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SOLID PHASE EXTRACTION OF BIOGENIC AMINES
USING IMMOBILIZED NEW HYDRAZONE LIGANDS
by
ABDASSALAM ABDELHAFIZ ALFERGANI
Thesis submitted in fulfillment of the requirements for the degree
of
Doctor of Philosophy
Universiti Sains Malaysia
February 2011
ii
ACKNOWLEDGMENTS
I am greatful to many people who have contributed to my graduate education and
experience. Since my first step in my PhD studies till now, many individuals helped to
make this thesis a reality. I would like to thank Sebha University and the Ministry of
Higher Education Libya for providing the scholaship and paid leave of absence during my
study.
Foremost, I am highly grateful to my supervisor Professor Bahruddin Saad for his
guidance, comments, motivation and his unwavering support over the last 5 years. I
would like to express my acknowledments and appreciation to my co-supervisor Dr.
Abdussalam Salhin for his help and dedication and patience in accomplishing this project.
Special thanks also goes to my co-supervisor Dato’ Professor Muhammad Idiris Saleh for
his help throughout the research.
It is a pleasure as well as an obligation to acknowledge the cordial working relationship
that I enjoyed with all the members of my research group, specially Ahmad Makahleh,
Khaldun Mohammad, Marwan Shalash, Elbaleeq Adam, Gan Hui Siang, Wong Yong,
Mardiana Saaid, Bahiyah Abubaker, and Jamalia Jaafar, for their support and friendship. I
consider my self very fortunate to have known them all and wish them the very best
wishes in all their endeavors.
I would also like to thank the staff of the School of Chemical Sciences, USM specially to
Mr Ariffin Majid and Mr Hashim Othman for Their great help and continuous assistance
during my research. I wish to thank Universiti Sains Malaysia, School of Chemical
Sciences for financial support throughout my studies.
iii
Not to be forgotten,
my best friend, (Abdurrahman Saif Annaesr, Abdulgader Almadani, Abdallah Ali,
Almahdi Albaroni, Ibrahim Albaroni, and Muftah Gamag) for their continuous support
and help throughout the last five years, I am grateful to thank all of them.
I am ever greatful indiebted to Dr. El-Sayed Negim for all his suggestions, instigation and
assistance throughout the final stage of my study. I also would like to thank Dr. Naser
Eltaher for his help and assistance.
My gratitude is also due to my wife, Burnia, children, (Ali, Mohamed, Nasser, Abdelhafiz
and Mohamed) and entire Alferjani family their unscelfish sacrifice, patience and
reassurance.
My beloved parents, Alhaj Abdelhafiz Alferjani and Alhaja Modallala Abobaker
have always been such amazing support. Thanks for always encouraging me in my
interests and teaching me about values and respect. I am totally grateful for you and I
would never be where I am, if you have not sacrificed everything in your life for me. That
is why this thesis is specially dedicated to you.
iv
TABLES OF CONTENTS
Title Page
ACKNOWLEDMENTS ii
TABLE OF CONTENTS iv
LIST OF TABLES ix
LIST OF SCHEMES AND FIGURES x
LIST OF ABBREVIATIONS xii
ABSTRAK xv
ABSTRACT xvii
CHAPTER ONE : INTRODUCTION 1
1.1 Hydrazones 1
1.2 Hydrazines 2
1.3 Synthesis of hydrazones 3
1.3.1 Condensation of functionalized aldehydes and ketones with
hydrazines
3
1.3.2 Coupling active methylenes with aromatic and heteroaromatic
diazonium salts
4
1.4 Applications of hydrazones 6
1.4.1 Analytical applications of hydrazones and their derivatives 6
1.4.2 Biological activity 16
1.4.2.1 Anti-convulsant activity 17
1.4.2.2 Anti-depressant activity 18
1.4.2.3 Anti-inflammatory activity 18
1.4.2.4 Anti-malarial activity
20
1.4.2.5 Anti-microbial activity
21
1.4.2.6 Anti-mycobactrial activity 24
v
1.4.2.7 Anti-tumoral activity 26
1.5 Biogenic amines 28
1.5.1 Functions of BA 29
1.5.2 Formation of BA
29
1.5.3 Prevalence of BA in food 30
1.6 Solid phase extraction 31
1.7 Research objectives 33
1.8 Thesis organization 33
CHAPTER TWO : EXPERIMENTAL 35
2.1 Chemicals and materials 35
2.2 Synthesis of hydrazone schiff base ligands 35
2.2.1 Synthesis of benzophenone-2,4-dinitrophenylhydrazone
(HL1)
35
2.2.2 Synthesis of formaldehyde-2,4-dinitrophenylhydrazone (HL2)
35
2.2.3 Synthesis of salicylaldehyde-2,4-dinitrophenylhydrazone
(HL3)
37
2.2.4 Synthesis of 3-hydroxy-4-nitrobenzaldehyde-2,4-
dinitrophenylhydrazone (HL4)
37
2.2.5 Synthesis of 3-hydroxy-4-nitrobenzaldehyde-4-
hydroxybenzoylhydrazone (HL5)
38
2.2.6 Synthesis of 2-hydroxy-5-nitrobenzaldehyde-2,4-
dinitrophenylhydrazone (HL6)
38
2.2.7 Synthesis of 2-hydroxy-5-nitrobenzaldehyde-4-
hydroxybenzoylhydrazone (HL7)
39
2.2.8 Synthesis of salicylaldehyde-4-hydroxybenzoylhydrazone
(HL8)
39
2.3 Preparation of sol-gel immobilized hydrazone sorbents 40
2.4 Antimicrobial activity 40
vi
2.4.1 Source of microbes 40
2.4.2 Half strength potato dextrose agar (PDA) 41
2.4.3 Preparation of hydrazone concentration 41
2.4.4 Sterilization of testing materials 41
2.4.5 Preparation of inoculum 41
2.4.6 Testing of hydrazone 42
2.5 Preparation of BA standard solutions 43
2.6 Derivatization procedure 43
2.7 Extraction of BA 43
2.8 Determination of BA in food samples 44
2.9 Instrumentation 44
2.9.1 Elemental analysis 44
2.9.2 Infrared measurements 44
2.9.3 Nuclear magnetic reasonance spectroscopic (NMR) studies 45
2.9.4 X-Ray crystallography 45
2.9.5 Thermal analysis 45
2.9.6 UV-Vis measurements 46
2.9.7 HPLC analysis 46
CHAPTER THREE: CHARACTERIZATION OF LIGANDS AND
SORBENTS
47
3.1 Characterization of ligands
47
3.1.1 Elemental analysis (CHN) 47
3.1..2 FTIR spectroscopic analysis 47
3.1.3 1H NMR spectroscopic analyses 49
3.1.4 X-Ray crystallographic analyses 54
3.1.5 Thermal analyses 59
3.2 Characterization of Sol-gel sorbents 60
vii
3.2.1 FT-IR spectroscopic analysis 60
3.2.2 UV-Vis analysis 61
3.2.3 Thermal analysis 62
CHAPTER FOUR: BIOLOGICAL ACTIVITY SCREENING 68
4.1 Biological species 69
4.1.1 Fungi 69
4.1.2 Bacteria 72
4.2 Results and discussion 73
4.2.1 Anti-fungal activity of hydrazones against A. flavus 73
4.2.2 Anti- fungal activity of hydrazones against Aspergillus
fumigatus
74
4.2.3 Anti-fungal activity of hydrazones against A.niger 74
4.2.4 Anti-bacterial activity of hydrazones against Enterobacter
aerogenes
76
4.2.5 Anti-bacterial activity of hydrazones against Staphylococcus
aureus
76
4.3 Structure activity relationship 78
CHAPTER FIVE: SOLID PHASE EXTRACTION OF BIOGENIC
AMINES USING SORBENTS BASED ON
IMMOBILIZED HYDRAZONE LIGANDS
80
5.1 Determination of BA 80
5.2 Results and discussion 88
5.2.1 HPLC conditions 88
5.2.2 Solid-phase extraction of BA (batch method) 90
5.2.2.1 Leaching studies 90
5.2.2.2 Optimized parameters for extraction 91
5.3 Summary 102
CHAPTER SIX: CONCLUSIONS AND SUGGESTIONS FOR
FUTURE WORK
103
viii
REFERENCES 106
THESIS OUTCOME 127
APPENDIXES 129
Appendix A The crystal packing of benzophenone 2,4-
dinitrophenylhydrazone HL1, formaldehyde 2,4-
dinitrophenylhydrazone HL2, 2-hydroxy-5-nitrobenzaldehyde
2,4-dinitrophenyl-hydrazone HL6, and 4-Hydroxy-N'-[(E)-(2-
hydroxyphenyl)methylidene]-benzo-hydrazide HL8
129
Appendix B
Thermograms of the studied compounds. 130
Appendix C Thermograms of the studied sol-gel sorbents. 131
Appendix D
1H NMR data for ligands (HL1-HL8) 132
ix
LIST OF TABLES
Table 1.1 Some examples of potentiometric sensors based on hydrazone
derivatives for the determination of cationic species.
14
Table 1.2 Structures of the BA studied.
28
Table 2.1 Chemicals and reagents used in the study.
36
Table 3.1 CHN results for hydrazone ligands HL1- HL8.
48
Table 3.2 Selected FT-IR data (cm-1) for ligands HL1-HL8.
48
Table 3.3 Chemical shifts of protons in hydrazone ligand (HL1-HL8).
50
Table 3.4 The crystal structure, data and refinement parameters for HL1 ligand.
55
Table 3.5 The crystal structure, data and refinement parameters for HL2 ligand.
56
Table 3.6 The crystal structure, data and refinement parameters for HL6 ligand.
57
Table 3.7 The crystal structure, data and refinement parameters for HL8 ligand.
58
Table 3.8 Selected angles and bond lengths for the studied hydrazone compounds.
59
Table 3.9 Thermal data for the decomposition processes of ligand HL1-HL8.
60
Table 3.10 Solid state UV-Vis data of free ligands (HL1-HL8), blank and sorbents
immobilized Sol-gel (Sol1-Sol8).
62
Table 3.11 Thermal data for the decomposition processes of the sorbents (Sol1-
Sol8).
66
Table 4.1 Chemical structure of the studied hydrazones.
71
Table 4.2 Collective measured diameter (mm) of the inhibition zones after the
action of the hydrazones on different microbes.
75
Table 5.1 Some derivatizing reagents and related experimental conditions used in
various methods for the determination of BA.
84
Table 5.2 Preparation procedures and HPLC conditions used for the determination
of BA in food.
86
Table 5.3 Analytical characteristics of the HPLC method.
89
Table 5.4 The capacity of sorbent (mmol g -1) with different concentrations of BA.
97
Table 5.5 Reusability of the sol-gel sorbent.
98
x
Table 5.6 Recoveries (%) of BA from spiked food samples. 98
LIST OF SCHEMES AND FIGURES
SCHEMES
Scheme 1.1 Nitrogen lone pair resonance. 2
Scheme 1.2 Condensation of functionalized aldehyde and ketone with
hydrazines.
3
Scheme 1.3 Coupling active methylenes with aromatic and heteroaromatic
diazonium salts.
4
Scheme 1.4 Coupling of oxo- and oxophenylbutanamide with aromatic
diazonium salts.
5
Scheme 1.5 Coupling of acetato- and cyano-compounds with aromatic
diazonium salts.
5
Scheme 1.6 Coupling of cyanoacetic acid with aromatic diazonium salts. 5
FIGURES
Figure 1.1 Structures of some hydrazone derivatives used as potentiometric
sensors for the determination of cation species.
15
Figure 2.1 Testing of hydrazone towards microbes by using filter paper
technique.
42
Figure 3.1 FTIR spectra of free hydrazone compounds HL1-HL8. 49
Figure 3.2 400 MHz 1H NMR spectra of hydrazone ligands (HL1-HL8) IN
DMSO-d6 at 25ºC.
51
Figure 3.3 UV-Vis spectra of (1) blank sol-gel sorbent, (2) free ligand, and (3)
sol-gel sorbent (a) HL1, (b) HL2, (c) HL3, (d) HL4, (e) HL5, (f)
HL6, (g) HL7 and (h) HL8.
63
Figure 4.1 Photograph (Enterobacter aerogens) showing antibacterial
screening of hydrazones. Cncentration of hydrazone on filter
paper from clockwise: 15 µg mL-1, 6 µg mL-1, control.
77
Figure 4.2 Photograph (Staphylococcus staphylococcus) showing
antibacterial screening of hydrazones.
77
Figure 4.3 Effect of hydrazones on fungi and bacteria at 15 µg mL-1
concentration.
79
Figure 5.1 Chromatogram of BA (20 mg L-1 each) standard solution.
TRY (1); PEA (2); PUT (3); CAD (4); HIS (5); TYR (6);
SPD (7).
89
Figure 5.2 UV-Vis absorption spectra of sorbent (Sol8) when exposed in
ethanol (1); methanol (2); acetonitrile (3); buffer, pH 9 (4);
water (5) and 0.1 M HCl (6).
91
xi
Figure 5.3 Effect of pH on the extraction of BA (20 mg L-1) with
Sol1Sol8 sorbents. Extraction time = 30 min.
93
Figure 5.4 Effect of contact time on the extraction of BA (20 mg L-1);
pH 9; Sol1.
94
Figure 5.5 Effect of ligand concentration on the extraction of BA (20 mg
L-1); pH 9; contact time 30 min; Sol1.
95
Figure 5.6 Effect of biogenic amine (BA) concentration on the
extraction. Extraction conditions: 25 mg sorbent, extraction
time; 30 min, pH; 9, and ligand concentration; 17 10-3 M .
96
Figure 5.7 Chromatograms of extracts of (a) ketchup, (b) ketchup spiked
with 5 mg L-1 BA before extraction and (c) ketchup spiked
with 5 mg L-1 BA after extraction. Peak identification: TRY
(1); PEA (2); PUT (3); CAD (4); HIS (5); TYR (6); SPD (7).
99
Figure 5.8 Chromatograms of extracts of (a) orange, (b) orange spiked
with 5 mg L-1 BA before extraction and (c) orange spiked
with 5 mg L-1 BA after extraction. Peak identification: TRY
(1); PEA (2); PUT (3); CAD (4); HIS (5); TYR (6); SPD (7).
100
Figure 5.9 Chromatograms of extracts of (a) soy bean, (b) soy bean
spiked with 5 mg L-1 BA before extraction and (c) soy bean
spiked with 5 mg L-1 BA after extraction. Peak identification:
TRY (1); PEA (2); PUT (3); CAD (4); HIS (5); TYR (6);
SPD (7).
101
xii
LIST OF ABBREVIATIONS
ACN Acetonitrile
AGM Agmatine
BA Biogenic amines
C18 Octadecylsilane groups
CAD Cadaverine
CE Capillary electrophoresis
CHN Carbon, hydrogen and nitrogen elemental analysis
DAD Diode array detector
DAPCH 1-(3,4–dihydroxybenzaldehyde)–2–acetylpyridiniumchloride hydrazone
DMSO Dimethylsulfoxide
Dn-Cl Dansyl chloride
ELISA Enzyme-linked immunosorbent assays
EtOH Ethanol
FL Fluorescence
FTIR Fourier transform infrared spectroscopy
GC Gas chromatography
GC-MS Gas chromatography-mass spectrometry
HBH 4-hydroxybenzoylhydrazine
HCl Hydrochloric acid
HIS Histamine
HL Hydrazone ligand
HPLC High performance liquid chromatography
LC Liquid chromatography
LC-MS Liquid chromatography-mass spectrometry
LIF Laser induced fluorescence
xiii
LLE Liquid-liquid extraction
LOD Limit of detection
LOQ Limit of quantification
LPME Liquid phase microextraction
MeOH Methanol
NaHCO3 Sodium hydrogen carbonate
NaOH Sodium hydroxide
NQS 1,2-naphthoquinone-4-sulfonatemethanol
NR Not reported
OPA o-phthalaldehyde
P.S.I Pounds per square inch
PDA Potato dextrose agar
PEA β-phenylethylamine
PSE 1-pyrenebutanoic acid succinimidyl ester
PUT Putrescine
PVC Polyvinylchloride
r2 Square of regression coefficient
RSD Relative standard deviation
SD Standard deviation
SPD Spermidine
SPE Solid phase extraction
SPM Spermine
SPME Solid phase microexteaction
TEOS Tetraethoxyorthosilane
TGA Thermogravimetric analysis
THF Tetrahydrofuran
TLC Thin layer chromatography
xiv
TRY Tryptamine
TYR Tyramine
UDLLME Ultrasound-assisted depressive liquid-liquid microextraction
UV-Vis Ultraviolet and visible
Wt Weight
1H NMR Proton nuclear magnetic resonance spectroscopy
xv
PENGEKSTRAKAN FASA PEPEJAL TERHADAP AMINA
BIOGENIK MENGGUNAKAN LIGAN HIDRAZON BARU
TERPEGUN
ABSTRAK
Dalam kajian ini, sebatian hidrazin dan hidrazid melalui tindak balas kondensasi
dengan keton dan terbitan berbeza aldehid menghasilkan hidrazon sepadan yang
dijangkakan. Kesemua ligan yang dipisahkan memperolehi hasil yang memuaskan (82-94
%). Lapan ligan hidrazon iaitu benzofenon-2,4-dinitrofenilhidrazon (HL1), formaldehid-
2,4-dinitrofenilhidrazon (HL2), salisilaldehid-2,4-dinitrofenilhidrazon (HL3), 3-hidroksi-
4-nitrobenzaldehid-2,4-dinitrofenilhidrazon (HL4), 3-hidroksi-4-nitrobenzaldehid-4-
hidroksibenzoilhidrazone (HL5), 2-hidroksi-5-nitrobenzaldehid-2,4-dinitrofenilhidrazon
(HL6), 2-hidroksi-5-nitrobenzaldehid-4-hidroksibenzoilhidrazon (HL7) dan
salisilaldehid-4- hidroksibenzoilhidrazon (HL8) telah disintesis dan dicirikan. Struktur
kimia ligan-ligan ini adalah konsisten dengan data analisis dan spektroskopi (CHN, FTIR,
UV-Vis, TGA dan 1H NMR). Ligan-ligan yang diperolehi diuji untuk aktiviti anti-
mikrobial terhadap fungi; Aspergillus niger, Aspergillus flavus dan Aspergillus fumigitus
dan bacteria; Staphylooccus sp dan Enterobakteria sp. Keputusan menunjukkan kesemua
sebatian ini muncul sebagai agen antibakteria berbanding antifungal.
Lapan bahan penjerap (Sol1-Sol8) berasaskan kepada ligan hidrazon yang
disintesis (HL1-HL8), telah disediakan secara pemegunan ligan-ligan tersebut ke atas
matrik silica sol-gel. Keupayaan bahan penjerap untuk mengekstrak tujuh amina biogenik
(BA) iaitu triptamina (TRY), -feniletilamina (PEA), putrescina (PUT), cadaverina
(CAD), histamina (HIS), tiramina (TYR) dan spermidina (SPD) telah dikaji
xvi
menggunakan kaedah serapan kelompok. Ekstrak dianalisis menggunakan kromatografi
cecair prestasi tinggi (HPLC) dan pengesan ultralembayung (254 nm). Keputusan yang
diperolehi menunjukkan penjeraap Sol1 berasaskan hidrazon (HL1) pada pH 9
menunjukkan kepilihan yang lebih terhadap amina alifatik (PUT, CAD, SPD) dan
heterosiklik (HIS) (%E > 96) berbanding aromatic (TRY, PEA, TYR) (%E 78.7, 46.4 dan
82.0 masing-masing). Keupayaan pengekstrakan bahan penjerap ini yang tinggi
diperolehi disebabkan oleh afiniti di antara penjerap sol-gel yang mengandungi ligan
hidrazin dan analit yang dikehendaki. Bahan penjerap ini boleh digunakan sehingga enam
kali pengekstrakan terhadap SPD, CAD dan PUT dan telah digunakan untuk penentuan
BA di dalam sampel makanan yang berbeza (sos, jus oren dan kicap) yang telah
ditambahkan dengan 5 mg L-1 BA piawai. Analit yang telah diekstrak diterbitkan
menggunakan dansil klorida sebelum penentuan HPLC. Perolehan semula yang
memuaskan (84.1 - 106.7%) telah diperolehi untuk kesemua analit dalam sampel
makanan yang telah diuji.
xvii
ABSTRACT
In this work, hydrazine and hydrazide compounds were subjected to condensation
reaction with ketone and different aldehyde derivatives affording the corresponding
expected hydrazones. All the ligands were isolated in satisfactory yields (82 94%).
Eight hydrazone ligands, namely, benzophenone - 2,4 - dinitrophenylhydrazone (HL1),
formaldehyde-2,4–dinitrophenylhydrazone (HL2), salicylaldehyde–2,4– dinitrophenyl-
hydrazone (HL3), 3-hydroxy-4-nitrobenzaldehyde-2,4-dinitrophenylhydrazone (HL4), 3-
hydroxy-4-nitrobenzaldehyde-4-hydroxybenzoylhydrazone (HL5), 2-hydroxy-5-nitro-
benzaldehyde-2,4-dinitrophenylhydrazone (HL6), 2-hydroxy-5-nitrobenzaldehyde-4-
hydroxybenzoylhydrazone (HL7) and salicylaldehyde-4-hydroxybenzoylhydrazone
(HL8) have been synthesized and characterized. Their chemical structures were found to
be consistent with both analytical and spectroscopic data (CHN, FTIR, UV/Vis, TGA and
1H NMR). The obtained ligands were evaluated for their anti-microbial activity towards
fungi, Aspergillus niger, Aspergillus flavus and Aspergillus fumigatus and bacteria,
Staphylooccus sp and Enterobacter sp. The results showed that these compounds are
mostly antibacterial rather than antifungal agents.
Eight sorbent materials (Sol1-Sol8) based on the newly synthesized hydrazone
ligand (HL1-HL8), were prepared by immobilizing the ligands into a silica sol-gel matrix.
The capability of the sorbent materials for the extraction of seven biogenic amines (BA),
i.e., tryptamine (TRY), β-phenylethylamine (PEA), putrescine (PUT), cadaverine (CAD),
histamine (HIS), tyramine (TYR), and spermidine (SPD) was studied by the batch
sorption method. The extracts were analysed by high-performance liquid chromatography
(HPLC) with ultraviolet detection (254 nm). Results obtained showed that sorbent Sol1
based hydrazone (HL1) at pH 9, exhibited preferential selectivity towards the aliphatic
xviii
(PUT, CAD, SPD) and the heterocyclic (HIS) (%E > 96) over the aromatic (TRY, PEA,
TYR) BA (%E 78.7, 46.4 and 82.0 respectively). The high extraction efficiency of the
sorbent was achieved due to the high affinity between the sol-gel sorbent that contained
the hydrazone ligand and the target analyte. The sorbent could be used up to six
extraction cycles for SPD, CAD and PUT and was applied to the determination of BA in
different food samples (ketchup, orange juice, soy sauce) that were spiked with 5 mg L–1
of the standard BA. The extracted analytes were derivatized with dansyl chloride before
the HPLC determination. Reasonable recoveries (84.1 - 106.7%) were found for all
analytes in the tested food samples.
1
CHAPTER ONE
INTRODUCTION
1.1 Hydrazones
Hydrazones are organic compounds of the formula R1CNNR2 where R1 and R2
represent H, aliphatic and aromatic group. They are usually formed by the condensation
reaction between hydrazine and ketones or aldehydes. The oxygen atoms in aldehydes and
ketones are replaced with the NNR2 functional group. Hydrazones are practically insoluble
in hot and cold water but some are partially soluble in cold ethyl alcohol and ether. They
possess a free amino group and can condense with another molecule of the carbonyl
component to form hydrazone derivative or azine (Fieser and Fieser, 1956). The hydrazone
unit offers a number of attractive features: a degree of rigidity, a conjugate π-system and an
NH unit that can readily participate in hydrogen bonding and may be a site of protonation and
deprotonation (Beves et al., 2009). The formation of hydrazone and its derivatives can be
presented by the following equation:
C O + H2NNH2 C
C O
NNH2 C NN C
Aldehydeor
ketone
Hydrazine Hydrazone Hydrazone derivatives
(1.1)
Derivatives of hydrazine in which this double condensation is obviated
by the presence of a substituent group have more practical value than hydrazine. One of them
is phenylhydrazone that are formed from phenylhydrazine and benzaldehyde. Replacement
of the oxygen atom of the carbonyl group by =NNHC6H5 is followed by the increase in
molecular weight and decrease in solubility. An aldehyde or a ketone can often be
precipitated from a dilute solution as phenylhydrazone and identified by the melting point of
this derivative (Fieser and Fieser, 1956).
2
Accordingly, various hydrazone derivatives with different applications and properties
were prepared by the reaction of hydrazine with different aldehydes and ketones. Hydrazone
derivatives have been demonstrated to possess antimicrobial, anticonvulsant, antidepressant,
analgesic, anti-inflammatory, anti-platelet, anti-tubercular, anti-tumoral activities, etc.
Hydrazones are organic compounds in which the carbon functional group exist in the
X and Y positions as CO2R, CN are usefull in dye industry (Hunger et al., 2003). Nitrogen
lone pair resonance (Scheme 1.1) made the hydrazone to have the carbon atom electron rich
and the nucleophilicity of this carbon atom has been used extensively in the synthesis
(Hunger et al., 2003). Due to their theoretical and practical importance, the structures and
synthetsis of these molecules have been extensively studied. In this part of our study we
review the chemistry of this class of compounds.
N
N
R2
YX
R1
N
N
R2
YX
R1
Scheme 1.1: Nitrogen lone pair resonance (R1 and R2 = H, aliphatic or aromatic group)
1.2 Hydrazines
Hydrazine compounds or derivatives are the main starting material in the synthesis
and preparation of hydrazone derivatives. Hydrazine, having the formula H2N–NH2 (N2H4),
can be considered as a nitrogen analog of hydrogen peroxide. It has an ammonia-like odour
and is derived from ammonia but its physical properties are similar to water. It is a weak base
which is protonated to give the N2H5+ ions according to equation (1.2):
N2H4 (aq) + H2O (l) N2H5 (aq) + OH (aq)
(1.2)
3
Hydrazine has an endothermic enthalpy of formation; therefore it is an unstable
compound (Brady, 1990). It is a powerful reducing agent and reacts vigorously with strong
oxidizing agents such as the halogens, nitric acid, dinitrogen tetraoxide and hydrogen
peroxide to give large volume of gaseous products according to equations (1.3) and (1.4). It
has been used as rocket fuels because the combustion of hydrazine is very exothermic
(Gillepie et al., 1989).
2 N2H4 (l) + N2O4 (l) 3 N2 (g) + 4 H2O (g) (1.3)
N2H4 (l) + 2 H2O2 (l) N2 (g) + 4 H2O (g) (1.4)
1.3 Synthesis of hydrazones
1.3.1 Condensation of functionalized aldehydes and ketone with hydrazines
The required α–functional aldehyde and/or ketone can be easily provided by this route
(Alexakis et al., 1991). Condensation of glyoxal 1a and substituted glyoxal 1b and 1c with
substituted hydrazines leads to the formation of the corresponding mono or bis hydrazones 2
or 3 basing on the molar ratio and on the applied reaction conditions (Huisman et al., 1997).
It has been reported that 1b reacts with phenylhydrazine to yield 4 which then rearranges into
2 (Scheme 1.2).
R
H
O
O
R2
N
R1
NH2
R
H
O
R2
N
R1N
R H
R2
N
R1
N
R2
N
R1
N
H
RO
NHArN
1a : R = Hb : R = alkylc : R = aryl
2 3
4
R = MeArNHNH2
Rearrangment
Scheme 1.2: Condensation of functionalized aldehyde and ketone with hydrazines
4
1.3.2 Coupling active methylenes with aromatic and heteroaromatic diazonium salts
Generally the coupling reaction is conducted at ambient temperature in protic solvents
in the presence of a base. The most commonly used base is sodium acetate but reaction in the
presence of sodium hydroxide or pyridine also has been reported (Al–Awadi et al., 1995). In
this step, acetylacetone 5a, benzoylacetone 5b, diphenylpentanedione 5c and 1,3 - diketones
(6a, 6b) have been coupled with aromatic diazonium salts in ethanolic sodium acetate
solution yielding the corresponding products 7a - c and 8a - b (Al–Awadi et al., 1995)
(Scheme 1.3).
R R1
O O
R R1
O O
N
NH
Ar
ArN NCl
NaOAc / EtOH
5a : R = R1 = Me
b : Ph, R = Me
c : R = R1 = CH2Ph
7a - c
OO
R2R1
ArN NCl
OO
R2R1
N
ArHN
NaOAc / EtOH
6 a : R1 = R2 = H
b : R1 = R2 = Me8a-b
Scheme 1.3: Coupling active methylenes with aromatic and heteroaromatic diazonium salts
Similar coupling of oxobutanamide 9a and oxophenylbutanamide can readily yield
arylhydrazones 10a & b (Sadanadam et al., 1991) and were usefull as dyes and pigments
(Scheme 1.4).
5
NHR
O O
NHR
O O
N
NH
Ar
ArN NCl
NaOAc / EtOH
9a : R = Hb : R = Ph 10a-b
Scheme 1.4: Coupling of oxo- and oxophenylbutanamide with aromatic diazonium salts
Ethyl acetoacetate 11a (Kumar et al., 2003), ethyl cyanoacetate 11b (Brecknell et al.,
1969), cyanoanilides 11c (Shawali and Abd El-Galil, 1971), have been reported to be readily
coupled with aromatic diazonium salt in the presence of sodium acetate to yield the
corresponding arylhydrazones 12a – c but the exact geometry has only been established
recently (Scheme 1.5).
ArN NClY R
NaOAc / EtOH
12a - c
Y
R
N
HN
Ar
ArN NClY R
NaOAc / EtOH
11a: R = COMe; Y = CO2Et
b: R = CN; Y = CO2Et
c: R = CN; Y = CONHAr
Scheme 1.5: Coupling of acetato- and cyno-compounds with aromatic diazonium salts
The reactivity of methylene functions in acetonitrile and benzylcyanide towards
aromatic diazonium salts is not sufficient. Attempted coupling of cyanoacetic acid with
aromatic diazonium salt affords only formazans 13 (Cankař and Slouka, 2003) (Scheme 1.6).
NC CH2 CO2HArN NCl
N NHAr
NHArN
NC
13
Scheme 1.6: Coupling of cynoacetic acid with aromatic diazonium salts
6
1.4 Applications of hydrazones
1.4.1 Analytical applications of hydrazones and their derivatives
Hydrazones have been intensively investigated and their chemical properties studied.
In inorganic chemistry (mainly due to their facile synthesis), tuneable electronic and steric
properties and good chelating ability (Carcelli et al., 2005 and Das et al., 2005) were
reported. Derivatives of these compounds were investigated and used for the determination of
glucose in biological fluids such as serum, plasma and urine (Gochman and Schmitz et al.,
1972). 3–methyl–2–benzothiazolone hydrazone was applied for the determination of glucose
in serum or plasma as a blue cationic dye with glucose in aqueous solution was formed
(Neeley and Cupas, 1973).
Hydrazone derivatives are widely used in various analytical applications (Zegota,
1999; Zlotorzynska and Lai, 1999; Cordis et al., 1998; Lamberton et al., 1974). They are also
found to have versatile coordinating abilities towards different metal ions giving well-
characterized metal complexes (Raj and Kurup, 2007). They act as multidentate ligands with
metal ions (e.g., transition elements) forming coloured chelates which are then used in the
selective and sensitive determination of metal ions (Vasilikiotis and Stratis, 1975). They are
also used to determine airborne aldehydes and ketones in air samples by using 2,4–
dinitrophenylhydrazine (DNPH) method in which the analytes were analyzed by passing a
stream of air through DNPH coated silica gel test tubes which are immediately eluted with
acetonitrile and injected into HPLC system (Vogel et al., 2000). In addition they were used
for the detection of formaldehyde in libraries (Hanoune et al., 2006).
Fernando et al. (1986) synthesized N–oxalylamine (salicylaldehyde hydrazone), 14.
The Al3+–14 system exhibited fluorescence properties when tested in ethanol solution (λex =
385 nm, λem = 470 nm) and in aqeous 4% v/v DMF (λex = 370 nm, λem = 470 nm). The
optimum concentration of the reagent was 3.86 10-4 M for 198 ppb of aluminum (Al). The
7
linear concentration range of Al was 0 – 160 ppb with relative error (P = 0.05) of ± 0.86%.
This method was applied for the determination of Al in mineral waters.
OH
CH N NH C C
O
NH2
O
14
Jose et al. (1984) synthesized N,N′-oxalylbis(salicylaldehyde hydrazone) 15, which
was used for the spectrophotometric and spectrofluorimetric determination of trace amounts
of aluminium. The complex absorbs maximally at 390 nm and is linear over 0.1 – 1.8 g mL-1
Al. The molar absorptivity of the 15 – aluminium complex was 14.5 103l mol-1 cm-1. Cations
such as Hg2+, Ni2+, Pb2+, In3+, Ga3+ Ti4+, V5+, Cu2+, Zr4+ and Fe3+ interfered. They were
minimized by using thiosulphate as a masking agent and also by retention on a cation –
exchange column.
The fluorimetric method for the determination of aluminium using 15 was linear over
0 - 200 ppb aluminium. Relative error is 6.7% (P = 0.05). Interferences of gallium, indium
and thorium, also the effects of Pd2+, Th4+, Bi3+, Ti4+, V5+, Cu2+, Fe3+ and Ti4+ on the
applicability of the reagent was also studied. The developed methods were applied for the
determination of Al in alloys, pharmaceutical preparations and mineral water.
OH
CH N NH C C
O O
HO
CHNNH
15
Silva and Valcàrcel, (1980) synthesized diphenylglyoxal bis(2– hydroxybonzoyl
hydrazone) 16, and was used for the spectrophotometric determination of calcium in water
and foods. This compound reacts with calcium (II) at pH 12 to produce a yellow complex.
8
Beer’s law was obeyed from 0.5 and 2.0 µg mL-1 and the molar absorptivity at 432 nm was
1.76 ± 0.14 104 L mol-1 cm-1.
Another reagent, 16 enables Beer’s law to be obeyed from 0.75 – 3.0 ppm of calcium.
Molar absorptivity was 1.03 ± 0.05 104 L mol-1 cm-1. The linear concentration range of
calcium was from 1.24 – 2.50 ppm with minimum error (P = 0.05) as ± 32%. Interferences
from strontium, barium and magnesium were also studied.
Chan and Cha, (1998) reported the synthesis of 2–hydroxybenzaldehyde–5–nitro–
pyridylhydrazone 17, and its application in the spectrophotometric determination of trace
amounts of cobalt in the presence of surfactants after a column separation containing
Amberlite resin. The method was linear over 0.02 – 2.0 mg L-1 and the limit of detection was
0.01 mg L-1.
C
C
N
N NH
NH
R
R
C
C
O
O
HO
HO
N
R
BSHB
BSHP
Compound
16
N
O2N
N
N
C
HO
H H
17
9
Zhang et al. (2009) synthesized a series of novel colorimetric anion sensors
18(a – f) bearing phenol O–H and hydrazone N–H groups. Compounds 18a, b and d showed
selective recognition for acetate ion in DMSO/H2O (7.5 : 2.5, v/v) solutions. Sensors 18 (a &
b) could selectively sense chloride ion in DMSO/H2O (9 : 1, v/v) binary solutions by the
collaboration of phenol O–H and hydrazone N–H groups. Comparison with sensor 18e
indicates that the introduction of acidic phenol O–H group significantly enhanced the sensor′s
affinity to the chloride ion, which also enhanced the sensitivity of the sensor for sensing
acetate ion. It was found that the O–H group plays a critical role in the anion-recognition
process
.
Cl
OH
NH
N
X1
X2
NH
N
X1
X2
OH
NH
N
X1
X2
NH
N
X1
X2
NH
N
X1
X2
18a & b
18c & d
18e & f
18a, c, e: X1 = NO2, X2 = H; b, d, f: X1 = X2 = NO2
Shang and Xub, (2009) synthesized a series of artificial receptors and hydrazone
derivatives containing anthracene (19a-c). The reactivity of these compounds towards
10
biologically important anions was studied by UV–vis, fluorescence and 1H NMR titration
experiments and theoretical investigations. Results showed that the reagent 19a in the
absence of NO2 has no binding ability for various anions. While other reagents 19(b & c)
showed the highest binding ability towards various anions. Moreover, the colour changes
took place during the binding between the host and guest, has made this system to be used as
colorimetric anion sensor. The interaction of this reagent with the studied anions depends on
the NO2 which acts as a colorimetric and electron withdrawing group. The reagent is not
affected by the presence of other anions.
N
N
H
R2 R1
19
a: R1 = R2 = H
b: R1 = NO2, R2 = H
c: R1 = R2 = NO2
Hamid et al. (2005) prepared polyvinylchloride (PVC) – based membrane electrodes
that are selective to lead ion, containing a neutral ionophore 1–phenyl–2-(2–quinolyl)–1,2–
dioxo–2–(4–bromo)phenylhydrazone 20. It was used as an indicator electrode for the
potentiometric titration of lead ion with potassium dichromate.
N
N
NH
O
Br
20
11
Ganjali et al. (2009) reported a highly selective and sensitive dysprosium (III) optical
sensor based on N'-[(2-hydroxyphenyl)methylene] benzohydrazide (BBH) immbilized on
triacetylcellulose 21. The effect of various ions present in the sample was studied. This sensor
was successfully applied for the direct determination of Dy (III) ions in urine and river water.
OH
N
NH
O
21
Jie Shao et al. (2008) investigated phenylhydrazone – based sensor 22 for the sensing
of acetate in DMSO and in aqueous solutions. The change in colour of the reagent from
yellow to red indicates its binding capability upon the addition of acetate. The intensity of the
colour can be compared by the naked eye in which its colour induced by acetate is deep red
while by phosphate or fluoride is light red. Acetate anion produced stronger complex than the
other anions which allows acetate anion to be selectively detected from other anions.
Jie Shao et al. (2009) reported that an anion receptor 23 based on a tripodal
benzaldehydic – phenylhydrazone can selectively sense acetate ion by using UV – vis
absorption spectroscopy in DMSO. The interaction of this reagent (host) with the acetate
O2N
NO2
NH
N C OH
Br22
12
anion (guest) was found to be in the ratio (1:3). The colour change disappears upon addition
of water which disturbs the anion binding. The reagent has proven as an excellent sensor for
anions such as fluoride and acetate in organic medium.
N
N
N
NH
HN
NH
NO2
O2N
NO2
NO2
NO2
NO2
23
El–Seify and El-Dossoki, (2006) studied the electronic spectra of [3–(2–thenoyl) 3–
(p–NO2– phenylhydrazone) ethyl pyruvate] 24, p–Br (II) and p–CH3 in ethanol and the
obtained spectra showed four absorption bands which are due to the corresponding electronic
transition. Conductometric titrations have also been conducted and the results showed that
this transition can be used for the determination of the metal ion and also the ligand
concentrations. Polymeric membrane lanthanum (III)-selective electrode based on N,N'-
adipylbis(5-plenylazosalicylaldehyde hydrazone) 24 was also reported (El-Seify and El-
Dossoki, 2006).
S
C C
N
C
O
OC2H5
O
NH
C
O
X
(I): (X = NO2; (II): (X = Br); (III): (CH3)
24
13
Hassanien et al. (2008) has successfully separated chromium III & VI from different
solutions using 1-(3,4–dihydroxybenzaldehyde)–2–acetylpyridinium chloride hydrazone 25
loaded on Duolite C20 in batch and column modes. The method is simple, fast and can easily
be applied for the separation and determination of two cations simultaneously. The separation
was done by using 100 mg modified resin with stirring time for 45 and 30 min. Cr(III) was
separated at pH 6 and Cr(IV) at pH 2, respectively. The method showed high recovery (97–
100%), satisfactory repeatability with RSD < 4% and it was applied successfully for the
determination of chromium in tap water and synthetic mixtures by using the column
technique.
N
HN
N
OH
OH
O
HC
SO3
H2C
25 DAPCH-Duolite C20
Hydrazones were used as potentiometric sensors for the determination of cation
species. Some of these sensors are summarized in Table 1.1 and their structures shown in
Figure 1.1.
14
Table 1.1 Some examples of potentiometric sensors based on hydrazone derivatives for the determination of cationic species.
Ligand
no.* Cation Hydrazone ionophore
Linear range
(M)
Slope
)1 -(mV decade
Interfering ions
2)-> sel(log K Reference
26 3+Er N-(2-hydroxy-1,2-diphenyl-
ethyldiene)benzohydrazide 2-10 -7-10 21.0 - Ganjali et al., 2006
27 3+Er Pyridine-2-carbaldehyde-2-(4methyl-1,3-
benzothiazol-2-yl)hydrazone 2-10 -5 -10 21.8 3+ , Gd3+, Tm3+Sm Ganjali et al., 2007
28 3+Eu Bis(thiophenol)butane-2,3-dihydrazone 2-10 -5 -10 19.8 3+ , Sm3+ , Gd3+La Ganjali et al., 2007
29 2+Hg Bis(2-hydroxy-benzophenone)- butane-
2,3-dihydrazone 1-10 -6 -10 29.7 3+ , La2+, Cd3+Fe Ganjali et al., 2007
30 3+La Bis(2-methylbenzaldehyde)-butane-2,3-
dihydrazone 1-10 -5 -10 19.8 3+, Pr3+, Ce2+Cu Ganjali et al., 2005
31 3+La 3-hydroxy- N -(pyridin-2-ylmethylene)-
2-naphthohydrazide 2-10 -7 -10 19.2 3+ , Ce3+Yb Ganjali et al., 2006
32 3+La N -(1-pyridine-2-y lmethylene)-2-
furohydrazide 1-10 -6 -10 19.2 3+, Nd3+Sm Zamani et al., 2006
33 3+Ce N’-[(2-hydroxyphenyl)methylidene]-2-
furohydrazide 1 -10 -5 -10 19.4 - Zamani et al., 2007
34 3+Pr N-(pyridin-2-ylmethylene)
benzohydrazide 6 -10 - 2-10 21.1 3+ , Er3+Sm Ganjali et al., 2006
35 3+Sm
3-{[2-oxo-1(2H)-
acenaphthylenyliden]amino}-2-thioxo-
1,3-thiazolidin-4-one
1-10 -6 -10 19.3 - Zamani et al., 2007
36 3+Tm Thiophene-2-carbaldehyde-(7-methyl-1,3-
benzothiazol-2-yl) hydrazone 2-10 -5 -10 19.5 3+Er Ganjali et al., 2005
37 3+Yb 3-hydroxy- N-[(2-hydroxy-phenyl)-
methylene]-2-naphthohydrazide 2-10 -7 -10 19.2 3+ , Gd2+, Pb3+Nd Ganjali et al., 2006
*= Please refer to Figure 1.1 for their structures.
15
N OHHN
O
26
O2N
N
NO2 N
H3C
H
NO2
N
NO2N
CH3
H
30
S
N
NH
N
CH3
N27
OH
NH
N
O
N
31
H3C CH3
N N NN
HH
SS
28
O
O
HNN
N
32
N
H3C
N
CH3
NN
PhPh
HOOH
29
O
HN
O
N
HO
33
Figure 1.1 Structures of some hydrazone derivatives used as potentiometric sensors for the
determination of cation species.
16
N
N
N
PhO
H34
N
S
NH
CH3
N
S36
ONN
S
O
O35
OH
NH
N
O
HO
37
Figure 1.1. Continued.
1.4.2 Biological activity
Hydrazones have been extensively studied due to their biologically active properties
such as anti-tumoral, analagesic, anti-platelet, anti-microbial, anti-tubercular and anti-
inflammatory activities. For example, isonicotinoyl hydrazones have been reported to have
anti-tubercular activity, also the activity of 4-hydroxybenzoic acid [(5-nitro-2-furyl)
methylene]-hydrazide (nifuroxazide) was investigated and the results showed that this
compound was active as an intestinal anti-septic. Moreover, Küçükgüzel et al. (1999) and
Rollas et al. (2002) have found that 4-fluorobenzoic acid[(5-nitro-2-furyl)methylene]-
hydrazide and 2,3,4-pentanetrione-3-[4-[[(5-nitro-2-furyl) methylene] hydrazino]carbonyl]
phenyl]-hydrazone have anti-bacterial activity against both Staphylococcus aureus and
Mycobacterium tuberculosis. In addition N1-(4-methoxybenzamido)benzoyl]-N2-[(5-nitro-2-
furyl)methylene]hydrazine was prepared and tested for its anti-bacterial activity (Küçükgüzel
et al., 2002). Recently the activities of some newly synthesized hydrazide hydrazones were
investigated against M. tuberculosis (Kaymakçıоğlu et al., 2006).
17
1.4.2.1 Anti-convulsant activity
Biological tests indicated that, the activity of acetylhydrazone 38 was higher than that
of oxamoylhydrazones 39 against convulsions. The results showed that the hydrogen bonding
capabilities and anti-convulsant properties of these compounds was enhanced by the electron-
donating groups (Dimmock et al., 2000).
H3C C N
O H
N C
R3
R2 R1
H2N C N
O
H
N C
R3
R2 R1
C
O
38 39
R1 = H, Cl, CH3, OCH3, Br;
R2 = R3 = H
A series of 4-aminobutyric acid (GABA) hydrazones 40 were synthesized and tested
for their anti-convulsant properties in different animal models of epilepsy such as the
maximal electroshock, the subcutaneous pentylenetetrazole, subcutaneous and intraperitonal
picrotoxin induced seizure tests. Some of these derivatives were active in these models.
Functionality of these compounds plays an important role in the anticonvulsant properties
(Ragavendran et al., 2007).
N
O
O
40
O
R1
N
HN
R2
R1 = H, 2-CH3, 4-Br, 3-Cl ; R2= 4-Cl, 2-CF, 3-F, 6-CH3, 2-Br
18
1.4.2.2 Anti-depressant activity
A new group of compounds of arylidenehydrazides 41 which were resulted from the
reaction of 3-phenyl-5-sulfonamidoindole-2-carboxylic acid hydrazide with different
aldehydes and their anti-depressant activity was studied. All the prepared compounds showed
anti-depressant activity at 100 mg kg-1 (Ergenç and Günay, 1998). Among this group, the
most active compound was 3,4-methylenedioxy – substituted derivative.
N
C
O
NH N CH
H2NO2S
H
C6H5
R
41
R= H, 4-Cl, 4-NO2, 2-OCH3, 4-N(CH3), 4-CH3, 2-OH, 4-F, 3,4-OCH2O
1.4.2.3 Anti-inflammatory activity
The biological activity of 2-(2-formylfuryl)pyridylhydrazone was studied and the
results reported that this compound is useful as anti-inflammatory agent and gives a 79%
inhibition of pleurisy at a dose of 80.1 µmol kg-1. The optimum concentration for
complexation of compound 42 with Ca2+ in vitro experiments was found to be 100 µM
concentration (Todeschinia et al., 1998).
N NH
NO
42
Lima and his co-workers, (2000) synthesized a new series of N-acylarylhydrazone
anti-nociceptive compounds from natural safrole. 43 was more active than dipyrone and
indomethacine, and are tested for their anti-inflammatory and anti-nociceptive activities.
19
O
O
N
N
O
H H
N
CH3
CH3
43
Salgin-Göşen et al. (2007) synthesized a series of 5-methyl-2-benzoxazolinone
hydrazones. The analgesic-anti-inflammatory studies of 2-[2-(5-methyl-2-benzoxazoline- 3-
yl)acetyl]-4-chloro-4-methyl benzylidene hydrazine 44a and 44b were conducted and the
results showed that these compounds are highly active than those of morphine and aspirin.
Moreover, 2-[2-(5-methyl-2-benzoxazoline-3-yl) acetyl]-4-methoxybenzylidene hydrazine
exhibited the most anti-inflammatory activity at 200 mg kg-1 dose.
Duarte et al. (2007) described results on the testing of N\-(3,5-di-tert-butyl-4-
hydroxybenzylidene)-6-nitro-1,3-benzodioxole-5-carbohydrazine 45 as a novel anti-
O
N
O
CH2 C
O
NH N CH Cl
44a
O
N
O
CH2 C
O
NH N CH CH3
44b
20
inflammatory compound. It was found that the activity of this compound depends on the 3,5-
di-tert-butyl-4-hydroxyphenyl group attached to the imine function. This compound was
tested by using 300 µmol kg-1 without any side effect of gastric irritation.
1.4.2.4 Anti-malarial activity
Due to the urgent need for more and highly effective anti-malarial drugs, the
aroyhydrazone chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone 46 have been
studied and the results showed the high activity of this compound against malaria than
desferrioxamine and chloroquine (Walcourt et al., 2004).
HO
N
NH
O
N 46
Bernardino et al. (2006) designed and synthesized a series of 1-substituted phenyl-
N`- [(substituted phenyl) methylene]-1H-pyrazole-4-carbohydrazides 47 and examined their
leishmanicidal and cytotoxic effects. In comparison with the ketoconazole, benznidazole,
allopurinol and pentamidine drugs in vitro, the 1H-pyrazole-4-carbohydrazide derivatives
with X = Br, Y = NO2 and X = NO2, Y = Cl showed the highest activity and more active
against L. amazonensis than on L. chagasi and L. braziliensis species.
O
ONO2
C
O
NH N CH
OH45