Huma Final Thesis File 20-1-2023 6666

Synthesis, Charaterization and In-silico Study of
Substituted Benzhydrazide and its Schiff Base

Analogues
By
Huma Sabir
CIIT/SP21-RCM-009/ATD
MS Thesis
In
Chemistry
COMSATS University Islamabad

Abbottabad Campus - Pakistan
Fall, 2022
i
COMSATS University Islamabad,
Abbottabad Campus

Analogues
A Thesis Presented to
COMSATS University Islamabad, Abbottabad Campus
In partial fulfillment
of the requirement for the degree of
MS (Chemistry)
By
Huma Sabir
Fall, 2022
ii
Synthesis, Charaterization, and In-silico Study of
Analogues
A Post Graduate Thesis submitted to the Department of Chemistry as partial

fulfillment of the requirements for the award of Degree of MS in Chemistry.
Name Registration Number
Huma Sabir CIIT/SP21-RCM-009/ATD
Supervisor
Dr. Syed Majid Bukhari

Associate Professor
Department of Chemistry
COMSATS University Islamabad
Abbottabad Campus
iii
Final Approval
This thesis titled

Analogues
By
Huma Sabir
Has been approved
For the COMSATS University Islamabad, Abbottabad Campus
External Examiner:____________________________________________________
Dr. Abdul Latif, Assistant Professor
Department of Chemistry, University of Malakand
Supervisor:___________________________________________________________
Dr. Syed Majid Bukhari, Associate Professor
Department of Chemistry, CUI, Abbottabad Campus
Co-supervisor:_________________________________________________________
Dr. Asma Zaidi, Assistant Professor
HoD:________________________________________________________________
Prof. Dr. Umar Farooq
iv
Declaration
I, Huma Sabir CIIT/SP21-RCM-009/ATD hereby declare that I have produced the

work presented in this thesis, during the scheduled period of study. I also declare that
I have not taken any material from any source except referred to wherever due that
amount of plagiarism is within acceptable range. If a violation of HEC rules on
research has occurred in this thesis, I shall be liable to punishable action under the
plagiarism rules of the HEC.
Date: _________________
________________________
Huma Sabir
v
Certificate
It is certified that Huma Sabir, CIIT/SP21-RCM-009/ATD has carried out all the
work related to this thesis under my supervision at the Department of Chemistry,
COMSATS University Islamabad, Abbottabad Campus and the work fulfills the
requirements for award of MS degree.
Date:___________________
Supervisor:
_________________________________
Dr. Syed Majid Bukhari
Associate Professor
Abbottabad Campus
Head of Department:
______________________________
Prof. Dr. Umar Farooq
Professor
Abbottabad Campus
vi
DEDICATION
I dedicate this thesis to my loving father Mr. Sabir
Sultan, whose utmost efforts and prayers are always with
me
vii
ACKNOWLEDGMENTS
All praises for Almighty Allah the most merciful, beneficent and the most gracious,
who is the entire and every source of knowledge and wisdom endowed to mankind
and who guides me in the darkness, help me in difficulties and blessed me with the
ability to do this work and his Prophet Hazrat Muhammad (Salallaho-Allaehe
Wasallim).
I would like to acknowledge all the wonderful people in my life, whose scientific,
financial, and most of all, moral support made this work possible specifically, I would
like to take this opportunity to express my sincere thanks to my worthy respectful and
dedicated gratitude to my supervisor Dr. Syed Majid Bukhari. Without their
constant help, deep interest and guidance, the completion of this thesis was not
possible. I wish to acknowledge the sincere gratitude to Prof. Dr. Umar Farooq,
Head of Chemistry Department COMSATS University Islamabad, Abbottabad
Campus.
I would like to place on record sincere and special thanks to Komal Abbasi, Faiza
Manzoor, Maryum Qureshi, Attia Irum, Urooj Shah, Kepdieu Tchebou Robert
Viani and rest of my fellows for their assistance, good company and marvelous
behaviour. At last, I really acknowledge and offer my heartiest gratitude to all
members of my family especially parents, sisters and brothers (Saima Sabir, Yasmin
Sabir, and Abid Sultan).
I am giving profound appreciation from the deepest core of my heart and highly best
regards to my beloved Father Sabir Sultan for his moral as well as financial support.
Huma Sabir
CUI/SP21-RCM-009/ATD
viii
ABSTRACT
Synthesis, Characterization and In-silico Study of Substituted

Benzhydrazide and its Schiff Base Analogues
Hydrazone Schiff base compounds were synthesized by subjecting 4-

hydroxybenzohydrazide to acid catalysed condensation reaction with corresponding
ortho and para substituted benzaldehyde. Hydrazone Schiff bases become the topic of
interest in the field of biological science and organic chemistry and also in the field of
pharmaceutical chemistry due to their anti-pathogenic, anti-inflamatory, anti-TB, and
anti-cancer activities and their use in medicine. Hydrazone Schiff bases are active
structural motifs with a wide range of pharmacological and anti-pathogenic
properties. Study reveals that hydrazones have shown α-glucosidase inhibition.
Different substituted derivatives of 4-hydroxybenzohydrazide were synthesized and
characterized by spectroscopic techniques. In-silico study was done for all the
synthesized compounds and all the compounds showed excellent inhibitory activities
against selected enzyme α-glucosidase and it was revealed that all the compounds
have high ability to interact with protein molecule and have high potential to bind
with the active site of enzyme. Benzohydrazones with electron donating groups like
(OH) at ortho and para show good interaction with enzyme than those with electron
withdrawing substituents attached to benzene ring.
ix
TABLE OF CONTENTS
1 Introduction................................................................................................. 2
1.1 Neurodegenerative diseases ...................................................................2
1.2 Hydrazides .............................................................................................2
1.3 Substituted hydrazides ...........................................................................3

1.3.1 Isonicotinic acid hydrazide ................................................................. 3
1.3.2 Phenylsulfonylhydrazide .................................................................... 4
1.3.3 Benzohydrazides ................................................................................. 4
1.3.4 Acid hydrazides .................................................................................. 5
1.3.5 Arylhydrazides ................................................................................... 5
1.3.6 Benzoic acid hydrazides ..................................................................... 6
1.4 Schiffʼs bases (hydrazones) ...................................................................6
1.4.1 Thiazole derivatives ............................................................................ 8
1.4.2 1, 3, 4-Oxadiazoles ............................................................................. 8
1.4.3 Metal complexes of hydrazones ......................................................... 8
1.4.4 Hydrazide hydrazones ........................................................................ 9
1.4.5 Acylhydrazones .................................................................................. 9
1.4.6 2, 2' bipyridyl-2- quinolyl hydrazone ............................................... 10
1.4.7 Aromatic hydrazones ........................................................................ 10
1.4.8 Coupling products ............................................................................ 10
1.4.9 Coordinattion complexes of hydrazones .......................................... 10
1.4.10 Biscyclohexanone oxalyl dihydrazone.......................................... 11
1.5 Phenolic compounds ............................................................................12
1.5.1 Phenylhydrazones ............................................................................. 12
1.6 Salicylate Hydrazones ..........................................................................12
1.7 Sulfonyl Hydrazones ............................................................................12
1.8 Applications of hydrazones .................................................................13

1.8.1 Anti-tubercular activities .................................................................. 13
1.8.2 Antibacterial characteristics of Schiff bases .................................... 13
1.8.3 Antifungal properties of Schiff bases ............................................... 14
1.8.4 Anti-inflammatory activity ............................................................... 14
1.8.5 Analgesic activity ............................................................................. 15
1.8.6 Antiviral activity ............................................................................... 15
1.8.7 Cardio-protective activities .............................................................. 16
x
1.9 Pharmacological activities of 1, 3, 4-oxadiazole-2- thioacetohydrazide-
hydrazones......................................................................................................16
1.9.1 Anti-TB ............................................................................................. 16
1.9.2 Anti-viral agents ............................................................................... 17
1.10 Applications in modern technologies ..................................................18
1.11 In Analytical Chemistry .......................................................................18

1.11.1 For the detection of metal ions ...................................................... 18
1.12 Biogenic amines ...................................................................................19
2 Material and methods............................................................................... 21

2.1 Chemicals used ....................................................................................21
2.2 Characterization ...................................................................................21

2.2.1 Chromatographic Technique (Thin layer chromatography) ............. 21
2.2.2 UV-Vis Spectroscopy ....................................................................... 21
2.2.3 Fourier Transform Infrared Spectroscopy (FT-IR) .......................... 22
2.2.4 Nuclear magnetic resonance (NMR) ................................................ 22
2.3 Method for the synthesis of benzohydrazide based Schiff base
analogues ........................................................................................................22
2.3.1 Schematic method for the synthesis of (E)-4-hydroxy-N-(4-
nitrobenzylidene) benzohydrazide .............................................................. 23
2.3.2 Synthesis of (E)-N-(3-ethoxybenzylidene)-4-hydroxybenzohydrazide
.......................................................................................................... 23
2.3.3 Synthesis of (E)-4-hydroxy-N'-(4-metoxybenzylidene)
benzohydrazide ........................................................................................... 24
2.3.4 Synthesis of (E)-N'-(3-ethoxy-2-hydroxybenzylidene)-3-
hydroxybenzohydrazide .............................................................................. 24
3 Results ........................................................................................................ 27
3.1 Characterization of hydrazones ...........................................................27
3.2 UV-Vis Spectroscopic Analysis ..........................................................27
3.3 FT-IR results ........................................................................................28

3.3.1 FT-IR analysis of compound 01 ....................................................... 28
xi
1
3.4 H NMR spectroscopic analysis ..........................................................30
1
3.4.1 H NMR Spectrum of hydrazine (Compound-01) ........................... 31
1
3.4.2 H NMR of compound 02................................................................. 31
1
3.4.3 H NMR of compound 03................................................................. 32
1
3.4.4 H NMR of compound 04................................................................. 33
3.5 Docking Studies ...................................................................................33
3.5.1 Compound 01 ................................................................................... 34
3.5.2 Compound 02 ................................................................................... 34
3.5.3 Compound 03 ................................................................................... 35
3.5.4 Compound 04 ................................................................................... 36
4 Discussion .................................................................................................. 38
4.1 Hydrazone Synthesis ............................................................................38
4.1.1 Mechanism of Hydrazones ............................................................... 38
4.2 UV-Vis results .....................................................................................38
4.3 FT-IR Analysis.....................................................................................38

4.3.1 Compound 01 ................................................................................... 39
4.3.2 Compound 02 ................................................................................... 39
4.3.3 Compound 03 ................................................................................... 39
4.3.4 Compound 04 ................................................................................... 40
4.4 NMR Spectroscopic analysis ............................................................... 40
1
4.4.1 H NMR characterization of hydrazone compound 01 .................... 40
1
1
1
4.5 Docking studies ....................................................................................41
4.6 Conclusion ...........................................................................................42
5 References .................................................................................................. 44
xii
LIST OF FIGURES
Figure 1.1 Structure of isonicotinic acid hydrazide ...................................................... 3
Figure 1.2 Hydrazine Schiff base .................................................................................. 6
Figure 1.3 N-(salicylidine)-2-hydroxyaniline ............................................................... 7
Figure 1.4 Oxadiazole derivative of hydrazone ............................................................ 8
Figure 1.5 Benzaldehyde 2-thiazolyl hydrazone......................................................... 13
Figure 1.6 N-(Salicylidine)-2-hydroxyaniline ............................................................ 14
Figure 1.7 Ciprofloxacin analogue.............................................................................. 14
Figure 1.8 Thiazine carbohydrazide............................................................................ 15
Figure 1.9 N-thiopheneacetyl hydrazine of isatin ....................................................... 15
Figure 1.10 Antiviral compound of schiff base .......................................................... 16
Figure 1.11 Analogue of safrole ................................................................................. 16
Figure 1.12 Structural analogue of thioacetohydrazide .............................................. 17
Figure 1.13 Substituted acetohydrazide ...................................................................... 17
Figure 1.14 2-(D-galactopyranosylidene) hydrazide .................................................. 17
Figure 1.15 Amino-3- hydroxyguinidine tosylate....................................................... 18
Figure 2.1 Scheme for the synthesis of benzohydrazone ............................................ 22
Figure 2.2 Synthesis of (E)-4-hydroxy-N-(4-nitrobenzylidene) benzohydrazide ....... 23
Figure 2.3 Synthesis of (E)-N'-(3-ethoxybenzylidene)-4-hydroxybenzohydrazide .... 24
Figure 2.4 Synthetic scheme of (E)-4-hydroxy-N-(4-metoxybenzylidene)
benzohydrazide ............................................................................................................ 24
Figure 2.5 Synthetic scheme of (E)-N'-(3-ethoxy-2-hydroxybenzylidene)-3-
hydroxybenzohydrazide ............................................................................................... 25
xiii
LIST OF SCHEMES
Scheme 4.1 Mechanism for the synthesis of Hydrazones ........................................... 38
xiv
LIST OF TABLES
Table 3.1 Physical data of synthesized compounds .................................................... 27

Table 3.2 Results of UV-Vis spectra ........................................................................... 27
Table 3.3 FT-IR table of compound 01 ....................................................................... 28
Table 3.7 1H NMR of compound 01 ........................................................................... 31
Table 3.10 1H NMR of compound 04 ......................................................................... 33
Table 4.1 Binding energies of docked compounds against α-glucosidase .................. 42
xv
LIST OF ABBREVIATIONS
NDDs Neurodegenerative disorders

AD Alzheimer’s disease
INH Isonicotinicacid hydrazide
PAPH Pyridyl hydrazone
Rf Retention factor
1
H NMR Proton nuclear magnetic resonance
FT-IR Fourier transform infrared
AChE Acetyl cholinesterase enzyme
BuChE Butyryl cholinesterase enzyme
HIV Human immune virus
EtOAc Ethyl acetate
MeOH Methanol
DMSO Dimethyl sulfoxide
TLC Thin layer chromatography
xvi
Chapter 1
Introduction
xvii
1 Introduction
1.1 Neurodegenerative diseases
Neurodegenerative diseases which are caused by changes in structure and function of
protein and DNA are becoming hazardous with the passage of time. Medicinal
chemists and researchers all over the world are doing constant efforts to combat and
control the spread of these diseases. New drugs are being designed with the passage
of time by the combined efforts of medicinal chemists and research pharmacist to
control the spread of these diseases with better potential and efficacy [1]. Different
hydrazine based aromatic and heterocyclic compound are used for development of
new drugs due to their greater use in biological field because of their potent ability to
fight against diseases. Naturally occurring compound in plants which are the source of
allopathic medicines and are also effective against diseases contain in their structure
heterocyclic rings with other hydrazine based moieties [2].
Alzheimer’s disease (AD) which is mainly caused by degeneration of nervous system

and causes slow and progression loss of memory, learning abilities, etc. is due to the
deposition of beta-amyloid in brain. Which effects hippocampus and leads to brain
cell death with symptoms of memory loss, mood imbalancement, etc. [3]. The
decrease in level of acetylcholine (a hydrazine based neurotransmitter) which help in
conduction of impulses in the nervous system causes brain cell damage and eventually
leads to death of patient suffering from Alzheimer’s [4].
1.2 Hydrazides
Hydrazine in organic chemistry are a class of organic compounds having general
formula RNHNH2.The pharmaceutical importance of these compounds is due to the
availability of proton. Because they are acylated derivatives of hydrazine they have
immense application in pharmaceutical chemistry and have vast biological activities
[5]. Hydrazides are used as surfactants as antifungal and antibacterial agents.
Hydrazides and their derivatives are used for biological assessment of fatty acids [6].
Derivatives of fatty acid hydrazides are used to obtain new antifungal and
antibacterial agents. Hydrazides and their derivatives are used for the direct synthesis
of extensive variety of heterocyclic rings [7]. Vast variety of carbohydrazides
including aliphatic, alicyclic, or aromatic possess wide range of biological activities.
Hydrazides are basic compounds possessing reducing properties [8]. Hydrazides are
2
used as intermediate for the production of different types of drugs which are used for
treatment of different illnesses [9]. During the past few years, hydrazides got the
attention of research chemists because of inhabiting antibacterial activities and also
act as antitumor agent, anticancer agent and anti-tuberculostatic agent [10].
Hydrazides are used for synthesis of hydrazones because of high reactivity of
azomethine group. Due to their high reactivity, hydrazides are considered as
important starting materials and intermediates in the synthesis of hydrazones and
heterocyclic compounds [8]. Hydrazides are produced by the hydrazinolysis of esters.
The condensation products of a large number of hydrazides with various aldehydes
and ketones were being reported [11]. The pharmacological activities of hydrazides
are related to azomethine functionality. Due to the presence of carbon-Nitrogen
double bond linkage in hydrazides they have got wide area of success in coordination
field [12]. Importance of hydrazides in organic chemistry is due to their vast
biological activities as antibacterial, anti-tubercular [13].
1.3 Substituted hydrazides
1.3.1 Isonicotinic acid hydrazide

Isonicotinic acid hydrazide shows selective anti-retroviruses activity against different
strains of viruses. Pyrazinamide drug derived from isonicotinic acid hydrazides are
used as first treatment of tuberculosis. Complexes of Schiff bases are used for
treatment of iron overload diseases [14].
Figure 1.1 Structure of isonicotinic acid hydrazide
Isoniazid an antituberculosis drug and hydralsane (an antihypertensive and vasodilator

drug) contain hydrazide intermediate in their synthesis. Hydralsane hydrochloride is
an active hypotensive drug used for reduction of blood pressure in hypertensive crisis
[15]. Derivatives of hydrazine are extensively used for treatment of mental disorder
3
and for treatment of tuberculosis. Substituted derivatives of hydrazide hydrazones are
used as antimicrobial, antihypertensive, antimalarial and antitumoral agents [16].
1.3.2 Phenylsulfonylhydrazide
New phenylsulfonyl hydrazide derivatives have been synthesized for structure–
activity relationships (SAR) study. Two regioisomers of phenoxycarboxyl group have
been determined [17]. Their biological evaluation showed that one regioisomer was
systematically more potent than the other [18]. Sulfonylchlorides are used for
preparing sulfohydrazides. Both acid hydrazides and sulfohydrazides reacts with
different substituted salicylaldehydes to give the corresponding hydrazones [19].
1.3.3 Benzohydrazides
A chalcone-like benzohydrazide derivative has been synthesized from natural
resources vanillin and Wintergreen oil. This compound is a source of natural
antibacterial agent. Vilsmier-Hack reaction is used for formylation of benzaldehyde
substituted phenyl carbonyl hydrazones [20]. Formyl derivatives of hydrazones were
screened for Antibacterial activity. In organic synthesis, Schiff bases are used as
corrosion inhibitors and polymer stabilizers [21]. Schiff bases containing azomethine
functional group, are used as pharmacophores and are used for development of
chemotherapeutic drugs [22]. Metal complexes of Schiff bases have immense
applications in the field of pharmacology as antibacterial, antifungal, anti-tubercular,
anti-malarial, anticancer, anti-cancer, anti-inflammatory, and anti-pyretic activities
[23]. Some of the benzohydrazide compounds are synthesized from benzohydrazine
derivatives and benzaldehyde chalcone [24]. In-silico studies shows that flavone
hydrazide Schiff base derivatives have excellent potential against diabetes mellitus
[25]. And they are α-glucosidase inhibitor. Schiff bases are commonly used in
medicine as antibacterial agents. Benzohydrazide possesses anti-leishmanial, anti-
anticancer, anti-mycobacterial activities etc. [26]. Benzohydrazides are easily
converted into hydrazones by treating with Aldehydes or ketone. Schiff base
synthesized compounds evaluated for molecular docking study, and antibacterial
activity [27]. Class of organic compounds containing Schiff bases have vast biological
activities such as antiglycation, urease, and alpha-glucosides [28]. Hydrazones are
involved in different enzymatic activities. 4-Hydroxybenzhyhydrazide is being used
as tyrosinase inhibitors. Benzohydrazide act as inhibitor of
acetylbutyrylcholinesterase [29]. Hydrazone bearing aryl functionalities are used as
4
inhibitor of laccase enzyme (a copper containing enzyme) [30]. 4-
Hydroxybenzohydrazide was used as modulating agent in solvothermal process to
enhance the crystalinity of covalent organic frameworks (COFs) [31].
1.3.4 Acid hydrazides

Acid hydrazides are produced by converting carboxylic acids to methyl esters. Which
then reacts with hydrazine to yield the corresponding hydrazide [32]. The reaction is
basically a condensation reaction. These hydrazones are potent antifungal inhibitors
and inhibit the growth of different types of fungi [33]. Hydrazide based drugs are way
to development of new therapeutically active drugs with potential antifungal activities
against different species of Candida [33]. Hydrazides of Isonicotinic acid are used as
chemotherapeutic agent for the treatment of tuberculosis [34]. Hydrazones of acid
hydrazides and aromatic aldehydes act as mono, bi, or tridentate anionic C, N and O
donor ligands to provide cyclometallated complexes [35].
1.3.5 Arylhydrazides
Molecules in the the aldehydes and ketones are replaced by NH – NR2 functional
group. Practically N-Aryl hydrazones are extensively synthesized in industry [36].
Arylhydrazides such as carbazoles, pyrazoles, indoles, indazoles and triazines are
used for the synthesis of biologically active heterocyclic ring compounds [37].
Reduction of N-nitrosoarylamines produces N-Arylhydrazides. N-nitrosoarylamines
which are formed by nitrosation of anilines, or by aminations of aryl Grignard
reagents, aryllithium reagents, and aryl zinc halide [38]. Hydrazones are substituted
organic compounds possessing aliphatic and aromatic groups and are formed by the
condensation of hydrazine with carbonyl compounds ketones or aldehydes [39].
Oxygen is insoluble in water but soluble in most of organic solvents like methanol,
ethanol, DMSO, and are partially soluble in cold ethyl alcohol and ether. Because of
the presence of free amino group in hydrazine they condense with aldehydes and
ketones to give hydrazones [40]. The hydrazone functionality possess various
attractive features like degree of rigidity, a conjugate π-system and a deprotonation
[41]. Transition metal complexes of hydrazones are investigated with variety of
biological activities [41]. The arylhydrazones containing (–CO–NH–N=C< group) act
as chelating agents and are known to possess fungicidal effects [42].
5
Hydrazides react with different nucleophilic and electrophilic reagents. These
hydrazides are used for production and synthesis of indoles, 4-thiazolidine-4-one,
azetidines by cycloaddition and in the synthetic mechanism of various membered
heterocyclic compounds. Different substituted triazoles and oxadiazoles are
synthesized by hydrazone intermediates [36].
1.3.6 Benzoic acid hydrazides

Hydrazides of benzoic acid found to inhibit the peroxidation activity of
myeloperoxidase [43]. A neutrophil protein which convert hydrogenperoxide and
chloride to HOCl. Myeloperoxidase is an enzyme help in killing bacteria and cause
inflammation. ABAH is a potent inhibitor and cause peroxidation [43].
1.4 Schiffʼs bases (hydrazones)

Schiff bases were first reported by Hugo Schiff at (1864) Reaction of primary amine
(in case of hydrazone the amine part will be of an appropriate hydrazide) with an
aldehyde or a ketone, produces Schiff bases and hydrazones. They are nitrogen
analogues of an aldehyde or ketone in which an imines group (C=N) has replaced the
carbonyl group (C=O) [44]. Benzohydrazide based Schiff’s base derivatives
(hydrazones) are excellent products for various pharmaceutical applications.Schiff
bases or imines are distinguished from oximes due to the presence of the two
interlinked (-N=N-) nitrogen atoms [45].
Figure 1.2 Hydrazine Schiff base
Due to the presence of reactive azomethine group hydrazones are key way to
Development of new drugs. Hydrazones are formed by reaction of aldehydes and
ketones with different hydrazide derivatives [46]. In organic synthesis hydrazines
constitutes an important class of organic compounds. They have been employed as
reagent for characterization and derivatization of carbonyl compounds [47]. Due to
their vast biological properties hydrazides are used as anti-bacterial, anti-fungal, anti-
6
inflammatory, anti-malarial and anti-tubercular activities. Hydrazide hydrazone
complexes act as inhibitor of α-glucosidase. Hydrazones have vast role in developing
new drugs for leishmaniasis and malaria at pharmaceutical level [48].
Figure 1.3 N-(salicylidine)-2-hydroxyaniline
Schiff bases are considered to be a significant intermediate in many of the enzymatic

reactions. Biochemical process, involving the primary amine condensation in enzyme
normally including lysine [49]. Schiff bases of the benzohydrazones are prepared in
labs by heating the aromatic hydrazides with substituted benzaldehydes, in organic
solvents like ethanol, methanol, tetrahydrofuran, butanol and sometimes in the
presence of glacial acetic acid which act as catalyst [50]. Benzohydrazones can also
be synthesized by the coupling reaction between aryldiazoniurn salts containing active
hydrogen. A number of new hydrazones like benzylidine benzohydrazone have been
synthesized because of their straightforward way of synthesis. Hydrazones gained by
condensation of substituted acid hydrazide with aromatic 2-hydroxyaldehyde,
effective complex forming agents [51]. Diverse coupling products can be
manufactured by using active hydrogen of azomethine group. Vast variety of
biologically active compounds has been manufactured by the reduction of
benzohydrazones by researchers, for example: iproniazide and isocarboxazide.
Iproniazide have structure similar to isoniazid and both are used in the treatment of
tuberculosis along with antidepressant and mood changing effect [52]. Due to great
ability of d-block metal ions to form complexes schiff bases of aromatic hydrazones
have been quite extensively investigated. Benzohydrazone complexes involving 0, N,
S donor ligands have excellent coordination capability, with vast pharmacological
activity and their uses in analytical chemistry as metal extracting agents.
Benzohydrazone metal complexes are more potent and less toxic in many cases as
compared to the parent compound [53].
7
1.4.1 Thiazole derivatives
Thiazole derivatives of hydrazides and hydrazones act as potential tyrosine kinase
inhibitor. Which show potential activity as antitumor activities. Hydranones bearing
amino hydrazides shows anticancer activities. Benzohydrazides showing anti-
glycation activities possess inhibitory potential against urease enzyme [54].
Hydrazine based Schiff bases are excellent alpha-glucosidase, xanthine oxidase

inhibitors, cyclooxygenase, inhibitors. Certain pain reducing drugs called NSAIDS
are designed by the creation of new derivatives of Schiff bases [55].
Substituted derivative of hydrazones are known to possess high antiviral activities

against African green monkey, herpes simplex virus 1 and hepatitis A virus. Some
ortho and para substituted hydrazone compounds possess high prophylactic activity
against vesicular stomatitis virus(VSV) [41].
1.4.2 1, 3, 4-Oxadiazoles
1, 3, 4-oxadiazoles are generally laboring as bioisosteric replacements of the amide
bond. Due to toxophoric imine group 1, 3, 4-oxadiazoles possess efficient
pharmacological activities [56].
Figure 1.4 Oxadiazole derivative of hydrazone
Molybdenum involve in the epoxidation of olefins, and isomerization of allylic

alcohols form important complexes on reaction with different derivatives of Schiff
bases [57].
1.4.3 Metal complexes of hydrazones

Complexes of isatinic quinolyl hydrazones are used for incorporation of quinolone
ring with indole ring and show broad variety of biological activities. Schiff base
8
ligands are simply synthesized by the process of condensation of carbonyl compounds
and primary amines [58]. Because of their excellent biological and physiochemical
properties Metal complexes of O-, S-, and N-chelating ligands have gained attention
of synthetic chemists in field of organic synthesis. Due to their metalloenzyme
activity metal complexes of Schiff bases are used as anticancer agents [59].
Complexes of Zn (II), Cu(II), and Cd(II) containing isonicotinic acid hydrazide and
paracetamol ligands have been investigated beside their physical and chemical
properties [60]. Schiff bases have important application in polymer chemistry.
Derivatives of the Oxazepine shows various biological activities such as antibacterial
and inhibitors for some enzymes action [57].
1.4.4 Hydrazide hydrazones

Hydrazide hydrazones consist of substituted acetic acids present high bactericidal
activity against Gram-positive bacterial strains. Hydrazides/hydrazones act as anti-
platelet agent. Furthermore, these hydrazones containing benzohydrazide based Schiff
base analogs show mind blowing anti-arthritis and acetylcholinesterase and
butyrylcholinesterase inhibitory activities. The designing of Flourine containing
derivatives of hydrazones help to act as antioxidant and anticholinesterase agents [61].
1.4.5 Acylhydrazones
Acylhydrazones because of their versatile biological activities and role in
coordination chemistry these compounds hare used by synthestic chemists in field of
medicine.Due to this versatility the acylhydrazones are good polydentate- chelating
agents,and have ability form a vast variety of complexes with different outer
transition and inner transition metals [62]. Acylhydrazones are the important class of
compounds used as starting material for development of new anti-fungal and
antibacterial agents. Oxadiazole derivatives of hydrazides have been known to
possess broad spectrum of biological activity, including: antibacterial, antifungal,
anti-tubercular and anticancer properties [63]. The acylhydrazone group dissociate at
faster rate in acidic conditions than in neutral or basic condition.Thus, PH is altered to
modulate the rate of exchange bonds of acylhydrazones, which is important to control
all the features of polymer networks including self-healing properties,and gel
formation time [64].
9
1.4.6 2, 2' bipyridyl-2- quinolyl hydrazone
Benzohydrazides containing 2, 2' bipyridyl-2- quinolyl hydrazone are used as spot test
reagent for the detection of cobalt, iron, and copper. 47Silver and mercury are being
detected by the complexes of Cobalt (III) with 2-pyridyl hydrazone (PAPH) [65]. Gas
phase concentration of low molecular weight aldehydes and ketones is carried out by
aromatic hydrazones.2-4, dinitrophenyihydrazine is used for determination of
aldehydes and ketones. Hydrazone functionality plays an important role in medicinal
chemistry for production of new drugs for treatment of different disease [66].
1.4.7 Aromatic hydrazones

Aromatic hydrazones are compounds formed by the reaction of phenolic acid
hydrazides and aromatic carbonyl compounds (aldehydes and ketones).
Stoichiometric amounts of the aromatic hydrazide and carbonyl compounds are
dissolved in a suitable solvent and refluxed for the preparation of benzohyrazide
based Schiff base analogs (hydrazones). The increasing work in the chemistry of
hydrazones is due to their broad spectrum of pharmacological and bioactivities [67].
1.4.8 Coupling products

Hydrazones intermediates form various coupling products and numerous biologically
active compounds like iproniazide and isocarboxazide which are formed by reduction
of hydrazones. Both iproniazid and isoniazid have similar structure and they are used
for the treatment of tuberculosis and also act as antidepressant [68].
Hydrazones form good chelating agents that can form different types of complexes
with various inner transition metals. Metal complexes of the hydrazones depicts
chemotherapeutic use in anticancer activities. Heterocyclic ligand of hydrazones react
with metal chlorides to yield complexes with anticancer effects [69].
1.4.9 Coordinattion complexes of hydrazones

In coordination chemistry vanadium complexes of hydrazones are of greatest interest
with great number of applications. Vanadium complexes of hydrazones are important
in catalytic oxidation- reduction reactions. Studies shows that they are effective in
nitrogen fixation, and in various biochemical reactions [70]. Hydrazone complexes
are effective against detoxification of vanadium Compounds. In pharmaceutical
chemistry they act as potential enzyme inhibitors. Hydrazones are class of organic
compounds prepared by reaction of aldehydes and ketones with hydrazide by
10
replacing oxygen with NNH2 functional group [71]. These hydrazones reacts with
various metals to form various kinds of ligands depending on the substituents attached
to the aromatic ring in hydrazone. keto-enol tautomerization can be observed in
hydrazido-hydrazones, R1R2C = NNH2CO-R3. Due to excellent coordination
chemistry Schiff bases are pigments in dyes industry and also act as catalysts in
various chemical reactions. Schiff test is used for detection of aldehydes and ketones.
Geometries of different coordination complexes are due to stability and structural
variability of Schiff bases [72]. Carbohydrazide are used as colour stabilizer in dyes.
They are also used as ammunition propellants, soap stabilizer and as reagent in
synthetic labs. Oranometallic componds are derivatized to potential enzyme inhibitor
by the involvement of Schiff bases [73].Because of dominant application of
macrocyclic ligands in applied sciences Schiff bases are used on advance levels due to
their vast application in industrial field. Schiff bases are important in organic
chemistry due to their involvement in different kinds of reaction like carbonization,
reduction and oxidation [74]. During recent years Schiff bases got attention of
synthetic labs due to their contribution in the field of material sciences, analytical and
organic chemistry and molecule-based magnetism [75]. Coordination metal
complexes consist of a central metal ion coordinates with ligands or complexing
agents to provide active sites in DNA cleavage system, and act as anticancer agents
[76].
1.4.10 Biscyclohexanone oxalyl dihydrazone

Biscyclohexanone oxalyl dihydrazone is used to evaluate copper in paper pulp
products. This molecule is also used to assess copper in human serum and is also used
to detect steel, plants, non-ferrous metals, and alloys. Amount in food has been
measured employing 2-pyridinyl hydrazone (PAPH). Since they can easily undergo
several ring closure processes, hydrazones are intensively researched as reactants or
reaction intermediates [77]. Direct action of hydrazide derivatives on brain enzymes
may lead to change in their anti-convulsant properties without interfering with other
cellular responses associated with potential toxic effects. Potential antiepileptic agents
are developed by hydrazide/hydrazone scaffold (a complex structural linker).With
development of new drugs various types of hydrazide/hydrazones and pyrazoles have
been developed with potential anticonvulsant activities. In acidic medium Schiff bases
are very effective against corrosion of zinc, aluminium, copper and mild steel [78].
11
1.5 Phenolic compounds
Phenolic compounds are proved to possess anti-oxidant and antibacterial activity
which might be due to the scavenging environment of antioxidant to inhibit bacterial
growth. Phenolic derivative of p-hydroxy benzoic acid, known to possess different
wide range of pharmacological activities, including antimicrobial, anti-sickling, anti-
mutagenic, anti-algal, anti-inflammatory, anti-oxidant, etc.Nʹ-(4-
methoxybenzylidene)-4-hydroxybenzohydrazide,Nʹ-benzylidene-4-hydroxy-
benzohydrazide have excellent antimicrobial activities [79].
1.5.1 Phenylhydrazones
Phenylhydrazones are used as alternative for exploration and development of new
drugs used for treatment of fungal infections. Benzohydrazides containing
salicylaldehyde hydrazones are known to possess antimicrobial activity [80].
Salicylaldehyde Schiff base analogs (hydrazones) act as a broad spectrum antifungal
agent. Antifungal activities shown by salicylaldehyde derivatives including
salicylaldehyde phenylhydrazones, benzohydrazides and sulfohydrazides for Candida
albicans and Candida glabrata. Salicylaldehyde conataining nitro group are potent
inhibitors of fungal growth with minimal cytotoxicity to liver or kidney cells
Hydrazones containing phenolic groups are used for free radical scavenging assay and
have remarkable anti-oxidants properties [81].
1.6 Salicylate Hydrazones

Salicylate Hydrazones are identified and characterize for its therapeutic use and for
cosmetic usage worldwide. Some substituted derivatives of benzohydrazide based
Schiff base analogs depict inhibitory activity of HIV-integrase. Some of the Schiff
bases were used as chelating agents, analytical reagents for transition metal analysis
and used as catalyst for epoxidation of olefins [82].
1.7 Sulfonyl Hydrazones

New Schiff bases of sulfonyl hydrazides have been synthesized containing two
moietoies (sulfonyl group and hydrazide group) which are chemically and
biologically important. Sulfonyl hydrazides present vast pharmacological activities
such anti-diabetic, anti-inflammatory, anticancer, analgesic and anti-tumour activities
[83]. Wide range of bacteriostatic activities has been shown by derivaties of Sulfonyl
hydrazide and sulfonyl semicarbazide. Phenelzine (2-phenylethyl) hydrazine) is a
12
powerful muscle relaxant. INH (isonicotinohydrazide) is anantituberculostatic drug,
whereas Iproniazid (N'-isopropylisonicotinohydrazide) a hydrazide based drug shows
antidepressant activity [84].
1.8 Applications of hydrazones
1.8.1 Anti-tubercular activities

The newly synthesized bithiazolyl hydrazones compounds have been known to
possess anti-tubercular activity. 4-(4-methoxyphenyl)-2-(2- (1-(4-methyl-2-phenyl-2,
5-dihydrothiazol-5-yl-ethylidene) hydrazineyl) thiazole has displayed potent
antitubercular activity but not effective if matched to standard drug rifampicin [85].
Figure 1.5 Benzaldehyde 2-thiazolyl hydrazone
Metal complexes of Schiff bases unveil immence pharmacological activities.4-N, N-

biscyano ethyl amino benzaldehyde show a high degree of anticancer activity.
Chlorine derivatives of benzohydrazide are known to possess high pesticidal
activities. Benzohydrazides containing heterocyclic Schiff bases possess bactericidal,
fungicidal, antipyretic, anti-tumour, and anticancer activities along with sterease
inhibitory activities [86].
1.8.2 Antibacterial characteristics of Schiff bases

Schiff bases have been found as suitable anti-bacterial agents. N-
salicylidinehydroxyaniline is an example of Schiff base known to possess anti-
bacterial activities against mycobacterium tuberculosis [87].
13
Figure 1.6 N-(Salicylidine)-2-hydroxyaniline
The method in which the hydrogen is generated through a chemical reaction is known
as chemical storage. Different materials which store hydrogen by this method are metal
hydrides, ammonia, formic acid, carbohydrates, synthetic hydrocarbons and the liquid
organic hydrogen carriers (LOHC) [88]. Compared to ciprofloxacin N'-(2-
hydroxybenzylidene)-2-(2-phenyl-1H-benzo[d]imidazol-1-yl) acetohydrazide possess
outstanding inhibitory properties of bacterial growth [89].
Figure 1.7 Ciprofloxacin analogue
1.8.3 Antifungal properties of Schiff bases

Rapid increase in the fungal infection have been observed worldwide in recent years
which is possibly life threatening. Therefore, research and synthesis of more potent
antifungal agents is the dire need of the time, and Schiff bases are determined to be
potential antifungal drugs [71]. Imine analogues of quinazolinones contain antifungal
characteristics against T. Mentagrophytes, Microsporum gypseum etc [90]. Schiff
bases synthesized from furan with various amine possess antifungal activities [91].
1.8.4 Anti-inflammatory activity

N'-benzylidene-4-hydroxy-2-methyl-2H-benzo[e][1,2]thiazine-3-carbohydrazide 1,1-
dioxide [92] isone of the benzothiazine N-acylhydrazone which were designed by the
structural change of piroxicam and is used for the treatment of anti-inflammatory
14
activities [93]. These series had shown improved activity in comparison to Piroxicam
[94].
Figure 1.8 Thiazine carbohydrazide
1.8.5 Analgesic activity

PHI [95] and THI [96] had been used effectively against analgesic effect in
inflammatory pain by suppressing pro-inflammatory cytokines and enhanced
antioxidant potential [97].
Figure 1.9 N-thiopheneacetyl hydrazine of isatin
1.8.6 Antiviral activity

N-(4-(2-(4-chlorobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)-3-(2-
chlorophenyl)-5-methylisoxa-zole-4-carboxamide [41] is pyrazole-hydrazone
derivative possessing an isoxazole group, which was examined against (TMV) and
had shown better antiviral action than Ningnanmycin [98].
15
Figure 1.10 Antiviral compound of Schiff base
1.8.7 Cardio-protective activities

N-acylhydrazone analogue of safrole, N'-(benzo[d] [1,3]dioxol-5-
ylmethylene)thiophene-2-carbohydrazide was determined to have high anti‐
hypertensive activity [99]
Figure 1.11 Analogue of safrole
1.9 Pharmacological activities of 1, 3, 4-oxadiazole-2-

thioacetohydrazide-hydrazones
1.9.1 Anti-TB
α-[5-(2-furyl)-1, 3, 4-oxadiazol-2- ylthio] acetohydrazide [100] has similar activity
like that of isoniazid. N'-benzylidene-2-((5-(furan-2-yl)-1, 3, 4-oxadiazol-2-yl) thio)
acetohydrazide was one among new derivatives of hydrazones [100].
16
Figure 1.12 Structural analogue of thioacetohydrazide
α-(5-(5-Nitro-2-furyl)-l, 3, 4-oxadiazol-2-ylthio] acetohydrazide [101] and N'-

benzylidene-2-((5-(5-nitrofuran-2-yl)-1,3,4-oxadiazol-2-yl)thio) acetohydrazide had
been tested against M. tuberculosis and show positive results [100].
Figure 1.13 Substituted acetohydrazide
1.9.2 Anti-viral agents

2-(D-galactopyranosylidene) hydrazide depicts anti-HCV activity. While 2-[5-
[(Naphthalen-1-yloxy) methyl]-1, 3, 4-oxadiazol-2- ylthio] acetic acid 2-(D-
xylopyranosylidene) hydrazide showed the highest activity as anti-HIV [102].
Figure 1.14 2-(D-galactopyranosylidene) hydrazide
R = D-galactopentitolyl, b; R = D-xylotetritolyl
The vaccines are used against pathogens called viruses, such as poliomyelitis,
smallpox etc [103]. A vast number of other medicinal way are used to do effort
against viral infections, current anti-viral agent do not have better efficacy, due to
which an increased rate of mutation of viruses can occur [102]. Schiff base of
17
salicylaldehyde synthesized from amino-3- hydroxyguinidine tosylate have been
reported to be good medium for the synthesis of novel antiviral agents [104].
Figure 1.15 Amino-3- hydroxyguinidine tosylate
1.10 Applications in modern technologies

Because of their photochemical properties schiff base compounds act as
photostablizers and solar filters. Some derivatives of Schiff base compounds are used
in recording technology of optical sound [105] In gas chromatography Schiff base
compounds are used as stationary phase due to their vast thermal stability. Due to the
non-linearity in the structure of some Schiff base compounds they can be used in
electronic components, photonic materials and optical switches [106].
1.11 In Analytical Chemistry

In analytical chemistry benzohydrazones acts as reagents for determination of Ni.
Hydrazone Schiff bases are used as metal extracting agents. Biscyclohexanone oxalyl
dihydrazone is used for the determining copper in paper pulp products, human serum,
non-ferrous metals and alloys [107]. Copper in foodstuff is determined by PAPH
(Pyridine-2aldehyde 2- pyridyl hydrazone). Hydrazones are extensively studied as
reactants or reaction intermediates since they can readily undergo various ring closure
reactions [108].
1.11.1 For the detection of metal ions

Derivatives of hydrazones are used as spot test reagents for determination of iron,
cobalt and copper. P-dimethylamino benzaldehyde isonicotinoyl hydrazone reacts
with mercury in acidic medium to form an intensely orange-yellow precipitate. The
complex of Co with (PAPH) has been used in the determination of silver and mercury
[109]. Dinitrophenylhydrazine is used for determining the concentration of low
molecular weight aldehydes and ketones by using HPLC [110]. Hydrazone Schiff
18
base derivatives are also synthesized by combination between 2, 4-
dinitrophenylhydrazine and ketone substituted bis-chalcone [111].
1.12 Biogenic amines

Different biological functions including cell growth and development and
stabilization of the cell membrane are under the control of biogenic amines [112].
Biogenic amines are found in a variety of food like fruit juices, fish, cheese etc.
Decarboxylation of aminoacids produces biogenic amines [113]. Toxicity of biogenic
amines causes migrane, hypertension, and intercereberal haemorhage. Biogenic
amines(putrescine and spermidine) are powerful cancer markers as rapid tumor
growth has been associated with changes in polyamine concentration [114]. N-acetyl
isatin derivatives of hydrazones are known to possess anti-convulsant effects.
Nmethyl-5-bromo-3-(p-chlorophenylimino) isatin act as carbonic anhydrase inhibitor.
Halogen substituted derivatives of isatins possess useful anticonvulsant properties
[115]. Amine substituted benzohydrazones are used as flavouring agents.
Indomethacin used for treating moderate to acute pain and arthritis contain
chlorobenzohydrazide [116].
19
Chapter 2
Material and Methods
20
2 Material and methods
2.1 Chemicals used
All the chemicals, solvents and reagents used in this research work were purchased
from Sigma Aldrich®. All the materials and solvents are of analytical grade with high
purity. The chemicals used are substituted benzaldehyde like 2-
methoxybenzaldehyde, 4-ethoxybenzaldehyde, 3-ethoxy-4methoxybenzaldehyde, P-
nitrobenzaldehyde, 3-ethoxysalicylaldehyde, 4-hydroxybenzohydrazide, methanol and
ethanol, ethyl acetate, n-hexane, and acetone.
2.2 Characterization
2.2.1 Chromatographic Technique (Thin layer chromatography)

According to their polarity, the components of a mixture are distributed differently
between a mobile phase and stationary phase during the separation process known as
chromatography. Thin layer chromatography is a method that allows the mobile phase
to traverse the stationary phase by capillary action. The most extensively used
stationary phases in TLC involve cellulose powder, alumina, kieselguhr, and silica
gel. Mobile phase is allowed to move over the stationary phase through capillary
action by a technique called thin layer chromatography. The most commonly used
stationary phases used in TLC are silica gel, alumina, kieselguhr and cellulose
powders.A small amount of product is dissolved in suitable solvent like methanol,
ethanol or DMSO. Apply a spot of solution with the help of jet made of capillary tube
on the appropriate cut TLC plate just 1 cm above the lower brink of the plate (base
line).Dry spot with the help of hot air blower. Mark the solvent front from 1cm from
upper brink of the plate. The spotted plate was then placed in the tank containing
different ratio of solution of n-hexane and ethyl acetate. The plate was left for
development. When the solvent front reaches up to the given mark, the plate was
taken out and air-dried. Observe TLC under UV lamp. By measuring the distance
travelled by the solute and solvent the Rf value was calculated.
2.2.2 UV-Vis Spectroscopy

The absorbance of synthesized compounds was observed in UV-Vis region by making
solution of all the synthesized compound in suitable solvent like DMSO using 10mm
cuvette of quartz in Elisa instrument (T80 +) UV-Vis spectrophotometer. Dimethyl
21
sulphooxide (DMSO) used as reference standard. Wavelength range of UV-Vis is set
from 200nm to 800nm with the interval difference of 5nm for each sample. The
absorption spectra of all the synthesized compounds were recorded.
2.2.3 Fourier Transform Infrared Spectroscopy (FT-IR)

FT-IR of the all the synthesized hydrazide based Schiff bases were measured on
Thermo-Nicolet 6700P FT-IR Spectrophotometer model 270 by using ATR diamond
window method.
2.2.4 Nuclear magnetic resonance (NMR)

1
H NMR spectra of all the synthesized compounds were obtained from
spectrophotometer of 300MHz by utilizing DMSO-d6 (deuterated) as solvent while
internal reference was TMS (tetramethylsilane).Heating magnetic stirrer/hot plate for
reflux was used, model ARE of VELP scintifica.
2.3 Method for the synthesis of benzohydrazide based Schiff base

analogues
Already prepared benzohydrazide (4-hydroxybenzohydrazide) is taken from lab. The
already prepared hydrazide were reacted with different types of substituted
benzaldehyde in suitable solvents like; ethanol and methanol to form the
corresponding hydrazine (a Schiff base).2-3 drops of Acetic acid is added to the
reaction mixture as a catalyst. The reaction mixture was refluxed for 6-7 hours with in
specific boiling point temperature range of solvent. The progress of the reaction was
checked by TLC. After complete conversion of all the reactants into our desired
products excess of the solvent were evaporated by using rotary evaporator. Work up
was done by using chiller cold distilled water to get good yield of our desired
products. Different color precipitates are formed after complete process which is
further confirmed by using different types of characterization techniques.
Figure 2.1 Scheme for the synthesis of benzohydrazone
22
2.3.1 Schematic method for the synthesis of (E)-4-hydroxy-N-(4-
nitrobenzylidene) benzohydrazide
Equimolar quantity (1:1mmol) of 3-nitrobenzaldehyde dissolved in 10ml of methanol
is made to react with 10ml methanol solution of 4-hydroxybenzohydrazide in two
neck round bottom flask. The reaction mixture was put on a reflux with continuous
stirring with the help of stirrer. The temperature of the reaction is set on 78-80.The
reaction is refluxed approximately for 1 week at constant temperature. The formation
of product was determined by TLC. After completion of reaction excess of methanol
is evaporated from mixture with the help of rotary evaporator. Work up was done by
adding cold distilled water in to the content of round bottom flask with continuous
shaking. Light yellow precipitates of our desired product were formed solution were
filtered with the help of filter paper and dried in air. Dried precipitates were collected
after 2 days.
Figure 2.2 Synthesis of (E)-4-hydroxy-Nˈ-(4-nitrobenzylidene)benzohydrazide
2.3.2 Synthesis of (E)-N-(3-ethoxybenzylidene)-4-hydroxybenzohydrazide

Equimolar quantity (1:1mmol) of 4-ethoxy-3-hydroxy-N-(4-hydroxybenzylidene)
benzohydrazide dissolved in 10ml of methanol is made to react with 10ml methanol
solution of 4-hydroxybenzohydrazide in two neck round bottom flask. The reaction
mixture was put on a reflux with continuous stirring with the help of stirrer. The
temperature of the reaction is set on 78-80.The reaction is refluxed approximately for
1 week at constant temperature. The formation of product was determined by TLC.
After completion of reaction excess of methanol is evaporated from mixture with the
help of rotary evaporator. Work up was done by adding cold distilled water in to the
content of round bottom flask with continuous shaking. Off white precipitates of our
desired product were formed. Solution were filtered with the help of filter paper and
dried in air. Dried precipitates were collected after 2 days.
23
Figure 2.3 Synthesis of (E)-Nˈ-(3-ethoxybenzylidene)-4-hydroxybenzohydrazide
2.3.3 Synthesis of (E)-4-hydroxy-N'-(4-metoxybenzylidene)

benzohydrazide
Equimolar quantity (1:1mmol) of 4-methoxy benzaldehyde dissolved in 10ml of
methanol is made to react with 10ml methanol solution of 4-hydroxybenzohydrazide
in two neck round bottom flask. The reaction mixture was put on a reflux with
continuous stirring with the help of stirrer. The temperature of the reaction is set on
78-80.The reaction is refluxed approximately for 1 week at constant temperature. The
formation of product was determined by TLC. After completion of reaction excess of
methanol is evaporated from mixture with the help of rotary evaporator. Work up was
done by adding cold distilled water in to the content of round bottom flask with
continuous shaking. White precipitates of our desired product were formed solution
were filtered with the help of filter paper and dried in air. Dried precipitates were
collected after 2 days.
Figure 2.4 Synthetic scheme of (E)-4-hydroxy-N'-(4-metoxybenzylidene)benzohydrazide
2.3.4 Synthesis of (E)-N'-(3-ethoxy-2-hydroxybenzylidene)-3-

hydroxybenzohydrazide
Equimolar quantity (1:1mmol) of 3-ethoxy-2-hydroxybenzaldehyde dissolved in 10ml of
methanol is made to react with 10ml methanol solution of 4-hydroxybenzohydrazide
24
in two neck round bottom flask. The reaction mixture was put on a reflux with
continuous stirring with the help of stirrer. The temperature of the reaction is set on
78-80.The reaction is refluxed approximately for 1 week at constant temperature. The
formation of product was determined by TLC. After completion of reaction excess of
methanol is evaporated from mixture with the help of rotary evaporator. Work up was
done by adding cold distilled water in to the content of round bottom flask with
continuous shaking. Light yellow precipitates of our desired product were formed
solution were filtered with the help of filter paper and dried in air. Dried precipitates
were collected after 2 days.
Figure 2.5 Synthetic scheme of (E)-N'-(3-ethoxy-2-hydroxybenzylidene)-3-hydroxybenzohydrazide
25
Chapter 3
Results
26
3 Results
3.1 Characterization of hydrazones
Table 3.1 Physical data of synthesized compounds
Sr. No Yield Rf M.P (°C) Physical

(%) Appearance
C-01 82 0.5 225-227 Yellow
C-02 80 0.6 222-224 White
C-03 78 0.4 226-228 Off white
C-04 60 0.4 220-222 White
3.2 UV-Vis Spectroscopic Analysis

The absorption spectra of all the synthesized compounds were observed within the
range of 200nm to the 800nm. Absorbance of all the synthesized compounds was
measured by making solution in DMSO (dimethyl sulfoxide) using 10mm quartz
cuvette. The UV-Vis spectra of compounds were observed at 306nm, 292nm, 330nm,
296nm, respectively.
Table 3.2 Results of UV-Vis spectra
Sr. Name UV-Vis λ Absorbance

No max (nm) (A.U)
1 (E)-4-hydroxy-N-(4-nitrobenzylidene) 306 0.952

benzohydrazide
2 (E)-4-hydroxy-N-(4-metoxybenzylidene) 292 0.826
benzohydrazide
3 (E)-N'-(3-ethoxy-2-hydroxybenzylidene)-3- 330 1.00
hydroxybenzohydrazide
4 (E)-4-ethoxy-3-hydroxy-N'-(4- 296 0.807
hydroxybenzylidene) benzohydrazide
27
3.3 FT-IR results
FT-IR spectra of all the synthesized compounds were obtained by using Thermo-
Nicolet6700 P FT-IR spectrophotometer in its ATR diamond window.
3.3.1 FT-IR analysis of compound 01

FT-IR data of compound (01) expressed strong absorption band at 1644 cm-1, 3350
cm-1, 1530 cm-1 for imine group (H-C=N), N-H group, and N=O group, respectively.
OH phenolic group is confirmed by the band at 3460 cm-1, absorption band at 1183
cm-1 confirms the existence of C-N group.
Table 3.3 FT-IR table of compound 01
Frequency (cm-1) Assignment

1183 C-N
1650 C=O
1644 HC=N
3350 N-H
1508 C=C
1530 N=O

cm-1, for imine group (H-C=N), N-H group, respectively. OH phenolic group is
confirmed by the band at 3075 cm-1, absorption band at 1320 cm-1 confirms the
existence of C-N group.
1320 C-N
1610 C=O
3075 OH phenolic
28
1550 N-H
1510 C=C
1636 C=N
3168 N-H

existence of C-N group.
Frequency (cm-1) Asignment
1184 C-N
1739 C=O
3263 OH phenolic
3350 N-H
1508 C=C
1499 C=N
2930 C-H
29
existence of C-H group.
1169 C-N
1736 C=O
3269 OH phenolic
3246 N-H
1530 C=C
1499 C=N
2880 C-H
1
3.4 H NMR spectroscopic analysis
In 1HNMR spectroscopic technique we determine the chemical shift, coupling
constant of different protons and carbons also the multiplicity pattern of different
protons is observed. The spectra were obtained from a spectrophotometer of 400 MHz
by taking DMSO-d6, CDCl3 solvents, and TMS as an internal reference.
30
1
3.4.1 H NMR Spectrum of hydrazine (Compound-01)
Table 3.7 1H NMR of compound 01
Chemical shift Multiplicity Coupling constant No of protons

(ppm) J (Hz)
11.80 s - 1
10.20 s - 1
8.48 s - 1
8.28-8.26 d 8.5 2
7.10 d 8.4 2
7.62 d 8.3 2
6.7 d 8.6 2
1
3.4.2 H NMR of compound 02

(ppm) J (Hz)
11.61 s - 1
10.08 s - 1
8.74 s - 1
31
6.82-6.79 d 8.4 2
6.99-6.95 t 8 1
7.06-7.04 d 8.8 1
7.38-7.34 t 7.2 1
7.78-7.76 d 8.4 1
7.82-7.80 d 8 2
1

(ppm) J (Hz)
11.94 s - 1
10.15 s - 1
8.86 s - 1
6.70-6.68 d 8.4 2
6.90-6.86 t 8 1
7.06-7.04 d 8.3 1
7.38-7.34 t 7.2 1
7.78-7.76 d 8.4 1
7.82-7.80 d 7 2
32
1

(ppm) J (Hz)
11.40 s - 1
10.76 s - 1
8.93 s - 1
6.82-6.79 d 8.4 2
6.95-6.91 t 8 1
7.08-7.06 d 8.7 1
7.36-7.33 t 7.3 1
7.78-7.76 d 8.2 1
7.85-7.81 d 6 2
3.5 Docking Studies

Molecular docking studies of all the four benzohydrazones were done by insilico
method using enzyme alpha-glucosidase (PDB code=3WY1). In order to
computationally demonstrate their binding mode with the active site of enzyme each
compound were docked with alpha-glucosidase.
33
3.5.1 Compound 01
α-glucosidase structure was employed for the docking study of (E)-4-hydroxy-N-(4-

nitrobenzylidene) benzohydrazide to study the favourable binding mode of
compound. The binding energy between protein alpha-glucosidase and C-01 is
observed as -6.21kcal/mol.GLU-432,THR-435,LYS-352 and GLN-439 form
conventional hydrogen bond with OH and N-H atom of the product. ALA-349 gives
pi-pi stacked interaction with abond distance of 4.36Å.
3.5.2 Compound 02
34
The binding energy between protein α-glucosidase and (E)-4-hydroxy-N'-(4-
methoxy-benzylidene) benzohydrazide is observed as -6.14kcal/mol. TYR-121
and SER-286 form conventional hydrogen bond with OH and N-H atom of the
product. TYR-334 gives pi-pi interaction with bond distance of 5.60Å.
3.5.3 Compound 03
The binding energy between protein α-glucosidase and (E)-4-ethoxy-3-

hydroxy-N'-(4-hydroxybenzylidene)benzohydrazide is observed as -
6.14kcal/mol. ASP-441, GLN-439 and ASN-443 form conventional hydrogen
bond with OH and N-H atom of the product. ASP-40 and ARG-450 provides
pi-pi stacked interaction with a bond distance of 4.98 and 4.05Å.
35
3.5.4 Compound 04
The binding energy between protein α-glucosidase and (E)-4-ethoxy-3-

hydroxy-N'-(4-hydroxybenzylidene) benzohydrazide is observed as -
6.14kcal/mol. ASP-440,SER-44 and ASN-46 form conventional hydrogen
bond with OH atom of the product with a bond distance of 2.49, 2.38 and
2.26Å. Apart from this PRO-442, and ARG-450 provides pi-pi stacked
interaction with a bond distance of 3.70Å and 4.25Å respectively. PRO-347
shows pi-alkyl interaction with bond distance of 4.85 and 4.29Å.
36
Chapter 4
Discussion
37
4 Discussion
4.1 Hydrazone Synthesis
Hydrazones are compounds that are easily synthesized by condensing substituted
carbonyl compounds with substituted hydrazines. Due to their remarkable biological
activities and diverse synthetic methodologies under moderate conditions, an
increasing number of innovative methodologies and procedures have been described.
Vilsmier hack reaction is used for synthesis of iminium ions (Schiff bases).
4.1.1 Mechanism of Hydrazones
Scheme 4.1 Mechanism for the synthesis of Hydrazones
4.2 UV-Vis results

By preparing the solution of all the synthesized compounds in DMSO and using
10 mm quartz cuvette in a PG instrument T80+UV-Vis spectrophotometer the
absorbance of all the compounds were measured in the UV-Vis region. UV-Vis
spectrum of compound 01 shows lambda max of 306 nm with absorbance of 0.952.
4.3 FT-IR Analysis

FT-IR of all the synthesized hydrazone Schiff base compounds were recorded on
Thermo-Nicolet 6700 P FTIR Spectrometer Model 270.IR absorption of all the
synthesized benzohydrazide Schiff bases showed the resultant functional groups
bending and stretching frequencies.
38
4.3.1 Compound 01
FT-IR spectra of the compound shows the deviation of peaks from starting reactants.
The characteristic peak of C=N stretching vibration of azomethine group of
benzohydrazone appears at 1510 cm-1 and ArOH appears at 3360cm-1 while N=O peak
at 1530 cm-1.
4.3.2 Compound 02
benzohydrazone appears at 1644 cm-1 and N=O peak at 1530 cm-1
4.3.3 Compound 03
benzohydrazone appears at 1499 cm-1 and C-N at 1184cm-1.
39
4.3.4 Compound 04
benzohydrazone appears at 1644 cm-1 and C=O at 1640 cm-1 C-N at 1117 cm-1.
4.4 NMR Spectroscopic analysis

1
4.4.1 H NMR characterization of hydrazone compound 01
Compound 01 was characterized by proton NMR spectroscopy. The characteristic

azomethine proton of HC=N appeared at 8.48 ppm with spin multiplicity of singlet
confirms the formation of desired compound. The peak due to CONH appeared at
11.80 ppm with spin multiplicity of singlet confirmed the formation of bond in the
product.
1

azomethine proton of C=NH appeared at 8.74 ppm with spin multiplicity of singlet
confirms the formation of desired compound. The peak due to CONHN= appeared at
40
11.61 ppm with spin multiplicity of singlet confirmed the formation of bond in the
product.
1

azomethine proton of C=NH appeared at 8.86ppm with spin multiplicity of singlet
11.94ppm with spin multiplicity of singlet confirmed the formation of bond in the
product.
1

azomethine proton of C=NH appeared at 8.93ppm with spin multiplicity of singlet
11.40ppm with spin multiplicity of singlet confirmed the formation of bond in the
product.
4.5 Docking studies

In-silico studies of all the four synthesized compounds were done in order to
demonstrate our experimental findings computationally. For this purpose molecular
docking studies were done for the synthesized compounds to reveal their binding
mode with in the active site of enzyme. The crystal structure of alpha-glucosidase was
first retrieve from protein data bank having a PDB id 3WY1. Docking studies were
41
done by using MOE and the best cluster poses were saved and analyzed by discovery
studio. All the compounds show good inhibitory potential against selected enzyme.
Table 4.1 Binding energies of docked compounds against α-glucosidase
Compounds Binding Energies Values (Kcal/mol)

Compound-01 -6.21
Compound-02 -6.14
Compound-03 -6.14
Compound-04 -6.029
Among all these compounds (compound 01) showed highest binding energy against
the target enzyme i.e α-glucosidase. The binding energy of compound 01 is -6.21
against α-glucosidase.
4.6 Conclusion
This research project was based on synthesis of new analogues of benzohydrazones
from corresponding benzohydrazide. The Hydrazones were synthesized in the
equimolar ratio of substituted hydrazide with substituted benzaldehyde. The
characterization of synthesized compounds was done by advance analytical
spectroscopic techniques. Inhibition potential of synthesized compound was checked
out through in-silico study. Molecular docking study was done to rationalize the
orientation and interaction of ligands at the receptor site.
42
Chapter 5
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43
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Synthesis, Charaterization and In-silico Study of

Substituted Benzhydrazide and its Schiff Base

COMSATS University Islamabad

Synthesis, Charaterization and In-silico Study of

COMSATS University Islamabad, Abbottabad Campus

of the requirement for the degree of

A Post Graduate Thesis submitted to the Department of Chemistry as partial

Name Registration Number

Huma Sabir CIIT/SP21-RCM-009/ATD

Dr. Syed Majid Bukhari

This thesis titled

Synthesis, Charaterization and In-silico Study of

For the COMSATS University Islamabad, Abbottabad Campus

I, Huma Sabir CIIT/SP21-RCM-009/ATD hereby declare that I have produced the

Synthesis, Characterization and In-silico Study of Substituted

Hydrazone Schiff base compounds were synthesized by subjecting 4-

1.2 Hydrazides .............................................................................................2

1.3 Substituted hydrazides ...........................................................................3

1.7 Sulfonyl Hydrazones ............................................................................12

1.8 Applications of hydrazones .................................................................13

1.11 In Analytical Chemistry .......................................................................18

2 Material and methods............................................................................... 21

2.2 Characterization ...................................................................................21

3.2 UV-Vis Spectroscopic Analysis ..........................................................27

3.3 FT-IR results ........................................................................................28

4.3 FT-IR Analysis.....................................................................................38

4.6 Conclusion ...........................................................................................42

Scheme 4.1 Mechanism for the synthesis of Hydrazones ........................................... 38

Table 3.1 Physical data of synthesized compounds .................................................... 27

NDDs Neurodegenerative disorders

Alzheimer’s disease (AD) which is mainly caused by degeneration of nervous system

1.3 Substituted hydrazides

1.3.1 Isonicotinic acid hydrazide

Figure 1.1 Structure of isonicotinic acid hydrazide

Isoniazid an antituberculosis drug and hydralsane (an antihypertensive and vasodilator

1.3.4 Acid hydrazides

1.3.6 Benzoic acid hydrazides

1.4 Schiffʼs bases (hydrazones)

Figure 1.2 Hydrazine Schiff base

Figure 1.3 N-(salicylidine)-2-hydroxyaniline

Schiff bases are considered to be a significant intermediate in many of the enzymatic

Hydrazine based Schiff bases are excellent alpha-glucosidase, xanthine oxidase

Substituted derivative of hydrazones are known to possess high antiviral activities

Figure 1.4 Oxadiazole derivative of hydrazone

Molybdenum involve in the epoxidation of olefins, and isomerization of allylic

1.4.3 Metal complexes of hydrazones

1.4.4 Hydrazide hydrazones

1.4.7 Aromatic hydrazones

1.4.8 Coupling products

1.4.9 Coordinattion complexes of hydrazones

1.4.10 Biscyclohexanone oxalyl dihydrazone

1.6 Salicylate Hydrazones

1.7 Sulfonyl Hydrazones

1.8 Applications of hydrazones

1.8.1 Anti-tubercular activities

Figure 1.5 Benzaldehyde 2-thiazolyl hydrazone

Metal complexes of Schiff bases unveil immence pharmacological activities.4-N, N-

1.8.2 Antibacterial characteristics of Schiff bases

Figure 1.7 Ciprofloxacin analogue

1.8.3 Antifungal properties of Schiff bases

1.8.4 Anti-inflammatory activity

Figure 1.8 Thiazine carbohydrazide