Extraction, isolation and characterization of oleanolic acid and its analogues from syzygium aromaticum (cloves) and evaluation of their biological activities.
A ―Dissertation submitted in Partial Fulfilment of the Requirements for the Degree of Masters of Science (MSc.) in Chemistry in the Faculty of Science and Agriculture, at the University of Fort Hare‖
By
Khwaza Vuyolwethu (201209227)
Supervisor: Prof. O. O. Oyedeji
Co-supervisor: Prof. B. A. Aderibigbe
2019
i DECLARATION
I, ―Khwaza Vuyolwethu, student number 201209227 declare that this Dissertation entitled,
Extraction, isolation and characterization of oleanolic acid and its analogues from
Syzygium aromaticum (cloves) and evaluation of their biological activities, which I hereby submit to the university of Fort hare in partial fulfilment of the requirements for the Masters of Science (Chemistry) is my own original work, it has never been submitted for any academic award to any other institution of higher learning.‖
……………………………. ……………………………………
Khwaza Vuyolwethu Date
……………………………. ……………………………………
Prof O.O Oyedeji Date
Supervisor
……………………………. ……………………………………
Prof. B. A. Aderibigbe Date
Co-Supervisor
ii
PLAGIARISM DECLARATION
I, Khwaza Vuyolwethu, student number 201209227 hereby declare that I am fully aware of the University of Fort Hare‘s policy on plagiarism and I have taken every precaution to comply with the regulations.
1. I declare that this dissertation is the results of my own original work. Where someone
else‘s work was used (others sources i.e internet, printed sources etc.) due
acknowledgment was given and reference was made according to the department
requirements.
2. I did not use another student‘s work and submit it as my own work.
3. I did not and will not allow anyone to copy my work with the intention of presenting
it as his/her own work.‖
iii
CONFERENCE AND PUBLICATIONS
Conferences
Vuyolwethu Khwaza, Opeoluwa O. Oyedeji and Blessing A. Aderibigbe (2018).
Isolation and Characterization of oleanolic acid and its analogues from syzygium
aromaticum. Poster presentation at the BIOAFRICA CONVENTION: International
conference Durban ICC South Africa.
Publications
Vuyolwethu Khwaza, Opeoluwa O. Oyedeji and Blessing A. Aderibigbe (2018).
Antiviral Activities of Oleanolic Acid and Its Analogues. Molecules 2300 (23) 1-14.
Khwaza V, Opeoluwa O. Oyedeji, Mike O.Ojemaye, Adebola O.Oyedeji and Francis
B.Lewu (2018) ―Assessment of the heavy metal content of wild and cultivated
Pelargonium inquinans: an herbal plant used for the treatment of divers ailments in
South Africa‖. Fresenius Environmental Bulletin 27(6) 3914-165.
iv
ACKNOWLEDGEMENTS
In Africa they say ―It takes a village to raise a child‖. Nothing in this world is ever
successful without a corporate of many talented people who are willing to work and
submit their talents, experiences, and passion for a common goal.
Firstly I would like thank God Almighty for making it possible for me to complete
this work in spite of many difficulties experienced along the way.
I wish to thank my supervisors Prof. O. O. Oyedeji and Prof. B.A. Aderibigbe for
their assistance, guidance and encouragement throughout the study, you both played a
major role in my life. Thank you and may you continue to make tremendous
difference in South Africa and the world at large. It is an honour to have been under
your supervision.
My Mentor ―from Sasol Dr Du Plessis Esna, thank you for your words of
encouragement you have carried me to where I am today. For that, God bless you and
your family.
I would like to thank Mr T. Mcako for helping with the FTIR spectroscopy.
I wish to thank Prof. R. Krause and Dr S. N. Xavier from Rhodes University for their
excellent assistance with NMR, and MS.
I would like to thank National Research Foundation (NRF), Sasol inzalo foundation
and Medical Research Council for funding.‖
The Department of chemistry, University of fort hare.
I would like to send my sincere gratitude to my brother Mtobeli Khwaza ―Madala‖
and my dear sister Tobeka Khwaza for your support, encouragement and for being
there for me throughout.
A special thanks to Khatywa Ongeziwe. You‘ve been a big brother to me since my
first day in UFH.
v
I want to thank all my lab mates for lending their expertise when required.
My family at large thank you for your prayers, it is not my by power that I managed
to finish, but through your prayers. From the bottom of my heart I love you so much.
vi
DEDICATION
I dedicate this work to my Mom (Nozuzile khwaza) for being the positive motivating force in my life. You‘ve struggled against all odds to get me educated. May God Almighty shower you with all the blessings that you deserve, I love you Mazaka.
vii
ABSTRACT
Pathogenic microorganisms have serious impact on people's lives. Every year, millions of people around the world die of bacterial infections. Resistance to common antibacterial drugs has proven to be a challenging problem in control of bacterial infections. In an attempt to develop an effective and affordable treatment for bacterial infections, oleanolic acid isolated from syzygium aromaticum conjugates incorporating other pharmaceutical scaffolds such as chloroquine derivatives, curcumin, and ergocalciferol etc have been developed. Based on the previous successes of testing combination of antimicrobial drugs and pharmaceutical drugs which appeared to be the promising strategy to overcome treatment failure; a series of hybrid compounds containing oleanolic acid and other pharmaceutical scaffolds were synthesized. 4-
Aminoquinoline derivatives were first hybridized with selected organic compounds to form a class of hybrid compounds containing either amide bond or ester bond as a linker between the precursor molecules. Analogues/hybrid compounds can overcome the disadvantages of combination therapy such as drug-drug interaction. The structural effects of this type of conjugation of oleanolic acid and other pharmaceutical scaffolds were characterised by FT-
IR, Mass Spec and NMR spectroscopy. These compounds were studied along with the mono- substituted oleanolic acid analogues and the organic components in order to compare the effects of the substitution on their biological response.‖ All the synthesized analogues were tested against 11 bacterial strains on both Gram-positive and Gram-negative bacteria. The synthesized compounds showed selectivity and higher activity against Enterococcus faecalis
(EF), Klebsiella oxytoca (KO), Escherischia coli (EC), Staphylococcus aureous (SA),
Proteus vulgaris (PV) and Bacillus subtilis (BS) with MIC values; ranging between of 1.25 mg/mL to 0.072 mg/mL.
Key words: Oleanolic acid, Antibacterial, Syzygium aromaticum, Analogues and
Pharmaceutical scaffolds.
viii
LIST OF FIGURES
Figure 2. 1: phenol (parent structure of all phenolic compounds) ...... 13
Figure 2. 2: examples of phenolic compounds ...... 14
Figure 2. 3: biosynthesis of different classes terpenes...... 16
Figure 2. 4: examples of monoterpenes ...... 17
Figure 2. 5: Sesqueterpene compounds ...... 18
Figure 2. 6: examples of diterpene compounds ...... 19
Figure 2. 7: different classes of triterpenes structures...... 20
Figure 2. 8: Examples of terpene compounds...... 21
Figure 2. 9: Common modern drugs derived from plant sources ...... 22
Figure 2. 10: antimicrobial drugs ...... 24
Figure 2. 11: 3-acetoxy, 28-methyloleanolic acid (2.42), 3-acetoxyoleanolic acid (2.43)...... 27
Figure 2. 12: oleanolic acid derivatives studied for PTP-1B inhibition...... 29
Figure 2. 13: Oleanolic derivatives with anti-HIVactivity ...... 31
Figure 2. 14: derivatives of oleanolic acid with anti-HIV activities...... 32
Figure 2. 15: Previously modified anti-HIV triterpene derivatives...... 32
Figure 3. 1: World‘s distribution of syzygium aromaticum (clove) ...... 52
Figure 3. 2: Fresh (A) and dried (B) syzygium aromaticum ...... 53
Figure 3. 3: Clove‘s oil constituents...... 54
Figure 3. 4: TLC plate showing the isolated three compounds from ethylacetate crude extract.
...... 56
Figure 3. 5: formula for calculating Rf value of a compound...... 57
Figure 3. 6: Structure of Eugenol (Eu)...... 58
ix
Figure 3. 7: Structure of oleanolic acid (OA)...... 60
Figure 3. 8: Structure of Maslinic acid...... 62
Figure 4. 1: IR spectrum of N-(3-aminopropyl)-7-chloroquinolin-4-amine...... 74
Figure 4. 2: IR spectrum of N-(2-aminopropyl)-7-chloroquinolin-4-amine (1.2 PDA-Q) ...... 76
Figure 4. 3: IR spectra of N-(2-(2-Aminoethylamino) ethyl)-7-chloroquinoline-4-amine...... 77
Figure 4. 4: FT-IR spectrum of 1-(7-Choloroquinolin-4-yl) hydrazine ...... 79
Figure 4. 5: FT-IR spectrum of 2-(7-chloroquinolin-4-ylamino) ethanol...... 80
Figure 4. 6: FT-IR spectrum of 2-(2-(-chloroquinolin-4-ylamino)ethoxy)ethanol (AEE-Q). . 82
Figure 4. 7: FT-IR spectrum of 4-(7-chloroquinolin-4-ylamino)-2-hydroxybenzoic acid ...... 83
Figure 5. 1: structural elucidation of compound VK1 ...... 101
Figure 5. 2: 1IR spectrum of compound VK1...... 102
Figure 5. 3: 1H-NMR spectrum of compound VK1 ...... 103
Figure 5. 4: 13C-NMR spectrum of compound VK1...... 103
Figure 5. 5: LC-MS results of compound VK1 ...... 104
Figure 5. 6: structural elucidation of compound VK2 ...... 104
Figure 5. 7: IR spectra of compound VK2...... 106
Figure 5. 8: 1H NMR Spectrum of compound VK2 ...... 106
Figure 5. 9: 13C NMR spectrum of compound VK2...... 107
Figure 5. 10: LC-MS results of compound VK2...... 107
Figure 5. 11: Structural elucidation of compound VK3 ...... 108
Figure 5. 12: IR spectra of compound VK3...... 109
x
Figure 5. 13: 1H NMR spectrum of compound VK3...... 110
Figure 5. 14: 13C NMR spectrum of compound VK3 ...... 110
Figure 5. 15:LC-MS results of compound VK3 ...... 111
Figure 5. 16: Structural elucidation of compound VK4 ...... 111
Figure 5. 17: IR spectrum of compound VK4...... 113
Figure 5. 18: 1H NMR spectrum of compound VK4 ...... 113
Figure 5. 19: 13C NMR spectrum of compound VK4...... 114
Figure 5. 20: LC-MS results of compound VK4...... 114
Figure 5. 21: Structural elucidation of compound VK5...... 115
Figure 5. 22: IR spectrum of compound VK5...... 116
Figure 5. 23:1H NMR spectrum of compound VK5 ...... 117
Figure 5. 24: 13C NMR spectrum of compound VK5 ...... 117
Figure 5. 25: LC-MS results of compound VK5 ...... 118
Figure 5. 26: Structural elucidation of compound VK6 ...... 118
Figure 5. 27: IR spectrum of compound VK6...... 119
Figure 5. 28: 1H NMR spectrum of compound VK6...... 120
Figure 5. 29: 13C NMR spectrum of compound VK6...... 120
Figure 5. 30: LC-MS results of compound VK6 ...... 121
Figure 5. 31: Structural elucidation of compound VK7 ...... 121
Figure 5. 32: IR spectrum of Compound VK7...... 122
Figure 5. 33: 1H NMR spectrum of compound VK7 ...... 123
Figure 5. 34: 13C NMR spectrum of compound VK7...... 123
Figure 5. 35: LC-MS results of compound VK7 ...... 124
Figure 5. 36: Structural elucidation of compound VK8 ...... 124
Figure 5. 37: IR spectrum of compound VK8...... 125
xi
Figure 5. 38: 1H NMR spectrum of compound VK8 ...... 126
Figure 5. 39: 13C NMR spectrum of compound VK8 ...... 126
Figure 5. 40: LC-MS results of compound VK8 ...... 127
Figure 5. 41: structural elucidation of compound VK9 ...... 127
Figure 5. 42: IR spectrum of compound VK9...... 128
Figure 5. 43: 1H NMR spectrum of compound VK9...... 129
Figure 5. 44: 13C NMR spectrum of compound VK9 ...... 129
Figure 5. 45: LC-MS results of compound VK9 ...... 130
Figure 5. 46: The 96 Well plates (MIC) showing the antibacterial activities against 11 bacterial strains...... 132
Figure 5. 47: Successful hybrid compounds tested for antibacterial activity...... 134
xii
LIST OF SCHEMES
Scheme 4. 1: N-alkylation of 4.7-dichloroquinoline mechanism...... 72
Scheme 4. 2: synthesis of N-(3-aminopropyl)-7-chloroquinolin-4-amine...... 73
Scheme 4. 3: Synthesis of N-(2-aminopropyl)-7-chloroquinolin-4-amine...... 75
Scheme 4. 4: Symthesis of N-(2-(2-Aminoethylamino) ethyl)-7-chloroquinoline-4-amine ... 76
Scheme 4. 5: Symthesis of 1-(7-Choloroquinolin-4-yl) hydrazine (Hyd-Q) ...... 78
Scheme 4. 6: Synthesis of 2-(7-chloroquinolin-4-ylamino) ethanol (EA-Q) ...... 79
Scheme 4. 7: Synthesis of 2-(2-(-chloroquinolin-4-ylamino)ethoxy)ethanol (AEE-Q) ...... 81
Scheme 4. 8: Synthesis of 4-(7-chloroquinolin-4-ylamino)-2-hydroxybenzoic acid ...... 82
Scheme 4. 9: Synthesis of compound VK1 ...... 85
Scheme 4. 10: Synthesis of compound VK2 ...... 86
Scheme 4. 11: Synthesis of compound VK3 ...... 88
Scheme 4. 12: Synthesis of compound VK4 ...... 89
Scheme 4. 13: unsuccessful hybrid compounds ...... 99
Scheme 4. 14: Synthesis of compound VK5 ...... 91
Scheme 4. 15: Synthesis of compound VK6 ...... 92
Scheme 4. 16: Synthesis of compound VK7 ...... 94
Scheme 4. 17: Synthesis of compound VK8 ...... 95
Scheme 4. 18: Synthesis of compound VK9 ...... 97
xiii
“LIST OF TABLES
Table 2. 1: Examples of different major classes of phenolic compounds...... 13
Table 2. 2: Some plant species where oleanolic acid was reported, their biological activities, and the plant parts used...... 26
Table 2. 3: Synthesis of Oleanolic acid...... 30
Table 3. 1: Extraction results...... 58
Table 3. 2: 1H and 13C-NMR data of Compound VK121 compared with literature data...... 59
Table 3. 3: 1H and 13C-NMR data of Compound VK122 compared with literature data...... 60
Table 3. 4: 1H and 13C-NMR data of Compound VK123 compared with literature data ...... 62
Table 5. 1: Antibacterial activities of synthesized compounds...... 131"
xiv
LIST OF ABBREVIATION
1.2 PDA: 1.2-diaminopropane
1.2 PDA-Q: N-(2-aminopropyl)-7-chloroquinolin-4-amine
1.3 PDA: 1.3-diaminopropane
1.3 PDA-Q: N-(3-aminopropyl)-7-chloroquinolin-4-amine
AEE: 2(2-Aminoethoxy) ethanol
AEE-Q: 2-(2-(-chloroquinolin-4-ylamino)ethoxy)ethanol
BA: Betulinic acid
CC: Column Chromatography
CHCl3: Chloroform cm-1: per centimetre
DMSO: Dimethylsulfoxide
DCC: N.N'-Dicyclohexylcarbodiimide
DCM: Dichloromethane
DET: Diethelenetriamine
DET-Q: N-(2-(2-Aminoethylamino) ethyl)-7-chloroquinoline-4-amine.
DMAP: 4-Dimethylaminopyridine
DMAPP: dimethylally pyrophosphate
DMF: Dimethylformide
xv
EA: Ethanolamine
EA-Q: 2-(7-chloroquinolin-4-ylamino) ethanol
EDA: Ethyldiamine
EDDA: 2-(2-(2-aminoethoxy)ethoxy)ethanamine
EtOAc: ethylcetate
Eu: Eugenol
FDA: food drug administration
FGPP: farnesyl geranyl pyrophosphate
FGPPS: farnesyl geranyl pyrophosphate synthase
FT-IR: Fourier-transorm infrared specroscopy
FPP: farnesyl pyrophosphate
FPPS: Farnesyl pyrophosphate synthase
GGPP: Geranylgeranyl pyrophosphate
GGPPS: Geranylgeranyl pyrophosphate synthase
GPPS: Geranyl pyrophosphate synthase
H2SO4: Sulfuric acid
HIV: Human Immunodeficiency Virus
HYD: Hydrazine hydrate
HYD-Q: 1-(7-Choloroquinolin-4-yl) hydrazine
xvi
Hz: Hertz.
IPP: Isopentenyl pyrophosphate
LC-MS: Liquid chromatography mass spectroscopy
LiAlH4: Lithium Aluminium hydride
MeOH: Methanol
MEP: 2C-Methyl-D- erythritol-4-phosphate
MIC: Minimum inhibition concentration
MVA: Mevalonate mmol: milimole
Mp: Melting point
Ms: Mass spectrometry
NME: New molecular entities
NMR: Nuckear magnetic resonance
OA: Oleanolic acid
OH: Hydroxyl ppm: parts per million
PTP: Protein-tyrosine phosphate
Rf: retardation factor
TEA: Triethylamine
xvii
TLC: Thin layer chromatography
SA: South Africa
UA: Ursolic acid
WHO: World Health Organisation.
xviii
TABLE OF CONTENTS
―
DECLARATION ...... ii
PLEGERISM DECLARATION ...... iii
CONFERENCE AND PUBLICATIONS ...... iv
Conferences ...... iv
Publications ...... iv
ACKNOWLEDGEMENTS ...... v
DEDICATION ...... vii
ABSTRACT ...... viii
LIST OF FIGURES ...... ix
LIST OF SCHEMES...... xiii
LIST OF ABBREVIATION ...... xv
CHAPTER ONE ...... 1
INTRODUCTION ...... 1
1.1. Background of the study ...... 1
1.2. Antibacterial drug resistance ...... 3
1.3. Justification of the study ...... 4
1.4. Null hypothesis...... 5
1.5. Aim and Objectives ...... 5
1.5.1. Overall objectives ...... 5
1.6. References ...... 7
xix
CHAPTER TWO ...... 11
LITERATURE REVIEW ...... 11
2.1. Antibacterial resistance...... 11
2.2. Plant natural products ...... 12
2.3. Classes of natural products ...... 13
2.3.1 Phenolic Compounds ...... 13
2.3.2. Terpenes...... 14
2.3.3. Biosynthesis of terpenes ...... 15
2.3.4. Monoterpenes ...... 17
2.3.5. Sesqueterpenes...... 17
2.3.6. Diterpenes ...... 18
2.3.7. Triterpenes ...... 19
2.4. Role of plants in westernised medicine ...... 21
2.5. Natural products as antimicrobial drugs ...... 22
2.6. Combinations of plant natural products with antimicrobial drugs ...... 24
2.6. Oleanolic acid and its analogues ...... 25
2.6.1. Oleanolic acid ...... 25
2.6.2. Analogues of OA ...... 27
2.7. References ...... 34
CHAPTER THREE ...... 52
PHYTOCHEMICAL EXAMINATION OF SYZYGIUM AROMATICUM (L.) MERR.
& PERRY ...... 52
xx
3.1. Introduction ...... 52
3.2. Medicinal use of syzygium aromaticum...... 53
3.3. Chemistry of syzygium aromaticum...... 54
3.4. EXPERIMENTAL ...... 55
3.4.1 Plant Identification ...... 55
3.4.2. Plant preparation and solvent extraction ...... 55
3.4.3. Isolation method ...... 55
3.4.4. 13C and 1 H NMR spectroscopic analysis ...... 57
3.5. Results ...... 58
3.5.1. Physical properties of compound (VK121) ...... 58
3.5.2. Physical properties of compound (VK122) ...... 60
3.5.3. Physical properties of compound VK123 ...... 62
3.6. Discussion ...... 64
3.6.1. Compound (VK121) ...... 64
3.6.2. Compound (VK122) ...... 65
3.6.3. ―Compound (VK123) ...... 65
3.7. References ...... 67
CHAPTER FOUR ...... 71
SYNTHESIS OF OLEANOLIC ACID ANALOGS ...... 71
4.1. Materials ...... 71
4.2. Characterization ...... 71
xxi
4.3. Introduction ...... 72
4.3. Synthesis of 4-aminoquinoline derivatives...... 73
4.4. Synthesis of N-(3-aminopropyl)-7-chloroquinolin-4-amine (1.3 PDA-Q) ...... 73
4.5. Synthesis of N-(2-aminopropyl)-7-chloroquinolin-4-amine (1.2 PDA-Q) ...... 75
4.6. Symthesis of N-(2-(2-Aminoethylamino) ethyl)-7-chloroquinoline-4-amine (DET.Q)
...... 76
4.7. Symthesis of 1-(7-Choloroquinolin-4-yl) hydrazine (HYD-Q) ...... 78
4.8. Synthesis of 2-(7-chloroquinolin-4-ylamino) ethanol (EA-Q) ...... 79
4.9. Synthesis of 2-(2-(-chloroquinolin-4-ylamino)ethoxy)ethanol (AEE-Q) ...... 81
4.10. Synthesis of 4-(7-chloroquinolin-4-ylamino)-2-hydroxybenzoic acid ...... 82
4.11. DERIVATIVES OF OLEANOLIC ACID...... 85
4.11.1. Synthesis of hybrid compounds with amide linkers ...... 85
4.11.2 Synthesis of compound VK1 ...... 85
4.11.3. Synthesis of compound VK2 ...... 86
4.11.4. Synthesis of compound VK3 ...... 88
4.11.5. Synthesis of compound VK4 ...... 89
4.12. Synthesis of hybrid compounds with ester linkers...... 91
4.12.1. Synthesis of compound VK5...... 91
4.12.2. Synthesis of compound VK6 ...... 92
4.12.3. Synthesis of compound VK7 ...... 94
4.12.4. Synthesis of compound VK8 ...... 95
4.12.5. Synthesis of compound VK9 ...... 97
xxii
4.12.6. Antibacterial assay ...... 100
4.12.7 Minimum inhibitory concentration (MIC) ...... 100
CHAPTER FIVE ...... 101
RESULTS AND DISCUSION ...... 101
5.1. Structural elucidation of compound VK1 ...... 101
5.2. Structural elucidation of compound VK2 ...... 104
5.3. Structural elucidation of compound VK3 ...... 108
5.4. Structural elucidation of compound VK4 ...... 111
5.5. Structural elucidation of compound VK5 ...... 115
6.6. Structural elucidation of compound VK6 ...... 118
5.7. Structural elucidation of compound VK7 ...... 121
5.8. Structural elucidation of compound VK8 ...... 124
5.9. Structural elucidation of compound VK9 ...... 127
5.10. Minimum inhibitory concentration ...... 130
5.11. Successful hybrid compounds ...... 133
CHAPTER SIX ...... 135
CONCLUSION AND RECOMMENDATIONS ...... 135
6.1. CONCLUSION ...... 135
6.2. RECOMMENDATIONS ...... 135
6.3. References ...... 137
APPENDIX ONE...... 141
xxiii
SPECTRA OF EUGENOL ...... 141
APPENDIX TWO ...... 145
Specta of oleanolic acid...... 145
APPENDIX THREE ...... 149
Specta of Maslinic acid ...... 149
xxiv
CHAPTER ONE
INTRODUCTION
1.1. Background of the study
Plants ―have been the significant source of medicinal treatments throughout history and still continue playing a fundamental role in the primary health of over 80% of the world's developing and underdeveloped countries1–4. Plants produce a large number of organic compounds, the majority of which is believed not to contribute in growth or development of the plant. These compounds are known as natural products or secondary metabolites. The primary metabolites in contrast, such as chlorophyll, simple carbohydrates, nucleotides, or amino acids are found in every plant kingdom and are essential for growth and normal functioning of the plant5. Plants use these natural products in order to defend themselves against herbivores and pathogenic microorganisms6,7.
Natural products have been generally seen as biologically unimportant and have historically received little attention from most plant biologists. Organic chemists, however, have for quite some time been interested in these novel phytochemicals and have examined their chemical properties widely since the 1850s. Investigations of natural products led to the improvement of isolation strategies, spectroscopic approaches to structure elucidation, and synthetic techniques that presently constitute the establishment of current organic chemistry5.
The study of natural products has attracted many researchers to carry out laborious analysis on the plants and to establish a relationship between phytochemicals/chemical composition and therapeutic activities. The isolation of morphine (1.1), as a pure secondary metabolite in
1806 from the extract of Papaver somniferum L. (opium poppy) historically used as a pain
1 reliever was a breaking ground to drug development from plants8. As a result around half of all drugs in medicinal use today are derived from medicinal plants worldwide. For instance, artemisinin (1.2) an antiviral drug isolated from Artimisia annua is used to treat multi-drug- resistant malaria9 and aspirin (1.3), an isolate of willow bark tree Salix alba L., commonly known as acetylsalicylic acid also considered to be one of the most effective analgesic, antipyretic and anti-inflammatory drugs which are commonly used in modern medicine10, other examples of important plant-derived pharmaceutical drugs in use today are codeine
(1.4), atropine (1.5) and quinine (1.6).
―
CH3 HO H O OH O N H C O O 3 O O H O H H O CH 3 1.3 1.1 HO O 1.2
O
HO N OH N O H O H N O
HO O N 1.6 1.4 1.5 ”
Figure 1. 1: Examples of pharmaceutical drugs isolated from plants.
2
1.2. Antibacterial drug resistance
The ―abusive and intensive use of antibacterial drugs has dramatically increased the occurrence of microbial resistance and has led to an increase of difficult-to-eradicate infections. To solve the problem, combination therapy of two or more antimicrobial drugs has emerged some years ago, in the belief that they can achieve a reversal of microbial resistance with lower quantities of each substance and can also lower the known antimicrobial drugs‘ toxic side-effects. In spite of the many advantages of combination therapy, several reports have proven that it has failed in several patients11. The clinical development of the good combination therapy is expensive and drug-drug interactions may cause additive toxic side effects. Thus modification of the existing drugs through synthesis of analogues could be a noble solution. Combining two or more active pharmacophores into a single molecule
(analogue) can overcome combination therapy while enhancing overall potency12.
In previous years, the testing of combination of antimicrobial drugs and non-antimicrobial compounds (drugs not initially designed for this reason) appears to be another new promising strategy to overcome treatment failures12–15. Therefore, synthesis of a class of analogues/ hybrid compounds containing a natural product oleanolic acid which has antibacterial activities and other pharmaceutical compounds such as 4-aminoquinoline derivatives,
Curcumin, 4-aminosalicylic acid, Ergocalciferol etc can result in a class of potent compounds with effective antibacterial activities.‖
3
1.3. Justification of the study
According ―to the World Health Organisation (WHO), bacterial resistance to various antibiotics is one of the best challenges to human health in this century because of the rising hospitalization and death rates of patients infected with clinically antibiotic resistant around the world16.‖
Plants ―contain a variety of natural products whose chemical structures often unlikely to be formed in laboratories. The pharmacological activities of plants could be based on the antiviral, antibacterial, antipyretic effects of the active compounds in them17,18. The therapeutic effect of plants has encouraged scientists and drug specialists to do a thorough analysis of the plants and to build up a connection between phytochemicals and their therapeutic activities. In spite of the fact that it has been recognized that plant-based drug is one of the surest means of achieving total health care coverage of the world‘s population.
Many natural products have been studied for their effectiveness and safety but their modification to improve their effectiveness is still lacking. An assessment of all food and drug administration (FDA)-approved and new molecular entities (NMEs) shows that natural products and their derivatives are approximately 33 % of all NMEs19. One of the natural products under investigation is oleanolic acid (OA) isolated from syzygium aromaticum, this compound has been reported to have many medical applications. However, to the best information of this researcher, there is no recorded data about a class of analogues/hybrid compounds containing oleanolic acid and other pharmaceutical compounds (such as 4- aminoquinoline derivatives, curcumin, 4-aminosalicilic acid, etc).‖
Recently, plants have become a major significant source of various biofertilizers, herbicides, fungicides20–22etc. These bioactive compounds may be better than synthetic agrochemicals23 and could be significantly safer from health and environmental perspective. Considering this
4 consistently developing demand of phytochemicals and the varieties that exist among them, it is important to develop a new methodology for modifying these compounds to improve their pharmacological activity. Natural products have played a significant role in designing novel drugs due to their massive structural diversity24. The synthetic transformation of phytochemicals to improve their pharmacological activity has attracted many researchers in organic synthesis and medicinal chemistry25.
1.4. Null hypothesis.
Hybridization of oleanolic acid with selected pharmaceutical scaffolds via the C28 carboxylic acid can result in a class of potent compounds with effective antibacterial activities.
1.5.Aim and Objectives
To isolate and characterize OA from Clove flower buds.
To prepare a class of hybrid compounds with enhanced biological activities
containing OA and other pharmaceutical scaffolds (e.g. 4-aminoquinoline derivatives,
4-Aminosalicylic acid, curcumin, etc.) via the C28 carboxylic acid to form ester or
amide linkers
1.5.1. Overall objectives
To ―extract crudes from Syzygium aromaticum flower buds (clove) using different
solvents such as n-Hexane, Dichloromethane(DCM), Ethylacetate (EtOAc), and
Methanol (MeOH).
To isolate compounds from fraction extracts using Thin Layer Chromatography
(TLC) and Column Chromatography (CC).
5
To characterize the isolates and semi-synthesize the analogues using spectroscopic
methods, such as Mass spectrometry (MS), Melting point (m.p), Fourier-transform
infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR).
To evaluate the biological activities of oleanolic acid and its analogues.‖
6
1.6. References
1. Khazir J, Mir BA, Pilcher L, Riley DL. Role of plants in anticancer drug discovery.
Phytochem Lett. 2014;7(1):173-181. doi:10.1016/j.phytol.2013.11.010
2. Sasidharan S, Chen Y, Saravanan D, Sundram KM, Yoga Latha L. Extraction,
isolation and characterization of bioactive compounds from plants‘ extracts. African J
Tradit Complement Altern Med. 2011;8(1):1-10. doi:10.4314/ajtcam.v8i1.60483
3. Canter PH, Thomas H, Ernst E. Bringing medicinal plants into cultivation :
opportunities and challenges for biotechnology. Trends Biotechnol. 2005;23(4):180-
185. doi:10.1016/j.tibtech.2005.02.002
4. Akhtar MS, Hossain MA, Said SA. Isolation and characterization of antimicrobial
compound from the stem-bark of the traditionally used medicinal plant Adenium
obesum. J Tradit Complement Med. 2017;7(3):296-300.
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5. Croteau R, Kutchan TM, Lewis NG. Secondary Metabolites. Biochem Mol Biol Plants.
2000;7(7):1250-1318. doi:10.1016/j.phytochem.2011.10.011
6. Anulika NP, Ignatius EO, Raymond ES, Osasere O, Hilda A. The Chemistry Of
Natural Product : Plant Secondary Metabolites. Int J Technol Enhanc Emerg Eng Res.
2016;4(8):1-8.
7. Dixon RA. Natural products and plant disease resistance. Nature. 2001;411(June):843-
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8. Rishton GM. Natural Products as a Robust Source of New Drugs and Drug Leads: Past
Successes and Present Day Issues. Am J Cardiol. 2008;101(10):43-49.
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9. Gurib-Fakim A. Medicinal plants: Traditions of yesterday and drugs of tomorrow. Mol
Aspects Med. 2006;27(1):1-93. doi:10.1016/j.mam.2005.07.008
10. Gilani AH, Atta-ur-Rahman. Trends in ethnopharmacology. J Ethnopharmacol.
2005;100(1-2):43-49. doi:10.1016/j.jep.2005.06.001
11. Taganna JC, Quanico JP, Perono RMG, Amor EC, Rivera WL. Tannin-rich fraction
from Terminalia catappa inhibits quorum sensing (QS) in Chromobacterium
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Pseudomonas aeruginosa. J Ethnopharmacol. 2011;134(3):865-871.
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12. Zacchino SA, Butassi E, Liberto M Di, Raimondi M, Postigo A, Sortino M. Plant
phenolics and terpenoids as adjuvants of antibacterial and antifungal drugs.
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13. Bush K, Courvalin P, Dantas G, Davies J, Eisenstein B, Huovinen P, George A.
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14. Ejim L, Farha MA, Falconer SB, Wildenhain J, Coombes BK,Tyers M. Combinations
of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat Chem Biol.
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19. Patridge E, Gareiss P, Kinch MS, Hoyer D. natural products and their derivatives.
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doi:10.1016/j.bmc.2014.04.061.
10
CHAPTER TWO
LITERATURE REVIEW
2.1. Antibacterial resistance
Antimicrobial drugs have for quite some time been instrumental in combating infectious diseases such as cancer, antioxidants, diabetes, etc. ―since the first discovery of antibiotic, penicillin from the plant in 1928 by Alexander Fleming. Antibiotics have proven to be highly effective for the control of infectious diseases, however, the increase in the number of resistant pathogenic microorganisms to antibiotics in the past few years has gradually rendered conventional drugs less effective1. The misuse or abusive of these drugs has also aided in lowering antimicrobial effectiveness. As a result of the increase in resistance, many patients in clinics are being left untreated. According to WHO most people who are being disappointed by orthodox medicine often turn to medicinal plant remedies2.
There are three different types of antibacterial resistance, namely intrinsic, acquired and clinical resistance. Intrinsic resistance is seen when the pathogens are not exposed to the antibiotic. Acquired resistance occurs among the microbes that have been exposed to the antibiotic when managing the infection, and is always due to a genetic mutation. .Clinical resistance occurs when there is a failure in drug, which could be affected by many factors, including immune status of the patients, and the pharmacokinetics of the treatment or the species of pathogens being treated3,4. The emergence of resistance to conventional antimicrobial drugs has become a major problem to public health4. This global concern has led to efforts being directed to finding solutions to the problem of antimicrobial resistance.
One of the strategies to overcome this problem is the identification of new, more effective
11 antimicrobial alternatives. Many studies are thus, being directed towards discovering new antimicrobials, to which no resistance has developed.‖
The ―need for new antibiotic improvement to confront the threat imposed by resistant pathogens has become a major global concern for human health. Most antibiotics in clinical use today are derived from natural products5. To confront the challenge there is a need for the discovery and development of a new class of antibiotics.‖
2.2. Plant natural products
Therapeutic ―plants have shown the great potential of giving powerful or effective drugs for prevention and treatment of infectious diseases with antimicrobial drugs. As a result, a number of researchers are concentrating on plant natural products with the aim to discover novel drugs. Due to limited accessibility or high cost of many antimicrobial drugs in developing countries, most people in these countries rely on traditional medicinal plants6,7.
South Africa (SA) is one of the countries where a number of people rely on medicinal plant especially in rural areas. Hence, there is also a great need to validate how plant isolates and their analogues could lead to the discovery of potent and novel drugs.‖
Plants ―produce a large number of different compounds that seem to have no direct function in the development and growth of the plant, but instead they help the plant to adapt to its environment for the purpose of self-defense and to interact with other organisms in the environment8, these compounds are collectively referred to as natural products or secondary metabolites. Natural products involved in the protection against microorganism such as bacteria, viruses, and fungi, also, some plants use natural products as signals for communication between symbiotic microorganisms and plants, and attraction of seed dispersers and pollinators via colour and scent9,10. Plant natural products are classified into three chemically major groups: phenolics, terpenes, and alkaloids.‖
12
2.3. Classes of natural products
2.3.1 Phenolic Compounds
Phenolic ―compounds are the most widely distributed secondary metabolites, naturally present in the plant kingdom11. The consumption of a diet rich in phenolic compounds has been hypothesized to be important in health promotion and disease prevention in humans and animals12. Phenolic compounds are characterized as aromatic metabolites that have one or more acidic hydroxyl (OH) groups attached to the phenyl ring.‖
OH
2.1
Figure 2. 1: Phenol (parent structure of all phenolic compounds) Polyphenols ―can be divided into different classes depending on their number of carbon atoms and basic chemical structure. Table 2.1 shows the basic chemical structure of different polyphenolic compounds.‖
Table 2. 1: ―Examples of different major classes of phenolic compounds.‖
Class Basic Skeleton Number of C-atoms
Simple phenols, and C6 6 Benzoquinoline
Naphthaquinones C6 – C4 10
Anthraquinones C6 – C2–C6 14
Flavonoids C6 – C3–C6 15
Phenylpropenes (C6 – C5–C6)n n
13
Phenolic ―compounds possess various health benefits including treatment and prevention of many diseases such as cardiovascular disease, cancer, diabetes, and obesity13,14. Other phenolic compounds such as Caffeic acid(2.2) in figure 2 are known to be the strong antiviral15, antioxidant16 and anticancer17 agent. Eugenol (2.3) an isolate of syzygium aromaticum and many other plants also possesses antibacterial and antioxidant activities18,19.‖
O
OH O
HO HO OH 2.3 2.2
Figure 2. 2: Examples of phenolic compounds
2.3.2. Terpenes
The second ―class of natural products or secondary metabolites are the terpenes also known as terpenoids, these compounds are structurally characterized by the presence of at least one or more isoprene units (C5H10 = C5) which are normally joined in a head-to-tail manner. The fusion of isoprene units produces subfamilies of terpenes named as follows: monoterpenes
(C10), sesquiterpenes (C15), diterpenes (C20), sesterpenee (25), triterpenes (C30), and
20 tetraterpenes (C40) and polyterpenes (C5)n, n is the number of isoprene units greater than 8 .‖
14
2.3.3. Biosynthesis of terpenes
Despite the ―massive structural differences between terpenes, they are all derived from isoprene skeleton units. There are two precursors for the synthesis of terpenes which includes isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). They are synthesized through a different number of rearrangement, cyclization reactions and repeats.
Two different pathways for the formation of both IPP and DMAPP have been reported, 2C- methyl-D-erythritol-4-phosphate (MEP) pathway and the classical mevalonate (MVA) pathway The MVA pathway is present in the chloroplasts of plants. The MVA pathway
(Figure 2.3) includes seven enzymatic reactions to convert acetyl-CoA precursor to IPP and
DMAPP while the MEP pathway converts pyruvate and glyceraldehyde-3-phosphate, to
DMAPP and IPP through eight different enzymatic reactions21,22. The linear prenyl diphosphates: geranylgeranyl pyrophosphate (GGPP), farnesyl pyrophosphate (FPP), farnesyl geranyl pyrophosphate (FGPP) and geranyl pyrophosphate (GPP), are synthesized from the both basic building blocks, DMAPP and IPP where a group of enzymes known as prenyltransferases frequently add the isoprene unit IPP to (DMAPP) or a prenyl diphosphate in consecutive head-to-tail condensations leading to the production of a range of molecules with fixed lengths and stereochemistry. Geranyl pyrophosphate synthase (GPPS) and farnesyl pyrophosphate synthase (FPPS) catalyze the condensation of IPP and DMAPP to produce
GPP (C10) and FPP (C15). Geranylgeranyl pyrophosphate synthase (GGPPS) and farnesyl geranyl pyrophosphate synthase (FGPPS) are responsible for the formation of GGPP (C20) and FGPP (C25). The precursors GPP, FPP, GGPP and FGPP, are rearranged by different terpene synthase enzymes to produce the different classes of terpenoids23,24.‖
15
Mevalonate pathway Non-mevalonate pathway
Pyruvate + D-Glyceraldehyde 3-phosphate
Acetyl-CoA + Acetyl-CoA DXS Acetyl-CoA 1-Deoxy-D-xylulose 5-phosphate (DOXP) thiolase IspC Acetoacetyl-CoA HMG-CoA 2-C-Methyl-D-erythritol 4-phosphate (MEP) synthesis
3-hydroxy-3-methylglutaryl-CoA IspD HMG-CoA reductase 4-diphosphocyltidyl-2C-methyl-D-erythritol
Mevalonate IspE Mevalonate 4-diphosphocyltidyl-2C-methyl-D-erythritol- kinase 2-phosphate Mevalonate 5-phosphate IspF Phosphomevalonate kinase 2C-methyl-D-erythritol-2,4-cyclodiphosphate Mevalonate 5-pyrophosphate IspG Mevalonate ,,,,,,,,,,,,pyrophosphate 1-Hydroxy-2-methyl-2-butenyl 4-diphosphate ,,,,,,,,,,,,,,,decarboxylate
Isopentyl pyrophosphate (IPP) Diethylally pyrophosphate (DMAPP)
IDI
GPPS Monoterpene synthesis Monoterpenes GPP IPP FPPS Sesqueterpene & triterpene synthesis Monoterpenes FPP triterpenes IPP GPPS Diterpenes & Tetrapene synthesis Tetrapernes GGPP Diterpenes 16
Figure 2. 3: Biosynthesis of different classes‖ terpenes25. 2.3.4. Monoterpenes
Monoterpenes (C25) are commonly found in the essential oils and these compounds are essential for the flavouring, cosmetic, and pharmaceutical industries26–28. Figure 2.4 shows examples of monoterpenes include ―camphor (2.5), α-pinene (2.6), 1,8-cineole (2.7), limonene (2.8) and Perillyl alcohol (2.9), and carvone(2.10) and geraniol (2.11).‖
O
O
2.5 2.6 2.7
OH
O OH
2.8 2.9 2.10 2.11
Figure 2. 4: Examples of monoterpenes 2.3.5. Sesquiterpenes
Sesquiterpenes―(C15) are secondary metabolites which are commonly found in various plants with essential oils. Most sesquiterpenes are known to possess a number of biological activities such as germacrene D (2.12) which is known to have anti-inflammatory properties29,30. One of the important sesquiterpene compounds is artemisinin (2.13), an antimalarial drug obtained from Artemisia annua L. (Asteraceae)31,32. This plant has been used to treat malaria and fevers in China for many years33. Other examples of sesquiterpenes
17 shown in figure 2.5 include humulene (2.14), caryophyllene (2.15) lactone (2.16) and δ- cadinene (2.17).”
H
O O O H H O 2.14 2.12 2.13 O
H O
O H H 2.17 2.16 2.15
Figure 2. 5: Sesquiterpene compounds
2.3.6. Diterpenes
Diterpenes (C20) are one of the largest groups of non-volatile terpenes formed from the fusion of four isoprene units. Recent studies showed that oral administration of diterpene trans-retinoic acid (2.18) could be used as alternative treatment for skin flap necrosis in diabetic patients undergoing flap surgery34. Diterpenes like taxol (2.19) possesses some biological activities such as anticancer to treat breast, prostate and ovarian cancers. Taxol, and forskolin(2.20) the isolates of Pacific Yew (Taxus brevifolia) and coleus plant
(Plectranthus scutellarioides) respectively are common major diterpenes obtained from nature28,35. Diterpenes are divided into different major classes such as acyclic, bicyclic, tricyclic, tetracyclic and the macrocyclic diterpenes. Jatrophone (2.21), an isolate of elliptica
18
J. and Jatrapus (J.) gossypifolia is an example of macrocyclic diterpene and was reported to inhibit tumor cells36. ―
O OH
COOH O
OH 2.18 H 2.20 O
OH O O O O O
O O O O O H O O O O O 2.21 O O O O 2.19
Figure 2. 6: Examples of diterpene compounds
2.3.7. Triterpenes
Triterpenes (C30) are formed from the fusion of 6 isoprene units and widely distributed in nature including plants, animals, microorganisms, and humans. Triterpenes are one of the largest classes of secondary metabolites synthesized through cyclization of squalene37.
Structurally, terpenes have four 6-membered rings (A, B, C, D) and E being 5-membered or
6-membered ring and are divided into 6 different common subgroups as shown in figure 6: oleanane (2.22), ursane (2.23), friedelane (2.24), gammacerane(2.25) and lupine (2.26), hopane (2.27)38.
19
E E E C D C D C D
A B A B A B
2.22 2.23 2.24
E E E C D C D C D A B A B A B
2.25 2.26 2.27
Figure 2. 7: Different classes of triterpenes structures.
Triterpenes possessed various therapeutic properties, such as antiviral39,40, antimicrobial41,42, antioxidant43–45, and anti-inflammatory46,47. Examples of triterpenes include ursolic acid UA
(2.28), oleanolic acid OA (2.29), lupeol (2.30) and betulinic acid BA (2.31) as shown in
Figure 2.8. OA and BA acid are triterpenes commonly known to have an inhibitory effect on the human immunodeficiency virus (HIV) pathogen48,49.
20
OH OH
O O
HO HO 2.29 2.28
OH
O
HO 2.30 HO 2.31
Figure 2. 8: Examples of terpene compounds.
2.4. Role of plants in westernized medicine
Most common modern drugs were derived from plant sources, including quinine (2.32), aspirin (2.33), morphine (2.34), and atropine (2.35), and codeine (2.36). According to
Phillipson (2001), worldwide about 50% of clinically used top 20 drugs, are derived from plants. Until now, novel modern drugs are still being derived from plants sources. The number of phytomedicines entering the market increases worldwide50.
21
HO HO N O OH O N O O H N 2.32 O 2.33 HO 2.34
O
N OH
O O H 2.36 H N 2.35 O HO
Figure 2. 9: Common modern drugs derived from plant sources
2.5. Natural products as antimicrobial drugs
Bacterial ―resistance to antibiotics is rapidly emerging around the world, endangering the effectiveness of antibiotics and now causing a serious problem to human health51–53. The extensive use and abusive/misuse of antibiotics in developing countries has increased the occurrence of antibiotic resistance to make it a worldwide‖issue54,55. To confront this challenge an urgent development of novel drugs and antimicrobial strategies are needed.
There are different potential next-generation therapeutic strategies such as antibacterial peptides produced by microorganisms56, metal nanoparticles57, bacteriophages58 and plant natural products, such as terpenes, phenols, and alkaloids obtained in many plants52.
In order to find more effective drugs for microbial therapy, research has been directed to the medicinal plant. These plants have been proven over centuries to be successfully inhibiting infectious diseases among our ancestors, they provide a promising source of novel
22 antimicrobial drugs59,60. In many developing countries such as South Africa, therapeutic plants play an important role in human health and ―about 80% of people in developing countries use medicinal plants because of its cultural acceptability and affordability61,62. It has been reported that plant-derived antimicrobial drugs are rarely associated with side-effects, and furthermore have the ability to treat many types of diseases63. The use of the plant as antimicrobial treatment was first reported in Europe with the discovery of ―Iceman‖ the body of a human being that was preserved by bracket fungus64.‖
Plants ―have been known for centuries as a source of medicine to treat‖ microbial diseases and the use of the plant as a medicine is well documented. Rios and Recio 2005 also noted an increased number of research articles being published on the use of plant-derived antimicrobial drugs65. The main focus of many studies is on the determination of antimicrobial efficacy of the plants and also included the antimicrobial screening of plant extracts66,67 and essential oils68–70. Other studies have gone as far to test the isolated antimicrobial active compounds from plants against different pathogens, many of these studies are listed in article reviews by Cowan (1999)71 and Van Vuuren (2008)72and (2017)73.
Most plant-derived natural products such as pentacyclic triterpenes have a great potential to inhibit microbial pathogens as they are active against various bacterial species of both Gram- negative and Gram-positive and they directly target the cell envelope52. Triterpenes are one of the largest and structurally diverse groups of secondary metabolites with approximately 200 different compounds74. These compounds are commonly found in medicinal plants and food in free form/bound to glycosides. Triterpenes and their analogues have been studied for their antimicrobial75,76, anti-inflammatory77, antivirals78,79, and antioxidant characteristics80.
Chemical compounds obtained from plants have always been investigated by the pharmaceutical companies as agents of biological properties, or as models for the synthesis of novel drugs such as antimicrobials81.‖
23
2.6. Combinations of plant natural products with antimicrobial drugs
Most natural products were found to be synergistic enhancers for antimicrobial drugs, even if the plant natural compounds are non-antimicrobial82. Other studies have investigated the effects of combining plant natural products (e.i plant extracts and essential oils) and antimicrobial drugs such as cefuroxime (2.37), tobramycin (2.38), tetracycline (2.39), amphotericin B (2.40), and nystatin (2.41).
HO NH2
NH HO 2 H H S O NH2 N HO O H2N N O OH O O OH O 2.38 O NH2 2.37 H2N OH
OH OH O OH O OH O O OH OH H NH2 HO O OH OH OH OH O OH
OH O H H HO H N O O 2.39 2.40
HO OH OH NH OH 2 O OH H HO O OH OH OH OH O OH
O H O O 2.41
HO OH
NH2
Figure 2. 10: Antimicrobial drugs.
24
These were tested on a range of resistant microbes, such as methicillin-resistant
Pseudomonas aeruginosa and Staphylococcus aureus (MRSA)83–85, a synergistic interaction has been noted in most cases. Some of these studies have focused on antibiotic combinations with plant extracts of Rosmarinus officinalis L.86, Melaleuca alternifolia87,88, Origanum vulgare, Thymus vulgaris, Mentha piperita89and Syzygium aromaticum90. The synergistic effect is shown by a reduced minimum inhibition concentration (MIC) for the antimicrobial and the reduced MIC indicates an improved antimicrobial effect, which can finally render an ineffective antimicrobial drug to be effective again and this has resulted to define other plant extracts as resistance modifying ―agents91. Van Vuuren et al. (2011) and Adwan et al. (2010) argued that the enhanced effect made by plant extracts on antimicrobial drugs has been ignored and it needs further investigation92,93.‖
2.6. Oleanolic acid and its analogues
2.6.1. Oleanolic acid
Oleanolic acid (3b-Hydroxy-olea-12-en-28-oic acid) (2.29), is a bioactive natural compound found in various plants and foods, it belongs to the triterpene family of‖ compounds. The role of OA in plants is often associated with the prevention of pathogens and water loss76.
Oleanolic acid possesses various interesting biological activities, such as anti-inflammatory, analgesic47,94, antibacterial75,95, antiviral78,96, antitumor97 anti-cancer98–101, anti- oxidation102,103, and cardioprotective activities101. Chen et al. 2014 also reported that OA offers extraordinary protection against chronic and acute injury, and could be used in oral administration for human liver disorders104,105. Oleanolic acid has been isolated from more than 1600 various plant species76,104,106–108, and is moderately water soluble and non-toxic47.
Some medicinal plants containing oleanolic acid as their active constituent are demonstrated in table 2.2 below.
25
Table 2. 2: Some plant species where oleanolic acid was reported, their biological activities, and the plant parts used.
Plant Species (Family) Biological Activity Plant Parts Used References
Aceriphyllum rossii Stems, leaves, Cytotoxic Anticomplement activity 109,110 (Saxifragaceae) roots
Astilbe chinensis Cytotoxic rhizomes 111 (Saxifragaceae)
Baccharis uncinella Antileishmanial Aerial 112,113 (Asteraceae)
Fabiana imbricata R. et Antiviral, antitumor, and Leaves and 114–116 P. (Solanaceae) antihyperlipidemic flowers
Fructus Ligustri Lucidi Anti-hepatitis Leaves 96,117 (FLL)
Gentiana lutea Dried root and Antimicrobial 118 (Gentianaceae) rhizome
Anti-inflammatory, antioxidative, Ligustrum lucidum Ait antiprotozoal, antimutagenic, and Fruits and leaves 119 (Oleaceae) anticancer
L. camara Anti-inflammatory, antioxidative, Leaves and 119 (Verbenaceae) antiprotozoal flowers
Oleaeuropaea L. Anticancer, antimicrobial, anti-diabetic Fruits and leaves 76,102,120 (Oleaceae)
Phyllanthus amarus Anti-diabetes Leaves or aerial 121 (Phyllanthaceae)
Punica granatum L. Antioxidant activity Fruit 101,102 (Punicaceae)
Rosa laevigata Anti-inflammatory Leaves 122 (Rosaceae)
Rosmarinus officinalis Hepatoprotective, Anti-inflammatory, Leaves, flowers, 102 L. (Lamiaceae) gastroprotective, antiulcer stems, branches.
Siphonodon celastrineus Anti-inflammatory Root bark, stem 123,124 (Celastraceae)
26
Syzygium aromaticum Antinociceptive, Anti-inflammatory, Flower buds and 47,96,114,125 (Myrtaceae) antioxidant and antihypertensive, leaves
Viburnum chingii Antimicrobial Leaves 126 (Asteraceae)
Viscum album Anti-tumor, anti-inflammatory and Leaves and stems 119,127 (Santalaceae) analgesic
2.6.2. Analogues of OA
OA contains three(3) active sites (i.e., the hydroxyl, alkene, and carboxylic acid), which can be decorated in order to improve its biological effects122,128,129. Most derivatives of OA have been synthesized or tested for many biological activities130. OA is a good precursor molecule for synthesis due to its various biological activities, availability, and low production cost131.
Nkeh-Chungag et al 2015 reported ―the acetylation and methylation of OA initially isolated from Syzygium aromaticum (clove), which resulted to the formation two compounds (3- acetoxyoleanolic acid and 3-acetoxy, 28-methyloleanolic acid) as shown in figure (2.11).
Both compounds exhibited better membrane-stabilizing properties and anti-inflammatory properties when compared to OA77.
Compound R OR H 2.42 CH O O 3 H O 2.43 H H
Figure 2. 11: 3-acetoxy, 28-methyloleanolic acid (2.42), 3-acetoxyoleanolic acid (2.43).
27
Modification of a compound OA has resulted in compounds with various biological activities such as ant-diabetic, antiviral antimicrobial activities, etc. Yolanda et al. 2014 synthesized derivatives of OA (3 ethers and 4 esters on C3 in ring A, 3 esters from C-28 and corresponding primary alcohol) by reduction of carboxylic acid (COOH) with LiAlH4.
Cinnamoyl ester (2.49) and ethyl ether (2.53) (Figure 2.12) were found to be the most PTP-
1B inhibitors. The in vitro inhibitory effect of compound (2.53) was significant and it substantially lowered blood glucose levels in vivo experiments when compared to OA.
Compound (2.53) exhibited better inhibitory activity and selectivity over the protein-tyrosine phosphatase 1B (PTP-1B) with advanced interaction with site B, in accordance with docking studies121,132.
Compound R1 R2
2.44 COCH3 H
2.45 H CH3
2.46 O H
2.47 H CH2CH3
2.48 CO(CH2)2CH3 H
2.49 O H
2.50 H H2C
28
2.51 H H
2.52 CH3 H
OR2 H 2.53 CH2CH3 H
O H 2.54 (CH2)2CH3 H
R1O H
Figure 2. 12: Oleanolic acid derivatives studied for PTP-1B inhibition.
The modification of oleanolic acid also resulted in potent antibacterial agents. Hichri et al. explored the effect of introducing an acyl substituent at the hydroxyl C-3 in ring A of OA. A sequence of diverse triterpenic acid esters was prepared from oleanolic acid using suitable cyclic anhydrides, acid chlorides and N,N-dimethyl-4-aminopyridine (DMAP) as a catalyst
(table 2.3)118.
Oleanolic acid and its acylated analogues were screened for their antimicrobial activity against five fungal plant pathogens, two Gram-positive and two Gram-negative bacteria.
Compound (2.55) with sulfur and chlorine atom(s): ((3b)-3-((thiophene-2-carbonyl)oxy)- olean-12-en-28-oic acid, was found to be an effective antibacterial agent and the most active antifungal compound. It exhibited good activity against A. niger, P. italicum, P. digitatum, A. flavus, and T. harzianum.
H H OH OH O H O X /DMAP H HO H RO H
29
Table 2. 3: Synthesis of Oleanolic acid.
Compound R X Yield(%)2
O 2.55 RCl 98 S
Cl O 2.56 RCl 94 Ph
Cl O 2.57 RCl 91
O Cl 2.58 RCl 95 Cl
2.59 RCl 94
O O 2.60 O O 92 HOOC
O O 2.61 82 O COOH O
O O 2.62 O 91 COOH O
O 2.63 85 HOOC
30
Synthesis of oleanolic acid, as well as other closely-related triterpenes, such as betulinic acid and dihydrobetulinic acid, has led to anti-HIV agents48,79,133 . Zhu et al. synthesized derivatives of OA, These authors modified the C12-C13 double bond of OA yielding compound 2.64, which was 3-fold more active than OA. Esterification of 2.64 with anhydrides resulted in compounds 2.65–2.67, which were 5-fold more active than OA with
2.67 showing remarkable activity49.
Compound R
2.64 H
H
2.65 HOOC H COOH O
2.66 HOOC
RO O
2.67 HOOC O
Figure 2. 13: Oleanolic derivatives with anti-HIV activity
31
Compound 2.64 was further modified by converting the C28-carboxyl group to an aminomethyl group, resulting in compounds 2.68 and 2.69, which were greater than 10-fold more active when compared to OA (Figure 2.13).
Compound R1 R2
H R2 2.68 HO O O HN O O OH
R1O
2.69 HO O O O O HN OH
Figure 2. 14: Derivatives of oleanolic acid with anti-HIV activities.
Yu et al. in their structure-activity relationship study of effective anti-HIV agents synthesized and evaluated new triterpene derivatives in vitro for antiviral activity. OA analogue compound (2.70) was inactive, while OA derivative (2.71) exhibited an EC50 value of 0.32
μM, indicating that OA is a promising anti-HIV inhibitor49,134. These compounds are potential therapeutics that would benefit from further studies in vivo (Figure 2.15).
Compound R OH 2.70 O O O OH H O RO O O O H 2.71 OH
Figure 2. 15: Previously modified anti-HIV triterpene derivatives.
32
Kashiwada et al. prepared several 3-O-acyl-ursolic acids and evaluated their anti-HIV
135 activity. The most potent compound indicated an EC50 value of 0.31 µM and a TI of 155.5 .
In another report by Kashiwada et al., OA derivatives inhibited HIV-1 replication in acutely infected H9 cells. OA-triterpenes isolated from the leaves of S. claviflorum exhibited potent anti-HIV activity, which further revealed the potential of OA derivatives for the treatment of
HIV136,137.‖
33
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51
CHAPTER THREE
PHYTOCHEMICAL EXAMINATION OF SYZYGIUM AROMATICUM (L.) MERR. &
PERRY
3.1. Introduction
Syzygium ―aromaticum (L.) Merr. & Perry belongs to the Myrtaceae family and is an evergreen tree that grows up to a height of 8-12 meters and is native in Maluku Island of
Indonesia1–4. This plant is also cultivated in many tropical countries such as India, Malaysia,
Zanzibar, and Sri Lanka5. The flower buds of this tree are commonly known as clove and used as a spice. Figure 3.1 shows the native and exotic areas of the plant Syzygium aromaticum.‖
Figure 3. 1: World‘s distribution of Syzygium aromaticum (clove)6
52
3.2. Medicinal use of Syzygium aromaticum.
The ―plant is used as a folk medicine in China and in many western countries against various diseases, such as oral diseases and dental complaints7. The plant is used to treat nausea and vomiting, diarrhea, cough, dyspepsia, stomach distension, flatulence, and gastrointestinal spasm8. According to Pandey and Singh 2011, the cloves are also used for stomachic, tonicardiac, and stimulant effects9. Essential oil obtained from this plant add aroma to the food and serves as a dietary antioxidant which should prevent some diseases caused by free radicals10. The plant is also known to have antibacterial activities and is used in many dental creams, mouth washes, tooth pastes, and throat sprays to cleanse bacteria4,11,12. Many studies revealed it to possess a variety of other biological activities such as anticancer5,13, antioxidant4,14, antifungal15,16, and antidiabetic17. Cloves contain a number of phytochemical compounds such as tannins, sesquiterpenes, Sterols, Flavonoids and triterpenoids11,18.‖
A B
Figure 3. 2: Fresh (A) and dried (B) Syzygium aromaticum6
53
3.3. Chemistry of Syzygium aromaticum.
Syzygium ―aromaticum is one of the major sources of phenolic compounds, like flavonoids, hydroxybenzoic acids, hydroxyphenyl propens and hydroxycinnamic acids, as well as terpenoids19,20. Eugenol constitutes 72 to 90% of the volatile oil in clove and is responsible for the aroma of cloves20. Other common constituents of clove essential oil include β- caryophyllene(3.1), eugenyl acetate(3.2), pinene(3.3), methyl salicylate(3.4), vanillin(3.5) and α-humulene (3.6)10,21.‖
O
O
3.1 3.2 3.3
H O
O
O O OH OH 3.4 3.5 3.6
Figure 3. 3: Clove‘s oil constituents.
54
3.4. EXPERIMENTAL
3.4.1 Plant Identification
―The dried flower buds of Syzygium Aromaticum (L.) were purchased from the spice market in Durban, South Africa. The taxonomic identification of S. aromaticum (L.) was done in the
School of Biological and Conservation Sciences, University of KwaZulu-Natal, Westville
Campus. The voucher specimen number OO4 was deposited at the University Herbarium.‖
3.4.2. Plant preparation and solvent extraction
―The plant material was grounded to a powder form using mortar and pestle. A dried powdered sample (500g) was taken into a 5000 ml Erlenmeyer flask, and sequentially extracted with 2000 ml organic solvents of different increasing polarity namely, n-hexane, ethyl acetate, dichloromethane and methanol. The mixture was placed on a rotating shaker for a period of about two weeks twice per solvent. The supernatant was filtered using filter paper
(Whatman size: 32.0 cm), the filtrate was concentrated on a rotary evaporator at reduced pressure, the concentrated extracts were collected in a pre-weighed beaker and allowed to air dry for complete evaporation of the extracting solvents then air dried in the fume hood. After drying, the weight and the percentage yield of each extract was determined.‖
3.4.3. Isolation method
―The ethyl acetate crude extract (15.535 g) was taken for further purification since the previous studies showed that it contains the mixtures of Maslinic and Oleanolic Acid22. The
Ethyl Acetate crude extract was purified by column chromatography using silica gel (Merck, silica gel 60 F254: 0.063-0.200 mm). The column was eluted with series of solvent systems: n- hexane: ethyl acetate (9:1, 8:2, 7:3 and 6:4) respectively. All fractions were collected and visualized on a Thin Layer Chromatography (TLC) plate with Anisaldehyde/sulphuric acid
55 spray reagent for spot development. All the fractions with similar behaviour on TLC plate, such as fraction 6-33, fraction 40-120, and fraction 123- 173 were combined together and further purified. Combination and purification of these fractions afforded a pale yellow oil pure compound labelled VK121, and two white powder pure compounds labelled VK122 and
VK123.‖
Solvent from
Origin
R vk121 vk122 vk123 CcC3 TLC plate showing the isolated compounds
Figure 3. 4: TLC plate showing the isolated three compounds from ethylacetate crude extract.
―The TLC plate reveals a mixture of compounds, which exhibited different colours when reacting to the anisaldehyde/H2SO4 spraying reagent. The classes of the isolated compounds include the terpenoids, which are bluish or purple in colour when spotted on a TLC plate
(VK122 and VK123)23. The isolated compounds labelled (VK121, VK122 and VK123) were characterized for structural elucidation using Mass Spectrometry, melting point (m.p),
Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectrometry (NMR). The retardation factor (Rf value) of all the isolated compounds was calculated using the formula in figure 3.5.‖
56
Distance travelled by compound
Rf value = Distance travelled by Solvent (Solvent Front)
Figure 3. 5: Formula for calculating Rf value of a compound.
3.4.4. 13C and 1 H NMR spectroscopic analysis
1H and 13C NMR experiments were performed at 400.0 MHz for hydrogen and 13C, using
CDCl3 as solvents. Solutions were prepared with between 15–20 mg of pure compounds in
0.5 ml of CDCl3.
57
3.5. Results
The table below shows the physical state and the % yields of the different crude extracts from cloves of syzygium aromaticum.
Table 3. 1: Extraction results.
Extracting solvents Physical state % Yield
Hexane Dark brown oil 15.6
Ethyl acetate Light orange powder 4.7
DCM light brown oil 1.52
Methanol Dark brown oil 9.7
3.5.1. Physical properties of compound (VK121)
Physical state: Pale yellow liquid.
% Yield: 0.42
Molecular weight:
IR vmax: OH stretch at 3452.37, C-H stretch at 2975 & 2938, ar C=C at 1511.36, C-O at 1033.59.
O 7 2 HO 1 3
6 4 9
5 8 10 Eu
Figure 3. 6: Structure of Eugenol (Eu).
58
Table 3. 2: 1H and 13C-NMR data of Compound VK121 compared with literature data24.
Position δCLit δHLit δCEu δHEu
1 144.3 ---- 142.7 ----
2 114.7 ---- 151.3 ---
3 121.7 6.74(s) 114.5 6.40(s)
4 138.1 ------131.1 ----
5 115.6 6.76 (d, 8.6 Hz) 122.8 6.45(d, 8 Hz)
6 146.9 6.92 (d, 8.6 Hz) 116.8 6.50(d, 8 Hz)
7 111.7 3.86 (s) 56.2 3.73(s)
8 56.3 3.38 (d, 6.6 Hz) 48.4 3.22 (d, 8 Hz)
9 40.3 5.96 (m) 136.5 6.30
10 132.1 5.12(d, 19.7 Hz) 117.2 4.96(d, 8)
5.19(d, 19.7 Hz) 4.93(d, 8)
OH 4.89(s) 5.0(s)
59
3.5.2. Physical properties of compound (VK122)
Physical state: White powdery
% Yield: 0.17
Melting point: 240 oC
Molecular weight: 456.3598
IR vmax: OH stretch at 3443, C-H at 2941 & 2870, C=O at 1693, ar C=C at 1468, C-O at
1031-998.
30 29
19 20 21 12 18 17 22 11 OH 25 26 13 1 9 14 16 28 2 O 10 8 15 3 5 4 HO 7 27 OA 6 23 24
Figure 3. 7: Structure of oleanolic acid (OA).
Table 3. 3: 1H and 13C-NMR data of Compound VK122 compared with literature data25.
25 25 Position δCOA δHOA δC δH
1 33.64 0.99, 1.62 38.37 0.98, 1.62
2 27.48 1.56, 1.60 27.15 1.56, 1.60
3 79.03 3.22(dd, 12, 4 Hz) 79.01 3.20(dd, 12.0, 4.3 Hz)
60
4 41.25 ------38.74 -----
5 55.24 0.81 55.18 0.75
6 22.97 1.36, 1.55 18.27 1.38, 1.54
7 35.3 1.29, 1.36 32.59 1.29, 1.44
8 39.9 ---- 39.23 -----
9 49.8 1.54 47.6 1.54
10 37.03 ------37.05 -----
11 25.88 0.91:1.79 23.37 0.91, 1.88
12 123.07 5.35(t, 4 Hz) 122.61 5.28 (t 3.6 Hz)
13 143.22 ----- 143.26 -----
14 41.25 ----- 41.59 -----
15 29.71 0.99, 1.63 27.66 1.10, 1.72
16 27.48 1.61, 1.92 22.91 1.61, 1.97
17 55.24 ------46.47 ------
18 41.78 2.51 40.98 2.81
19 47.64 1.15, 1.63 45.84 1.16, 1.63
20 30.67 ------30.66 ------
21 39.43 1.22, 1.23 33.77 1.22, 1.33
22 31.45 1.57, 1.63 32.41 1.58, 1.77
23 18.43 0.87 28.08 0.98 s
24 18.32 0.78 15.52 0.75 s
25 23.46 0.91 15.52 0.91 s
26 17.18 0.81 17.06 0.77 s
27 23.63 1.21 25.91 1.13 s
28 172.93 ------182.66 ------
61
29 27.19 0.89 33.05 0.92 s
30 27.48 0.89 23.66 0.90 s
3.5.3. Physical properties of compound VK123
Physical state: white powder
Yield: 220 mg
Melting point: 233 oC
Molecular weight: 473.0778
IR vmax: OH stretch at 3444, C-H stretch at 2942 & 2895, C=O at 1695, ar C=C at 1468,
(COOH) at 1746, C-O at 1032cm-1.
30 29
19 20 21 12 18 17 22 11 OH 25 26 13 1 HO 9 14 16 28 2 O 10 8 15 3 5 4 HO 7 27 MA 6 24 23
Figure 3. 8: Structure of Maslinic acid. Table 3. 4: 1H and 13C-NMR data of Compound VK123 compared with literature data
26,27 27 Position δCMA δHMA δCLit δHLit
1 46.01 0.92, 1.99 46.2 0.91: 1.99
2 68.47 3.21(m) 68.3 3.70(m)
3 83.46 3.09 (d,12 Hz) 82.3 3.01 (d, 9.5 Hz)
4 39.10 ------39.1 -----
62
5 55.01 0.85 55.1 0.85
6 18.12 1.36, 1.55 18.27 1.40, 1.55
7 32.73 1.23, 1.34 32.59 1.32, 1.46
8 39.05 ---- 39.23 -----
9 47.45 1.62 47.6 1.62
10 38.03 ------37.05 -----
11 23.25 2.04:1.79 23.37 0.90, 1.95
12 121.92 5.35(t, 4 Hz) 122.61 5.30(t, 3.65 Hz)
13 143.71 ----- 143.26 -----
14 41.63 ----- 41.59 -----
15 27.4 1.09, 1.61 27.66 1.09, 1.71
16 23.0 1.62, 1.99 22.91 1.62, 1.99
17 46.43 ------46.47 ------
18 41.0 2.51 40.98 2.88
19 45.74 1.63, 1.65 45.84 1.63, 1.66
20 30.67 ------30.66 ------
21 33.64 1.22, 1.33 33.77 1.22, 1.35
22 31.45 1.57, 1.60 32.41 1.59, 1.78
23 28.3 0.80 28.08 1.03 s
24 16.67 0.83 15.52 0.83 s
25 16.48 0.99 15.52 0.99 s
26 16.18 0.90 17.06 0.77 s
27 23.2 1.15 25.91 1.14 s
28 178.5 ------183.66 ------
29 32.71 0.85 33.05 0.91 s
63
30 23.147 0.85 23.28 0.93 s
3.6. Discussion
3.6.1. Compound (VK121)
The Rf value of compound VK121 was determined using a TLC plate and hexane-ethyl acetate (7:3 v/v) as mobile phase. The Rf value (0.75) of VK121 was calculated by the formula described in Figure 3.5.
―FT-IR spectrum of compound VK121 (figure 3.9 APPENDIX ONE) indicated a broad peak
1 -1 2 around 3536-3469 cm- (OH stretch) and two aliphatic peaks at 3084-3012 cm (-CH sp
-1 3 -1 stretch), 2926-2856 cm (-CH sp stretch), 1513-1203 cm (-C=C, aromatic stretch), 1610 cm-1 (C=C alkene), 1267 cm-1 (C-O ether) and 1034 cm-1 (C-O alcohol).
For NMR analysis, compound VK121 was dissolved in spectroscopic grade deuterated
1 13 1 chloroform (CDCl3) to record H & C NMR Spectra. H NMR spectrum (figure 3.10,
APPENDIX ONE) showed various signals with OH proton at 5.01 (1H, s), aromatic protons ring at 6.40 (1H, s), 6.45 (1H, d), 6.50 (1H, d), two doublets at 3.22 (2H, d), 4.93 and 4.96 (1H, each d), a singlet with three protons at 3.73 (3H, s) which belongs to the methoxy group (-OCH), and a multiplet with one proton at 6.30 (1H, m).
13C NMR spectrum (figure 3.11, APPENDIX ONE) indicated 10 different signals which correspond to 10 carbon atoms. It indicated a signal at 142.7 ppm for C attached to hydroxyl
(OH), five aromatic carbons at 116.8, 122.8, 131.1, 114, 151.3, alkane carbon at 48, 56.2 (C-
O), and two carbons for alkene at 136.5, 117.2. The complete 1H and 13C NMR data is given in Table 3.2 which correlated well with the literature reported by Kumar L 2010 thus
Compound VK121 was identified as eugenol. The structure of eugenol is shown in Figure
64
3.6. The LC-MS results =264.0616 in figure 3.12 APPENDIX ONE are in good agreement with the theoretical molecular weight of eugenol.‖
3.6.2. Compound (VK122)
―Compound (VK122) was obtained as white powder with Rf value of 0.45, and a m.p. of 240 oC. The FT-IR spectrum (Figure 3.13, APPENDIX TWO) showed the presence of hydroxyl group (-OH) at 3443 cm-1, a carbonyl group at 1693 cm-1(-C=O), 2941 - 2870 cm-1 (-CH),
1468 cm-1 (-C=C) and 1031-998 cm-1 (-C-O).
The 1H NMR spectrum (figure 3.14, APPENDIX TWO) of compound VK122 showed signals of an olefinic proton at 5.35 cm-1 and seven methyls at 0.87, 0.78, 0.91, 0.81, 1.21,
0.89 and 0.89 which confirmed the characteristics of the oleanane skeleton28. This structural type was further supported by the 13C NMR spectrum (figure 3.15, APPENDIX TWO), which also contained resonance for olefinic carbons at (143.22 and 123.07 ppm) and one oxygenated carbons at 79.03 ppm and a carboxylic acid at 172.93 ppm. Compound VK122 was identified as oleanolic acid. The complete 1H and 13C NMR data is given in Table 3.3 and APPENDIX TWO, Full characterization of compound VK122 was done by comparison with literature reported data for oleanolic acid. The LC-MS results =456.3598 in figure 3.16
APPENDIX TWO are in good agreement with the theoretical molecular weight of oleanolic acid. The structure of VK122 is given in figure 3.7.‖
3.6.3. “Compound (VK123)
―Compound VK123 with Rf value of 0.14 was also isolated as a white powder. The spectral data of this compound were almost similar to that of compound VK122 however; 13C NMR
(figure 3.18, APPENDIX THREE) of VK123 indicated an extra oxygenated carbon signal at
68.47 ppm due to the presence of –OH group at C2 of its chemical structure. The FTIR
65 spectrum (figure 3.16, APPENDIX THREE) also indicted peaks of hydroxyl group (OH), carbonyl(C=O) and alkene at 3412, 1690 cm-1 and 1468 cm-1 respectively.
The 13C NMR spectrum exhibited signals for 30 carbon atoms with one carbonyl at 178.4 ppm, two olefinic carbons at (121.92 and 143.71). The 1H NMR spectrum (figure 3.16,
APPENDIX THREE) showed an olefinic proton signal at 5.50 cm-1. The complete 1H and 13C
NMR data is given in Table 3.3 and APPENDIX THREE. Compound VK123 was identified as maslinic acid by comparison of the obtained 1H and 13C NMR spectra with those of maslinic acid reported in literature and LC-MS confirmed the molecular weight=473.0778.
The structure of VK123 is given in figure 3.8.‖
66
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14. Sobeh M, Esmat A, Petruk G, Abdelfattah MAO, Dmirieh M, Montic DM, Abdel-
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70
CHAPTER FOUR
SYNTHESIS OF OLEANOLIC ACID ANALOGS
4.1. Materials
―All chemicals and―reagents used were purchased from Sigma-Aldrich and used without further purification. The solvents used for synthesis were of high grade solvents and they were dried over molecular sieves with pore size of 4 Å and particle size of 4-8 Mesh.‖
4.2. Characterization
―The fingerprint identification of specific functional groups was achieved by assigning of group frequencies (cm-1) of particular peaks within the IR spectra. The IR spectra were obtained from
Perkin Elmer model 100 Hz and the percentage (%) transmittance was recorded against the wave length (cm-1) for the reported signals in the range of 4000- 400 cm-1.
The 1H and 13C NMR spectra were recorded on Bruker Nuclear Magnetic Resonance
Spectrometer 400 MHz at room temperature using deuterated CDCl3. The chemical shifts (δ) are reported in ppm relative to internal solvent peaks and the coupling constants (J) were measured in Hertz (Hz). The 1H signals splitting pattern abbreviation were reported in the following order: singlet (s), (doublet), triplet (t), quartet (q), doublet of doublet (dd), and multiplet (m).
The high resolution mass spectra were recorded on Bruker Compact Liquid Chromatography
Mass Spectrometry (LC-MS).
71
Finally, the melting points (m.p) were determined on a variable heat Gallenkamp melting point apparatus (temperature range 50–350 °C) equipped with the laboratory thermometer.‖
4.3. Introduction
―The synthesis of hybrid compounds containing oleanolic acid and other pharmaceutical scaffolds was performed by first synthesizing 4-aminoquinoline derivatives by amination reaction of either amines or amino alcohols with 4.7-dichloroquinoline resulting in compounds with targeted functional groups. The synthesis of 4-aminoquinoline derivatives was carried out via a nucleophilic substitution on the suitably substituted 4.7-dichloroquinoline scaffold. This was achieved by using amines and amino alcohols as nucleophils.‖
R NH2
Cl H R Cl N R HN -H -Cl
Cl N Cl N Elimination of a leaving group Cl N H H neuclephilic addition B on Cloroquinolium ring
Scheme 4. 1: N-alkylation of 4.7-dichloroquinoline mechanism. The hybrid compounds containing oleanolic acid were then prepared from the reaction of oleanolic acid with isolated 4-aminoquinoline derivatives and other selected organic compounds such as curcumin or ergocalciferol via amidation or esterification reactions. The reactions were performed at 75-95oC and monitored by TLC plate to ensure the completion of reaction. The synthesized hybrid compounds were further purified by column chromatography and characterized by using FT-IR, NMR, m.p, and LC-MS.‖
72
4.3. Synthesis of 4-aminoquinoline derivatives.
―Chloroquine is a 4-aminoquinoline ―derivative with a wide range of pharmacological activities such as, anti-malarial1, anti-bacterial2, clonorchis sinesis, anti-fungal3, and rheumatoid arthritis.
Chloroquine derivatives with various alkyl amino side chain were synthesized by one-step substitution reaction of an appropriate amine and 4,7-dichloroquinoline. Some studies have proven that lengthening and shortening of the linker of the alkyl amine side chain in chloroquine leads to compounds that remain effective against drug-resistant strains4.‖
4.4. Synthesis of N-(3-aminopropyl)-7-chloroquinolin-4-amine (1.3 PDA-Q)
HN Cl H N NH Cl Cl 2 2 NH2 900C 24 hr N N N-(3-aminopropyl)-7-chloroquinolin-4-amine
Scheme 4. 2: synthesis of N-(3-aminopropyl)-7-chloroquinolin-4-amine. ―A solution of 4,7-dichloroquinoline (1 g, 5.05 mmol) in 1,3-diaminopropane(1.3 PDA) (1.9 mL, 22.7 mmol) was refluxed for 24 hours at 90 oC with stirring, and the reaction was monitored by TLC using methanol: Triethanolamine (TEA): hexane (6:3:1) as eluent solvent system to ensure the completion of reaction, then allowed to cool at room temperature. The mixture was diluted with 60 mL dichloromethane (DCM) (20 mL x 3) and the resulting mixture was successfully washed with sodium hydroxide (NaOH) (1 M, 10 mL x 3) followed by 10 mL brine, to give an aqueous layer, an organic layer, and a white course particulate precipitate. The organic layer was filtered through a cotton plug and concentrated in a rotary evaporator. N-(3-
o aminopropyl)-7-chloroquinolin-4-amine (Yield 62 %, m.p 129-131 C, Rf value 0.34) was
73 obtained as pale yellow crystals. The physical characteristics were almost the same as previously described by Sunduru et al., 20095.
The FTIR spectrum showed the successful synthesis of N-(3-aminopropyl)-7-chloroquinolin-4- amine by indicating the peaks of N-H stretch for secondary and primary amine at 3357 cm-1,
3279 cm-1 respectively, CH stretch at 2947 cm-1, C-Cl stretch at 601 cm-1 and C=C stretch for aromatic at 1582 cm-1 .‖
95
85
75
65 Transmitance % 55
45
35 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1
Figure 4. 1: ―IR spectrum of N-(3-aminopropyl)-7-chloroquinolin-4-amine.”
74
4.5. Synthesis of N-(2-aminopropyl)-7-chloroquinolin-4-amine (1.2 PDA-Q)
Cl HN H2N NH NH2 2
0 Cl N 90 C 24 hr Cl N
N-(2-aminopropyl)-7-chloroquinolin-4-amine
Scheme 4. 3: Synthesis of N-(2-aminopropyl)-7-chloroquinolin-4-amine. ―A mixture of 4,7-dichloroquinoline (1 g, 0.05 mmol) and 1,2-diaminopropane (1.9 mL, 22.7 mmol) was heated to reflux at 90 oC for 24 hours with stirring, and the reaction was monitored by TLC using MeOH: TEA: EtOAc (6:3:1) as eluent solvent system to ensure the completion of reaction, then allowed to cool to room temperature. The mixture was diluted with 60 mL DCM
(20 mL x 3) and the resulting mixture was successfully washed with sodium hydroxide (NaOH)
(1 M, 10 mL) and brine (10 mL), to give an aqueous layer, an organic layer, and a white course particulate precipitate. The organic layer was filtered through a cotton plug and concentrated in a rotary evaporator. N-(2-aminopropyl)-7-chloroquinolin-4-amine (Yield 66 %, m.p 128–130 oC,
Rf value 0.64) was obtained as pale yellow crystals.
The FTIR spectra for N-(2-aminopropyl)-7-chloroquinolin-4-amine molecule, peaks that were observed are N−H stretch visible at 3366 cm-1, C−H stretch alkane at 2901 cm-1, C=C stretch aromatic at 1587 cm-1 and C−Cl stretch at 756.5 cm-1 respectively which confirmed the successful linkage of ethyl diamine to the quinoline moiety.‖
75
EDAQ 100
90
80
70
60
Transmittance (%) Transmittance 50
40
30
4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1
Figure 4. 2: IR spectrum of N-(2-aminopropyl)-7-chloroquinolin-4-amine (1.2 PDA-Q)
4.6. Synthesis of N-(2-(2-Aminoethylamino) ethyl)-7-chloroquinoline-4-amine (DET.Q)
H Cl N H HN NH2 N H2N NH2 850C 24 h Cl N Cl N N-(2-(2-Aminoethylamino) ethyl)-7-chloroquinoline-4-amine
Scheme 4. 4: Synthesis of N-(2-(2-Aminoethylamino) ethyl)-7-chloroquinoline-4-amine
76
―N-(2-aminopropyl)-7-chloroquinolin-4-amine was prepared using a modified procedure described by Musonda at al 20066. A solution of 4,7-dichloroquinoline (1.00 g, 5.05 mmol) in diethelenetriamine (DET) (1.05 g, 10.1 mmol) was heated to reflux at 85°C for 24 hours with stirring, the reaction was monitored by TLC using MeOH: TEA: Hex (6:3:2) as eluent solvent system to ensure the completion of reaction, then allowed to cool to room temperature. The mixture was basified with 20 ml 1 M NaOH, extracted with 60 ml hot EtOAc (20 ml x 3) and the resulting mixture was successfully washed with Na2SO4 (10 ml). The separated organic layer was filtered and concentrated in a rotary evaporator. N-(2-aminopropyl)-7-chloroquinolin-4-
0 amine (Yield 85 %, m.p 112–114 C, Rf value 0.56) was obtained as yellow crystals.
The FTIR spectra showed a successful synthesis of N-(2-(2-Aminoethylamino) ethyl)-7- chloroquinoline-4-amine (DET-Q) by showing the visible peaks of N-H stretch at 3302 cm-1, C-
H stretch for alkene at 3005 cm-1, C-H stretch at 2947, cm-1, C=C for the aromatic at 1579 cm-1 and C-Cl at 647 cm-1 . The obtained IR results were in good agreement with results reported in literature6,7.‖
100
90
80
70
60
50
40 Transmitance %
30
20 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1 Figure 4. 3: IR spectra of N-(2-(2-Aminoethylamino) ethyl)-7-chloroquinoline-4-amine.
77
4.7. Symthesis of 1-(7-Choloroquinolin-4-yl) hydrazine (HYD-Q)
NH2 Cl HN
H2N NH2 950C 24 h Cl N Cl N 1-(7-Choloroquinolin-4-yl) hydrazine
Scheme 4. 5: Synthesis of 1-(7-Choloroquinolin-4-yl) hydrazine (Hyd-Q)
A solution of 4.7- dichloroquinoline (1g, 5. 05 mmole) and Hydrazine (0.75 ml, 30.30 mmole) in
3 ml ethanol as solvent was refluxed at 95 oC for 24 hr. the reaction was monitored by TLC using toluene: EtOAc: meOH (6:3:1) as eluent solvent system to ensure the completion of reaction, the obtained solution was filtered and allowed to dry in fume-cardboard then later it was recrystallized with 10 ml ethanol. 1-(7-Choloroquinolin-4-yl) hydrazine (Yield 72 %, m.p 129–
0 132 C, Rf value 0.64) was obtained as pale yellow crystals. The FTIR spectrum shown in figure
4.4 confirmed a successful synthesis of 1-(7-Choloroquinolin-4-yl) hydrazine by indicating N-H stretch for secondary amine at 3278 cm-1, C-H stretch for alkene at 3120 cm-1, C-H stretch for alkane at 2945 cm-1, C=C 1550 cm-1 for aromatic and also C-Cl for alkyl halide at 643 cm-1. The obtained results of 1-(7-Choloroquinolin-4-yl) hydrazine were in good agreement with results reported by Pretorius et al 20138.
78
90 85 80
75 70 65 60
55 transmitance% 50 45 40 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1
Figure 4. 4: FT-IR spectrum of 1-(7-Choloroquinolin-4-yl) hydrazine
4.8. Synthesis of 2-(7-chloroquinolin-4-ylamino) ethanol (EA-Q)
OH Cl HN OH H2N 850C 24 h Cl N Cl N 2-(7-chloroquinolin-4-ylamino)ethanol
Scheme 4. 6: Synthesis of 2-(7-chloroquinolin-4-ylamino) ethanol (EA-Q)
―4,7-dichloroquinoline (1000 mg, 5.05mmol) was dissolved in EA (3.05ml). The reaction was refluxed over a period of 24 hours at 85oC. the reaction was monitored by TLC using toluene:
EtOAc: MeOH (6:3:1) as eluent solvent system to ensure the completion of reaction, After 24 hours, the reaction was allowed to cool at room temperature and 30ml of distilled water was added into the solution and the resultant was filtered, the solids were then allowed to dry at room temperature. Then later it was recrystallized with 20 ml of methanol. 2-(7-chloroquinolin-4- ylamino) ethanol (Yield 65%, m.p 112–114 oC, Rf value 0.72) was obtained as pale yellow crystals.
79
The FTIR results confirmed a successful incorporation of 4.7 dichloroquine and ethanolamine
(EA), it indicated N-H stretch for secondary amine at 3300 cm-1, O-H stretch for alcohol at 3190 cm-1, C-H stretch for alkene at 3111 cm-1, also for C-H stretch for alkane at 2802 cm-1, C=C for aromatic at 1550 cm-1, C-N stretch for amine at 1083 cm-1 and also C-Cl at 750 cm-1. The above peaks confirmed the successful isolation of compound 2-(7-chloroquinolin-4-ylamino) ethanol. 9
90
80
70
60 Transmitance Transmitance %
50
40 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1 Figure 4. 5: FT-IR spectrum of 2-(7-chloroquinolin-4-ylamino) ethanol
80
4.9. Synthesis of 2-(2-(-chloroquinolin-4-ylamino)ethoxy)ethanol (AEE-Q)
O Cl HN OH O H2N OH 850C 24 h Cl N Cl N 2-(2-(7-chloroquinolin-4-ylamino)ethoxy)ethanol
Scheme 4. 7: Synthesis of 2-(2-(-chloroquinolin-4-ylamino)ethoxy)ethanol (AEE-Q)
―4,7-dichloroquinoline (1000 mg, 5.05mmol) was dissolved in AEE (3.05ml. The reaction was refluxed over a period of 24 hours at 85oC. After 24 hours, the reaction was allowed to cool at room temperature and 30ml of distilled water was poured into it and the resultant was filtered, the solids were dried at room temperature, 1ml of methanol and 5ml of ethyl acetate was used to boil those solids and ice was used to cool them after solids were collected. 2-(2-(-chloroquinolin-
4-ylamino)ethoxy)ethanol (Yield 75%, m.p 122–124 oC, Rf value 0.62) was obtained as brownish orange crystals. The FTIR revealed the presence of the expected functional groups in
AEE-Q. it revealed N-H stretch for secondary amine at 3310 cm-1, C-H stretch for alkane at 2803 cm-1, C=C for aromatic at 1580 cm-1, C-N for amine at 1108 cm-1, C-O for ester at 1050 cm-1 and
-1 9 also C-Cl at 530 cm . (6:2:2 methanol/TEA/Hexane, Rf = 0.54) .‖
81
95
85
75
65
55 Transittance %
45
35 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1
Figure 4. 6: FT-IR spectrum of 2-(2-(-chloroquinolin-4-ylamino)ethoxy)ethanol (AEE-Q).
4.10. Synthesis of 4-(7-chloroquinolin-4-ylamino)-2-hydroxybenzoic acid
O O OH Cl OH HN OH
H2N OH
DMSO 850C 24 hrs Cl N Cl N 4-(7-chloroquinolin-4-ylamino)-2-hydroxybenzoic acid
Scheme 4. 8: Synthesis of 4-(7-chloroquinolin-4-ylamino)-2-hydroxybenzoic acid ―4-aminosalicylic acid (70 mg, 0.45 mmol) was dissolved in 5 mL DMSO followed by addition of 4.7-dichloroquinoline (100 mg, 0.45 mmol). The reaction was allowed to stir for approximately 10 minutes until all the solute were completely dissolved followed by the addition of DMAP (55 mg, 0.45 mmol). The reaction was allowed to stir for 10 minutes followed by the addition of DCC (103 mg, 0.50 mmol) in portion within a period of 5 minutes. The reaction was
82 allowed to stir overnight at 85oC temperature. It was monitored by TLC using (6:4 toluene/ethyl acetate, and Rf = 0.24). The obtained product was extracted three times using 20 mL dichloromethane and 20 mL cold distilled water. The organic layer was dried over anhydrous sodium sulphate, filtered and then concentrated on the roti- evaporator. A viscous liquid was obtained which was further purified by column chromatography (6:4:1 Toluene/Ethyl acetate/Methanol). (0.108 g), Yield: (68%). FT-IR (cm-1): 3382 (N−H), 2981 (C−H), 15621
(C=C aromatic), 1695 (C=O), 1288 (N−H bending) and 1176 (C−O)10.‖ PDAQ 100
90
80
70
60
50
Transmittance (%) Transmittance 40
30
20
10 4000 3500 3000 2500 2000 1500 1000 500
Wavenumber cm-1
Figure 4. 7: FT-IR spectrum of 4-(7-chloroquinolin-4-ylamino)-2-hydroxybenzoic acid
83
―The first step of the proposed strategy was to synthesise chloroquine derivatives with various alkyl amino side chain. The synthesis was carried out via a nucleophilic substitution reaction on the suitably substituted 4,7-dichloroquinoline scaffold. This was successfully achieved by using amine or amino alcohol as nucleophiles.
―According to the reported experimental methods in literature2,6,11, a mixture of chloroquine and amine is commonly homogenised at 80 oC, followed by reflux at high temperature(i.e 120-140 oC). Similarly, the experiments were refluxed at 120 °C and monitored by TLC plate. However, the reactions at 120 °C resulted in decomposition of the precursors.
From this observation, it was concluded that the high temperatures became unfavourable for the reactions. Decomposition has been confirmed as the major problem for similar reactions and milder reaction conditions have been considered useful for these reactions to be successful, i.e. reactions at 75-95°C rather than high temperatures12. All the synthesised 4-aminoquinoline derivatives were characterized with FT-IR which indicated the successful isolation of compounds as reported by some researchers.‖
84
4.11. DERIVATIVES OF OLEANOLIC ACID.
4.11.1. Synthesis of hybrid compounds with amide linkers
4.11.2 Synthesis of compound VK1.
Cl N Cl N
H OH N NH H2N NH O O HO HSU, DCC, ref 120 0C HO Scheme 4. 9: Synthesis of compound VK1
39 N 35 Cl ―Oleanolic acid (200 mg, 0.4mmol) and 1.3 23 30 40 36 34 43 21 22 20 41 37 33 42 38 PDA.Q (103 mg, 0.4mmol) was dissolved in 5 13 18 19 H 14 12 17 N 46 NH 25 24 27 48 47 45 44 6 10 11 16 mL of DMSO. N-Hydroxysuccinimide (HSU) 1 5 9 15 O31 2 4 8 29 HO 3 7 VK1 (50.40mg, 0.4mmol) and N,N‘- 32 28 26 Dicyclohexylcarbodiimide (DCC) (99.39 mg, 0.5 mmol) portions were added and stirred at room temperature for 10 minutes then heated to reflux at 120 0C for two days, the reaction was monitored by TLC to ensure the completion of reaction, after which the resultant solution was diluted with DCM (20 mL) and the mixture was washed with 30 mL of ice water to afford an organic and aqueous layer. The organic layer was then collected and dried over anhydrous sodium sulphate, filtered and concentrated on the rotary evaporator to obtain a light brown powder. The crude compound was purified by column chromatography using silica gel (Merck, silica gel 60 F254: 0.063-0.200 mm). The column was eluted with EtOAc: MeOH (9:1) to afford a
0 white powder (Yield 54%, m.p 131–133 C, Rf value 0.42). IR vmax 3296 (NH), 3076 (OH),
1 2932-2873 (aliphatic CH), 1626(C=O), 1545(C=C), 1337(C-N). H NMR (400 MHz, CDCl3): δ
(ppm) 8.28-8.26 (d, J=8.0 Hz, 1H, H-40), 7.32 (s, 1H, C-48), 7.18-7.16 (d, j=8.0 Hz, 1H, H-38),
85
7.08-7.06 (d, j=8.0 Hz, 1H, H-33), 5.66-5.64 (d, j=8.0 Hz, 1H, H-41), 5.28 (t, J=4.0 Hz, 1H, H-
13) 4.06-4.00 (q, j=8.0 Hz, 3H, H-2 & 47), 3.39(s, 1H, H-48), 3.17-3.13 (dd, j=4.0 Hz, 2H, H-
13 45), 2.48-2.43 (dd, j=4.0 Hz, 1H, H-18), 2.19 (s, 1H, H-32). C NMR (400 MHz, CDCl3): δ
(ppm) 177.13 (C27), 155.23 (C42), 152.43 (C40), 148.21 (C36), 144.73 (C12), 135.43 (C34),
129.11 (C35), 125.77 (C33), 122.53 (C38), 121.34 (C13), 119.41 (C37), 114.24 (C41), 78.97
+ + (C2). MS (ESI ) 673.44 calculated for C42H60ClN3O2 [M+H] , found 673.33.‖
4.11.3. Synthesis of compound VK2
N Cl N Cl
OH H NH N NH O H2N O HO HSU, DCC, ref 120 0C HO
Scheme 4. 10: Synthesis of compound VK2
38 N 34 Cl ―Oleanolic acid (400 mg, 0.8 mmol) and 23 29 39 35 33 42 21 22 20 40 36 32 41 37 HYD.Q (200 mg, 0.8 mmol) was dissolved in 18 13 19 H 14 12 17 N NH 24 26 44 43 5 mL of DMSO. HSU (100.8 mg, 0.8 mmol) 6 10 11 16 1 5 9 15 O
2 4 8 28 VK2 and DCC (99.39 mg, 0.5 mmol) portions were HO 3 7 31 27 25 added and stirred at room temperature for 10 minutes then heated to reflux at 120 0C for 24 hr, the reaction was monitored by TLC to ensure the completion of reaction, after which the obtained solution was diluted with DCM (20 mL) and the mixture was washed with 30 mL of ice water to afford an organic and aqueous layer. The organic layer was then collected and dried over anhydrous sodium sulphate, filtered and
86 concentrated on the rotary evaporator to obtain off-white crystals. The crude was purified by column chromatography using silica gel (Merck, silica gel 60 F254: 0.063-0.200 mm). The column was eluted with EtOAc: Hex (7:3) to afford a white powder (Yield 66 %, m.p 114–115 o C, Rf value 0.33). IR vmax 3548 (NH), 3383 (OH), 2929-2857 (CH aliphatic), 1709(C=O),
1 1631(C=C alkene), 1455(C=C aromatic), 1217(C-N), 628 (C-Cl). H NMR (400 MHz, CDCl3):
δ (ppm) 8.06-8.04 (d, J=8.0 Hz, 1H, H-39),7.89(s,1H, C34), 7.62-760 (d, J=8.0 Hz, 1H, C-37),
7.44-7.42 (d, J= 8.0 Hz, 1H, H-32), 5.65-5.63 (d, J=8.0 Hz, 1H, H-40), 5.28 (t, J=4.0 Hz 1H, H-
13), 3.40 (s, 1H, H-44), 3.17-3.13 (dd, J=4.0 Hz, 1H, H-2), 2.48-2.43 (dd, J=4.0 Hz, 1H, H-18),
13 2.10 (s, 1H, H-31). C NMR (400 MHz, CDCl3): δ (ppm) 181.52 (C26), 152.12 (C39), 149.97
(C41), 148.24 (C35), 144.74 (C12), 135.43 (C33), 129.65 (C34), 127.61 (C32), 122.52 (C37),
121.99 (C13), 118.42 (C36), 113.65 (C40), 78.97 (C2). MS (ESI+) 631.39 calculated for
+ C39H54ClN3O2 [M+H] , found 631.35.‖
87
4.11.4. Synthesis of compound VK3
N
H2N OH N H N H N Cl N O H O HO HSU, DCC, ref 120 0C Cl HO Scheme 4. 11: Synthesis of compound VK3
34 27 ―Oleanolic acid (200 mg, 0.4 mmol) 25 26 24 44 45 N 43 17 22 23 and 1.2 PDA.Q (103 mg, 0.4 mmol) H 18 16 21 N 1 46 40 29 31 4 2 N 41 39 10 14 15 20 H 48 5 9 13 19 42 38 was dissolved in 5 mL of DMSO. HSU O 3 37 Cl 6 8 12 47 HO 7 11 36 VK3 (50.40 mg, 0.4 mmol) and DCC (99.39 32 30 mg, 0.5 mmol) portions were added and stirred at room temperature for 10 minutes then heated to reflux at 120 0C for 24 hours, the reaction was monitored by TLC to ensure the completion of reaction, after which the obtained solution was diluted with DCM (20 mL) and the mixture was washed with 30 mL of ice water to afford an organic and aqueous layer. The organic layer was then collected and dried over sodium sulphate, filtered and concentrated on the rotary evaporator to obtain a white powder. The crude was purified by column chromatography using silica gel
(Merck, silica gel 60 F254: 0.063-0.200 mm). The column was eluted with EtOAc: Hex (7:3) to
0 afford a white powder (Yield 51%, m.p 122–124 C, rf value 0.52). IR vmax 3546 (NH), 3359
(OH), 2928-2857 (CH aliphatic), 1712 (C=O), 1637(C=C alkene), 1456 (C=C aromatic),
1 1223(C-N), 626 (C-Cl). H NMR (400 MHz, CDCl3): δ (ppm) 7.92 (s, 1H, H-4), 7.62 (s, 1H, H-
39), 7.56-7.54 (d, J= 8.0 Hz, 1H, H-42), 7.41-7.39 (d, J=8.0 Hz, 1H, H-37), 5.64-5.62 (d, J=8.0
Hz, 1H, H-45), 5.28 (t, J=4.0 Hz 1H, H-17), 4.45 (s, 1H, H-48), 4.32-4.30 (d, J=8.0 Hz, 1H, H-
88
2), 3. 17- 3.13 (dd, J=4.0 Hz, 1H, H-6), 2.48- 2.44(dd, J=4.0 Hz, 1H, H-22), 2.19 (s, 1H, H-36).
13 C NMR (400 MHz, CDCl3): δ (ppm) 177.03 (C31), 155.75 (C38), 150.23 (C42), 148.23 (C40),
144.00 (C16), 133.75 (C46), 128.43 (C47), 126.41 (C45), 122.50 (C44), 121.13 (C17), 119.23
+ + (C39), 114.34 (C43), 78.96 (C6). MS (ESI ) 673.44 calculated for C42H60ClN3O2 [M+H] , found
671.09.‖
4.11.5. Synthesis of compound VK4
N Cl N Cl
OH H H N NH N NH 2 N N O H H O HO 0 HSU, DCC, ref 120 C HO Scheme 4. 12: Synthesis of compound VK4
7 N 3 Cl ―Oleanolic acid (200 mg, 0.4 mmol) 48 41 8 4 2 11 39 40 38 9 5 1 10 6 and DET.Q (103 mg, 0.4 mmol) was 31 36 37 H 32 30 35 N 16 15 14 NH 43 42 45 18 17 N 13 12 dissolved in 5 mL of DMSO. HSU 24 28 29 34 H 19 23 27 33 O 49 20 22 26 47 (50.40 mg, 0.4 mmol) and DCC HO 21 25 VK4 50 46 44 (99.39 mg, 0.5 mmol) portions were added and stirred at room temperature for 10 minutes then heated to reflux at 120 0C for two days, the reaction was monitored by TLC to ensure the completion of reaction, after which the obtained solution was diluted with DCM (20 mL) and the mixture was washed with 30 mL of ice water to afford an organic and aqueous layer. The organic layer was then collected and dried over anhydrous sodium sulphate, filtered and concentrated on the rotary evaporator to obtain a white powder. The crude compound was purified by column chromatography using silica gel
(Merck, silica gel 60 F254: 0.063-0.200 mm). The column was eluted with EtOAc: MeOH (9:1) to
89
0 afford a white powder (Yield 62%, m.p 128–130 C, rf value 0.52). IR vmax 3356 (NH), 3265
(OH), 2943-2857 (CH aliphatic), 1712 (C=O), 1637(C=C alkene), 1456 (C=C aromatic),
1 1223(C-N), 626 (C-Cl). H NMR (400 MHz, CDCl3): δ (ppm) 7.74-7.72 (d, J=8.0 Hz, 1H, H-8),
7.14(s, 1H, H-3), 7.04-7.02(d, J=8.0 Hz, 1H, H-6), 6.74-6.72(d, j=8.0, 1H, H-1), 6.42-6.40(d,
J=8.0, 1H, H-9), 5.28 (t, J=4.0, 1H, H-31), 4.45 (s, 1H, H-12), 4.04 (m, 4H, H-13& H-17), 3.66
(q, J=8.0, 4H, H-14& 16), 3.40 (s, 1H, H-18), 3.14 (dd, J=8.0, 1H, H-36), 2.45 (dd, 4.0 Hz, 1H,
13 H-20), 1.98 (s, 1H, H-50). C NMR (400 MHz, CDCl3): δ (ppm) 177.07 (C45), 154.65 (C10),
151.30 (C8), 148.44 (C4), 144.75 (C30), 134.50 (C2), 129.13 (C3), 127.41 (C1), 122.50 (C6),
121.99 (C31), 119.09 (C5), 113.21 (C9), 78.96 (C20), 48.08 (C13), 47.57 (C17), 46.81 (C14),
+ + 46.23 (C16). MS (ESI ) 702.46 calculated for C43H63ClN3O2 [M+H] , found 701.38.‖
90
4.12. Synthesis of hybrid compounds with ester linkers.
4.12.1. Synthesis of compound VK5.
HO NH OH Cl
O N O NH HO O Cl DMSO, DCC,DMAP, reflux 120 0C 24 h HO N Scheme 4. 13: Synthesis of compound VK5
30 23 ―Oleanolic acid (400 mg, 0.8 mmol) and EA.Q 21 22 20
13 18 19 (195.02 mg, 0.8 mmol) was dissolved in 5 mL 14 12 17 O 34 25 24 27 33 34 36NH 6 10 11 16 of DMSO. 4-Dimethylaminopyridine (DMAP) 1 5 9 15 O31 37 46 Cl 38 42 45 47 2 4 8 29 HO 3 7 39 41 44 32 N 43 (97.74 mg, 0.8 mmol) and DCC (181.56 mg, 28 26 40 VK5 0.88 mmol) portions were added and stirred at room temperature for 10 minutes then heated to reflux at 120 0C for 28 hours, the reaction was monitored by TLC to ensure the completion of reaction, after which the obtained solution was diluted with DCM (20 mL) and the mixture was washed with 30mL of ice water to afford an organic and aqueous layer. The organic layer was then collected and dried over anhydrous sodium sulfate, filtered and concentrated on the rotary evaporator to obtain a light brown powder. The crude was purified by column chromatography using silica gel (Merck, silica gel 60
F254: 0.063-0.200 mm). The column was eluted with EtOAc: MeOH (9:1) to afford white crystals
0 (Yield 54%, mp 131–133 C, Rf value 0.43).
IR vmax 3450 (NH), 3307 (OH), 2943-2897 (CH aliphatic), 1694 (C=O), 1468(C=C alkene), 1398
1 (C=C aromatic), 1274(C-N), 660 (C-Cl). H NMR (400 MHz, CDCl3): δ (ppm) 7.94-7.92 (d,
91
J=8.0 Hz, 1H, H-39), 7.78(s, 1H, H-46), 7.58-7.56 (d, J=8.0 Hz, 1H, H-44), 5.64-5.62(d, J=8.0
Hz, 1H, H-38), 5.38(t, J=4.0 Hz,1H, H-13) 4.45(s, 1H, H-36), 4.34-4.29(dd, J=8.0 Hz, 1H, H-
34), 4.05-4.00 (dd, J=8.0 Hz, 2H, H-35), 3.17-3.13(dd, J=4.0 Hz, 1H, H-2), 2.48-2.44(dd, J=4.0
Hz, 1H, H-18).
13 C NMR (400 MHz, CDCl3): δ (ppm) 177.05 (C27), 152.43 (C37), 149.20 (C39), 145.74
(C41), 144.51 (C12), 130.50 (C45), 130.21 (C44), 130.01 (C43), 122.50 (C42), 121.72 (C13),
107.50 (C46), 103.43 (C38), 78.96 (C2), 58.44 (C34), 55.13 (35). MS (ESI+) 660.41 calculated
+ for C41H57ClN2O3 [M+H] , found 660.38.‖
4.12.2. Synthesis of compound VK6
O HO NH Cl OH Cl H O O N N O HO O N DMSO, DCC,DMAP, reflux 120 0C 24 h HO Scheme 4. 14: Synthesis of compound VK6
30 23 47 Cl ―Oleanolic acid (400 mg, 0.8 mmol) 21 22 20 45 46 44 and AEE.Q (220.43 mg, 0.8 mmol) 13 18 19 H 14 12 17 O 49 48 34 N 42 43 25 24 27 33 50 O 35 36 37 41 6 10 11 16 were dissolved in 5 mL of DMSO. 1 5 9 15 O 38 N 31 39 40 2 4 8 29 VK6 DMAP) (97.74 mg, 0.8 mmol) and HO 3 7 32 28 26 DCC (181.56 mg, 0.88 mmol) portions were added and stirred at room temperature for 10 minutes then heated to reflux at 120
0C for 24 hrs, the reaction was monitored by TLC to ensure the completion of reaction, after which the obtained solution was diluted with DCM (20 mL) and the mixture was washed with 30
92 mL of ice water to afford an organic and aqueous layer. The organic layer was then collected and dried over anhydrous sodium sulphate, filtered and concentrated on the rotary evaporator to obtain off-white crystals. The crude compound was purified by column chromatography using silica gel (Merck, silica gel 60 F254: 0.063-0.200 mm). The column was eluted with EtOAc:
0 MeOH (9:1) to afford an off white powder (Yield 51%, m.p 132–133 C, rf value 0.45). IR vmax
3535 (NH), 3391 (OH), 2932-2855 (CH aliphatic), 1704 (C=O), 1626 (C=C alkene), 1451 (C=C
1 aromatic), 1274 (C-N), 628 (C-Cl). H NMR (400 MHz, CDCl3): δ (ppm) 7.10-7.08 (d, J=8.0
Hz, 1H, H-39), 6.85-6.83 (d, J = 8.0 Hz, 1H, H-43), 6.80 (s, 1H, H-46), 6.51-6.50 (d, J = 8.0 Hz,
1H, 44-H), 5.72-5.70 (d, J=8.0, 1H, H-38), 5.37 (t, J = 4.0 Hz, 1H, H-13), 4.16 (q, 4H, H-50),
3.19 (s, 1H, H-36), 3.74 (q, 4H, H-49-34), 3.48 (dd, J=4.0,8.0 Hz, 2H, H-35), (dd, J = 12.0, 4.0
13 Hz, 1H, 18-H), 2.54 (dd, J=4.0,12 Hz, 1H, H-18). C NMR (400 MHz, CDCl3): δ (ppm) 176.99
(C27), 152.48 (C37), 150.11 (C39), 145.13 (C41), 144.41 (C12), 130.21 (C43), 122.48 (C42),
121.99 (C13), 118.46 (C46), 113.43 (C45), 112.48 (C44), 78.96 (C2), 70.23 (C34), 68.11(C49),
+ + 65.13 (C50). MS (ESI ) 704.43 calculated for C43H61ClN2O4 [M+H] , found 704.11.‖
93
4.12.3. Synthesis of compound VK7
O O HO OH OH O O O O O O OH HO O DMSO, DCC,DMAP, reflux 120 0C 24 h HO O O Scheme 4. 15: Synthesis of compound VK7
30 23 ―Oleanolic acid (200 mg, 0.4 21 22 20 59 56 O 58 O55 13 18 19 mmol) and Curcumin (162.09 14 12 17 O 51 36 OH 25 24 27 32 52 50 37 35 57 6 10 11 16 53 47 45 43 41 38 34 mg, 0.44 mmol) was dissolved 1 5 9 15 O 54 31 46 44 42 40 39 2 4 8 29 3 7 O O HO 49 33 48 in 5 mL of DMSO. DMAP) 28 26 VK7 (48.87 mg, 0.4 mmol) and DCC (90.78 mg, 0.4 mmol) portions were added and stirred at room temperature for 10 minutes then heated to reflux at 120 0C for 24 hrs, the reaction was monitored by TLC to ensure the completion of reaction, after which the obtained solution was diluted with
DCM (20 mL) and the mixture was washed with 30 mL of ice water to afford an organic and aqueous layer. The organic layer was then collected and dried over anhydrous sodium sulphate, filtered and concentrated on the rotary evaporator to obtain a light brown powder. The crude compound was purified by column chromatography using silica gel (Merck, silica gel 60 F254:
0.063-0.200 mm). The column was eluted with EtOAc:MeOH (9:1) to afford a pale yellow
0 powder (Yield 53%, m.p 132–133 C, rf value 0.62). IR vmax 3354 (OH), 2929-2898 (CH
1 aliphatic), 1735 (C=O), 1469 (C=C), 1156- 1031(C-O ester). H NMR (400 MHz, CDCl3): δ
(ppm) 7.34-7.32 (d, J=8.0 Hz, 1H, H-46), 6.82-6.80 (d, J = 8.0 Hz, 1H, H-53), 6.78 (s, 1H, H-
50), 6.72-6.70 (d, J=8.40 Hz, 1H, H-54), 6.65-6.63(d, J=8.0 Hz, 1H, H-46), 5.24(t, J=4.0 Hz,
94
1H, H-13), 4.99(s. 1H, H-57), 4.65(s, 1H, H-43), 3.45(s, 6H, H-56 & 59), 3.16-3.12(dd, J=4.0,
13 1H, H-2), 2.83-2.79 (dd, J=4.0 Hz, 1H, H-18). C NMR (400 MHz, CDCl3): δ (ppm) 196.14
(C44), 196.13 (C42), 177.63 (C27), 158.34 (C51), 152.02 (C36), 143.58 (C12), 142.25 (C46),
141.51 (C40), 139.04 (C52), 133.52 (C47), 129.25 (C38), 127.61 (C45), 127.60 (C41),
122.52(C13), 114.13 (C34), 113.24(C37), 113.23(C50), 79.02(C2), 67.96(C59), 58.47(C56). MS
+ + (ESI ) 806.48 calculated for C51H66O8 [M+H] , found 801.14.‖
4.12.4. Synthesis of compound VK8
H OH H O O HO H O H HO 0 DMSO, DCC,DMAP, reflux 120 C 24 h HO Scheme 4. 16: Synthesis of compound VK8
30 23 ―Oleanolic acid (200 mg, 21 58 22 20 47 59 56 48 46 57 13 18 19 32 60 54 0.4 mmol) and ergocalciferol 52 55 14 12 17 O 34 41 43 45 53 44 25 24 27 35 39 42 51 63 6 10 11 16 H 38 49 50 H 1 5 9 15 O 36 61 62 (162.09 mg, 0.44 mmol) 31 37 42 2 4 8 29 VK8 HO 3 7 33 were dissolved in 5 mL of 28 26 DCM. DMAP) (48.87 mg, 0.4 mmol) and DCC (90.78 mg, 0.4 mmol) portions were added and stirred at room temperature for 10 minutes then heated to reflux at 70 0C for 24 hrs, the reaction was monitored by TLC to ensure the completion of reaction, after which the obtained solution was diluted with DCM (20 mL x 3) and the mixture was washed with 30 mL of ice water to
95 afford an organic and aqueous layer. The organic layer was then collected and dried over anhydrous sodium sulfate, filtered and concentrated on the rotary evaporator to obtain white crystals. The crude was purified by column chromatography using silica gel (Merck, silica gel 60
F254: 0.063-0.200 mm). The column was eluted with Hexane: EtOAc (7:3) to afford white
0 crystals (Yield 77%, m.p 120–122 C, rf value 0.64). IR vmax 3553-3462 (OH), 2930-2854 (CH aliphatic), 1709 (C=O), 1626 (C=C alkene) 1488(C=C aromatic), 1214(C-O ester). 1H NMR
(400 MHz, CDCl3): δ (ppm) 6.22-6.20 (d, J=8.0 Hz, 1H, H-41), 5.86-5.84 (d, J= 8.0 Hz, 1H, H-
42), 5.44-5.42 (d, J=8.0 Hz, 1H, H-53), 5.24 (t, J=4.0 Hz, 1H, H-13), 4.94 (s, 2H, H-40), 4.32
(m, 1H, H-35), 3.16-3.12 (dd, J=4.0 Hz. 1H, H-2), 2.78-2.73 (dd, J=4.0 Hz, 1H, H-18), 2.19(s,
13 1H, H-33). C NMR (400 MHz, CDCl3): δ (ppm) 172.92 (C27), 145.12 (C38), 144.20 (C43),
143.92 (C12), 141.17 (C39), 134.99 (C54), 130.25 (C53), 126.42 (C41), 124.05 (C42), 123.37
+ (C13), 111.12 (C40), 79.02 (C2), 74.75 (C35). MS (ESI ) 834.69 calculated for C58H90O3
[M+H]+, found 834.69.‖
96
4.12.5. Synthesis of compound VK9
HO O C O
O O N OH C HO HN HO N O H Cl N HO HO DMSO, DCC,DMAP, reflux 120 0C 24 h Cl
Scheme 4. 17: Synthesis of compound VK9
29 23 ―Oleanolic acid (200 mg, 0.4 mmol) and 21 22 20 HO O 52 53 51 13 18 19 4-(7-chloroquinolin-4-ylamino)-2- 14 12 17 O 47 24 54 26 50 46 48 6 10 11 16 hydroxybenzoic acid (162.09 mg, 0.44 1 5 9 15 O 45 49 30 44 2 4 8 26 mmol) were dissolved in 5 mL of DMSO. HO 3 7 HN 43 40 31 41 39 27 25 VK9 36 N DMAP) (48.87 mg, 0.4 mmol) and DCC 38 37 35
32 34 (90.78 mg, 0.4 mmol) portions were added 33
Cl 42 and stirred at room temperature for 10 minutes then heated to reflux at 120 0C for 24 hrs, the reaction was monitored by TLC to ensure the completion of reaction, after which the obtained solution was diluted with DCM (20 mL) and the mixture was washed with 30 mL of ice water to afford an organic and aqueous layer. The organic layer was then collected and dried over anhydrous sodium sulfate, filtered and concentrated on the rotary evaporator to obtain a light brown powder. The crude compound was purified by column chromatography using silica gel (Merck, silica gel 60 F254: 0.063-0.200 mm).
The column was eluted with Hexane: EtOAc (7:3) to afford a white powder (Yield 57%, m.p
0 134–135 C, rf value 0.25). IR vmax 3535 (NH), 3391 (OH), 2932-2855 (CH aliphatic), 1704
97
(C=O), 1626 (C=C alkene), 1451 (C=C aromatic), 1274 (C-N), 628 (C-Cl). 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.96-7.94 (d, J=8.0 Hz, 1H, H-39), 7.80 (s, 1H, H-34), 7.56-7.54 (d, J=8.40 Hz,
1H, H-48), 7.43-7.41 (d, J=8.0, 1H, H-37), 7.34- 7.32 (d, J=8.0 Hz, H-32), 5.74 (s, 1H, H-45),
5.64-5.63 (d, J=4.0 Hz, 1H, H- 40 & 49), 5.28 (t, J=4.0 Hz, 1H, H-13), 3.40 (s, 1H, H-43), 3.17-
3.13 (dd, J=4.0 Hz, 1H, H-13), 2.48-2.44(dd, J=4.0 Hz, 1H, H-18), 2.10 (s, 1H, H-31). 13C NMR
(400 MHz, CDCl3): δ (ppm) 176.34 (C26), 171.44 (C51), 157.22 (C46), 151.74 (C39), 149.21
(C41), 148.99 (C44), 148.58 (C35), 144.76 (C12), 136.42 (C33), 128.61 (C48), 127.42 (C34),
122.50 (C13), 118.40 (C37), 112.72 (C40), 112.71 (C49), 111.23 (C47), 105.40 (C45), 78.96
+ + (C2). MS (ESI ) 752.4 calculated for C46H57ClN2O5 [M+H] , found 755.08.‖
98
Unsuccessful hybrid compounds
O
HN
O N H HO HO O OH H H H N O N O O O H2N H O N H HO HSU, DCC, ref 120oC X HO
H H N OH H OH 2 NH O OH H O H HO HSU, DCC, ref 120oC X HO OH OH
N H N NH H HN 2 H OH N O N O H H O O H HO HSU,X DCC, ref 120oC HO
NH2
OH H O H OH O P P OH OH NH HO OH O O H H o OH HO HSU, DCC, ref 120 C HO O O P P OH X OH HO OH
Scheme 4.19: unsuccessful hybrid compounds
99
4.12.6. Antibacterial assay
―The synthesized hybrid compounds were tested against 11 reference bacterial strains namely:
Gram-positive bacteria: Bacillus subtilis (ATCC19659), Enterococcus faecalis (ATCC13047),
Staphylococcus epidermidis (ATCC14990), Staphylococcus aureous, and Mycobaterium smegmatis (MC2155). Gram-negative strains: Enterobacter cloacae (ATCC13047), Proteus vulgaris (ATCC6380), Klebsiella oxytoca (ATCC8724), Proteus vulgaris (ATCC6380),
Pseudomonas aeruginosa (ATCC27853), Proteus mirabilis (ATCC7002) and Escherichia coli
(ATCC25922).‖
4.12.7 Minimum inhibitory concentration (MIC)
―The minimum inhibitory concentration (MIC) of the synthesized hybrid compounds MIC of the tested compounds was carried out following Fonkui et al., (2018). Stock solutions were prepared by adding 4 mL of DMSO to each tube containing in average 20 mg of the synthesized compounds. These solutions were then serially diluted in 100 uL of nutrient broth in a 96 well plates to the desired concentrations (2.5, 1.25, 0.625, 0.3125, 0.1562, 0.0781 mg/mL). Then after, 100 µL of each of these solutions was placed in duplicate and seeded with 100 µL of an overnight bacterial culture brought to 0.5 Mc Farland in nutrient broth. Streptomycin and nalidixic acid were used as positive control and the negative control was prepared to contain
50% nutrient broth in DMSO.
100
CHAPTER FIVE RESULTS AND DISCUSION
5.1. Structural elucidation of compound VK1
39 N 35 Cl 30 23 40 36 34 43 21 22 20 41 37 33 42 38 13 18 19 H 14 12 17 N 46 NH 25 24 27 48 47 45 44 6 10 11 16 1 5 9 15 O31 2 4 8 29 HO 3 7 32 28 26
Figure 5. 1: structural elucidation of compound VK1
The ―IR spectroscopic skeletal vibrations were first used to confirm the important functional groups of compound VK1 (Figure 5.2): 3296 cm-1 (NH stretch) was assigned to the aliphatic
-1 -1 -1 amine; 3076 cm OH stretch; 2932-2873 cm (CH2-CH3) aliphatic stretch; 1626 cm C=O stretch; and peak at 1545 cm-1 frequency further confirmed the C=C aromaticity.
In deuterated chloroform, the structural elucidation of compound VK1 by 1H NMR spectroscopy
(Figure 5.3) showed the expected signals for 4-aminoquinoline aromatic rings in the region
8.28-700 ppm, the olefinic proton of oleanolic acid at 5.28 ppm and hydroxyl proton at 3.16 ppm of oleanolic acid. The doublet at 8.28 ppm with coupling constant J=8.0 Hz and the singlet at
7.32 ppm were assigned to the two protons H-40 and H-48 experiencing the inductive deshielding effect from the ring nitrogen, N-39 and the substituted N-48 respectively. The doublets at 7.18-7.16, 7.08-7.06 and 5.66-5.64 ppm were assigned to the protons H-38, H-33, and H-41 of the aromatic rings respectively. The triplet at 5.28 ppm and two doublets of a
101 doublet at 3.17-3.13 and 2.48-2.43 ppm were assigned to the proton H-13, H-45 and H-18 of oleanolic acid. The multiplet at 4.06-4.00 ppm was assigned to both protons H-2 of oleanolic acid and H-47 of the installed amine group. A singlet at 3.39 ppm was also assigned to the H-48 of the installed amine group.
13C NMR spectrometry was also used to substantiate the structural elucidation of VK1 (Figure
5.4). To the oleanolic acid 13C NMR spectrum; additional aromatic carbon signals were observed. The aromatic signals at 155.23, 152.43, 148.21, 135.43, 129.11, 125.77, 122.53, 119.4, and 114.24 were assigned to the carbons C42, C40, C36, C34, C35, C33, C38, C37 and C41 respectively.
In addition to the IR and NMR spectroscopy experiments, the LC-MS was used to confirm the expected isotopic mass of compound VK1.‖ The determined experimental mass of VK1 [M+H]+
= 673.33 (Figure 5.5) and ―was found to be in good agreement with the theoretical 673.44.‖
100 95 90
85 80 75 70
65 trasmittance% 60 55 50 4000 3500 3000 2500 2000 1500 1000 500 wavenumber cm -1
Figure 5. 2: 1IR spectrum of compound VK1.
102
Figure 5. 3: 1H-NMR spectrum of compound VK1
Figure 5. 4: 13C-NMR spectrum of compound VK1
103
Figure 5. 5: LC-MS results of compound VK1
5.2. Structural elucidation of compound VK2
38 N 34 Cl 23 29 39 35 33 42 21 22 20 40 36 32 41 37 13 18 19 H 14 12 17 N NH 24 26 44 43 6 10 11 16 1 5 9 15 O 2 4 8 28 HO 3 7 31 27 25
Figure 5. 6: structural elucidation of compound VK2
The ―IR spectrum of compound VK2 (Figure 5.7) displayed almost similar peaks with compound VK1, compound VK2 displayed the absorption bands for NH stretch at 3548 cm-1,
OH at 3383 cm-1, CH aliphatic stretch at 2929-2857 cm-1, C=O stretch at 1709 cm-1, C=C aromatic stretch 1631 cm-1 at 1455 cm-1, C-N stretch at 1217 cm-1, and C-Cl stretch at 628 cm-1 which clearly confirms the successful synthesis of compound VK28,13.
104
The 1H NMR and 13C NMR spectra (Figure 5.8 and Figure 5.9) prove that the target compound
1 was achieved. H NMR (400 MHz, CDCl3) spectrum of compound VK2 indicated three doublets at 8.06-8.04 ppm, 7.62-760 ppm and 7.44-7.42 ppm assigned to protons H-39, H-37 and H-32 of the aromatic ring. A singlet at 7.89 ppm belongs to proton H-34. It also indicated another doublet at 5.65-5.63 ppm linked to proton H-40. The triplet at 5.28 ppm is allocated to proton H-13. The two singlets at 3.40 ppm and 2.10 ppm are assigned to proton H-44 of the linker and H-31 hydroxyl of oleanolic acid respectively. Two doublets of doublet at 3.27-3.13 and 2.48-2.43 ppm are linked to protons H-2 and H-18.
13 C NMR (400 MHz, CDCl3) spectrum of compound VK2 (Figure 5.9) showed a signal at
181.52 ppm belonging to the carbonyl carbon (C26). The signals at 152.12 ppm, 149.97 ppm,
148.24 ppm, 135.43 ppm, 129.65 ppm, 127.61 ppm, 122.52 ppm, 118.42 ppm, and 113.65 ppm are allocated to aromatic carbons C39, C41, C35, C33, C34, C32, C37, C36, and C40 respectively. The signals at 144.74 ppm and 121.99 ppm arise from the olefinic carbons C12 and
C13 of oleanolic acid. An oxygenated carbon C2 of oleanolic acid is allocated at 78.97 ppm.
The LC-MS analysis confirmed that compound VK2 was successfully 6,13. MS (ESI+) 631.39
+ calculated for C39H54ClN3O2 [M+H] , found 631.35 (Figure 5.10).‖
105
105
100
95
90
85
80 Transitance % 75
70
65
60 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1
Figure 5. 7: IR spectra of compound VK2.
Figure 5. 8: 1H NMR Spectrum of compound VK2
106
Figure 5. 9: 13C NMR spectrum of compound VK2.
Figure 5. 10: LC-MS results of compound VK2.
107
5.3. Structural elucidation of compound VK3
34 27 25 26 24 44 45 N43 17 22 23 H 18 16 21 N 1 46 40 29 31 4 2 N 41 39 10 14 15 20 H48 5 9 13 19 42 38 O 3 35 37 Cl 6 8 12 33 47 HO 7 11 36 32 30
Figure 5. 11: Structural elucidation of compound VK3
―The IR spectrum of compound VK3 (Figure 5.12) confirmed the successful formation of VK3 by showing the peaks of the expected functional groups at 3546 (NH), 3359 (OH), 2928-2857
(CH aliphatic), 1712 (C=O), 1637 and 1456 (C=C aromatic), 1223(C-N), 626 (C-Cl).
1 H NMR (400 MHz, CDCl3) spectrum of compound VK3 (Figure 5.13) indicated two singlets with one proton each at 4.45 ppm and 7.62 ppm attributed to protons H-4 of the linker, and H-39 of the aromatic ring. Three doublets at 7.56-7.54, 7.41-7.39, and 5.64-5.62 ppm were assigned to aromatic protons H-42, H-37 and H-45. The triplet with one proton at 5.28 ppm is ascribed to olefinic proton H-17 of oleanolic acid. The singlet with one proton at 3.40 ppm is assigned to proton H-48 of the linker. A multiplet with one proton at 4.32-4.30 ppm is attributed to proton H-
2 of the linker. The two doublets of doublet at 3. 17- 3.13 ppm and 2.48-2.44 ppm were assigned to protons H-6 and H-22 of the oleanolic acid. A singlet with one proton at 2.19 ppm is ascribed to hydroxyl proton H-366,14.
108
13 C NMR (400 MHz, CDCl3) spectrum of compound VK3 (Figure 5.14) showed a signal for carbonyl (C31) at 177.03ppm. It also indicated signals at 155.75, 150.23, and 148.23 ppm attributed to aromatic carbons C38, C42 and C40 respectively. The signals at 144.00 ppm and
121.13 ppm are assigned to olefinic carbons C16 and C17 of oleanolic acid. The signals at
133.75, 128.43, 126.41, 122.50, 119.23, and 114.34 ppm are linked to aromatic carbons C46,
C47, C45, C44, C39, and C43 respectively.
Finally, the LC-MS was further used to confirm the mass of the compound KV3. The determined experimental mass of VK8 [M+H]+ = 671.09 (Figure 5.15) and MS (ESI+) 673.44 calculated for
C42H60ClN3O2.‖
105 100
95 90 85 80
75 Transmitttance% 70 65 4000 3500 3000 2500 2000 1500 1000 500
Wavenumber cm-1
Figure 5. 12: IR spectra of compound VK3.
109
Figure 5. 13: 1H NMR spectrum of compound VK3.
Figure 5. 14: 13C NMR spectrum of compound VK3
110
Figure 5. 15:LC-MS results of compound VK3
5.4. Structural elucidation of compound VK4
7 N 3 Cl 48 41 8 4 2 11 39 40 38 9 5 1 10 6 31 36 37 H 32 30 35 N 16 15 14 NH 43 42 45 18 17 N 13 12 24 28 29 34 H 19 23 27 33 O 49 20 22 26 47 HO 21 25 50 46 44
Figure 5. 16: Structural elucidation of compound VK4
―The IR spectrum of compound VK4 (Figure 5.17) confirmed the successful formation of VK4 by showing the peaks of the expected functional groups at 3356 cm-1 (NH), 3265 cm-1 (OH),
2943-2857 cm-1 (CH aliphatic), 1712 cm-1 (C=O), 1637 cm-1(C=C alkene), 1456 cm-1 (C=C aromatic), 1223 cm-1(C-N), 626 cm-1(C-Cl).
111
The 1H NMR and 13C NMR spectra prove that the target compound VK4 was successfully achieved. 1H NMR (400 Hz) spectrum hybrid compound VK4 (Figure 5.18) presented a doublet with one proton at 7.74-7.72 ppm assigned to proton H-8 of the aromatic ring. A singlet with one proton at 7.14 ppm is assigned toaroamtic proton H-3. Three doublets at 7.04-7.02, 6.74-6.72 and
6.42-6.40 ppm are assigned to aromatic protons H-6, H-1, and H-9. A triplet with one proton at is assigned to the olefinic proton of oleanolic acid H-31. A singlet with one proton at 3.40 ppm is assigned to N-H proton H-12. The multiplet with 4 protons at 4.04 ppm is assigned to both protons H-13 and H-17 of the linker DET. A quartet with 4 protons at 3.66 ppm is linked to both protons H-14 and H-16. The two singlets at 4.45 and 1.98 ppm are linked to protons H-18 and H-
50. Lastly, the two doublets of doublet at 3.14 and 2.45 ppm are assigned to both protons H-36 and H-20.
13 C NMR (400 MHz, CDCl3) spectrum of compound VK4 (Figure 5.19 indicated signals
177.07, 154.65, 151.30, 148.44, 144.75, 134.50, 129.13, 127.41, 122.50, 121.99, 119.09, 113.21,
78.96, 48.08, 47.57, 46.81, 46.23 assigned to carbons C45, C10, C8, C4, C30, C2, C3, C1, C6,
+ + C31, C5, C9, C20, C13, C17, C14, C16. MS (ESI ) 702.46 calculated for C43H63ClN3O2 [M+H] , found 701.38 (Figure 5.20).‖
112
95
85
75
65
55 Transmittance%
45
35 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1
Figure 5. 17: IR spectrum of compound VK4.
Figure 5. 18: 1H NMR spectrum of compound VK4
113
Figure 5. 19: 13C NMR spectrum of compound VK4.
Figure 5. 20: LC-MS results of compound VK4.
114
5.5. Structural elucidation of compound VK5
30 23 21 22 20
13 18 19 14 12 17 O 35 25 24 27 33 34 36NH 6 10 11 16 1 5 9 15 O31 37 46 Cl 38 42 45 47 2 4 8 29 HO 3 7 39 41 44 32 N 43 28 26 40
Figure 5. 21: Structural elucidation of compound VK5.
―The IR spectrum of compound VK5 (Figure 5.22) confirmed the successful formation of VK5 by showing the peaks of the expected functional groups at 3450 cm-1 (NH), 3307 cm-1 (OH),
2943-2897 cm-1 (CH aliphatic), 1694 cm-1 (C=O), 1468 cm-1(C=C alkene), 1398 cm-1 (C=C aromatic), 1274 cm-1(C-N), 660 cm-1 (C-Cl).
1 H NMR (400 MHz, CDCl3) spectrum of compound VK5 (figure 5.23) indicated a doublet with one proton at 7.94-7.92 ppm assigned to aromatic proton H-39. A singlet with one proton at 7.78 ppm is linked toaroamtic proton H-46. Two doublets with one proton each at 7.58-7.56 and 5.64-
5.62 ppm are attributed to aroamtic protons H-44 and H-38 respectively. A triplet at 5.28 ppm with one proton is assigned to olefinic proton H-13 of oleanolic acid. A singlet with one proton at 4.45 ppm is assigned to proton H-36 of the linker. Three doublets of doublet at 4.34-4.29,
4.05-4.00, 3.17-3.13, and 2.48-2.44 ppm are assigned to protons H-34, H-35, H-2 and H-18 respectively.
115
13 C NMR (400 MHz, CDCl3) spectrum of hybrid compound VK5 (figure 5.24) presented signals at 177.05, 152.43, 149.20, 145.74, 144.51, 130.50, 130.21, 130.01, 122.50, 121.72, 107.50,
103.43, 78.96, 58.44, and 55.13 ppm assigned to carbons C27, C37, C39, C41, C12, C45, C44,
C43, C42, C13, C46, C38, C2, C34, and C35 respectively. MS (ESI+) 660.41 calculated for
+ C41H57ClN2O3 [M+H] , found 660.38 (figure 5.25).‖
100
95
90
85 transmitance%
80
75 4000 3500 3000 2500 2000 1500 1000 500 wavenumber cm-1
Figure 5. 22: IR spectrum of compound VK5.
116
Figure 5. 23:1H NMR spectrum of compound VK5
Figure 5. 24: 13C NMR spectrum of compound VK5
117
Figure 5. 25: LC-MS results of compound VK5
6.6. Structural elucidation of compound VK6
30 23 47 Cl 21 22 20 45 46 44 13 18 19 H 14 12 17 O 49 48 34 N 42 43 25 24 27 33 50 O 35 36 37 41 6 10 11 16 1 5 9 15 O 38 N 31 39 40 2 4 8 29 HO 3 7 32 28 26
Figure 5. 26: Structural elucidation of compound VK6 ―The IR spectrum of compound VK6 (figure 5.27) confirmed the successful formation of VK6 by showing the peaks of the expected functional groups at 3535 cm-1 (NH), 3391 cm-1 (OH),
2932-2855 cm-1 (CH aliphatic), 1704 cm-1 (C=O), 1626 cm-1 (C=C alkene), 1451 cm-1 (C=C aromatic), 1274 cm-1 (C-N), 628 cm-1 (C-Cl).
1 H NMR (400 MHz, CDCl3) spectrum of compound VK6 (figure 5.28) indicated two doublets with one proton each at 7.10-7.08 ppm and 6.85-6.83 ppm assigned to aoamitic protons H-39 and
H-43. A singlet at 6.80 ppm attributed to aromatic proton H-46. Other doublets at 6.51-6.50, and
118
5.72-5.70 ppm linked to proton H-44, and H-38. A triplet with one proton at 5.37 ppm is assigned to olefinic proton H-13 of the oleanolic acid. Two quartets at 4.26 and 3.74 ppm are assigned to protons H-50 and H-34 of the linker respectively. A singlet at 3.92 ppm is linked to proton H-36 of the linker. Three doublets of doublet at 3.48, 3.14, and 2.54 ppm linked to proton
H-35, H-49, and H-18.
13 C NMR (400 MHz, CDCl3) spectrum of hybrid compound VK6 (figure 5.29) presented signals at 176.99, 152.48, 150.11, 145.13, 144.41, 130.21, 122.48, 121.99, 118.46, 113.43, 112.48,
78.96, 70.23, 68.11, and 65.13 ppm linked to carbons C27, C37, C39, C41, C12, C43, C42, C13,
+ + C46, C45, C44, C2, C34, C49, and C50. MS (ESI ) 704.43 calculated for C43H61ClN2O4 [M+H] , found 704.11(figure 5.30).‖
100
95
90
85 Transmitance % 80
75 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1
Figure 5. 27: IR spectrum of compound VK6.
119
Figure 5. 28: 1H NMR spectrum of compound VK6.
Figure 5. 29: 13C NMR spectrum of compound VK6.
120
Figure 5. 30: LC-MS results of compound VK6
5.7. Structural elucidation of compound VK7
30 23 21 22 20 59 56 O 58 O55 13 18 19 14 12 17 O 51 36 OH 25 24 27 32 52 50 37 35 57 6 10 11 16 O 53 47 45 43 41 38 34 1 5 9 15 54 31 46 44 42 40 39 2 4 8 29 3 7 O O HO 49 33 48 28 26
Figure 5. 31: Structural elucidation of compound VK7 ―The IR spectrum of compound VK7 (figure 5.32) confirmed the successful formation of
VK7 by showing the peaks of the expected functional groups at 3354 cm-1 (OH), 2929-2898 cm-1 (CH aliphatic), 1735 cm-1 (C=O), 1469 cm-1 (C=C), 1156- 1031 cm-1 (C-O ester).
1 H NMR (400 MHz, CDCl3) spectrum of hybrid compound VK7 (figure 5.33) indicated two doublets with one proton each at 7.34-7.32 and 6.82-6.80 ppm linked to proton H-46 and aromatic proton H-53 of the substituted curcumin. A singlet with one proton at 6.78 ppm
121 assigned to aromatic proton H-50. Another two doublets at 6.72-6.70 ppm and 6.65-6.63 ppm linked to aromatic protons H-54, and H-53. A triplet with one proton at 5.24 ppm assigned to olefinic proton H-13 of oleanolic acid. Another three singlets attributed to protons H-57 at
4.99 ppm with one proton, H-43 with one proton at 4.65 ppm and H-56 & 59 at 3.45 ppm with 6 protons. Two doublets of doublet at 3.16-3.12, and 2.83-2.79 ppm assigned to proton
H-2 and H-18 of oleanolic acid.
13 C NMR (400 MHz, CDCl3) spectrum of hybrid compound VK7 ( figure 5.34) presented signals at 196.14, 196.13, 196.13, 177.63, 158.34, 152.02, 142.25, 141.51, 139.04, 133.52,
129.25, 127.61, 127.60, 122.52, 114.13, 113.24, 113.23, 79.02, 67.96, and 58.47 ppm assigned to carbons C44, C42, C27, C51, C36, C12, C46, C40, C52, C47, C38, C45, C41,
C13, C34, C37, C50, C2, C59, and C56 respectively 13,15. MS (ESI+) 806.48 calculated for
+ C51H66O8 [M+H] , found 801.14 (figure 5.35).‖
100
95
90
85 Transmitance %
80
75 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1 Figure 5. 32: IR spectrum of Compound VK7.
122
Figure 5. 33: 1H NMR spectrum of compound VK7
Figure 5. 34: 13C NMR spectrum of compound VK7.
123
Figure 5. 35: LC-MS results of compound VK7
5.8. Structural elucidation of compound VK8
30 23
21 58 22 20 47 59 56 48 46 57 13 18 19 60 54 52 55 14 12 17 O 34 41 43 45 53 44 25 24 27 35 39 42 51 63 6 10 11 16 H 38 49 50 H 1 5 9 15 O 36 37 40 2 4 8 29 HO 3 7 28 26
Figure 5. 36: Structural elucidation of compound VK8
The IR spectrum of compound VK8 (figure 5.37) confirmed a successful isolation of VK8 by showing the peaks of the expected functional groups at 3553-3462 cm-1 (OH), 2930-2854 cm-1 (CH aliphatic), 1709 cm-1 (C=O), 1626 cm-1 (C=C alkene) 1488 cm-1 (C=C aromatic), and 1214 cm-1(C-O ester).
1H NMR (400 Hz) spectrum (figure 5.38) presented two doublets at 6.22-6.20 ppm and 5.86-
5.84 ppm assigned to protons H-41 and H-42 respectively. It also indicated a triplet with one proton at 5.24 ppm which is assigned to olefinic proton H-13 of oleanolic acid. A singlet with
124 two protons at 4.94 ppm is assigned to proton H-40. A multiplet with one proton at 4.32 ppm is assigned to proton H-35. Two doublets of a doublet at 3.16-3.12 ppm and 2.78-2.73 ppm were assigned to protons H-2 and H-18 on oleanolic acid. It also indicated a singlet with one proton at 2.19 ppm which is assigned to proton H-33.
13 C NMR (400 MHz, CDCl3) spectrum of hybrid compound VK8 (figure 5.39) presented signals at 172.92, 145.12, 144.20, 143.92, 141.17, 134.99, 130.25, 126.42, 124.05, 123.37,
111.12, 79.02, and 74.75 ppm assigned to the carbons C27, C38, C43, C12, C39, C54, C53,
C41, C42, C13, C40, C2, and C35 respectively. The determined experimental mass of VK8
[M+H]+ = 834.69 (figure 5.40) and was found to be in good agreement with the theoretical
834.69.‖
100
90
80 %Transmitance 70
60 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm-1)
Figure 5. 37: IR spectrum of compound VK8.
125
Figure 5. 38: 1H NMR spectrum of compound VK8
Figure 5. 39: 13C NMR spectrum of compound VK8
126
Figure 5. 40: LC-MS results of compound VK8
5.9. Structural elucidation of compound VK9
29 23 21 22 20 HO O 52 53 51 13 18 19 14 12 17 O 47 24 54 26 50 46 48 6 10 11 16 1 5 9 15 O 45 49 30 44 2 4 8 HO 3 7 HN 43 40 31 41 39 27 25 36 N 38 37 35
32 34 33
Cl 42
Figure 5. 41: structural elucidation of compound VK9
―The IR spectrum of compound VK9 in figure 5.42 confirmed the successful formation of
VK7 by showing the peaks of the expected functional groups at 3535 cm-1 (NH), 3391cm-1
(OH), 2932-2855 cm-1 (CH aliphatic), 1704 cm-1 (C=O), 1626 cm-1 (C=C alkene), 1451 cm-1
(C=C aromatic), 1274 cm-1 (C-N), 628 cm-1 (C-Cl).
127
1 H NMR (400 MHz, CDCl3): spectrum of compound VK9 (figure 5.43) showed a doublet at
7.96-7.94 ppm assigned to aromatic proton H-39. A singlet with one proton at 7.80 ppm linked to aromatic proton H-34. It indicated another two doublets at 7.56-7.54 ppm and 7.43-
7.41 ppm, and 7.34- 7.32 ppm attributed to aromatic protons H-48, H-37 and H-32 respectively. The two singlets at 5.74 ppm and 5.38 ppm assigned to proton H-45 and H-43.
A doublet and triplet at 5.64-5.63 ppm and 5.28 ppm are assigned to protons H-40/49 and H-
13 respectively. The two doublets of doublet and a singlet at 3.17-3.13, 2.48-2.44 and 2.10 ppm are attributed to protons H-2, H-18 and H-31 of oleanolic acid respectively10,14,16.
13 C NMR (400 MHz, CDCl3) spectrum of hybrid compound VK9 (figure 5.44) presented signals at 176.34, 171.44, 157.22, 151.74, 149.21, 148.99, 148.58, 144.76, 136.42, 127.42,
128.61, 122.50, 118.40, 112.72, 112.71, 111.23, 105.40, and 78.96 ppm assigned to carbons
C26, C51, C46, C39, C41, C44, C35, C12, C33, C48, C34, C13, C37, C40, C49, C47, C45,
+ + and C2 respectively. MS (ESI ) 752.4 calculated for C46H57ClN2O5 [M+H] , found 755.08
(figure 5.45).‖
100
95
90
85
80
75 Transmitance % 70
65
60 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber cm-1
Figure 5. 42: IR spectrum of compound VK9.
128
Figure 5. 43: 1H NMR spectrum of compound VK9.
Figure 5. 44: 13C NMR spectrum of compound VK9
129
Figure 5. 45: LC-MS results of compound VK9
The melting points and the percentage yields of the synthesized compounds range from of
114-135℃ and 51-66% respectively. Hao et al 201317 synthesized a number of hybrid compounds containing oleanolic acid and other scaffolds with the melting point ranging from
120-138%.
LC-MS results successfully confirmed the isolation of hybrid compounds by showing the expected molecular weights that correlate well with calculated molecular weight of the synthesized compounds and some of the compounds were visible as isotopes. The differences on expected molecular weights are linked to the impurities or fragmentation during compound separation via column chromatography.
5.10. Minimum inhibitory concentration
The MIC results of the synthesized compounds are presented in Table 5.1 and Figure 5.46 shows the 96 micro-well plates of the synthesized compounds against the 11 bacterial strained mentioned above.‖
130
Table 5. 1: Antibacterial activities of synthesized compounds.
Minimum inhibitory concentration (MIC, mg/mL)
Test Gram-positive Gram-negative compound
BS EF SE SA MS ECL PV KO PA PM EC
VK1 2.5 1.25 1.25 2.5 2.5 2.5 2.5 1.25 2.5 2.5 1.25 VK2 2.5 1.25 1.25 2.5 1.25 2.5 2.5 1.25 2.5 1.25 1.25 VK3 2.5 2.5 2.5 2.5 2.5 1.25 2.5 2.5 2.5 2.5 2.5 VK4 2.5 1.25 1.25 1.25 1.25 1.25 1.25 1.25 2.5 1.25 1.25 VK5 2.5 1.25 1.25 1.25 1.25 1.25 2.5 1.25 2.5 2.5 2.5 VK6 2.5 1.25 2.5 2.5 1.25 2.5 2.5 2.5 2.5 1.25 1.25 VK7 2.5 1.25 1.25 2.5 1.25 2.5 2.5 1.25 2.5 1.25 1.25 VK8 2.5 1.25 1.25 2.5 1.25 1.25 2.5 1.25 1.25 1.25 2.5 VK9 1.25 2.5 2.5 0.078 1.25 1.25 0.078 1.25 1.25 2.5 1.25 Oleanolic 2.5 2.5 1.25 2.5 1.25 1.25 2.5 2.5 0.078 1.25 2.5 acid STM 16 128 8 256 4 512 128 16 16 128 64 NLD 16 >512 64 64 512 16 128 8 256 32 512
131
Figure 5. 46: The 96 Well plates (MIC) showing the antibacterial activities against 11 bacterial strains. ―MIC values of the synthesized hybrid compounds against eleven bacterial strains are recorded in Table 5.1. The synthesized compounds exhibited significant antibacterial activity against six bacterial strains such as Enterococcus faecalis (EF), Klebsiella oxytoca (KO),
Escherischia coli (EC), Staphylococcus aurous (SA), Proteus vulgaris (PV) and Bacillus subtilis (BS) with greater activity than did the precursor oleanolic acid.
132
VK1, VK2 and VK7 were more effective against Klebsiella oxytoca (KO), Enterococcus faecalis (EF), and Enterobacter cloacae (EC) with the MIC value of 1.25 mg/mL. VK4 was more effective against Staphylococcus aureous (SA), Proteus vulgaris (PV), Klebsiella oxytoca, Enterococcus faecalis, and Enterobacter cloacae with the MIC value 1.25 mg/mL.
VK5 had higher activity against Enterococcus faecalis, Staphylococcus aurous and
Klebsiella oxytoca with MIC value 1.25 mg/mL. VK6 had higher activity against
Enterococcus faecalis and Enterobacter cloacae with MIC value 1.25 mg/mL. VK7 was effective against EF, KO and EC with MIC value of 1.25 mg/mL. VK8 had higher activity against EF and KO with MIC value 1.25 mg/mL. VK9 was synergistic against Bacillus subtilis (BS), SA, PV, KO and EC with MIC value ranging from 1.25 mg/mL to 0.078 mg/mL.
5.11. Successful hybrid compounds
The chemical structures displaced below represent the final hybrid compounds that were successfully synthesized and evaluated for antibacterial activities.
N Cl N Cl
H H N NH N NH
O O HO VK1 HO VK2
N Cl
H N N H N N NH H N O Cl H HO O VK3 HO VK4
133
Cl O NH H O N O Cl O HO O N VK5 N VK6 HO
HO O C O O O O OH O O HO HN O O HO N VK7 VK9
Cl
O H O H
HO VK8
Figure 5. 47: Successful hybrid compounds tested for antibacterial activity.
134
CHAPTER SIX
CONCLUSION AND RECOMMENDATIONS
6.1. Conclusion
Oleanolic acid possesses various biological properties which include anti-bacteria18, antitumor19 and anticancer20. ―Recently, OA was documented as a promising lead compound for new drug formulation19. Indeed, Shirahata et al. (2012)21 demonstrated the improvement of anti-influenza effect of OA by decorating C28 position carboxylic with glycosyl ester moiety, again Pattnaik et al.(2017)22 also decorated C28 of oleanolic acid and their compounds exhibited highly potent activities against the human breast cancer cell lines
(MCF-7 & MDA-MB-231). The present study corroborates the fact that modification of OA in C28 results in enhancement of biological properties. The primary objectives of this work were successfully achieved. The oleanolic acid precursor was successfully isolated from plant
Syzygium aromaticum. The synthesis of hybrid compounds containing the isolated oleanolic acid and other pharmaceutical scaffolds was successfully accomplished and obtained using different reaction routes as described in the dissertation. The characterization and structural elucidation of the novel compounds was carried out using IR, 1H NMR, and 13C NMR spectroscopic methods supported by LC-MS. The antibacterial activity of the synthesized compounds was lastly investigated on various bacterial strains using Minimum inhibition concentration (MIC) method. The synthesized hybrid compounds were more effective compared to oleanolic acid when tested against 11 bacterial strains on both Gram-positive and Gram-negative bacteria. The synthesized compounds were selective and more active against Enterococcus faecalis (EF), Klebsiella oxytoca (KO), Escherischia coli (EC),
Staphylococcus aureous (SA), Proteus vulgaris (PV) and Bacillus subtilis (BS). The
135 synthesized compounds might be helpful in the future development of oleanolic acid analogs as novel antibacterial agents.
6.2. Recommendations
The antibacterial results were excellent indicating the isolated compounds are potent compounds and it also suggests they would exhibit excellent anticancer activity. All the successful hybrid compounds will be taken for anticancer studies. However, more studies are required in order to understand the mode of action of these hybrid compounds.
The synthetic approach needs to be reviewed for the synthesis of the unsuccessful compounds.
136
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140
APPENDIX ONE
SPECTRA OF EUGENOL 100
90 3536 3012 2856
3084 1610
2926 80
70 Transmitance %
60 1034 1513 1267
50 4000 3500 3000 2500 2000 1500 1000 500 wavenumber cm--1
Figure 3.9: FTIR spectrum of eugenol.
141
Figure 3. 10: 1H NMR spectrum of eugenol
142
Figure 3.11: 13C NMR spectrum of eugenol.
143
Figure 3.12: : LC-MS results of eugenol.
144
APPENDIX TWO
Spectra of oleanolic acid 100
95
3443 90
85 1466 transmitance% 2870 998
1031 80 1693 2941 75 4000 3500 3000 2500 2000 1500 1000 500 wavenumber/mc-1
Figure 3.13: FT-IR of oleanolic acid.
145
Figure 3.14: 1H NMR spetrum of oleanolic acid.
146
Figure 3.15: 13C NMR spectrum of oleanolic acid
147
Figure 3.16: : LC-MS results of eugenol.
148
APPENDIX THREE
Spectra of Maslinic acid 100
95
3411 90
2884 1464 85 Transmittance%
1279 1053 2940 80
1691
75 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber mc-1
Figure 3.17: FT-IR Spectrum of Maslinic acid.
149
Figure 3.18: 1H NMR Spectrum of Maslinic acid
150
Figure 3.19: 13C NMR Spectrum of Maslinic acid
151
Figure 3.20: 13C NMR Spectrum of Maslinic acid
152
153