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SYNTHESIS OF SOME VANILLIN SEMICARBAZONES
AND EVALUATION OF THEIR ANTI-DIABETIC AND
ASSOCIATED HYPOCHOLESTREMIC ACTIVITIES
Thesis Submitted To
THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY, GUINDY,
CHENNAI As a partial fulfillment of the requirement for the award of the degree of
DOCTOR OF PHILOSOPHY
Submitted by
N.Venkateshan, M.Pharm.,
Under the supervision of
Prof. Dr. V. Ravichandiran, M.Pharm., Ph.D., Principal
Vels College of Pharmacy
Velan Nagar Pallavaram,
Chennai-600117 Tamilnadu, India
JANUARY – 2011
Prof. Dr. V. Ravichandiran., M.Pharm., Ph.D., Guide Principal Vels College of Pharmacy Velan Nagar Pallavaram, Chennai – 600117 Tamil Nadu, India
CERTIFICATE
This is to certify that the thesis entitled “Synthesis of Some Vanillin
Semicarbazones and Evaluation of their Anti-diabetic and Associated
Hypocholestremic Activities” is submitted to The Tamilnadu Dr. M.G.R. Medical
University, Chennai in partial fulfillment of the requirements for the award of degree
of Doctor of Philosophy is the record of original research work done by
Mr. N. Venkateshan, M.Pharm., for the academic year 2007 – 2010 under my
supervision and guidance and the thesis has not formed the basis for the award of
any degree, diploma, associateship, fellowship or other similar title.
Date : Place : Prof. Dr. V. RAVICHANDIRAN
DECLARATION
This is to certify that the thesis, entitled “Synthesis of Some Vanillin
Semicarbazones and Evaluation of their Anti-diabetic and Associated
Hypocholestremic Activities” is submitted to The Tamilnadu Dr. M.G.R. Medical
University, Chennai in partial fulfillment of the requirements for the award of degree
of Doctor of Philosophy is the record of original research work done by me under
the guidance and supervision of Prof. Dr. V. Ravichandiran. Principal, Vels
College of Pharmacy, Pallavaram, Chennai-600117 for the academic year 2007 –
2010 and the thesis has not formed the basis for the award of any Degree, Diploma,
Associateship, Fellowship or other similar title.
Date: Place : N. VENKATESHAN
ACKNOWLEDGEMENT
“The joy, satisfaction and euphoria that come along with the successful
completion of any work would be incomplete unless we mention the names of the
people who made it possible, whose constant guidance and encouragement served as
a beam of light and crowned our efforts.”
First and foremost I would like to thank THE DIVINE for his grace which
fetched the strength and understanding to surmount the difficulties during the tenure
of my project work and enabled me to complete this herculean task during this
journey. I owe to my father Mr. P.A. Narayanan, my mother
Mrs. N. Athilakshmi, and my beloved wife Dr.V.Hema who have stood as pillars of
support in all my endeavors, whose love and care is always there with me during all
my difficult times. They are the source of my inspiration always wishing the best for
me from the core of their heart.
I feel privileged to thank my adorable project guide
Prof. Dr. V. Ravichandiran, M.Pharm., Ph.D., Principal, Vels College of Pharmacy
for his valuable guidance, constructive criticism, constant encouragement and
intelligent decisions made my work easy. It is my privilege to express my deep sense
of gratitude to my Chairman, Dr. Isari Ganesh, Vels College of Pharmacy,
Pallavaram, Chennai.
No words can substitute the timely help and valuable suggestions extended
by Dr. K. Chinnasamy during the course of my work. My heartful of thanks to
Mr. A. Sarangapani, Drug Control (Retd), Dr. T. Elango, Registrar, Tamilnadu
Pharmacy Council, Chennai for his mentorship, innovative ideas, constant
inspiration and encouragement for successful completion of this work.
My heartful thanks to Dr. A. Nirmala, R. Vishnuvardh, R. Ajitha Nayac for
their kind support throughout the study.
I express my gratitude to Dr. J. Anbu, Head, Department of Pharmacology,
Dr. K.F.H. Nazeer Ahamed, Department of Pharmacology, Vels University,
Chennai, Dr. D. Selvakumar, Director, R&D, AVN Madurai, and
Mr. K. Anandarajagopal, Senior Lecturer, Masterskill University College of Health
Sciences, Malaysia without whose support my work would not be completed.
I would like to place on record my deepest gratitude to Mr. A.Ponraj,
Mr. V. Lavakumar, Mr. K.Masilamani and Mr.P.Balaji for their moral support
during my project work.
At this juncture, I would like to express my deep sense of gratitude to
Dr. A. Rajalakshmi, (former), Professor and Head, Dept of transfusion medicine,
The Tamil Nadu. Dr. M.G.R. Medical University, Chennai supported during
histopathology report.
I would like to thank all my beloved family members,
Mr.T.Sundararajan, Mrs.S.Chitra, Mr.G.Kumar, Mr.K.Muthusamy,
Mrs.M.Packiam Mr.T.Parthasarathy, Mr.G.Bala, Mr.K.Senthilkumar, for their
support and constant encouragement and motivation in all walks of my life.
I would like to thank all Faculty members and the non teaching staff, Vels
College of Pharmacy, Chennai, for their scholarly guidance and constant
encouragement for carrying out this work successfully.
Last but not the least, I express my sincere thanks to one and all and also to
those whom I might have missed to mention, for contributing their help directly and
indirectly for successful completion of this work.
List of Abbreviations Used
% - Percentage
µg - Microgram
µL - Microlitre
ALT - Alanine transferase
ATP - Adenosine triphosphate
BF - Beaf fat
BID - twice daily
BUF - Butter fat
BUN - Blood urea nitrogen 0C - degree celsius
CHD - Coronary Heart Disease
CI - Confidence interval
CIMT - Carotid intimal–medial thickness
Cm - Centimetre
CMC - Carboxy methyl cellulose
CVD - Cardiovascular disease
CNS - Central nervous system
DMSO-d6 - Dimethyl sulfoxide deuterated
DB - Direct bilirubin
dL - Decilitre
DM - Diabetes mellitus
ECG - Electro cardio gram
eg - Example
FT-IR - Fourier transform infra red
GLN - Glutamine
GLUT-4 - Glucose transporter-4
GPCR - G-protein-coupled receptor
HbA1C - Glycated hemoglobin
HCl - Hydrochloric acid
HDL - High density lipoprotein
HFM - High fat meal
HIS - Histidine
HMG-CoA - 3-hydroxy-3-methylglutanyl-Coenzyme A 1H NMR - Proton nuclear magnetic resonance
HLA - Human Leukocyte Antigen
hr - Hours
i.p - Intraperitoneal
IS - Internal standard
KBr - Potassium bromide
kg - Kilogram
L - Litre
LDL - Low density lipoprotein cholesterol
µg - Microgram
mg - milligram
MgSO4 - Magnesium sulphate
MHz - Megahertz
min - Minutes
mL - Millilitre
mmole - Millimoles
NaOH - Sodium hydroxide
NS - Not significance
OGTT - Oral glucose tolerance test
p.o. - per oral
PPARs - Peroxisome proliferator-activated receptors
QSAR - Quantitative structural activity relationship
RBC - Red blood cell
rpm - Revolution per minute
S - Significant
SAR - Structural activity relationship
SD - Standard deviation
SEM - Standard error mean
SER - Serine
SGOT - Serum glutamic oxaloacetic transaminase
SGPT - Serum glutamic pyruvic transaminase
STZ - Streptozotocin
TBARS - Thiobarbituric acid reactive substances
TG - Triglycerides
TB - Total bilirubin
TC - Total cholesterol
TLC - Thin layer chromatography
TP - Total protein
TYR - Tyrosine
U - Unit
V/V - Volume / Volume
VLDL - Very low density lipoproteins
WBC - White blood cell
WHO - World health organization
W/V - Weight / Volume
W/W - Weight / Weight
CCOONNTTEENNTTSS
CHAPTER TITLE PAGE
I INTRODUCTION 1
1.1 SEMICARBAZONES 3
1.2 DIABETES MELLITUS 4
1.2.1 Types of Diabetes mellitus 4
1.2.2 Signs and symptoms 5
1.2.3 Causes 6
1.2.4 Pathophysiology 7
1.2.5 Diagnosis 9
1.2.6 Treatment of Diabetes mellitus 10
1.3 HYPERCHOLESTEROLEMIA 12
1.3.1 Classification 13
1.3.2 Signs and Symptoms 13
1.3.3 Causes 14
1.3.4 Diagnosis 14
1.3.5 Screening 15
1.3.6 Lifestyle Changes 16
1.3.7 Medication 16
1.4 PEROXISOME PROLIFERATOR- ACTIVATED RECEPTORS
17
II REVIEW OF LITERATURE 20
III AIM AND OBJECTIVE OF WORK 40
IV MOLECULAR DOCKING STUDIES 42
4.1 INTRODUCTION 42
4.2 EXPERIMENTAL 44
4.2.1 Ligand preparation 45
CHAPTER TITLE PAGE
4.2.2 Protein preparation 45
4.2.3 Docking protocol 45
V SYNTHETIC METHODOLOGY 56
5.1 MATERIALS AND METHODS 57
5.2 SYNTHETIC METHODOLOGY 59
5.2.1 Synthesis of Aryl Carbamates 59
5.2.2 Hydrazinolysis and formation of
semicarbazides 60
5.2.3 Synthesis of vanillin
semicarbazones 60
5.2.4 Nomenclature of synthesized compounds
61
5.2.5 Physical Properties and Spectral
Data of Synthesized compounds 62
VI QSAR STUDIES 70
6.1 INTRODUCTION 70
6.2 PARAMETERS- Log P 70
VII PHARMACOLOGICAL EVALUATION 73
7.1 INTRODUCTION 73
7.2 ACUTE TOXICITY STUDIES 74
7.2.1 Animals 74
7.2.2 Formulations and stock solution
preparation 74
7.2.3 Drug treatment and assessment
of toxicity 74
7.2.4 Hematological, biochemical and
histological studies 75
7.2.5 Hemoglobin concentration of
whole blood 75
CHAPTER TITLE PAGE
7.2.6 Erythrocyte Count 76
7.2.7 Total Leukocyte Count 76
7.2.8 Packed cell volume 76
7.2.9 Total Protein 77
7.3 STATISTICAL ANALYSIS 77
7.4 ANTI DIABETIC STUDIES OF VANILLIN SEMI CARBAZONES
78
7.4.1 Induction of Experimental Diabetes
78
7.4.2 Effect of Vanillin semicarbazones (VSC I – IV) on glucose tolerance
in rats
79
7.4.3 Blood sugar estimation 79
7.4.4 Determination of Total Cholesterol and Triglycerides
80
7.4.5 Histopathological Studies 80
7.5 STATISTICAL ANALYSIS 80
7.6 ANTIHYPERLIPIDEMIC ACTIVITY OF
VANILLIN SEMICARBAZONES 81
7.6.1 Drug stock solution Preparation 81
7.6.2 Experimental animals 81
7.6.3 Diet preparation 81
7.6.4 Extraction for cholesterol and triacyl glycerol (TAG)
82
7.6.5 Extraction for thibarbituric acid reacting system (TBARS)
82
7.6.6 Extraction for HMGCoA reductase enzyme
82
7.6.7 Extraction for AST and ALP 82
CHAPTER TITLE PAGE
VIII RESULTS AND DISCUSSION 84
8.1 MOLECULAR DOCKING STUDIES 84
8.2 SYNTHETIC METHODOLOGY 86
8.3 CHARACTERIZATION 86
8.4 QSAR STUDIES 87
8.5 PHARAMACOLOGICAL EVALUATION
87
8.5.1 Effect of Vanillin Semicarbazones derivatives in acute oral toxicity test in mice
87
8.5.2 Effect of Vanillin Semicarbazones derivatives in Oral Glucose
Tolerance Test (OGTT)
88
8.5.3 Effect of Vanillin Semicarbazones derivatives in STZ induced
diabetic rats
88
8.5.4 Effect of Vanillin Semicarbazones derivatives in body weight changes
in diabetic animals
89
8.5.5 Effect of Vanillin Semicarbazones derivatives Serum total Cholesterol and Triglycerides levels
89
8.5.6 Effect of Vanillin semicarbazones derivatives in diabetic animal
pancreas
89
8.5.7 Effect of Vanillin Semicarbazones derivatives in High fat meal treated
hyperlipidemic rats
90
IX CONCLUSION 93
X REFERENCES 146
IR, NMR & MASS SPECTRA OF COMPOUNDS
COMPOUND
NO NAME OF THE COMPOUND PAGE
1. (E)-1-(4-hydroxy-3- methoxy benzylidene)-4-(4-chlorophenyl) semicarbazide
95-97
2. (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(4-bromophenyl) semicarbazide
98-100
3. (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(4-fluorophenyl) semicarbazide
101-103
4. (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(4-nitrophenyl) semicarbazide
104-106
5. (E)-1-(4-hydroxy-3- methoxy benzylidene)-4-(4-(hydroxyl methyl) phenyl) semicarbazide
107-109
6. (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-p-tolyl semicarbazide
110-112
7. (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(3-chloro-4-methylphenyl) Semicarbazide
113-115
8. (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(sulfonamido phenyl) semicarbazide
116-118
HISTOPATHOGICAL DIAGRAMS OF VARIOUS ORGANS
FIGURE ORGANS PAGE
7.1.1 HEART 130, 131
7.1.2 LUNGS 132, 133
7.1.3 LIVER 134, 135
7.1.4 KIDNEY 136, 137
7.1.5 STOMACH 138, 139
7.1.6 SPLEEN 140, 141
7.1.7 TESTIS 142, 143
7.1.8 PANCREAS 144, 145
LIST OF TABLES
TABLE NO PAGE
7.1 Effect of Compounds VSC I-IV on Haematological Parameters in mice
119
7.2 Effect of Compounds VSC I-IV on Biochemical Parameters in mice
120
7.3 Effect of Compounds on Organ Weight (in gm) in mice
121
7.4 Effect of Compounds VSC I - IV on Oral glucose tolerance test
122
7.5 Effect of Compounds VSC I - IV on Fasting serum Glucose concentration in normal and STZ-induced diabetic rats
123
7.6 Measurement of Body weight changes after the treatment of compounds VSC I -IV
124
7.7 Effect of Compounds VSC I - IV Serum Total Cholesterol and Triglyceride levels in normal and STZ- induced diabetic rats
125
7.8 Effect of Compounds VSC I - IV on Serum TAG. Cholesterol, VLDL+LDL. HDL cholesterol (mg/ml) and ALP activities
126
7.9 Effect of Compounds VSC I - IV on TC, TAG, TBARS, HMG CoA reductase, ALP and AST level in heart tissue
127
7.10 Effect of Compounds VSC I - IV on TC, TAG, TBARS, ALP and AST level in liver tissue
128
7.11 Effect of Compounds VSC I - IV on TC, TAG, TBARS and HMG CoA reductase level in kidney tissue
129
1
CCHHAAPPTTEERR II
INTRODUCTION
In recent years, there have been significant developments made in the way
new drugs are being discovered and developed. Such changes are driven by new
technologies that have expanded the opportunities to prepare and screen large
libraries of compounds in a rapid time frame by the use of high-throughput synthesis
(HTS) and screening techniques1. Investigations in medicinal chemistry are
undertaken as an adventure of the human spirit, stimulated largely by curiosity and
served by disciplined imagination. Synthetically, organic compounds have generated
a great deal of interest in exploiting more than one proximal functional group in
designing novel structures for performing a variety of synthetic functions in
transformations and synthesis2.
A promising new approach to drug discovery concerns with synthesis and
screening of combinational libraries in order to identify new compounds that express
high affinity and specificity for a pharmacologically relevant, bimolecular target.
Advances in molecular biology, automated chemical synthesis and robotics have
facilitated the formulation of vast libraries of structurally related molecules. An
essential aspect of screening large combinational libraries is the ability to identify
the active components in these complex mixtures, which is usually based on the
strength of binding to a selected target macromolecule3.
The recent advancement like drug designing, combinatorial chemistry,
computational chemistry, molecular biology and genetic engineering make
interesting the chemistry approach4.
Molecular modeling technique became popular to study the drug excipient
interaction which helps to visualize the type and site of interaction on a computer
monitor5. These strategies are driven by the need to shorten time lines for bringing
discovery to the market. As a result the role and needs of synthetic chemistry in the
discovery and development of new therapeutic agents has been altered6.
2
Carbocyclic or heterocyclic ring systems comprise the core of chemical structures of the vast majority of therapeutic agents. This finding results in the majority of drugs exerting their effect by their actions at receptor or receptor-like sites on cells, enzymes, or related entities. These interactions depend on the receiving site being presented with a molecule that has a well-defined shape, distribution of electron density, and array of ionic or ionizable sites, which complement features on the receptor. These requirements are readily met by the relatively rigid carbocyclic or heterocyclic molecules.
Free-standing benzene rings have provided the core for a very large number of biologically active compounds. Over the past few years, it has been established that several apparently quite unrelated drug classes owe their activity to effects on a shared biochemical system. A number of compounds have been found that treat
elevated lipid levels by other diverse mechanisms7.
Similar groups/structures often exhibit similar biological activities. However, they usually exhibit different potency. The traditional structure activity relationship (SAR) investigations are a useful tool in the search for new drugs. However, SAR is usually determined by making minor changes to the structure of the existing compound and assessing the effect on its biological activity.
In general, clinically used drugs are not discovered. What is more likely discovered is known as a lead compound8. The lead is a prototypic compound that has the desired biological or pharmacological activity, but it may have many undesirable effects. The structure of the lead compound is then modified by synthesis to amplify the desired activity and to eliminate the unwanted properties. Most of the drugs have been investigated and developed based on results obtained from the screening of potential drugs. There are a variety of approaches used to identify a lead compound and these include random screening, non-random
screening, drug metabolism studies, clinical observations and rational approaches.
Structure modifications of the lead compound are designed to achieve
specific goals over the prototypic molecule by the following improvements.
The development of more potent drugs
To eliminate or minimize toxic effects
3
To discover the pharmacophore and to separate the molecular features
responsible for the desired activity and the undesirable or toxic effects
and
Modification of the pharmacokinetic properties of the compound
The process of rational drug design has three fundamental steps such as
Identification and molecular level understanding of a specific etiologic/
pathogenic mechanism to be exploited in the drug discovery
Identification of a class of molecules to be exploited as the molecular
template for the new drug and
Identification of appropriate techniques for determining the properties of
the prototypic drug and related analogues.
1.1 SEMICARBAZONES
Semicarbazones have proved the efficiency and efficacy in combating
various diseases9. Semicarbazone is a derivative of an aldehyde or ketone formed by
a condensation reaction between a ketone or aldehyde and semicarbazide. It serves
as important synthetic intermediates and can be preferably used for isolation,
purification, characterization and protection of aldehydes and ketones10. Several
semicarbazones, as well as their sulfur analogs and its derivatives, have proved the
efficiency and efficacy in combating various diseases11. Semicarbazones are
associated with diverse pharmacological activities, such as antibacterial, antifungal,
antihypertensive, hypolipidemic, antineoplasic, hypnotic and anticonvulsant.
Several studies have reported the anticonvulsant activity of semicarbazones
derived from aromatic and unsaturated carbonyl compounds. Anticonvulsant activity
was displayed by most of the compounds in the maximal electroshock screen after
administration in rats. Some of these compounds provided greater protection than
phenytoin, carbamazepine and sodium valproate, three known anticonvulsant
drugs.Some semicarbazones, such as nitrofurazone, and thiosemicarbazones are
4
known to have anti-viral and anti-cancer activity, usually mediated through binding
to copper or iron in cells.
Many semicarbazones are crystalline solids, useful for the identification of
the parent aldehydes/ketones by melting point analysis. It has been shown that 4-[4-
fluorophenoxy] benzaldehyde semicarbazone, a member of the semicarbazone
family, acts as a Na+ channel blocker and inhibits allodynia and hyperalgesia in a rat
model of peripheral neuropathy.
In some cases complexation to metal ions can improve properties of these
ligands, such as lipophilicity and pharmacological activity12. Semicarbazones and
their metal complexes exhibit a wide range of bioactivities13. Complexes of
semicarbazones with a variety of metal ions have been extensively studied but
vanadium complexes have received less attention. Vanadium complexes of
semicarbazones of low molecular weight could in principle is useful as potential
biomimetic drugs14.
As the number of people with diabetes multiply world wide, the diseases
takes an ever increasing proportion of national and international healthcare budgets.
It is projected to become one of the worlds main disables within the next 25 years. It
is very popularly known in medical history as “silent killer”. Regions with greatest
potential are Asia and Africa, where diabetes mellitus rates could rise to two to three
–folds than the present rates.
1.2 DIABETES MELLITUS
Diabetes mellitus refers to a group of disorders characterized by absent or
deficient insulin secretion or peripheral insulin resistance, resulting impaired
metabolism and leads hyperglycemia, vascular and nerve complications.15
1.2.1 Types of Diabetes mellitus
Diabetes mellitus occurs in two major forms16
5
a. Type I: Insulin-Dependent Diabetes mellitus (IDDM)
Diabetes mellitus, is formerly known as juvenile-onset or ketosis-prone
diabetes. This form is most common in children and in adults up to age 30 years but
may occur at any age. Disease onset is sudden. Beta cells, insulin-producing cells of
pancreatic islets of langerhands , are destroyed, causing absolute insulin deficiency
b. Type II: Non-Insulin-Dependent Diabetes mellitus (NIDDM)
NIDDM is formerly called as adult-onset diabetes. Most type II diabetic
patients are over 40 years old and obese. In most case type II DM is characterized by
insensitivity to insulin in the target tissues, deficient response of pancreatic beta cells
to glucose, or both
1.2.2 Signs and symptoms
The classical symptoms of diabetes are polyuria (frequent urination),
polydipsia (increased thirst) and polyphagia (increased hunger)17. Symptoms may
develop rapidly (weeks or months) in type 1 diabetes while in type 2 diabetes they
usually develop much more slowly and may be subtle or absent.
Fig.1.1. Overview of the most significant symptoms of diabetes
6
Prolonged high blood glucose causes glucose absorption, which leads to
changes in the shape of the lenses of the eyes, resulting in vision changes; sustained
sensible glucose control usually returns the lens to its original shape. Blurred vision
is a common complaint leading to a diabetes diagnosis; type 1 should always be
suspected in cases of rapid vision change, whereas with type 2 change is generally
more gradual, but should still be suspected.
People (usually with type 1 diabetes) may also present with diabetic
ketoacidosis, a state of metabolic dysregulation characterized by the smell of
acetone; a rapid, deep breathing known as Kussmaul breathing; nausea; vomiting
and abdominal pain; and an altered states of consciousness.
A rarer but equally severe possibility is hyperosmolar nonketotic state, which
is more common in type 2 diabetes and is mainly the result of dehydration. Often,
the patient has been drinking extreme amounts of sugar-containing drinks, leading to
a vicious circle in regard to the water loss. A number of skin rashes can occur in
diabetes that are collectively known as diabetic dermadromes.
1.2.3 Causes
The cause of diabetes depends on the type. Type 2 diabetes is due primarily
to lifestyle factors and genetics18. Type 1 diabetes is also partly inherited and then
triggered by certain infections, with some evidence pointing at Coxsackie B4 virus.
There is a genetic element in individual susceptibility to some of these triggers
which has been traced to particular HLA genotypes (i.e., the genetic "self"
identifiers relied upon by the immune system). However, even in those who have
inherited the susceptibility, type 1 diabetes mellitus seems to require an
environmental trigger.
7
1.2.4 Pathophysiology
Fig.1.2. The fluctuation of blood sugar (red) and the sugar-lowering hormone
insulin (blue) in humans during the course of a day with three meals
One of the effects of a sugar-rich vs a starch-rich meal is highlighted.
Fig.1.3. Mechanism of insulin release in normal pancreatic beta cells
Insulin production is more or less constant within the beta cells, irrespective
of blood glucose levels. It is stored within vacuoles pending release, via exocytosis,
which is primarily triggered by food, chiefly food containing absorbable glucose.
The chief trigger is a rise in blood glucose levels after eating
8
Insulin is the principal hormone that regulates uptake of glucose from the
blood into most cells (primarily muscle and fat cells, but not central nervous system
cells). Therefore deficiency of insulin or the insensitivity of its receptors plays a
central role in all forms of diabetes mellitus.
Humans are capable of digesting some carbohydrates, in particular those
most common in food; starch, and some disaccharides such as sucrose, are converted
within a few hours to simpler forms most notably the monosaccharide glucose, the
principal carbohydrate energy source used by the body. The most significant
exceptions are fructose, most disaccharides (except sucrose and in some people
lactose), and all more complex polysaccharides, with the outstanding exception of
starch. The rest are passed on for processing by gut flora largely in the colon. Insulin
is released into the blood by beta cells (β-cells), found in the Islets of Langerhans in
the pancreas, in response to rising levels of blood glucose, typically after eating.
Insulin is used by about two-thirds of the body's cells to absorb glucose from the
blood for use as fuel, for conversion to other needed molecules, or for storage.
Insulin is also the principal control signal for conversion of glucose to
glycogen for internal storage in liver and muscle cells. Lowered glucose levels result
both in the reduced release of insulin from the beta cells and in the reverse
conversion of glycogen to glucose when glucose levels fall. This is mainly
controlled by the hormone glucagon which acts in the opposite manner to insulin.
Glucose thus forcibly produced from internal liver cell stores (as glycogen) re-enters
the bloodstream; muscle cells lack the necessary export mechanism. Normally liver
cells do this when the level of insulin is low (which normally correlates with low
levels of blood glucose).
Higher insulin levels increase some anabolic ("building up") processes such
as cell growth and duplication, protein synthesis, and fat storage. Insulin (or its lack)
is the principal signal in converting many of the bidirectional processes of
metabolism from a catabolic to an anabolic direction, and vice versa. In particular, a
low insulin level is the trigger for entering or leaving ketosis (the fat burning
metabolic phase).
9
If the amount of insulin available is insufficient, if cells respond poorly to the
effects of insulin (insulin insensitivity or resistance), or if the insulin itself is
defective, then glucose will not have its usual effect so that glucose will not be
absorbed properly by those body cells that require it nor will it be stored
appropriately in the liver and muscles. The net effect is persistent high levels of
blood glucose, poor protein synthesis, and other metabolic derangements, such as
acidosis.
When the glucose concentration in the blood is raised beyond its renal
threshold (about 10 mmol/L, although this may be altered in certain conditions, such
as pregnancy), reabsorption of glucose in the proximal renal tubuli is incomplete,
and part of the glucose remains in the urine (glycosuria). This increases the osmotic
pressure of the urine and inhibits reabsorption of water by the kidney, resulting in
increased urine production (polyuria) and increased fluid loss. Lost blood volume
will be replaced osmotically from water held in body cells and other body
compartments, causing dehydration and increased thirst.
1.2.5 Diagnosis
2006 WHO Diabetes criteria
Condition 2 hour glucose Fasting glucose
mmol/l(mg/dl) mmol/l(mg/dl)
Normal <7.8 (<140) <6.1 (<110)
Diabetes mellitus ≥11.1 (≥200) ≥7.0 (≥126)
Diabetes mellitus is characterized by recurrent or persistent hyperglycemia,
and is diagnosed by demonstrating any one of the following
• Fasting plasma glucose level ≥ 7.0 mmol/L (126 mg/dL).
• Plasma glucose ≥ 11.1 mmol/L (200 mg/dL) two hours after a 75 g oral
glucose load as in a glucose tolerance test.
10
• Symptoms of hyperglycemia and casual plasma glucose ≥ 11.1 mmol/L
(200 mg/dL).
• Glycated hemoglobin (HbA1C) ≥ 6.5%
A positive result, in the absence of unequivocal hyperglycemia, should be
confirmed by a repeat of any of the above-listed methods on a different day. It is
preferable to measure a fasting glucose level because of the ease of measurement
and the considerable time commitment of formal glucose tolerance testing, which
takes two hours to complete and offers no prognostic advantage over the fasting
test19. According to the current definition, two fasting glucose measurements above
126 mg/dL (7.0 mmol/L) is considered diagnostic for diabetes mellitus.
People with fasting glucose levels from 100 to 125 mg/dL (5.6 to
6.9 mmol/L) are considered to have impaired fasting glucose. Patients with plasma
glucose at or above 140 mg/dL (7.8 mmol/L), but not over 200 mg/dL
(11.1 mmol/L), two hours after a 75 g oral glucose load are considered to have
impaired glucose tolerance. Of these two pre-diabetic states, the latter in particular is
a major risk factor for progression to full-blown diabetes mellitus as well as
cardiovascular disease.
1.2.6 Treatment of Diabetes mellitus
The goal in treating diabetes is to keep the patient's blood sugar level in the
normal range.
A. INSULIN
Insulin replacement therapy is indicated for all patients with type I DM and
for those with type II DM whose hyperglycemia doesn’t respond to dietary or oral
antihyperglycemic drug therapy. There are three major types of insulin which differ
in onset and duration of action (fast acting-semilente, intermediate acting-lente and
long acting-ultralente)
11
B. ORAL HYPOGLYCEMIC AGENTS
a. Sulfonylureas
First generation drugs - Tolbutamide, Chlopropamide, Tolazamide,
Acetohexamide,
Second generation drugs - Glipizide, Glyburide
Third generation drugs - Glimepiride
Mechanism of action
They act by increasing insulin release from the beta cells in the pancreas.
Side effects
Induce hypoglycemia as a result of intermittent excesses in insulin
production and release
Induce weight gain, mainly as a result of edema
Reduction of the osmotic diuresis
b. Biguanides
Metformin HCl
Mechanism of action:
It improves hyperglycemia primarily through its suppression of hepatic
glucose production (hepatic gluconeogenesis).
Side effects
Lactic acidosis
Gastrointestinal upset, including diarrhoea, cramps, nausea, vomiting
Long-term use - increased homocysteine levels
12
c. Meglitinides
Repaglinide and Nateglinide
Mechanism of action
Bind to an ATP-dependent K+ (KATP) channel on the cell membrane of
pancreatic beta cells in a similar manner to sulfonylureas but at a separate binding
site.
Side effects
Weight gain and hypoglycemia
d. Thiazolidinediones
Pioglitazone
Rosiglitazone
Mechanism of action
It is through activation of the intracellular receptor class of the peroxisome
proliferator-activated receptors (PPARs), specifically PPARγ
Side effects
Macular Edema
1.3 HYPERCHOLESTEROLEMIA
Hypercholesterolemia (literally: high blood cholesterol) is the presence of
high levels of cholesterol in the blood. It is not a disease but a metabolic
derangement that can be secondary to many diseases and can contribute to many
forms of disease, most notably cardiovascular disease. It is closely related to the
terms "hyperlipidemia" (elevated levels of lipids) and "hyperlipoproteinemia"
(elevated levels of lipoproteins).
13
Elevated cholesterol in the blood is due to abnormalities in the levels of
lipoproteins, the particles that carry cholesterol in the bloodstream. This may be
related to diet, genetic factors (such as LDL receptor mutations in familial
hypercholesterolemia) and the presence of other diseases such as diabetes and an
under active thyroid. The type of hypercholesterolemia depends on which type of
particle (such as low density lipoprotein) is present in excess.
High cholesterol levels are treated with diets low in cholesterol, medications,
and rarely with other treatments including surgery (for particular severe subtypes).
This has also increased emphasis on other risk factors for cardiovascular disease,
such as high blood pressure20.
1.3.1 Classification
Classically, hypercholesterolemia was categorized by lipoprotein
electrophoresis and the Fredrickson classification. Newer methods, such as
"lipoprotein subclass analysis" have offered significant improvements in
understanding the connection with atherosclerosis progression and clinical
consequences.
If the hypercholesterolemia is hereditary (familial hypercholesterolemia),
there is more often a family history of premature, earlier onset atherosclerosis, as
well as familial occurrence of the signs mentioned above.
1.3.2 Signs and symptoms
Elevated cholesterol does not lead to specific symptoms unless it has been
longstanding. Some types of hypercholesterolemia lead to specific physical findings:
xanthoma (deposition of cholesterol in patches on the skin or in tendons),
xanthelasma palpabrum (yellowish patches around the eyelids) and arcus senilis
(white discoloration of the peripheral cornea).
Longstanding elevated hypercholesterolemia leads to accelerated
atherosclerosis; this can express itself in a number of cardiovascular diseases:
14
coronary artery disease (angina pectoris, heart attacks), stroke and short stroke-like
episodes and peripheral vascular disease21.
1.3.3 Causes
There are a number of secondary causes for high cholesterol:
• Diabetes mellitus and metabolic syndrome
• Kidney disease (nephrotic syndrome)
• Hypothyroidism
• Cushing's syndrome
• Anorexia nervosa
• Sleep deprivation
• Zieve's syndrome
• Family history
• Antiretroviral drugs, like protease inhibitors and nucleoside reverse
transcriptase inhibitors.
• Diet
• Body weight
• Physical activity
1.3.4 Diagnosis
There is no specific level at which cholesterol levels are abnormal.
Cholesterol levels are found in a continuum within a population. Higher cholesterol
levels lead to increased risk of several diseases, most notably cardiovascular
diseases. Specifically, high levels of small LDL cholesterol particles are associated
with increased risk of heart disease22. Larger LDL particles do not carry the same
risk.
When measuring cholesterol, it is important to measure its sub-fractions
before drawing a conclusion as to the cause of the problem. The sub-fractions are
15
LDL, HDL and VLDL. In the past, LDL and VLDL levels were rarely measured
directly due to cost concerns. VLDL levels are reflected in the levels of triglycerides
(generally about 45% of triglycerides are composed of VLDL). LDL was usually
estimated as a calculated value from the other fractions (total cholesterol minus
HDL and VLDL); this method is called the Friedewald calculation; to be specific:
LDL ~= Total Cholesterol - HDL - (0.2 x Triglycerides).
Less expensive (and less accurate) laboratory methods and the Friedewald
calculation have long been used because of the complexity, labor, and expense of the
electrophoretic methods developed in the 1970s to identify the different lipoprotein
particles that transport cholesterol in the blood. In 1980, the original methods,
developed by research work in the mid-1970s cost about $5,000, in US 1980 dollars,
per blood sample/person.
With time, more advanced laboratory analyses that do measure LDL and
VLDL particle sizes and levels have been developed, and at far lower cost. These
have partly been developed and become more popular as a result of the increasing
clinical trial evidence that intentionally changing cholesterol transport patterns,
including to certain abnormal values compared to most adults, often has a dramatic
effect on reducing, even partially reversing, the atherosclerotic process. With
ongoing research and advances in laboratory methods, the prices for more
sophisticated analyses have markedly decreased, to less than $100, US 2004, by
some labs, and with simultaneous increases in the accuracy of measurement for
some of the methods.
1.3.5 Screening
Screening for a disease refers to testing for a disease, such as
hypercholesterolemia, in patients who have no signs or symptoms of the disease.
In patients without any other risk factors, moderate hypercholesterolemia is
often not treated. According to Framingham Heart Study, people with an age greater
than 50 years have no increased overall mortality with either high or low serum
cholesterol levels. There is, however, a correlation between falling cholesterol levels
16
over the first 14 years and mortality over the following 18 years (11% overall and
14% CVD death rate increase per 1 mg/dL per year drop in cholesterol levels). This,
however, does not mean that a decrease in serum levels is dangerous, as there has
not yet been a recorded heart attack in the study in a person with a total cholesterol
below 150 mg/dL. The U.S. Preventive Services Task Force (USPSTF) has
evaluated screening for hypercholesterolemia.
The goal of treatment is to reduce the risk of atherosclerotic heart disease.
Those who inherit only one copy of the defective gene may respond well to diet
changes combined with statin drugs.
1.3.6 Lifestyle Changes
Lifestyle Changes can reduce the saturated fat intake by:
• Decreasing amounts of beef, chicken, pork, and lamb
• Substituting low-fat dairy products for full-fat ones
• Eliminating coconut and palm oils
• eliminating egg yolks and organ meats
Dietary counseling is often recommended to help people make these
adjustments to their eating habits. Weight loss and regular exercise may also aid in
lowering cholesterol levels.
1.3.7 Medications
If lifestyle changes do not change your cholesterol levels, your doctor may
recommend medication. There are several types of drugs available to help lower
blood cholesterol levels, and they work in different ways. Some are better at
lowering LDL cholesterol, some are good at lowering triglycerides, while others
help raise HDL cholesterol.
The most commonly used and effective drugs for treating high LDL
cholesterol are called statins. The include lovastatin (Mevacor), pravastatin
17
(Pravachol), simvastatin (Zocor), fluvastatin (Lescol), atorvastatin (Lipitor), and
rosuvastatin (Crestor).
Other cholesterol-lowering medicines include:
• Bile acid-sequestering resins
• Ezetimibe
• Fibrates (such as gemfibrozil)
• Nicotinic acid
Those with more severe forms of this disorder may need a treatment called
extracorporeal apheresis. This is the most effective treatment. Blood or plasma is
removed from the body. Special filters then remove the extra LDL-cholesterol, and
the blood plasma is then returned.
1.4. PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS
Research in Peroxisome proliferator-activated receptors (PPARs) has
attained great medical significance because of its multiple effects on metabolic
disorders and the fact that developing countries. Research targeted at PPARs has led
to several novel hypoglycemic agents, which are unrelated structurally to drugs
previously used to treat diabetes7. The PPAR agonists can help to improve blood
glucose levels and levels of blood lipids (fats and cholesterol) and may also reduce
risks of atherosclerosis because PPARs regulate the expression of genes that affect
blood lipid metabolism, the generation of adipocytes (fat cells), and blood glucose
control.
PPARs are ligand-activated transcription factors belonging to the nuclear
hormone receptor superfamily. There are three PPAR subtypes, which are the
products of distinct genes and are commonly designated PPARα, PPARγ, and
PPARδ.
18
PPARα is a key factor in fatty acid metabolism, and is responsible for
mediating the lipid-lowering effects of fibrate drugs (e.g., fenofibrate and
gemfibrozil). PPARγ is expressed most abundantly in adipose tissue and mediates
the antidiabetic activity of the insulin-sensitizing drugs belonging to the
thiazolidindione. A number of compounds have been found that treat elevated
glucose levels and/or lipid levels by other diverse mechanisms.
Fig.1.4. Mechanisms of PPAR activation and regulation of target gene
expression
19
The phosphonic acid derivative ibrolipim, synthesized from substituted
aniline leads to the hypolipidemic agent and is believed to lower those levels by
accelerating fatty acid oxidation7. The blood lipid lowering effect of the fibrates,
such as, clofibrate, and the hypoglycemic action of the recently introduced
hypoglycemic thiazolidinediones both trace back to action on subtypes of the
peroxisome proliferator activated receptors (PPAR), which regulates lipid and
glucose metabolism.
Ibrolipim
In this present thesis, we focused on the arylcarbamates moiety, followed by
vanillin semicarbazones, which are apt to form up to 4 pivotal hydrogen bonds with
serine, tyrosine and histidine of the PPARα and PPARγ, as the acidic warhead; such
a strong hydrogen acceptor is indispensable for obtaining potent agonists. For the
cyclic tail, partly solvent exposed and in general quite tolerant with respect to
structural variations, we focused and well-tried in countless PPAR ligands.
Regarding the aromatic center, close inspection of several X-ray structures revealed
that a simple phenyl ring does not optimally fit the cavity of the receptor.
Fig.1.4. Pharmacophore model of PPAR agonists
(The aromatic centre can be substituted to access additional subpockets in the receptor)
We therefore decided to explore the potential of some novel vanillin
semicarbazones synthesized from substituted aryl carbamates specifically which had
already proven to meet the structural requirements essential for antidiabetic activity
associated with lipid lowering effect.
20
CCHHAAPPTTEERR IIII
REVIEW OF LITERATURE
Various semicarbazones and related compounds have been great interest
because of their chemistry and potentially beneficial biological activities. The
development of semicarbazones as potential therapeutic agents evolved from the
modifications of functional groups present on compounds. Numerous
semicarbazones and thiosemicarbazones have been found to possess anticonvulsant
activity (Dimmock et al., 1995; Pandeya et al., 2000; Yogeeswari et al., 2004;
Thirumurugan et al., 2006)). Some vanadium (V) complexes with salicylaldehyde
semicarbazone derivatives exhibited insulin-mimetic activity (Pabla et al., 2004;
Bastos et al., 2008). Many complexes of 2-hydroxyacetophenone semicarbazones
has been proposed as being necessary for superoxide dismutase mimetic action
(Safavi et al., 2010).
Previous studies on semicarbazones reported that the structural requirements
for the activity and have possessed wide variety of biological activities such as
antinociceptive, anti-inflammatory, anticonvulsant, sedative and hypnotic, anti-
parkinsonism, antiarrhythmic, insulin-mimic, uterotrophic, antiviral, antimalarial,
antitubercular, cytotoxic, antibacterial and antifungal activities.
Ozair et al., (2010) reported that synthesis, anticonvulsant and toxicity
screening of newer pyrimidine semicarbazone derivatives23. A number of N-
(4,6-substituted diphenylpyrimidin-2-yl) semicarbazones were synthesized
and tested for their anticonvulsant activity against the two seizure models,
maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole
(scPTZ). All the synthesized compounds possessed the four essential
pharmacophoric elements for good anticonvulsant activity. Most of the
compounds displayed good anticonvulsant activity with lesser neurotoxicity.
To assess the unwanted effects of the compounds on liver, estimation of
enzymes and proteins was carried out.
21
Mohammad et al., (2010) reported that synthesis of two novel 3-amino-5-[4-
chloro-2-phenoxyphenyl]-4H-1,2,4-triazoles with anticonvulsant Activity24.
Two novel 3-amino-5-(4-choloro-2-phenoxyphenyl)-4H-1,2,4-triazole
derivatives were prepared and their anticonvulsant activity was measured by
evaluation of the ability of these compounds to protect mice against
convulsion induced by lethal doses of pentylenetetrazole (PTZ). Diazepam
was considered as a positive control drug with anticonvulsant effect.
Compound, 3-amino-5- [4-chloro-2-(2-flurophenoxy)phenyl]-4H-1,2,4-
triazole, showed potent anti-convulsant activity compared to diazepam.
Amir et al., (2010) reported that synthesis of N1-(3-chloro-4-fluorophenyl)-
N4-substituted semicarbazones as novel anticonvulsant agents25. Several 3-
chloro-4-flourophenyl substituted semicarbazones have been synthesized in
three steps involving aryl urea and aryl semicarbazide formation and selected
compounds have been evaluated for anticonvulsant activity by using
maximal electroshock seizure (MES) test. The compounds have also been
screened for their neurotoxicity and CNS depressant activity. Compound N1-
(3-chloro-4-fluorophenyl)-N4-(4-N,N-dimethylamino-benzaldehyde)
semicarbazone is found most active of the series without neurotoxicity and
less CNS depressant effect as compared to standard drug.
Hemendra et al., (2010) reported that chalconsemicarbazone: A new Scaffold
for antiepileptic drug discovery26.Investigation in the area of epileptic drug
discovery, the present work have applied hybridization of pharmacophore
strategy of drug design and developed a new pharmacophore, designed a
scheme for synthesizing such pharmacophore and performed their
pharmacological screening for the protection of seizures, behavioral study
and CNS activity. Compound 1-[1-(2,4-dihydroxyphenyl)-3-(2-
hydroxyphenyl)allylidene]-4-(2-fluorophenyl) semicarbazide emerged as the
most active prototype molecule in all the models.
Nadeem et al., (2009) reported that synthesis of some new coumarin
incorporated thiazolyl semicarbazones as anticonvulsants27. Several
22
heteroaryl semicarbazones were prepared by the reaction of heteroaryl
hydrazine carboxamide with aryl aldehydes or ketones. Compounds were
tested for anticonvulsant activity utilizing pentylenetetrazole induced seizure
(PTZ) and maximal electroshock seizure (MES) tests at 30, 100 and 300
mg/kg dose levels. Neurotoxicity of the compounds was also assessed at the
same dose levels. Three compounds of the series, exhibited significant
anticonvulsant activity at 30 mg/kg dose level comparable to the standard
drug, phenytoin.
Navneet et al., (2009) reported that anticonvulsant and neurotoxicity
evaluation of some N4 phenyl substituted pyridyl semicarbazones28. A series
of 4-aryl substituted semicarbazones of pyridyl carbaldehyde and pyridyl
methyl ketone were designed and synthesized to meet the structural
requirements essential for anticonvulsant activity. All the compounds were
evaluated for neurotoxicity and anticonvulsant activity by maximal
electroshock (MES) and subcutaneous metrazol (ScMet) induced seizure
methods and minimal motor impairment was determined by rotorod test.
Majority of compounds exhibited significant anticonvulsant activity after
intraperitoneal administration. Some of them also showed good activity after
oral administration. In this study (Methyl-4- pyridyl) ketone -N4- (p- chloro
phenyl) substituted semicarbazones emerged as most active derivative
showing activity at 100 mg/kg in both the test with prolonged duration of
action. In the present study semicarbazones of pyridyl containing carbonyl
compounds emerges as the lead molecule, showing broad spectrum of
activity with low neurotoxicity and prolong duration of action on oral
administration.
Thirumurugan et al., (2006) reported that 2,4-dimethoxyphenylsemi-
carbazones with anticonvulsant activity against three animal models of
seizures: Synthesis and pharmacological evaluation29. Various 2,4-
dimethoxyphenylsemicarbazones were synthesized starting from 2,4-
dimethoxyaniline via a phenylcarbamate intermediate and characterized. The
anticonvulsant activity of the synthesized compounds was established after
23
intraperitoneal administration in three seizure models in mice which include
maximal electroshock seizure, subcutaneous pentylenetetrazole, and
subcutaneous strychnine-induced seizure screens. Nine compounds exhibited
protection in all the three seizure models, and N1-(2,4-dimethoxyphenyl)-N4-
(propan-2-one) semicarbazone emerged as the most active compound with
no neurotoxicity. These compounds were found to elevate c-aminobutyric
acid (GABA) levels in the midbrain and medulla oblongata regions
equipotent to clobazam.
Yogeeswari et al., (2006) reported that synthesis of N4-(2,4-dimethylphenyl)
semicarbazones as 4-aminobutyrate aminotransferase inhibitors30. Several
2,4-dimethylphenyl substituted semicarbazones were synthesized in three
steps involving aryl urea and aryl semicarbazide formation and
characterized. All the compounds were evaluated for anticonvulsant activity
by using a series of test models, including maximal electroshock seizure,
subcutaneous pentylenetetrazole and subcutaneous strychnine seizure
threshold tests. The compounds were also evaluated for behavioural
impairement and depression activity. In the neurochemical investigation,
potent compounds were evaluated for their effects on rat brain γ-
aminobutyric acid (GABA) levels and in vitro γ -aminobutyrate transaminase
(Pseudomonas fluorescens) activity. Preliminary studies suggest that these
compounds exhibit anticonvulsant activity via a GABA-mediated
mechanism.
Yogeeswari et al., (2005) reported that discovery of N-(2,6-dimethylphenyl)-
substituted semicarbazones as anticonvulsants: Hybrid Pharmacophore-
Based Design31. In the present work various N4-(2,6-dimethylphenyl)
semicarbazones were designed as pharmacophore hybrids between the aryl
semicarbazones and ameltolide. A three-dimensional four-point
pharmacophore model was developed for anticonvulsants, and the title
compounds were found to match with ralitoline. All of the compounds
exhibited anticonvulsant activity in the maximal electroshock test when
administered by both intraperitoneal and oral routes. Compound N1-(2,6-
24
dimethylphenyl)-N4-(2-hydroxybenzaldehyde) semicarbazone emerged as a
prototype with wide spectrum anticonvulsant agent active in five models of
seizure with no neurotoxicity and hepatotoxicity which increased the 4-
aminobutyric acid (GABA) level by 118% and inhibited the GABA
transaminase enzyme both in vitro and ex vivo.
Navneet et al., (2005) reported that synthesis and evaluation of 4-substituted
semicarbazones of levulinic acid for anticonvulsant activity32. A series of 4-
aryl substituted semicarbazones of levulinic acid (4-oxo pentanoic acid) was
designed and synthesized to meet the structural requirements essential for
anticonvulsant activity. All the compounds were evaluated for anticonvulsant
activity by maximal electroshock (MES) and subcutaneous metrazol (ScMet)
induced seizure methods and minimal motor impairment was determined by
rotorod test. A majority of the compounds exhibited significant
anticonvulsant activity after intraperitoneal administration. In the present
study 4-(4′-fluoro phenyl) levulinic acid semicarbazone emerged as the most
active molecule, showing broad spectrum of activity with low neurotoxicity.
Unsubstituted levulinic acid semicarbazone was found to be inactive in all
the screens. The results obtained validate the hypothesis that presence of an
aryl group near the semicarbazone moiety is essential for anticonvulsant
activity. The results also indicate that the hydrophilic-hydrophobic site can
accommodate hydrophilic groups.
Yogeeswari et al., (2004) reported that 4-sulphamoylphenyl semicarbazones
with anticonvulsant activity33. A series of 4-sulphamoylphenyl
semicarbazone derivatives were prepared starting from sulphanilamide and
screened for anticonvulsant activity. The results indicated that greater
protection was obtained in the maximal electroshock screen (MES) and
subcutaneous strychnine (scSTY) than the subcutaneous pentylenetetrazole
(scPTZ) tests. All the compounds showed low neurotoxicity when compared
to the clinically used drugs. Compounds with substituted acetophenone
showed good activity in the rat oral MES screen. Seven compounds
exhibited anticonvulsant activity greater than sodium valproate.
25
Navneet et al., (2004) reported that synthesis of 4-aryl substituted
semicarbazones of some terpenes as novel anticonvulsants34. A series of 4-
aryl substituted semicarbazones of citral and R- (-) carvone were designed
and synthesized to meet the structural requirements essential for
anticonvulsant activity. Seventy two percent of the compounds exhibited
protection in ScMet test. Some of them also showed good activity after oral
administration. The results showed that anticonvulsants with cyclic and
acyclic terpenoid moiety retain activity in MES as well as ScMet test. The p-
fluoro aryl substituted semicarbazones emerged as the most active analogue
in both cyclic and acyclic terpenes. Semicarbazones with terpenoid as the
lipophilic moiety resulted in compounds with broad spectrum of
anticonvulsant activity. The results also validated pharmacophore model with
four binding sites essential for anticonvulsant activity.
Yogeeswari et al., (2004) reported that 3-chloro-2-methylphenyl-substituted
semicarbazones: Synthesis and anticonvulsant activity35. A series of 3-
chloro-2-methylphenyl substituted semicarbazones was synthesized and
evaluated for anticonvulsant and CNS activities. The aryl urea and the
semicarbazide showed anticonvulsant activity in the MES and scPTZ screens
with acute neurotoxicity, whereas the semicarbazone derivatives showed
good anticonvulsant potency in the scSTY screen with moderate activity
against MES and scPTZ screens. Some title compounds exhibited lesser
CNS depression and neurotoxicity compared to phenytoin or carbamazepine
as was evident from the CNS studies.
Pandeya et al., (2000) reported that synthesis and anticonvulsant activity of
4-bromophenyl substituted aryl semicarbazones36. A number of 4-
bromophenyl semicarbazones were synthesized and evaluated for
anticonvulsant and sedative–hypnotic activities. All the compounds showed
anticonvulsant activity in one or more test models. Three compounds showed
greater protection than sodium valproate. The essential structural features
responsible for interaction with receptor site are established within a
suggested pharmacophore.
26
Ramanan et al., (1998) reported that anticonvulsant activity of various aryl,
arylidene and aryloxyaryl semicarbazones37. A number of aryl, arylidene and
aryloxyaryl semicarbazones were evaluated as anticonvulsants. In particular,
insertion of an olefinic group between the carbimino carbon atom and an aryl
ring (referred to as the proximal ring) led to a series in which there was
retention in activity. At the doses utilized, neurotoxicity was absent in these
compounds when given orally to rats. Attachment of a 2-naphthyloxy group
at 4 position of the proximal ring gave a compound whose high activity in
the rat oral maximal electroshock (MES) screen suggested that the binding
site of the second aryl ring was capable of accommodating groups with
molecular refractivity values of over 40. The greatest activity was displayed
by a series of aryloxyaryl semicarbazones which had oral activity in the MES
screen substantially greater than phenytoin and with protection indices of
over 100. A binding site hypothesis formulated as a result of the biodata
generated was in accord with the information obtained by X-ray
crystallography.
Dimmock et al., (1995) reported that some aryl semicarbazones possessing
anticonvulsant activity38. A number of aryl semicarbazones displayed
anticonvulsant activity in the maximal electroshock (MES) and subcutaneous
pentylenetetrazole (scPTZ) screens when administered intraperitoneally to
mice. When given by the oral route to rats, protection was afforded in the
MES but not scPTZ tests. Correlations were noted between the σ and σ*
values of the aryl substituents, the interplanar angles made by the aryl rings
with the adjacent carbimino groups and the shapes of certain semicarbazones
determined by X-ray crystallography, and the activities in the rat oral MES
screen. Molecular modeling studies revealed a number of statistically
significant descriptors which contributed to anticonvulsant activity.
Dimmock et al., (1995) reported that evaluation of the semicarbazones,
thiosemicarbazones and bis-carbohydrazones of some aryl alicycylic ketones
for anticonvulsant and other biological properties39. A number of aryl
alicyclic ketones were converted to their corresponding semicarbazones,
27
thiosemicarbazones and bis-carbohydrazones. Anticonvulsant activity was
displayed by most of the compounds in the maximal electroshock (MES) and
subcutaneous pentylenetetrazole (scPTZ) screens when given
intraperitoneally to mice. However, on oral administration to rats, a marked
selective activity in the MES screen only was noted. X-ray crystallography
on five semicarbazones was undertaken in order to find correlations between
the shapes of these molecules and anticonvulsant properties. The
thiosemicarbazones displayed greater cytotoxicity to P388D1 and L1210
cells than the semicarbazones while a number of human tumors and different
viruses were, in general, insensitive to representative compounds
Smitha et al., (2008) reported that anticonvulsant and sedative-hypnotic
activities of N-acetyl / methyl isatin derivatives40. A series of N-
methyl/acetyl 5-(un)-substituted isatin-3-semicarbazones were screened for
anticonvulsant and sedative-hypnotic activities. The results revealed that
protection was obtained in Maximal electroshock (MES), subcutaneous
pentylene tetrazole (scPTZ) and subcutaneous strychnine (scSTY) screens.
Three compounds possessed anti-MES activity and all the compounds were
less neurotoxic than phenytoin, carbamazepine and phenobarbital. All the
compounds were completely non-toxic at 4h when compared to phenytoin,
carbamazepine and phenobarbital, which were toxic at 100 and 300 mg/kg
respectively. Selected compounds were evaluated for quantification studies
in MES, scPTZ and neurotoxicity screens after i. p and oral administration in
rats.
Krishan et al., (2009) reported that synthesis and pharmacological activity of
some substituted menthone semicarbazone and thiosemicarbazone
derivatives41. A series of Menthone semicarbazone and thiosemicarbazone
derivatives were synthesized and characterized by their spectral data and
screened for anticonvulsant and analgesic activity. Compounds investigated
showed significant anticonvulsant and analgesic activity.
28
Rocha et al., (2006) reported that antinociceptive, antiedematogenic and
antiangiogenic effects of benzaldehyde semicarbazone42. Semicarbazones
induce an anticonvulsant effect in different experimental models. As some
anticonvulsant drugs also have anti-inflammatory activity, the effects of
benzaldehyde semicarbazone (BS) on models of nociception, edema and
angiogenesis were investigated. BS (10, 25 or 50mg/kg, i.p.) markedly
inhibited the second phase of nociceptive response induced by formaldehyde
(0.34%, 20μl) in mice, but only the highest dose inhibited the first phase of
this response. The thermal hyperalgesia and mechanical allodynia induced by
carrageenan (1%, 50μl, i.pl.) in rats were also inhibited by BS (50mg/kg,
i.p.). However, treatment of mice with BS did not induce an antinociceptive
effect in the hot-plate model. The paw edema induced by carrageenan (1%,
50μl, i.pl.) in rats was inhibited by BS (25 or 50 mg/kg, i.p.). Treatment of
mice with BS (0.25, 0.5 or 2.5mg/kg/day, i.p., 7 days) also inhibited
angiogenesis induced by subcutaneous implantation of a sponge disc. It is
unlikely that the antinociceptive effect induced by BS results from motor
incoordination or a muscle relaxing effect, as the mice treated with this drug
displayed no behavioral impairment in the rotarod apparatus. The results
concluded that BS possesses antinociceptive, antiedematogenic and
antiangiogenic activities.
Manmohan et al., (2010) reported that evaluation of anti-phlogistic activity
of synthesized chalconesemicarbazone derivatives43. A series of
chalconesemicarbazones was synthesized and evaluated for their anti-
phlogistic activities. Most of the compounds were found to be potent anti-
phlogistic agent in formalin induced paw edema and cotton pellet induced
granuloma in rats. Based on the results, 1-[1-(2-hydroxyphenyl)-3-(2-
hydroxyphenyl)allylidene]-4-(2-methylphenyl) semicarbazide was the most
active compound. It was found that hydroxyl substituted
chalconesemicarbazones were potent antiphlogistic agents and unsubstituted
compound 1-[1-(2-hydroxyphenyl)-3-phenylallylidene]-4-(2-methylphenyl)
29
semicarbazide and 1-[1-(2-hydroxyphenyl)-3-phenyl allylidene]-4-(4-
methylphenyl)semicarbazide showed very less activity.
Bernard et al., (1995) reported that selective and potent monoamine oxidase
type B inhibitors: substituted semicarbazones and acylhydrazones of
aromatic aldehydes and ketones44. The synthesis and the evaluation of the
monoamine oxidase A and B inhibitory activities of 21 new substituted
acylhydrazones of various aromatic aldehydes and 4-
(benzyloxy)acetophenone, and four substituted semicarbazones of
benzaldehyde and 4-(benzyloxy)benzaldehyde, are described. The 4-
(benzyloxy)phenyl group contributing to a high lipophilicity led to the most
active compounds. One of these, compound (IC50 = 3 nM, MAO A/MAO B
selectivity > 33000), was found to act as a reversible and probably tight-
binding inhibitor. The studied acyclic hydrazones and semicarbazones are
structurally related to other reversible and potent inhibitors, eg, heterocyclic
compounds such as 1,3,4-oxadiazol-2(3H)-one derivatives in which the
hydrazono group is intracyclic. Some of these new inhibitors might find use
in the symptomatic treatment of neurodegenerative processes.
Shebl et al., (2010) reported that ligational behavior of thiosemicarbazone,
semicarbazone and thiocarbohydrazone ligands towards VO(IV), Ce(III),
Th(IV) and UO2(VI) ions: Synthesis, structural characterization and
biological studies45. Mono- and binuclear VO(IV), Ce(III), Th(IV) and
UO(2)(VI) complexes of thiosemicarbazone, semicarbazone and
thiocarbohydrazone ligands derived from 4,6-diacetylresorcinol were
synthesized and characterized. The antibacterial and antifungal activities
were also tested against Rhizobium bacteria and Fusarium-Oxysporium
fungus. The metal complexes of H(4)L(1) ligand showed a higher
antibacterial effect than the free ligand while the other ligands (H(4)L(2) and
H(3)L(3)) showed a higher effect than their metal complexes. The antifungal
effect of all metal complexes is lower than the free ligands.
30
Maria et al., (2010) reported that evaluation of the antimicrobial activity of
some chloro complexes of imidazole-2-carbaldehyde semicarbazone: X-ray
crystal structure of cis-NiCl2(H2L)(H2O) 46. Some first row transition metal
(II) complexes of imidazole-2-carbaldehyde semicarbazone (H2L) have been
synthesized and characterized. The non-deprotonated semicarbazone ligand
behaves as an N,N,O-donor, through the imidazole and imine N atoms and
the O keto atom. The coordinative behaviour of H2L in CuCl2(H2L)(H2O),
ZnCl2(H2L)2·0.5EtOH and CoCl2(H2L)2·0.5H2O is reported as only N,O-
donor. The antimicrobial activity of the semicarbazone ligand and its metal
complexes has been tested against some representative bacteria and fungi. A
moderate inhibitory activity of the cobalt complex was detected towards the
assayed phytopathogenic fungi Alternaria tenuis and Sclerotinia minor (MIC
50 μg/mL).
Noriko et al., (2006) reported that Syntheses, crystal structures and
antimicrobial activities of 6-coordinate antimony(III) complexes with
tridentate 2-acetylpyridine thiosemicarbazone, bis(thiosemicarbazone) and
semicarbazone ligands47. Five novel antimony(III) complexes with the
mono- and bis(thiosemicarbazone) ligands of 2N1S or 4N2S donor atoms,
N'-[1-(2-pyridyl)ethylidene]morpholine-4-carbothiohydrazide (Hmtsc, L1)
and bis[N'-[1-(2-pyridyl)ethylidene]]-1,4-piperazinedicarbothiohydrazide
(H(2)ptsc, L7), and the tridentate semicarbazone ligand of 2N1O donor
atoms, 2-acetylpyridine semicarbazone (Hasc, L2b), were prepared by
reactions of SbCl(3) or SbBr(3), and characterized. Water-soluble
antimony(III) complexes showed moderate antimicrobial activities against
Gram-positive (Bacillus subtilis and Staphylococcus aureus) and -negative
bacteria (Escherichia coli and Pseudomonas aeruginosa), yeasts (Candida
albicans and Saccharomyces cerevisiae) and molds (Aspergillus niger and
Penicillium citrinum).
Kenji et al., (2004) reported that Syntheses, crystal structures and
antimicrobial activities of monomeric 8-coordinate, and dimeric and
monomeric 7-coordinate bismuth (III) complexes with tridentate and
31
pentadentate thiosemicarbazones and pentadentate semicarbazone ligands48.
Novel bismuth(III) complexes 1-4 with the tridentate thiosemicarbazone
ligand of 2N1S donor atoms [Hmtsc (L1); 2-acetylpyridine (4N-morpholyl
thiosemicarbazone)], the pentadentate double-armed thiosemicarbazone
ligand of 3N2S donor atoms [H2dmtsc (L3); 2,6-diacetylpyridine bis(4N-
morpholyl thiosemicarbazone)] and the pentadentate double-armed
semicarbazone ligand of 3N2O donor atoms [H2dasc (L4b); 2,6-
diacetylpyridine bis(semicarbazone)], were prepared and characterized.
Bismuth(III) complexes showed selective and effective antibacterial
activities against Gram-positive bacteria.
Noriko et al., (2003) reported that synthesis, structural characterization and
antimicrobial activities of 12 zinc(II) complexes with four thiosemicarbazone
and two semicarbazone ligands49. Twelve zinc(II) complexes with
thiosemicarbazone and semicarbazone ligands were prepared and
characterized. Their antimicrobial activities were evaluated by MIC against
four bacteria (B. subtilis, S. aureus, E. coli and P. aeruginosa), two yeasts
(C. albicans and S. cerevisiae) and two molds (A. niger and P. citrinum). The
zinc(II) complexes with 4N-substituted ligands showed no antimicrobial
activities. In contrast to the previously reported nickel(II) complexes,
properties of the ligands such as the ability to form hydrogen bonding with a
counter anion or hydrated water molecules or the less bulkiness of the 4N
moiety would be a more important factor for antimicrobial activities than the
coordination number of the metal ion for the zinc(II) complexes.
Hemalatha et al., (2008) reported that synthesis, antibacterial and antifungal
activities of some N-nitroso-2,6-diarylpiperidin-4-one semicarbazones and
QSAR analysis50. A series of N-nitroso-2,6-diarylpiperidin-4-one
semicarbazones and thiosemicarbazones were synthesized and characterized.
All the compounds were screened for their antibacterial activity against
Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus and Gram-
negative bacteria Escherichia coli and fungi Candida albicans. These
compounds have showed moderate and very good antibacterial activity.
32
Quantitative Structure Activity Relationship (QSAR) analysis was performed
for these compounds by the application of Semiempirical calculations and
molecular modeling.
Majed (2009) reported that synthesis and antibacterial activity of some
transition metal complexes of oxime, semicarbazone and phenylhydrazone51.
Co, Ni and Cu complexes of oxime, semicarbazone and phenylhydrazone
have been prepared and their antibacterial activity have been studied and
compared with their ligands against E. coli which gave significant results of
activity.
Patel et al., (2010) reported that synthesis, characterization and chelating
properties of 4-butyrylsemicarbazone-1-phenyl-3-methyl-2-pyrazolin-5-
one52. 4-butyrylsemicarbazone-1-phenyl-3-methyl-2-pyrazolin-5-one and its
metal chelates of Cu2+, Ni2+, Co2+, Mn2+, Fe2+, Fe3+,Cr3+, UO2 and OV were
prepared and characterized. The compounds also were screened for their
antimicrobial activity.
Yang et al., (2010) reported that synthesis and antiviral activity of
phthiobuzone analogues53. A series of phthiobuzone analogs, prepared from
potassium phthalimide or phthalandione, have been evaluated for their
antiviral activities. Among the candidates, compounds which contain the
substituted 4-halogenated phenyl ring at N-4’,4” position, show more potent
antiviral activity than phthiobuzone against herpes simplex virus 1 and
herpes simplex virus 2. Compounds with a propylene linker between the
phthalimide and bisthiosemicarbazone moieties display similar antiviral
potency against herpes simplex virus 1.
Joanna et al., (2010) reported that organotin compound derived from 3-
hydroxy-2-formylpyridine semicarbazone: Synthesis, crystal structure, and
antiproliferative activity54. The novel diphenyltin (IV) compound
[Ph2(HyFoSc)Sn], where H2HyFoSc is 3-hydroxy-2-formylpyridine
semicarbazone, was prepared and characterized. Compounds have been
33
evaluated for antiproliferative activity in vitro against the cells of three
human tumor cell lines: MCF-7 (human breast cancer cell line), T24 (bladder
cancer cell line), A549 (nonsmall cell lung carcinoma), and a mouse
fibroblast L-929 cancer cell line.
Christian et al., (2007) reported that effect of metal ion complexation and
chalcogen donor identity on the antiproliferative activity of 2-acetylpyridine
N,N-dimethyl(chalcogen) semicarbazones55. Three
chalcogensemicarbazones, viz., 2-acetylpyridine N,N-
dimethylsemicarbazone(HL(1)), 2-acetylpyridine N,N-dimethyl thio
semicarbazone(HL(2)) and 2-acetylpyridine N,N-
dimethylselenosemicarbazone (HL(3)), their corresponding gallium(III)
complexes [Ga(L(1-3))(2)]PF(6) and the ruthenium(III) compound
[Ru(L(2))(2)]PF(6) have been prepared and characterized in order to
elucidate the effect of metal ion complexation and chalcogen donor identity
on the cytotoxicity of chalcogensemicarbazones in two human tumour cell
lines 41M (ovarian carcinoma) and SK-BR-3 (mammary carcinoma).
Amir et al., (2009) reported that evaluation of a [67Ga]-thiosemicarbazone
complex as tumor imaging agent56. [67Ga] labeled 2-acetylpyridine 4,4-
dimethylthiosemicarbazone ([67Ga]-[APTSM2]2+) was prepared using freshly
prepared [67Ga]GaCl3 and 2-acetylpyridine 4,4-dimethylthiosemicarbazone.
Stability of the complex was checked in human serum for 37°C. The
biodistribution of the labeled compound in vital organs of normal and
fibrosarcoma bearing mice were compared with that of free Ga3+ cation up to
24h. Initial SPECT images and biodistribution results showed significant
tumor uptake in fibrosarcoma-bearing mice after 2 hour post injection.
Pabla et al., (2005) reported that Vanadium(V) complexes with
salicylaldehyde semicarbazone derivatives bearing in vitro anti-tumor
activity towards kidney tumor cells (TK-10): Crystal structure of [VVO2(5-
bromosalicylaldehyde semicarbazone)]57. New dioxovanadium(V)
semicarbazone complexes, cis-VO(2)L (where L=5-bromosalicylaldehyde
34
semicarbazone and 2-hydroxynaphtalen-1-carboxaldehyde semicarbazones)
have been synthesized and characterized. Results were compared with those
previously reported for other three analogous complexes of this series. The
five complexes were tested in three different human tumor cell lines for
bioactivity as potential anti-tumor agents, showing selective cytotoxicity on
TK-10 cell line. Results showed that structural modifications on the
semicarbazone moiety could have a significant effect on the anti-tumor
activity of the vanadium complexes.
Violeta et al., (2010) reported that synthesis, structural studies and biological
activity of a dioxovanadium(v) complex with pyridoxal semicarbazones58.
Reaction between the NH4VO3 and pyridoxal semicarbazone (PLSC) in a
methanol/ammonia solution forms NH4[VO2(PLSC–2H)] complex in which
vanadium is in the oxidation state +5, and pyridoxal semicarbazone is
coordinated in its dianionic form. The complex was characterized and
evaluated for in vitro cytotoxicity.
Pingaew et al., (2010) reported that synthesis, cytotoxic and antimalarial
activities of benzoyl thiosemicarbazone analogs of isoquinoline and related
compounds59. Thiosemicarbazone analogs of papaveraldine and related
compounds were synthesized and evaluated for cytotoxic and antimalarial
activities. The cytotoxic activity was tested against HuCCA-1, HepG2, A549
and MOLT-3 human cancer cell lines. Thiosemicarbazones displayed
cytotoxicity toward all the tested cell lines. Significantly, N(4)-phenyl-2-
benzoylpyridine thiosemicarbazone exhibited the most potent activity against
HuCCA-1, HepG2, A549 and MOLT-3 cell lines with IC50 values of 0.03,
4.75, 0.04 and 0.004 μg/mL, respectively. In addition, 2-benzoylpyridine
thiosemicarbazones showed antimalarial activity against Plasmodium
falciparum.
Pavan et al., (2010) reported that thiosemicarbazones, semicarbazones,
dithiocarbazates and hydrazide/hydrazones: Anti – mycobacterium
tuberculosis activity and cytotoxicity60. This study was aimed to identify a
35
candidate drug for the development of anti-tuberculosis therapy from
previously synthesized compounds based on the thiosemicarbazones,
semicarbazones, dithiocarbazates and hydrazide/hydrazones compounds. The
minimal inhibitory concentration (MIC) of these compounds against
Mycobacterium tuberculosis was determined and their in-vitro cytotoxicity
to J774 cells (IC<sub>50</sub>) was determined to establish a selectivity
index (SI) (SI = IC<sub>50</sub>/MIC). The results are comparable to or
better than those of "first line" or "second line" drugs commonly used to treat
TB, suggesting these compounds as anti-TB drug candidates.
Al et al., (2010) reported that Synthesis of some new 4(3H)-quinazolinone-2-
carboxaldehyde thiosemicarbazones and their metal complexes and a study
on their anticonvulsant,analgesic,cytotoxic and antimicrobial activities61.
Novel 3-aryl-4(3H)-quinazolinone-2-carboxaldehydes and their
corresponding schiff's base and thio-semicarbazone derivatives were
synthesized from the starting 5-iodo anthranilic acid. Copper(II),zinc(II)
complexes of some thiosemicarbazone derivatives were also synthesized,
characterized and screened for some selected compounds to probe their
potential anticonvulsant, analgesic, cytotoxic as well as their antimicrobial
activities.
Sriram et al., (2004) reported that antituberculous activity of some aryl
semicarbazone derivatives62. N1-(4-acetamido phenyl)-N4-(2-nitro
benzylidene) semicarbazones was synthesized and inhibited in vitro
Mycobacterium tuberculosis H(37)Rv; 100% inhibition at 1.56 microg/mL.
This newly synthesized aryl semicarbazones are reported as first of its kind
to possess antimycobacterials potency greater than p-aminosalicylic acid,
ethionamide, ethambutol, ciprofloxacin and kanamycin.
Julio et al., (2009) reported that design of vanadium mixed-ligand complexes
as potential anti-protozoa agents63. In the search for new therapeutic tools
against Chagas' disease (American Trypanosomiasis) four novel mixed-
ligand vanadyl complexes, [V(IV)O(L(2)-2H)(L(1))], including a bidentate
36
polypyridyl DNA intercalator (L(1)) and a tridentate salycylaldehyde
semicarbazone derivative (L(2)) as ligands were synthesized, characterized
by a combination of techniques, and in vitro evaluated. Data obtained by
electrophoretic analysis suggest that the mechanism of action of these
complexes could include DNA interactions.
Xiaohui et al., (2002) reported that Synthesis and structure-activity
relationship study of potent trypanocidal thio semicarbazone inhibitors of the
trypanosomal cysteine protease cruzain64. A novel series of potent
thiosemicarbazone small-molecule inhibitors of the Trypanosoma cruzi
cysteine protease cruzain have been identified. Some of these inhibitors have
been shown to be trypanocidal. We initially discovered that 3¢-
bromopropiophenone thio semicarbazone inhibited cruzain and could cure
mammalian cell cultures infected with T. cruzi. 3¢-Bromopropiophenone
thio semicarbazones showed no toxicity for mammalian cells at
concentrations that were trypanocidal. Following this lead, more than 100
compounds were designed and synthesized. A specific structure activity
relationship was established, and many potent analogues with IC50 values in
the low nanomolar range were identified. Eight additional analogues were
trypanocidal in a cell culture assay, and this indicates that aryl thio
semicarbazone is a productive scaffold for killing the parasites. Kinetic
studies show that these are time-dependent inhibitors. Molecular modeling
studies of the enzyme-inhibitor complex have led to a proposed mechanism
of interaction as well as insight into the SAR of the thio semicarbazone
series. The nonpeptide nature of this series, small size, and extremely low
cost of production suggest this is a promising direction for the development
of new antitrypanosome chemotherapy.
Hugo et al., (2000) reported that synthesis and anti-trypanosomal evaluation
of E-isomers of 5-nitro-2-furaldehyde and 5-nitrothiophene-2-
carboxaldehyde semicarbazone derivatives; Structure–activity
relationships65. Several novel semicarbazone derivatives were prepared from
5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde and
37
semicarbazides bearing a spermidine-mimetic moiety. All derivatives
presented the E-configuration, as determined by NMR-NOE experiments.
These compounds were tested in vitro as potential antitrypanosomal agents,
and some of them, together with the parent compounds, 5-nitro-2-
furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone
derivatives, were also evaluated in vivo using infected mice. Structure-
activity relationship studies were carried out using voltammetric response
and lipophilic-hydrophilic balance as parameters. Two of the compounds
displayed the highest in vivo activity. A correlation was found between
lipophilic-hydrophilic properties and trypanocidal activity, high R(M) values
being associated with low in vivo effects.
Hugo et al., (1998) reported that synthesis and anti-trypanosomal activity of
novel 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde
semicarbazone derivatives66. Several novel semicarbazones derivatives were
prepared from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde,
and tested in vitro as potential anti-trypanosomal agents. Some derivatives
were found to be active against Trypanosoma cruzi with an activity similar to
that of Nifurtimox.
Safavi et al., (2010) reported that Complexes of 2-hydroxyacetophenone
semicarbazones: A novel series of superoxide dismutase mimetics67. A series
of copper(II) and zinc complexes of 2-hydroxyacetophenone semicarbazones
have been prepared and evaluated as superoxide dismutase (SOD) mimetics.
The SOD-like activity of parent ligands and complexes were determined by
the inhibition of nitroblue tetrazolium (NBT) reduction method, using
xanthine/xanthine oxidase as the superoxide radical generator. The obtained
results indicate that Cu(II) complexes exhibited the most potent SOD-like
activities. Among copper complexes, 2-hydroxy-4-methoxyacetophenone
semicarbazone analog was the most active compounds.
Omar et al., (1992) reported that synthesis and evaluation for uterotrophic
and antiimplantation activities of 2-substituted estradiol derivatives68. Two
38
novel series of 2-substituted estradiol derivatives have been synthesized and
evaluated for uterotrophic and antiimplantation activities. Among the
compounds tested in the rat, 2-acetylestradiol 17β-acetate, 2-(3'-
dimethylamino-1'-propionyl)estradiol 3,17β-diacetate, 2-(3'-diethylamino-1 '-
propionyl)estradiol 3,17β-diacetate, 2-(3'-piperidino-1'-propionyl)estradiol
3,17β-diacetate, 1'-(2-estradiol 3,17β-diacetate)-3'-diethylaminopropionyl
thiosemicarbazone, and 1'-(2-estradiol 3,17β-diacetate)-3'-
morpholinopropionyl thiosemicarbazone displayed estrogenic activity. At
dosages of 4μg/rat/day, none of the tested compounds elicited
antiimplantation activity. All compounds shared a similar characteristic:
nuclear substitution at the C-2 position of the steroid nucleus, a property
previously thought to be markedly inhibitory for estrogenic activity.
Jose et al., (2008) reported that Molecular modeling optimization of
anticoagulant pyridine derivatives69. Eleven pyridine derivatives (oximes,
semicarbazones, N-oxides) were synthesized and tested as anticoagulants on
pooled normal plasma using the prothrombin time (PT) protocol. The best
anticoagulant within the oxime series was compound AF4, within the oxime
N-oxide series was compound AF4-N-oxide, and within the semicarbazone
series, compound MD1-30Y. Molecular modeling approach found that there
was good correlation between coagulation data and computational energy
scores.
Wu et al., (2006) reported that discovery and synthesis of tetrahydroindolone
derived semicarbazones as selective Kv1.5 blockers70. A novel class of
tetrahydroindolone-derived semicarbazones has been discovered as potent
Kv1.5 blockers. In in vitro studies, several compounds exhibited very good
potency for blockade of Kv1.5.
Pabla et al., (2004) reported that new vanadium (V) complexes with
salicylaldehyde semicarbazone derivatives: synthesis, characterization, and
in vitro insulin-mimetic activity - crystal structure of [VvO2 (salicylaldehyde
semicarbazone)]71. The new dioxo(semicarbazone)vanadium(V) complexes
39
cis-VO2L (where L = salicylaldehyde semicarbazone (L1), salicylaldehyde 4-
n-butylsemicarbazone (L2), or salicylaldehyde 4-(2-naphthyl)semicarbazone
(L3)) have been synthesized, characterized and tested for bioactivity as
potential insulin-mimetic agents. All dioxovanadium(V) complexes
exhibited essentially no in vitro insulin-mimetic activity, but the VO2L2
complex developed activity in the presence of ascorbic acid, similar to that of
vanadyl sulfate.
Anwar et al., (2003) reported that Actions of benzaldehyde hydrazones and
semicarbazones on biogenic amine receptors in the silkworm Bombyx mori72.
Four hydrazones (HZs) and six semicarbazones (SCZs) of substituted
benzaldehydes were synthesized and examined for their ability to control
insect adenylate cyclase through their interaction with biogenic amine
receptors. Among the compounds synthesized, two with a hydroxyl group at
the 4-position of the phenyl moiety, HZ-01 and SCZ-03, were found to
reduce the basal levels of cAMP in head membrane homogenates of fifth
instar larvae of the silkworm Bombyx mori. The semicarbazone SCZ-03
dose-dependently attenuated not only basal but also forskolin-stimulated
cAMP levels. Tyramine (TYR) and dopamine (DPM) also produced a dose-
dependent reduction in basal cAMP levels. DPM and TYR receptor
antagonists abolished the attenuating effects of SCZ-03. These findings
suggest that SCZ-03 acts as a non-selective agonist for DPM and TYR
receptors to inactivate adenylate cyclase in B. mori larvae.
40
CCHHAAPPTTEERR IIIIII
AIM AND OBJECTIVE OF THE WORK
Vanillins are of considerable pharmacological interest since a number of
derivatives have been reported to possess various biological activities.
Semicarbazones have proved the efficiency and efficacy in combating various
diseases. It is of great interest because of their chemistry and potentially beneficial
biological activities such as antibacterial, antifungal, antiviral, cytotoxic,
antimalarial antinociceptive, anticonvulsant, antiarrhythmic and insulin-mimic
activities. A wide variety of compounds related to aniline were tested as lipid-
lowering agents some five decades ago when association between heart disease and
hyperlipidemia was established.
In continuation of the earlier work on semicarbazones derivatives and above
observation prompted me to synthesize the novel vanillin semicarbazones since
there is no extensive and individual scientific reports are available for the
incorporation of vanillin into aryl substituted semicarbazides to synthesis vanillin
semicarbazones which are apt to form up to 4 pivotal hydrogen bonds with serine,
tyrosine and histidine of the PPARα and PPARγ which is essential for anti diabetic
activity associated with lipid lowering effect
Based upon the hypothesis, this modification would enhance the efficacy of
antidiabetic potential associated with hypolipidemic activity.
This present thesis embarks on the following objectives:
First objective is to study the molecular docking of human PPAR with
X-ray crystallography.
As per the outcome of the docking studies it proposed to synthesis of
vanillin semicarbazones derivatives by involving the following three
steps (a) Synthesis of aryl carbamates, (b) Synthesis of aryl
semicarbazides and (c) Synthesis of semicarbazones of vanillin.
41
Physical Characterization of title compounds
All the titled compounds will be subjected to physical characterization
such as melting point and Rf value
All the titled compounds will be subjected to various analytical
techniques such as IR, 1H NMR and MASS spectral studies
Pharmacological Screening of title compounds
a. Acute toxicity study
b. Anti-diabetic potential study
c. Hypocholestremic study
42
CCHHAAPPTTEERR IIVV
MOLECULAR DOCKING STUDIES
4.1 INTRODUCTION
The process of finding novel leads for a new target is the most important and
undoubtedly one of the most crucial steps in identifying a drug and its development
program73. Most drugs act at a specific site such as an enzyme or receptor.
Compounds with similar structures often tend to have similar pharmacological
activity. However, they usually exhibit differences in potency, unwanted side effects
and in some cases different activities. The study of the structure-activity
relationships of a lead compound and its analogues can be used to determine the
parts of the structure of the lead that are responsible for its biological activity
(Pharmacophore).
The pharmacophore summarizes the important binding groups which are
required for activity, and their relative positions in space with respect to each other.
In order to identify the 3D pharmacophore, it is necessary to know the active
confirmation of the molecule. There are various ways in which this might be done.
Rigid analogues of the flexible compound could be synthesized and tested to see
whether activity is retained. Alternatively, it may be possible to crystallize the target
with the compound bound to the binding site. X-ray crystallography could be used to
identify the structure of the complex as well as active confirmation of the bound
ligand74.
The number of potential therapeutic target proteins is proliferating rapidly,
making it increasingly important to develop techniques for rapidly discovering and
optimizing novel therapeutic agents for these new targets. Experimental
combinatorial chemistry has provided enormous libraries with millions of potential
ligands quickly accessible for experimental tests to find positive lead compounds
against specific target proteins75. Plasma protein binding of drugs is of great interest
as it influences their pharmacokinetic and pharmacodynamic properties, and may
43
also lead to interference with the binding of other endogenous and/or exogenous
ligands as a result of overlap of binding sites and/or conformational changes76.
Molecular-docking methodologies ultimately seek to predict (or often
retrospectively reproduce) the best mode by which a given compound will fit into a
binding site of a macromolecular target and it has caught the attention of many
pharmaceutical and biotechnology companies eager to discover novel chemical
entities77.
From the past decades, there has been an extensive research focused on
Peroxisome proliferator-activated receptors (PPARs) which are ligand-activated
transcription factors belonging to the nuclear hormone receptor superfamily. There
are three PPAR subtypes, which are the products of distinct genes and are
commonly designated PPARα, PPARγ, and PPARδ78. PPARα is a key factor in fatty
acid metabolism, and is responsible for mediating the lipid-lowering effects of
fibrate drugs (e.g., fenofibrate and gemfibrozil)79. PPARγ is expressed most
abundantly in adipose tissue and mediates the antidiabetic activity of the insulin-
sensitizing drugs belonging to the thiazolidindione80. It plays a pivotal role in
regulating adipogenesis, insulin sensitivity and glucose homeostasis81. The clinical
potential of targeting PPARδ isotype has not been clearly determined and the clinical
potential of targeting this isotype remains to be clearly determined82. Dual-acting
PPARα/γ agonists have been developed and have a very attractive option in the
treatment of dyslipidemic type 2 diabetes83, 84.
The identification of the novel ligand-receptor interaction modalities
represents a new hallmark of the partial agonist action of certain ligands and could
be exploited for the design of new antidiabetic agents appropriately targeting the
PPARs. Some analogues characterized by the presence of a linker, with different
length and stereoelectronic properties, between the aromatic ring system that could
allow the complete occupation of the entire cavity with the aim of evaluating the
effects in terms of potency, efficacy, and subtype selectivity82. Sheraer et al
described that a typical PPAR agonist consists of an acidic head attached to an
aromatic scaffold, a linker, and a hydrophobic tail85. With respect to structural
44
modifications, molecular docking studies were performed to reveal the potency of
the title compounds and to predict the structural requirements for the dual agonist
action of PPARα/γ. For this purpose, the present investigation was aimed to develop
new ligands as vanillin semicarbazones with antidiabetic associated with
hypolipidemic activities on PPARs.
4.2 EXPERIMENTAL
Because the process of finding a novel compound showing bioactivity can be
time-consuming and expensive, structure based drug design has been established as
a vital first step to therapeutic development86. Receptor-based design requires the
availability of the receptor structure, which is used to examine the interactions that
occur with any members of a large database of ligands87.
Docking procedures aim to identify correct poses of ligands in the binding
pocket of a protein and to predict the affinity between the ligand and the protein. In
other words, docking describes a process by which two molecules fit together in a
three-dimensional space. Molecular docking has contributed important proceedings
to drug discovery for many years88. A large number of docking and dynamics
software packages are available for academic research. Each program is slightly
different in terms of its variations in calculation methods and results89.
No single program was deemed the best docking software, but the study
demonstrated that the characteristics of the ligand and the target have a significant
effect on the efficiency of the docking program used90.
In this study, docking software programs such as protein preparation wizard
(Maestro 8.5, Schrodinger, LLC), Marvin sketch-5.0.6.1 (Chemaxon), MGL Tools-
1.4.6 and AutoDock4 (The Scripps Research Institute) have been used. Molecular
docking studies were performed with X-ray crystal structure of human PPARα and
PPARγ using AutoDock4 (The Sripps Research Institute). X-ray crystallographic
models 3G8I for hPPARα and 3HOD for hPPARγ were downloaded from Protein
data bank (www.rcsb.org). Docking procedures were carried out by the method
developed by Agnes et al.91
45
4.2.1 Ligand preparation
Structures of ligands (Compound 01 to Compound 32) were sketched using
Marvin Sketch-5.0.6.1 (Chemaxon), 3D-geometry optimized and saved in PDB
format for AutoDock compatibility. MGLTools-1.4.6 (The Sripps Research
Institute) was used to convert ligand.pdb files to ligand.pdbqt files.
Fig. 4.1 Pharmacophore model of PPAR agonists
(The aromatic centre can be substituted to access additional subpockets in the receptor)
4.2.2 Protein preparation
For each protein target, the system expert selected a representative protein
structure to be used for all docking calculations. The system expert therefore took
special care to select a structure that both was a high-quality structure of good
resolution. X-ray crystal structure of hPPARα (3G8I) and hPPARγ (3HOD) were
downloaded from Protein data bank (www.rcsb.org). Protein preparation wizard
(Maestro 8.5, Schrodinger, and LLC) was used to prepare protein. Through which
hydrogens were added, water molecules were removed, side chains were optimized
for hydrogen bonding and finally energy minimized using OPLS2001 force field.
The energy minimized protein was then saved in PDB format. Using MGLTools-
1.4.6 nonpolar hydrogens were merged, AutoDock atom type AD4 and Gasteiger
charges were assigned and finally saved in protein.pdbqt format.
4.2.3 Docking protocol
Grid parameter file (protein.gpf) and docking parameter files (ligand.dpf)
were written using MGLTools-1.4.6. Receptor grids were generated using 60x60x60
46
grid points in xyz with grid spacing of 0.375 Å. Grid box was centered
cocrystallized ligand. Map types were generated using autogrid4. The ‘Grid
Parameter File’ (protein.gpf) was used for generating map types of compound 13
with 3G8I (PPARα) and compound 31 with 3HOD (PPARγ).
Grid parameter file for generating map types for 3G8I (PPARα) 3G8I.gpf ------------------------------------------------------------------------------------------------------ npts 60 60 60 # num.grid points in xyz gridfld 3G8I_rigid.maps.fld # grid_data_file spacing 0.375 # spacing(A) receptor_types A C HD N NA OA SA # receptor atom types ligand_types A C Cl Br F HD N NA OA S SA # ligand atom types receptor 3G8I_rigid.pdbqt # macromolecule gridcenter 47.81 34.59 34.302 # xyz-coordinates or auto smooth 0.5 # store minimum energy w/in rad(A) map 3G8I_rigid.A.map # atom-specific affinity map map 3G8I_rigid.C.map # atom-specific affinity map map 3G8I_rigid.Cl.map # atom-specific affinity map map 3G8I_rigid.Br.map # atom-specific affinity map map 3G8I_rigid.F.map # atom-specific affinity map map 3G8I_rigid.HD.map # atom-specific affinity map map 3G8I_rigid.N.map # atom-specific affinity map map 3G8I_rigid.NA.map # atom-specific affinity map map 3G8I_rigid.OA.map # atom-specific affinity map map 3G8I_rigid.S.map # atom-specific affinity map map 3G8I_rigid.SA.map # atom-specific affinity map elecmap 3G8I_rigid.e.map # electrostatic potential map dsolvmap 3G8I_rigid.d.map # desolvation potential map dielectric -0.1465 # <0, AD4 distance-dep.diel;>0, constant Grid parameter file for generating map types for 3G8I (PPARα) 3HOD.gpf ------------------------------------------------------------------------------------------------------ npts 60 60 60 # num.grid points in xyz gridfld 3HOD_rigid.maps.fld # grid_data_file spacing 0.375 # spacing(A) receptor_types A C HD N NA OA SA # receptor atom types
47
ligand_types A C Cl Br F HD N NA OA S SA # ligand atom types receptor 3HOD_rigid.pdbqt # macromolecule gridcenter 20.406 66.263 17.626 # xyz-coordinates or auto smooth 0.5 # store minimum energy w/in rad(A) map 3HOD_rigid.A.map # atom-specific affinity map map 3HOD_rigid.C.map # atom-specific affinity map map 3HOD_rigid.Cl.map # atom-specific affinity map map 3HOD_rigid.Br.map # atom-specific affinity map map 3HOD_rigid.F.map # atom-specific affinity map map 3HOD_rigid.HD.map # atom-specific affinity map map 3HOD_rigid.N.map # atom-specific affinity map map 3HOD_rigid.NA.map # atom-specific affinity map map 3HOD_rigid.OA.map # atom-specific affinity map map 3HOD_rigid.S.map # atom-specific affinity map map 3HOD_rigid.SA.map # atom-specific affinity map elecmap 3HOD_rigid.e.map # electrostatic potential map dsolvmap 3HOD_rigid.d.map # desolvation potential map dielectric -0.1465 # <0, AD4 distance-dep.diel;>0, constant
Docking was carried out with default parameters such as number of runs: 50,
population size: 150, number of evaluations: 2500000 and number of generations:
27000, using autodock4. The ‘Docking Parameter File’ (ligand.dpf) was used for
molecular docking of compound 13 with 3G8I (PPARα) and compound 31 with
3HOD (PPARγ).
Docking parameter file for docking compound 13 with 3G8I (PPARα) compound_13.dpf ------------------------------------------------------------------------------------------------------ outlev 1 # diagnostic output level intelec # calculate internal electrostatics seed pid time # seeds for random generator ligand_types A C HD N OA # atoms types in ligand fld 3G8I_rigid.maps.fld # grid_data_file map 3G8I_rigid.A.map # atom-specific affinity map map 3G8I_rigid.C.map # atom-specific affinity map map 3G8I_rigid.HD.map # atom-specific affinity map
48
map 3G8I_rigid.N.map # atom-specific affinity map map 3G8I_rigid.OA.map # atom-specific affinity map elecmap 3G8I_rigid.e.map # electrostatics map desolvmap 3G8I_rigid.d.map # desolvation map move compound_13.pdbqt # small molecule about 2.3782 2.4859 -4.4255 # small molecule center tran0 random # initial coordinates/A or random quat0 random # initial quaternion ndihe 6 # number of active torsions dihe0 random # initial dihedrals (relative) or random tstep 2.0 # translation step/A qstep 50.0 # quaternion step/deg dstep 50.0 # torsion step/deg torsdof 6 0.274000 # torsional degrees of freedom and coefficient rmstol 2.0 # cluster_tolerance/A extnrg 1000.0 # external grid energy e0max 0.0 10000 # max initial energy; max number of retries ga_pop_size 150 # number of individuals in population ga_num_evals 2500000 # maximum number of energy evaluations ga_num_generations 27000 # maximum number of generations ga_elitism 1 # number of top individuals to survive to next generation ga_mutation_rate 0.02 # rate of gene mutation ga_crossover_rate 0.8 # rate of crossover ga_window_size 10 # ga_cauchy_alpha 0.0 # Alpha parameter of Cauchy distribution ga_cauchy_beta 1.0 # Beta parameter Cauchy distribution set_ga # set the above parameters for GA or LGA sw_max_its 300 # iterations of Solis & Wets local search sw_max_succ 4 # consecutive successes before changing rho sw_max_fail 4 # consecutive failures before changing rho sw_rho 1.0 # size of local search space to sample sw_lb_rho 0.01 # lower bound on rho ls_search_freq 0.06 # probability of performing local search on individual set_sw1 # set the above Solis & Wets parameters compute_unbound_extended # compute extended ligand energy ga_run 50 # do this many hybrid GA-LS runs analysis # perform a ranked cluster analysis
49
Docking parameter file for docking compound 31 with 3HOD (PPARγ) compound_31.dpf ------------------------------------------------------------------------------------------------------ outlev 1 # diagnostic output level intelec # calculate internal electrostatics seed pid time # seeds for random generator ligand_types A C HD N NA OA S # atoms types in ligand fld 3HOD_rigid.maps.fld # grid_data_file map 3HOD_rigid.A.map # atom-specific affinity map map 3HOD_rigid.C.map # atom-specific affinity map map 3HOD_rigid.HD.map # atom-specific affinity map map 3HOD_rigid.N.map # atom-specific affinity map map 3HOD_rigid.NA.map # atom-specific affinity map map 3HOD_rigid.OA.map # atom-specific affinity map map 3HOD_rigid.S.map # atom-specific affinity map elecmap 3HOD_rigid.e.map # electrostatics map desolvmap 3HOD_rigid.d.map # desolvation map move compound_31.pdbqt # small molecule about 2.7008 2.6444 -5.1956 # small molecule center tran0 random # initial coordinates/A or random quat0 random # initial quaternion ndihe 7 # number of active torsions dihe0 random # initial dihedrals (relative) or random tstep 2.0 # translation step/A qstep 50.0 # quaternion step/deg dstep 50.0 # torsion step/deg torsdof 7 0.274000 # torsional degrees of freedom and coefficient rmstol 2.0 # cluster_tolerance/A extnrg 1000.0 # external grid energy e0max 0.0 10000 # max initial energy; max number of retries ga_pop_size 150 # number of individuals in population ga_num_evals 2500000 # maximum number of energy evaluations ga_num_generations 27000 # maximum number of generations ga_elitism 1 # number of top individuals to survive to next generation ga_mutation_rate 0.02 # rate of gene mutation ga_crossover_rate 0.8 # rate of crossover
50
ga_window_size 10 # ga_cauchy_alpha 0.0 # Alpha parameter of Cauchy distribution ga_cauchy_beta 1.0 # Beta parameter Cauchy distribution set_ga # set the above parameters for GA or LGA sw_max_its 300 # iterations of Solis & Wets local search sw_max_succ 4 # consecutive successes before changing rho sw_max_fail 4 # consecutive failures before changing rho sw_rho 1.0 # size of local search space to sample sw_lb_rho 0.01 # lower bound on rho ls_search_freq 0.06 # probability of performing local search on individual set_sw1 # set the above Solis & Wets parameters compute_unbound_extended # compute extended ligand energy ga_run 50 # do this many hybrid GA-LS runs analysis # perform a ranked cluster analysis
51
Table 4.1. Compounds with their Docking scores
Code R
PPAR-δ 3HOD
PPAR-α 3G8I
Estimated Free energy of binding (Kcal/mol)
Estimated Ki
(μM)
Estimated Free energy of binding (Kcal/mol)
Estimated Ki
(μM)
Compound_01 H -6.93 8.33 -6.84 9.63 Compound_02 2-Cl -6.65 13.42 -7.07 6.60 Compound_03 3-Cl -7.66 2.42 -7.23 4.98 Compound_04 4-Cl -7.37 3.93 -7.37 3.93 Compound_05 2-Br -7.35 4.10 -7.34 4.17 Compound_06 3-Br -7.86 1.72 -7.40 3.78 Compound_07 4-Br -7.48 3.27 -6.67 12.92 Compound_08 2-F -6.94 8.18 -6.76 11.04 Compound_09 3-F -7.01 7.23 -6.89 8.98 Compound_10 4-F -6.81 10.12 -6.84 9.65 Compound_11 2-NO2 -7.95 1.50 -6.93 8.29 Compound_12 3-NO2 -7.78 1.98 -8.19 0.9945 Compound_13 4-NO2 -7.18 5.49 -6.58 15.00 Compound_14 2-CH3 -7.15 5.74 -7.02 7.10 Compound_15 3-CH3 -7.48 3.27 -7.09 6.33 Compound_16 4-CH3 -7.24 4.96 -7.34 4.16 Compound_17 2-OCH3 -6.89 8.93 -6.93 8.36 Compound_18 3-OCH3 -7.09 6.36 -7.14 5.88 Compound_19 4-OCH3 -7.04 6.88 -7.45 3.46 Compound_20 2-OC2H5 -6.39 20.82 -7.65 2.47 Compound_21 3-OC2H5 -6.39 20.59 -7.78 1.99 Compound_22 4-OC2H5 -7.51 3.15 -6.36 21.62 Compound_23 2-C2H5 -6.73 11.68 -7.46 3.40 Compound_24 3-C2H5 -7.50 3.21 -7.17 5.57 Compound_25 4-C2H5 -7.44 3.50 -7.63 2.54 Compound_26 2-OH -7.52 3.09 -7.09 6.35 Compound_27 3-OH -6.84 9.65 -6.91 8.57 Compound_28 4-OH -6.87 9.17 -6.86 9.29 Compound_29 2-SO2NH2 -7.57 2.83 -7.89 1.65 Compound_30 3-SO2NH2 -7.62 2.59 -7.73 2.16 Compound_31 4-SO2NH2 -8.01 1.34 -7.25 4.87 Compound_32 3-Cl-4-CH3 -7.13 5.98 -7.16 5.66
52
Table 4.2. Interaction for PPARγ
Code No. of
H-bonds Ligand groups
Protein residues Binding Energy
(Kcal/mol) a
Ki (µM)b remarks
GLN286 HIS323 HIS449 TYR473
Compound 4 No interaction
Compound 4 2 H-bonds Van-OH HN1 (Imz) -OH -5.77 58.57µM Compound 7 No
interaction
Compound 10 2 H-bonds Van-OH HN1 (Imz) -OH -5.92 46.10µM Compound 13 3 H-bonds Van-OH
Nit-O HN1 (Imz) -OH -6.78 10.71µM GLU343 (amide
NH)
Compound 16 No interaction
Compound 19 2 H-bonds Van-OH HN1 (Imz) -OH -6.37 21.47µM Compound 31 3 H-bonds Van-OH
Sul-O HN1 (Imz) -OH -6.59 14.73µM GLU343 (amide
NH)
Compound 32 2 H-bonds Van-OH HN1 (Imz) -OH -6.15 31.00µM
53
Table 4.3. Interaction for PPARα
Code No. of
H-bonds Ligand groups
Protein residues Binding Energy
(Kcal/mol) a
Ki (µM)b remarks SER280
TYR314 HIS440 TYR464
Compound 4 No interaction
Compound 7 HN1 (Imz) -OH -6.31 23.57 Compound 10 No
interaction
Compound 13 5 H-bonds
(1) Van-OH (2) Van-OCH3 (3) NO2
-OH
-OH
HN1 (Imz) -7.25 4.82µM TYR334&GLY335 (amide NH)
Compound 16 HN1 (Imz) -OH -6.79 10.52µM Compound 19 No
interaction
Compound 31 2 H-bonds
SO2-NH2 HN1 (Imz) -OH -6.86 9.32µM
Compound 32 No interaction
54
Fig. 4.2a. Interaction of Compound 13 with PPARγ (PDB Code: 3HOD), H-
bond interaction were shown in green dots and atoms establishing H-
bonds were colored by atom type
Fig. 4.2b. Interaction of Compound 31 with PPARγ (PDB Code: 3HOD), H-
bond interaction were shown in green dots and atoms establishing H-
bonds were colored by atom type
55
Fig.4.3a. Interaction of Compound 13 with PPARα (PDB Code: 3G8I), H-
bond interaction were shown in green dots and atoms establishing H-
bonds were colored by atom type
Fig.4.3b. Interaction of Compound 31 with PPARα (PDB Code: 3G8I), H-
bond interaction were shown in green dots and atoms establishing H-
bonds were colored by atom type
56
CCHHAAPPTTEERR VV
SYNTHETIC METHODOLOGY
The strategies used for the development of novel vanillin semicarbazones
include application of the following chemical approaches to structural modification
of the lead compound linked with molecular docking studies.
i. The synthesis of a series of homologous compounds or modifications
which causes changes in lipophilicity and structural features.
ii. The concept of molecular simplification which involves the synthesis
and evaluation of analogues of the prototypic compound.
This semicarbazones based pharmacophoric model comprises of following
essential binding sites with PPAR receptors: (i) An aryl centre (ii) A linker, (iii) An
acidic head, hydrophobic-hydrophilic site regulating the pharmacokinetic properties
of the required pharmacological profiles
Fig.5.1. Pharmacophore model of PPAR agonists
As per the approach by molecular docking studies on vanillin
semicarbazones, the most potential compounds for the desired pharmacological
properties were identified and have been synthesized.
57
5.1 Materials and methods
All reagents and chemicals were analytical grade. Weighing was carried out
on Shimadzu BL-220H, an analytical balance. Thin layer chromatography (TLC)
was performed for the all synthesized compounds (VSC I to VSC VIII) by using
TLC plate silica gel G (0.25 mm thickness) as stationary phase and mobile phase
methanol, chloroform 9:1 ratio after development of chromatogram. The spots were
identified by iodine chamber92 method and Rf values are calculated and reported. All
compounds reported were homogeneous by TLC. Melting points were observed
visually on Electrothermal 9100 instrument and were uncorrected. All samples were
dried and stored in glass desiccators over silica at room temperature before being
used.
The structure of the synthesized compounds was established by spectral
studies via IR, 1H NMR and MASS spectrums93, 94. IR spectra (KBr disc method)
were recorded on Shimadzu 8400 series FT-IR Spectrophotometer. 1H NMR spectra
were obtained on Bruker AV spectrophotometer at 300 MHz in DMSO-d6. Chemical
shifts were reported in δ units (ppm) relative to an internal standard of
tetramethylsilane. Mass spectra were recorded on JEOL GCmate. Microanalyses for
C, H and N were performed in Heraeus CHN Rapid Analyzer and analyses.
58
SCHEME
R'
NH2
+
O Cl
O
R'
HN O
O
H2N NH2 .H2O
R'
HN
HN
NH2
O
R'
HN
HN
N
O
HO
OH3CO
OH
OCH3
Phenyl chloroformateAniline/substituted aniline
Phenyl N-aryl carbamates
EtherNaoH
Reflux 2 hrsEthanol
Stir 2 hrs
Aryl Semicarbazides
Glacial acetic acid
Aryl semicarbazones of vanillin
R
R
R
R
1-8
Compound Code
R R'
1 H Cl 2 H Br 3 H F 4 H NO2 5 H CH2OH 6 H CH3 7 Cl CH3 8 H SO2NH2
59
5.2 SYNTHETIC METHODOLOGY
The procedure for the synthesis of vanillin semicarbazones involves 3 steps.
STEP I
5.2.1 Synthesis of Aryl Carbamates
R'
NH2
+
O Cl
O
R'
HN O
O
Phenyl chloroformateAniline/substituted aniline
Phenyl N-aryl carbamates
EtherNaoHStir 2 hrs
R
R
0.01 mol of phenyl chloroformate is added drop wise at 0˚C to a well stirred
solution of 0.01 mol of aniline/substituted aniline in 100ml of anhydrous ether.
During the second phase of addition, a solution of 0.01mol of NaOH in 10ml of
distilled water is added simultaneously. The mixture is stirred vigorously for 1 hr at
0˚C and again for 1 hr at 20˚C. The organic layer is washed with 25ml of 0.2M HCl
and water and finally dried over MgSO4. Evaporation of the solvent yields the
desired Phenyl N-aryl carbamates95.
60
STEP II
5.2.2 Hydrazinolysis and formation of semicarbazides
R'
HN O
O
H2N NH2 .H2O
R'
HN
HN
NH2
O
Phenyl N-aryl carbamates
Reflux 2 hrsEthanol
Aryl Semicarbazides
R
R
A solution of desired carbamates, 0.01mol, 85% hydrazine hydrate (4ml) and
ethanol (16ml) was heated under reflux for 2 hrs. The solvent is evaporated under
reduced pressure and the product is recrystallized from ethanol.
STEP III
5.2.3 Synthesis of vanillin semicarbazones
R'
HN
HN
NH2
O
R'
HN
HN
N
O
HO
OH3CO
OH
OCH3
Aryl Semicarbazides
Glacial acetic acid
Aryl semicarbazones of vanillin
R
R
1-8
61
A solution of phenyl semicarbazides (0.01mol) and an equimolar quantity of
the appropriate carbonyl compound were refluxed for 30 minutes in the presence of
glacial acetic acid (1-1.5ml). The product obtained after cooling was filtered and
recrystallized from 95% ethanol36.
5.2.4. Nomenclature of synthesized compounds
S.No Structure IUPAC Name
1.
HN
O
NHN
OCH3
OHCl
(E)-1-(4-hydroxy-3- methoxy benzylidene)-4-(4-chlorophenyl) semicarbazide
2.
HN
O
NHN
OCH3
OHBr
(E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(4-bromophenyl) semicarbazide
3.
HN
O
NHN
OCH3
OHF
(E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(4-fluorophenyl) semicarbazide
4.
HN
O
NHN
OCH3
OHNO2
(E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(4-nitrophenyl) semicarbazide
5.
HN
O
NHN
OCH3
OHHOH2C
(E)-1-(4-hydroxy-3- methoxy benzylidene)-4-(4-(hydroxyl methyl) phenyl) semicarbazide
6.
HN
O
NHN
OCH3
OHH3C
(E)-1-(4-hydroxy-3-methoxy benzylidene)-4-p-tolyl semicarbazide
7.
HN
O
NHN
OCH3
OHH3C
Cl
(E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(3-chloro-4-methylphenyl) Semicarbazide
8.
HN
O
NHN
OCH3
OHH2NO2S
(E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(sulfonamido phenyl) semicarbazide
62
5.2.5 PHYSICAL PROPERTIES AND SPECTRAL DATA OF
SYNTHESIZED COMPOUNDS
Compound-1
(E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-chlorophenyl)semicarbazide
HN
O
NHN
OCH3
OHCl
Molecular formula : C15H14ClN3O3
Mol. Wt. : 319.74
% yield : 69
Rf : 0.7414 [Chloroform-Methanol (9:1)]
Melting point : 198-2010C
Elemental analysis : Anal. Calc. : C 56.35%, H 4.41%, N 13.14%
Found : C 56.34%, H 4.38%, N 13.12%
IR (KBr, cm-1) : 3427 (OH), 3311 (NH), 1653 (CONH), 1609 (C=C),
1588 (C=N), 1231 (C-N),1091 (C-O), 730 (C-Cl) 1HNMR (DMSO, δ) : 3.42 (s, 1H, CH), 3.85 (s, 3H, OCH3), 6.79 - 7.74 (m, 7H,
ArH), 7.84 (s, 1H, CONH), 8.97 (s, 1H, NH-N), 10.59 (s,
1H, OH)
EI-MS m/z : 288.2 (M-31), 124, 89, 62.
IR spectra showed C=N peak at 1588 cm-1 and absence of characteristic peak
for –NH2. 1H NMR spectrum showed singlet at δ 8.97 for hydrazine (-HN-N) proton
attached to the -CH proton. Mass spectrum revealed that the peaks for the respective
compounds are in agreement with their molecular weight. All these data confirms
the formation of compound 1.
63
Compound-2
(E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-bromophenyl) semicarbazide
HN
O
NHN
OCH3
OHBr
Molecular formula : C15H14BrN3O3
Mol. Wt. : 364.19
% yield : 72
Rf : 0.6186 [Chloroform-Methanol (9:1)]
Melting point : 208-2100C
Elemental analysis : Anal. Calc. : C 49.47%, H 3.87%, N 11.54%
Found : C 49.44%, H 3.82%, N 11.52%
IR (KBr) cm-1 : 3406 (OH), 3300 (NH), 1652 (CONH), 1609
(C=C), 1583 (C=N), 1248 (C-N),1071 (C-O), 682 (C-Br) 1HNMR (DMSO, δ) : 3.43 (s, 1H, CH), 3.85 (s, 3H, OCH3), 6.79 - 7.69 (m, 7H,
ArH), 7.84 (s, 1H, CONH), 8.96 (s, 1H, NH-N), 10.60 (s,
1H, OH)
EI-MS m/z : 243.6 (M-122), 167, 89, 62.
The presence of C=N peak at 1583 cm-1 and absence of characteristic peak
for –NH2 in IR spectrum and singlet at δ 8.97 for hydrazine (-HN-N) proton attached
to the -CH proton in 1H NMR spectrum confirms the formation of compound 2.
Mass spectrum revealed that the peaks for the respective compounds are in
agreement with their molecular weight.
64
Compound-3
(E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-fluorophenyl)semicarbazide
HN
O
NHN
OCH3
OHF
Molecular formula : C15H14FN3O3
Mol. Wt. : 303.29
% yield : 67
Rf : 0.6824 [Chloroform-Methanol (9:1)]
Melting point : 211-2120C
Elemental analysis : Anal. Calc. : C 59.40%, H 4.65%, N 13.85%
Found : C 59.44%, H 4.68%, N 13.82%
IR (KBr cm-1) : 3469 (OH), 3249 (NH), 2886 (OCH3), 1668 (CONH),
1511 (C=N), 1240 (C-N), 1071 (C-O) 1HNMR (DMSO, δ) : 3.32 (s, 1H, CH), 3.85 (s, 3H, OCH3), 6.79 - 7.69 (m, 7H,
ArH), 7.83 (s, 1H, CONH), 8.92 (s, 1H, NH-N), 10.58 (s,
1H, OH)
EI-MS m/z : 303.9 (M+), 274, 168, 125, 81, 54.
IR spectra showed C=N peak at 1511 cm-1 and absence of characteristic peak
for –NH2. 1H NMR spectrum showed singlet at δ 8.92 for hydrazine (-HN-N) proton
attached to the -CH proton. Mass spectrum revealed that the peaks for the respective
compounds are in agreement with their molecular weight. All these data confirms
the formation of compound 3.
65
Compound-4
(E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-nitrophenyl) semicarbazide
HN
O
NHN
OCH3
OHNO2
Molecular formula : C15H14N4O5
Mol. Wt. : 330.3
% yield : 79
Rf : 0.5982 [Chloroform-Methanol (9:1)]
Melting point : 202-2050C
Elemental analysis : Anal. Calc. : C 54.55%, H 4.27%, N 16.96%
Found : C 54.52%, H 4.28%, N 16.92%
IR (KBr cm-1) : 3866 (OH), 3515 (NH), 3103 (OCH3), 1725 (CONH),
1491 (C=N), 1186 (C-N), 1061 (C-O) 1HNMR (DMSO, δ) : 3.35 (s, 1H, CH), 3.87 (s, 3H, OCH3), 6.80 – 8.224 (m,
7H, ArH), 8.229 (s, 1H, CONH), 9.49 (s, 1H, NH-N),
10.92 (s, 1H, OH)
EI-MS m/z : 330.8 (M+), 189, 125, 81, 67.
The presence of C=N peak at 1491 cm-1 and absence of characteristic peak
for –NH2 in IR spectrum and singlet at δ 9.49 for hydrazine (-HN-N) proton attached
to the -CH proton in 1H NMR spectrum confirms the formation of compound 4.
Mass spectrum revealed that the peaks for the respective compounds are in
agreement with their molecular weight.
66
Compound – 5
(E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-(hydroxymethyl) phenyl)
semicarbazide
HN
O
NHN
OCH3
OHHOH2C
Molecular formula : C16H17N3O4
Mol. Wt. : 315.32
% yield : 71
Rf : 0.7692 [Chloroform-Methanol (9:1)]
Melting point : 224-226°C
Elemental analysis : Anal. Calc. : C 60.94%, H 5.43%, N 13.33%
Found : C 60.92%, H 5.48%, N 13.32%
IR (KBr, cm-1) : 3792 (OH), 3412 (NH), 3074 (OCH3), 1676 (CONH),
1515 (C=N), 1115 (C-N), 1033 (C-O) 1HNMR (DMSO, δ) : 3.32 (a, 1H, CH), 3.73 (s, 3H, OCH3-Ar), 3.85 (s, 3H,
OCH3), 6.78 – 7.54 (m, 7H, ArH), 7.82 (s, 1H, CONH),
8.73 (s, 1H, NH-N), 10.47 (s, 1H, OH)
EI-MS m/z : 315 (M+), 279, 182, 123, 71, 53.
IR spectra showed C=N peak at 1515 cm-1 and absence of characteristic peak
for –NH2. 1H NMR spectrum showed singlet at δ 8.73 for hydrazine (-HN-N) proton
attached to the -CH proton. Mass spectrum revealed that the peaks for the respective
compounds are in agreement with their molecular weight. All these data confirms
the formation of compound 5.
67
Compound-6
(E)-1-(4-hydroxy-3-methoxybenzylidene)-4-p-tolylsemicarbazide
HN
O
NHN
OCH3
OHH3C
Molecular formula : C16H17N3O3
Mol. Wt. : 299.32
% yield : 73
Rf : 0.8396 [Chloroform-Methanol (9:1)]
Melting point : 214-2160C
Elemental analysis : Anal. Calc. : C 64.20%, H 5.72%, N 14.04%
Found : C 64.24%, H 5.78%, N 14.10%
IR (KBr, cm-1) : 3400 (OH), 3311 (NH), 2900 (OCH3), 1653 (CONH),
1593 (C=N), 1109 (C-N), 1024 (C-O) 1HNMR (DMSO, δ) : 2.27 (s, 3H, CH3), 3.32 (s, 1H, CH), 3.85 (s, 3H, OCH3),
6.79 – 7.55 (m, 7H, ArH), 7.82 (s, 1H, CONH), 8.77 (s,
1H, NH-N), 10.53 (s, 1H, OH)
EI-MS m/z : 198.9 (M-108), 134, 86.
The presence of C=N peak at 1593 cm-1 and absence of characteristic peak
for –NH2 in IR spectrum and singlet at δ 8.77 for hydrazine (-HN-N) proton attached
to the -CH proton in 1H NMR spectrum confirms the formation of compound 6.
Mass spectrum revealed that the peaks for the respective compounds are in
agreement with their molecular weight.
68
Compound-7
(E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(3-chloro-4-methylphenyl)
Semicarbazide
HN
O
NHN
OCH3
OHH3C
Cl
Molecular formula : C16H16ClN3O3
Mol. Wt. : 333.77
% yield : 78
Rf : 0.5472 [Chloroform-Methanol (9:1)]
Melting point : 240-2430C
Elemental analysis : Anal. Calc. : C 57.58%, H 4.83%, N 12.59%
Found : C 57.54%, H 4.80%, N 12.56%
IR (KBr, cm-1) : 3429 (OH), 3301 (NH), 1652 (CONH), 1615 (C=C),
1588 (C=N), 1051 (C-N),1033 (C-O), 698 (C-Cl) 1HNMR (DMSO, δ) : 2.49 (s, 3H, CH3), 3.32 (s, 1H, CH), 3.84 (s, 3H, OCH3),
6.79 – 7.872 (m, 7H, ArH), 7.877 (s, 1H, CONH), 8.96 (s,
1H, NH-N), 10.65 (s, 1H, OH)
EI-MS m/z : 332 (M+), 262, 205, 176, 104
IR spectra showed C=N peak at 1588 cm-1 and absence of characteristic peak
for –NH2. 1H NMR spectrum showed singlet at δ 8.96 for hydrazine (-HN-N) proton
attached to the -CH proton. Mass spectrum revealed that the peaks for the respective
compounds are in agreement with their molecular weight. All these data confirms
the formation of compound 7.
69
Compound-8
(E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(sulfonamido phenyl)
semicarbazide
HN
O
NHN
OCH3
OHH2NO2S
Molecular formula : C15H16N4O5S
Mol. Wt. : 364.38
% yield : 75
Rf : 0.5822 [Chloroform-Methanol (9:1)]
Melting point : 190-1920C
Elemental analysis : Anal. Calc. : C 49.44%, H 4.43%, N 15.38%
Found : C 49.40%, H 4.38%, N 15.32%
IR (KBr, cm-1) : 3346 (OH), 3200 (NH), 1682 (CONH), 1639 (C=C), 1586
(C=N), 1115 (C-N), 1030 (C-O) 1HNMR (DMSO, δ) : 3.33 (s, 1H, CH), 3.86 (s, 3H, OCH3), 6.80 – 7.86 (m, 7H,
ArH), 7.87 (s, 2H, NH2), 7.88 (s, 1H, CONH), 9.16 (s, 1H,
NH-N), 10.70 (s, 1H, OH)
EI-MS m/z : 256 (M-108), 138, 80.
The presence of C=N peak at 1586 cm-1 and absence of characteristic peak
for –NH2 in IR spectrum and singlet at δ 9.16 for hydrazine (-HN-N) proton attached
to the -CH proton in 1H NMR spectrum confirms the formation of compound 8.
Mass spectrum revealed that the peaks for the respective compounds are in
agreement with their molecular weight.
70
CCHHAAPPTTEERR VVII
QSAR STUDIES
6.1 INTRODUCTION
Determination of Molecular and Drug-likeness properties is one of the most
important aspects of Quantitative Structural Activity Relationship (QSAR). Drug-
likeness is defined as a complex balance of various molecular properties and
structural features, which determine whether particular molecule is a drug or non-
drug96. These properties are mainly hydrophobicity, electronic distribution,
hydrogen bonding characteristics such as transport, affinity to proteins, reactiviy,
toxicity, metabolic stability etc., These properties can be assessed by calculating
molecular weight, log P and number of hydrogen bond donors / acceptors or by
calculating the important drug targets namely GPCR ligands, ion-channel
modulators and kinase inhibitors.
QSAR studies in organic and biochemistry essentially answers two questions
namely,
i. What feature of a molecule affects its activity?
ii. What can be modified to enhance the desired properties or functions?
If the answers to these questions can be formulated in the form of
mathematical expression, then these expressions could be effectively utilized for
formulations, design and synthesis of new compounds with desired properties and
functions.
6.2 PARAMETER - Log P
The properties of molecules can be in the form of philic or phobic
interactions arising out of electronic and steric considerations. In organic molecules
hydrophobicity is definable quantitatively in terms of partition coefficients. The
commonly used term to definite the partition coefficient is Log P or п.
71
Concentration of drug in octanol P =
Concentration of drug in aqueous solution
Partition coefficient (log P) in octanol-water system is used in QSAR studies
and rational drug design as a measure of molecular hydrophobicity. Hydrophobicity
affects drug absorption, bio-availability, hydrophobic drug-receptor interactions,
metabolism of molecules, as well as toxicity. According to this definition, high
value of log P denotes high hydrophobic character and low value of log P
indicates hydrophilic character. The binding of the drugs to serum albumin has been
quantitatively expressed as
Log(1/C) = 0.75 log P + 2.3
From this one can able to estimate how much of the drug is unavailable for
bonding with receptor. Generally the relationship between log P and log (1/C) is a
parabolic in nature as shown in the given below the figure. Most of the anesthetics,
which will enter cell membrane and affect CNS activity, are not available for drug
receptor interactions. Normally it is considered that any compound with a log P
value close to 2 can be efficiently entering CNS.
The hydrophobicity of the molecule (log P) will be expected to affect the
rate/extent of transportation to a site of action and may align with the liphophilic
72
pocket of the binding site. The separation and orientation of the liphophilic pocket
and hydrogen bonding surface influence activity97.
A molecule editor, that is program for input and editing of molecules, is an
indispensable part of every cheminformatics or molecular processing system. The
JME editor has been released to the public and is currently probably the most
popular molecular entry system on the web. Log P calculation is based on the
methodology published by Ertl.et.al.98.
Table 6.1. Molecular and Drug-likeness properties
Compound code Log P
1 3.08
2 3.35
3 2.68
4 2.22
5 1.95
6 3.01
7 3.57
8 1.27
73
CCHHAAPPTTEERR VVIIII
PHARMACOLOGICAL EVALUATION
7.1 INTRODUCTION
Hyperglycemia and hyperlipidemia are two important characters of Diabetes
mellitus, an endocrine disorder based disease. A number of patterns of lipid
abnormalities are encountered in patients with diabetes. Given the common presence
of both diabetes and hypercholesterolemia in patients with coronary artery disease
many patients are found to have elevated levels of low-density lipoprotein (LDL)
cholesterol. However, in many other patients with diabetes LDL cholesterol seems
to be in ‘normal’ limits. In these patients the predominant lipid abnormalities include
hypertriglyceridemia, low levels of high-density lipoprotein (HDL) cholesterol and
the presence of small, dense LDL particles99.
The precise pathogenesis of diabetic dyslipidemia is not known;
nevertheless, a large body of evidence suggests that insulin resistance has a central
role in the development of this condition. The main cause of the three cardinal
features of diabetic dyslipidemia is the increased free fatty-acid release from insulin-
resistant fat cells100. In modern medicine, no satisfactory effective therapy is still
available to cure diabetes mellitus associated with hyperlipedemia101.
Thiazolidinediones are agonists of the PPAR-γ receptor with beneficial
effects on insulin sensitivity, lipids, and inflammatory markers. The combination of
improving glycemic control with raising HDL cholesterol by 15–20% and lowering
of triglycerides by 30–50% and C-reactive protein by 40–50% with pioglitazone is
associated with a beneficial impact on progression of carotid intimal–medial
thickness (CIMT)102 and coronary atherosclerosis103.
Although a number of small studies have reported potentially beneficial
effects of rosiglitazone at the level of the vessel wall, a meta-analysis suggested a
potential increase in myocardial infarction104. This observation has not been
74
replicated in subsequent clinical trials105 and requires ongoing investigation. There
remains considerable interest in the development of more potent PPAR-α agonists or
use of agents that target more than one PPAR-α subtype.
However, this field has been challenged by a lack of incremental efficacy or
toxicity with a large number of experimental agents.
7.2 ACUTE TOXICITY STUDIES
7.2.1 Animals
Adult albino male and female mice were used for this study and procured
from animal facility department. they were fed with standard pellet feeds (sai durga
feeds ltd., bangalore, india) and water ad libitum. they were housed in poly-
propylene cages (6 per cage) with dust free rice husk as bedding material. the
animals were fasted prior to dosing according to organization for economic
cooperation development (oecd) guidelines, food but not water was withheld for 3-4
h106. all the animal experiment protocols of this study were approved by the
institutional animal ethics committee (iaec, 38-pcol 290/cpcsea/12.12.2000) of vel's
college of pharmacy, chennai. tamil nadu, india.
7.2.2 Test drugs and stock solution preparation
Suspension of vanillin semicarbazones (compound 1 - 8) were prepared
separately by mixing with 2% CMC to achieve 100 mg/ml concentration as the stock
solution and used for acute toxicity studies.
7.2.3 Drug treatment and assessment of toxicity
All mice were observed at the first, second, fourth and sixth hours and
thereafter once daily over 14 days for clinical signs of toxicity such as respiratory
pattern, color of body surfaces, frequency and nature of movement, marked
involuntary contraction or seizures of contraction of voluntary muscle, and loss of
reflex etc107. After the experimental period, blood was collected from the retro
75
orbital vein using non heparinized tube for hematological studies. i.e., complete
blood count, red blood cell count, platelet count and red cell indices108.
The serum was separated for determining the concentrations of biochemical
parameters like glucose, blood urea nitrogen (BUN), creatinine, total protein (TP),
albumin, total bilirubin (TB), direct bilirubin (DB), serum glutamic-oxaloacetic
transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT) and alkaline
phosphatase (ALP) using standard analytical methods109.
They were killed by euthanasia and vital organs including heart, lungs,
livers, kidneys, spleen, adrenals, sex organs and brain were dissected out and
weighed and taken for gross pathological examination. Organ to body weight ratio,
and various hematological and biochemical variables were studied. Tissues of vital
organs viz., lung, liver, kidney, spleen, heart and testes, stomach were fixed in 10%
buffered formalin for microscopic examination. Standard procedures were used for
the evaluation of hematological, biochemical and histological parameters110,111.
7.2.4 Hematological, biochemical and histological studies
Twenty-four hours after the oral dosing the animals were lightly
anaesthetized with ether and blood was withdrawn from the orbital plexus112.
7.2.5 Hemoglobin concentration of whole blood113,114
The concentration of hemoglobin was measured by the usual procedure using
Shali’s haemometer. Blood sample was drawn into the pipette up to the 20 cumm
mark and transferred to the rectangular cell containing a little amount of N/10 HCl
placed in haemometer (Hellige Shali haemometer No. 304-B, Hellige, USA). After 5
minutes, a color comparison was made with standard color prism of haemometer. If
the color of the solution was high, distilled water was added to this solution and
mixed using a stirrer until a good color match was obtained.
The final reading of the solution in the tube was noted. From the cuvette
reading, hemoglobin in g/100ml of blood or its percentage was calculated.
76
7.2.6 Erythrocyte Count113, 114
Blood was taken up to 0.5 mark in the RBC pipette and excess blood was
wiped off from the tip. The pipette was then filled to 101 marks with RBC diluting
fluid. The RBC pipette was horizontally shaken and a drop of resultant mixture was
discharged under the cover glass of a Neubauer counting chamber (Neubauer,
Feinoptic, Germany). Number of erythrocytes in 80 small squares was counted
under the light microscope. The number of cells in 1 ml of undiluted blood was
calculated using the standard formula:
Erythrocyte count per ml = N/80 ×1 × 2000 × 0.02
Where, N= number of cells in 80 small squares (dilution)
7.2.7 Total Leukocyte Count113,114
Blood was drawn up to 0.5 mark in the WBC pipette, diluted with WBC
diluting fluid up to 11 mark and mixed properly. The resultant mixture was charged
under the cover slip in the Neubauer chamber and the number of cells in four-corner
block (each block is sub divided into 16 squares) was counted. The total leucocytes
count per ml of blood was calculated by multiplying the average number of cells in
the four blocks by 200.
7.2.8 Packed cell volume113,114
Using a Pasteur pipette, the wintrobe tube was filled with blood, starting at
its bottom and withdrawing the pipette as the tube is filled from below upwards.
The blood column was brought to the ‘O’ mark air bubbles, if any were removed
from the top of the column of blood so that it stands exactly at ‘O’. The tube was
centrifuged for about 20 minutes at 25.60 rpm. The reading of the packed cells was
taken, the tubes again centrifuged for 5 minutes and the reading was noted. Final
reading was recorded when three consecutive readings were identical i.e., when the
red cells have been fully packed
77
7.2.9 Total Protein113,114
To 0.1 ml of serum suitably diluted with 0.9 ml of water and 4.5 ml of
alkaline copper reagent were added and kept at room temperature for 10 min. Then
0.5 ml of Folin’s reagent was added and the colour developed was read after 20 min
at 640 nm. The level of protein was expressed as mg/dl of serum.
7.3 STATISTICAL ANALYSIS
In pharmacological evaluation, all the data (results) obtained at the end of the
experiments were subjected to statistical analysis to determine whether the effect
produced by a compound under study is genuine and not due to chance. Hence, the
analysis usually attaches a test of statistical significance. The first step in such a test
is to state the null hypothesis. A null hypothesis is only useful if it is possible to
calculate the probability of observing a data set with particular parameters from it. In
general it is much harder to be precise about how probable the data would be if the
alternative hypothesis is true. When the null hypothesis is accepted, the difference
between the two groups is not significant. In other words, both samples were indeed
drawn from a single population and the difference observed between the two groups
was due to chance.
If experimental observations contradict the prediction of the null hypothesis,
it means that either the null hypothesis is false, or we have observed an event with
very low probability. This gives us high confidence in the falsehood of the null
hypothesis, which can be improved by increasing the number of trials. However,
accepting the alternative hypothesis only commits us to a difference in observed
parameters; it does not prove that the theory or principles that predicted such a
difference is true, since it is always possible that the difference could be due to
additional factors not recognized by the theory.
For example, rejection of a null hypothesis (that, say, rates of symptom relief
in a sample of patients who received a placebo and a sample who received a
medicinal drug will be equal) allows us to make a non-null statement (that the rates
differed); it does not prove that the drug relieved the symptoms, though it gives us
more confidence in that hypothesis.
78
If the null hypothesis is rejected, then the difference is significant. A
difference between the treated and the control group which would have arisen by
chance in less than 5% of cases, is considered as statistically significant (P<0.05);
that arising in less than 1% of cases as highly significant (P<0.01); while that arising
in less than 0.1% of cases as very highly significant (P<0.001).
All the results from pharmacological experiments were expressed as mean ±
SEM. The data were statistically analyzed by one-way ANOVA followed by
Dunnett’s test. The data of haematological parameters were analyzed using ANOVA
followed by Tukey multiple comparison test. INSTAT -3 was used for all the
statistical analyses. A probability value less than 0.05 were taken as statistically
significant115.
7.4 ANTI DIABETIC STUDIES OF VANILLIN SEMI CARBAZONES
Among the compounds tested for the toxicity profile the following
compounds are selected for the further pharmacological study.
VSC I. Vanillin semicarbazone having fluoro substituent (300mg/kg),
VSC II. Vanillin semicarbazone having nitro substituent (500mg/kg),
VSC III. Vanillin semicarbazone having chloro substituent (500mg/kg),
VSC IV. Vanillin semicarbazone having 3-chloro 4-methyl (500mg/kg)
7.4.1 Induction of Experimental Diabetes
Male wistar rats (200-250gm) were fasted for 16 hours before the induction
of diabetes with Streptozotocin (STZ), a valuable agent for induction of Diabetes
mellitus116. Animals (n=48) were injected intraperitoneally with 0.22 - 0.25ml of
freshly prepared solution of STZ (60 mg/ml in 0.01 M citrate buffer, pH 4.5) at a
final dose of 60 mg/kg body wt. The diabetic state was assessed in STZ - treated rats
by measuring the non-fasting serum glucose concentration after 48 hours. Only rats
with serum glucose levels greater than 300 mg/dl were selected and used in this
experiment117.
79
7.4.2 Effect of Vanillin semicarbazones (VSC I – IV) on glucose tolerance in
rats
Fasted rats were divided into 5groups of six rats each.
Group l served as a control, received distilled water.
Group 2 – 5 received VSC I (300mg/kg), VSC II (500mg/kg), VSC III
(500mg/kg), and VSC IV (500mg/kg) body weight orally as a fine CMC aqueous
suspension orally as doses fixed after performing the toxicity study.
The rats of all groups were given glucose (2g/kg body weight, p.o) 30 min
after administration of the drug. Blood samples were collected from the tail vein just
prior to glucose administration and at 30 and 90 min after the glucose loading.
Serum was separated and blood glucose levels were measured immediately by using
one touch glucometer118
7.4.3 Blood sugar estimation
Diabetic rats (n = 36) were randomly divided into 8 groups of 6 rats each.
Group 1 served as a normal control receiving 0.5ml of saline.
Group 2 served as diabetic control
Group 3 - 6 served as test groups treated with VSC I (300mg/kg), VSC II
(500mg/kg), VSC III (500mg/kg), and VSC IV (500mg/kg) body weight orally, as
doses fixed after performing the toxicity study.
Group 7 treated with drugs - Glibenclamide (0.5mg/kg)
Group 8 treated with standard Zinc insulin (4U).
Fasting serum glucose concentrations were determined in mg/dl, from rat tail
vein by using one touch glucometer (Ultra Co.) device. The rats were dosed daily by
80
gavage with saline, standard drug and VSC I - IV for 15 days for respective groups.
On day 14, blood samples were collected from retro orbital vein and serum was
separated by centrifuging the blood samples at 3000rpm for 15mins and biochemical
parameters like total cholesterol and triglyceride levels were estimated. The
periodical body weight difference of the individual animals was also measured.
On the evening of day 14, all fasted rats were sacrificed by decapitation. The
abdomen was cut opened. Liver and Kidney were dissected out and homogenized
using Polystrin homogenizer. From these samples the malondialdehyde and other
antioxidant enzyme levels were measured. Pancreases were removed carefully
devoiding of adhering tissues from each group and its microanatomical changes was
studied119,120.
7.4.4 Determination of Total Cholesterol and Triglycerides
Serum total cholesterol and triglycerides concentration were analyzed using
cholesterol diagnostic estimation kit and triglycerides estimation kit121.
(QUALIGEN Chemicals, Mumbai).
7.4.5 Histopathological Studies
The dissected sample of pancreas from each group of diabetic animals were
collected in 10% formalin solution and stained with hemotoxylin and eosin for
preparation of section by using of microtome122.
7.5 STATISTICAL ANALYSIS
The results are expressed as means ± SEM. Data were analyzed by using one
way ANOVA followed by Tukey’s multiple comparison tests. P values of <0.05
were considered as significant115.
81
7.6 ANTIHYPERLIPIDEMIC ACTIVITY OF VANILLIN
SEMICARBAZONES
The various chemicals employed for different procedures were of analytical
grade supplied by BDH Glaxo laboratories, E.Merck and Sigma Diagnostic (india)
Pvt. Ltd. Beef fat was bought from a butcher’s shop. It was heated on a pan and the
melted fat was filtered through a cloth and collected for the use. Commercially
available butter fat (BUF) was purchased for the present work from a shop.
7.6.1 Drug stock solution Preparation
Suspension of selected vanillin semicarbazones were prepared separately by
mixing with 2% CMC to achieve 100 mg/ml concentration
7.6.2 Experimental animals
Adult albino rats 9-12 months old and weighing around 250g were selected
and divided into seven groups containing 5 rats in each group, for different
derivatives studies.
7.6.3 Diet preparation
Normal rat feed (Lipton India Ltd., Calcutta) was fed to control of group 1 in
measured quantities and it was found that a rat consumed an average weight of 14 g
feed daily. The normal rat feed was powdered and mixed with each type of fat so as
to fix 21% fat in the diet for control of groups 2 and 5, and similar high fat diets
mixed with VSC derivatives, VSC I (300mg/kg), VSC II (500mg/kg), VSC III
(500mg/kg), and VSC IV (500mg/kg) body weight in 100g of feed (Average of
15g/rat and n=6). The mixture of feeds were wetted with a little water and made into
balls and dried in an oven for feeding it daily. Water was supplied in bottles to each
group so that controls and tests were paired fed.
After three months of feeding the rats, they were sacrificed after overnight
fasting. Their blood was collected in centrifuge tubes by punching the retro orbital
vein and the serum was separated after an hour. It was used for the estimation of
82
lipid parameters and enzyme activities. Their liver heart and kidneys were also
collected and preserved in ice cold beakers for various estimations. Kits provided by
sigma diagnostics Pvt. Ltd. were used for lipid and enzyme estimations according to
standard methods. Extractions of tissues were carried out for various estimations122.
7.6.4 Extraction for cholesterol and triacyl glycerol (TAG)
Accurately weighed (0.5g) tissue was ground with 4g of anhydrous sodium
sulphate using mortar and pestle and diluted to 20ml and centrifuged. 2ml of this
supernatant was evaporated and redissolved in 1ml acetic acid 0.05ml of this extract
was used for the estimation of total cholesterol. 0.01ml of the extract was used for
the estimation of TAG. Serum VLDL+LDL cholesterol was determined by
subtracting HDL cholesterol from total cholesterol123,124.
7.6.5 Extraction for thibarbituric acid reacting system (TBARS)
Accurately weighed 0.5g tissue was ground in a mortar with a pestle. 4.5ml
of 0.5% cold TCA was added and mixed well. 1.0ml of this homogenate was used
for TBARS which is that part of the products of lipid peroxidation viz;
malondialdehyde (MDA) that reacts with thiobarbituric acid117,125.
7.6.6 Extraction for HMGCoA reductase enzyme
Accurately weighed 0.5g tissue was ground in a mortar with a pestle under
cold conditions. A 10% homogenate was prepared by adding 4.5ml of saline
arsenate (1g of sodium arsenate/L of normal saline). This homogenate was used for
the assay of the enzyme122.
7.6.7 Extraction for AST and ALP
Accurately weighed 0.5g tissue was ground in a mortar with pestle under
cold conditions. 2ml of phosphate buffer (pH 7.4) was added and centrifuged in a
refrigerated centrifuge at 2000g. The supernatant was used for the assay of enzyme.
Serum lipid parameters such as triacyl glycerol (TAG), total cholesterol, HDL
cholesterol and VLDL+LDL cholesterol and serum enzyme such as aspartate
83
transaminase (AST) and alkaline phosphate (ALP) were estimated by standard
methods.
Tissue homogenates from liver, hearts and kidney prepared as above were
used for the reductase. A modification for expressing the activity of the last enzyme
was used by which the ratio of Mevalonate/HMG CoA is calculated which gives the
activity of the enzyme directly126.
84
CCHHAAPPTTEERR VVIIIIII
RESULTS AND DISCUSSION
Pharmacophore modeling provides a useful frame work for better
understanding of the existing data which can be used as a predictive tool in the
design of compounds with improved potency selectivity and/or pharmacokinetic
properties.
8.1 MOLECULAR DOCKING STUDIES
Molecular docking studies of the compounds delineated structure-activity
relationships and to evaluate the viability of the binding sites of the selected
receptors hypothesis.
Thirty two vanillin derived phenyl semicarbazones were designed showing
variation in phenyl ring of semicarbazide. The designed molecules were found to be
fitting better in the proposed pharmacophore model for PPAR agonists since they
pocess an acidic head (Vanillyl phenol group mimicking carboxylic acid group), a
linker (semicarbazide) and an aromatic centre (substituted phenyl group) as depicted
in the Figure 4.1. As per the previous reports vanillin was designed for acidic head,
which are apt to form up to 4 pivotal hydrogen bonds with serine, tyrosine and
histidine of the protein, as the acidic warhead; such a strong hydrogen acceptor is
indispensable for obtaining potent agonists.
Based on the conceived idea, the library of thirty two molecules were docked
against X-ray crystal structure of hPPARα (PDB code: 3G8I) and hPPARγ (PDB
code: 3HOD). Autodock4 was employed for the purpose and the docking protocol
was validated by redocking with the co-crystallized ligand. Top scoring conformer
from the largest cluster has shown 1.27 and 0.99 for 3G8I and 3HOD respectively.
Molecular docking was performed for all the thirty molecules against
hPPARα (PDB code: 3HOD). Top scoring molecules from the largest cluster were
85
presented in Table 4.1. Acidic head of an agonist were reported to show hydrogen
bonding interaction with GLN286, HIS323, HIS449 and TYR473. But none of the
thirty two molecules was showing required interaction while analyzing top scoring
conformer in the largest cluster. While analyzing other cluster for the reported
interaction only six molecules, compound 4, compound 10, compound 13,
compound 19, compound 31 and compound 32, were found to exhibit them. The
interactions and docking scores of the corresponding conformers were reported in
Table 4.2. None of the six has shown all the four H-bonding interaction and hence
may act as partial agonist of PPARγ. Interaction of compound 13 and compound
31with PPARγ were shown in figure 4.2a and 4.2b.
Molecular docking was performed for all the thirty molecules against
hPPARα (PDB code: 3G8I). Top scoring molecules from the largest cluster were
presented in Table 4.1. Acidic head of an agonist were reported to show hydrogen
bonding interaction with SER280, TYR314, HIS440 and TYR464. But none of the
thirty molecules was showing required interaction while analyzing top scoring
conformer in the largest cluster. While analyzing other cluster for the reported
interaction only four molecules compound 7, compound 13, compound 16 and
compound 31, were found to exhibit them. Compound 31 found to establish required
H-bonding interaction through acidic sulphonamide group instead of vanillyl
moeity. The interactions and docking scores of the corresponding conformers were
reported in Table 4.3. None of the four molecules has shown all the four H-bonding
interaction and hence may act as partial agonist of PPARα. Interaction of compound
13 and compound 31 with PPARα was shown in figure 4.3a and 4.3b.
On the basis of molecular docking reports eight compounds, Compound 4(4-
Cl-), Compound 10 (4-F), Compound 19 (4-OCH3) and Compound 32 (3-Cl, 4-CH3)
showed partial agonist characteristics against PPARγ; Compound 7 (4-Br) and
Compound 16 (4-CH3) showed partial agonist characteristics against PPARα
whereas Compound 13 (4-NO2) and Compound 31 (4-SO2NH2) showed partial
agonist characteristics against both PPARα/γ. Thus, the dual agonist action of
compound 13 and 31 by structural biology and the docking studies was clearly
indicated. The distance and placement of the acidic head in relation to the oxygen
86
atom on the aromatic centre correlate with the binding affinity of the ligand to the
PPAR α/γ, and that the aromatic centre substituted at C-4 with substituent as NO2
and SO2NH2 provides the optimum distance, explaining its better potency as
compared to 2- or 3-substituted aromatic centres and aromatic centres having other
substituents such as Cl, Br, F, CH3, C2H5.
8.2 SYNTHETIC METHODOLOGY
The newly synthesized vanillin semicarbazones become a useful drug if it
posses anticipated pharmacological activities and free from toxicity. Keeping this
view in mind, the synthesis of compounds 1 to 8 was accomplished successfully.
The synthesis of vanillin semicarbazones was achieved as depicted in
scheme 1. A same experimental condition was followed for synthesis of vanillin
semicarbazones except for the difference in substituent. All the compounds were
synthesized with good yield.
8.3 CHARACTERIZATION
Melting points were determined by open-ended capillary tube and are
uncorrected and to purity of the compounds were checked by TLC using silica gel G
as stationary phase and visually detected by iodine vapor. The structure of the
compounds was elucidated by FT-IR in KBr disc method, 1H NMR and MASS
spectral data.
The Rf value of the newly synthesized vanillin semicarbazones indicted the
formation of new chemical analogues which was further confirmed by their different
melting points. The structure of the synthesized compounds was established by IR, 1H NMR and MASS spectral data. IR spectra revealed that the formation of new
vanillin semicarbazones by characteristic peak for C=N at 1593–1491 cm-1;
characteristic amide group at 3515–3200 cm-1 and 1725–1652 cm-1; and absence of
characteristic peak for –NH2. The presence of hydrazine (-HN-N) proton at δ 8.92-
9.49 attached to the methine (-CH) proton; amide (-CONH-) proton at δ 7.82-8.229
attached to the phenyl ring; methine (-CH-) proton at δ 2.27-3.43 attached to the
87
phenyl ring were confirmed by 1H NMR spectrum. Mass spectrum revealed that the
peaks for the respective compounds are in agreement with their molecular weight.
Elemental analyses for final compounds were performed on Heraeus CHN Rapid
Analyzer and the observed values were within the acceptable limits (±0.4%). All the
above data confirmed the formation of the title compounds.
8.4 QSAR STUDIES
QSAR studies of the test compounds are predicted by the methodology
developed by mol inspiration software program (edited by P.Ertil.Novartis) as a sum
of fragment based contribution and correction factor.
The prediction of molecular and drug-likeness properties was based on the
description of “Rule of 5” properties given by Lipinski. The rule states, that most
“drug-like” molecules have logP<=5 and molecular weight<=500. Based on this, it
was found that all the newly synthesized vanillin semicarbazones have molecular
and drug-likeness properties.
8.5 PHARAMACOLOGICAL EVALUATION
8.5.1 Effect of Vanillin Semicarbazones derivatives in acute oral toxicity test
in mice
The results from the acute oral toxicity studies suggested that mice
administered orally with 3g/kg of vanillin semicarbazone (VSC I) and with 5g/kg of
vanillin semicarbazones (VSC II-IV) did not show any toxic signs and mortality.
Results from the hematological and biochemicals measured in serum clearly
indicates that there is no significant changes in the hematological and biochemical
parameters in the serum of mice treated with 5g/kg of vanillin semicarbazones (VSC
II-IV) except vanillin semicarbazones VSC I. However animals administered with
substituted vanillin semicarbazones (VSC I-IV) significantly (P<0.01) affect the
polymorphs, Lympocytes (P<0.01) (vanillin semicarbazones (VSC II-IV)),
Eosinophils (P<0.01) (vanillin semicarbazones) (VSC II & IV) when compared with
control vehicle treated mice. There is a significant change in Hb % and PCV (%)
88
observed in mice administered with tolyl substituted vanillin semicarbazones
(Table 7.1).
Table 7.2 represents the effect of test drugs in biochemical parameters.
There is mild significant elevation in the biochemical markers such as Urea, AST
and ALP (P<0.01) (vanillin semicarbazones (VSC I-IV)), Cholesterol, protein
(P<0.01) (vanillin semicarbazones (VSC I, VSC II and VSC IV), Potassium
(P<0.01) (vanillin semicarbazones (VSC II, VSC III and VSC IV) were observed in
mice treated with vanillin semicarbazones (VSC I-IV). There is a significant
(P<0.05 to P<0.01) in visceral organs weight between the control and drug treated
groups. However pathological examinations of these tissues indicate that there are
no detectable abnormalities such as edema, atrophy or lesion in the tested organs
(Table 7.3). The remaining vanillin semicarbazones are exhibited 100 % mortality
at 3g/kg b.wt. when given single oral dose to mice, so these derivatives are excluded
for pharmacological studies.
8.5.2 Effect of Vanillin Semicarbazones derivatives in Oral Glucose Tolerance
Test (OGTT)
Table 7.4 depicts the effect of test drugs in OGTT. It is interesting to
observe that vanillin semicarbazones (V-VIII) significantly affect (P<0.01) the blood
glucose levels at 30 and 90 min after 2 grams of glucose ingestion.
8.5.3 Effect of Vanillin Semicarbazones derivatives in STZ induced diabetic
rats
The present data shows that intraperitoneal injection 60 mg/kg of STZ
induces diabetes in animals. In this study, among 50 numbers of animals are treated
with STZ in which only 45 animals exhibited diabetic (blood glucose >280mg/dl).
Diabetic animals are randomly divided in seven groups of 6 animals for further
studies.
Table 7.5 describes the anti-diabetic effect of semicarbazones in diabetic rats
at various day intervals. In diabetic animals treated with semicarbazones (I-IV)
89
significantly (P<0.001) reduced the blood glucose level tested at 7, 14, 21 and 28th
day when compared with respective diabetic control rats. The fasting blood glucose
level was found to be in normal range. The anti-diabetic effect of test drugs (VSC I-
IV) is comparable to that of standard glibenclamide (0.5:40mg/kg) and Zinc Insulin.
8.5.4 Effect of Vanillin Semicarbazones derivatives in body weight changes in
diabetic animals
Induction of diabetes with STZ is significantly (P<0.01) decreased the body
weight measured at regular intervals as compared with non diabetic control rats.
Decrease in body weight from 4th day to end of the study period. However there is
no effect on body weight of the diabetic rats treated with semicarbazones (VSC I-
IV) on from day 1 to day 4. There is significant change P<0.05 (at day 8) as well as
P<0.01 (at Day 14) in increasing body weight was noted in semicarbazone treated
diabetic rats when compared with diabetic animal group (Table 7.6).
8.5.5 Effect of Vanillin Semicarbazones derivatives Serum total Cholesterol
and Triglycerides levels
Increase in serum total cholesterol and triglyceride is noted in diabetic
animals which statistically P<0.01 as compared with normal control animal group.
This increase in total cholesterol and triglycerides was significantly decreased in
diabetic animals administered with vanillin semicarbazone (I-IV) when compared
with vehicle treated diabetic animals (Table 7.7). However the cholesterol and
triglycerides lowering effect of test drugs is not comparable to that of glibenclamide
and insulin which is found to highly significant (P<0.001) when compared with
diabetic control.
8.5.6 Effect of Vanillin semicarbazones derivatives in diabetic animal
pancreas
From the micro anatomical section of pancreas, it was observed that, the
VSC treated animals showing β–cell regeneration but not in diabetic control. The
90
standard drug Glibenclamide (0.5:40mg/kg) and Zinc insulin (4U) doesn’t showed
any remarkable regeneration of β-cells.
8.5.7 Effect of Vanillin Semicarbazones derivatives in High fat meal treated
hyperlipidemic rats
It is observed that animals fed with high fat meal containing butter fat (BUF)
and beef fat (BF) increased the triacylglycerol (TAG), Total cholesterol VLDL +
LDL level which is statistically significant compared with normal diet fed rats
(Table 7.8). Oral administration of vanillin semicarbazone (I-IV) to fat meal treated
rats significantly reduced the TAG (P<0.001), Total cholesterol (P<0.05) and
VLDL+LDL (P<0.001) when compared with high fat meal (HFM) treated group
animals. However none of the test drugs are affects the HDL level in high fat meal
treated animals. In addition, vanillin semicarbazone treatment significantly
decreased (P<0.01) the serum AST level as compared with normal and high fat meal
treated animal group.
Effect of high fat meal and semicarbazone in total cholesterol, TAG, TBARS
and HMG CoA reductase ALP and AST level in heart tissue is shown in Table 7.9.
In heart, there is an increase in total cholesterol, TAG, TBARS, HMG Co A
reductase, ALP and AST observed which is statistically significant (P<0.001)
compared with normal diet fed animals. Oral administration of vanillin
semicarbazone to high fat diet treated animals significantly lowered the TC, TAG,
TBARS, and ALP levels. Interestingly, test drugs exhibit the inhibition of HMG Co
A reductase level in heart tissue. The effect is significant (P<0.01) as compared with
high fat diet fed animals.
Effect of high fat meal and semicarbazone in total cholesterol, TAG,
TBARS, ALP and AST level in liver tissue is shown in Table 7.10. In heart, there is
an increase in total cholesterol, TAG, and TBARS, observed which is statistically
significant (P<0.001) compared with normal diet fed animals. Oral administration
of vanillin semicarbazone to high fat diet treated animals significantly lowered the
TC, TAG, TBARS, and ALP levels.
91
Effect of high fat meal and semicarbazone in total cholesterol, TAG,
TBARS, and HMG Co A reductase level in kidney tissue is shown in Table 7.11. In
heart, there is an increase in total cholesterol, TAG, and TBARS, observed which is
statistically significant (P<0.001) compared with normal diet fed animals. Oral
administration of vanillin semicarbazone to high fat diet treated animals
significantly lowered the TC, TAG, TBARS, and ALP levels.
The pharmacological evaluation of vanillin semicarbazones highlights the
anti-diabetic and hypolipidemic effect in diabetogenic and high fat diet induced
diabetes and hyperlipidemia. The present study provided the fist experimental
evidence that vanillin semicarbazones possess anti-diabetic and hypolipidemic
property. In this study, over 8 vanillin semicarbazones have been synthesized and
only four vanillin semicarbazones tested for anti-diabetic and hypolipidemic
properties. The data from safety profile studies revealed that rodents treated with
vanillin semicarbazone (VSC I) at 3g/kg p.o. and vanillin semicarbazones (VSC I-
IV) at 5g/kg per oral route is devoid of any toxicity except there is a mild change in
biochemical enzymes levels.
However, animals treated with other vanillin semicarbazoness (VSC V-VIII)
showed mortality tested at 3g/kg, so these semicarbazones are excluded for
pharmacological screening. In oral glucose tolerance test (OGTT), rats treated with
vanillin semicarbazones (VSC I-IV) are shown to decrease the hyperglycemic
response at 30min and 60min time interval induced by 2g of glucose. However none
of the vanillin semicarbazones are exhibited hypoglycemic response in OGTT test.
The data from the anti-diabetic studies revealed that oral administration of
vanillin semicarbazones (VSC I-IV) significantly reduced high blood glucose level
in streptozotocin (STZ) induced diabetes. The onset of anti-diabetic effect of was
observed from 1st day onwards and the same effect was observed throughout the
study period. However there is no hypoglycemic response was observed in any of
the tested semicarbazones. It is interesting to observe that administration of vanillin
semicarbazones (VSC I-IV) attenuates total cholesterol and triglycerides in diabetic
animals. This beneficial effect could explain the possible putative effect of
92
semicarbazones in hyperglycemia induced dyslipidemia. Further the histological
investigation of pancreas is in line with biochemical effect. Vanillin semicarbazone
treated diabetic rats pancreas showed augmentation in streptozotocin induced lesion.
The investigation from the high fat induced hyperlipidemic study revealed
that the treatment with vanillin semicarbazones affect total cholesterol and
lipoprotein levels. There is inverse relationship is observed in HDL and LDL level
in animals treated with high fat meal. This effect was significantly reversed by
increasing HDL level and decreasing LDL+VLDL level. The observed benefit
effect of vanillin semicarbazone is partially due to reduction in the total cholesterol,
LDL, TBARS level as well as inhibition of HMG Co A reductase level in the animal
visceral organs fed with high fat meal.
This present study warrants further exploration and exploitation of vanillin
semicarbazones in well validated animal model of hyperlipidemia associated insulin
resistance that lead to development of unified new chemical entity for the
management of Type II diabetes mellitus associated with hyperlipidemia.
Our results validated that the pharmacophoric model with the binding sites is
vital for the antidiabetic associated with hypocholestremic activities. These new
facts might be expedient in the future research and development of vanillin
semicarbazones as novel antidiabetic associated with hypocholestremic agents.
93
CCHHAAPPTTEERR IIXX
CONCLUSION
Superimposition of docking structures of compound 13 and 31 with
PPARα/γ revealed that these analogues superimposed well with each other, whereas
the linker and the tail parts adopt different conformations. The 4-substituted
aromatic centre allows the best fit to the binding site by providing the optimum
distance between the acidic head and oxygen at C-4 position of aromatic centre, thus
moving the hydrophobic tail on aromatic centre close to the hydrophobic region of
protein consisting of serine, tyrosine and histidine, resulting in strong hydrophobic
interactions with these residues. From these findings, it can be suggested that the
designing of new chemical analogues of vanillin semicarbazones and its structural
modification with a very interesting pharmacological profile lead the necessity of
further research.
A series of eight vanillin semicarbazones were synthesized from
aniline/substituted aniline and their proposed structure was established by various
analytical techniques such as IR, 1H NMR and MASS spectral studies. Log p value
was found that all the newly synthesized vanillin semicarbazones have molecular
and drug-likeness properties.
Acute oral toxicity studies concluded that vanillin semicarbazones (I and II
to IV) administered orally with 3g/kg and 5g/kg respectively did not show any toxic
signs and mortality in mice. Anti-diabetic studies concluded that oral administration
of vanillin semicarbazones (I-IV) significantly reduced high blood glucose level in
streptozotocin (STZ) induced diabetes and attenuates total cholesterol and
triglycerides in diabetic animals. This beneficial effect could explain the possible
putative effect of vanillin semicarbazones in hyperglycemia induced dyslipidemia.
The investigation from the high fat induced hyperlipidemic study concluded
that there was a significant reduction in the total cholesterol, LDL, TBARS level as
well as inhibition of HMG Co A reductase level. This present study warrants the
94
further exploration and exploitation of unified new vanillin semicarbazones for the
management of hyperlipidemia associated Type II diabetes mellitus. Such a study
provides precious information for drug development and also serving to highlight
the areas for future research. Further characterization and advanced pharmacological
studies on vanillin semicarbazones are under pipeline.
95
Compound-1 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-chlorophenyl)semicarbazide
96
Compound-1 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-chlorophenyl)semicarbazide
97
Compound-1 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-chlorophenyl)semicarbazide
98
Compound-2 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-bromophenyl) semicarbazide
99
Compound-2 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-bromophenyl) semicarbazide
100
Compound-2 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-bromophenyl) semicarbazide
101
Compound-3 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-fluorophenyl)semicarbazide
102
Compound-3 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-fluorophenyl)semicarbazide
103
Compound-3 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-fluorophenyl)semicarbazide
104
Compound-4 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-nitrophenyl) semicarbazide
105
Compound-4 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-nitrophenyl) semicarbazide
106
Compound-4 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-nitrophenyl) semicarbazide
107
Compound – 5 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-(hydroxymethyl) phenyl) semicarbazide
108
Compound – 5 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-(hydroxymethyl) phenyl) semicarbazide
109
Compound – 5 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(4-(hydroxymethyl) phenyl) semicarbazide
110
Compound-6 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-p-tolylsemicarbazide
111
Compound-6 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-p-tolylsemicarbazide
112
Compound-7 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(3-chloro-4-methylphenyl) Semicarbazide
113
Compound-7 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(3-chloro-4-methylphenyl) Semicarbazide
114
Compound-7 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(3-chloro-4-methylphenyl) Semicarbazide
115
Compound-7 (E)-1-(4-hydroxy-3-methoxybenzylidene)-4-(3-chloro-4-methylphenyl) Semicarbazide
116
Compound-8 (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(sulfonamido phenyl) semicarbazide
117
Compound-8 (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(sulfonamido phenyl) semicarbazide
118
Compound-8 (E)-1-(4-hydroxy-3-methoxy benzylidene)-4-(sulfonamido phenyl) semicarbazide
130
FIG 7.1.1. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE HEART TREATED WITH VANILLIN SEMICARBAZONES ORALLY
Shows hemorrhage, No changes in muscle fibres, Look normal Muscle bundles
a b
c d
e f
131
FIG 7.1.1. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE HEART TREATED WITH VANILLIN SEMICARBAZONES ORALLY
g h
Normal
a. Fluro b. Nitro c. Chloro d. 3-Chloro 4-Methyl e. Bromo f. Hydroxyl methyl g. P-tolyl h. Sulfonamide Description: Magnification 400X Staining – Hematoxylin and eosin
132
FIG 7.1.2. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE LUNGS TREATED WITH VANILLIN SEMICARBAZONES ORALLY
Mild emphysematous changes, Few inter alveolar septae shows edema in some
areas, chronic inflammatory cells in septal area
a b
c d
e f
133
FIG 7.1.2. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE LUNGS TREATED WITH VANILLIN SEMICARBAZONES ORALLY
g h
Normal
a. Fluro b. Nitro c. Chloro d. 3-Chloro 4-Methyl e. Bromo f. Hydroxyl methyl g. P-tolyl h. Sulfonamide Description: Magnification 400X Staining – Hematoxylin and eosin
134
FIG 7.1.3. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE LIVER TREATED WITH VANILLIN SEMICARBAZONES ORALLY
Shows nuclear clearing cells appear normal in some cells, nuclear fragment in some
cells
a b
c d
e f
135
FIG 7.1.3. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE LIVER TREATED WITH VANILLIN SEMICARBAZONES ORALLY
g h
Normal
a. Fluro b. Nitro c. Chloro d. 3-Chloro 4-Methyl e. Bromo f. Hydroxyl methyl g. P-tolyl h. Sulfonamide Description: Magnification 400X Staining – Hematoxylin and eosin
136
FIG 7.1.4. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE KIDNEY TREATED WITH VANILLIN SEMICARBAZONES ORALLY
The Glomeruli show mild shrinkage, normal tubules
a b
c d
e f
137
FIG 7.1.4. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE KIDNEY TREATED WITH VANILLIN SEMICARBAZONES ORALLY
g h
Normal
a. Fluro b. Nitro c. Chloro d. 3-Chloro 4-Methyl e. Bromo f. Hydroxyl methyl g. P-tolyl h. Sulfonamide Description: Magnification 400X Staining – Hematoxylin and eosin
138
FIG 7.1.5. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE STOMACH TREATED WITH VANILLIN SEMICARBAZONES ORALLY
Gastric and intestinal mucosa appears normal
a b
c d
e f
139
FIG 7.1.5. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE STOMACH TREATED WITH VANILLIN SEMICARBAZONES ORALLY
g h
Normal
a. Fluro b. Nitro c. Chloro d. 3-Chloro 4-Methyl e. Bromo f. Hydroxyl methyl g. P-tolyl h. Sulfonamide Description: Magnification 400X Staining – Hematoxylin and eosin
140
FIG 7.1.6. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE SPLEEN TREATED WITH VANILLIN SEMICARBAZONES ORALLY
Looks normal histology (Giant cells)
a b
c d
e f
141
FIG 7.1.6. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE SPLEEN TREATED WITH VANILLIN SEMICARBAZONES ORALLY
g h
Normal
a. Fluro b. Nitro c. Chloro d. 3-Chloro 4-Methyl e. Bromo f. Hydroxyl methyl g. P-tolyl h. Sulfonamide Description: Magnification 400X Staining – Hematoxylin and eosin
142
FIG 7.1.7.SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE TESTIS
TREATED WITH VANILLIN SEMICARBAZONES ORALLY
Spermatogenesis partially arrested, sperms are not seen in the lumens
a b
c d
e f
143
FIG 7.1.7. SHOWS HISTOPATHOLOGICAL SECTIONS OF MICE TESTIS TREATED WITH VANILLIN SEMICARBAZONES ORALLY
g h
Normal
a. Fluro b. Nitro c. Chloro d. 3-Chloro 4-Methyl e. Bromo f. Hydroxyl methyl g. P-tolyl h. Sulfonamide Description: Magnification 400X Staining – Hematoxylin and eosin
144
FIG 7.1.8. SHOWS HISTOPATHOLOGICAL SECTIONS OF RAT
PANCREAS TREATED WITH VANILLIN SEMICARBAZONES ORALLY
a b
c d
e f
145
FIG 7.1.8. SHOWS HISTOPATHOLOGICAL SECTIONS OF RAT
PANCREAS TREATED WITH VANILLIN SEMICARBAZONES ORALLY
Normal
Normal:- Pancreatic morphology Description: a. Fluro b. Nitro c. Chloro d. 3-chloro 4-methyl e. Glibeclamide f. STZ treated pancreas show degenerated (atrophic and small islets) Magnification value 400X Staining – Hemotoxylin and eosin Standard treated pancreas shows less degeneration
146
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