Republic of Iraq Kurdistan Regional Government Ministry of Higher Education and Scientific Research University of Sulaimani Faculty of Medical Sciences School of Pharmacy
SYNTHESIS AND CHARACTERIZATION OF NEW COUMARIN DERIVATIVES CONTAINING VARIOUS MOIETIES WITH ANTI BACTERIAL ACTIVITIES
A THESIS SUBMITTED TO THE COUNCIL OF THE SCHOOL OF PHARMACY AT UNIVERSITY OF SULAIMANI IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACEUTICAL CHEMISTRY
By Shokhan Jamal Hamid B.Sc. Pharmacy 2010
Supervised by Dr. Ammar A. Mahmood Kubba PhD Pharmaceutical Chemistry
Acknowledgements First and foremost, thanks to Almighty God for giving me countless blessing and strength to carry out this study. I would like to express my special appreciation and thanks to my supervisor Dr. Ammar A. Kubba for his guidance, support and patience throughout the study. I owe my sincere and heartfelt thanks to my beloved husband Safeen for his encouragement and countless help in every moment of my study. My great appreciation to Dr. Mohammed Nawzad Sabir, the head of department of Pharmacognosy & Pharmaceutical Chemistry for his tremendous help, support and encouragement. Words fail to express my humble gratitude to Dr. Emad Manhal Al-Khafaji in Faculty of Science/ School of Chemistry for his unlimited help and guidance during my study. I am deeply grateful to public health laboratory of Sulaimani health directorate for allowing me to conduct some works in their laboratories. Many thanks to my friends Swara J. Mohammed and Lana S. Sabir for their help during my study. Last but not least, I would like to thank my family and dear friends. Without their love and affection it would not have been possible to complete this project.
Shokhan J. Hamid
I
Abstract Coumarin is one of the six membered oxygen containing heterocyclic compound and a member of benzo pyrone family. Because of their broad spectrum of biological activities, various coumarin derivatives have been synthesized. In this study some new coumarin derivatives have been synthesized by adding the formyl group to carbon 8 of 7-hydroxy-4-methyl coumarin, using Duff reaction. The series of the reactions for the preparation of new coumarin derivatives was as follows:
Synthesis of 7-hydroxy-4-methyl coumarin [S1] by reaction of resorcinol with ethylaceto acetate, using concentrated sulfuric acid at (0-10) ˚C, then preparation of 8-formyl-7-hydroxy-4methyl coumarin [S2] by the reaction of compound [S1] with hexamine in glacial acetic acid at (85-90) ˚C using 20% hydrochloric acid. Schiff bases [S3-S5] were prepared by the reaction of compound [S2] with different aromatic amines in ethanol without using acid or base, while amino acid Schiff bases [S6-S8] were prepared by the reaction of compound [S2] with different amino acids in basic medium using alcoholic sodium hydroxide solution. The chalcone derivatives [S9-S11] were synthesized by aldol condensation reaction in which compound [S2] was reacted with different aromatic ketones at a temperature not exceeding 25 ˚C using 10% sodium hydroxide solution. Hydrazone derivatives of coumarin [S12-S14] were prepared by refluxing compound [S2] with different hydrazines in absolute ethanol. Compound [S15] which is a hydrazinyl thiazol derivative of coumarin, prepared by the reaction of compound [S14] with chloroacetic acid in glacialacetic acid as solvent containing anhydrous sodium acetate. The progress of the reactions and purity of the synthesized compounds were checked by thin layer chromatography. Structure elucidation was confirmed by physical properties and spectrometric analysis using FT-IR, 13C-NMR and Mass spectroscopy. The new coumarin derivatives have been screened for their antibacterial activity by serial broth dilution method against two gram positive bacteria Staphylococcus epidermidis and Staphylococcus hemolyticus and two gram negative bacteria Escherichia coli and Klebsiella II
pneumoniae. All the synthesized compounds have been found to exhibit considerable antibacterial activity in vitro. Among all the derivatives, compound [S9] showed the highest rate of inhibition against Escherichia coli, while compound [S12] showed greatest anti-bacterial activity against Staphylococcus hemolyticus.
III
Table of Contents Subjects
Page
Acknowledgements
I
Abstract
II
Table of Contents
IV
List of Tables
VII
List of Figures
IX
List of Schemes
XII
List of Abbreviations
XIV
Chapter One: Literature Review 1.1: Heterocyclic Compounds
1
1.2: Coumarin
2
1.3: Structure of Coumarin
2
1.4: Occurrence
3
1.5: Synthesis of Coumarin
5
1.5.1: Perkin Reaction
5
1.5.2: Pechmann Reaction
6
1.5.3: Knoevenagel Condensation
7
1.5.4: Wittig Reaction
8
1.5.5: Kostanecki-Robinson Reaction
8
1.5.6: Reformatsky Reaction
9
1.6: Reactions of Coumarin
10
1.6.1: Reaction with Electrophilic Reagents
10
1.6.2: Reaction with Nucleophilic Reagents
11
1.6.3: Photodimerization of Coumarin
11
1.6.4: Reaction with Dienes (Diels-Alder Reaction)
12
1.6.5: Mannich Reaction
13
1.7: Pharmacokinetics of Coumarin
13
1.7.1: Absorption and Distribution
14
IV
1.7.2: Metabolism
14
1.7.3: Excretion of Coumarin
16
1.8: Toxicities of Coumarin
16
1.9: Biological Activity of Simple Coumarins and Analogues
17
1.9.1: Antimicrobial
18
1.9.2: Antiviral
19
1.9.3: Anticancer
19
1.9.4: Enzyme Inhibition
21
1.9.5: Antioxidant
21
1.9.6: Anti-Inflammatory
22
1.9.7: Anticoagulant
22
1.9.8: Antihyperlipidemic Activity
23
1.9.9: Central Nervous System
24
1.10: Aim of the Study
25
Chapter Two: Experimental 2.1: Chemicals
26
2.2: Instruments and Analytical Techniques
29
2.2.1: Melting Point Apparatus
29
2.2.2: Thin Layer Chromatography (TLC)
29
2.2.3: Infrared Spectrometer
29
2.2.4: 13C-NMR Spectrometer
29
2.2.5: Mass Spectrometer
29
2.3: Procedures
30
2.3.1: Synthesis of 7-hydroxy-4-methyl Coumarin [S1]
30
2.3.2: Synthesis of 8-formyl-7-hydroxy-4-methyl Coumarin [S2]
31
2.3.3: Synthesis of Aromatic Amine Schiff Bases [S3-S5]
32
2.3.4: Synthesis of Amino Acid Schiff Bases [S6-S8]
33
2.3.5: Synthesis of Chalcones [S9-S11]
34
2.3.6: Synthesis of 8-(hydrazonomethyl)-7-hydroxy-4-methyl Coumarin [S12]
35
2.3.7: Synthesis of 8-((2-(2,4-dinitrophenyl)hydrazono)methyl)-7-hydroxy-4-methyl V
Coumarin [S13]
36
2.3.8: Synthesis of 2-((7-hydroxy-4-methyl-2-oxo-2H-chromen-8yl)methylene)
37
hydrazine carbothioamide [S14] 2.3.9: Synthesis of 2-(2-((7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl)methylene)
38
hydrazinyl)thiazol-4(5H)-one [S15]
Chapter Three: Results and Discussion Results and Discussion
39
3.1: Synthesis of 7-hydroxy-4-methyl Coumarin [S1]
42
3.2: Synthesis of 8-formyl-7-hydroxy-4-methyl Coumarin [S2]
43
3.3: Synthesis of Aromatic Amine Schiff Bases [S3-S5]
48
3.4: Synthesis of Amino Acid Schiff Bases [S6-S8]
55
3.5: Synthesis of Chalcones [S9-S11]
62
3.6: Synthesis of Hydrazone Derivatives [S12-S14]
70
3.7: Synthesis of 2-(2-((7-hydroxy-4- methyl-2-oxo-2H-chromen-8-yl)methylene)
77
hydrazinyl)thiazol-4(5H)-one [S15] 3.8: Anti-microbial Activity
81
3.8.1: Antibacterial Susceptibility Tests
81
3.8.1.1: Dilution methods
81
3.8.1.2: Disk Diffusion Method
82
Chapter Four: Conclusions and Recommendations 4.1: Conclusions
108
4.2: Recommendations
108
References References
109
VI
List of Tables Table
Title
Page
1-1
4
2-1
The four main coumarin subtypes, the main structural features and examples of each coumarin subtype Name of chemicals and their suppliers
26
2-2
physical properties of compound [S1]
30
2-3
physical properties of compound [S2]
31
2-4
physical properties of compound [S3-S5]
32
2-5
physical properties of compound [S6-S8]
33
2-6
physical properties of compound [S9-S11]
34
2-7
physical properties of compound [S12]
35
2-8
physical properties of compound [S13]
36
2-9
physical properties of compound [S14]
37
2-10
physical properties of compound [S15]
38
3-1
FT-IR spectral data of compound [S1]
43
3-2
FT-IR spectral data of compound [S2]
46
3-3
13
48
3-4
FT-IR spectral data of compound [S3-S5]
54
3-5
13
55
3-6
FT-IR spectral data of compound [S6-S8]
61
3-7
13
62
3-8
FT-IR spectral data of compound [S9-S11]
69
3-9
13
70
C-NMR and MS spectral data of compound [S2]
C-NMR and MS spectral data of compound [S3-S5]
C-NMR and MS spectral data of compound [S6-S8]
C-NMR and MS spectral data of compound [S9-S11]
VII
3-10
FT-IR spectral data of compound [S12-S14]
75
3-11
13
76
3-12
FT-IR spectral data of compound [S15]
79
3-13
13
81
3-14
Anti-bacterial activity of compound [S2-S15]
83
3-15
Mean values of MIC
85
C-NMR and MS spectral data of compound [S12-S14]
C-NMR and MS spectral data of compound [S15]
VIII
List of Figures Figure
Title
Page
1-1
Examples of heterocyclic compounds
1
1-2
3
1-3
Chemical structures of benzopyrone subclasses, with the basic coumarin structure (benzo- α -pyrone) [A], and flavonoid (benzo- γ -pyrone) structure [B] Chemical structure of Novobiocin and Clorobiocin
19
1-4
Natural coumarin geiparvarin
20
1-5
Natural coumarin osthenol
21
1-6
Structures of dicoumarol and warfarin
23
1-7
A-coumarin-indole derivative, B-coumarin chalcone fibrate
23
1-8
Natural coumarin, fraxetin
24
3-1
Anti-bacterial activity of compound [S9] against Escherichia coli
84
3-2
Anti-bacterial activity of compound [S15] against Klebsiella pneumoniae
85
3-3
Disc diffusion method of anti-bacterial susceptibility test
86
3-4
Anti-bacterial activity of some coumarin derivatives
87
3-5
FT-IR spectrum of compound [S1]
88
3-6
FT-IR spectrum of compound [S2]
88
3-7
FT-IR spectrum of compound [S3]
89
3-8
FT-IR spectrum of compound [S4]
89
3-9
FT-IR spectrum of compound [S5]
90
3-10
FT-IR spectrum of compound [S6]
90
3-11
FT-IR spectrum of compound [S7]
91
3-12
FT-IR spectrum of compound [S8]
91
IX
3-13
FT-IR spectrum of compound [S9]
92
3-14
FT-IR spectrum of compound [S10]
92
3-15
FT-IR spectrum of compound [S11]
93
3-16
FT-IR spectrum of compound [S12]
93
3-17
FT-IR spectrum of compound [S13]
94
3-18
FT-IR spectrum of compound [S14]
94
3-19
FT-IR spectrum of compound [S15]
95
3-20
13
96
3-21
13
96
3-22
13
97
3-23
13
97
3-24
13
98
3-25
13
98
3-26
13
99
3-27
13
99
3-28
13
100
3-29
Mass spectrum of compound [S2]
100
3-30
Mass spectrum of compound [S3]
101
3-31
Mass spectrum of compound [S4]
102
3-32
Mass spectrum of compound [S5]
102
3-33
Mass spectrum of compound [S6]
103
3-34
Mass spectrum of compound [S7]
104
3-35
Mass spectrum of compound [S10]
105
C-NMR spectrum of compound [S2] C-NMR spectrum of compound [S3] C-NMR spectrum of compound [S5] C-NMR spectrum of compound [S6] C-NMR spectrum of compound [S7] C-NMR spectrum of compound [S10] C-NMR spectrum of compound [S11] C-NMR spectrum of compound [S14] C-NMR spectrum of compound [S15]
X
3-36
Mass spectrum of compound [S11]
105
3-37
Mass spectrum of compound [S13]
106
3-38
Mass spectrum of compound [S14]
106
3-39
Mass spectrum of compound [S15]
107
XI
List of Schemes Scheme
Title
Page
1-1
Perkin synthesis of coumarin
5
1-2
Pechmann synthesis of coumarin
6
1-3
Knoevenagel condensation
7
1-4
Knoevenagel synthesis of coumarin
7
1-5
Wittig synthesis of coumarin
8
1-6
Kostanecki-Robinson synthesis of coumarin
9
1-7
Reformatsky synthesis of coumarin
10
1-8
Reaction of coumarin with electrophile
10
1-9
Reaction of coumarin with nucleophile
11
1-10
Reaction of coumarin with alkali
11
1-11
Photodimerization of coumarin
12
1-12
Photodimerization of coumarin-3-carboxylic acid in the solid state
12
1-13
Addition of dienes to coumarin
13
1-14
Mannich reaction of coumarin
13
1-15
Metabolism of coumarin
15
3-1
Synthesis of coumarin derivatives [S1 and S2]
39
3-2
Synthesis of coumarin derivatives [S3-S11]
40
3-3
Synthesis of coumarin derivatives [S12-S15]
41
3-4
Synthesis of compound [S1]
42
3-5
Mechanism of synthesis of compound [S1]
42
XII
3-6
Synthesis of compound [S2]
43
3-7
Mechanism of synthesis of compound [S2]
44
3-8
Resonance stability of 7-hydroxy-4-methyl coumarin
45
3-9
Synthesis of compounds [S3-S5]
48
3-10
Mechanism of synthesis of aromatic amine Schiff bases [S3-S5]
49
3-11
Synthesis of compounds [S6-S8]
56
3-12
Mechanism of synthesis of amino acid Schiff bases [S6-S8]
57
3-13
Synthesis of compounds [S9-S11]
63
3-14
Mechanism of synthesis of compounds [S9-S11]
64
3-15
Synthesis of compounds [S12-S14]
71
3-16
Mechanism of synthesis of hydrazone derivatives [S12-S14]
71
3-17
Synthesis of compound [S15]
77
3-18
Mechanism of synthesis of compound [S15]
78
XIII
List of Abbreviations Abbreviations
Meaning
µg
Micro gram
µL
Micro liter
3-D
Type of dopaminergic receptor
5HT1A
5-hydroxy tryptamine receptor
Å
Angstrom
CYPs
Cytochrome P family of metabolizing enzyme
D2A
Type of dopaminergic receptor
D3
Type of dopaminergic receptor
DMSO
Dimethyl sulfoxide
DNA
Deoxyribonucleic acid
EFSA
European Food Safety Authority
FDA
Food and Drug Administration
FT-IR
Fourier transform infrared spectroscopy
FT-NMR
Fourier transform -Nuclear magnetic resonance
G+ve
Gram positive
G-ve
Gram negative
HBV
Hepatitis B virus
HIV
Human immunodeficiency virus
HIV-PR
Human immunodeficiency virus protease
LD50
Lethal dose 50
XIV
LNCaP cells
Androgen-sensitive human prostate adenocarcinoma cells
m/z
Mass to charge ratio
MAO
Monoamine oxidase
MHz
Megahertz
MIC
Minimum inhibitory concentration
MP
Melting point
MS
Mass spectrometry
NIOSH
National Institute for Occupational Safety and Health
NMR
Nuclear magnetic resonance
OSHA
Occupational Safety and Health Administration
PABA
Para amino benzoic acid
ppm (δ)
Part per million
QSAR
Quantitative structure activity relationship
Rf
Retention factor
S1-S15
Codes of the synthesized coumarin derivatives
SAIF
Sophisticated analytical instrument facility
SAR
Structure activity relationship
TDI
Tolerable daily intake
TLC
Thin layer chromatography
XV
Chapter One Literature Review 1.1: Heterocyclic Compounds: A cyclic compound containing all carbon atoms in ring formation is referred to as a carbocyclic compound. If at least one atom other than carbon forms apart of the ring system, it is designated as a heterocyclic compound. That is 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. Since non-carbons are usually considered to have replaced carbon atoms, they are called heteroatoms. The structures may consist of either aromatic or non-aromatic rings. (Figure1-1) shows some examples of heterocyclic compounds (1, 2).
Figure (1-1) Examples of heterocyclic compounds
These compounds generally consist of small (3- and 4- membered) and common (5 to 7 membered) ring system. The aromatic heterocyclic compounds are those which have a heteroatom in the ring and behave in a manner similar to benzene in some of their properties. Furthermore, these compounds obey with the general rule proposed by Hückel. This rule states that aromaticity is obtained in cyclic conjugated and planar systems containing (4n+2) π electrons. The conjugated cyclic rings contain six π-electrons as in benzene. This forms a conjugated molecular orbital system which is thermodynamically more stable than the non-cyclic conjugated system (3).
1
This extra stabilization results in a diminished tendency of the molecule to react by addition but a large tendency to react by substitution in which the aromatic ring remains intact (4). A heterocyclic ring may comprise of three or more atoms which are saturated or unsaturated. Also, the ring may contain more than one heteroatom which may be similar or dissimilar. The chemistry of heterocyclic compounds is as logical as that of alphabetic or aromatic compounds. A large number of heterocyclic compounds are essential to life. Various compounds such as alkaloids, antibiotics, essential amino acids, vitamins, haemoglobin, hormones and a large number of synthetic drugs and dyes contain heterocyclic ring system. Knowledge of heterocyclic chemistry is useful in biosynthesis and drug metabolism as well (2, 5).
1.2: Coumarin: Coumarin is one of six-membered oxygen containing heterocyclic compounds. It is a fragrant organic chemical compound which is a natural substance found in many plants
(6)
. The name
comes from a French term for tonka bean, coumarou, one of the sources from which coumarin was first isolated as a natural product in 1820 (7).
1.3: Structure of Coumarin: Coumarin is classified as a member of the benzopyrone family of compounds which consist of a benzene ring joined to a pyrone ring. The benzopyrones can be subdivided into the benzo-αpyrones to which the coumarins belong and the benzo-γ-pyrones, of which the flavonoids are principal members (Figure 1-2) (8).
2
Figure (1-2) Chemical structures of benzopyrone subclasses with the basic coumarin structure (benzo- α -pyrone) [A], and flavonoid (benzo- γ -pyrone) structure [B]
1.4: Occurrence: The coumarins can be categorized into four sub-types: the simple coumarins, furanocoumarins, pyranocoumarins and the pyrone-substituted coumarins (Table 1-1). The simple coumarins e.g. coumarin, 7-hydroxycoumarin and 6,7-dihydroxycoumarin, are the hydroxylated, alkoxylated and alkylated derivatives of the parent compound coumarin, along with their glycosides. Furanocoumarins consist of a five-membered furan ring attached to the coumarin nucleus, divided into linear or angular types with substituents at one or both of the remaining benzoid positions. Pyranocoumarin members are analogous to the furanocoumarins, but contain a six-membered ring. Coumarins substituted in the pyrone ring include 4hydroxycoumarin (7, 8).
3
Table (1-1) the four main coumarin subtypes, and the main structural features and examples of each coumarin subtype
Classification
Features
Examples
Simple coumarins
Hydroxylated, alkoxylated or alkylated on benzene ring 7-hydroxycoumarin
Furanocoumarins
5-membered furan ring attached to benzene ring. Linear or angular Psoralen
Pyranocoumarins
Angelicin
6-membered pyran ring attached to benzene ring. Linear or angular Seselin
Pyrone-substituted
Substitution on pyrone ring,
coumarins
often at 3-C or 4-C positions
Xanthyletin
Warfarin
4
1.5: Synthesis of Coumarin: The biosynthesis of
coumarin
and cyclization of cinnamic acid
in
plants
is
via hydroxylation, glycolysis,
(9)
. Coumarins chemically can be classically synthesized by the
Perkin, Pechmann or Knoevenagel reactions. Recently, the Wittig, the Kostanecki–Robinson and Reformatsky (9-11). However the two most important methods for the synthesis of coumarin derivatives are Perkin and Pechmann reactions (11).
1.5.1: Perkin Reaction: In 1868, Perkin for the first time reported the synthesis of coumarin (12). In the Perkin reaction coumarin was synthesized by aldol condensation of aromatic salicylaldehyde and acid anhydrides, in the presence of an alkali salt of the acid such as sodium acetate, as shown in (Scheme 1-1) (13, 14).
Scheme (1-1) Perkin synthesis of coumarin
5
1.5.2: Pechmann Reaction: A very valuable method for the synthesis of coumarins is the Pechmann reaction. In general, the coumarins were obtained by condensation of phenols with β-ketoesters, in the presence of acid catalysts. The reaction is often called Pechmann-Duisberg, when acetoacetic esters and derivatives are used (Scheme 1-2) (9, 10).
The reaction is conducted with a strong Brønstedt acid such as Lewis acid; AlCl3. The acid catalyses transesterification as well as keto-enol tautomerisation:
A Michael Addition leads to the formation of the coumarin skeleton. This addition is followed by rearomatisation:
Subsequent acid-induced elimination of water gives the product:
Scheme (1-2) Pechmann synthesis of coumarin 6
1.5.3: Knoevenagel Condensation: Condensation of aldehydes with active methylene compounds, in the presence of ammonia or amines, is a reaction known as Knoevenagel. Usually, the reaction is catalyzed by weak bases or by suitable combinations of amines and carboxylic or Lewis acids under homogeneous conditions. Moreover, when malonic acid and pyridine, with or without traces of pipiridine, are used the reaction is often named as Doebner modification (Scheme 1-3). A large list of coumarins has been synthesized using this method, for instance, coumarin-3-carboxylic acids, amino- and alkyl amino coumarins and 3-acetylcoumarins (15).
Scheme (1-3) Knoevenagel condensation
In the early 1900s, the Knoevenagel reaction emerged as an important synthetic method to synthesize coumarin derivatives which was substituted at the 3-position (Scheme 1-4) (16, 17).
Scheme (1-4) Knoevenagel synthesis of 3-substituted coumarin 7
1.5.4: Wittig Reaction: In the Wittig reaction, the alkene formation occurs from carbonyl compounds and phosphonium ylides, proceeding primarily through betaine and/or oxaphosphetane intermediates. When the ylide is replaced by a phosphine oxide carbanion or by a phosphonate carbanion, the reaction is referred to Horner or Horner-Emmons- Wadsworth, respectively
(15)
. This type of
olefination of ortho-hydroxy carbonyl aromatic compounds, followed by further lactonization, is a well-known method for the preparation of coumarin derivatives (Scheme 1-5) (17).
Scheme (1-5) Wittig synthesis of coumarin
1.5.5: Kostanecki-Robinson Reaction: The formation of coumarins, usually 3- and 4-substituted coumarins, by this reaction proceeds via acylation of ortho-hydroxy aryl ketones with aliphatic acid anhydrides, followed by cyclization (Scheme 1-6) (15).
8
Scheme (1-6) Kostanecki-Robinson synthesis of coumarin
1.5.6: Reformatsky Reaction: Condensation of aldehydes or ketones with organozinc derivatives of α-halo esters to yield βhydroxy esters is known as the Reformatsky reaction. In appropriate reaction conditions, lactonization could occur with the formation of coumarins (Scheme 1-7) (18).
9
Scheme (1-7) Reformatsky synthesis of coumarin
1.6: Reactions of Coumarin: 1.6.1: Reaction with Electrophilic Reagents: Coumarin and its derivatives undergo reactions at the activated C-3,4 double bond, or they undergo electrophilic aromatic substitution on the benzene ring most preferably at C-6 and C-8 (19)
. Chlorosulphonation with chlorosulphonic acid gives coumarinsulphonyl chloride in good
yield (Scheme 1-8) (20).
Scheme (1-8) Reaction of coumarin with electrophile
10
1.6.2: Reaction with Nucleophilic Reagents: Severel kinds of nucleophile may react with coumarin either at C-2 (a) or C-9 (b). Some of these reactions involve ring opening (Scheme 1-9) (21).
Scheme (1-9) Reaction of coumarin with nucleophile
The substituted coumarin was converted into benzofuran-2-carboxylic acid upon heating with alkali at 100 ˚C as outlined in (Scheme 1-10) (20).
Scheme (1-10) Reaction of coumarin with alkali
1.6.3: Photodimerization of Coumarin: It is usually difficult to control the regio- and stereoselective [2+2] photodimerization of coumarins both in solution and in the solid state (Scheme 1-11). For example, the direct photoirradiation of coumarin in benzene afforded a mixture of syn–head–head, anti–head–head, syn–head–tail and anti–head–tail (22, 23).
11
Scheme (1-11) Photodimerization of coumarin
More recently, when monomer pairs of coumarin-3-carboxylic acid are arranged in crystals such that the C=C double bonds are related by an inversion center and separated by 3.632 Å, the [2+2], cycloaddition reaction was achieved upon irradiation in the solid state (Scheme 1-12) (23).
Scheme (1-12) Photodimerization of coumarin-3-carboxylic acid in the solid state
1.6.4: Reaction with Dienes (Diels-Alder Reaction): Numerous atempts to bring about the addition of dienens to coumarin as [2+4] cycloaddition, which would yield directly hydroginated di-benzopyrons were failed under normal conditions. The reaction was finally accomplished in the case of 2,3-dimethyl-1,3-butadiene, but not with
12
butadiene or isoprene, by using a large excess of reagent and a tempeature of 260 ˚C (Scheme 113) (24).
Scheme (1-13) Addition of dienes to coumarin
1.6.5: Mannich Reaction: Mannich
reported
that
4-hydroxy coumarin
reacts
with
secondary amines
and
paraformaldehyde to form substituted 3-methyl amino coumarin (Scheme 1-14) (25).
Scheme (1-14) Mannich Reaction of coumarin
1.7: Pharmacokinetics of Coumarin: The pharmacokinetics of coumarin, including the excretion of various metabolites, was elucidated over many years. Coumarin is rapidly and almost completely metabolised with little unchanged compound excreted (8).
13
1.7.1: Absorption and Distribution: Following oral administration, coumarin is rapidly absorbed from the gastrointestinal tract and is distributed throughout the body
(26)
. Coumarin and 7-hydroxycoumarin are both poorly
soluble in water 0.22 and 0.031 %, respectively. However, both compounds have high partition coefficients 21.5 for coumarin and 10.4 for 7-hydroxycoumarin, which are considered favorable for the rapid absorption of compounds once they are in solution. This coupled with the fact that coumarin is non-polar. It also suggests that theoretically coumarin should cross lipid bilayers easily by passive diffusion (8, 27). Pharmacokinetic studies in humans have demonstrated that coumarin is completely absorbed from the GIT after oral administration, and extensively metabolised by the liver in the first pass with only between 2-6 %, reaching the systemic circulation intact. The low bioavailability of coumarin, in addition to its short half-life (1.02 hrs. per oral and 0.8 hrs. intravenous), has brought into question its importance in vivo. At normal therapeutic plasma concentrations, many drugs exist in the plasma mainly in the bound form. It has been shown that 35% of coumarin and 47% of 7-hydroxycoumarin bind plasma proteins
(28)
. Availability of the compounds at their
target tissues should not be problematic since the proportions that bound are well below the accepted critical value of 80% binding. The pharmacokinetics of coumarin has been studied in a number of species including the rat, dog, rhesus monkey and in man (27).
1.7.2: Metabolism: Traditionally coumarin has been viewed by pharmacologists as the ideal model for studying the complex metabolism of a structurally simple organic molecule. Therefore, its metabolic fate has been extensively studied. Determining the metabolic fate of coumarin is important in order to utilize the fact that it is metabolized at several sites, and to access the possible dependence of coumarin-induced toxicity on metabolism (29). The superfamily of cytochromes P450 (CYPs) consists of microsomal haemoproteins that catalyse the oxidative, peroxidative and reductive metabolism of a wide variety of endogenous and exogenous compounds. The CYP superfamily is divided into families and subfamilies according to their nucleotide sequence homology. Coumarin is metabolized initially by specific cytochrome P-450-linked mono-oxygenase enzyme CYP2A6 system in liver microsomes resulting in hydroxylation to form 7-hydroxycoumarin. After 7-hydroxylation, coumarin 14
undergoes a phase II conjugation reaction resulting in a glucuronide conjugation associated with 7-hydroxycoumarin. The 7-hydroxylase activity is exceptionally high in human liver microsomes compared with its activity in the livers of other animal species. The activity of coumarin 3hydroxylase is very high in rodent microsomes, but is absent in human microsomes. Although coumarin may be metabolised by hydroxylation at all six possible positions (carbon 3, 4, 5, 6, 7, and 8), the most common routes of hydroxylation are at positions 7 and 3 to yield 7hydroxycoumarin and 3- hydroxycoumarin respectively (Scheme 1-15). 7-hydroxylation has received the most attention among the various metabolic steps. This is predominantly because it is the major metabolic route in humans and is easily analyzed (8, 29).
Scheme (1-15) Metabolism of Coumarin
15
1.7.3: Excretion of Coumarin: According to the studies done on the excretion of coumarin, 7-hydroxycoumarin,and 4hydroxycoumarin in chickens by use of the Sperber technique indicates that the chicken excreted these coumarins almost entirely in conjugated forms (30). Both 7-hydroxycoumarin and 4-hydroxycoumarin appeared in the urine only as the corresponding glucuronide. During infusion, 84% of excreted radioactivity was identified as 7hydroxycoumarin -glucuronide with only 8% appearing as a conjugate of open-ring metabolite ohydroxyphenylacetic acid (30, 31).
1.8: Toxicities of Coumarin: Coumarin is moderately toxic to the liver and kidneys with a median lethal dose (LD50) of 275 mg/kg. Coumarin is hepatotoxic in rats but less so in mice. Rodents metabolize it mostly to 3,4-coumarin epoxide, a toxic and unstable compound that on further differential metabolism may cause liver cancer in rats and lung tumors in mice. Humans metabolize it mainly to 7hydroxycoumarin, a compound of lower toxicity (26). The European Food Safety Authority (EFSA) adopted in 2004 an opinion in which it was concluded that coumarin was not genotoxic in experimental animals and therefore, a tolerable daily intake (TDI) could be allocated (32). The German Federal Institute for Risk Assessment has established a tolerable daily intake (TDI) of 0.1 mg coumarin per kg body weight, but also advises that higher intake for a short time is not dangerous. The Occupational Safety and Health Administration (OSHA) of the United States does not classify coumarin as a carcinogen for humans (6, 33). Coumarin
is
found naturally in
currants, apricots, and cherries
many edible plants
such as strawberries, black
(7)
. Coumarin is often found in artificial vanilla substitutes,
despite having been banned as a food additive in numerous countries since the mid-20th century. In 1954 the United State Food and Drug Administration (FDA) classified coumarin as a toxic substance and banned its use in foods, mainly because of the hepatotoxicity results in rodents. Coumarin is currently listed by the Food and Drug Administration (FDA) of the United States among substances generally prohibited from direct addition or use as human food. This
16
move was not copied by their European counterparts until 20 years later. The FDA action was taken based on results from routine toxicity tests which showed that coumarin initiated toxic liver damage in rats. No pathological effects were observed in any other organs (26). Clinically, studies carried out on humans have shown little evidence of liver dysfunction. An idiosyncratic-type hepatotoxicity was observed in a clinical trial of 2163 patients, but with only a 0.37% incidence. In addition, cessation of coumarin therapy returned the elevated liver enzymes to normal levels in patients with this response (34). Coumarin has been cited as a chemical carcinogen by National Institute for Occupational Safety and Health (NIOSH), and has been reported as a probable carcinogen in rodent carcinogenicity bioassays. However, as precaution needs to be taken in extrapolating this information to human situations as these tests were carried out in rodents. Various tests have shown that coumarin and its metabolites are non-mutagenic. Although coumarin can cause chromosome and DNA breaks in plant cells, it offers anti-mutagenic protection in E. coli cells exposed to U.V. radiation or 4-nitro-quinoline-l-oxide
(35)
. In cultured Chinese hamster ovary
cells, neither coumarin nor 7-hydroxycoumarin induces sister chromatid exchange. Coumarin is also found to be non-teratogenic and non-phototoxic (36).
1.9: Biological Activities of Simple Coumarins and Analogues: The coumarins are of great interests due to their biological properties. In particular, their physiological, bacteriostatic and anti-tumor activities make these compounds attractive for further backbone derivatization and screening as novel therapeutic agents (37). Coumarin and its derivatives have antimicrobial, antiviral, anti-inflammatory, antioxidant, anticoagulant and enzyme inhibition activities
(38-44)
. It has been shown that coumarin and its
metabolite 7-hydroxycoumarin have antitumor activity against several human tumor cell lines. In addition, it has been shown that 4-hydroxycoumarin and 7-hydroxycoumarin inhibited cell proliferation in a gastric carcinoma cell line (44).
17
1.9.1: Antimicrobial: Novobiocin and clorobiocin (Figure 1-3) are coumarin antibiotics of natural origin, which are inhibitors of DNA gyrase, and have a broad spectrum towards Gram-positive bacteria, including methicillin resistant strains of staphylococci species. Due to some limitations of these compounds, particularly with regard to solubility, toxicity and development of resistance, a novel series of coumarin analogues has been synthesized (15). Over the past years, several efforts were directed towards the design of effective, orally bioavailable coumarin antibiotic inhibitors of bacterial DNA gyrase. From these studies, a series of coumarin congeners has appeared that bear the coumarin part of the molecule and isosteres, like carboxyl or basic amino groups, in order to improve the antibacterial activity. Molecular modeling has been used in conjunction with crystallographic and biological data. In an attempt to design compounds that mimic the structure and activity of the coumarin antibiotic novobiocin, possessing a better profile. The novobiocin analogues, bearing different amino groups, displayed excellent inhibitory activity against DNA gyrase supercoiling. The introduction of dialkyl substituents at the 5,5´-position of novobiose leads to coumarin analogues with improved in vitro antibacterial activity incorporation of alkyl, alkylamino and aryl substituents afford new analogues of coumarin containing antibiotics with interesting activities. A series of novobiocinlike coumarin carboxylic acids has also been synthesized bearing L-rhamnosyl moiety, instead of L-novobiose, as the sugar portion of the molecule. It was found that when alkyl side-chains at C5 of coumarin were introduced, an enhancement of the in vitro antibacterial properties was observed. Several coumarin derivatives were also tested for their antifungal activity. A free 6OH on the coumarin nucleus was found to be important for its antifungal activity. Also, a free 7OH on the same nucleus, was important for antibacterial activity (39, 41, 42, 45-49).
18
Figure (1-3) Chemical structure of Novobiocin and Clorobiocin
1.9.2: Antiviral: The antiviral activity of simple coumarins focuses essentially on the inhibition of HIV-1 protease (HIV-PR) and HIV-1 integrase. The recent advances in the development of coumarin derivatives as potent anti-HIV agents concerning the discovery, structural modification and structure-activity relationships studies have been topics of different reviews or updated articles. With this goal, the inhibitory activity of various coumarins towards HIV-1 protease has been investigated and classified as a class of drugs of interest as antiviral agents. From this, phenprocoumon, warfarin and substituted 4-hydroxy-2-pyrone derivatives are actually referred to as first generation of HIV-PR inhibitors. It was also found that certain coumarin dimers, particularly those containing hydrophobic moieties, display potent inhibitory activity against HIV-1 integrase. In addition, some coumarin derivatives were tested for their activity against Herpes simplex virus. From these studies, 5,7,4´-trihydroxy-4-styrylcoumarin was found to exhibit a significant antiviral activity. It is worthwhile to note that the natural collinin; greveal has significant anti-HBV, DNA replication activity (50, 51).
1.9.3: Anticancer: Among the coumarins screened for anticancer activity, geiparvarin, (Figure 1-4) was found to be the most representative. Geiparvarin is a natural coumarin based structure, isolated from the leaves of Geijera parviflora, which is known for its significant in vitro cytostatic activity. The compound is constituted of three units: a furan, an unsaturated alkenyloxy substituent and a 19
coumarin moiety. The first mentioned unit is essential for the activity as it could work as an alkylating agent of bionucleophiles through a Michael-type reaction. In that way, geiparvarin became a challenging lead compound, for the synthesis of analogues that could also be promising candidates for anticancer activity. (52).
Figure (1-4) Natural coumarin, geiparvarin
Moreover, the inhibitory effects of several simple coumarins, synthesized or isolated from plants, on cytotoxic activity have also been reported (44, 53-58). The in vitro effects of 4-hydroxycoumarin were studied, employing melanoma and the nonmalignant fibroblastic cell lines. It was proposed that the compound might be a useful adjuvant for melanoma therapy (59-61). Hydroxycoumarins with a nitro group in the aromatic ring have been shown to be selective anti-proliferative agents that mediate apoptosis in renal carcinoma cells, through modulation of mitogen-activated protein kinases. It is important to note that the chemo preventive effects of several natural coumarins have been evaluated (7). The mutagenicity of simple coumarins has also been studied. It was found that the antimutagenicity of this class of compounds was linked to the presence of polar functions at carbons 3, 4, and 7 (47, 62).
20
1.9.4: Enzyme Inhibition: Some natural and synthetic coumarins have been found to be cholinesterase inhibitors, which are considered to be a promising approach for the treatment of Alzheimer´s disease, and for possible therapeutic applications in the treatment of Parkinson’s disease. Steroid 5α-reductase type I inhibitory activities of a series of umbelliferone derivatives were evaluated in cell culture systems. A SAR study was performed in order to elucidate the essential structural requirements for the activity. A natural prenylated coumarin, osthenol was also revealed to be a new potential inhibitor of 5α-reductase type I in LNCaP cells (63).
Figure (1-5) Natural coumarin, osthenol
1.9.5: Antioxidant: Various synthetic simple coumarins with various hydroxyl groups and other substituents were tested for their antioxidant activity namely, in relation to the ability to inhibit lipid peroxidation and to scavenge reactive species, for instance, hydroxyl and superoxide radicals, and hypochlorous acid (44, 64, 65). Several
coumarins
have
shown
beneficial
biochemical
profiles
in
relation
to
pathophysiological processes dependent upon reactive oxygen species. Classic and three dimensional (3-D) QSAR analyses of radical scavengers, structurally based on coumarin, have also been performed. The photodynamic damage prevention done by some hydroxy coumarins was evaluated, and compared with that of p-aminobenzoic acid (PABA) as a model sun screen. The activity could be related to their antioxidant action which could minimize skin photo aging (66-69)
.
21
1.9.6: Anti-Inflammatory: It has been found that several coumarins isolated from plants or of synthetic origin possess significant anti-inflammatory and/or analgesic activities
(40, 70-72)
. In a QSAR study for lead
optimization in the design of coumarins as potent non-steroidal anti-inflammatory agents, it was found that substituents in the coumarin positions C-4 and C-7 contributed to the high activity. New diamino ether coumarin derivatives with anti-inflammatory and antioxidant activity were also synthesized. Lipophilicity and ionization were found to be important physicochemical parameters which correlated with the biological activity (15).
1.9.7: Anticoagulant: Warfarin (Fig. 1-5) is a synthetic derivative of dicoumarol, a 4-hydroxycoumarin derived mycotoxin anticoagulant originally discovered in spoiled sweet clover-based animal feeds. It was initially introduced in 1948 as a pesticide against rats and mice, and is still used for this purpose. In the early 1950s, warfarin was found to be effective and relatively safe for preventing thrombosis and thromboembolism in many disorders. It was approved for use as a medication in 1954, and has remained popular ever since. It is considered, even today, the dominant coumarin anticoagulant, owing to its excellent potency and good pharmacokinetic profile.
Warfarin
blood coagulation by
and
related 4-hydroxycoumarin-containing
inhibiting vitamin
K
epoxide
reductase.
molecules It
is
decrease
an enzyme that
recycles oxidized vitamin K1 to its reduced form, after it has participated in the carboxylation of several blood coagulation proteins, mainly prothrombin and factor VII (73, 74). In the search for platelet aggregation inhibitors, several coumarins, either of natural or synthetic origin has been tested. A number of coumarin compounds possessing anticoagulant activity, like their prototype dicoumarol (Figure 1-5), have been synthesized as potential drugs for the management of myocardial infarction (26, 70).
22
Figure (1-6) Structures of dicoumarol and warfarin
1.9.8: Antihyperlipidemic Activity: Statins constitute the major therapeutic force for treatment of hyperlipidemia. However, these have some side effects like muscle fatigue, type 2 diabetes, liver damage and digestive problems. Naturally derived coumarins such as esculetin inhibit oxidative low density lipoproteins and umbelliferone possess good lipid lowering potential. Many synthetic derivatives of coumarins have also emerged as good leads (75). Hybridization of coumarin with an indole moiety (Figure 1-6), which is present in various synthetic statins like fluvastatin has yielded coumarin–indole hybrids which at the dose of 10 mg/kg significantly decrease the plasma triglycerides and total cholesterol by 55% and 20%, respectively. Coumarin-chalcone fibrates (Figure 1-6), has also been synthesized which inhibit the biosynthesis of cholesterol and enhances the activity of lipolytic enzyme, lipoprotein lipase, to facilitate early clearance of lipids from circulation in triton induced hyperlipidemia (66).
Figure (1-7) A-coumarin-indole derivative
B-coumarin chalcone fibrate
23
1.9.9: Central Nervous System: Phenylpiperazine coumarin derivatives were synthesized, and the affinities of these compounds for dopaminergic (D2A, D3) and serotonergic (5HT1A) receptors were evaluated. The results obtained confirm the importance of the N-arylpiperazine fragment in the modulation of this type of activity. SAR studies on antipsychotic compounds were also performed, in which several 4-cyclohexyl hydroxy coumarins were engaged. The potential neuro-protective effects of the natural coumarin; fraxetin (Figure 1-7) on the signalling pathways using a neuronal cell model of Parkinson´s disease were also studied (26, 50, 76).
Figure (1-8) Natural coumarin, fraxetin
24
1.10: Aim of the Study: Coumarin and its derivatives are important organic compounds that have significant biological activities. On the bases of earlier reviews that proved this fact, we synthesized some new (Schiff bases, chalcones, hydrazones and cyclized thiosemicarbazone) derivatives at C-8 of coumarin nucleus, and evaluated their anti-bacterial activity by serial broth dilution method.
25
Chapter Two 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) Name of chemicals and their suppliers Chemicals
Structure
Supplier
2,4-dinitro phenyl hydrazine
Alpha Chemika/ India
2-hydroxy acetophenone
Sigma-Aldrich
4-nitro aniline
Sigma-Aldrich
7-hydroxy-4-methyl coumarin
Sigma-Aldrich
Absolute ethanol
J.T.Baker/ Netherland
Acetophenone
Sigma-Aldrich
Alanine
Merck
Anhydrous calcium chloride
Ca-Cl2 26
Alpha Chemika/ India
Anhydrous sodium acetate
Scharlau/ Spain
Aniline
AVONCHEM/ UK
Chloroacetic acid
Sigma-Aldrich
Chloroform
Merck
Dehydroacetic acid
Sigma-Aldrich
Diethyl ether
Alpha Chemika/ India
Ethyl acetate
Merck
Ethyl acetoacetate
Merck
Glacial acetic acid
AVONCHEM/ UK
Glycine
Merck
Hexamethylene tetramine
Sigma-Aldrich
(Hexamine)
Hydrazine hydrate 99%
NH2-NH2.H2O
Sigma-Aldrich
Hydrochloric acid
Alpha Chemika/ India
Methanol
Alpha Chemika/ India 27
Para amino benzoic acid
Sigma-Aldrich
Phenyl alanine
Sigma-Aldrich
Resorcinol
Merck
Sodium hydroxide
NaOH
Fluka
Sulphuric acid
Alpha Chemika/ India
Thiosemicarbazide
Sigma-Aldrich
Note: The solvents used during the work were purified by distillation.
28
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 reaction were determined using Thin Layer Chromatography on aluminum silica gel 60 florescent plate254 nm (Fluka) detected by UV light (302 nm).
2.2.3: Infrared Spectrometry Infrared spectra were recorded as KBr disk using Perkin-Elmer FT-IR spectrometer in the Faculty of Science/ School of Chemistry at University of Sulaimani.
2.2.4: 13C-NMR Spectrometry The
13
C-NMR spectra were recorded on Bruker FT-NMR spectrophotometer-500 MHz in
SAIF (Sophisticated Analytical Instrument Facility) research center in India/ Chennai, using deuterated Dimethyl sulfoxide (DMSO-d6) and deuterated Chloroform (CDCl3) as a solvent.
2.2.5: Mass Spectrometry Mass spectra were done at: -
Central research laboratory of Tehran/ Iran using Agilent Technology (HP), GC/MS model 5973 network mass selective detector.
-
Genetic and research center at University of Koya using Waters 2998 HPLC/Mass photodiode array detector.
29
2.3: Procedures: 2.3.1: Synthesis of 7-hydroxy-4-methyl Coumarin [S1] (77):
A solution of (0.01mole, 1.1 gm) resorcinol and (0.01mole, 1.3 ml) Ethyl acetoacetate was added drop wise over a period of 30 min. with stirring to (10ml) of conc. sulfuric acid in the ice bath so that the temperature of the mixture did not rise above 10 ˚C. The reaction mixture was kept at room temperature for 3 hours, and then poured with vigorous stirring into mixture of ice and water. The precipitate was filtered off and washed with water. After drying recrystallized from ethanol and creamy colored needles were obtained. The physical properties of compound [S1] are listed in table (2-2).
Table (2-2) physical properties of compound [S1]
Comp.
MP
Yield %
No.
Recrystallization
Color
solvent
TLC Solvent system
Molecular Rf
formula
value
S1
181-183
78
Ethanol
creamy
˚C
Ethylacetate:chloroform
0.68
1:9
One spot
30
C10H8O3
2.3.2: Synthesis of 8-formyl-7-hydroxy-4-methyl Coumarin [S2] (78-80):
A mixture of 7-hydroxy-4-methyl-coumarin (0.025 mole, 5.1 g) and hexamethylene tetramine (0.07 mole, 9.8 g) in glacialacetic acid (40 mL) was heated at 85-90 ˚C on water bath for 7 hr. The hexamine adduct so formed was hydrolyzed with 20% HCl (75 mL) and the mixture was heated for another 30 min. After cooling, the reaction mixture was extracted with diethyl ether (50 mL) twice. The ether layer was evaporated by using rotary evaporator and the pale yellow colored crystals obtained which were recrystallized from ethanol. The physical properties of compound [S2] are listed in table (2-3).
Table (2-3) physical properties of compound [S2]
Comp.
MP
Yield %
No.
S2
177-179
22
Recrystalliza-
Molecular
tion solvent
formula
Ethanol
C11H8O4
˚C
TLC Solvent
Rf
reagent
system
value
test
Ethylacetate:
0.727
+ve
chloroform
One
1:9
31
Tollens
spot
2.3.3: Synthesis of Aromatic Amine Schiff Bases [S3-S5] (81-83):
To a solution of (0.005) mole compound [S2] in (10 ml) absolute ethanol, appropriate aromatic amine [(0.01) mole of aniline and (0.005) mole of 4-amino benzoic acid and 4-nitro aniline] was added with continuous stirring and refluxed for (30 min). The orange colored precipitate was formed which was filtered, dried and recrystallized from ethanol. The physical properties of compound [S3-S5] are listed in table (2-4).
Table (2-4) physical properties of compound [S3-S5] Comp.
MP
No S3
168-171
Yield
Recrystalliza-
Molecular
%
tion solvent
formula
91
Ethanol
C17H13NO3
˚C
Solvent
Rf
system
value
Ethylacetate:
0.645
chloroform
one
1:9
S4
241-245
89
Ethanol
C18H13NO5
˚C
262-266
90
Ethanol
C17H12N2O5
˚C
0.82
chloroform
one
32
Bright orange
spot
Ethylacetate:
0.672
chloroform
one
1:9
Orange
spot
Ethylacetate:
1:1
S5
Color
TLC
spot
Intense orange
Rgroup
2.3.4: Synthesis of Amino Acid Schiff Bases [S6-S8] (84-86):
To a homogenous solution of (0.01 mole) amino acid (glycine, alanine and phenyl alanine) and (0.01 mole) sodium hydroxide in (20 ml) absolute ethanol; (0.01 mole) of compound [S2] which was also dissolved in absolute ethanol, was added drop by drop with continuous stirring. After 2-3 minute of the addition, 20% of the mixture was evaporated and (1 ml) of acetic acid was added. The mixture was left at room temperature for 2-3 hr. the obtained precipitate was collected by filtration, washed with cold ethanol, dried and recrystallized from suitable solvent. The physical properties of compound [S6-S8] are listed in table (2-5).
Table (2-5) physical properties of compound [S6-S8] Comp. No.
S6
MP
200-209
Yield %
72
˚C (Dec.) S7
148-151
231-237 ˚C (Dec.)
Molecular
solvent
formula
Ethanol: Water
C13H11NO5
7:3 67
˚C S8
Recrystallization
Ethanol: Water
Color
Faint yellow
C14H13NO5
yellow
C20H17NO5
Intense
7:3 53
Ethanol: Water 7:3
33
yellow
R group
2.3.5: Synthesis of Chalcones [S9-S11] (56, 87-89):
A mixture of compound [S2] (0.01 mole) and appropriate aromatic ketones [0.02 mole acetophenone, 2-hydroxy acetophenone and 0.01 mole dehydoacetic acid] was dissolved in (25 ml) absolute ethanol in a 100 ml round-bottom flask equipped with a magnetic stirrer. Then sodium hydroxide solution (10 ml, 10 %) was added drop wise to the reaction mixture on vigorous stirring for 30 minutes when the solution became turbid. The reaction temperature was maintained between 20-25 ˚C using a cold water bath on the magnetic stirrer. After vigorous stirring for 4-5 hours, the reaction mixture was left to stand for overnight at refrigerator. The precipitate obtained was filtered, washed with ice cold water, and recrystallized from suitable solvent. The physical properties of compound [S9-S11] are listed in table (2-6).
Table (2-6) physical properties of compound [S9-S11] Comp.
MP
Yield %
No. S9
170-173
Recrystallization
Molecular
solvent
formula
Color
70
Ethyl acetate
C19H14O4
Chartreuse
64
Ethyl acetate
C19H14O5
Chartreuse
36
Ethyl acetate
C19H14O7
Chartreuse
˚C S10
187-189 ˚C
S11
207-210 ˚C
34
R group
2.3.6: Synthesis of 8-(hydrazonomethyl)-7-hydroxy-4-methyl Coumarin [S12] (82) :
(0.005 mole) of compound [S2] dissolved in minimum amount of ethanol to which (0.01 mole) of 99% hydrazine hydrate was added with continuous stirring and refluxed for (30 min). A creamy colored precipitate formed which was collected by filtration and recrystallized from ethanol. The physical properties of compound [S12] are listed in table (2-7).
Table (2-7) physical properties of compound [S12]
Comp.
MP
Yield %
No. S12
201-207
91
Recrystalliza-
Molecular
tion solvent
formula
Solvent system
Rf value
Ethanol
C11H10N2O3
Ethylacetate:chloroform
0.75
1:1
one
˚C (Dec.)
TLC
spot
35
2.3.7: Synthesis of 8-((2-(2,4-dinitrophenyl)hydrazono)methyl)-7-hydroxy-4methyl Coumarin [S13] (90):
(0.005 mole) of compound [S2] and (0.005 mole) 2,4-dinitro phenyl hydrazine was refluxed in round bottom flask for 1 hr. in the presence of (20 ml) absolute ethanol as a solvent, the intense orange colored precipitate collected by filtration and recrystallized from suitable solvent. The physical properties of compound [S13] are listed in table (2-8).
Table (2-8) physical properties of compound [S13]
Comp.
MP
Yield %
No. S13
234-241
88
Recrystalliza-
Molecular
tion solvent
formula
Solvent system
Rf value
Ethanol
C17H12N4O7
Ethylacetate:chloroform
0.806
1:9
one
˚C (Dec.)
TLC
spot
36
2.3.8: Synthesis of 2-((7-hydroxy-4-methyl-2-oxo-2H-chromen-8 yl)methylene) hydrazinecarbothioamide [S14] (58, 91):
(0.005 mole) of compound [S2] and (0.005 mole) Thiosemicarbazide dissolved in (20 ml) absolute ethanol and mixed in round bottom flask with continuous stirring and refluxed for (30 min). A faint yellow colored precipitate was formed which was collected by filtration and recrystallized from ethanol. The physical properties of compound [S14] are listed in table (2-9).
Table (2-9) physical properties of compound [S14]
Comp.
MP
Yield %
No. S14
218-227
93
Recrystalliza-
Molecular
tion solvent
formula
Solvent system
Rf value
Ethanol
C12H11N3O3S
Ethylacetate:chloroform
0.503
1:9
one
˚C (Dec.)
TLC
spot
37
2.3.9: Synthesis of 2-(2-((7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl) methylene)hydrazinyl)thiazol-4(5H)-one [S15] (59, 92):
A mixture of compound [S14] (0.01 mole) and chloroacetic acid (0.01 mole) in glacial acetic acid (30 mL) containing anhydrous sodium acetate (0.04 moles) was refluxed for 17 hr. (monitored by TLC). The reaction mixture was cooled; the resulting pale yellow colored precipitate was filtered off and recrystallized from ethanol. The physical properties of compound [S15] are listed in table (2-10).
Table (2-10) physical properties of compound [S15]
Comp.
MP
Yield %
No. S15
221-234
83
Recrystalliza-
Molecular
tion solvent
formula
Solvent system
Rf value
Ethanol
C14H11N3O4S
Ethylacetate:chloroform
0.708
1:9
one
˚C (Dec.)
TLC
spot
38
Chapter Three Results and Discussion Coumarin and its derivatives are very important organic compounds possessing a wide spectrum of biological activity. They are the structural unit of several natural products. Their applications range from pharmaceuticals, optical brighteners, food additives, perfumes, cosmetics to laser dyes. Many of these compounds have proven to be active as antibacterial, antifungal, anti-inflammatory, anticoagulant, anti-HIV and antitumor agents. These properties have made coumarin interesting target for pharmaceutical chemists. 7-hydroxy-4-methyl coumarin is chosen as a starting material for the synthesis of different derivatives. The prepared derivatives include Schiff bases, chalcones, and hydrazones. All the synthesized compounds were identified by their physical properties and some spectral analysis such as FT-IR,
13
C-NMR, and MS spectroscopy and evaluated for their anti-microbial
properties. The reaction sequence of synthesized coumarin derivatives are outlined in scheme (3-1, 3-2 and 3-3) respectively.
Scheme (3-1) Synthesis of coumarin derivatives [S1 and S2] 39
Scheme (3-2) Synthesis of coumarin derivatives [S3-S11]
40
Scheme (3-3) Synthesis of coumarin derivatives [S12-S15]
41
3.1: Synthesis of 7-hydroxy-4-methyl Coumarin [S1]: Synthesis of compound [S1], scheme (3-4), was achieved by Pechmann-Duisberg reaction of ethyl acetoacetate with equimolar amount of substituted phenol; resorcinol.
Scheme (3-4) synthesis of compound [S1]
The reaction mechanism of resorcinol with ethyl acetoacetate which proceeds through electrophilic aromatic substitution using concentrated sulfuric acid to remove water and ethanol is outlined in scheme (3-5) below (10).
Scheme (3-5) mechanism of synthesis of compound [S1] The physical properties of compound [S1] are listed in table (2-2).
42
The FT-IR spectrum of compound [S1], figure (3-5), shows the O-H stretching frequency at (3155) cm-1, C=C stretching of alkene at (1678) cm-1, C=C stretching of aromatic ring at (1599) cm-1. Other significant bands were observed at (1238, 1076) that may be assigned to the C-O-C stretching frequencies. The characteristic bands of compound [S1] are listed in table (3-1).
Table (3-1) FT-IR spectral data of compound [S1] Characteristic bands of FT-IR (cm-1, KBr)
Comp. No.
[S1]
ʋ O-H
3155
ʋ C=C
ʋ C=C
alkene
aromatic
1678
1599
Others
-C-O-C (1238, 1076) - O-H bending or C-H bending of CH3 at 1450
3.2: Synthesis of 8-formyl-7-hydroxy-4-methyl Coumarin [S2]: Synthesis of compound [S2], scheme (3-6), was achieved by the Duff reaction or hexamine aromatic formylation, which is used for the synthesis of aromatic aldehyde with hexamine as the formyl carbon source, in the presence of acid (glacialacetic acid) at 85-90 ˚C, the reaction was monitored by TLC using chloroform and ethyl acetate (9:1) solvent system.
Scheme (3-6) synthesis of compound [S2]
43
The electrophilic species in this electrophilic aromatic substitution reaction is iminium ion (CH2+NR2). The initial reaction product is an iminium which is hydrolyzed to the aldehyde as shown in scheme (3-7) below (78, 79).
Scheme (3-7) mechanism of synthesis of compound [S2] 44
The reaction mechanism demonstrates how hexamine donates a methine group to an aromatic substrate via a series of equilibria reactions, with iminium ion intermediates. Initially, addition to the aromatic ring results in an intermediate at the oxidation state of a benzylamine. An intramolecular redox reaction ensues, raising the benzylic carbon to the oxidation state of an aldehyde. The oxygen atom is kindly provided by water on acid hydrolysis in the final step (93). The reaction requires strongly electron donating substituents on the aromatic ring such as in a phenol. Formylation occurs ortho to the electron donating substituent preferentially, unless the ortho positions are blocked, in which the formylation occurs in the para position (93). In case of 7-hydroxy-4-methyl coumarin formylation occur at C8 rather than C6 due to the higher resonance stability of C8 than C6 as outlined in scheme (3-8) below (94-96).
Scheme (3-8) resonance stability of 7-hydroxy-4-methyl coumarin The physical properties of compound [S2] are listed in table (2-3). 45
The FT-IR spectrum of compound [S2], figure (3-6), shows the O-H stretching frequency at (3437) cm-1, C=O stretching of ester (lactone) at (1745) cm-1, and appearance of a new band at (1644) cm-1 which is C=O stretching frequency of aldehyde with reduced frequency due to the conjugation or inter-molecular hydrogen bonding, C=C stretching of aromatic ring at (1594 and 1479) cm-1. The characteristic FT-IR bands of compound [S2] are listed in table (3-2).
Table (3-2) FT-IR spectral data of compound [S2] Characteristic bands of FT-IR (cm-1, KBr)
Comp. No.
ʋ O-H
ʋ C-H
ʋ C=O
Stretching [S2]
3437
3080 (aromatic)
2930, 2990 (aliphatic)
ʋ C=C
Others
Aromatic 1745 (lactone)
1644 (aldehyde)
1594
- OH bending at 1430
1479
-C-H bending of CH3 at 1390 -C-O-C (1228, 1072)
The 13C-NMR (ppm) spectrum of compound [S2], which is recorded in CDCl3, figure (3-20) shows the following bands of carbon: 193.399 (C=O aldehyde), 165.266 (C=O ester or lactone), 159.224, 156.14, 152.708, 132.928, 114.3, 112.055, 111.975, and 108.663 (C 18.963 (CH3). The 13C-NMR peaks of compound [S2] are listed in table (3-3).
46
aromatic and alkene),
The mass spectrum of compound [S2], figure (3-29), shows an intense molecular ion peak at m/z 204 corresponding to the molecular formula C11H8O4. The other significant bands at m/z 176, 175, 147, 143, 130 and other fragments are listed in table (3-3).
The above data are also in agreement with those published in the following research papers (79, 80, 93) .
47
Table (3-3) 13C-NMR and MS spectral data of compound [S2] C-NMR, ppm (δ)
13
Comp.
Mass spectroscopy data
No.
C=O
C aromatic and alkene
C aliphatic
[S2]
193.399
159.224, 156.14, 152.708, 132.928, 114.3, 112.055, 111.975, 108.663
18.963
204 [M] +, 176, 175, 147, 143,
(CH3)
130.
(aldehyde) 165.266
m/z
(lactone)
3.3: Synthesis of Aromatic Amine Schiff Bases [S3-S5]: The Schiff bases of aromatic amine [S3-S5], scheme (3-9), have been synthesized in good yield by condensation of compound [S2] with appropriate aromatic amines in absolute ethanol as a solvent without addition of any reagents like acid or base. The completion of the reaction and purity of the synthesized compounds were monitored by TLC.
Scheme (3-9) synthesis of compounds [S3-S5] The mechanism of reaction, as outlined in scheme (3-10), proceeds via nucleophilic attack of the lone pair of electron of amine nitrogen on the carbonyl carbon of the aldehyde with the loss of a water molecule by the application of heat (97). 48
Scheme (3-10) mechanism of synthesis of aromatic amine Schiff bases [S3-S5]
The physical properties of compound [S3-S5] are listed in table (2-4). The FT-IR spectrum of compound [S3], figure (3-7), shows a broad band of O-H stretching frequency at (3450) cm-1, C-H stretching of aromatic ring or alkene at (3080) cm-1, C-H stretching aliphatic for CH3 at (2923 and 2860) cm-1, C=O stretching of ester (lactone) at (1736) cm-1. The IR spectra also show the disappearance of C=O stretching of aldehyde at (1644) cm -1 and appearance of a new band at (1618) cm-1 which is according to the Hooke’s law (discussed below) it belongs to the C=N stretching of Schiff base. Other bands at (1577 and 1488) cm -1 belong to C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S3] are listed in table (3-4).
49
Hooke’s law which can be used to estimate the wave number of light that will be absorbed by different types of chemical bonds states that:
Ʋ bar = 1/2π * (K/μ)1/2 Where: K= force constant in (dynes/ cm), K for double bond = 10 * 105 dynes/cm μ = M1M2/ (M1+M2) (M1 and M2 are molar masses of atoms involved in the bond) According to the Hooke’s law C=N stretching frequency is nearly equal to 1627 cm-1. The 13C-NMR (ppm) spectrum of compound [S3], which is recorded in CDCl3, figure (3-21), shows the following bands of carbon: 166.877 (C=O lactone), 160.29 (C=N), 156.451, 154.55, 153.292, 146.413, 129.581, 129.317, 127.726, 121.292, 115.066, 110.985, 110.934, and 106.859 (C aromatic and alkene), 18.973 (CH3). 13C-NMR peaks of compound [S3] are listed in table (3-5).
The mass spectrum of compound [S3], figure (3-30), shows an intense molecular ion peak (M+1) at 280 corresponding to the molecular formula C17H13NO3 +1 or (M+H), and other band at m/z 202, results are listed in table (3-5).
50
The FT-IR spectrum of compound [S4], figure (3-8), shows a broad band of O-H stretching frequency at (3420) cm-1 which either belongs to the phenol group of coumarin or carboxylic acid group of the aromatic amine, C-H stretching of aromatic ring or alkene at (3070 and 3075) cm-1, C-H stretching for CH3 (aliphatic) at (2982 and 2926) cm-1, the dimer sign of carboxylic acid has appeared at nearly 2500 cm-1, C=O stretching of ester (lactone) at (1723) cm-1, C=O stretching of carboxylic acid at (1700) cm-1, C=C stretching of alkene at (1633) cm-1, C=N stretching frequency of Schiff base at (1616) cm-1. Other bands such as (1578 and 1489) are C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S4] are listed in table (3-4).
The mass spectrum of compound [S4], figure (3-31), shows an intense molecular ion peak at m/z 323 corresponding to the molecular formula C18H13NO5 . Significant bands of m/z 295, 278, 202, 175 and other fragments are listed in table (3-5).
51
The FT-IR spectrum of compound [S5], figure (3-9), shows a broad band of O-H stretching frequency at (3460) cm-1, C-H stretching of aromatic ring or alkene at (3120 and 3090) cm-1, CH stretching for CH3 (aliphatic) at (2920) cm-1, C=O stretching of ester (lactone) at (1744) cm-1, C=C stretching of alkene at (1630) cm-1, C=N stretching frequency of Schiff base at (1615) cm-1. Other bands such as (1570 and 1339) are NO2 stretching of the p-nitro group attached to the aromatic ring. The characteristic FT-IR bands of compound [S5] are listed in table (3-4). The 13C-NMR (ppm) spectrum of compound [S5], which is recorded in CDCl3, figure (3-22) shows the following bands of carbon: 165.368 (C=O lactone), 165.305 (C=N), 159.857, 154.662, 153.177, 152.99, 146.52, 132.913, 126.346, 125.277, 122.217, 114.536, 114.321, 113.355, and 112.049 (C
aromatic and alkene),
18.977 (CH3). The
13
table (3-5). 52
C-NMR peaks of compound [S5] are listed in
The mass spectrum of compound [S5], figure (3-32), shows the molecular ion peak at m/z 324 corresponding to the molecular formula C17H12N2O5 and other significant band at m/z 292; results are listed in table (3-5).
53
Table (3-4) FT-IR spectral data of compound [S3-S5] Characteristic bands of FT-IR (cm-1, KBr)
Comp. No.
[S3]
ʋ O-H
3450
ʋ C-H Stretching
3080(aromatic)
ʋ C=O
1736 (lactone)
ʋ
ʋ C=C
C=N
Aromatic
1618
1577 1488
2923& 2860
Others
- O-H bending or C-H bending of CH3 at 1388
(aliphatic) -C-O-C (1236, 1074) [S4]
3420
3070&3075
1723 (lactone)
1616
1578 1489
(aromatic)
O-H bending of carboxylic acid at 1427
1700 2982&2926
(carboxylic
O-H bending of phenol or C-H bending of CH3 at 1394
acid)
(aliphatic)
C=C alkene at 1633
-C-O-C at (1289,1077) [S5]
3460
3120&3090
1744 (lactone)
(aromatic) 2920 (aliphatic)
1615
1516
1570&1339 (NO2 stretching) O-H bending at 1390 -C-O-C at (1220,1074)
54
Table (3-5) 13C-NMR and MS spectral data of compound [S3-S5] C-NMR, ppm (δ)
13
Comp.
Mass spectroscopy
No.
C=O
C=N
C aromatic and alkene
C aliphatic
[S3]
166.877
160.29
156.451, 154.55, 153.292, 146.413, 129.581, 129.317, 127.726, 121.292, 115.066, 110.985, 110.934, 106.859.
18.973 (CH3)
280 [M+1]+ , 202.
323 [M]+, 295, 278, 202, 175.
(lactone)
[S4]
-
-
-
-
[S5]
165.368
165.305
159.857, 154.662, 153.177, 152.99, 146.52, 132.913, 126.346, 125.277, 122.217, 114.536, 114.321, 113.355, 112.049.
18.977
(lactone)
(CH3)
m/z
324 [M]+.
3.4: Synthesis of Amino Acid Schiff Bases [S6-S8]: The Schiff bases of amino acid [S6-S8], scheme (3-11), have been synthesized by reaction of compound [S2] with appropriate amino acids in absolute ethanol as a solvent by using sodium hydroxide, (NaOH) and acid, (glacial acetic acid).
55
Scheme (3-11) synthesis of compounds [S6-S8] Sodium hydroxide is used to make the NH2 group of the amino acid free since it is in resonance with the carboxylic acid group continuously. NaOH is also used to convert the carboxylic acid group of the amino acid to its sodium salt.
The mechanism of reaction as outlined in scheme (3-12), proceeds via nucleophilic attack of the lone pair of electron of amine nitrogen on the carbonyl carbon of the aldehyde with the loss of a water molecule by the use of acid; glacial acetic acid. The acid is also used to convert back the sodium salt of amino acid to carboxylic acid (86).
56
Scheme (3-12) mechanism of synthesis of amino acid Schiff bases [S6-S8] The physical properties of compound [S6-S8] are listed in table (2-5). The FT-IR spectrum of compound [S6], figure (3-10), shows a broad band of O-H stretching frequency at (3421) cm-1 which either belongs to the phenol group of coumarin or carboxylic acid group of the amino acid (glycine), C-H stretching of aromatic ring or alkene at (3070 and 3080) cm-1, C-H stretching for CH3 (aliphatic) at (2910) cm-1, C=O stretching of ester (lactone) 57
at (1724) cm-1 with reduced frequency due to the conjugation or hydrogen bonding, C=O stretching of carboxylic acid at (1650) cm-1 with reduced frequency, which might be due to intermolecular hydrogen bonding, C=N stretching frequency of Schiff base at (1582) cm-1 also with reduced frequency. Other bands such as (1531 and 1502) cm-1 are C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S6] are listed in table (3-6). The 13C-NMR (ppm) spectrum of compound [S6], which is recorded in DMSO-d6, figure (323) shows the following bands of carbon: 178.862 (C=O carboxylic acid), 172.416 (C=O lactone), 162.799 (C=N), 159.531, 157.179, 154.923, 132.077, 120.607, 106.335, 106.211, and 102.765 (C
aromatic and alkene),
53.51 (CH2), 18.147and 18.088 (CH3). The
13
C-NMR peaks of
compound [S6] are listed in table (3-7).
The mass spectrum of compound [S6], figure (3-33), shows the molecular ion peak (M+1) at 262 corresponding to the molecular formula C13H11NO5 +1 or (M+H), and other band at m/z 216 for (M- COOH). Results are listed in table (3-7).
58
The FT-IR spectrum of compound [S7], figure (3-11), shows a broad band of O-H stretching frequency at (3430) cm-1 which either belongs to the phenol group of coumarin or carboxylic acid group of the amino acid (alanine), C-H stretching of aromatic ring or alkene at (3002) cm-1, C-H stretching for CH3 (aliphatic) at (2910) cm-1, C=O stretching of (lactone) at (1712) cm-1 with reduced frequency due to the conjugation or hydrogen bonding, C=O stretching of carboxylic acid at (1642) cm-1 with reduced frequency, which might be due to intermolecular hydrogen bonding, C=N stretching frequency of Schiff base at (1581) cm-1 also with reduced frequency. Other bands such as (1520 and 1508) cm-1 are C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S7] are listed in table (3-6). The 13C-NMR (ppm) spectrum of compound [S7], which is recorded in DMSO-d6, figure (324) shows the following bands of carbon: 178.791 (C=O carboxylic acid), 175.869 (C=O lactone), 162.842 (C=N), 157.85, 157.19, 154.967, 132.074, 120.646, 106.392, 106.303, and 102.806 (C
aromatic and alkene),
50.506 (CH), 18.224 (CH3 attached to aromatic ring), 16.154 (CH3).
The 13C-NMR peaks of compound [S7] are listed in table (3-7).
The mass spectrum of compound [S7], figure (3-34), shows the molecular ion peak (M+1) at 276 corresponding to the molecular formula C14H13NO5 +1 or (M+H), and other band at m/z 230 for (M- COOH). Results are listed in table (3-7).
59
The FT-IR spectrum of compound [S8], figure (3-12), shows a broad band of O-H stretching frequency at (3434) cm-1 which either belongs to the phenol group of coumarin or carboxylic acid group of the amino acid (phenyl alanine), C-H stretching of aromatic ring or alkene at (3080 and 3085) cm-1, C-H stretching for CH3 (aliphatic) at (2930) cm-1, C=O stretching of ester (lactone) at (1716) cm-1 with reduced frequency, due to the conjugation or hydrogen bonding, C=O stretching of carboxylic acid at (1630) cm-1 with reduced frequency which might be due to intermolecular hydrogen bonding
(84)
, C=N stretching frequency of Schiff base at (1583) cm-1
also with reduced frequency. Other band such as (1503) cm-1 is C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S8] are listed in table (3-6).
60
Table (3-6) FT-IR spectral data of compound [S6-S8] Characteristic bands of FT-IR (cm-1, KBr)
Comp. No.
[S6]
ʋ O-H
3421
ʋ C-H Stretching
ʋ C=O
3080&3070
1724 (lactone)
(aromatic)
1650 (carboxylic acid)
2910(aliphatic)
ʋ
ʋ C=C
C=N
Aromatic
1582
1531 1502
Others
- O-H bending or C-H bending of CH3 at 1413 -C-O-C (1224, 1064)
[S7]
3430
3002 (aromatic)
1712 (lactone)
1581
1520 1508
2910 (aliphatic)
1642 -C-O-C at
(carboxylic
(1230,1050)
acid) [S8]
3434
3080&3085
1716 (lactone)
(carboxylic
O-H bending or C-H bending of CH3 at 1412
acid)
-C-O-C at
(aromatic) 2930 (aliphatic)
O-H bending or C-H bending of CH3 at 1412
1630
1583
1503
(1220,1073)
61
Table (3-7) 13C-NMR and MS spectral data of compound [S6-S8] C-NMR, ppm (δ)
13
Comp.
Mass spectroscopy
No.
C=O
C=N
C aromatic and alkene
C aliphatic
[S6]
178.862
162.799
159.531, 157.179, 154.923, 132.077, 120.607, 106.335, 106.211, 102.765.
53.51
(carboxy lic acid)
[S7]
18.147& 18.088
(lactone)
(CH3) 162.842
(carboxy lic acid)
[S8]
(CH2) C-N 262 [M+1]+, 216.
172.416
178.791
157.85, 157.19, 154.967, 132.074, 120.646, 106.392, 106.303, 102.806.
50.506 (CH) C-N
6.154
(lactone)
(CH3) -
-
276 [M+1]+, 230.
18.224&1
175.869
-
m/z
-
-
3.5: Synthesis of Chalcones [S9-S11]: The chalcones or α, β unsaturated ketones [S9-S11], scheme (3-13), are synthesized by reaction of compound [S2] with appropriate aromatic ketones by using ethanol as a solvent and using sodium hydroxide (NaOH) as a reagent.
62
Scheme (3-13) synthesis of compounds [S9-S11]
The mechanism of the reaction proceeds via aldol condensation reaction in which, an enol or an enolate ion reacts with a carbonyl compound (8-formyl-7-hydroxy-4-methyl coumarin) to form a β-hydroxyketone, followed by a dehydration to give a conjugated enone (α, β unsaturated ketones). The mechanism of the reaction consists of four main steps as outlined in scheme (3-14) (98): -
Step one is an acid-base reaction, hydroxide functions as a base and removes an acidic αhydrogen giving a carbanion intermediate or reactive enolate.
-
Step two is alkoxide formation; the nucleophilic enolate attacks the carbonyl carbon of 8formyl-7-hydroxy-4-methyl coumarin in a nucleophilic addition process giving an intermediate alkoxide.
-
Step three is protonation of alkoxide, the alkoxide deprotonates a water molecule producing β-hydroxyketone.
-
Step four is dehydration an elimination reaction in which a water molecule is removed by the action of base leading to the final product α, β unsaturated ketones.
63
Scheme (3-14) mechanism of synthesis of compounds [S9-S11]
The physical properties of compound [S9-S11] are listed in table (2-6). The FT-IR spectrum of compound [S9], figure (3-13), shows a broad band of O-H stretching frequency at (3394) cm-1 , C-H stretching frequency of aromatic ring or alkene at (3080) cm-1, CH stretching for CH3 (aliphatic) at (2922 and 2870) cm-1, C=O stretching of ester (lactone) at 64
(1719) cm-1 with reduced frequency, due to the conjugation or hydrogen bonding, C=O stretching of ketone at (1684) cm-1 with reduced frequency due to the conjugation (α,β unsaturated), C=C stretching frequency of conjugated alkene at (1640) cm-1 also with reduced frequency. Other band such as (1584) cm-1 is C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S9] are listed in table (3-8). The FT-IR spectrum of compound [S10], figure (3-14), shows a broad band of O-H stretching frequency at (3420) cm-1 , C-H stretching frequency of aromatic ring or alkene at (3095 and 3005) cm-1, C-H stretching for CH3 (aliphatic) at (2950 and 2890) cm-1, C=O stretching of ester (lactone) at (1719) cm-1 with reduced frequency due to the conjugation or hydrogen bonding, C=O stretching of ketone at (1685) cm-1 with reduced frequency due to the conjugation (α,β unsaturated), C=C stretching frequency of conjugated alkene at (1640) cm-1 also with reduced frequency. Other band such as (1585) cm-1 is C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S10] are listed in table (3-8). The 13C-NMR (ppm) spectrum of compound [S10], which is recorded in DMSO-d6, figure (325) shows the following bands of carbon: 190.705 (C=O α,β unsaturated ketone) (C=O lactone), 164.301, 157.049, 155.991, 131.438(C
aromatic),
121.626 (C
(88)
, 178.226
aromatic and alkene),
111.525, 106.392, 105.306 (C aromatic), 18.255 (CH3). The 13C-NMR peaks of compound [S10] are listed in table (3-9).
65
The mass spectrum of compound [S10], figure (3-35), shows the molecular ion peak at m/z 322 corresponding to the molecular formula C19H14O5. Other significant bands at m/z 201, 175 and other fragments are listed in table (3-9).
The FT-IR spectrum of compound [S11], figure (3-15), shows a broad band of O-H stretching frequency at (3393) cm-1 , C-H stretching frequency of aromatic ring or alkene at (3040) cm-1, C-H stretching for CH3 (aliphatic) at (2960 and 2910) cm-1, C=O stretching of ester (lactone) at (1716) cm-1 with reduced frequency due to the conjugation or hydrogen bonding. Both (1685) cm-1 are C=O stretching of ketones with reduced frequency, due to the conjugation (α,β unsaturated) and intermolecular hydrogen bonding, C=C stretching frequency of conjugated alkene at (1611 and 1640) cm-1. Other band such as (1584) cm-1 is C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S11] are listed in table (3-8).
66
The 13C-NMR (ppm) spectrum of compound [S11], which is recorded in DMSO-d6, figure (326) shows the following bands of carbon: 190.905 (C=O α,β unsaturated ketone) (C=O lactone), 165.036, 164.446, 157.215, 156.076 (C 111.638, 106.557, 105.422 (C
aromatic and alkene),
aromatic),
131.598 (C=C
(88)
alkene),
, 178.312 121.634,
37.871, 37.704 (CH3), 18.299 (CH3). The
13
C-
NMR peaks of compound [S11] are listed in table (3-9).
The mass spectrum of compound [S11], figure (3-36), shows the molecular ion peak at m/z 354 corresponding to the molecular formula C19H14O7. Other significant bands at m/z 201, 175 and other fragments are listed in table (3-9).
67
68
Table (3-8) FT-IR spectral data of compound [S9-S11] Characteristic bands of FT-IR (cm-1, KBr)
Comp. No.
ʋ O-H
ʋ C-H Stretching
ʋ C=O
ʋ C=C
Others
[S9]
3394
3080 (aromatic or
1719 (lactone)
1640 (alkene)
1481 (C-H bending of CH2)
1684 (ketone)
1584 (aromatic)
alkene) 2922&2870
1434 (C-H bending of CH3)
(aliphatic) 1416 (O-H bending) 1220&1072 (C-O-C) [S10]
3420
3095&3005
1719 (lactone)
1640 (alkene)
1685 (ketone)
1585 (aromatic)
(aromatic or alkene)
1481 (C-H bending of CH2) 1435 (C-H bending of CH3)
2950&2890 1416 (O-H bending)
(aliphatic)
1220&1072 (C-O-C) [S11]
3393
3040 (aromatic or
1716 (lactone)
alkene) 2960&2910
1611and 1640 (alkene)
1685 (ketone) 1584 (aromatic)
(aliphatic)
1483 (C-H bending of CH2) 1436 (C-H bending of CH3) 1415 (O-H bending) 1222&1072 (C-O-C)
69
Table (3-9) 13C-NMR and MS spectral data of compound [S9-S11] C-NMR, ppm (δ)
13
Comp.
Mass spectroscopy
No.
C=O
C=C
C aromatic and alkene
C aliphatic
m/z
[S9]
-
-
-
-
-
[S10]
190.705
121.626
164.301, 157.049, 155.991, 131.438, 121.626, 111.525, 106.392, 105.306.
18.255
165.036, 164.446, 157.215, 156.076, 121.634, 111.638, 106.557, 105.422.
37.871&
(ketone) 178.226
322 [M]+, 201, 175.
(CH3)
(lactone) [S11]
190.905 (ketone) 178.312
131.598
(lactone)
354 [M]+, 201, 175.
37.704 (CH3) 18.299 (CH3)
3.6: Synthesis of Hydrazone Derivatives [S12-S14]: The hydrazone derivatives of coumarin [S12-S14], scheme (3-15), have been synthesized in a good yield by condensation of compound [S2] with hydrazine hydrate 99% [S12], 2,4-di-nitro phenyl hydrazine [S13], and thiosemicarbazide [S14] in absolute ethanol as a solvent by the action of heat without addition of any reagents like acid or base.
70
Scheme (3-15) synthesis of compounds [S12-S14] The mechanism of reaction as outlined in scheme (3-16), proceeds via nucleophilic attack of the lone pair of electron of hydrazine nitrogen (which is more nucleophilic than a regular amine due to the presence of the adjacent nitrogen) on the carbonyl carbon of the aldehyde with the loss of a water molecule by the action of heat (83).
Scheme (3-16) mechanism of synthesis of hydrazone derivatives [S12-S14] 71
The physical properties of compound [S12-S14] are listed in table (2-7, 8 and 9). The FT-IR spectrum of compound [S12], figure (3-16), shows a sharp band of O-H stretching frequency at (3381) cm-1 which indicates that the O-H group is free, NH2 stretching frequency gave a double peak at (3295 and 3218) cm1, C-H stretching for CH3 at (2910 and 2865) cm-1, C=O stretching of ester (lactone) at (1695) cm-1 with reduced frequency due to the conjugation or intermolecular hydrogen bonding, C=N stretching frequency at (1605) cm-1. Other bands such as (1579) and (1491) cm-1 belong to N-H bending and C=C stretching of aromatic ring, respectively. The characteristic FT-IR bands of compound [S12] are listed in table (3-10). The FT-IR spectrum of compound [S13], figure (3-17), shows a broad band of O-H stretching frequency at (3420) cm-1, N-H stretching frequency at (3231) cm1, C-H stretching frequency of aromatic ring or alkene at (3095) cm-1, C-H stretching for CH3 at (2910) cm-1, C=O stretching of ester (lactone) at (1736) cm-1, C=C stretching frequency of alkene at (1640) cm-1, C=N stretching frequency at (1607) cm-1. Other bands such as (1590) cm-1 C=C stretching of aromatic ring, N-H bending frequency at (1550) cm-1 and NO2 stretching at (1519 and 1386) cm-1. The characteristic FT-IR bands of compound [S13] are listed in table (3-10). The mass spectrum of compound [S13], figure (3-37), shows an intense molecular ion peak at m/z 384 corresponding to the molecular formula C17H12N4O7, and other significant bands at m/z 367, 323, 202, 188, 175 and other fragments are listed in table (3-11).
72
The FT-IR spectrum of compound [S14], figure (3-18), shows a broad band of O-H stretching frequency which is mixed with NH2 and N-H stretching region at nearly (3400- 3200) cm-1, NH2 stretching frequency gave a double peak at (3390 and 3283) cm1 N-H stretching at (3185) cm-1, C-H stretching of aromatic or alkene at (3010)cm-1, C=O stretching of ester (lactone) at (1727) cm-1 with reduced frequency, C=N stretching frequency at (1599) cm-1. Other bands such as (1524 and 1492) cm-1 belong to N-H bending and C=C stretching of aromatic ring respectively. The characteristic FT-IR bands of compound [S14] are listed in table (3-10). The 13C-NMR (ppm) spectrum of compound [S14] which is recorded in DMSO-d6, figure (327) shows the following bands of carbon: 178.319 (C=O lactone), 164.141 (C=N), 160.034 (C=S), 159.728, 159.232, 154.075, 153.922, 133.729, 121.697, 113.667, 112.572, and 111.14
73
(C
aromatic and alkene),
55.551 (C-N), 18.791 (CH3). The
13
C-NMR peaks of compound [S14] are
listed in table (3-11).
The mass spectrum of compound [S14], figure (3-38), shows intense molecular ion peak at m/z 277 corresponding to the molecular formula C12H11N3O3S. Other significant bands at m/z 202, 188, 175 and other fragments are listed in table (3-11).
74
Table (3-10) FT-IR spectral data of compound [S12-S14] Characteristic bands of FT-IR (cm-1, KBr)
Comp. No.
[S12]
ʋ O-H
3381
ʋ N-H
ʋ C-H
Stretching
Stretching
3295&3218
2910&2865 (aliphatic)
(NH2)
ʋ C=O
ʋ C=N
Others
1695
1605
1579 (N-H bending) 1491 (C=C aromatic) 1385 (O-H or C-H bending of CH3) 1228& 1072 (C-O-C)
[S13]
3420
3231 (N-H)
3095 (aromatic or alkene)
1736
1607
1640 (C=C alkene) 1590 (C=C aromatic) 1550 (N-H bending)
2910 (aliphatic)
1519&1386 (NO2) stretching 1226& 1068 (C-O-C)
[S14]
3400-
3390&3283
3010
3200
(NH2)
(aromatic or alkene)
3185 (N-H)
1727
1599
1524 (N-H bending) 1492 (C=C aromatic) 1431(O-H bending) 1217& 1080 (C-O-C)
75
Table (3-11) 13C-NMR and MS spectral data of compound [S12-S14] C-NMR, ppm (δ)
13
Comp.
Mass spectroscopy
No.
C=O
C=N
C aromatic and alkene
others
m/z
[S12]
-
-
-
-
-
[S13]
-
-
-
-
384 [M]+, 367, 323, 202, 188, 175.
[S14]
178.319 (lactone)
164.141
159.728, 159.232, 154.075, 153.922, 133.729, 121.697, 113.667, 112.572, 111.14.
160.034 (C=S) 55.551 (C-N) 18.791 (CH3).
76
277 [M]+, 202, 188, 175.
3.7: Synthesis of 2-(2-((7-hydroxy-4-methyl-2-oxo-2H-chromen-8-yl) methylene)hydrazinyl)thiazol-4(5H)-one [S15]: Synthesis of compound [S15], scheme (3-17), was achieved by condensation of compound [S14] with chloroacetic acid in the presence of sodium acetate in glacial acetic acid as a solvent.
Scheme (3-17) synthesis of compound [S15]
77
The mechanism of the reaction is outlined in scheme (3-18) below (92):
Scheme (3-18) mechanism of synthesis of compound [S15] The physical properties of compound [S15] are listed in table (2-10).
78
The FT-IR spectrum of compound [S15], figure (3-19), shows a band of O-H stretching frequency at (3446) cm-1. Disappearance of NH2 band while N-H stretching frequency appeared at nearly (3300) cm1, C-H stretching of aromatic or alkene at (3000)cm-1, C-H stretching of CH3 at (2930) cm-1, C=O stretching of ester (lactone) at (1731) cm-1, the appearance of new band of C=O stretching frequency of amide at (1687) cm-1, C=N stretching frequency at (1624) cm-1, NH bending at (1561) cm-1. Other bands (1520) cm-1 demonstrates C=C stretching of aromatic ring. The characteristic FT-IR bands of compound [S15] are listed in table (3-12).
Table (3-12) FT-IR spectral data of compound [S15] Characteristic bands of FT-IR (cm-1, KBr)
Comp. No.
[S15]
ʋ O-H
3446
ʋ N-H
ʋ C-H
Stretching
Stretching
3300
3000 (aromatic or alkene) 2930 (aliphatic)
ʋ C=O
ʋ C=N
Others
1731 (lactone)
1624
1561 (N-H bending)
1687 (amide)
1520 (C=C aromatic) 1385 (O-H or C-H bending of CH3) 1239-1085 (C-O-C)
The 13C-NMR (ppm) spectrum of compound [S15], which is recorded in DMSO-d6, figure (328) shows the following bands of carbon: 174.162 (C=O amide), 166.609 (C=O lactone), 161.939 (C=N), 159.827 (C
aromatic
attached to OH group), 159.497 (C=N), 154.212, 153.432,
129.445, 116.611, 113.672, 112.448, 112.4 (C aromatic and alkene), 34.137 (C-S), 18.792 (CH3). The 13
C-NMR peaks of compound [S15] are listed in table (3-13).
79
The mass spectrum of compound [S15], figure (3-39), shows an intense molecular ion peak at m/z 317 corresponding to the molecular formula C14H11N3O4S. Other significant bands at m/z 202, 175, 160 and other fragments are listed in table (3-13).
80
Table (3-13) 13C-NMR and MS spectral data of compound [S15] C-NMR, ppm (δ)
13
Comp.
Mass spectroscopy
No.
C=O
C=N
C aromatic and alkene
others
m/z
[S15]
174.162
161.939
159.827, 154.212, 153.432, 129.445, 116.611, 113.672, 112.448, 112.4.
34.137 (C-S)
317 [M]+, 202, 175,
(amide) 166.609 (lactone)
159.497
18.792
160.
(CH3).
3.8: Anti-microbial Activity: Antimicrobial agents are chemicals that kill or inhibit the growth of micro-organisms and are used to treat microbial infections. Some are produced naturally by microbes but many are synthetic. Antimicrobials include antibiotics, antivirals, antifungals and other natural plant bioactive compounds (99).
3.8.1: Antibacterial Susceptibility Tests: Antibacterial susceptibility testing may be performed by either dilution or diffusion methods. The choice of methodology is often based on many factors, including relative ease of performance, flexibility, use of automated or semi-automated devices for both identification and susceptibility testing (100). There are many methods implied to test antimicrobial activity of chemical compounds. Standard laboratory procedures which are applied are three main methods that are still used by standard institutes for setting guidelines. Conventional methods of testing antimicrobial activities are broth dilution, agar dilution and disc diffusion methods (101).
3.8.1.1: Dilution Methods: Dilution susceptibility testing methods are used to determine the minimal concentration, usually expressed in micrograms per milliliter of antimicrobial agents required to inhibit the 81
growth of a microorganism. Procedures for determining the antimicrobial inhibitory activity are carried out by either agar or broth based methods. Antimicrobial agents are usually tested as (twofold) serial dilutions, but any other concentrations can be set in between. The lowest concentration that inhibits visible growth of an organism in vitro after overnight incubation is recorded as the MIC (101). Minimum inhibitory concentrations (MICs) are considered the gold standard for determining the susceptibility of organisms to antimicrobials and are, therefore, used to judge the performance of all other methods of susceptibility testing. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent. MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism, which provides a quantitative result (102,103)
.
The general approaches for broth methods include macro-broth dilution, in which the broth volume for each antimicrobial concentration is ≥ 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 macro-broth dilution broth method is a well standardized and reliable reference method that is useful for research purposes. However, because of the laborious nature of the procedure and the availability of more convenient dilution systems (micro dilution), this procedure is generally not useful for routine susceptibility testing in clinical laboratories (102).
3.8.1.2: Disk Diffusion Method: Disc diffusion tests were introduced mainly because they were less cumbersome technically when large numbers of organisms were tested against many antimicrobials including antibiotics. The disk diffusion method of susceptibility testing allows categorization of bacterial isolates as susceptible, resistant, or intermediate to a variety of antimicrobial agents. It is technically simple to perform and very reproducible. Moreover, it does not require any special equipment and provides category results that are easily interpreted by clinicians. The primary disadvantage of disk diffusion susceptibility testing is that, it provides a qualitative result (103,104). In the present study the macro-broth dilution testing method is performed for screening antibacterial activity of new coumarin derivatives against two Gram positive (G+ve) bacteria 82
Staphylococcus epidermidis and Staphylococcus hemolyticus, and two Gram negative (G-ve) bacteria Escherichia coli and Klebsiella pneumoniae to determine their MIC in vitro. A series of 6 to 9 broth tubes containing 1 mL Muller Hinton autoclaved and cooled then inoculated with (50 µL) bacterial inoculum of bacterial suspension at McFarland turbidity of 0.5. Then a defined concentration of coumarin derivatives are added to each broth of a serial doubling concentration except the negative control in which the solvent (DMSO) was added and a positive control with bacterial inoculum without adding any derivatives. After those broths are incubated aerobically at 37 ˚C for 24 hour, MIC is determined by visual observation as the lowest concentration that inhibits bacterial growth and appears as clear broth tube. All the synthesized compounds except compound [S1] are tested for their anti-microbial (antibacterial) activities. Results are listed in table (3-14). Table (3-14) Anti-bacterial activity of compound [S2-S15] Compound
Staphylococcus
Staphylococcus
Escherichia
Klebsiella
No.
epidermidis (G+ve)
hemolyticus (G+ve)
coli (G-ve)
pneumoniae (G-ve)
S2
100 µg/mL
100 µg/mL
150 µg/mL
200 µg/mL
S3
100 µg/mL
75 µg/mL
100 µg/mL
150 µg/mL
S4
150 µg/mL
100 µg/mL
100 µg/mL
150 µg/mL
S5
100 µg/mL
75 µg/mL
150 µg/mL
150 µg/mL
S6
75 µg/mL
100 µg/mL
100 µg/mL
150 µg/mL
S7
50 µg/mL
100 µg/mL
100 µg/mL
150 µg/mL
S8
50 µg/mL
75 µg/mL
75 µg/mL
150 µg/mL
S9
75 µg/mL
75 µg/mL
25 µg/mL
100 µg/mL
S10
50 µg/mL
50 µg/mL
75 µg/mL
150 µg/mL
S11
100 µg/mL
100 µg/mL
100 µg/mL
150 µg/mL
S12
50 µg/mL
25 µg/mL
50 µg/mL
150 µg/mL
S13
75 µg/mL
75 µg/mL
100 µg/mL
100 µg/mL
S14
100 µg/mL
100 µg/mL
100 µg/mL
150 µg/mL
S15
50 µg/mL
50 µg/mL
100 µg/mL
100 µg/mL
83
From the results illustrated in table (3-14) it is clear that all the synthesized coumarin derivatives have anti-bacterial activity against both gram positive and gram negative bacteria in vitro, the MIC ranges between 25-200 µg/mL in which they varies according to the derivatives and bacteria. The lowest MIC is 25 µg /mL for both compounds S9 against Escherichia coli and S12 against Staphylococcus hemolyticus, and the highest MIC of 200 µg /mL for compound S2 against Klebsiella pneumoniae, figures (3-1 and 3-2) shows anti-bacterial activity of some of the synthesized compounds.
Figure (3-1) Anti-bacterial activity of compound [S9] against Escherichia coli
84
Figure (3-2) Anti-bacterial activity of compound [S15] against Klebsiella pneumoniae According to the statistical analysis and the mean values, table (3-15), the synthesized coumarin derivatives have greater activity against G+ve bacteria than G-ve bacteria.
Table (3-15) Mean values of MIC
Mean MIC
Staphylococcus
Staphylococcus
Escherichia
Klebsiella
epidermidis (G+ve)
hemolyticus (G+ve)
coli (G-ve)
pneumoniae (G-ve)
80.35 µg/mL
78.57 µg/mL
94.64 µg/mL
142.85 µg/mL
Generally, the MIC of all synthesized compounds is higher for Klebsiella pneumoniae compared to other bacteria which might be due to its prominent poly saccharide capsule (which is composed of 63% capsular polysaccharide, 30% lipopolysaccharide, and 7% protein) around bacterial cell which may play a protective role for the bacteria (105). Disk diffusion method for antimicrobial activity has been tried for above coumarin derivatives. However, due to the lack of diffusion of almost all of compounds over the petridish agar media rendered this technique not useful method for testing their anti-bacterial activity, and this derived us to use serial broth dilution method to test their anti-bacterial activity, as illustrated in figure (3-3) below. 85
(a)
(b)
Figure (3-3) Disc diffusion method of anti-bacterial susceptibility test a- Disc diffusion method of synthesized coumarin derivatives showing no inhibition zone. b- Disc diffusion method of synthesized coumarin derivatives with standard 10 µg Imipenem disc, with clear zone of inhibition against Escherichia coli
While direct drops containing 50-200 µg of compounds which was placed over the inoculated petridish that spread out about 1cm over the stricken bacterial inoculum showed inhibited zone of growth. It gives a clue of having antibacterial activity over the area of direct contact but not beyond drop contact. It also comes back to mind that minimal or lack of diffusion or solubility of chemical compounds beyond border of drop contact, as shown in figure (3-4).
86
Figure (3-4) Anti-bacterial activity of some coumarin derivatives
87
Figure (3-5) FT-IR spectrum of compound [S1]
Figure (3-6) FT-IR spectrum of compound [S2]
88
Figure (3-7) FT-IR spectrum of compound [S3]
Figure (3-8) FT-IR spectrum of compound [S4]
89
Figure (3-9) FT-IR spectrum of compound [S5]
Figure (3-10) FT-IR spectrum of compound [S6]
90
Figure (3-11) FT-IR spectrum of compound [S7]
Figure (3-12) FT-IR spectrum of compound [S8]
91
Figure (3-13) FT-IR spectrum of compound [S9]
Figure (3-14) FT-IR spectrum of compound [S10]
92
Figure (3-15) FT-IR spectrum of compound [S11]
Figure (3-16) FT-IR spectrum of compound [S12]
93
Figure (3-17) FT-IR spectrum of compound [S13]
Figure (3-18) FT-IR spectrum of compound [S14]
94
Figure (3-19) FT-IR spectrum of compound [S15]
95
Figure (3-20) 13C-NMR spectrum of compound [S2]
Figure (3-21) 13C-NMR spectrum of compound [S3] 96
Figure (3-22) 13C-NMR spectrum of compound [S5]
Figure (3-23) 13C-NMR spectrum of compound [S6] 97
Figure (3-24) 13C-NMR spectrum of compound [S7]
Figure (3-25) 13C-NMR spectrum of compound [S10] 98
Figure (3-26) 13C-NMR spectrum of compound [S11]
Figure (3-27) 13C-NMR spectrum of compound [S14] 99
Figure (3-28) 13C-NMR spectrum of compound [S15]
A
b u
n d
a n
c
e
S 1 8
0 0
0 0
0
1 7
0 0
0 0
0
1 6
0 0
0 0
0
1 5
0 0
0 0
0
1 4
0 0
0 0
0
1 3
0 0
0 0
0
1 2
0 0
0 0
0
1 1
0 0
0 0
0
1 0
0 0
0 0
0
9 0
0 0
0 0
8 0
0 0
0 0
7 0
0 0
0 0
6 0
0 0
0 0
5 0
0 0
0 0
4 0
0 0
0 0
3 0
0 0
0 0
2 0
0 0
0 0
c
a
n
1
4
( 0 . 2 8 1
) :
6
4 0
1 5
1 2
1 4
8 . D
8
1 7
6
9 1
5 5
6 5
7 7 1 1
0 0
in
2 0
4 3
1 0
m
8 4
1 0 9 8
0 0
9
5
1 3 1 1
0
2
1 5 1 3
1 6
5
1 8
7
9
3 1 9
0
1 9
0
1 9
7
0 4 0 m
/
5 0
6 0
7 0
8 0
9 0
1 0
0
1 1
0
1 2
0
1 3
0
1 4
0
1 5
0
1 6
0
z - - >
Figure (3-29) Mass spectrum of compound [S2]
100
1 7
0
1 8
0
2 0
0
4
Figure (3-30) Mass spectrum of compound [S3]
101
A b u n d a n c e
S c a n 3 0 0 0 0
4 4 7
(2 .3 0 6
m in ) : 6 4 0 1 5 1 2 9 . D
4 5 6 5
2 5 0 0 0
7 7
2 0 0 0 0
1 5 0 0 0 9 1
3 2 3
1 0 0 0 0 2 9 5 1 0 3 5 0 0 0
1 2 1
1 7 2
1 4 6
1 3 3
1 5 8
1 8 7
2 0 2 2 2 02 3 2
2 5 0 2 6 62 7 8
0 4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
2 0 0
2 2 0
2 4 0
m / z -->
Figure (3-31) Mass spectrum of compound [S4]
Figure (3-32) Mass spectrum of compound [S5]
102
2 6 0
2 8 0
3 0 0
3 2 0
Figure (3-33) Mass spectrum of compound [S6]
103
Figure (3-34) Mass spectrum of compound [S7]
104
Figure (3-35) Mass spectrum of compound [S10]
Abundanc e S c a n 3 3 3 (1 . 7 6 4 m in ): 6 4 0 1 5 1 3 4 . D 14000
43 63
12000 10000
91 77
8000 6000 4000 105 119
2000
147
172 202 188
0 40
60
80
232
266
289
335 354
100 120 140 160 180 200 220 240 260 280 300 320 340
m / z -->
Figure (3-36) Mass spectrum of compound [S11]
105
A
b
u
n
d
a
n
c
e
S 6
2
5
0
0
0
2
4
0
0
0
2
3
0
0
0
2
2
0
0
0
2
1
0
0
0
2
0
0
0
0
1
9
0
0
0
1
8
0
0
0
1
7
0
0
0
1
6
0
0
0
1
5
0
0
0
1
4
0
0
0
1
3
0
0
0
1
2
0
0
0
1
1
0
0
0
1
0
0
0
0 4
9
0
0
0
8
0
0
0
7
0
0
0
6
0
0
0
5
0
0
0
4
0
0
0
3
0
0
0
2
0
0
0
1
0
0
0
c
a
n
7
5
6
( 3
. 7
7
5
m
in
) :
6
4
0
1
5
1
3
0
. D
3 7
7
9
1
1
7
2
2
0
2
1
3
1
1
8
4
8
1
1
3
4
6
2
2 1
8
2
0
8
3 2
3
4
2
4
2
4
8 2
6
2
0
5
2
8
0 2
9
5
2
8
0
3
0
2
3 3
4
0
3
4
0
3
6
7
0 4 m
/
0
6
0
8
0
1
0
0
1
2
0
1
4
0
1
6
0
1
8
0
2
0
0
2
2
0
0
6
0
3
2
0
3
6
z - - >
Figure (3-37) Mass spectrum of compound [S13] A b u n d a n c e
S c a n
3 9 6
(2 .0 6 4
m in ) :
6 4 0 1 5 1 3 1 .D
5 1
3 0 0 0 0 2 8 0 0 0 2 6 0 0 0 4 1 2 4 0 0 0
6 3
2 2 0 0 0 2 0 0 0 0 7 7
1 8 0 0 0
8 9
1 7 3
1 6 0 0 0 1 4 0 0 0 1 2 0 0 0 1 0 0 0 0 1 0 3
8 0 0 0 6 0 0 0
1 1 7
4 0 0 0
2 0 2 1 3 1
1 4 7 1 6 0
2 0 0 0
1 8 9
2 1 8
2 3 92 4 9
2 6 62 7 7
0 4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
2 0 0
2 2 0
m / z -->
Figure (3-38) Mass spectrum of compound [S14] 106
2 4 0
2 6 0
0
3
8
0
A b u n d a n c e
S c a n
3 9 0
(2 . 0 3 5
m
in ) :
6 4 0 1 5 1 3 2 . D
7 5 0 0 0 4 2 7 0 0 0 0 6 5 0 0 0 6 0 0 0 0 5 5 0 0 0 5 0 0 0 0 4 5 0 0 0 4 0 0 0 0 6 3
3 5 0 0 0 3 0 0 0 0 2 5 0 0 0 2 0 0 0 0
1 3 1
1 6 0
1 5 0 0 0
3 1 7
1 0 0 0 0 7 7 5 0 0 0
1 4 5 1 7 2
9 1 1 0 3 1 1 7
1 8 9
2 0 2
0 4 0 m
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
2 0 0
2 2 6 2 2 0
/ z -->
Figure (3-39) Mass spectrum of compound [S15]
107
2 9 2
2 4 82 6 0 2 4 0
2 6 0
2 8 0
3 0 0
Chapter Four Conclusions and Recommendations
4.1: Conclusions: In the present study some new coumarin derivatives have been synthesized from 7-hydroxy-4methyl coumarin by applying Duff reaction to insert the formyl group to carbon 8 of coumarin nucleus. The synthesized compounds include Schiff bases, Chalcones, hydrazones, and cyclized thiosemicarbazone derivative. The compounds [S2-S15] have been evaluated for anti-bacterial activity by determining their minimum inhibitory concentration (MIC) by serial broth dilution method, in which they demonstrated antibacterial activity against gram positive more than the gram negative bacteria with varying ranges of MIC.
4.2: Recommendations: 1- Further spectral analysis such as 1H-NMR and CHN analysis. 2- Antifungal activity of the synthesized compounds. 3- Animal and cell line studies of the synthesized compounds for determining anti-cancer, antioxidant and anti-inflammatory activity and studying their pharmacokinetic properties.
108
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