, 113.24, 111.48, and 103.40 (C aromatic and alkene). 13CNMR peaks of compound [D6] are listed in table (3-4).

60

Chapter Three

Results and Discussion

The mass spectrum of compound [D6], figure (3-33), shows a molecular ion peak at 305 [M]+.corresponding to the molecular formula (C12H7N3O3S2) and other bands at m/z (304, 272, 188 and 161), results are listed in table (3-4).

61

Chapter Three

Results and Discussion

Table (3-3): FR-IR spectral data of compounds [D2-D6]

Comp. No. [D2]

[D3]

[D4]

IR Characteristic bands (cm-1) υ O-H stretching 3481

3359

3300

υ C=O stretching 1720 of lactone

1704 lactone / ketone

1718 , C=O of lactone

υ C=N stretching 1624

υ C=C stretching 1592 Aromatic C=C 1682 C=C 1589 C=C aromatic

1610

3350

1500 Asymmetric N-O str. 1389 Symmetric N-O str. 1068 (C-O) str., Ar (CH) str. at 3080 1388 (O-H) bend.

1670 C=C 1652 C=C stretching

1607

1507 and C=C aromatic

[D5]

Others,

1640

1682 C=C

1068 (C-O) str. of lactone 3065 Ar(CH )str. 1403 (OH ) phenolic bend. 1368 (CH3) bend, 1137( C-N )str. of diazole, 1070 (C-O) str. of lactone, 3080 Ar(CH)str. 2978 aliph.( CH3 ) str.

1597 and 1517 C=C of aromatic

[D6]

3380

1760 Lactone

1639 C=N

1676 C=C

1474 (CH2) bend. 1389 (O-H) bend. 1069 (C-O s)tr of lactone and 1044 (C-O )str. of ester. 3074 Ar(CH) str. 2550 (SH ) w.str.

1598 and 1505 C=C str. of aromatic

62

1066 (C-O) str. of lactone

Chapter Three

Results and Discussion

Table (3-4): 13C-NMR and MS spectral data of compounds [D2-D6] 13

Comp. No.

[D2]

[D3]

C=O 162.61 (lactone)

C=N 160.46

C aromatic and alkene 155.02, 154.02,154.00, 152.78, 138.92, 128.52, 126.41, 125.99, 113.26, 111.07, 102.96.

C aliphatic

197.51 (ketone)

160.90

155.09, 153.86, 151.66, 133.5,131.05, 129.93, 125.90, 113.52, 112.53, 110.92, 103.31. 158.1, 151.10, 129.50, 125.73, 113.28, 111.12, 103.25.

26.07 (CH3-C=O)

158.63, 155.03, 150.76, 131.60, 130.77, 125.90, 112.48, 111.29, 103.26

60.56 (CH2-O)

162.52 (lactone) [D4]

[D5]

166.28 (C=O lactone 161.8 (C=O amide) 166.97 (C=O ester)

160.80

160.74 (C=N)

162.58 (C=O lactone)

[D6]

C-NMR, (δ)=ppm

162.17 (C=O lactone)

159.88 (C=N)

155.09, 153.46, 113.24, 111.48, , 103.40

-

34.7( CH3-N) 18.73 (CH3).

14.42 (CH3).

-

Mass spectroscopy data m/z 310[M] +. (31.2%), 293[MOH] (23.7%); 264 [M-NO2] (6.2%); 188[MC6H4-pNO2] (3.7%); 161 (7.5%).

307 [M] +. (8.9%), 292 (3.8%), 264 (12.8%), 188 (5.1%), 161 (3.8%). 375[M] +. (7%), 374 (3.3%), 188 (6.6%), 161 (13.3). 337[M] +. (32.4%) ; 320 [M-OH] (9.5%);308[MCH3CH2] (16.2%) ;292[MCH3CH2O] (20.2%); 264[MCH3CH2CO2] (33.7%). 305 [M]+. (4%); 304 (4%), 272[M-SH] (6.5%), 188 (9.2%), 161 (30.2%).

63

Chapter Three

3.3: Synthesis of Amino Acid Schiff Bases [D7-D8]:

Results and Discussion

Amino acid Schiff bases [D7-D8] have been synthesized by the reaction of compound [D1] with the appropriate amino acids using absolute ethanol as a solvent in the presence of sodium hydroxide and acetic acid, scheme (3-8).

Scheme (3-8): Synthesis of compounds [D7-D8]

The rationale behind the use of sodium hydroxide is to free the NH2 group of the amino acid, as it is in continuous resonance with the carboxylic acid group continuously, also to convert the carboxylic acid group to its sodium salt.

As outlined in scheme (3-9), the mechanism of the reaction proceeds via the nucleophilic attack of the lone pair of electrons of amine nitrogen on the carbonyl carbon of the aldehyde with the loss of a water molecule (84).

64

Chapter Three

Results and Discussion

Scheme (3-9): Mechanism of synthesis of amino acid Schiff bases [D7-D8]

The physical properties of compounds [D7-D8] are listed in table (2-3). The FT-IR spectrum of compound [D7], figure (3-7), show O-H stretching frequency of phenol at (3645) cm-1, a broad O-H stretching frequency of carboxylic acid at (3465-2601) cm-1, (3031) C-H stretching of aromatic ring, C-H stretching of aliphatic CH2 at (2987,2871) cm-1, C=O stretching of carboxylic acid or lactone at (1704) cm-1, with reduced frequency due to conjugation, C=C stretching at (1640) cm-1, C=N stretching frequency at (1600) cm-1, and aromatic ring at (1500) cm-1, C-H bending of CH2 at (1470) cm-1, O-H bending of phenol at (1410) cm-1 and C-O stretching of at (1071) cm-1. The characteristic FT-IR bands of compound [D7] are listed in table (3-5).

65

Chapter Three

Results and Discussion

The 13C-NMR (ppm) spectrum of compound [D7], which is recorded in DMSO-d6, figure (3-22) shows the following bands: 174.16 (C=O carboxylic acid) 163.25 (C=O lactone), 161.01 (C=N), 156.91, 154.16, 126.85, 113.89, 111.69, and 106.05 (C aromatic and alkene), 56.52 (CH2-COOH). 13C-NMR peaks of compound [D7] are listed in table (3-6).

The mass spectrum of compound [D7], figure (3-34), shows a molecular ion peak at 247 [M]+.,corresponding to the molecular formula (C12H9NO5) and other bands at m/z (246, 202, 188, 161), results are listed in table (3-6).

The FT-IR spectrum of compound [D8], figure (3-8), shows O-H stretching frequency of phenol at (3364) cm-1, a broad O-H stretching frequency of carboxylic acid at (3253-2503) cm-1 , aromatic C-H stretching at (3078)cm-1, C-H 66

Chapter Three

Results and Discussion

stretching of CH3 at (2948) cm-1, C=O stretching of lactone or carboxylic acid at (1720) cm-1, with reduced frequency due to conjugation, C=C stretching at (1645) cm-1 , C=N stretching frequency at (1617) cm-1, C=C stretching of aromatic ring at (1585 and 1519) cm-1, O-H bending of phenol at (1411) cm-1 ,C-H bending of CH3 at (1361) cm-1 and C-O stretching at (1065) cm-1. The characteristic FT-IR bands of compound [D8] are listed in the table (3-5). The 13C-NMR (ppm) spectrum of compound [D8], which is recorded in DMSO-d6, figure (3-23), shows the following bands: 174.20 (C=O carboxylic acid) 165.1 (C=O lactone), 161.18 (C=N), 155.83, 154.17, 126.64, 114.56, 110.65, 102.82 (C 13 C-NMR peaks of compound [D8] aromatic and alkene), 56.8(CH-COOH), 18.60 (CH3). are listed in table (3-6).

The mass spectrum of compound [D8], figure (3-35), shows a molecular ion peak at 261[M]+., corresponding to the molecular formula (C13H11NO5) and other bands at m/z (260, 246, 229, 216, 188), results are listed in table (3-6).

67

Chapter Three

Results and Discussion

Table (3-5): FR-IR spectral data of compounds [D7-D8] Comp. No. υ O-H stretching

IR Characteristic bands ,ʋ= cm-1 υ C-H υ C=O υ C=N υ C=C stretching stretching stretching stretching

Others

[D7] 3645 (phenol) (3465-2601) (carboxylic acid)

3031 aromatic, 2987,2871 aliphatic (CH2)

(1704) (C=O) of carboxylic acid

(1600)

(1640) (C=C)

(1410) (OH) bend.

(1500) C=C aromatic

1470 (CH2) bend.

or

[D8]

3364 (phenol) (3253-2503) (carboxylic acid)

2948 (CH3)

lactone 1720 (lactone or carboxylic acid)

1071(C-O) str.

1617 (C=N)

1645 (C=C)

1361 (CH3) bend.,

1585, 1519 (C=C) aromatic

1411,(OH) phenol bend . 1065(C-O) str.1.

68

Chapter Three

Results and Discussion

Table (3-6): 13C-NMR and MS spectral data of compounds [D7-D8] 13

Comp. No.

[D7]

C-NMR, (δ)=ppm

C=O

C=N

C aromatic and alkene

174.16 or (C=O carboxylic acid)

161.01

156.91, 155.49, 154.16, 126.85, 116.24, 113.89, 111.69, 109.19, 107.47, 106.05, 102.76

161.18

155.83, 154.17, 126.64, 114.56, 110.65, 102.82

C aliphatic 56.52 (CH2COOH)-

Mass spectroscopy data m/z 247[M]+. (13.7%), 246 (7.5%), 202 (12.5%), 188 (15%), 161 (82.55%).

163.25 (C=O lactone)

[D8]

174.20 (C=O carboxylic acid) 165.1 (C=O lactone)

56.8 (CH-), 18.60 (CH3)

261 [M]+. (6%), 260 (4.8%), 246[M-CH3] (5.4%), 229 (42.1%), 216[M-CO2H] (12%), 188 (10.8%).

3.4: Synthesis of Chalcone derivatives [D9-D11]: Chalcones or α, β unsaturated ketones [D9-D11], scheme (3-10), are synthesized by the reaction of compound [D1] with appropriate aromatic ketones by using ethanol as a solvent and sodium hydroxide as a reagent.

69

Chapter Three

Results and Discussion

Scheme (3-10): Synthesis of compound [D9-D11]

The mechanism of the reaction proceeds via claisen-schmidt condensation in which enolate anion is first formed by the action of sodium hydroxide as a base. There is only one site for enolization as there is only one set of α -proton, on the methyl group of the ketone. The aldehyde has no α-protons at all. To get the desired product, the enolate attacks the aldehyde to give an aldol, which then dehydrates (102). The mechanism of chalcone formation is outlined in the scheme (3-11).

70

Chapter Three

Results and Discussion

Scheme (3-11): The mechanism of synthesis of compound [D9-D11]

The physical properties of compounds [D9-D11] are listed in table (2-5). The FT-IR spectrum of compound [D9], figure (3-9), shows (C=O) stretching of lactone at (1755) cm-1, (C=O) stretching of chalcone at (1682) cm-1, (C=C)stretching at (1640) cm-1, aromatic (C=C) stretching at (1582) cm-1, O-H bending of phenol at (1408) cm-1, and C-O stretching of lactone at (1068) cm-1. The results are listed in table (3-7). 71

Chapter Three

Results and Discussion

The mass spectrum of compound [D9], figure (3-36), shows the molecular ion peak at m/z 292, [M]+.,corresponding to the molecular formula (C18H12O4) and other significant bands at m/z (291, 275, 187 and 161).The results are listed in table (38).

The FT-IR spectrum of compound [D10], figure (3-10), shows a broad (O-H) stretching frequency at (3586) cm-1,(C=O) stretching of lactone at (1755) cm-1, (C=O) stretching of chalcone at (1685) cm-1 with reduced frequency due to conjugation, C=C stretching at (1652) cm-1, aromatic (C=C) stretching at (1575 and 1396) cm-1, (O-H) bending of phenol at (1407) cm-1 and ( C-O) stretching of lactone at ( 1214) cm-1. The results are listed in table (3-7). The mass spectrum of compound [D10], figure (3-37), shows the molecular ion peak at m/z 308[M].+ ,corresponding to the molecular formula ( C18H12O5), and other bands at m/z (187 and 161). The results are listed in table (3-8).

72

Chapter Three

Results and Discussion

The FT-IR spectrum of compound [D11], figure (3-11), O-H stretching frequency of phenol at (3526) cm-1, N-H stretching frequency of primary amine at (3420 and 3330) cm-1, aromatic C-H stretching at (3002) cm-1 , C=O stretching of lactone at (1733) cm-1 , C=O stretching of chalcone at (1683) cm-1 with reduced frequency due to conjugation, C=C stretching (1646) cm-1, aromatic C=C stretching at (1595 and 1395) cm-1 , (O-H) bending of phenol at (1410) cm-1 and C-O stretching of lactone at ( 1215) cm-1. The results are listed in table (3-7). The mass spectrum of compound [D11], figure (3-38), shows the molecular ion peak at m/z 307, [M]+., corresponding to the molecular formula (C18H13NO4), and other bands at m/z ( 306, 290, 187 and 161). The results are listed in table (3-8).

73

Chapter Three

Results and Discussion

Table (3-7): FR-IR spectral data of compounds [D9-D11] Comp. No. υ O-H stretching

IR Characteristic bands (cm-1) υ C=O υ C=C stretching

Others

[D9]

-

1755 (C=O) of lactone

1640 (C=C)

1408 (OH) bend. of phenol

1682 (C=O) of chalcone

1582 Aromatic (C=C)

1068 (C-O) str. of lactone

74

[D10]

Chapter Three

3586 (phenol)

1755 C=O of lactone 1685 C=O of chalcone

[D11]

3536

1652 (C=C) 1575 and 1396 (C=C) of aromatic ring

1733 C=O of lactone

1646 (C=C)

1683 C=O of chalcone

(1595 and 1395) aromatic (C=C)

Results and Discussion

1407 (OH) bend. of phenol

1214 (C-O) str. of lactone

3420 and 3300 (NH2) stretching of primary amine 3002 C-H of aromatic 1410 O-H bend. of phenol 1215 (C-O) str. of lactone

Table (3-8): MS spectral data of compounds [D9-D11] Comp. No. [D9]

Mass spectroscopy data m/z 292[M]+. (3.8%), 291 (10.2%), 275 (5.1%), 187 (12.8%), 161 (15.3%)

[D10]

308[M]+. (3%), 187 (2.4%), 161 (6.1%)

[D11]

307[M]+. (5.1%), 306 (5.7%), 290 (4.6%), 187 (8%),161 (13.8%)

3.5: Synthesis of 2-((7-hydroxy-2-oxo-2H-chromen-4-yl)methylene) hydrazine carbothioamide [D12]: Thiosemicarbazone derivative scheme (3-12) is obtained by condensation of compound [D1] with thiosemicarbazide, by the action of heat, using ethanol as solvent, without using any acid or base (103).

75

Chapter Three

Results and Discussion

Scheme (3-12): Synthesis of compound [D12]

The mechanism of the reaction as outlined in scheme (3-13), starts with the nucleophilic attack of the lone pair electron of the (NH2) of hydrazine group, which is more nucleophilic as compared to that of regular amines, since it is adjacent to nitrogen on the thione group, then the loss of water molecule by the action of heat.

Scheme (3-13): The mechanism of synthesis of compound [D12]

The physical properties of compound [D12] are listed in table (2-6) The FT-IR spectrum of compound [D12], figure (3-12), shows (O-H) stretching of phenol at (3500) cm-1, NH2 stretching frequency at (3360 and 3260) cm-1, (C-H) 76

Chapter Three

Results and Discussion

stretching of aromatic at (3177) cm-1, (C=O) stretching of lactone at (1755) cm-1 (C=N) stretching frequency at (1610) cm-1 (1593 and 1490) cm-1 (C=C) stretching of aromatic, (N-H) bending at (1525) cm-1, (1408) cm-1 (O-H) bending of phenol, C=S stretching at (1069) cm-1. The FT-IR bands of compound [D12] are listed in table (3-9). The 13C-NMR (ppm) spectrum of compound [D12], which is recorded in DMSO-d6, figure (3-24), shows the following bands: 179.08 (C=O lactone), 161.79 (C=S), 160.79 (C=N), 156.06, 155.29, 154.04, 127.08, 113.34, 112.48, and 102.64 (C 13 aromatic and alkene). C-NMR peaks of compound [D12] are listed in table (3-10).

The mass spectrum of compound [D12], figure (3-39), shows the molecular ion peak at m/z 263,[M]+., corresponding to the molecular formula (C11H9N3O3S), and other bands at m/z (188 and 161). The results are listed in table (3-10).

77

Chapter Three

Results and Discussion

Table (3-9): FR-IR spectral data of compound [D12] IR Characteristic bands (cm-1)

Comp. No.

υ O-H stretching

υ NH2 stretching

υ C=O stretching

υ C=N stretching

υ C=C aromatic stretching

Others

3500

3360 3260

1755

1610

1593 1490

1069 C=S str.

[D12]

1273 C=S Str. 1525 N-H bend.

78

Chapter Three

Results and Discussion

Table (3-10): 13C-NMR and MS spectral data of compound [D12] 13

Comp. No.

C=O

D12

179.08

C=S 161.79

C=N 160.79

C-NMR, (δ)= ppm

Mass spectroscopy data m/z

C aromatic and alkene 156.06, 155.29, 154.04, 127.08, 113.34, 112.48, 102.64

263 (11.5%), 188 (10.3%), 161 (18.4%).

3.6: Synthesis of 4-((2-(2,4-dinitrophenyl)hydrazono)methyl)-7-hydroxy-2Hchromen-2-one [D13]: 2,4-Dinitro phenylhydrazine derivative, scheme (3-14) is obtained by the reaction of compound [D1] with 2,4-Dinitro phenyl hydrazine, by the action of heat, using ethanol as solvent in an acidic condition, this reaction can be named as condensation reaction, since the nucleophilic addition of the NH2 group is followed by the removal of a water molecule (104).

Scheme (3-14): Synthesis of compound [D13]

As outlined in the scheme (3-15), mechanism of the reaction starts with the protonation of the aldehyde carbonyl, followed by the nucleophilic attack of the lone pair electron (NH2) of hydrazine group, and the loss of a water molecule.

79

Chapter Three

Results and Discussion

Scheme (3-15): The mechanism of synthesis of compound [D13]

The physical properties of compound [D13] are listed in table (2-7).

80

Chapter Three

Results and Discussion

The FT-IR spectrum of compound [D13], figure (3-13), shows O-H stretching of phenol at (3460) cm-1, (N-H) stretching frequency at (3321) cm-1, aromatic (C-H) stretching at (3103) cm-1 , (C=C) stretching at (1681) cm-1, (C=N) stretching frequency at (1645) cm-1, (C=C) stretching of aromatic at (1599) cm-1, (N-H )bending at (1573) cm-1, N-O stretching of (NO2) at (1504 and 1310) cm-1, (C-O) stretching of lactone at (1107) cm-1. The FT-IR bands of compound [D13] are listed in table (3-11). The 13C-NMR (ppm) spectrum of compound [D13], which is recorded in DMSO-d6, figure (3-25), shows the following bands: 161.64 (C=O lactone), 160.76 (CH=N), 155.28, 153.99, 149.63, 134.72, 130.00, 127.99, 123.93, 116.02, 113.31, 112.41, and 102.62 (C aromatic and alkene). 13C-NMR peaks of compound [D13] are listed in table (3-12).

The mass spectrum of compound [D13], figure (3-40), shows the molecular ion peak at m/z 370, [M]+., corresponding to the molecular formula (C16H10N4O7), and other bands at m/z (324, 174 and 161), The results are listed in table (3-12).

81

Chapter Three

Results and Discussion

Table (3-11): FR-IR spectral data of compound [D13] IR Characteristic bands (cm-1)

Comp. No. υ O-H stretching

υ N-H stretching

υ C-H aromatic stretching

3460

3321

3103

[D13]

υ C=N stretching

1645

υ C=C stretching

Others

1681 (C=C)

1573 N-H bend.,

(1599) C=C of aromatic

1504 Asymmetric N-O Str. 1310 Symmetric N-O str. 1107 C-O str. of lactone

82

Chapter Three

Results and Discussion

Table (3-12): 13C-NMR and MS spectral data of compound [D13] 13

Comp. No.

D13

C-NMR, (δ)= ppm

C=O

CH=N

C aromatic and alkene

161.64

160.76

155.28, 153.99, 149.63, 134.72, 130.00, 127.99, 123.93, 116.02, 113.31, 112.41, 102.62

Mass spectroscopy data m/z 370[M]+. (7.1%), 324 (1.4%), 174 (7.1%), 161 (4.2%).

3.7: Synthesis of 1-((7-hydroxy-2-oxo-2H-chromen-4-yl)methylene) thiourea [D14]: Thiourea derivative synthesis, scheme (3-16), has been obtained by the reaction of compound [D1] with thiourea, using glacial acetic acid as a solvent and HCl as a catalyst. The physical properties of compound [D14] are listed in table (2-8)(105).

Scheme (3-16): Synthesis of compound [D14]

As outlined in the mechanism, scheme (3-17), HCl is used to protonate the aldehyde carbonyl, then the nucleophilic attack of the lone pair of NH2 group of thiourea on the carbonyl group that takes place, as shown below.

83

Chapter Three

Results and Discussion

Scheme (3-17): The mechanism of synthesis of compound [D14]

The physical properties of compound [D14] are listed in table (2-8). The FT-IR spectrum of compound [D14], figure (3-14), shows N-H stretching frequency at (3327) cm-1, (C=O) stretching frequency of lactone at (1710) cm-1,( C=C) stretching frequency at (1670) cm-1, (C=N) stretching at (1652) cm-1, (C=C) stretching of aromatic at (1593 and 1507) cm-1, (O-H) bending at (1388) cm-1, (C=S) stretching at (1067) cm-1 and (C-O) stretching of lactone at (1067) cm-1. The FR-IR spectral data of compound [D14] are listed in table (3-13). 84

Chapter Three

Results and Discussion

The 13C-NMR (ppm) spectrum of compound [D14], which is recorded in DMSO-d6, figure (3-26), shows the following bands: 184.29 (C=O lactone), 161.76 (C=S), 160.34 (CH=N), 155.27, 154.07, 127.03, 113.40, 112.41, 102.66 (C aromatic and alkene). 13 C-NMR peaks of compound [D14] are listed in table (3-14).

The mass spectrum of compound [D14], figure (3-41), shows the molecular ion peak at m/z 248,[M]+., corresponding to the molecular formula (C11H8N2O3S), and other bands at m/z (247, 232, 188, 174 and 161), the results are listed in table (314).

85

Chapter Three

Comp. No. [D14]

Results and Discussion

Table (3-13): FR-IR spectral data of compound [D14] IR Characteristic bands (cm-1) υ N-H stretching

υ C=O stretching

υ C=N stretching

υ C=C aromatic stretching

Others

3327

(1710) (C=O) of lactone

(1652)

(1670) (C=C)

1067 C=S Str.

(1593 and 1507) (C=C) of aromatic

86

Chapter Three

Results and Discussion

Table (3-14): 13C-NMR and MS spectral data of compound [D14] 13

Comp. No.

C=O

D14

C=S

184.29 161.76

C-NMR, (δ)= ppm

C=N

C aromatic and alkene

160.34

155.27, 154.07, 127.03, 113.40, 112.41, 102.66

Mass spectroscopy data m/z 248[M]+. (3.9%), 247 (3.2%), 232 (2.6%), 188 (13%), 174 (26%), 161 (13%)

3.8: Synthesis of 2-(2-((7-hydroxy-2-oxo-2H-chromen-4yl)methylene)hydrazinyl)thiazol-4(5H)-one [D15]: Synthesis of compound [D15], scheme (3-18), was achieved by condensation of compound [D12] with chloroacetic acid in the presence of sodium acetate in glacial acetic acid as a solvent.

Scheme (3-18): Synthesis of compound [D15]

The mechanism of the reaction is outlined in the scheme (3-19).

87

Chapter Three

Results and Discussion

Scheme (3-19): The mechanism of synthesis of compound [D15]

The physical properties of compound [D15] are listed in table (2-9). The FT-IR spectrum of compound [D15], figure (3-15), shows (N-H) stretching frequency appeared at (3150) cm-1, (C-H) stretching of aromatic at (3129) cm-1, (CH) stretching of (CH2) at (2918) cm-1, (C=O) stretching of lactone at (1760) cm-1, (C=O) stretching of amide at (1674) cm-1, (C=C) stretching at (1651) cm-1, (C=N) stretching of Schiff base at (1615) cm-1 , aromatic (C=C) stretching at (1590 and 1515) cm-1, (C-O) stretching of lactone at (1066) cm-1. The results are listed in table (3-15).

88

Chapter Three

Results and Discussion

The 13C-NMR (ppm) spectrum of compound [D15], which is recorded in DMSO-d6, figure (3-27), shows the following bands: 175.68 (C=O) of amide, 162.35 (C=O) of lactone, 160.89 (CH=N), 155.33, 154.08, 126.88, 113.59, 112.11, 102.72 (C 13 aromatic and alkene), 44.82 (CH2). C-NMR peaks of compound [D15] are listed in table (3-16).

The mass spectrum of compound [D15], figure (3-42), shows the molecular ion peak at m/z 303 [M]+. , corresponding to the molecular formula (C13H9N3O4S), and other bands at m/z (203, 188 and 161), the results are listed in table (3-16).

89

Chapter Three

Table (3-15): FR-IR spectral data of compound [D15] IR Characteristic bands (cm-1)

Comp. No. [D15]

Results and Discussion

υ N-H stretching

υ C-H stretching

υ C=O stretching

3150

3129 Ar(CH),

(1760) (C=O) of lactone

2918 Aliph.(CH)

υ C=N stretching

υ C=C stretching

Others

(1651) (C=C)

(1066) (C-O) str.of lactone

(1590 and 1515)

(708) (C-S)

(1615) (C=N)

(1674) (C=O) of amide

90

Chapter Three

Results and Discussion

Table (3-16): 13C-NMR and MS spectral data of compound [D15] 13

Comp. No. D15

C-NMR, ppm (δ)

C=O

CH=N

C aromatic and alkene

CH2

175.68 (C=O) lactone

160.89

155.33, 154.08, 126.88, 113.59, 112.11, 102.72

44.82

Mass spectroscopy data m/z 303[M]+. (4%), 203 (13.1%), 188 (26.3%), 161 (32.9%).

162.35 (C=O) lamide

3.9: Antibacterial activity test results: All of the newly synthesized coumarin derivatives are tested for their antibacterial activities except compound [D1]. The results are listed in table (3-17). Table (3-17): Antibacterial activity of title coumarin derivatives[D2-D15] Compound No. Staphylococcus Micrococcus Escherichia coli Pseudomonas aureus (G+ve) luteus (G+ve) (G-ve) aeruginosa (G-ve) Conc.µg/ml Conc.µg/ml Conc.µg/ml Conc.µg/ml D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 D15

79 63 40 98 40 40 300 192 123 154 98 123 123 123

40 50 63 123 40 40 240 79 98 79 63 154 79 192

123 98 192 154 98 31 192 240 192 240 123 192 154 79

192 154 240 192 79 50 123 123 154 300 63 98 98 98

Note:C- =negative control (solvent,DMSO), C+ = positive control indicates abroth with adding title coumarin derivatives.

91

Chapter Three

Results and Discussion

The results illustrated in table (3-17) clarify that all the newly synthesized coumarin derivatives have antibacterial activity in vitro against both Gram positive and Gram negative bacteria with MICs ranging between 31-300 µg/ml in which they vary according to the derivatives and bacteria. The lowest MIC is 31µg/ml for compound [D7] against Escherichia coli and also MIC 40 µg/ml is showed for the compounds [D6] and [D7] against gram-positive Staphylococcus aureus and Micrococcus luteus, respectively.While the highest MIC is 300µg/ml for both compounds [D8] and [D11] against both Staphylococcus aureus and Pseudomonas aeruginosa , respectively.

92

Chapter Three

Results and Discussion

Figure (3-1): FT-IR spectrum of compound [D1]

Figure (3-2): FT-IR spectrum of compound [D2] 93

Chapter Three

Results and Discussion

Figure (3-3): FT-IR spectrum of compound [D3]

Figure (3-4): FT-IR spectrum of compound [D4] 94

Chapter Three

Results and Discussion

Figure (3-5): FT-IR spectrum of compound [D5]

Figure (3-6): FT-IR spectrum of compound [D6]

95

Chapter Three

Results and Discussion

Figure (3-7): FT-IR spectrum of compound [D7]

Figure (3-8): FT-IR spectrum of compound [D8]

96

Chapter Three

Results and Discussion

Figure (3-9): FT-IR spectrum of compound [D9]

Figure (3-10): FT-IR spectrum of compound [D10] 97

Chapter Three

Results and Discussion

Figure (3-11): FT-IR spectrum of compound [D11]

Figure (3-12): FT-IR spectrum of compound [D12] 98

Chapter Three

Results and Discussion

Figure (3-13): FT-IR spectrum of compound [D13]

Figure (3-14): FT-IR spectrum of compound [D14] 99

Chapter Three

Results and Discussion

Figure (3-15): FT-IR spectrum of compound [D15]

100

Chapter Three

Results and Discussion

Figure (3-16): 13C-NMR spectrum of compound [D1]

Figure (3-17): 13C-NMR spectrum of compound [D2] 101

Chapter Three

Results and Discussion

Figure (3-18): 13C-NMR spectrum of compound [D3]

Figure (3-19): 13C-NMR spectrum of compound [D4] 102

Chapter Three

Results and Discussion

Figure (3-20): 13C-NMR spectrum of compound [D5]

Figure (3-21): 13C-NMR spectrum of compound [D6]

103

Chapter Three

Results and Discussion

Figure (3-22): 13C-NMR spectrum of compound [D7]

Figure (3-23): 13C-NMR spectrum of compound [D8]

104

Chapter Three

Results and Discussion

Figure (3-24): 13C-NMR spectrum of compound [D12]

Figure (3-25): 13C-NMR spectrum of compound [D13]

105

Chapter Three

Results and Discussion

Figure (3-26): 13C-NMR spectrum of compound [D14]

Figure (3-27): 13C-NMR spectrum of compound [D15]

106

Chapter Three

Results and Discussion

Figure (3-28): Mass spectrum of compound [D1]

Figure (3-29): Mass spectrum of compound [D2] 107

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Results and Discussion

Figure (3-30): Mass spectrum of compound [D3]

Figure (3-31): Mass spectrum of compound [D4] 108

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Results and Discussion

Figure (3-32): Mass spectrum of compound [D5]

Figure (3-33): Mass spectrum of compound [D6] 109

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Results and Discussion

Figure (3-34): Mass spectrum of compound [D7]

Figure (3-35): Mass spectrum of compound [D8]

110

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Results and Discussion

Figure (3-36): Mass spectrum of compound [D9]

Figure (3-37): Mass spectrum of compound [D10] 111

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Results and Discussion

Figure (3-38): Mass spectrum of compound [D11]

Figure (3-39): Mass spectrum of compound [D12] 112

Chapter Three

Results and Discussion

Figure (3-40): Mass spectrum of compound [D13]

Figure (3-41): Mass spectrum of compound [D14] 113

Chapter Three

Results and Discussion

Figure (3-42): Mass spectrum of compound [D15]

114

Chapter Four Conclusions and Recommendations

References

Chapter Four Conclusions and Recommendations

4.1: Conclusions: In the present work a series of new coumarin derivatives have been synthesized from 7-hydroxy-4-methylcoumarin by the oxidation of methyl group at C4 of the parent coumarin nucleus, using SeO2, to produce formyl group, then the formation of different derivatives under conventional method. The title compounds were screened for their preliminary antibacterial activity using broth dilution method, by determining their minimum inhibitory concentrations (MIC), and showed moderate to highest antibacterial activity against Gram-positive and Gram-negative bacteria. Compound 7 showed the highest antibacterial activity against Gram- positive Staphylococcus aureus and Micrococcus luteus, and potent antibacterial activity against Gram-negative E.coli.

4.2: Recommendations: 1. Calculation of octanol/water partition coefficient to estimate the lipophilicity of the final title coumarin derivatives. 2. In vitro studies including cell line screening study to evaluate the anticancer activity of selected synthesized coumarin derivatives as ligands and complexes with transition metals. 3. Synthesis more coumarin derivatives using green chemistry approach. 4. Conducting density functional theory (DFT), using molecular structures with optimized geometry, and calculation of some parameters like electronic chemical potential, highest occupied molecular orbital(HOMO)/lowest unoccupied molecular orbital/(LUMO) and etc.

115

References

References

1. McNaught AD, Wilkinson A. IUPAC. Compendium of Chemical Terminology,(the "Gold Book"). 2nd ed: Blackwell Scientific Publications, Oxford 1997. 2. Katritzky AR, Denisko OV. Heterocyclic compound. Encyclopædia Britannica, inc. 2008. 3. Walsh EJ. Book Notes: Heterocyclic Chemistry, 3rd ed. (Gilchrist, Thomas L.). J Chem Educ. 1997;74(12). 4. Valencia D, Klimova T, García-Cruz I. Aromaticity of five- and sixmembered heterocycles present in crude oils –An electronic description for hydrotreatment process. Fuel. 2012;100:177-85. 5. Estevez V, Villacampa M, Menendez JC. Multicomponent reactions for the synthesis of pyrroles. Chem Soc Rev. 2010;39. 6. Altaf AA, Shahzad A, Gul Z, Rasool N, Badshah A, Lal B, et al. A Review on the Medicinal Importance of Pyridine Derivatives. JDDMC. 2015;1(1):1-11. 7. Eicher T, Hauptmann S, Speicher A. The chemistry of heterocycles: Structures, reactions, synthesis, and applications , completely revised and enlarged edition. 3rd ed2012. 8. Musa MA, Cooperwood JS, Khan MOF. A review of coumarin derivatives in pharmacotherapy of breast cancer. Curr Med Chem. 2008;15(26):2664–79. 9. Egan D, O’Kennedy R, Mow E, Cox D, Prosser E, Thrones RD. The pharmacology, metabolism, analysis, and applications of coumarin and coumarinrelated compounds. Drug Metab Rev. 1990;22(5):503-29. 10. Borges F, Roleira F, Milhazes N, Santana L, Uriarte E. Simple coumarins and analogues in medicinal chemistry: Occurrence, synthesis and biological activity. Curr Med Chem. 2005;12(8):887-916. 11. Sairam KV, Gurupadayya BM, Chandan RS, Nagesha DK, Vishwanathan B. A review on chemical profile of coumarins and their therapeutic role in the treatment of cancer. Curr Drug Deliv. 2016;13(2):186-201. 12. Jain PK, Joshi H. Coumarin: Chemical and pharmacological profile. JAPS. 2012;02(06):236-40. 13. Lazar T. Chemistry of Natural Products. By S. V. Bhat, B. A. Nagasampagi and M. Sivakumar. ChemBioChem. 2005;6(6):1127-8. 14. Bubolsa GB, Vianna DdR, Medina-Remón A, Poser Gv, Lamuela-Raventos RM, Eifler-Lima VL, et al. The antioxidant activity of coumarins and flavonoids. Mini Rev Med Chem. 2012;13(3). 15. Venugopala KN, Rashmi V, B.Odhav. Review on natural coumarin lead compounds for their pharmacological activity. BioMed Res Int. 2013;2013:1-14. 16. Kostova I. Synthetic and natural coumarins as cytotoxic agents. Curr Med Chem - Anti-Cancer Agents. 2005;5(1):29-46. 116

References

17. Bourgaud F, Hehn A, Larbat R, Doerper S, Gontier E, Kellner S, et al. Biosynthesis of coumarins in plants: A major pathway still to be unravelled for cytochrome P450 enzymes. Phytochem Rev. 2006;5:293–308. 18. Hussien FA-H, Keshe M, Alzobar K, Merza J, Karam A. Synthesis and nitration of 7-hydroxy-4-methyl coumarin via Pechmann condensation using ecofriendly medias. ILCPA. 2016;69:66-73. 19. Phadtare SB, Shankarling GS. Greener coumarin synthesis by Knoevenagel condensation using biodegradable choline chloride. Environ Chem Lett. 2012;10:363–8. 20. Shaabani A, Ghadari R, Rahmati A, Rezayan AH. Coumarin synthesis via Knoevenagel condensation reaction in 1,1,3,3-N,N,N',N'-tetramethylguanidinium trifluoroacetate ionic liquid. J Iran Chem Soc. 2009;6(4):710-4. 21. Ranu BC, Jana R. Ionic liquid as catalyst and reaction medium – A Simple, efficient and green procedure for Knoevenagel condensation of aliphatic and aromatic carbonyl compounds using a task-specific basic ionic liquid. Eur J Org Chem. 2006;16:3767–70. 22. Verdía P, Santamarta F, Tojo E. Knoevenagel reaction in [MMIm][MSO4]: Synthesis of coumarins. Molecules. 2011;16:4379-88. 23. Palaniappan S, Shekhar RC. Synthesis of 7-hydroxy-4-methyl coumarin using polyaniline supported acid catalyst. J Mol Catal A: Chem 2004;209:117–24. 24. Khan SA, Khan SB, Asiri AM, Ahmad I. Zirconia-based catalyst for the one-pot synthesis of coumarin through Pechmann reaction. Nanoscale Res Lett. 2016;11(345). 25. Borges F, Roleira F, Milhazes N, Santana L, Uriarte E. Simple coumarins and analogues in medicinal chemistry: Occurrence, synthesis and biological activity. Curr Med Chem. 2005;12(8):887-916. 26. Musa MA. Applications of the Baylis-Hillman reaction in the synthesis of coumarin derivatives. Grahamstown, Rhodes University, Department of Chemistry, PhD Thesis2002. 27. Desai VG, Shet JB, Tilve SG, Mali RS. Intramolecular Wittig reactions: A new synthesis of coumarins and 2-quinolones. J Chem Research. 2003;5:628–9. 28. Eisenbeiss J, Schmid H. Struktur des erosnin (Norton & Hansberry's). Helvetica Chimica Acta. 1959;12(4):61-6. 29. El-Sayed EH, El-Moneim MA, El-Deen IM, Mossalam4 AM. Ring opening of coumarin derivatives with nitrogen nucleophilic reagents and their biological activity. IJIRSET. 2013;2(12):7232-43. 30. Asghari S, Baharfar R, Darabi SA, Mohammadian R. Three-component reactions of 7-hydroxy coumarin derivatives, acetylenic esters and aromatic aldehydes in the presence of NEt3. J Braz Chem Soc. 2015;26(2):218-23. 117

References

31. Katritzky AR, Boulton AJ. Advances in heterocyclic chemistry. New York & London: Academic press Inc.; 1966. 32. Cain JC, Greenawa AJ. Nitration of alkyl coumarins. J Chem Soc. 1910;17(1). 33. Zhou MX. The use of Selenium (IV) oxide to oxidize aromatic methyl groups. Emporia, Emporia State University, Department of Chemistry, MSc Thesis1992. 34. Clayden J, Greeves N, Warren S. Organic Chemistry Second Edition2012. 35. Kulkarni A, Patil SA, Badamib PS. Synthesis, characterization, DNA cleavage and in vitro antimicrobial studies of La(III), Th(IV) and VO(IV) complexes with Schiff bases of coumarin derivatives. Eur J Med Chem. 2009;44(7):2904–12. 36. Gupta P, Jat K, Singh V, Shrivastava R. An efficient synthesis, characterization and antibacterial activity of novel N'-arylidene-4- (2-(piperidin-1yl) ethoxy) benzohydrazide derivatives Schiff bases. IOSR-JAC. 2015;8(12):06-11. 37. Mashelkar UC, Audi AA. Synthesis of some novel 4-substituted coumarins having potential biological activity (Part III). Indian J Chem. 2006;45B(06):14639. 38. PN B, Seleena S, Vidya G, Himateja T, Neelima B, Lakshmiteja T. Synthesis and charecterisation of schiff bases derived from acetyl coumarin and evaluated for anti-microbial activity. Res J Pharm, Biol Chem Sci. 2013;4(2):9-15. 39. Creaven BS, Devereux M, Karcz D, Kellet A, McCann M. Copper(II) complexes of coumarin-derived schiff bases and their anti-candida activity. J Inorg Biochem. 2009;103(9):1196–203. 40. Mashelkar UC, JHA MS, Masheklar BU. Synthesis of 2-azetidinones substituted coumarin derivatives. J Serb Chem Soc. 2012;77(10):1339–44. 41. Hamid SJ, Kubba AARM. Synthesis and charactrization of new coumarin derivatives containing various moieties with antibacterial activities. Int J Pharm Pharm Sci. 2015;7(8):70-4. 42. Vazquez-Rodriguez S, Serra S, Santos Y, Santana L. Efficient synthesis of coumarin-chalcones hybrids as new scaffold with antibacterial interest. . In: Seijas JA, Tato MPV, editors. The 14th International Electronic Conference on Synthetic Organic Chemistry; University of Santiago de Compostela-MDPI-RSEQ, Spain2010. 43. Pingaew R, Saekee A, Mandi P, Nantasenamat C, Prachayasittikul S, Ruchirawat S, et al. Synthesis, biological evaluation and molecular docking of novel chalconeecoumarin hybrids as anticancer and antimalarial agents. Eur J Med Chem. 2014;85:65-76. 44. Singh P, Anand A, Kumar V. Recent developments in biological activities of chalcones: A mini review. Eur J Med Chem. 2014;85:758-77. 118

References

45. Sashidhara KV, Kumar A, Kumar M, Sarkar J, Sinha S. Synthesis and in vitro evaluation of novel coumarin-chalcone hybrids as potential Anticancer agents. Bioorg Med Chem Lett. 2006;20(24):7205-11. 46. Vazquez-Rodriguez S, Guíñez RF, Matos MJ, Olea-Azar C, Maya JD, Santana L, et al. Synthesis and trypanocidal properties of new coumarin-chalcone derivatives. Med chem. 2015;5(4):173-7. 47. Siddiqui ZN. A convenient synthesis of coumarinyl chalcones using HClO4–SiO2: A green approach. Arabian J Chem 2015;6(3):1-10. 48. Mohamed HM, El-Wahab AHFA, Ahmed KA, El-Agrody AM, Bedair AH, Eid FA, et al. Synthesis, reactions and antimicrobial activities of 8-ethoxycoumarin derivatives. Molecules. 2012;17:971-88. 49. Trkovnik M, KekiĆ M, KuleŠ M, Gorenc A. 3-Formyl-4-hydroxycoumarin derivatives syntheses of semicarbazone, phenylsemicarbazone, hydrazino(oxazolin-4-one-2-yl) hydrazone, urea derivatives, and their sulfur analogues. Z Naturforsch. 1975;30(b):237-8. 50. Siddqui N, Arshad MF, Khan SA. Synthesis of some new coumarin incorporated thiazolyl semicarbazones as anticonvulsants. Acta Pol Pharm. 2009;6(2):161-7. 51. Vekariya RH, Patel KD, Rajani DP, Rajani SD, Patel HD. A one pot, three component synthesis of coumarin hybrid thiosemicarbazone derivatives and their antimicrobial evolution. JAAUBAS. 2016;20(1):1-10. 52. Refat MS, El-Deen IM, Anwerb ZM, El-Ghol S. Bivalent transition metal complexes of coumarin-3-yl thiosemicarbazone derivatives: Spectroscopic, antibacterial activity and thermogravimetric studies. J Mol Struc. 2009;920:149-62. 53. Hasanen JA. Synthesis and mass spectra of some new 3-substituted coumarin derivatives. Der Pharma Chemica. 2012;4(5):1923-34. 54. Ranade DS, Bapat AM, Ramteke SN, Joshi BN, Roussel P, Tomas A, et al. Thiosemicarbazone modification of 3-acetyl coumarin inhibits Aβ peptide aggregation and protect against Aβ-induced cytotoxicity. Eur J Med Chem. 2015;8(12):1-28. 55. Jayashree BS, Nigam S, Pai A, Chowdary PVR. Overview on the recently developed coumarinyl heterocycles as useful therapeutic agents. Arabian J Chem. 2014;7:885-99. 56. Hamama WS, Berghot MA, Baz EA, Gouda MA. Synthesis and antioxidant evaluation of some new 3-substituted coumarins. Arch Pharm Chem Life Sci. 2011;344:710-8. 57. Nagamallu R, Kariyappa AK. Synthesis and biological evaluation of novel formyl-pyrazoles bearing coumarin moiety as potent antimicrobial and antioxidant agents. Bioorg Med Chem Lett. 2013;23:234-41. 119

References

58. Idemudia OG, Sadimenko AP, Hosten EC. Metal complexes of new bioactive pyrazolone phenylhydrazones; crystal structure of 4-acetyl-3-methyl-1phenyl-2-pyrazoline-5-one phenylhydrazone Ampp-Ph. Int J Mol Sci. 2016;17(687):1-24. 59. Souza SMd, Monache FD, Jr AS. Antibacterial Activity of Coumarins. Z Naturforsch. 2005;60(c):693-700. 60. Wagner HKM. Phytochemistry of medicinal plants. 1 ed. Arnason JT, Mata R, Romeo JT, editors: Springer US; 1995. 61. Hassan MZ, Osman H, Ali MA, Ahsan MJ. Therapeutic potential of coumarins as antiviral agents. Eur J Med Chem. 2016;123:236-55. 62. Hirsch A-M, Longeon A, Guyot M. Fraxin and esculin: two coumarins specific to Actinidia chinensis and A. deliciosa (kiwifruit). Biochem Syst Ecol. 2002;30:55-60. 63. Kim S-h, Kang K-a, Zhang R, Piao M-j, Ko D-o, Wang Z-h, et al. Protective effect of esculetin against oxidative stress-induced cell damage via scavenging reactive oxygen species. Acta Pharmacol Sin. 2008;29(11):1319-26. 64. Piller NB. A comparison of the effectiveness of some anti-inflammatory drugs on thermal oedema. Br J exp Path. 1975;56(6):554-9. 65. Witaicenis A, Seito LN, Stasi LCD. Intestinal anti-inflammatory activity of esculetin and 4-methylesculetin in the trinitrobenzenesulphonic acid model of rat colitis. Chem Biol Interact. 2010;186(2):211-8. 66. Huang G-J, Deng J-S, Liao J-C, Hou W-C, Wang S-Y, Sung P-J, et al. Inducible nitric oxide synthase and cyclooxygenase-2 participate in antiinflammatory activity of imperatorin from Glehnia littoralis. J Agric Food Chem. 2012;60(7):1673-81. 67. Kwon OS, Choi JS, Islam MN, Kim YS, Kim HP. Inhibition of 5lipoxygenase and skin inflammation by the aerial parts of Artemisia capillaris and its constituents. Arch Pharm Res. 2011;34(9):1561-9. 68. Emami S, Dadashpour S. Current developments of coumarin-based anticancer agents in medicinal chemistry. Eur J Med Chem. 2015;102:611-30. 69. Luo K-w, Sun J-g, Chan JY-W, Yang L, Wu S-h, Fung K-P, et al. Anticancer effects of imperatorin isolated from Angelica dahurica: induction of apoptosis in HepG2 cells through both death-receptor- and mitochondria-mediated pathways. Chemotherapy. 2011;57(6):449-59. 70. Whang WK, Park HS, Ham I, Oh M, Namkoong H, Kim HK, et al. Natural compounds,fraxin and chemicals structurally related to fraxin protect cells from oxidative stress. Exp Mol Med. 2005;37(5):436-46. 71. Hirsh J, Dalen JE, Anderson DR, Poller L, Bussey H, Ansell J, et al. Oral anticoagulants: mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest. 1998;114(5):8-21. 120

References

72. Mead JAR, Smith JN, Williams RT. The metabolism of coumarin and of ocoumaric acid. Biochem J. 1985;68(1):67-74. 73. Lei L, Xue Y-b, Liu Z, Peng S-s, He Y, Zhang Y, et al. Coumarin derivatives from Ainsliaea fragrans and their anticoagulant activity. Scientefic Reports. 2015;5:1-9. 74. Kharasch ED, Hankins DC, Taraday JK. Single-dose methoxsalen effects on human cytochrome P-450 2A6 activity. Drug Metab Dispos. 2000;28(1). 75. Ezeanyika LUS, Obidoa O. Comparative effects of scopoletin and cyanide on glucose-6-phosphatase and glutathione S-transferase activities of rat liver microsomes. Nutr Res. 2000;20(2):309-13. 76. Lake BG. Coumarin metabolism, toxicity and carcino genicity: relevance for human risk assessment. Food Chem Toxicol. 1999;37(4):423-53. 77. Vassallo JD, Hicks SM, Daston GP, Lehman-McKeeman LD. Metabolic detoxification determines species differences in coumarin-induced hepatotoxicity. Toxicol Sci. 2004;80(2):249-57. 78. Al-Amiery AA, Al-Majedy YK, Kadhum AAH, Mohamad AB. Hydrogen Peroxide Scavenging Activity of Novel Coumarins Synthesized Using Different Approaches. PLoS ONE. 2015;10(7):1-9. 79. Kini SG, Choudhary S, Mubeen M. Synthesis, docking study and anticancer activity of coumarin substituted derivatives of benzothiazole J Comput Methods Mol Des. 2012;2(1):51-60. 80. Lin W, Long L, Feng J, Wang B, Guo C. Synthesis of meso-CoumarinConjugated Porphyrins and Investigation of Their Luminescence Properties. Eur J Org Chem. 2007(26):4301-4. 81. Dömötöra O, Tuccinardi T, Karcz D, Walshd M, Creaven BS, Enyedy ÉA. Interaction of anticancer reduced Schiff base coumarin derivatives with human serum albumin investigated by fluorescence quenching and molecular modeling. Bioorg Chem. 2014;28(52):16–23. 82. Tan X-J, Hao X-Q, Zhao Q-Z, Cheng S-S, Xie W-L, Xing D-X, et al. MonoSchiff-base or di-Schiff-base? Synthesis, spectroscopic, X-ray structural and DFT study of a series of Schiff-bases derived from benzil dihydrazone. J Mol Struct. 2015;5(1099):373-87 83. Yang Z, Sun P. Compare of three ways of synthesis of simple Schiff base. Molbank. 2006;6:1-3. 84. A.S.P.Azzouz, Ali RT. Synthesis of Schiff Bases Derived From Benzaldehyde and Salicylaldehyde With Some Amino Acids by a New Develop Method. NJC. 2010;37:158-68.

121

References

85. Kumar DN, Sharma P, pareek A. Synthesis of new Schiff-Base complexes and their applications. IJARS. 2013;Vol. II(2):1-6. 86. Rîmbu C, Danac R, Pui A. Antibacterial activity of Pd(II) complexes with salicylaldehyde-amino acids Schiff Bases ligands. Chem Pharm Bull. 2014;62(1):12–5. 87. Mielcke TR, Mascarello A, Filippi-Chiela E, Zanin RF, Lenz G, Leal PC, et al. Activity of novel quinoxaline-derived chalcones on in vitro glioma cell proliferation. Eur J Med Chem. 2012;48:255-64. 88. Sashidhara KV, Kumara A, Kumara M, Sarkar J, Sinha S. Synthesis and in vitro evaluation of novel coumarin–chalcone hybrids as potential anticancer agents. Bioorg Med Chem Lett. 2010;20(24):7205–11. 89. Smit FJ, N’Da DD. Synthesis, in vitro antimalarial activity and cytotoxicity of novel 4-aminoquinolinyl-chalcone amides. Bioorg Med Chem Lett. 2014;22:1128–38. 90. Zuo Y, Yu Y, Wang S, Shao W, Zhou B, Lin L, et al. Synthesis and cytotoxicity evaluation of biaryl-based chalcones and their potential in TNFainduced nuclear factor-kB activation inhibition. Eur J Med Chem. 2012;50:393404. 91. Trkovnik M, ekiĆ MK, uleŠ MK, Gorenc A. 3-Formyl-4-hydroxycoumarin Derivatives Syntheses of Semicarbazone, Phenylsemicarbazone, Hydrazino(oxazolin-4-one-2-yl) Hydrazone, Urea Derivatives, and their Sulfur Analogues. Z Naturforsch. 1975;30 b(3-4):237-8. 92. Mohamed HM, El-Wahab AHFA, Ahmed KA, El-Agrody AM, Bedair AH, Eid FA, et al. Synthesis, Reactions and Antimicrobial Activities of 8Ethoxycoumarin Derivatives. Molecules. 2012;17(1):971-88. 93. Gomha SM, Khalil KD. A convenient ultrasound-promoted synthesis of some new thiazole derivatives bearing a coumarin nucleus and their cytotoxic activity. Molecules. 2012;17(8):9335-47. 94. Aarestrup FM, Pedersen K, Seyfarth AM, Emborg H-D, Hendriksen RS, Bager F. Effect of abolishment of the use of antimicrobial agents for growth promotion on occurrence of antimicrobial resistance in fecal Enterococci from food animals in Denmark. Antimicrob Agents Chemother. 2001;45(7):2054–9. 95. Reller LB, Weinstein M, Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing: A review of general principles and contemporary practices. Clin Infect Dis. 2009;49(11):1749-55. 96. Tille P. Bailey & Scott's Diagnostic Microbiology. Thirteenth edition ed2014. 97. Joyce M, Woods CW. Antibacterial susceptibility testing in the clinical laboratory. Infect Dis Clin N Am. 2004;18:401-3. 122

References

98. Boundless. Boundless Microbiology, Minimal Inhibitory Concentration (MIC). USA2016. 99. Barchiest F, Colombo AL, McGough DA, Michael MGR. Comparative study of broth macrodilution and microdilution techniques for In Vitro antifungal susceptibility testing of yeasts by using the national committee for clinical laboratory standards' proposed standard. J Clin Microbiol. 1994;32(10):2494-500. 100. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. JPA. 2016;6:71–9. 101. Hamid SJ. Synthesis and characterization of new coumarinn derivatives containing various moities with antibacterial activities [M.Sc.]. Sulaimani: University of Sulaimani; 2010. 102. Clayden J, Greeves N, Warren S. Organic Chemistry Second Edition2012. 103. El-Fattah MEA. Synthesis and investigation of mass spectra of 3substituted-2-thioxo-imidazolidin-4-one derivatives. Indian J Chem. 2006;45B:2523-33. 104. Kadam SS, ST T, ND G, DD G. Review article on: Chemical importance of brady's reagent. IJRPC. 2012;2(4):1086-92. 105. RP P, SA D, VS S, WN J. Synthesis and antimicrobial and antifungal activities of novel Bis-imine derivatives. RJPBCS. 2013;4(2):1411-8.

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        ، 4 7   4    4   7       4   7 [D1]   12  [D1][D6-D2]    [D1]  [D7-D8]   [D1][D9-D11] 10 25   [D1]         [D12]     2،4  [D1]         [D13]    [D1][D14] 

 [D12]           [D15]                               13      ،  ،               [D8]

 [D7]

 [D11]

      

4- ، .4 7    

 ٢٠١١  

  ١٤٣٨ ٢٧١٧  ٢٠١٧

   4 74      47     [D1]   4 7 12        [D1]    [D2-D6]     [D1]   [D7-D8]        [D1]      [D9-D11]         25           10  [D1] [D12]  2,4 [D1]  [D13] 2,4        [D1]     [D14]  

  [D12]      [D15]           Thin Layer           FT-IR,

13

C-NMR, Mass spectroscopy    Chromatography  

             Pseudomonas  Escherichia coli serial broth dilution  Micrococcus luteusStaphylococcus aureus      ǃ aeruginosa  ‫[ و‬D8] Escherichia coli  MIC [D7]  Staphylococcus aureus       MIC  [D11]  Pseudomonas aeruginosa

      

 ،  4 74

   



2011  

   ١٤٣٨٢٧١٧ 

٢٠١٧

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