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I
ABSTRACT
A Natural product is a chemical compound or substance
produced by a living organism found in nature that usually has a
Pharmacological/ Biological activity for use in Pharmaceutical Drug
discovery and drug design.
The main advantage of semi synthetic drug is they can act with
higher potency than their original natural products such as onset of
action, potency, site of action etc.
Based on the above facts Four Pharmacologically potential
compounds were selected. The various derivatives of this compound
were synthesized by using simple synthetic procedures. The total
Nineteen semi synthetic derivatives were synthsized and their
structures are conformed by physical and spectral analysis.
All the synthesized compounds were subjected for different
activities, the Anti-bacterial activity of the synthesized compounds were
performed against two Gram positive and Gram negative bacteria. The
compound II, IX and XIII has potent Anti-bacterial activity. Further more
studies on these derivatives for safer and potent Anti-bacterial drug.
The Anti-fungal activity of these derivatives compound I, IX, XV
and XVII has shown moderated activity, thus in future more works has
to be carried out on these derivatives to come up with potent moiety.
II
The compound III to XVIII showed good potent Anti-inflamatory
activity when compared with standard drug.
The acute toxicity studies showed that all theNineteen derivatives
were safe even up 1000mg/kg and thus a dose of 300mg/kg i.p. was
used as safer dose in experimental animals.
Based on the above it could be concluded that compound
III, IV, IX and XIII were found to have good potency in all activity
performed. Thus structure of these derivatives has to be optimized to
explore the desired Pharmacological activity
III
LIST OF ABBREVIATIONS
ALT - Alanine Amino Transferase
ACE - Angiotensin Converting Enzyme
ASA - Acetyl Salicylic Acid
AST - Aspartate Amino Transferase
COPD - Chronic Obstructive Pulmonary Disease
MeOH - Methanol
CDK - Cyclin Dependent Kinase
GABA - Gama Amino Butyric Acid
IC50 - 50% Inhibitory Concentration
gm - gram
FDA - Food Drug Administration
cfu - Colony Forming Units
TLC - Thin Layer Chromatography
DSB - Bromovanin-induced DNA double-strand breaks
FAH - Fumaryl Acetoacetate Hydro-Lase
MGYP - Maltose, glucose, yeast extract and peptone
DMSO - Dimethyl Sulfoxide
HDAC - Histone Deacetylase
HDL-C - High-Density Lipoprotein-Cholesterol
HPPD - p -Hydroxyphenylpyruvate Dioxygenase (HDL-C
HMG-CoA - 3-Hydroxy-3-Methylglutaryl Coenzyme A
IV
IMPDH - Inosine Monophosphate Dehydrogenase
LDL-C - Low-Density Lipoprotein-Cholesterol
d - Doublet
m - Multiplet
s - Singlet
Et2O - Diethyl Ether
Ar-H - Aromatic Protons
CAT - Catalase
GGT - Gamma – glutamyltransferase
GST - GLUTATHIONE-S-TRANSFERASE
GSH - Glutathione
MTT - MICROCULTURE TETRAZOLIUM
MRSA - Methicillin Resistant Staphylococcus Aureus
NCE - New Chemical Entity
FBS - Fetal Bovine Serum
ROS - Reactive Oxygen Species
PMR - Proton Magnetic Resonance
IR - Infrared
COX-2 - Cyclo-Oxygenase-2
PGE2 - Prostaglandin E2
NO - Nitric oxide
iNOS - Inducable Nitric Oxide Synthase
LSD - Lysergic Acid Diethylamide
V
LPS - Lipopolysaccharide
SOD - Superoxide Dismutase
ACE - Angiotensin Converting Enzyme
w/w - Weight/Weight
ml - Milli litre
conc. - Concentrated
mg/g - Milli gram / gram
mg/dl - Milli gram / deci litre
BUN - Blood Urea Nitrogen
MDA - Malondialdehyde
G - Gram
h - Hour
min - Minutes
mg / kg - Milli gram / kilogram
CMC - Carboxy Methyl Cellulose
b.w - Body Weight
TMS - Tetra Methyl Silane
mmol/L - Milli moles / litre
N - Normality
M - Molarity
TCA - Trichloro Acetic Acid
DPPH - 2, 2- Diphenyl 1-Picryl Hydrazyl
ABTS - 2, 2΄Azino Bis (3-Ethylbenzo- Thiazoline – 6-
Sulphuric Acid)
PBS - Phosphate Buffer Saline
VI
LIST OF TABLESTable No TITLE Page
No
1 Various uses of herbals in treatment and diagnosis 03
2 List of chemicals and their manufactures used for synthesis 80
3 List of Equipments used during the Experiments 81
4 Structure and nomenclature of Citral derivatives(Compound I, II) 94
5 Structure and nomenclature of Vanillin derivatives(Compound III - V) 95
6 Structure and nomenclature of Vanillin derivatives(Compound VI - VIII ) 96
7 Structure and nomenclature of Vanillin derivatives(Compound IX - XI) 97
8 Structure and nomenclature of Carvone derivatives(Compound XII -XIV ) 98
9 Structure and nomenclature of Carvone derivatives(Compound XV - XVI) 99
10 Structure and nomenclature of Camphor derivatives(Compound XVII - XIX) 100
11 Physical properties of the synthesized compounds (I-XIX) 101
12 TLC profile of the synthesized compounds (I – XIX) 102
13 Elemental analysis of novel semisynthetic compounds (I –XIX) 103
14 FT-IR Spectral Datas of the Semisynthetic Compounds (I –XIX) 104-07
15 1HNMR Spectral Datas of compounds (I – XIX) 108-111
16 Antibacterial Activity of the Compounds (I – IX) 187
17 Antifungal Activity of Compounds (I – IX). 188
18 Antioxidant activity of compounds (I-IX). 189
19 Anti-inflammatory activity of the compounds (I –XIX). 190
20 Analgesic activity of the compounds (I –XIX) 191
21 Anthelmintic activity of compounds (I –XIX) 192
22 List of the Newly Synthesized Derivatives along with theirIUPAC Name 219
VII
LIST OF FIGURES (INCLUDING FT-IR, HNMR AND MASS SPECTRAS).
FIGURENO. PARTICULARS PAGE
NO.1 Precursor and Derivative of Salicylic Acid. 9
2 Artemisinin 10
3 Cocaine and Quinine 11
4 Vincristine 12
5 New drugs from terrestrial plants 16
6 Plant-derived drug candidates. 19
7 New drugs from terrestrial microorganisms (2000 to2005). 23
8 Vincristine 33
9 Pictorial representation of various parts of Vanillin 34,35
10 Carvone 36
11 Camphor 37
12 Isomers of Citral 39
13 Schemes for synthesis of compound I-III 74
14 Schemes for synthesis of compound IV-VI 75
15 Schemes for synthesis of compound VII-IX 76
16 Schemes for synthesis of compound X – XII 77
17 Schemes for synthesis of compound XIII – XVI 78
18 Schemes for synthesis of compound XVII –XIX 79
19 FT-IR Spectrum of Compound I 112
20 HNMR Spectrum of Compound I 113
21 MASS Spectrum of Compound I 114
22 FT-IR Spectrum of Compound II 115
23 HNMR Spectrum of Compound II 116
24 MASS Spectrum of Compound II 117
25 FT-IR Spectrum of Compound III 118
VIII
26 HNMR Spectrum of Compound III 119
27 MASS Spectrum of Compound III 120
28 FT-IR Spectrum of Compound IV 121
29 HNMR Spectrum of Compound IV 122
30 MASS Spectrum of Compound IV 123
31 FT-IR Spectrum of Compound V 124
32 HNMR Spectrum of Compound V 125
33 MASS Spectrum of Compound V 126
34 FT-IR Spectrum of Compound VI 127
35 HNMR Spectrum of Compound VI 128
36 MASS Spectrum of Compound VI 129
37 FT-IR Spectrum of Compound VII 130
38 HNMR Spectrum of Compound VII 131
139 MASS Spectrum of Compound VII 132
40 FT-IR Spectrum of Compound VIII 133
41 HNMR Spectrum of Compound VIII 134
42 MASS Spectrum of Compound VIII 135
43 FT-IR Spectrum of Compound IX 136
44 HNMR Spectrum of Compound IX 137
45 MASS Spectrum of Compound IX 138
46 FT-IR Spectrum of Compound X 139
47 HNMR Spectrum of Compound X 140
48 MASS Spectrum of Compound X 141
49 FT-IR Spectrum of Compound XI 142
50 HNMR Spectrum of Compound XI 143
51 MASS Spectrum of Compound XI 144
52 FT-IR Spectrum of Compound XII 145
53 HNMR Spectrum of Compound XII 146
54 MASS Spectrum of Compound XII 147
55 FT-IR Spectrum of Compound XIII 148
IX
56 HNMR Spectrum of Compound XIII 149
57 MASS Spectrum of Compound XIII 150
58 FT-IR Spectrum of Compound XIV 151
59 HNMR Spectrum of Compound XIV 152
60 MASS Spectrum of Compound XIV 153
61 FT-IR Spectrum of Compound XV 154
62 HNMR Spectrum of Compound XV 155
63 MASS Spectrum of Compound XV 156
64 FT-IR Spectrum of Compound XVI 157
65 HNMR Spectrum of Compound XVI 158
66 MASS Spectrum of Compound XVI 159
67 FT-IR Spectrum of Compound XVII 160
68 HNMR Spectrum of Compound XVII 161
69 MASS Spectrum of Compound XVII 162
70 FT-IR Spectrum of Compound XVIII 163
71 HNMR Spectrum of Compound XVIII 164
72 MASS Spectrum of Compound XVIII 165
73 FT-IR Spectrum of Compound XIX 166
74 HNMR Spectrum of Compound XIX 167
75 MASS Spectrum of Compound XIX 168
76 A broad classification of reactive oxygen species 172
1
INTRODUCTION
Nature always stands as a golden mark to exemplify the
outstanding phenomenon of symbiosis. The history of herbal medicines
is as old as human civilization. The documents, many of which are of
great antiquity, revealed that plants were used medicinally in China,
India, Egypt and Greece long before the Christian era. The oldest
known herbal is Pen-t`sao written by emperor Shen nung around 300
B.C It contains 365 drugs, one for each day of the year (Satyajit D
Sarker, 2004).
Ancient Chinese and Egyptian papyrus writings describe
medicinal uses for plants. Indigenous cultures (such as African and
Native American) used herbs in their healing rituals, while others
developed traditional medical systems (such as Ayurveda and
Traditional Chinese Medicine) in which herbal therapies were used.
Researchers found that people in different parts of the world tended to
use the same or similar plants for the same purposes. In the early 19th
century, when chemical analysis first became available, scientists
began to extract and modify the active ingredients from plants. Later,
chemists began making their own version of plant compounds, and over
time, the use of herbal medicines declined in favor of drugs (Kokate C
K, 2007).
2
Herbal medicine also called botanical medicine or phytomedicine
refers to using a plant's seeds, berries, roots, leaves, bark, or flowers for
medicinal purposes. Herbalism has a long tradition of use outside of
conventional medicine. Herbs having Medicinal Properties in nature,
there are a huge variety of herbs, having medicinal properties and they
are used to prepare the herbal medicines. They can be used directly in
the form of extracts or tea, or used to produce the drugs. Herbs such as
St. John’s Wort, ginkgo, echinacea, and ginseng are among the most
popular herbs. In 1999, echinacea was reported to make up 38% of the
U.S. market, with ginkgo a close second at 34%. The efficacy of these
herbs is being investigated in many laboratories, and efforts are also
being made to isolate and identify any active constituents. It is
becoming more main stream as improvements in analysis and quality
control along with advances in clinical research show the value of
herbal medicine in the treating and preventing disease (Newman DJ,
2007).
Several herbs consist of powerful ingredients, which are helpful to
cure a number of health problems. They can be used safely, without
causing any side effects. Some of the commonly used herbs are
American ginseng, bee pollen, astragalus, cat's claw, black cohosh,
bladder wrack, chamomile, feverfew, damiana, dong quai, flaxseed,
ginger, garlic, ginkgo biloba, grape seed, green tea, licorice, muira
3
puama, saw palmetto, suma, schizandra, tea tree, turmeric, soy
isoflavones, white willow, etc (Dan Bensky, 2004).
Ephedra is an appetite suppressant. It is used to treat asthma or
bronchitis. Echinacea helps strengthen immune system and protects
from flu and common cold. Feverfew is used to prevent migraine
headache and also helps manage allergies, rheumatic disease and
arthritis. Kava-kava is used to treat stress, anxiety and restlessness.
Ginkgo increases oxygenation and blood circulation and helps improve
memory and concentration. Valerian is a muscle relaxant and a mild
sedative and helps deal with insomnia. Ginger decreases and prevents
vertigo, nausea and vomiting( Hernan Garcia, 1999).
Interest in the United States had been growing in the recent years
from the reported success stories from the use of herbs. For example,
St. John's Wort is widely used in the treatment of mild depression
without the need for Prozac. St. John's Wort does not have the side
effects such as that of Prozac. There are some Ayurvedic herbs that are
very useful for reducing cholesterol, diabetes etc. Similarly the
popularity of Ginseng and Ginkgo biloba (ginkgo) is rising due to its
beneficial effects ( El- Shemy HA, 2003).
From the review, it is obvious that there is growing economic
value of medicinal plants that the developing countries need to harness
in order to improve their economic and health care delivery systems.
4
The “pharmergeing” nations appear to understand this economic
dynamics and are living up to the challenges. In particular, developing
counties from African region need to put in more effort in order to face
these health and economic challenges especially in the face of
resurgence and emergency of resistint strains of pathogenic micro-
organisums and cancers that had become serious threat to our
collective survival (Bukata B. Bukar. May 2016).
Table No. 1 various uses of herbals in treatment and diagnosis
Plant Family Uses
Digitalis
purpurea
Scrophulariaceae As a cardiotonic
Chondrodendron
tomentosum
Loganiaceae Neuromuscular blocker
Panex ginseng Araliacea Immunomodulatory,
sedativeCinchona
officinalis
Rubiacea Antimalarial
Rauwolfia
serpentina
Apocynaceae Antihypertensive
Datura metel Solanaceae Anticholinergic
Papaver
somniferum
Papaveraceae Hypnotic,sedative,analgesic
Curcuma longa Zingiberaceae Anti-inflammatory,
condimentAloe
barbadensis
Liliaceae In cosmetic preparations
Catharanthus
roseus
Apocynaceae Anticancer herb
Areca catechu Palmae Respiratory, stimulant.
NtihelmenthicHolarrhena Apocynaceae Antiamoebic
Veratrum album Liliaceae Cardiac depressant
5
Advantages of Herbal Medicines
Allopathic medicines are very costly. In contrast, herbal medicines
are very cheap. This cost effectiveness makes them all the more
alluring. Herbal medicines can be brought without prescription
and they are available in all most all health stores. Some herbs
can even be grown at home.
For certain ailments, herbal medicines are considered to be more
effective than allopathic medicines.
Herbal medicines do not have any side effects, as they are free
from chemicals. They are also milder than allopathic medicines.
The natural detoxification process of the body is effectively
enhanced by herbal medicines. They can be used to cleanse the
colon, improve digestion and food absorption. Herbal medicines
are also very good in boosting the immune system.
Herbal medicines are very effective in curing various digestive
disorders like colitis, indigestion, peptic ulcers, and irregular
bowel movements.
These types of medicines are best for people who are allergic to
various types of drugs.
Herbal medicines are also effective in boosting the mental health.
6
Most of the ailments related to blood circulation like high blood
pressure, varicose ulcers, and many others can be controlled
through herbal medicine.
Some herbal medicines are very good in reducing the cholesterol
level in the blood stream. They are also used to treat coronary
artery diseases.
Herbal medicine can be used to reduce weight by regulating
appetite.
An example may be seen with herbs and alternative remedies
used to treat arthritis. Vioxx, a well-known prescription drug uses
to treat arthritis, was recalled due to increased risk of
cardiovascular complications. Alternative treatments for arthritis,
on the other hand, have few side effects. Adjusting the diet to
remove vegetables from the nightshade family, reducing white
sugar consumption, and adding simple herbs to the diet have few
side effects. Most herbal medicines are well tolerated by the
patient, with fewer unintended consequences than
pharmaceutical drugs.
Another advantage to herbal medicine is cost. Herbs cost much
less than prescription medications. Research, testing, and
marketing add considerably to the cost of prescription medicines.
Herbs tend to be inexpensive compared to drugs.
7
The chemical medicine prescribed by a pharmacist could have
certain negative side effects. However, many of the herbal
medicines and remedies do not have negative side effects. If any,
they are softer than allopathic medicine.
Herbal medicine can be effectively used for body’s natural
detoxification process. The herbs such as Plantago psyllium
seed, rhubarb juice powder, aloe vera, alfalfa juice, chlorella,
carrot concentrate and garlic can be used to cleanse the colon,
improve digestion and food absorption and boost your immune
system. Some digestive disorders such as colitis, indigestion,
peptic ulcers and irritable bowel syndrome can be cured using the
herbs.
Herbal medicine which includes herbs such as ginger, capsicum,
garlic and motherwort help to control the ailments related to blood
circulation such as high blood pressure, varicose ulcers and so
on. Many of the herbal medicines are used to treat coronary
artery disease and to reduce cholesterol level in the blood stream
and
Obesity is the cause of many of the health problems. Herbal
medicine can help reduce excess weight and regulate appetite.
8
Disadvantages of Herbal Medicine (Merrilield RB, 1975)
Herbs are not without some disadvantages. For sudden, serious
illnesses, mainstream medicine still reigns supreme. An herbalist
would not be able to treat serious trauma, such as a broken leg,
nor would be able to heal appendicitis or a heart attack as
effectively as a conventional doctor using modern diagnostic
tests, surgery, and drugs. Modern medicine treats sudden illness
and accidents much more effectively than herbal or alternative
treatments.
Herbal medicines take too much time to act. The entire process is
very slow.
There is also a remote chance that herbal medicine may not give
the desired result.
Some plant chemicals can be toxic to the body. In addition,
certain ingredients react differently with different people. So, it is
always necessary to test the herbal medicine to check that it is
not allergic to the body.
Some herbal medicines can cause negative side effects. These
side effects may also take a long time to revel.
Herbal medicines are also not properly regulated and so they do
not carry any quality assurance.
9
Herbal medicines require very good practitioners and these are
very few. Most of the ‘Doctors’ that populate the commercial
herbal remedy market are not qualified and so people must stay
away from them.
Another disadvantage of herbal medicine is the very real risks of
doing oneself harm through self-dosing with herbs. While one can
argue that the same thing can happen with medications, such as
accidentally overdosing on cold remedies, many herbs do not
come with instructions or package inserts. There’s a very real risk
of overdose. Harvesting herbs in the wild is risky, if not foolhardy,
yet some people try to identify and pick wild herbs. They run a
very real risk of poisoning themselves if they don’t correctly
identify the herb, or if they use the wrong part of the plant.
Herbal treatments can interact with medications. Nearly all herbs
come with some warning, and many, like the herbs used for
anxiety such as Valerian and St. John’s Wort, can interact with
prescription medication such as antidepressants. It’s important to
discuss your medications and herbal supplements with your
Doctor and
Because herbal products are not tightly regulated, consumers
also run the risk of buying inferior quality herbs. The quality of
herbal products may vary among batches, brands or
10
manufacturers. This can make it much more difficult to prescribe
the proper dose of an herb.
Phytochemicals which are chemicals derived from plants.
Specifically, phytochemistry describes the large number of
secondarymetabolic compounds found in plants. The
inconsistencies in chemicals are observed due to environmental
conditions like soil, temperature, light, rainfall and humidity.
Recently, the World Health Organization estimated that 80% of
people worldwide rely on herbal medicines for some part of their
primary health care. In Germany, about 600 - 700 plant-based
medicines are available and are prescribed by some 70% of German
physicians. In the last 20 years in the United States, public
dissatisfaction with the cost of prescription medications, combined with
an interest in returning to natural or organic remedies, has led to an
increase in herbal medicine use.
A natural product is a chemical compound or substance produced by
a living organism - found in nature that usually has a pharmacological or
biological activity for use in pharmaceutical drug discovery and drug
design. A natural product can be considered as such even if it can be
prepared by total synthesis.
Natural products are products from various natural sources, plants,
microbes and animals. Natural products can be an entire organism (e.g.
11
a plant, an animal or a micro-organism), a part of an organism (e.g.
leaves or flowers of a plant, an isolated animal organ), an extract of an
organism or part of an organism and an exudate, or pure compound
(e.g. alkaloids, coumarins, flavonoids, lignans, steroids and terpenoids)
isolated from plants, animals or micro-organisms. However, in practice,
the term natural product refers to secondary metabolites, small
molecules (molecular weight < 1500 amu), produced by an organism,
but not strictly necessary for the survival of the organism.
These small molecules provide the source of inspiration for the
majority of FDA- approved agents and continue to be one of the major
sources of inspiration for drug discovery. In particular, these compounds
are important in the treatment of life-threatening conditions.
Natural products may be extracted from tissues of terrestrial
plants, marine organisms or microorganism fermentation broths. A
crude (untreated) extract from any one of these sources typically
contains novel, structurally diverse chemical compounds, which the
natural environment is a rich source of Chemical diversity in nature is
based on biological and geographical diversity, so researchers travel
around the world obtaining samples to analyze and evaluate in drug
discovery screens or bioassays. This effort to search for natural
products is known as bioprospecting.
12
In the past, traditional peoples or ancient civilizations depended greatly
on local flora and fauna for their survival. They would experiment with
various berries, leaves, roots, animal parts or minerals to find out what
effects they had. As a result, many crude drugs were observed by the
local healer or shaman to have some medical use. Although some
preparations may have been dangerous, or worked by a ceremonial or
placebo effect, traditional healing systems usually had a substantial
active pharmacopoeia, and in fact most western medicines up until the
1920s were developed this way. Some systems, like traditional Chinese
medicine or Ayurveda were fully as sophisticated and as documented
systems as western medicine, although they might use different
paradigms.
As a result of rapid development of phytochemistry and
pharmacological testing methods in recent years, new plant drugs are
finding their way into medicine as purified phytochemicals, rather than in
the form of traditional galenical preparations.
The earliest pure compounds discovered were salicin (1), isolated
from the bark of the white willow, Salix alba, in 1825-26. It was
subsequently converted to salicylic acid (2) via hydrolysis and oxidation,
and proved as successful as an antipyretic (fever reducing) that it was
actively manufactured and used worldwide. The use of salicylic acid,
however, often led to severe gastrointestinal toxicity. This was
13
overcome when Felix Hoffmann of Bayer Company converted salicylic
acid into acetylsalicylic acid (3,ASA) via acetylation. Bayer then began
marketing ASA under the trade name aspirin in 1899. Today, aspirin is
still the most widely used analgesic and antipyretic drug in the world.
O
OOH
HH
OH
OH
HHH
OH
OH OH
OH O
OH
OAc O
Salicin(1) Salicylic Acid (2) Acetyl Salicylic Acid (3)
Fig. No. 1. Precursor and Derivative of Salicylic Acid.
Many of aqueous, ethanolic, distilled, condensed or dried extracts do
indeed have a real and beneficial effect, and a study of ethno botany
can give clues as to which plants might be worth studying in more
detail. Rhubarb root has been used as a purgative for many centuries.
In China, it was called "The General" because of its "galloping charge"
and was only used for one or two doses unless processed to reduce its
purgative qualities. (Bulk laxatives would follow or be used on weaker
patients according to the complex laxative protocols of the medical
system.) The most significant chemicals in rhubarb root are
anthraquinones, which were used as the lead compounds in the design
of the laxative dantron.
The extensive records of Chinese medicine about response to
Artemisia preparations for malaria also provided the clue to the novel
14
antimalarial drug, Artemisinin (4). The therapeutic properties of the
opium poppy (active principle morphine) were known in Ancient Egypt,
were those of the solanaceae plants in ancient Greece (active principles
atropine and hyoscine).
O
CH3
H
CH3H
CH3
H
OO
Fig.No.2. Artemisinin ( 4)
The snakeroot plant was well regarded in India (active principle
reserpine), and herbalists in medieval England used extracts from the
willow tree(salicin) and foxglove (active principle digitalis - a mixture of
compounds such as digitoxin, digitonin, digitalin). The Aztec and Mayan
cultures of Mesoamerica used extracts from a variety of bushes and
trees including the ipecacuanha root (active principle emetine), coca
bush (active principle cocaine ,5), and cinchona bark (active principle
quinine) (James J, Knittel, 2008).
O
N
CH3
OOCH3
O
N
NOH
H3CO
CH2
Cocaine(5) Quinine(6)
Fig.No.3. Cocaine and Quinine
15
Some of the natural drugs may not have potency to treat diseases. So
there the concept of semi synthetic chemistry araised and for the first
time in 1869 Brown and Fraser while working on relationship between
molecular structure and biological activity, identified that N-Methyl
morphine and N-Methylatropin are muscle relaxants instead their parent
natural compounds morphine is an analgesic and atropine is an
mydriatic agent. Then after working on the semi synthetic compounds
increased and further investigations were carried out ( El- Shemy HA et
al., 2003).
Not all natural products can be fully synthesized and many natural
products have very complex structures that are too difficult and
expensive to synthesize on an industrial scale. These include drugs
such as penicillin, morphine and paclitaxel (Taxol). Such compounds
can only be harvested from their natural source - a process which can
be tedious, time consuming, and expensive, as well as being wasteful
on the natural resource. For example, one yew tree would have to be
cut down to extract enough paclitaxel from its bark for a single dose.
Furthermore, the number of structural analogues that can be obtained
from harvesting is severely limited (Friedrich Wohler et al., 1828).
A further problem is that isolates often work differently than the
original natural products which have synergies and may combine, say,
antimicrobial compounds with compounds that stimulate various
16
pathways of the immune system. Many higher plants contain novel
metabolites with antimicrobial and antiviral properties. However, in the
developed world almost all clinically used chemotherapeutics have been
produced by in vitro chemical synthesis. Exceptions, like taxol and
vincristine, were structurally complex metabolites that were difficult to
synthesize in vitro. Many non-naturals, synthetic drugs produce severe
side effects that were not acceptable except as treatments of last resort
for terminal diseases such as cancer. The metabolites discovered in
medicinal plants may avoid the side effect of synthetic drugs, because
they must accumulate within living cells ( Merrilield RB, 1975).
Many higher plants contain novel metabolites with antimicrobial
and antiviral properties. However, in the developed world almost all
clinically used chemotherapeutics have been produced by in vitro
chemical synthesis. Exceptions, like Taxol and Vincristine, were
structurally complex metabolites that were difficult to synthesize in vitro.
Many non-natural, synthetic drugs produce severe side effects that
were not acceptable except as treatments of last resort for terminal
diseases such as cancer. The metabolites discovered in medicinal
plants may avoid the side effect of synthetic drugs, because they must
accumulate within living cells.
17
NH
N
OH C H 3
N
N C H 3
COOMe
COOMe
OH
CHOMeO H
Fig. No.4. Vincristine (7)
The antimicrobial activity of plants can sometimes be attributed to
the low molecular weight phenolic compounds that are present within
them.
Semi synthetic procedures can sometimes get around these problems.
This often involves harvesting a biosynthetic intermediate from the
natural source, rather than the final (lead) compound itself. The
intermediate could then be converted to the final product by
conventional synthesis. This approach can have two advantages. First,
the intermediate may be more easily extracted in higher yield than the
final product itself. Second, it may allow the possibility of synthesizing
analogues of the final product. The semi synthetic penicillins are an
illustration of this approach. Another recent example is that of paclitaxel.
It is manufactured by extracting 10-deacetylbaccatin III from the needles
of the yew tree, then carrying out a four-stage synthesis (Muthuswamy
Raghunathan, 2009).
18
The main advantage of the semi-synthetic drugs is they can act
with higher potency than their original natural compounds and also
helps in overcoming the problems of the natural products such as onset
of action , potency, site of action, etc,.
Natural product medicines have come from various source materials
including terrestrial plants, terrestrial microorganisms, marine
organisms, and terrestrial vertebrates and invertebrates (Newman DJ,
2000). The importance of natural products in modern medicine has
been discussed in recent reviews and reports (Jones WP, 2006). The
value of natural products in this regard can be assessed using 3 criteria:
(1) The rate of introduction of new chemical entities of wide structural
diversity, including serving as templates for semisynthetic and total
synthetic modification.
(2) The number of diseases treated or prevented by these substances.
(3) Their frequency of use in the treatment of disease.
An analysis of the origin of the drugs developed between 1981 and
2002 showed that natural products or natural product- derived drugs
comprised 28% of all new chemical entities (NCEs) launched onto the
market (Newman DJ, 2003). In addition, 24% of these NCEs were
synthetic or natural mimic compounds, based on the study of
pharmacophores related to natural products.11 This combined
percentage (52% of all NCEs) suggests that natural products are
19
important sources for new drugs and are also good lead compounds
suitable for further modification during drug development. The large
proportion of natural products in drug discovery has stemmed from the
diverse structures and the intricate carbon skeletons of natural
products. Since secondary metabolites from natural sources have been
elaborated within living systems, they are often perceived as showing
more “drug-likeness and biological friendliness than totally synthetic
molecules” (Koehn FE et al., 2005) making them good candidates for
further drug development (Drahl C, 2005).
Scrutiny of medical indications by source of compounds has
demonstrated that natural products and related drugs are used to treat
87% of all categorized human diseases (48/55), including as
antibacterial, anticancer, anticoagulant, antiparasitic, and
immunosuppressant agents, among others. There was no introduction
of any natural products or related drugs for 7 drug categories
(anesthetic, antianginal, anti histamine, anxiolytic, chelator and antidote,
diuretic, and hypnotic) during 1981 to 2002.2 In the case of antibacterial
agents, natural products have made significant contributions as either
direct treatments or templates for synthetic modification. Of the 90
drugs of that type that became commercially available in the United
States or were approved worldwide from 1982 to 2002, 79% can be
traced to a natural product origin.
20
Frequency of use of natural products in the treatment or
prevention of disease can be measured by the number or economic
value of prescriptions, from which the extent of preference and/or
effectiveness of drugs can be estimated indirectly. According to a study
by Grifo and colleagues (Friedrrich Wohler, 1828), 84 of a
representative 150 prescription drugs in the United States fell into the
category of natural products and related drugs. They were prescribed
predominantly as anti-allergy/ pulmonary/respiratory agents, analgesics,
cardiovascular drugs, and for infectious diseases. Another study found
that natural products or related substances accounted for 40%, 24%,
and 26%, respectively, of the top 35 worldwide ethical drug sales from
2000, 2001, and 2002. of these natural product-based drugs, paclitaxel
(ranked at 25 in 2000), a plant-derived anticancer drug, had sales of
$1.6 billion in 2000.10,11 The sales of categories of plant-derived
cancer chemotherapeutic agents were responsible for approximately
one third of the total anticancer drug sales worldwide, or just under $3
billion dollars in 2002; namely, the taxanes, paclitaxel and docetaxel,
and the camptothecin derivatives, irinotecan and topotecan (Thayer A et
al., 2003).
New drugs derived from natural sources launched in the 6-year
period from 2000 to 2005, and drug candidates from natural sources in
clinical trials during the same time period arranged according to their
21
origin (terrestrial plants, terrestrial microorganisms, marine organisms,
and other natural sources). For drug candidates in clinical trials (Butler
MS, 2005), only examples of new chemical templates of potential
cancer chemotherapeutic drugs will be mentioned.
Drug Discovery from Terrestrial Plants:
Terrestrial plants, especially higher plants, have a long history of
use in the treatment of human diseases. Several well-known species,
including Licorice (Glycyrrhiza glabra), Myrrh (Commiphora species),
and Poppy capsule latex (Papaver somniferum), were referred to by the
first known written record on clay tablets from Mesopotamia in 2600 BC,
and these plants are still in use today for the treatment of various
diseases as ingredients of official drugs or herbal preparations used in
systems of traditional medicine.11 Furthermore, morphine, codeine,
noscapine (narcotine), and papaverine isolated from P. somniferum
were developed as single chemical drugs and are still clinically used.
Hemisuccinate carbenoxolone sodium, a semi-synthetic derivative of
glycyrrhetic acid found in licorice, is prescribed for the treatment of
gastric and duodenal ulcers in various countries (Dewick PM, 2002).
22
Fig. No. 5. New drugs from terrestrial plants
Historical experiences with plants as therapeutic tools have
helped to introduce single chemical entities in modern medicine. Plants,
especially those with ethno- pharmacological uses, have been the
primary sources of medicines for early drug discovery. In fact, a recent
analysis by Fabricant and Farnsworth showed that the uses of 80% of
122 plant-derived drugs were related to their original
ethnopharmacological purposes (Fabricant DS et al., 2001). Current
drug discovery from terrestrial plants has mainly relied on bioactivity-
23
guided isolation methods, which, for example, have led to discoveries of
the important anticancer agents, paclitaxel from Taxus brevifolia and
camptothecin from Camptotheca acuminate (Kinghorn AD, 1994). Other
NCEs discovered or modified from terrestrial plants between 2000 –
2005 are summarized in Fig.No.5.
Approved Drugs:
Apomorphine hydrochloride(8), a short-acting dopamine D1 and
D 2 receptor agonist, is a potent dopamine receptor agonist used to
treat Parkinson ’s disease, a chronic neurodegenerative disease caused
by the loss of pigmented mesostriatal dopaminergic neurons linking the
substantia nigra (pars compacta) to the neostriatum (caudate nucleus
and putamen). Apomorphine is a derivative of morphine isolated from
poppy (Papaver somniferum). Subcutaneous apomorphine is currently
used for the management of sudden, unexpected and refractory
levodopa induced off states in fluctuating Parkinson’s disease (Deleu D
et al., 2004).
Tiotropium bromide (9) has been approved by the United States
Food and Drug Administration (FDA) for the treatment of bronchospasm
associated with (COPD). Tiotropium, a derivative of atropine from
Atropa belladonna (Solanaceae) and related tropane alkaloids from
other solanaceous plants, is a potent reversible nonselective inhibitor of
muscarinic receptors. Tiotropium is structurally analogous to
24
ipratropium, a commonly prescribed drug for COPD, but has shown
longer-lasting effects (Koumis T et al., 2005).
Nitisinone (10) is a derivative of leptospermone, an important new
class of herbicides from the bottlebrush plant (Callistemon citrinus), and
exerts an inhibitory effect for p -hydroxyphenylpyruvate dioxygenase
(HPPD) involved in plastoquinone synthesis (Hall MG, 2001). This drug
has been used successfully as a treatment of hereditary tyrosinaemia
type 1 (HT-1), a severe inherited disease of humans caused by a
deficiency of fumaryl acetoacetate hydrolase (FAH), leading to
accumulation of fumaryl and maleyl acetoacetate, and progressive liver
and kidney damage (Mitcnell G, 2001).
Galantamine hydrobromide (11) is an Amaryllidaceae alkaloid
obtained from Galanthus nivalis that has been used traditionally in
Bulgaria and Turkey for neurological conditions (Howes M-JR, 2003)
(Heinrich M, 2004), and was launched onto the market as a selective
acetylcholinesterase inhibitor for Alzheimer’s disease treatment, slowing
the process of neurological degeneration by inhibiting
acetylcholinesterase as well as binding to and modulating the nicotinic
acetylcholine receptor.
Arteether (11), an antimalarial agent, has been developed from
artemisinin, a sesquiterpene lactone isolated from Artemisia annua
(Asteraceae), a plant used in traditional Chinese medicine as a remedy
25
for chills and fevers. Other derivatives of artemisinin are in various
stages of clinical development as antimalarial drugs in Europe (Van
agtmael et al., MA, 1999).
Plant-derived Compounds Currently in Clinical Trials:
Plant derived secondary metabolites, several new chemical
entities (Fig.No.6) are undergoing clinical trials including four that are
derivatives of known anticancer drugs (camptothecin, paclitaxel,
epipodophyllotoxin, and vinblastine).In addition, combretastatin A4,
isolated from the South African medicinal tree, Combretum caffrum
(Combretaceae), was derivatized to combretastatin A4 phosphate (12)
and AVE-8062 ( 13) (Cirla A, 2003, Pinney KG 2005). These analogs
bind to tubulin leading to morphological changes and then disrupt tumor
vasculature, and are in phase II trials (West CML et al., 2004).
26
Fig.No.6. Plant-derived drug candidates.
Homoharringtonine (14), a cephalotaxus alkaloid from the tree,
Cephalotaxus harringtonia found in mainland China (Powell RG, 1970),
is an inhibitor of protein synthesis and is reported to have activity
against hematologic malignancies (Kantarjian HM et al., 2001). Ingenol
3-Oangelate
(15), an analog of the polyhydroxy diterpenoid, ingenol, which was
originally obtained from Euphorbia peplus (known as “ petty spurge ” in
England or “ radium weed ” in Australia), is a potential topical
27
chemotherapeutic agent for skin cancer and exhibits its action through
activation of protein kinase C (Kedei N, 2004). Phenoxodiol (16), a
synthetic analog of daidzein, a well known isoflavone from soybean
(Glycine max), is being developed as a therapy for cervical, ovarian,
prostate, renal, and vaginal cancers, and induces apoptosis through
inhibition of anti-apoptotic proteins including XIAP and FLIP. (Kamsteeg
M, 2003)Phenoxodiol is currently undergoing clinical studies in the
United States and Australia (Constantinou AI, 2003).
Protopanaxadiol (17), a derivative of a triterpene aglycone of
several saponins from ginseng (Panax ginseng), exhibits its apoptotic
effects on cancer cells through various signaling pathways, and is also
reported to be cytotoxic against multidrug resistant tumors (Shibata S,
1963). Triptolide, a diterpene triepoxide, was isolated from Tripterygium
wilfordii, and has been used for autoimmune and inflammatory diseases
in the People’s Republic of China (Kiviharju TM, et al., 2002). PG490 –
88 (18), 14-succinyl triptolide sodium salt), a semisynthetic analog of
triptolide, exerts antiproliferative and proapoptotic activities on primary
human prostatic epithelial cells as well as tumor regression of colon and
lung xenografts (Fidler JM et al., 2003).
Thus an attempt was done in this study to derive some semi
synthetic derivatives of well known compounds like citral, vanillin,
carvone and camphor.
28
Citral:Citral (C10H16O), is present in the oils of several plants, including
lemon myrtle (90-98%), Litsea citrata (90%), Litsea cubeba (70-85%),
lemongrass (65-85%), lemon tea-tree (70-80%), Ocimum gratissimum
(66.5%), Lindera citriodora (about 65%), Calypranthes parriculata
(about 62%), petitgrain (36%), lemon verbena (30-35%), lemon ironbark
(26%), lemon balm (11%), lime (6-9%), lemon (2-5%), and orange.
Citral also called 3,7-dimethyl-2,6-octadienal, a pale yellow liquid, with a
strong lemon odour, that occurs in the essential oils of plants. It is
insoluble in water but soluble in ethanol (ethyl alcohol), diethyl ether,
and mineral oil. It is used in perfumes and flavourings and in the
manufacture of other chemicals. Chemically, citral is a mixture of two
aldehydes that have the same molecular formula but different
structures. Citral is present as two isomers citral α (31, Geranial), and
Citral β ( 32, Neral).
Geranial(31) Neral (32)
Fig.No.12. Isomers of Citral
29
Lemongrass oil contains 70–80 percent citral, which may be
isolated by distillation. Other natural sources include the oils of verbena
and citronella. Citral can be synthesized from myrcene
Uses:Geranial (31) has a strong lemon odor. Neral (32) has a lemon
odor that is less intense, but sweeter. Citral is therefore an aroma
compound used in perfumery for its citrus effect. Citral is also used as a
flavor and for fortifying lemon oil. It also has strong anti- microbial
qualities140 and pheromonal effects in insects. Further it is used
topically as analgesic, and to relieve nasal obstructions. 141 Citral is
used in the synthesis of vitamin A, ionone, and methylionone, and to
mask the smell of smoke.
Vanillin:
Vanilla beans Vanilla plantifolia
30
Vanilla fragrans Dried Vanilla fruits
Fig.No.9. Pictorial representation of various parts of Vanillin
Vanillin is a single molecule, 4-hydroxy-3-methoxybenzaldehyde,
is a white crystalline solid, which melts at 81°C. Vanillin was first
isolated from vanilla pods family (Orchidaceae) by Nicholas-Theodore
Gobley in 1858. The biosynthetic pathway of vanillin starts with
phenylalanine. 90% of vanillin currently in use is synthetically produced
(nature identical) from lignin, eugenol or guaiacol.
Vanillin has generally recognized as safe status and is used as a
flavoring/ aroma compound in foods and fragrance industries. Currently,
approximately 50% of the worldwide production of synthetic vanillin is
used as an intermediate in the chemical and pharmaceutical industries
for the production of herbicides, antifoaming agents or drugs such as
papaverine, l-dopa, l-methyldopa and the antimicrobial agent,
trimethoprim. Moreover, vanillin exhibits strong antimicrobial properties
with activity demonstrated against a number of yeast and mould strains
in laboratory media, fruit-based agar systems, fruit purees and fruit
31
juices. However, no reports have yet detailed the mode of action of
vanillin inhibition. Vanillin was found to posses various pharmacological
activities like antimicrobial, antifungal, analgesic activity etc, thus vanillin
was chosen for the study.
Carvone:
Carvone (29) is a member of a family of chemicals called
terpenoids Carvone is found naturally in many essential oils, but is most
abundant in the oils from seeds of caraway (Carum carvi) and dill.
Fig.no. 10 Carvone (29)
Carvone forms two mirror image forms or enantiomers: S-(+)-carvone
smells like caraway. Its mirror image, R-(–)-carvone, smells like
spearmint. The fact that the two enantiomers are perceived as smelling
differently is proof that olfactory receptors must contain chiral groups,
allowing them to respond more strongly to one enantiomer than to the
other. Not all enantiomers have distinguishable odors. The two forms
are also referred to by older names, with dextro-, d- referring to S-
carvone, and laevo-, l- referring to R-carvone.
32
S-(+)-Carvone is the principal constituent (50-70%) of the oil from
caraway seeds (Carum carvi ) which is produced on a scale of about 10
tonnes per year It also occurs to the extent of about 40-60% in dill seed
oil (from Anethum graveolens), and also in mandarin orange peel oil. R-
(–)-Carvone is present at levels greater than 51% in spearmint oil
(Mentha spicata), which is produced on a scale of around 1500 tonnes
annually. This isomer also occurs in kuromoji oil. Some oils, like
gingergrass oil, contain a mixture of both enantiomers. Many other
natural oils, for example peppermint oil, contain lower concentrations of
carvones (Hernan Grarcia, 1999).
Uses:
Carvone are used in the food and flavor industry .Carvone is also
used for air freshening products and, like many essential oils, oils
containing carvone are used in aromatherapy and alternative medicine.
Further the compound was found to be used as analgesics,
antimicrobial agent, antifungal agent, and also relieves many respiratory
tract infections.
Camphor:
Camphor (30) is a waxy, white or transparent solid with a strong,
aromatic odor. It is a terpenoid with the chemical formula C10H16O. It is
found in wood of the camphor laurel (Cinnamomum camphora), a large
evergreen tree found in Asia (particularly in Borneo and Taiwan) and
33
also of Dryobalanops aromatica, a giant of the Bornean forests. It also
occurs in some other related trees in the laurel family, notably Ocotea
usambarensis. It can also be synthetically produced from oil of
turpentine.
O
CH3
Fig.No.11. Camphor (30)
Camphor is widely used in Hindu religious ceremonies. Hindus
worship a holy flame by burning camphor, which forms an important
part of many religious ceremonies. Camphor is used in the
Mahashivratri celebrations of Shiva, the Hindu god of destruction and
(re)creation. As a natural pitch substance, it burns cool without leaving
an ash residue, which symbolizes consciousness. Of late, most temples
in southern India have stopped lighting camphor in the main Sanctum
Sanctorium due to heavy deposits of carbon; however, open areas do
use camphor. It also acts as rubifacient, counter irritant for inflamed
joints, sprains, rheumatic and other inflamed conditions like cold. It may
be used as mild nasopharyngeal decongestant. It is also found in
clarifying masks used for skin.
34
In ancient and medieval Europe camphor was used as an
ingredient in sweets. It was also used as a flavoring in confections
resembling ice cream in China during the Tang dynasty (AD 618–907).
It was used in a wide variety of both savory and sweet dishes in
medieval Arabic language cookbooks, compiled in the 10th century and
An Anonymous Andalusian Cookbook of the 13th Century. And it
appears in sweet and savory dishes in a book written in the late 15th
century for the sultans of Mandu, the Ni'matnama. Currently, camphor is
used as a flavoring, mostly for sweets, in Asia. It is widely used in
cooking, mainly for dessert dishes, in India where it is known as Kachha
(raw/crude) karpooram ("crude camphor" in Tamil and is available in
Indian grocery stores where it is labeled as "Edible Camphor". It is also
used as preservatives, irritant, etc (Hirota N et al., 1967).
The structural activity relationship (SAR) is the relationship
between the chemical or 3D structure of a molecule and its biological
activity. The analysis of SAR enables the determination of the chemical
groups responsible for evoking a target biological effect in the organism.
This allows modification of the effect or the potency of a bioactive
compound by changing its chemical structure. Medicinal chemists use
the techniques of chemical synthesis to insert new chemical groups into
the biomedical compound and test the modifications for their biological
effects.
35
Antioxidant Activity (Blois MS, 1958)
A free radical exists with one or more unpaired electron in atomic
or molecular orbital. Free radicals are generally unstable, highly
reactive, and energized molecules. Normally it steals an electron from
weakly bonded structures. The molecule, which loses an electron, also
becomes a free radical giving rise to a self-perpetuating chain system.
Free radical often attack DNA, protein molecules, enzymes and cells
leading to alterations in genetic material and cell proliferation
Reactive oxygen species in biological systems are related to free
radicals, even though there are non-radical compounds in reactive
oxygen species such as singlet oxygen and hydrogen peroxide 242.
Reactive Species
Reactive Nitrogen Species Reactive Oxygen Species
●Nitric Oxide
●Nitric Dioxide Oxygen centered radical Oxygen Centered(NO2˙) non-radical
●Superoxide anion(˙O2). ●Hydrogen peroxide●Hydroxyl radical (˙OH) ●Singlet oxygen (O2)●alkoxyl radical (RO˙)●peroxyl radical (ROO˙)
Fig.No.76. A broad classification of reactive oxygen species.
Clinical studies reported that reactive oxygen species are
associated with many age related degenerative diseases, including
36
atherosclerosis, cancers, trauma, stroke, asthma, hyperoxia, arthritis,
heart attack, dermatitis, retinal damage, hepatitis and liver injury.
Sources of free radicals
a) Prooxidative enzymes such as lipoxygenase can generate free
radicals 45. Lipoxygenase can react with free forms of fatty acids,
which can be released from glycerides by membrane bound
phospholipase A2.
b) Environmental sources, such as ultraviolet (UV) irradiation,
ionizing irradiation and pollutants also produce reactive oxygen
species.
c) Injured cells and tissues can stimulate the generation of free
radicals.
d) Reactive oxygen species can be formed in foods through lipid
oxidation and photosensitizers exposed to light.
e) Non enzymatic lipid oxidation requires the presence of free forms
of bivalent metal ions such as copper and iron, which are not
common for healthy adults.
Defense systems against free radicals
The human body although continuously produces free radicals, it
possess several defense systems, which are constituted of enzymes
and radical scavengers. These are called “first – line antioxidant
defense system”, but are not completely efficient because almost all
37
components of living bodies, tissues, cells and genes undergo free
radical destructions.
The “second line defense systems” are constituted of repair
systems of bio molecules which are damaged by the attack of free
radicals. The functions of these enzymes involved in repairing directly
damaged biomolecules such as lipids, polysaccharides, proteins,
nucleic acids, etc., or in eliminating oxidized compounds are illustrated
below.
Antioxidants
Non-enzymatic Enzymatic
Fig.No.77. Classification of Antioxidants
Antioxidants are compounds which act as inhibitors of the
oxidative process. They are quite large in number and diverse in
Natural Compounds Synthetic Compounds
●Vitamin C ●Melatonin ●Superoxide
dismutase(SOD)
●Vitamin E ●Dihydroepiandrosterone ●Peroxidase
●β-carotene (DHEA) ●Catalase
●Uric acid ●Glutathionedisufide
reductase
●Ubiquinone ●Glutathione S transferase
●Methionine sulfoxide
Reductase
38
nature, which opposes the process of oxidation largely by neutralizing
free radicals. Antioxidants at relatively small concentration have the
potential to inhibit the oxidants chain reactions. Antioxidants are also of
paramount importance in pharmaceutical formulations because there
are innumerate medicinal agents possessing diverse chemical functions
and are known to undergo oxidative decomposition.
Reactive Oxygen Species (ROS)
Superoxide anion (.O2-)
It is a reduced form of molecular oxygen created by receiving one
electron. Superoxide anion is an initial free radical from mitochondrial
electron transport systems.
The superoxide anion plays an important role in the formation of
other reactive oxygen species such as hydrogen peroxide, hydroxyl
radical or singlet oxygen. The superoxide anion can react with nitric
oxide (NO˙) and form peroxynitrite (ONOO-) which can generate toxic
compounds such as hydroxyl radical and nitric dioxide.
(ONOO-+H+ OH +˙NO2).
Hydroxyl radical (.OH)
e- e- e- e-O2
H+ HO˙2 H+ H+
H2O2˙OH H2O
39
Hydroxyl radical is the most reactive free radical and can be
formed from superoxide anion and hydrogen peroxide in the presence
of metal ions such as copper or iron.
˙OH + OH- + O2 O2- + H2O2
In general, aromatic compounds or compounds with carbon-
carbon multiple bonds undergo addition reaction with hydroxyl radicals
resulting in the hydroxylated free radicals. In saturated compounds, a
hydroxyl radical abstracts a hydrogen atom from the weakest C-H bond
to yield a free radical. The resulting radicals can react with oxygen and
generate other free radicals.
Hydroxyl radicals react with lipid, polypeptides, proteins and DNA,
especially thiamine and guanosine. Hydroxyl radicals also add readily to
double bonds. When a hydroxyl radical reacts with aromatic
compounds, it can add on across a double bond, resulting in
hydroxycyclohexadienyl radical. The resulting radical can undergo
further reactions, such as reaction with oxygen, to give peroxyl radical
or decompose to phenoxyl-type radicals by water elimination.
Hydrogen peroxide (H2O2)
Hydrogen peroxide can be generated through a dismutation
reaction from superoxide anion by superoxide dismutase. Enzymes
such as amino acid oxidase and xanthine oxidase also produce
hydrogen peroxide from superoxide anion.
40
It is the least reactive molecule among reactive oxygen species and is
stable under physiological pH and temperature in the absence of metal
ions. It can generate the hydroxyl radical in the presence of metal ions
and superoxide anion
˙O2- +H2O2˙OH+OH-+O2
Singlet Oxygen
Singlet oxygen is a non - radical and excited status. Singlet
oxygen can be formed from hydrogen peroxide, which reacts with
superoxide anion or with HOCl or chloramines in cells and tissues.
Compared with other reactive oxygen species, singlet oxygen is rather
mild and non-toxic for mammalian tissue. However, singlet oxygen has
been known to be involved in cholesterol oxidation.
Peroxyl and alkoxy radicals.
Peroxyl radicals (ROO˙) are formed by a direct reaction of oxygen
with alkyl radical (R˙), for example the reaction between lipid radicals
and oxygen. Decomposition of alkyl peroxide (ROOH) also results in
peroxyl (ROO˙) and alkoxyl (RO˙) radicals. Irradiation of UV light or the
presence of transition metal ions can cause homolysis of peroxides to
produce peroxyl and alkoxyl radicals.
ROOH ROO˙ + H˙
ROOH + Fe3+ ROO˙ + Fe 2+ + H+
Reactive Nitrogen Species (RNS)
41
a. Nitric oxide (NO˙)
Nitric Oxide (NO˙) is a free radical with a single unpaired
electron. Nitric oxide is formed from L-arginine by NO synthase. Nitric
oxide itself is not a very reactive free radical, but the overproduction of
NO is involved in ischemia reperfusion and neurodegenerative and
chronic inflammatory diseases such as rheumatoid arthritis and
inflammatory bowel disease. Nitric oxide exposed in human blood
plasma, can deplete the concentration of ascorbic acid and uric acid
and initiate lipid peroxidation.
b. Nitrogen dioxide (NO2˙)
Nitrogen dioxide (NO2˙) is formed from the reaction of peroxyl
radical and NO, polluted air and smoking. Nitrogen dioxide adds to
double bonds and abstract liable hydrogen atoms initiating lipid
peroxidation and production of free radicals.
c. Peroxynitrite
Reaction of NO and superoxide anion can generate peroxynitrite
˙O2 - + NO˙ OONO –
Peroxynitrite is a cytotoxic species and cause tissue injury and oxidizes
low-density lipoprotein (LDL). Peroxynitrite appears to be an important
tissue-damaging species generated at the sites of inflammation and is
involved in various neurodegenerative disorders and several kidney
diseases.
42
In the present study, the antioxidant activity involved using three
standard methods. They are
Scavenging of ABTS radical cation
Scavenging of DPPH
Scavenging of Nitric oxide radical
43
REVIEW OF LITERATURE
Citral:
Annamaria Buschini et al., synthesized some new metal-
complexes with thio-semicarbazones derived from natural aldehydes
which showed peculiar biological activities. In particular, a nickel
complex [Ni(S-tcitr)2] (S-tcitr = S-citronellal thiosemicarbazonate) was
observed to induce an antiproliferative effect on U937, a human
histiocytic lymphoma cell line, at low concentrations (IC50 = 14.4 μM)
and suggest that [Ni(S-tcitr)2] could be a good model for the synthesis of
new metal thiosemicarbazones with specific biological activity
(Annamaria Buschini et al., 2009).
United States Patent 4547361 studied that improved stability
against discoloration upon aging comprising an unsaturated aldehyde
flavoring agent selected from the group consisting of cinnamic aldehyde
and citral and an effective amount in excess of 5% and preferably about
10-45% of a color stabilizer.
The Volatile Oils Vol1", by E. Gildemeister study revealed that
derivatives of citral with hydroxylamine, phenyl hydrazine, and ammonia
are liquid; they cannot be utilized for the characterization of citral. When
dehydrated with the aid of acetic acid anhydride, the oxime is converted
into the nitrile of geranic acid. When acted upon by semicarbazide, citral
yields several well crystallizable semicarbazones. Citrylidene cyanacetic
44
acid, C9H15CHC(CN)COOH, obtained bycondensation of citral with
cyanacetic acid, is another derivative melting at 122° that crystallizes
well and hence can be used for the identification of citral. Condensing
15.2 g. citral and 20 g. of acetyl acetone at room temperature with the
aid of piper dine obtained in light yellow wart-like crystals that melt at 46
to 48°). In small amounts of citral or other aldehydes, a-methyl-/i-
naphtho-cinchonic acid is formed by the interaction of pyrotartaric acid
and B-naphthylamine (Jildemeister E et al., 1994).
US Patent 7309795 study revealed that citral derivatives that
maintain the fragrance characteristics and lemony flavor of citral, while
lowering the allergic properties, providing a longer shelf-life than citral,
and/or increasing the odor intensity relative to citral are disclosed.
In one embodiment, the citral derivatives are prepared by
replacing one or more double bonds in the parent molecule with a
cyclopropyl group, which can be unsubstituted, or substituted with one
or two lower alkyl, preferably methyl groups. The alkyl groups can
optionally be substituted, for example, with electron donating groups,
electron withdrawing groups, groups which increase the hydrophilicity or
hydrophobocity, and in another embodiment, the derivatives are
prepared by replacing one or more aldehyde groups in citral with a nit
rile, methyl ether or acetal group. The acetal groups can provide the
45
compounds with a long lasting flavor or fragrance, where the acetals
slowly hydrolyze to provide the parent aldehyde compounds.
Hierro i. et al., Studied that In vivo larvicidal activity of monoterpenic
derivatives from aromatic plants against L3 larvae of Anisakis simplex.
The aldehydic monoterpene citral and the alcoholic citronellol, when
they are administered together to the larvae of the nematode at the
concentration of 46.90mg/0.5ml in olive oil, achieve 85.90% and
67.53% dead L3, respectively, and also stop rats suffering
gastrointestinal hemorrhages produced by the larvae.
Olga I. Yarovaya et al., study revealed 6, 7-epoxides of citral are
isomerization reactions, resulting in keto-aldehydes, substituted
tetrahydrofurans and dicyclic esters. Dicyclic ketals 4,8,8-trimethyl-7,9-
dioxabicyclo[4.2.1]non-4-ene and 2,2,6-trimethyl-3,9- di-oxa-bicyclo
[4.2.1]non-4-ene have obtained are in fact the structural counterparts of
the known pheromons products of acetal type have pleasant odour and
can be investigated as odorous substances (olga I Yaroveya et al.,
2002).
Xiao Xiao Jin et al., studied that the Schiff base of chitosan was
synthesized by the reaction of chitosan with citral working under high-
intensity ultrasound. The antimicrobial activities of chitosan and the
Schiff base of chitosan were investigated against E. coli, L aureus, and
46
A. Niger. The results indicate that the Schiff base of chitosan has better
antimicrobial activities than chitosan.
Grace O. Onawunmi, found that Citral showed appreciable
antimicrobial activity against Gram-positive and Gram-negative bacteria
as well as fungi. Media composition and inoculum size had no
observable effect on activity but alkaline pH increased citral activity. The
growth rates of Escherichia coli cultures were reduced at concentrations
of citral ≥0·01% v/v while concentrations ≥0·03% v/v produced rapid
reduction in viable cells followed by limited regrowth. In a non-growth
medium, 0·08% and 0·1% v/v showed rapid bactericidal effects. Citral
may therefore be of preservative use in addition to its other uses in the
food, soap and cosmetic industries.
Grace O. Onawunmi, et al., found that Cymbopogon citratus
(DC.) Stapf, commonly known as lemon grass and used, over many
years, for medicinal purposes in West Africa, produces a volatile oil on
steam extraction of its leaves. While the α-citral (geranial) and β-citral
(neral) components individually elicit antibacterial action on gram-
negative and gram-positive organisms, the third component, myrcene,
did not show observable antibacterial activity on its own.
Sang wan, Naresh. K et al., Nematicidal activity of the essential
oils of three Cymbopogon grasses (C. martini var. motia, C. flexuous
and C. winterianus) and their major constituents, geraniol, citral,
47
citronellol and citronellal was determined with second stage juveniles of
four nematodes, seed-gall nematode (Anguina tritici), citrus nematode
(Tylenchulus semipenetrans), root-knot nematode (Meloidogyne
javanica) and cereal cyst-nematode (Heterodera avenae). The essential
oils and their constituents were found to be nematicidal and their
activities (Sang Wang, 1985).
Hayes J. and Markovic B. Found that antimicrobial and
toxicological properties of the Australian essential oil, lemon myrtle,
(Backhousia citriodora) were investigated. Lemon myrtle oil was shown
to possess significant antimicrobial activity against the organisms
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa,
Candida albicans, methicillin-resistant S. aureus (MRSA), Aspergillus
Niger, Klebsiella pneumoniae and Propionibacterium acnes comparable
to its major component-citral.
Amna A. Saddiq and Suzan A. Khayyat found that 6, 7-Citral-
epoxy derivative (a mixture of E and Z isomers with respect to the
C2 = C3 double bond) could be react with DNA base producing a major
adduct. Antifungal and antibacterial studies were carried out on citral
and citral-epoxide. Studies on the antifungal especially Penicillium
italicum and Rhizopus stolonifer showed that citral and citral-epoxide
have good antibacterial action. Antimicrobial studies of P. italicum and
R. stolonifer explained also that citral and citral-epoxide have good
48
antimicrobial activity. Citral epoxide shows high activity against the
growth of bacteria methicillin resistant Staphylococcus aureus (MRSA)
and fungi comparing by citral(Amna A et al., 2005).
Nativ dudai et al., found that Lemongrasses (Cymbopogonspp,
Poaceae) are a group of commercially important C4tropical grasses. To
specifically locate the sites of citral accumulation in lemongrass we
employed Schiff's reagent, which reacts with aldehydes and gives a
purple-red coloration with citral and suggested that citral accumulation
takes place in individual oil cells within the leaf tissues (Nativ Dudai et
al., 1998).
Renu Sharma, Santosh K. et al., reported that the ligand cis-3, 7-
dimethyl-2, 6-octadiensemicarbazone Complexes yields: [ML2 Cl2] and
[ML2 Cl2] Cl type complexes, where M = CrIII, MnII, FeIII, CoII, NiII, CuII,
ZnII, CdII and HgII. All the newly synthesized metal complexes, as well as
the ligand, were screened for their antibacterial activity. All the
complexes exhibit strong inhibitory action against Gram (+) bacteria
Staphylococcus aureus and Gram (−) bacteria.
Francesca Di Renzo et al., found that the clinically used
antimycotic fluconazole (flu co) is teratogenic in rodents. Citral is a
retinoic acid synthesis inhibitor. The co-exposure to fluco + citral was
significantly effective in reducing bronchial arch and cranial nerve
49
defects; supporting the hypothesis that citral balances the fluco-induced
RA concentration increase.
P.A. Paranagama et al., found that Aspergillus flavus Link. Was
isolated from stored rice and identified as an aflatoxigenic strain.
Lemongrass oil was tested against A. flavus and the test oil was fungi
static and fungicidal against the test pathogen at 0·6 and 1·0 mg ml−1,
respectively. Aflatoxin production was completely inhibited at
0·1 mg ml−1. The results obtained from the thin layer chromatographic
bioassay and gas chromatography indicated citral a and b as the
fungicidal constituents in lemongrass oil (Parangama PA, 2003).
J.m. Kim, et al., study revealed that Carvacrol, citral and geraniol
showed potent antibacterial activity against Salmonella typhimurium and
its rifampicin-resistant (RifR) strain as determined in txyptic soy broth
and by zone of inhibition on agar-based medium.
Tamonud Modak and Abhilash Mukhopadhaya. The effect of citral
on adipose tissue. Representative stained sections of perinephric
adipose tissue from drug control group and experimental group. The
drug treated group shows smaller adipocyte than the drug control
(Indian J. Pharmacol.2011).
Vanillin:
Vanillin is an organic compound with the molecular formula
C8H8O3. Its functional groups include aldehyde, ether, and phenol. It is
50
the primary component of the extract of the vanilla bean. It is also found
in roasted coffee and the Chinese red pine. Synthetic vanillin, instead of
natural vanilla extract, is sometimes used as a flavoring agent in foods,
beverages, and pharmaceuticals.
Natural vanillin is extracted from the seed pods of Vanilla
planifola, a vining orchid native to Mexico, but now grown in tropical
areas around the globe. Madagascar is presently the largest producer
of natural vanillin.
Natural "vanilla extract" is a mixture of several hundred different
compounds in addition to vanillin. Artificial vanilla flavoring is a solution
of pure vanillin, usually of synthetic origin. Because of the scarcity and
expense of natural vanilla extract, there has long been interest in the
synthetic preparation of its predominant component.
The first commercial synthesis of vanillin began with the more
readily available natural compound eugenol. Today, artificial vanillin is
made from either guaiacol or from lignin, a constituent of wood which is
a byproduct of the paper industry.
Vanillin was first isolated as a relatively pure substance in 1858
by Nicolas-Theodore Gobley, who obtained it by evaporating a vanilla
extract to dryness, and recrystallizing the resulting solids from hot
water.
51
In 1874, the German scientists Ferdinand Tiemann and Wilhelm
Haarmann deduced its chemical structure, at the same time finding a
synthesis for vanillin from coniferin, a glycoside of isoeugenol found in
pine bark. Tiemann and Haarmann founded a company, Haarmann &
Reimer (now part of Symrise) and started the first industrial production
of Vanillin using their process in Holzminden (Germany).
In 1876, Karl Reimer synthesized vanillin from guaiacol.
OH
OCH3
HO
O
OH
CH3
Guaiacol Vanillin
KOH/CHCl3
Lignin-based artificial vanilla flavoring is alleged to have a richer
flavor profile than oil-based flavoring; the difference is due to the
presence of acetovanillone in the lignin-derived product, an impurity not
found in vanillin synthesized from guaiacol.
D.F. Taber et.al., given has recently been described, a very
convenient laboratory synthesis involving electrophilic bromination of 4-
hydroxybenzaldehyde, followed by copper-catalysed methoxylation
which is suitable for synthesysing vanillin in few grams (Taber DF et al.,
2007).
52
OH
OH
OH
OH
CH3
OH
OH
O
CH3
Br2
M eOH
NaOM e/CuBrEtOAc
4-hydroxybenzaldehyde 4-hydroxy-3-m ethylbenzaldehyde Vanillin
As far as large-scale industrial syntheses go, a classic early
method starts from eugenol, which occurs naturally in cloves, nutmeg
and cinnamon. This isomerises to isoeugenol in alkaline solution, and
this in turn can be oxidised (by nitrobenzene) to vanillin.
O H
O
C H 3
C H 2
O H
O
C H 3
C H 3
O H
O
C H 3
OH
E u g e n o l Iso e u g e n o l V a n i l l in
Today most of the vanillin was synthesized by reacting guaiacol
which is obtained from catechol with glyoxalic acid.
O H
O
C H 3
O H
O
C H 3
H
OHO H
O
O H
O
C H 3
OO H
O
O H
O
C H 3
O HH
OHC
COOH+
O H - O 2
C a ta lys t
H + -C O 2
V a n i l l in
53
The freshly harvested green vanilla pods do not smell "vanilla" as
the vanillin is locked up inside them as a β-D-glycoside. The pods have
to be cured, during which time they become deep brown, and enzymes
release the vanillin from the glycoside, along with over a hundred other
molecules, all of which contribute to the authentic vanilla aroma. Vanillin
itself makes up about 2% of the final mass of the "cured" beans.
Vanillin as β-D-glycoside
Vanillin has been used as a chemical intermediate in the
production of pharmaceuticals and other fine chemicals. In 1970, more
than half the world's vanillin production was used in the synthesis of
other chemicals, but as of 2004 this use accounts for only 13% of the
market for vanillin.
The vanilla plant, Vanilla planifola originates in subtropical forests
in Mexico and parts of Central America, and it was the Mayan and
Aztec civilisations which first realised the potential of vanilla, using it to
lighten up the flavour of the chocolate that they drank. The Aztec leader
Moctezuma hinted to Hernán Cortés (who led the Spanish invaders)
that the Aztecs' vanilla flavoured chocolate drinks were a very useful
54
aphrodisiac. After their conquest of these areas, the Spaniards brought
vanilla to Western Europe in the early 16th century.
Vanillin is in the class of vanilloids, that includes – surprisingly –
capsaicin (8-methy-N-vanillyl noneamide) from chile pepper and
eugenol from cloves, cinnamon and other spices, and zingerone from
ginger. The vanilloid receptors of the central and peripheral nervous
systems bind with these compounds, resulting in different sensory
effects. Thus, capsaicin can cause a burning sensation while eugenol
results in mild anesthesia; vanillin itself is neutral. Vanilla is an aromatic
stimulant, with a tendency towards the nervous system. It has also been
regarded as an aphrodisiac. It has been employed as a remedy in
hysteria, low fevers, impotency, etc.
Vanillin (4-hydroxy-3-methoxybenzaldehyde), a potent nuclear
factor-κB (NF-κB) inhibitor, was evaluated in mice with trinitrobenzene
sulfonic acid (TNBS)-induced colitis. Vanillin not only prevented TNBS-
induced colitis but also ameliorated the established colitis. Vanillin
reduced the expressions of proinflammatory cytokines [interleukin (IL)-
1β, IL-6, interferon-γ, and tumor necrosis factor-α] and stimulated the
expression of anti-inflammatory cytokine (IL-4) in colonic tissues.
Vanillin, the active ingredient in vanilla, has shown some
interesting anti-cancer properties. Not only does it prevent mutations,
the changes in the cell’s DNA that lead to cancer, but it also stops
55
growth of cancer cells in a laboratory setting. A study conducted on
mice showed that vanillin stopped the metastasis or spread of breast
cancer cells to the lungs and decreased their ability to invade new
tissue. Bromovanin, a derivative of vanillin, also shows some promise
for the treatment of cancer and could be used in the development of
new cancer treatments.
Vanillin, the active component of vanilla, has antioxidant activity
and appears to offset some of the oxidative damage that occurs in the
brains of patients with Alzheimer’s disease – particularly the formation
of a compound called peroxynitrite. Peroxynitrite plays a role in other
degenerative diseases of the brain such as Parkinson’s disease.
Although research in this area is still in its infancy, it may hold future
promise for people dealing with these debilitating diseases.
Studies have shown that vanillin can stop the sickling of red blood
cells that leads to problems for people with sickle cell anemia.
Unfortunately, vanilla can’t be used directly since it would be destroyed
by the acid in the stomach. Researchers are hoping that a drug using
vanillin can be developed to treat sickle cell disease in the near future.
Vanilla has been used historically as far back as the seventeenth
century to treat a variety of conditions including stomach ulcers and
sleep difficulties. The essential oil reportedly has sedative-like
properties. Some alternative practitioners use vanilla essential oil to
56
treat insomnia, anxiety, and depression. It’s also thought to be an
aphrodisiac although there’s little scientific evidence to support this.
Z. Yuxia et,al., synthesized some new Schiff bases from 4-
aminoantipyrine and vanillin and determining their antibacterial activity.
Literature survey shows that Schiff bases show bacteriostatic and
bactericidal activity. Antibacterial, antifungal, antitumor, anticancer
activity has been reported and they are also active against a wide range
of organisms, e.g. C. albicans, E. coli, S.aureus, B. polymyxa, P.
viticola, etc. Many Schiff bases are known to be medicinally important
and are used to design medicinal compounds (Yogesh Kumar, 2004).
Vanillin, a food additive, has been evaluated as a potential agent
to treat sickle cell anemia. Earlier studies indicated that vanillin had
moderate antisickling activity when compared with other aldehydes.
In particular the detection of volatile organic compounds in low
concentration, has become of interest, because they are widely used as
ingredients house-hold products. These compounds vaporize at normal
room temperature, sometimes causing adverse health effects. Abdullah
M. Asiri, et.al, synthesized six new dyes derived from vanillin and active
methylene have been prepared and these dyes were tested for use as
sensors for volatile organic compounds (VOCs namely Triethylamine
and diethyl amine). The electronic spectra of these dyes were examined
57
and gave color depending on the acceptor groups (Abdullah M et al.,
2009).
Potkin V, et.al, performed Reaction between 4-formyl -2 –
methoxy phenyl 4, 5-dichloro isothiazole -3-carboxylate with various
aromatic amines led to azomethins formation. By treatment of
azomethins with sodium triacetoxyborohydride corresponding amine
were obtained. During the bioassays of new vanillin derivatives in
mixtures with insecticides remarkable synergetic effect was discovered.
Currently, approximately 50% of the worldwide production of synthetic
vanillin is used as an intermediate in the chemical and pharmaceutical
industries for the production of herbicides, antifoaming agents or drugs
such as papaverine, l-dopa, l-methyldopa and the antimicrobial agent,
trimethoprim.
Sandra S, et,al stated Azomethines are of considerable interest
because of their chemistry and potentially beneficial biological activities,
such as antitumor, antibacterial, antiviral and antimalarial activities,
Considering this, the vanillin azomethines were synthesized and tested
for their antimicrobial activity. The antifungal activity of the obtained
compounds show that vanillin azomethines have enhanced the activity
more than intermediates due to C=N group.
Vanillin, a naturally occurring food component, has been reported
to have anti-mutagenic and anti-metastatic potentials, and to inhibit
58
DNA-PKcs activity. However, vanillin itself exhibits very weak
antiproliferative activity.
Yu-Qian Yan, et.al., studied effects of bromovanin (6-bromine-5-
hydroxy-4-methoxybenzaldehyde), a novel vanillin derivative, on
survival and cell-cycle progression of human Jurkat leukemia cells.
Treatment with >10 μM bromovanin significantly elicited apoptosis and
G2/M arrest in Jurkat cells in a dose- and timedependent manner.
Bromovanin-induced DNA double-strand breaks (DSB) were
demonstrated by means of comet assay as well as detection of
phosphorylated H2AX, a sensitive indicator of DNA DSBs.
CHO
O H
O
C H 3
CHO
O
OH
B r
CH 3 V a n il l in B ro m o v a n il l in
Vanillin can be iodinated in an aqueous solution of sodium
triiodide (NaI3.NaI), made in situ from sodium iodide and iodine, forming
5-iodovanillin. Refluxing 5-iodovanillin in a strong sodium hydroxide
solution produces 5-hydroxyvanillin, which is a useful precursor for 3, 4,
and 5-trimethoxy- benzaldehyde (used in the synthesis of mescaline) as
well as 3-methoxy-4, 5-methylenedioxy- benzaldehyde
(myristicinaldehyde, used in the synthesis of MMDA-2). 5-Iodovanillin
can also be treated with sodium methoxide to form syringaldehyde (4-
59
hydroxy-3, 5-dimethoxybenzaldehyde, used in the synthesis of escaline,
proscaline and other mescaline derivatives (Dikusar et al., 2006).
CHO
OH
OCH3
CHO
OH
OCH3
I
CHO
OH
OCH3
OH
1N NaOH
NaI3,H2SO4
NaOH
vanillin 5-iodo vanillin
5-hydroxy vanillin
Ortho-Vanillin is harmful if ingested, irritating to eyes, skin and
respiratory system, but has an unmistakable high LD50 of 1330 mg/kg in
mice. It is a weak inhibitor of tyrosinase, and displays both
antimutagenic and comutagenic properties in Escherichia Coli.
However, its net effect makes it a potent comutagen. Ortho-Vanillin
possesses moderate antifungal and antibacterial properties. Most ortho-
vanillin is used in the study of mutagenesis and as a synthetic precursor
for pharmaceuticals.
CHO
OH
OCH3
ortho-vanillin(2-hydroxy-3-methoxybenzaldehyde)
Condensed tannins can be detected by vanillic acid, as the tannin
will react and make a red complex. Any fluid has its own characteristic
absorption profile, which is recorded on a disc by VIS-
spectrophotometry. Tannin detenorates, the absorption profile due to
60
change in its physical characteristics. VIS-spectrophotometry is
therefore inapplicable for the identification of much detenorated tannins.
Shrinkage temperature (Ts) is an expression for the hydrothermal
stability of leather. A high shrinkage temperature indicates stable
leather, with lot of bindings between the collagen fibers.
Thiosemicarbazones of natural aldehydes and ketones are easily
characterized and very stable crystalline compounds which can be
valuable synthons, especially for heterocyclic synthesis.
Thiosemicarbazones are widely used as insecticides, inhibitors,
zoocides, and pharmaceuticals exhibiting antimicrobial, antiviral, and
antitumor activity. So new thiosemicarbazones are synthesized on the
basis of our previously prepared esters of natural aldehydophenols,
such as vanillin and vanilla.
Ethers and esters of oximes exhibit anti-inflammatory,
antimicrobial, pesticidal, insecticidal, fungicidal, and other types of
physiological activity. Oximes of plant phenols prepared from vanillin
and vanillal are convenient and available synthons for the synthesis of
new biologically active compounds and fragrances and can be used as
reagents for separating and concentrating chemical elements.
Douglass F Taber, Shweta Patel. Vanillin Synthesis from 4-
Hydroxybenzaldehyde. A simple and safe preparation of vanillin from 4-
hydroxybenzaldehyde is described (J. Chemical education 2012.)
61
Carvone:
Anand Akhila et al., worked on Biosynthesis of carvone in Mentha
spicata by degradation of, and measurement of isotope ratios in, (−)-
carvone that had been biosynthesized in Mentha spicata from 3H- and
14C-labelled geraniol and mevalonate. These results enable the
mechanisms for the introduction of the carbonyl group and for the
formation of the isopropenyl side-chain to be delimited (Anand Akhila et
al., 2008).
Gerardo C. Torres et al., worked on Hydrogenation of carvone on
Pt–Sn/Al2O3 catalysts the effect of Sn concentration in Pt–Sn/Al2O3
catalysts prepared by different procedures on the catalytic behavior in
the carvone hydrogenation in liquid phase was studied. Results
indicated that the increase of the Sn amount added to Pt modified the
catalytic behavior, favoring the formation of unsaturated ketones and
the simultaneous production of small quantities of unsaturated alcohols
as reaction intermediaries. On the other hand, Pt/Al2O3 catalyst only
produced carvomenthone as the main reaction intermediary (Gerar C et
al., 2009).
Kathleen McBride et al., assessed whether the enantiomers of
terpinen-4-ol, odorants that activate nearly identical areas of the
olfactory bulb, are more difficult to discriminate than those of carvone,
odorants that activate different areas of the olfactory bulb, and whether
62
olfactory bulb lesions that disrupt the pattern of bulbar activation
produced by these enantiomers degraded the ability of rats to
discriminate between them.
Richard A. Kjonaas et al., worked on Acid-Catalyzed
Isomerization of Carvone to Carvacrol this experiment demonstrates
several important concepts including (i) formation of a carbocation by
protonation of an alkene, (ii) rearrangement of a carbocation, (iii)
deprotonation of a carbocation, (iv) acid-catalyzed enolization, and (v)
aromaticity. The experiment is especially suitable for use with low-field
permanent-magnet FT–NMR (Richard A, 1984).
Goncalves Juan Carlos Ramos et al., worked on Antinociceptive
Activity of Carvone it is a monoterpene ketone that is the main active
component of Mentha plant species. This study aimed to investigate the
antinociceptive activity of carvone using different experimental models
of pain and to investigate whether such effects might be involved in the
nervous excitability elicited by others monoterpenes.
Mariet J. van der Werf et al., worked on a novel nicotinoprotein,
catalyzing the dichlorophenolindophenol-dependent oxidation of carveol
to carvone, was purified to homogeneity from Rhodococcus erythropolis
DCL14. The enzyme is specifically induced after growth on limonene
and carveol. Dichlorophenolindophenol-dependent carveol
dehydrogenase (CDH) is a homotetramer of 120 kDa with each subunit
63
containing a tightly bound NAD (H) molecule. The enzyme is optimally
active at pH 5.5 and 50 °C and displays broad substrate specificity with
a preference for substituted cyclohexanols.
Paul McGeady, et al., worked on Carvone and perillaldehyde
were shown to inhibit the transformation of Candida albicans to a
filamentous form at concentrations far lower and more biologically
relevant than the concentrations necessary to inhibit growth. This
morphological transformation is associated with C. albicans
pathogenicity; hence these naturally occurring monoterpenes are
potential lead compounds in the development of therapeutic agents
against C. albicans infection (Paul Mc Geady et al., 1999).
B. Slotnick et al., worked on Rats with lesions of dorsal and dorsolateral
bulbar sites known to be differentially responsive to carvone
enantiomers were tested for their ability to detect (+)-carvone, to
discriminate between (+)-carvone from (−)-carvone, and to discriminate
(+)-carvone from mixtures of both enantiomers after they had been pre-
trained or not pre-trained on these tasks prior to surgery. These results
indicate that removal of most bulbar sites known to be differentially
responsive to carvone enantiomers and the consequent disruption of
normal patterns of bulbar input produced in response to carvones are
largely without effect on the ability of rats to discriminate between these
odors.
64
Michael Schiendorfer et al., worked on provided the lower rim of
resorc[4]arenes with stereogenic centers as well as with functional
groups. For the synthesis of these lower rim functionalized chiral
resorc[4]arenes we have used aldehydes derived from citronellal and
carvone. In general, the functional group was introduced into the
aldehyde compound prior to the final cyclization step. Another strategy
is the introduction of the iodo group at the lower rim of a resorc[4]arene,
which can be easily substituted by good nucleophiles and mild bases
without protection of the upper rim (Micheal Schiendorfer, 2005) .
Fernando de Sousa Oliveira et al., studied on
hydroxydihydrocarvone (HC) is a monoterpene analog prepared as a
semi synthetic intermediate by hydration of the carvone monoterpene.
Recent reports from studies carried out on HC have demonstrated its
antinociceptive effect. HC exerts a central antinociceptive effect without
causing pharmacological tolerance, and no significant toxicological
alterations were observed during treatment.
Renata Zawirska-Wojtasiak et al., worked on Solid-phase micro
extraction was examined for its suitability for isolation of volatiles from
seeds of dill in comparison with the traditional steam distillation
procedure. Two main dill seeds volatiles, carvone and limonene, were
taken into consideration. Two Supelco SPME fibers were used for the
extraction: polyacrylic (PAc) and polydimethylsiloxane (PDMS). The
65
time required to saturate the fibers was 3 min, while distillation took 3 h.
Gas chromatography (GC) separation was reduced to 5 min by use of
microcapillary column HP-5 cross-linked 5% Ph Me Siloxan.
Diego S. Pisoni et al., worked on indium trichloride based on
Journal of the Brazilian Chemical Society Indium trichloride promotes
the chlorination of terminal olefins in the presence of sodium
hypochlorite with good results. Carvone was chosen as a model
compound to examine some of the general features of this reaction,
such as stoichiometry, temperature, reaction time and product
conversion. Treatment of b-pinene with sodium hypochlorite in the
presence of indium trichloride resulted in a facile rearrangement to
selectively yield perillyl chloride, which is an important precursor for C-7
oxygenated limonenes.
Anja A. Verstegen-Haaksma et al., worked on Application of S-
(+)-carvone in the synthesis of biologically active natural products using
chemical transformations and bioconversions.
Dohm Michlle et al., worked on Frequency-dependent, complex
refractive indices for carvone in the mid-infrared from 750 to 5000 cm-1
have been inverted from the Fourier transform extinction spectra of
laboratory-generated aerosols recorded at room temperature. Such
data can be used to elucidate the optical properties of a substance,
which are of critical importance in the interpretation of remote sensing
66
data and in the evaluation of how atmospheric particulate matter
consisting of organic compounds may affect climate change (Dohn
Michelle et al., 2003).
IsaTelci et al., worked on Agronomical and Chemical
Characterization of Spearmint Spicata Originating in Turkey and the
components were determined by using gas chromatography. Two
chemo types were identified; one high in carvone and the other is
pulegone. Agronomic and essential oil properties of cultivated landraces
of M. spicata were also investigated under field conditions.
Asturias JA, et al., worked on the evolutionary relationship of
biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6
to multicomponent dioxygenases from gram-negative bacteria.
Jeronimo S. Costa et al., worked on the reactivity and
diastereoselectivity of conjugate addition of different nitronates ions to
(R)-carvone was systematically studied. The nitro adducts were
transformed via Nef reaction into (R)-carvone ketone derivatives and
nitro adducts led to (R)-carvone alkylated derivatives via a denitration
reaction.
T. J. Raphael et al., worked on the immunomodulatory activity of
some naturally occurring monoterpenes were studied in Balb/c mice.
Administration of various monoterpenes such as carvone, limonene and
perillic acid were found to increase the total white blood cells (WBC)
67
count in Balb/c mice. Administration of terpenoids increased the total
antibody production, antibody producing cells in spleen, bone marrow
cellularity and α-esterase positive cells significantly compared to the
normal animals indicating its potentiating effect on the immune system
(Raphael TJ et al., 2003).
Y. S. R. Krishnaiah, et al., worked on the purpose of this study
was to investigate the effect of carvone on the permeation of nicardipine
hydrochloride across the excised rat abdominal epidermis from 2% w/w
hydroxypropyl cellulose gel system. The results suggest that carvone
may be useful for enhancing the skin permeability of nicardipine
hydrochloride from transdermal therapeutic system containing HPC gel
as a reservoir (Krishnaiah Y, 19984).
Fatemeh Rafii et al, worked on Piperitone from plant essential oils
enhance bactericidal activities of nitrofurantoin and furazolidone against
bacteria from the family Enterobacteriaceae. In this study, the essential
oils of spearmint, dill and peppermint were screened for augmentation
of nitrofurantoin activity and the most active components were
determined. Pure carvone and piperitone equally increased the
bactericidal activity of nitrofurantoin. Other ingredients of essential oils,
including camphor, limonene and menthone, were less effective.
Paul McGeady, et al., worked on Carvone and perillaldehyde
were shown to inhibit the transformation of Candida albicans to a
68
filamentous form at concentrations far lower and more biologically
relevant than the concentrations necessary to inhibit growth. This
morphological transformation is associated with C. albicans
pathogenicity.
Camila M.S. Silva, Carlos W.S. Carvone (R )-(-) and (S)-(+)
enantiomers inhibits upper gastrointestinal motility in mice. These
effects were accompanied by a reduction of the propulsive behavior of
small intestine. The retarding effects of carvone on gastric pressure
waves (Flavour and Fragrance Journal. 2015)
Camphor:
Warren P, Bishop MD, Kathleen D and Sanders MD et al., worked
on hepato toxicity in a 2-month-old baby after a camphor-containing
cold remedy was applied dermally. Liver function tests returned to
normal after the application of the cold remedy was discontinued.
Ingestion of camphor can cause severe liver and central nervous
system injury, and neurotoxicity has been observed after exposure to
camphor through the skin. Hepato toxicity after dermal application of
camphor has never been reported. This report emphasizes the common
use of cold remedies that are usually not beneficial and may be
potentially dangerous.
N S Vostrikov, A V Abutkov et al., worked on new camphor
derivative functionalized by Base-catalyzed condensation of 10
69
methylene camphor with diethyl oxalate gave the corresponding (Z)-3-
ethoxycarbonyl (hydroxyl) methylene derivative which was converted
into methyl ether and acetate. The Z-methyl ether undergoes
isomerization into the E-methyl ether on treatment with N-
bromosuccinimide in the presence of radical initiator [azobis
(isobutyrodinitrile)]. (Z)-3-Ethoxycarbonyl (hydroxy) methylene-10-
methylenecamphor smoothly reacts with N-bromosuccinimide to afford
stereo isomeric 3-bromo derivatives.
Mauro L. Mellão L and Mario L A, Vasconcellos et al., worked on
new camphor derivatives for enantio selective syntheses. New 1,3 diols
3a→3c were efficiently prepared in the enantiopure form in 50–68%
yield (2 steps), from the available 1-(R)-(+)-camphor (Kumar M, et al.,
1995).
Uzi Ravid, Eli Putievsky, Irena Katzir, et all worked on the
enantiomeric differentiation of camphor isolated from natural essential
oils and samples from commercial sources was determined using a
fused-silica Lipodex E capillary column. Enantiomerically pure (S)(-)-
camphor was detected in Chrysanthemum parthenium (L.) Bernh. oil.
The (S)(-)-enantiomer, with high enantiomeric purity was detected in
two types of Salvia offtcinalis L., and (R)(+)-camphor with high
enantiomeric purity was detected in two other types of S. officinalis and
in S. glutinosa L (Medoff G et al., 1999),.
70
Chihliang Chang, Kwunmin Chen et al., worked on Practical and
convenient synthetic routes for the synthesis of a new class of
pyrrolidinyl-camphor derivatives. These novel compounds were
screened as catalysts for the direct Michael addition of symmetrical , -
disubstituted aldehydes to -nitro alkenes. When this asymmetric
transformation was catalyzed by organo catalyst, the desired Michael
adducts were obtained in high chemical yields, with high to excellent
stereo selectivities and 99 % enantiomeric excess. The synthetic
application was demonstrated by the synthesis of a tetrasubstituted-
cyclohexane derivative from (S)-citronellal, with high stereo selectivity.
Peter Weyerstahl Christian Gansau, Tilo Claußen et al., worked
on Studies on the patchouli character of camphor derivatives. From
camphor the ketones were prepared by -alkylation, and the tertiary
alcohols by Grignard reaction. The disubstituted (iso) borneols could be
obtained by Grignard reaction with MtMgI or allyl magnesium halide.
Reaction with EtMgl afforded the secondary alcohols by reduction.
Olfactive evaluation showed that the -monoalkylated camphor’s as well
as the 2-alkylisoborneols remained more or less within the camphor-
eucalyptus odour profile. However, and the disubstituted (iso) borneols
possess a strong patchouli scent.
V Z Vasiko and M S Miftakhov et al., worked on Synthetic routes
to precursors of tricyclic camphor derivatives fused at the 2, 10-
71
positions, the corresponding halohydrins and dimethyl acetal, are
discussed (Bishop, 2000).
Antonio García Martínez, Enrique Teso Vilar, Amelia García
Fraile, et al., worked on the new, general and straightforward method
for the enantiospecific synthesis of 9,10-dihalocamphors (including
mixed derivatives) is reported and exemplified for the preparation of (+)-
9,10-dibromocamphor (a well-known chiral intermediate) as well as (+)-
9-bromo-10-chlorocamphor and (+)-9-bromo-10-iodocamphor (novel
mixed dihalides). Our approach is based on a key stereo controlled
tandem electrophilic addition - Wagner-Meerwein rearrangement of
optically pure 3-endo-(halo methyl)-3-methyl-2-methylenenorbornan-1-
ols, which are easily obtained from readily accessible 9-halocamphors.
William J, Phelan M.D, et al., worked on Camphor Poisoning
Over-the-Counter Dangers. Intoxication from camphor has been
reported frequently in the literature for decades, most cases involving
the accidental ingestion of camphorated oil, mistaken for castor oil or
other similar products. Over 20 years ago, Smith and Margolis collected
130 nonfatal and 18 fatal cases from literature dating back to 1833.
Recent data from the National Clearinghouse for Poison Control
Centers reveal an increasing proportion of ingestions of other over-the-
counter camphor-containing preparations.
72
Julian A Peterson et al., worked on large-scale purification from
the bacterium Pseudomonas putida of two components of the camphor
methylene hydroxylase system, cytochrome P-450 and putidaredoxin.
The heme iron of cytochrome P-450 is in a high-spin state with five
unpaired electrons in the presence of camphor and a low-spin state with
one unpaired electron in the absence of camphor as determined by
magnetic susceptibility and electron paramagnetic resonance
measurements, respectively.
J L Urai and F J Humphreys, et al., worked on Thin polycrystalline
specimens of rhombohedra camphor in pure shear in the temperature
range 283–343K. The deformation processes, dynamic recrystallization
and the development of microstructure were followed by transmission
polarized light microscopy. The development of microstructure changed
drastically above 310–320K.This change, probably due to the
development of a marked anisotropy in grain boundary motilities, and
the activation of new slip systems made the development of shear
zones a more frequently occurring phenomenon above 310–320K.
ChanI Ping, chiu ShuPing, WuFu Ming, et al., worked on acute
camphor oil intoxicosis in cats. Three cats were treated with camphor oil
due to flea infestation 2 weeks prior to presentation. These cats showed
signs of depression, anorexia and lethargy. On physical examination, all
3 cats had symptoms of dehydration and jaundice. Moreover, they
73
smelled of camphor lightly or heavily. The 3 cats died from days 1 to 4
after hospitalization. One of the cats was subjected to necropsy.
Regeneration of the renal tubular epithelial cells and hepatic tissue was
observed from many areas, and urine bladder had severely damaging
lesions from the clinical signs, laboratory and histopathological findings,
a diagnosis of acute camphor oil intoxication was made.
Sonja Frölich, Carola Sch ubert et al., worked on In vitro
antiplasmodial activity of prenylted chalcone derivatives of hops
(Humulus lupulus) and their interaction with haemin. There is an urgent
need to discover new antimalarials, due to the spread of chloroquine
resistance and the limited number of available drugs. Chalcones are
one of the classes of natural products that are known to possess
antiplasmodial properties. Therefore, the in vitro antiplasmodial activity
of the main hop chalcone xanthohumol and seven derivatives was
evaluated. In addition, the influence of the compounds on glutathione
(GSH)-dependent haemi-n degradation was analysed.
Matherine, Achanta Geetha et al., worked on2.Anticancer
activityes odzelewska Aneta; Pettit C of novel chalcone and bis-
chalcone derivatives.A series of novel chalcones and bis-chalcones et
against the human breast cancer MDA-MB-231 (estrogen receptor-
negative) and MCF7 (estrogen receptor-positive) cell lines and against
two normal breast epithelial cell lines, MCF-10A and MCF-12A. These
74
molecules inhibited the growth of the human breast cancer cell lines at
low micromolar to nanomolar concentrations, with five of them (1-4, 9)
showing preferential inhibition of the human breast cancer cell lines.
Furthermore, bis-chalcone 8 exhibited a more potent inhibition of colon
cancer cells expressing wild-type p53 than of an isogenic cell line that
was p53-null (Selipe Herencia et al., 1996).
G.Thirunarayanan and G. Vanangamudiet al., worked on
Synthesis of Some Aryl Chalcones Using Silica-Sulphuric Acid Reagent
under Solvent Free Conditions.There are two series of unsaturated
ketones derived from Biphenyl and 9H-Fluorenyl, and ketones with
various substituted benzaldehydes under solvent free conditions using
silica-sulphuric acid as a reagent in an oven. The catalyst silica is
reusable and the yields of chalcones are more than 90%. These
chalcones are characterized by physical constants.
Y.K.Srivastava et al., worked on Ecofriendly microwave assisted
synthesis of some chalcone. Claisen –Schmidt condensation has been
carried out for the synthesis of some o-hydroxchalcone, using
microwave assisted solid phase, solvent free method. Instead of normal
bases like NaOH, KOH the condensation has been carried out in
presence of anhydrous K2CO3 as catalyst which makes the process
eco-friendly, economic and easy and becomes a part of e-chemistry.
75
P.M. Gurubasavaraja Swamy et al., worked on Synthesis and
Antimicrobial Activity of Some Novel Chalcones Containing 3-Hydroxy
Benzofuran.2-Acetyl-3-hydroxy benzofuran were allowed to react
separately with different aromatic aldehydes in presence of 50%
alkaline medium to yield the corresponding 3-hydroxy benzofuran
substituted chalcones. The compounds obtained were identified by
spectral data and screened for antimicrobial activity (Shivakumar PM et
al., 2005).
Jian Wang, Shaojie et al., worked on .Chalcone Derivatives Inhibit
Glutathione S-Transferase P1-1 Activity: Insights into theInteraction
Mode of a, b-Unsaturated CarbonylCompounds. Resistance to
chemotherapeutic drugs has long been a considerable barrier to
successful treatmentof many cancers and over-expression of
glutathioneS-transferase P1-1 is correlated tocarcinogenesis and
resistance of cancer cellsagainst chemotherapeutic agents. This study
throws light on the role of chalcone derivatives.
Bahar Ahmeda and Tawfeq.A et al., worked on Two New Hydroxy
Chalcone Derivatives from Thymus cilicicus. The aerial part of Thymus
cilicicus Linn. (Labiatae) has afforded two new hydroxy chalcone
derivatives, characterized as 4,2,4,6,7, 8-hexahydroxy-7 (8)-dihydro-
chalcone (1), and 3, 4, 2, 4, 6,7, 8-hepta hydroxy-7 (8)-diydro-chalcone
76
(2). The structures of the isolated compounds have beenelucidated
based on various spectral studies.
Ms. Rashmi Jain, O.P chourasia et al., worked on Synthetic and
Antimicrobial Studies of Some New Chalcones of 3-Bromo-4-(p-tolyl
sulphonamido)acetophenone.Eleven new chalcones have been
sysnthesised by condensing 3-bromo-4-(p-tolyl sulphonamido)
acetophenone with different aromatic aldehydesusing the method or
Rohrman et al. The antimicrobial activity of these chalconeshas been
tested by adopting “paper disc diffusion plate method”, against
variouspathogenic fungi(10) and bacteria (9). It has been found that the
chalcones haveconsiderable antifungal activity but less antibacterial
activity. The results showthat these chalcones may find use as
antifungal agents.
Li Feng, Saeed R. Khan et al., worked on Chalcones and their
derivatives have been shown to have potent anticancer activity.
However, the exact mechanisms of cytotoxic activity remain to be
established. In this study, we have evaluated a series of boronic
chalcones for their anticancer activity and mechanisms of action.
Among the eight chalcone derivatives tested, 3,5-bis-(4-boronic acid-
benzylidene)-1-methyl-piperidin-4-one (AM114) exhibited most potent
growth inhibitory activity with IC50 values of 1.5 and 0.6 μM in 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony
77
formation assay, respectively. The cytotoxic activity of AM114 was
shown to be associated with the accumulation of p53 and p21 proteins
and induction of apoptosis.
Y.Rajendra Prasad, A.lakshmana Rao et al., worked on Synthesis
and Antimicrobial Activityof Some Chalcone Derivatives.In an effort to
develop antimicrobial agents, a series of chalconeswere prepared by
Claisen-Schmidt condensation ofappropriate acetophenoneswith
appropriate aromatic aldehydes in the presence of aqueous solution
ofpotassium hydroxide andethanol at room temperature. The
synthesized compounds were characterized by means of their IR, 1H-
NMR spectral data and elemental analysis. All the compounds were
tested for their antibacterial and antifungal activities by the cup plate
method (Yamakawa T et al., 1990).
Beom-Tae and Kim, Kwang-Joong O et al., worked on Synthesis
of Dihydroxylated Chalcone Derivatives with Diverse Substitution
Patterns and Their Radical Scavenging Ability toward DPPH Free
Radicals. A series of dihydroxylated chalcone derivatives with diverse
substitution patterns on a phenyl ring B and the para-substituents on a
phenyl ring A were prepared, and their radical scavenging activities
were evaluated by simple DPPH test to determine quantitative
structure-activity relationship in these series of compounds. The
chalcone compounds with the ortho-(i.e. 2',3'- and 3',4'-) and para-(i.e.
78
2,5'-) substitution patterns show an excellent antioxidant activities (80-
90% of control at the concentration of 50 μM) which are comparable to
those of ascorbic acid and α-tocopherol as positive reference materials.
A Rahaman, Y Rajendra Pasad et al., worked on synthesis and
anti histamic activity of some novel pyrimidines. Novel Pyrimidines were
prepared by the condensation of Chalcones of 4΄-
piperazineacetophenone with guanidine HCl. The structures of the
synthesized compounds RP 1-5were assigned on the basis of
Elemental analysis, IR, 1H NMR and Mass spectroscopy. These
compounds were also screened for antihistaminic activity. The recorded
% of histamine inhibition showed significant antihistaminic activity when
compared to the reference antihistaminic drug mepiramine.
N Domínguez, Caritza León, Juan.Rodrigues et al., worked on
Synthesis and antimalarial activity of sulfonamide chalcone
derivatives.A series of sulfonamide chalcone derivatives were
synthesized and investigated for their abilities to inhibit beta-hematin
formation in vitro and their activity against cultured Plasmodium
falciparum parasites. Inhibition of beta-hematin formation was minimal
in the aromatic ring of the chalcone moiety as it appeared for
compounds 4b, 4d-f, and greatest with compounds 4g (IC50 0.48
microM) and 4k (IC50 0.50 microM) with a substitution of 3,4,5-
trimethoxyl and 3-pyridinyl, respectively. In this study, the most active
79
compound resulted 1[4'-N(2'',5''-dichlorophenyl) sulfonyl-amidephenyl]-
3-(4-methylphenyl)-2-propen-1-one 4i, effective as antimalarial by the
inhibition of cultured P. falciparum parasites (1 microM).
Li-Ping Guan, Ji-Xing Nan, Xue-Jun Jin et al., worked on
Protective Effects of Chalcone Derivatives for Acute Liver. The
hepatoprotective effects of chalcone derivatives were evaluated in D-
galactosamine/lipopolysaccharide (D-GalN/LPS)-induced fulminant
hepatic failure in mouse. Thirteen chalcone derivatives were
synthesized for study and their hepatoprotective effects were evaluated
by assessing aspartate amino transferase (AST) and alanine amino
transferase (ALT) levels inserum.
Pramod Singh, Jagmohan S. Negi et al., worked on5-(3-
Nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde.A novel
1-formyl-2-pyrazoline was synthesized by reaction of an α,β-
unsaturated ketone with hydrazine hydrate and formic acid. The
structure of the title compound was established by UV, IR, 1H NMR,
13C NMR and microanalysis.
Felipe Herencia, M. Pilar López-García et al., worked on Nitric
Oxide-Scavenging Properties of Some Chalcone Derivatives.The
implication of NO in many inflammatory diseases has been well
documented. We have previously reported that some chalcone
derivatives can control the iNOS pathway in inflammatory processes. In
80
the present study, we have assessed the NO-scavenging capacity of
three chalcone derivatives (CH8, CH11, and CH12) in a competitive
assay with HbO2, a well-known physiologically relevant NO scavenger.
Our data identify these chalcones as new NO scavengers. The
estimated second-order rate constants (ks) for the reaction of the three
derivatives with NO is in the same range as the value obtained for
HbO2, with CH11 exerting the greatest effect. These results suggest an
additional action of these compounds on NO regulation (Brogden KA et
al., 2005).
Rafie Hamidpour, Soheil Hamidpour. Camphor a traditional
remedy with the history of trating several diseases.our focus is on the
use of Camphor as a remedy for daily minor problems as well as
reporting some information about the new applications of this traditional
medicine to treat or prevent some serious life threatening diseases such
as cancer and diabetes (IJCRI. 2012)
81
NEED FOR THE STUDY
The compounds used as starting materials are, Citral, Vanillin, Carvone
and Camphor; these compounds are volatile in nature. Hence in this
study we synthesize non-volatile derivatives of these substances with
better Pharmacological activity. By which we derive good potent
moieties as well as we can save the compounds from vaporization.
The major drawback of drugs derived from natural products is that
we cannot produce in a bulk quantity. Since the compounds
synthesized in the present study are very simple structures these can
be synthesized easily by synthesis.
The starting materials of the study are used in day to day life so
the derivatives may not have any major side effects in long term use.
To convert from volatile compounds to non-volatile compound is
very difficult. They are mild in action.
Volatile compounds in pureform is difficult to get and it is very
costly. The Volatile compounds are unstable in nature.
Some compounds shows side effects on use.
82
OBJECTIVES AND HYPOTHESIS
The newly approved drugs mentioned in previous chapter were
derived from natural sources and have been launched on the market
during 2000 – 2005. These new drugs have been approved for the
treatment of cancer, neurological diseases, infectious diseases,
cardiovascular and metabolic diseases, immunological, inflammatory
and related diseases, and genetic disorders, which encompass many of
the common human diseases. Besides new drugs launched on the
market from 2000 to the present, there are a variety of new chemical
entities from natural sources undergoing clinical trials. Thus further
research on these compounds at industrial, governmental, and
academic institutions is seen as vital for the enhancement of human
health.
Based on this concept the present work was designed to synthesize
semisynthetic derivatives of some commonly used phytoconstituent’s. In
the present study semisynthetic derivatives of citral, carvone, camphor
and vanilla were synthesized. These derivatives are chosen due to their
wide pharmacological activity and are used in our day to day life.
Eventhough these compounds are safer but it is not used for their
pharmacological activity due to less potency. Thus the objective of the
present work is
83
To optimize the biological activity of the compounds selected in
the study.
The compounds selected for the study are volatile in nature,
formation of non volatile derivative with a better activity avoids
the wastage through vaporization and also increases the potency.
The compounds to be synthesized in this study are very simple
derivatives and follow one or two step reactions, thus there is very
less chance for the degradation of the starting compound.
The compounds synthesized in this study will be subjected for
TLC, physical and spectral analysis in order to identify purity, and
to interpret the structure of the derivatives synthesized.
The phytoconstituents used in this study has the following
biological activities in common, thus all the derivatives
synthesized in the study will be subjected for the below
mentioned biological activities.
1. Acute toxicity studies
2. Antibacterial activity
3. Antifungal activity
4. Anthelmintic activity
5. In-vitro antioxidant activity
6. Anti-inflammatory activity
7. Analgesic activity
84
METHADOLOGY ADOPTED
Latest trends in drug discovery focuses a lot on the semi synthetic
derivatives, synthesized from the intermediate or the final lead
compound in order to reduce the toxic side effects of synthetically
obtained organic compound or synthesize some complicated drugs like
Vincristine, Vinblatine, Taxols etc., or to optimize the pharmacological
activity, ADME properties of a drug obtained from a natural origin.
Even though lot’s of drugs are available for treating the infections
caused by various bacteria’s, viruses, and fungus. The synthetically
derived compounds gets resistance for these pathogens, thus there is a
need of new molecules in future to treat the infections caused by these
resistant pathogens.
The present study was designed to synthesize some novel
derivatives of some selected compounds. The compounds selected for
the present study are Vanillin, Camphor, Carvone, and Citral. The
above mentioned compound was reported for various pharmacological
activities. Various derivatives of the above mentioned compounds were
designed with a motto to optimize the pharmacological activity and
should be less expensive for synthesis of those compounds. The
derivatives which we have planned for synthesis are smaller molecules
with less chiral centres, thus the total synthesis of these compounds will
be quiet easier.
85
Since the above mentioned compounds are used regularly in day
to day life and derived from natural product it could be safer than the
other synthetically derived compounds.
Various derivatives of the above compounds are prepared by various
reactions like alkylation, benzoylation, mannich condensation reaction
etc. The schemes for the synthesis are represented in the next section
of this thesis. The derivatives synthesized above are subjected for thin
layer chromatography for identifying the purity of the derivatives. The
derivatives will be subjected for physical and spectral analysis like
melting point, FTIR, HNMR, MASS spectroscopy for structural
interpretation.
Further the derivatives will be subjected for pharmacological evaluation.
The pharmacological activities to be carried out are
1. Acute toxicity studies
2. Antibacterial activity.
3. Antifungal activity
4. Antihelmentic activity
5. In-vitro antioxidant activity
6. Anti-inflammatory activity
7. Analgesic activity
86
Schemes for synthesis of Citral Derivatives
Compound 1:
CHO
CH3
CH3CH3
NH2 NH C
O
NH2 C2H5OH
SHAKE
+
hydrazinecarboxamide
MeCOONa+
CH3
CH3CH3
N NH C
O
NH2
2-[(2Z)-3,7-dimethylocta-2,6-dien-1-ylidene]hydrazinecarboxamideCitralCompound I
Compound 2:
OHC
CH3
CH3
CH3
C2H5OH
PYRIDINE
N-hydroxy-1,1-diphenylmethanimine
+
NOH
(2Z)-N-(diphenylmethylidene)-3,7-dimethylocta-2,6-dienamide
Citral
CH3
CH3
O
CH3
Compound II
Synthesis of Vanillin derivatives
Compound 3:
CHO
OH
OCH3
NH2 NH2CH3COONa
OH
OCH3
N
NH2
+
4-[(E)-hydrazinylidenemethyl]-2-methoxyphenolVanillin
Hydrazine
Compound III
Fig.No.13. Schemes for synthesis of compound I-III
87
Compound 4:
CHO
OH
OCH3
+
NHNH2
CH3COONa
OH
OCH3
N
NH
2-methoxy-4-[(E)-(2-phenylhydrazinylidene)methyl]phenol
Vanillin
Phenylhydrazine
Compound IV
Compound 5:
CHO
OH
OCH3
NHNH2
O2N
NO2
OH
OCH3
N
NH
O2N NO2
+CH3COONa
Vanillin
4-{(E)-[2-(2,4-dinitrophenyl)hydrazinylidene]methyl}-2-methoxyphenol
2,4 Dinitro Phenyl Hydrazine
Compound V
Compound 6:
CHO
OCH3
OH
+NH2 NH
NH2
O
CH3COONa
OCH3
OH
N NH
O
NH2
N-[(Z)-(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinecarboxamideVanillin Semicarbazide
Compound VI
Fig.No.14. Schemes for synthesis of compound IV-VI
88
Compound 7:
N N
OH
O
CH3
OH
O
CH3
4,4'-{benzene-1,2-diylbis[nitrilo(E)methylylidene]}bis(2-methoxyphenol)
NH2
NH2
benzene-1,2-diamine
+
CHO
OH
OCH3
Vanillin
Compound VIICompound 8:
HO
OH
O
CH3
+
CH3O
1-phenylethanone
O
O
OH CH3(2E)-3-(3-hydroxy-2-methylphenyl)-1-phenylprop-2-en-1-one
Compound VIIIVanillin
Compound 9:
OH
O
CH3OH
+CH3
OCH3
H
O
CH3OH
O
CH3
butan-2-one
(1Z)-1-(4-hydroxy-3-methoxyphenyl)pent-1-en-3-oneCompound IX
Vanillin
Fig.No.15. Schemes for synthesis of compound VII - IX
89
Compound 10:
HO
OH
O
CH3
+O
diphenylmethanoneOH
O
CH3
O
{3-[(Z)-(3-hydroxy-2-methoxycyclohexa-2,4-dien-1-ylidene)methyl]phenyl}(phenyl)methanone
Compound XVanillin
Compound 11:
OH
O
CH3OH
+
H
O
CH3OH
N
CH3 N
CH3
OH
(2Z,3E)-N,N'-dihydroxybutane-2,3-diimine
4-[(Z)-2-{[(2E,3Z)-3-(hydroxyimino)butan-2-ylidene]amino}ethenyl]-2-methoxyphenol
OH N
N
CH3
OH
CH3
VanillinCompound XI
Synthesis of Carvone derivatives
Compound 12:
CH3
CH3 CH2
O
CH4
CH3
CH3
CH2
N
NH
O2N
NO2
(2E)-1-(2,4-dinitrophenyl)-2-[2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidene]hydrazine
Carvone
NHNH2
N+
O-
O
N+
O-
O
/ Ethanol
Warm
+
2,4 Dinitro Phenyl Hydrazine Compound XII
Fig.No.16. Schemes for synthesis of compound X - XII
90
Compound 13:
C H 3
CH 3 C H 2
O
Carvone
Sod.acetate
Ethanol
Warm
C H 3
CH 3 C H 2
N N H 2
(1 E )-[2 -m ethyl-5 -(p ro p -1 -en-2 -yl)cyc lo hex-2 -en-1 -ylid ene]hyd raz ine
+ NH 2 N H 2
Hydrazine
Compound XIII
Compound 14:
O
CARVONE
Sod.acetate
Shake
N NHCONH2
(2E)-2-[2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidene]hydrazinecarboxamide
semicarbazide
NH2 NH
O
NH2+
Compound XIVCompound 15:
O
Carvone
+NH2
NH2
EthanolReflux
o'-Phenylenediamine
N NCH3
CH3 CH2 CH3CH2
N,N'-Bis[(1e)-2-Methyl-5-(Prop-1-En-2-Yl)Cyclohex-2-En-1-Ylidine]Benzene-1,2-DiamineCompound XV
Compound 16.:
CH3
O
CH3 CH2
+ EthanolPyridineWarm
CH3
N
CH3 CH2
OH
Carvone (1E)-N-hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-im ine
NH2 OH
hydroxylam ine
Compound XVI
91
Fig.No.17. Schemes for synthesis of compound X111 – XVI
Synthesis of Camphor Derivatives
Compound17:
CH3
O
CH3 CH3
1-methylbicyclo[2.2.1]heptan-2-one
+
NH2
NH2
benzene-1,2-diamine
CH3
N
N
CH3
CH3CH3
CH3CH3
N,N'-bis-[(2E)-1-methylbicyclo[2.2.1]hept-2-ylidene]benzene-1,2-diamine-ethane(1:1)
Compound XVII
Compound18:
+Anhydrous ether
CH3
O
CH3 CH3
CH3O
O
CH3 CH3
1-methylbicyclo[2.2.1]heptan-2-one
MgCl
Benzyl megnesium chloride Compound XVIII
2 - benzyl -1,7,7 -methyl[2.2.1]hept -2-ylbenzoate
Compound 19:
CH3
O
+
NH2
aniline
CH3
N
CamphorCompound XIX
N-[(2E)-1,7,7-trimethylbicyclo[2.2.1]hept-2-ylidene]aniline
Fig.No.18. Schemes for synthesis of compound XVII -XIX
The above mentioned derivatives are synthesized, purified using
Thin Layer Chromatography and Column chromatography. The purified
compounds are further processed for physical analysis like meking
92
point, solubility etc and spectral analysis using FTIR, MASS, HNMR for
confirming the structure of the compound.
MATERIALS AND METHODSTable No.2. List of chemicals and their manufactures used for
synthesis
S.No Chemicals required Manufacturers
1 Carvone Gift Sample from Director,OTRI-JNTUA
2 Citral Gift Sample from Director,OTRI-JNTUA
3 Vanillin S.D.Fine Chem Pvt.Ltd, Boisar.
4 Camphor Finar chemical limited ahmedabad
5 Sodium acetate S.d-fine Chem. Ltd, Mumbai
62,4-DinitroPhenylhydrazine S.d-fine Chem. Ltd, Mumbai
7 Hydrazine Sulphate IDPL, Hyderabad8 Semicarbazine Sisco research laboratories, Mumbai
9 O-phenyl diamine Oxford lab, Mumbai
10 Hydroxylamine Sisco research laboratories, Mumbai11 Pyridine Merck specialties pvt.ltd
12 Ethanol Changshu yang Yuan chemical ltdChina
13 Benzophenone S.d-fine Chem. Ltd, Mumbai
14 Phenylhydrazine .HCl S.D.Fine Chem. Pvt.Ltd, India.
15 Iodine S.D.Fine Chem. Pvt.Ltd, India.
16 Hydroxylamine.HCl Sisco research lab, India.
17 Aniline Finar chemical limited ahmedabad
18 Formaldehyde Merck limited Mumbai
19 Conc. Hcl Finar chemical limited ahmedabad
20 Acetone S.D-Fine Chem.Ltd, Mumbai
21 Phenyl acetylene S.D-Fine Chem.Ltd, Mumbai
22 Anhydrous ether Finar chemical limited, Ahmedabad
93
23 Magnesium Merck limited, Mumbai
24 Ammonium Chloride Finar chemical limited,Ahmedabad
25 Benzoyl Chloride Finar chemical limited AhmedabadTable No 3. List of Equipments used during the Experiments:
S.No Equipment MAKE
1. IR SpectroPhotometer
Thermo Nicolet nexus 670spectrophotometer and SHIMADZU, FTIR-8400S.
2. Mass spectrophotometer Micromass Quatro II
3. Elemental Analyser Carlo Erba EA 1108
4. 1H NMR Gemini 300 MHz
5. Melting pointapparatus Toshniwal and Cintex
Instrumentation and general methodology:
All the melting points recorded in this thesis were determined in
open capillaries, using Toshniwal and Cintex melting point apparatus,
expressed in 0C and are uncorrected. The elemental analysis of the
synthesized compounds were determined by using Carlo Erba EA 1108
elemental analyzer expressed in percentage found. The IR spectra of
the compounds were recorded on Thermo Nicolet nexus 670
spectrophotometer using KBr discs and SHIMADZU, FTIR-8400S
expressed in cm-1. 1H NMR spectra were recorded on a Gemini 300
MHz spectrophotometer using TMS as an internal standard and the
values are expressed in δ ppm. Mass spectra of the compounds were
94
recorded by Micromass Quatro II Mass Spectrophotometer operating in
the ESI mode expressed in m/z.
Ia.Synthesis of Citral Derivatives
Procedure for synthesis of compound 1 (2-[(2z)-3,7-dimethylocta-
2,6-dien-1-ylidene]hydrazine carboxamide ) from citral. (Furniss BS,
2008)
1gm of Semicarbazine and 1.5gm of crystallized sodium acetate
was dissolved in 8-10ml of water, to this 0.5gm of the citral was added
and shaken for a while. To the above solution alcohol was added until
the clear solution was obtained. The mixture was shaken for few
minutes and allowed to stand for some time. Semicarbazone
crystallized from the cold solution on standing. Filtered off the crystals
and washed with cold water and recrystalized from dilute ethanol.
Procedure for synthesis of compound II ((2z)-N-
Diphenylmethylidene-3,7-Dimethylocta-2,6-Dienamide ) from
citral.(Brain’s and Furniss, 2008)
A mixture of 0.5gm of citral, 0.5gm of benzophenone oxime, 5ml
of ethanol and 0.5ml of pyridine was refluxed on a water bath for
30minutes. Ethanol was removed by evaporation in a stream of air. 5ml
of water was added to the cooled residue and kept in an ice bath and
stirred until the oxime crystallized out. The solid was filtered and
95
washed with a little water and dried. The dried product was
recrystallized using ethanol.
The structure and nomenclature of citral derivatives are represented in
Table No.4.
Ib.Synthesis of Vanillin Derivatives
Procedure for synthesis of compound III (4[(E)-
Hydrazinylidenemethyl]-2-Methoxyphenol) from Vanillin.
4-[(E)-hydrazinylidenemethyl]-2-methoxyphenol ( Compound III)
was synthesized by dissolving 0.5 gm of hydrazine sulphate and 0.8 gm
of sodium acetate in 5 ml of water. To the above mixture a solution of 2-
5gm of aldehyde (vanillin) in ethanol free from aldehydes and ketones
was added. The solution was shaken until a clear solution is formed
(add little more ethanol if necessary), warm on a water bath for 10-15
minutes and cool until the crystals appear, filter off the crystals and
recrystallize from dilute ethanol.
Procedure for synthesis of compound IV (2-Methoxy-4-[(E)-(2-
Phenylhydraziny lidene ) Methyl]Phenol ) from Vanillin.
2-methoxy-4-[(E)-(2-phenylhydrazinylidene)methyl]phenol was
prepared by dissolving 0.5 gm of colourless phenylhydrazine
hydrochloride and 0.8 gm of sodium acetate in 5 ml of water. To the
above mixture a solution of 2-5gm of aldehyde (vanillin) in ethanol free
from aldehydes and ketones was added. The solution was shaken until
96
a clear solution is formed (add little more ethanol if necessary), warm on
a water bath for 10-15 minutes and cool until the crystals appear, filter
off the crystals and recrystallize from dilute ethanol.
Procedure for synthesis of compoundV(4-{(E)[2-(2,4-Dinitrophenyl)
Hydrazinyl idene]Methyl}-2-Methoxy Phenol) from Vanillin.
4-{(E)-[2-(2,4-dinitrophenyl)hydrazinylidene]methyl}-
2methoxyphenol was prepared by dissolving 0.5 gm of 2,4
dinitrophenyhydrazine hydrochloride and 0.8 gm of sodium acetate in 5
ml of water. To the above mixture a solution of 2-5gm of aldehyde
(vanillin) in ethanol free from aldehydes and ketones was added. The
solution was shaken until a clear solution is formed (add little more
ethanol if necessary), warm on a water bath for 10-15 minutes and cool
until the crystals appear, filter off the crystals and recrystallise from
dilute ethanol.
Procedure for synthesis of compoundVI N-[(Z)-(4-Hydroxy-3-
Methylphenyl) Methylidene]Hydrazinecarboxamide from Vanillin.
N-[(Z)-(4 - hydroxy - 3 - methoxy phenyl) methylidene] hydrazine
carboxamide was prepared by dissolving 1.0 gm of semicarbazine
hydrochloride and 10.5gm of crystalline sodium acetate in 8-10 ml of
water. To the above mixture a solution of 2-5gm of aldehyde (vanillin) in
ethanol free from aldehydes and ketones was added. The solution was
shaken until a clear solution is formed (add little more ethanol if
97
necessary), warm on a water bath for 10-15 minutes and cool until the
crystals appear, filter off the crystals and recrystallise from dilute
ethanol.
Procedure for synthesis of compound VII 4,4'-{benzene-1,2-
diylbis[nitrilo(E) methylylidene]}bis(2-methoxyphenol from Vanillin.
4,4'-{benzene-1,2-diylbis[nitrilo(E)methylylidene]}bis(2-
methoxyphenol) was prepared by the reaction of Vanillin (0.010mol) and
o-Phenylenediamine (0.005mol) in ethanol under reflux for 3 h. The
precipitated product was filtered and recrystallised from the ethanol and
dried in vacuum over CaCl2.
Procedure for synthesis of compound VIII (2E)-3-(3-Hydroxy-2-
Methylphenyl)-1-Phenylprop-2-en-1-One from Vanillin.
2gm of NaoH in 18ml of water and 9ml of rectified spirit was taken
in a 500ml of bolt head flask provide with a mechanical stirrer. The flask
was immersed in a bath of a crushed ice, then 4.8 ml of freshly distilled
acetophenone was added and the stirring was started, During stirring
add 4.2gm of pure vanillin and the temperature of mixture was
maintained at about 250C and vigorous stirring was continued until
mixture becomes so thick that stirring is no longer effective. The stirrer
was removed and left overnight in an ice chest. The product was filtered
with suction and washed with cold water until the washings are neutral
to litmus, and then with 20ml of ice cold rectified spirit.
98
Procedure for synthesis of compound IX (1Z)-1-(4-Hydroxy-3-
Methoxyphenyl) Pent-1-en-3-one from Vanillin.
2gm of NaoH in 18ml of water and 9ml of rectified spirit was taken
in a 500ml of bolt head flask provide with a mechanical stirrer. The flask
was immersed in a bath of a crushed ice, then 4.8 ml of freshly distilled
ethylmethylketone was added and the stirring was started, During
stirring add 4.2gm of pure vanillin and the temperature of mixture was
maintained at about 250C and vigorous stirring was continued until
mixture becomes so thick that stirring is no longer effective. The stirrer
was removed and left overnight in an ice chest. The product was filtered
with suction and washed with cold water until the washings are neutral
to litmus, and then with 20ml of ice cold rectified spirit.
Procedure for synthesis of compound X {3-[(Z)-(3-Hydroxy-2-
Methoxycyclohexa-2,4-Dien-1-Ylidene)MethylPhenyl}(Phenyl)
Methanone from Vanillin.
2gm of NaoH in 18ml of water and 9ml of rectified spirit was taken
in a 500ml of bolt head flask provide with a mechanical stirrer. The flask
was immersed in a bath of a crushed ice, then 4.8gms of pure
benzophenone was added and the stirring was started, During stirring
add 4.2gm of pure vanillin and the temperature of mixture was
maintained at about 250C and vigorous stirring was continued until
99
mixture becomes so thick that stirring is no longer effective. The stirrer
was removed and left overnight in an ice chest. The product was filtered
with suction and washed with cold water until the washings are neutral
to litmus, and then with 20ml of ice cold rectified spirit. The crude
chalcone after drying in the air weigh 2.7gms and melts at 950c.
Recrystallised using rectified spirit at to 50oC.
Procedure for synthesis of compound XI 4-[(Z)-2-{[(2e,3z)-3-
(Hydroxyimino) Butan-2-Ylidene]Amino}Ethenyl]-2-Methoxyphenol
from Vanillin.
2gm of NaoH in 18ml of water and 9ml of rectified spirit was taken
in a 500ml of bolt head flask provide with a mechanical stirrer. The flask
was immersed in a bath of a crushed ice, then 4.8 ml of freshly distilled
dimethyl aldehyde was added and the stirring was started, During
stirring add 4.2gm of pure vanillin and the temperature of mixture was
maintained at about 250C and vigorous stirring was continued until
mixture becomes so thick that stirring is no longer effective. The stirrer
was removed and left overnight in an ice chest. The product was filtered
with suction and washed with cold water until the washings are neutral
to litmus, and then with 20ml of ice cold rectified spirit. Recrystallised
using rectified spirit, m.p. 1000C
The Structure and nomenclature of vanillin derivatives are represented
in Table No.5 and 6.
100
Ic.Synthesis of Carvone Derivatives
Procedure for synthesis of compound XII (2E)-1-(2,4-
Dinitrophenyl)-2-[2-Methyl-5-(Prop-1-en-2-yl)cyclohex-2-en-1-
ylidene]Hydrazine from Carvone.
0.5gm 2,4-dinitro phenyl hydrazine and 0.8gm of sodium acetate
was dissolved in 5ml of water, and a solution of 0.2-0.4g of carvone in a
little ethanol was added and the mixture was stirred until clear solution
appears. Then warm on water bath for 10-15minutes and cooled to
obtain product. The product was filtered using Buchnaer funnel and
washed with cold water and dried. Recrystallize by using ethanol.
Procedure for synthesis of compound XIII (1E)-[2-Methyl-5-(Prop-1-
en-2-yl) cyclohex-2-en-1-ylidene]Hydrazine from Carvone.
0.5gm hydrazine and 0.8gm of sodium acetate was dissolved in
5ml of water, and a solution of 0.2-0.4g of carvone in a little ethanol was
added and the mixture was shaken until clear solution appears. Then
warm on water bath for 10-15minutes and cooled to obtain product. The
product was filtered using Buchnaer funnel and washed with cold water,
dried and recrystallized using ethanol.
Procedure for synthesis of compound XIV (2E)-2-[2-Methyl-5-(Prop-
1-en-2-yl)Cyclohex-2-en-1-Ylidene]Hydrazine Carboxamide from
Carvone.
101
1gm of semi carbazine and 1.5gm of crystallized sodium acetate
was dissolved in 8-10ml of water to this mixture, 0.5gm of the carvone
was added and shaken for a while, then minute quantity of alcohol was
added and continued shaking until a clear solution was obtained. Then
the mixture was allowed to stand for crystallization of semicarbazone.
The crystals were filtered and washed with cold water and recrystalized
from dilute ethanol.
Procedure for synthesis of compound XV N,N’-Bis[(1E)-2-Methyl-5-
(Prop-1-en-2-yl)cyclohex-2-En-1-ylidine]Benzene-1,2-Diamine from
Carvone.
Carvone and ortho phenylenediamine (0.005mol) in ethanol was
taken in a 250ml RBF and refluxed under for 3 hours. Allow the mixture
to cool. The precipitated product was filtered and recrystalized from the
ethanol and dried in vacuum over calcium chloride.
Procedure for synthesis of compound XVI (1E)-n-Hydroxy-2-
Methyl-5-(Prop-1-en-2-yl)cyclohex-2-en-1-Imine from Carvone.
A mixture of 0.5gm of carvone, 0.5gm of hydroxylamine, 5ml of
ethanol and 0.5ml of pyridine was refluxed on a water bath for
30minutes. Remove the ethanol by evaporation of the hot solution in a
stream of air, cool.. To the above mixture 5ml of water was added and
kept in an ice bath and stirred until the oxime crystallizes out. The solid
102
was filtered and washed with little water, dried and recrystalized using
ethanol.
The structure and nomenclature of carvone derivatives are represented
in Table No.7 and 8.
Id.Synthesis of Camphor Derivatives
Procedure for synthesis of compound XVII N,N’-Bis-[(2E)-1-
Methylbicyclo [2.2.1] Hept-2-ylidene]Benzene-1,2-Diamine-
Ethane(1:1) from Camphor.
1 gm of Orthophenylene diamine dissolved in 10ml of ethanol and
1gm of camphor was taken in a 50ml round bottom flask. The mixture
was heated for 3hrs on a water bath with occasional stirring. The
product precipitates out as fine crystals. The crystals were filtered out
and recrystallized using ethanol and dried in vacuum over calcium
chloride.
Procedure for synthesis of compound XVIII 2-Benzyl-1-
Methylbicyclo [2.2.1] Hept-2-Benzoate from Camphor
A solution of Chloro (phenyl) magnesium in 50ml of anhydrous
ether was prepared from 27.3gm of ethyl bromide and 6gm of
magnesium. After cooling, 25.5gms phenyl acetylene in 30ml of
anhydrous ether was added drop wise. The reaction mixture was gently
refluxed for 2hrs and cooled to room temperature. Then the mixture was
slowly stirred and a solution of 45gm of benzophenone in 50ml of
103
anhydrous ether was added and continued stirring at room temp for 1.5
hrs. Then the mixture was refluxed for 1hr and cooled in an ice bath.
55gm of ammonium chloride as a saturated agues solution was added
and the product starts liberating out, the residue oil was kept in ice and
triturate with light petroleum until the buttery phenyl propynol crystallizes
out. Recrystallize using a mixture of benzyl and light petroleum.
Procedure for synthesis of compound XIX N-[(2E)-1-
Methylbicyclo[2.2.1]Hept-2-Ylidene]Aniline from Camphor
5.3gm of dry aniline, 2gm of powdered paraformaldehyde and
6gm of camphor was taken in a 50ml round bottom flask attached to a
reflux condenser. 8ml of 95% ethanol to which 2-3 drops of Conc.HCl
acid have been added was introduced into the reaction mixture and
refluxed on a water bath for 1 hour. The reaction mixture was almost
clear and homogenous. The yellowish solution was filtered through a
pre heated Buchner funnel and the filterate was transfered to a 100ml
wide mouthed conical flask and still warm. 40ml of acetone was added
and allowed to cool room temperature and placed on an ice bath to
obtain crystals. Crystals are filtered at the pump and washed with 2-3ml
of acetone. Acetone was drained and crystals are dried in steam oven
for 30min. Crude product obtained was recrystallized using hot rectified
spirit and the yield of product was 7.4gm, m.p-153-1550C
104
The structure and nomenclature of camphor derivatives are represented
in Table No.4.
Identification and Characterization:
The identification and characterization of synthesized compounds
were carried out by the following procedure to ascertain that all
prepared compounds had different chemical nature than the respective
parent compounds. The yields of the synthesized derivatives are
represented in Table No.11.
Melting point
Solubility
Elemental analysis
Thin layer chromatography
Infrared spectroscopy
Nuclear Magnetic Resonance spectroscopy (N.M.R)
Mass spectroscopy
Melting point determination:
The melting points of the organic compounds were determined by
open capillary tube method. Melting point is a valuable criterion of purity
for an organic compound as a pure crystal is having definite and sharp
melting point. The purity should not be assumed but must be
established by observation of any changes in the melting point when the
compound is subjected to purification by recrystallisation. The
synthesized compounds showed a minute change in melting point after
105
recrystallisation. The melting points of the compounds were reported in
Table No.11.
Solubility:
The solubility of the synthesized compounds was tested in
various solvents. All the synthesized compounds are soluble in Dimethyl
sulphoxide, Dimethyl formamide, Chloroform and Methanol.
Elemental analysis:
The elemental analysis(C, H and N) of the synthesized
compounds were determined by using Carlo Erba EA 1108 elemental
analyzer. The calculated and found percentages of the elements are
presented in Table No.13.
Thin layer chromatography:
Chromatography is an important technique to identify the
formation of new compounds and also to determine the purity of the
compound. The Rf value is characteristic for each of the compound.
Preparation of chromatography plate:
Clean and dry glass plates were taken. Uniform slurry of silica
Gel-G in water was prepared in the ratio of 1:2. The slurry was then
poured into the chamber of the TLC applicator, which was fixed and the
thickness was set to 0.5mm, glass plates were moved under the
applicator smoothly to get uniform coating of slurry on the plates.
106
The plates dried first at room temperature and then kept for
activation at 110oC for 1 hour.
Preparation of solvent system and saturation of chamber:
The solvent system used for the development of chromatogram
was prepared carefully by mixing ethyl acetate: chloroform (0.5ml:
1.5ml).
Application of sample:
The solution of the parent compound and its derivatives were
taken in small bored capillary tube and spotted at 2 cm from the base
end of the plate. After spotting the plates were allowed to dry at room
temperature and plates were transferred to chromatographic chamber
containing solvent system for development.
Development of chromatogram:
Plates were developed by ascending technique when solvent
front had reached a distance of 10-12 cm. They were taken out and
dried at room temperature.
Detection of spots:
The developed spots were detected by exposing them to iodine
vapours.
Calculation of Rf values:
The Rf values of compounds were calculated using the formula.
107
Rf value = Distance moved by sample from origin line / Distance
moved by solvent front from origin line
In all these cases the distance traveled by the sample was found to be
different from that of the parent compound spotted along with it. Thus
confirming that the compound formed was entirely different from that of
the parent compound. Moreover since the entire sample gave a single
spot, the compounds were taken to be free from impurities. The Rf
values of compounds and solvent system used are presented in Table
No.12.
FT-IR Spectra:
The peaks in IR spectrum gives an idea about the probable
structure of the compound, IR region ranges between 4000-666cm-1
quanta of radiation from this region of the spectrum corresponds to
energy differences between different vibrational levels of molecules.
The compounds were recorded on Thermo Nicolet nexus 670,
and SHIMADZU, FTIR-8400S spectrophotometer by using KBr pellet
technique showed different vibration levels of molecules.
The characteristic absorption bands of the few of the
synthesized compounds are presented in Table No.14.
1H NMR spectra:
NMR spectroscopy enables us to record differences in magnetic
properties of the various magnetic nuclei present and to deduce in the
108
large measure about the position of these nuclei within the molecule.
We can deduce how many different kinds of environments are there in
the molecules and also which atoms are present in neighboring groups.
The proton NMR spectra enable us to know different chemical
and magnetic environments corresponding to protons in molecules.
The samples were analyzed on Gemini 300 MHz spectrometer.
The proton NMR spectrums of the synthesized compounds are
presented in Table No.15.
Mass spectroscopy:
Mass spectroscopy enables us to know;
a) Relative molecular masses (molecular weights) with very high
accuracy, from this exact molecular formula can be deduced.
b) To detect within the molecules the places at which it prefers
fragmentation, from this we can deduce the presence of
recognizable groups within the molecule.
c) As a method of identifying analytes by comparison of their mass
spectra with libraries of digitalized mass spectra of known
compounds.
Mass spectra of title compounds were recorded on Micromass Quatro II
MassSpectrophotometer.
109
Table No.4 Structure and nomenclature of Citral derivatives (Compound I, II)
S. No Compound Mol.formula Chemical name Structure
1 I C11H19N3O
2-[(2Z)-3,7-DIMETHYLOCTA-2,6-DIEN-1-
YLIDENE] HYDRA ZINE CARBOXAMIDE
CH3
CH3CH3
N NH C
O
NH2
2 II C23H25NO
(2Z)-N-DIPHENYLMETHYLIDENE-3,7-
DIMETHYLOCTA-2,6-DIENAMIDE N
CH3
CH3
CH3
O
110
Table No.5 Structure and nomenclature of Vanillin derivatives (Compound III - V).
S. No Compound Mol.formula Chemical name Structure
3 III C8H10N2O2
4[(E)-HYDRAZINYLIDENEMETHYL]-2-
METHOXY PHENOL
OH
OCH3
N
NH2
4 IV C14H14N2O2
2-METHOXY-4-[(E)-(2-
PHENYLHYDRAZINYLIDENE) METH
YL]PHENOL
OH
OCH3
N
NH
5 V C14H12N4O6
4-{(E)[2-(2,4-
DINITROPHENYL)HYDRAZINYLIDENE]
METH YL}-2-METHOXY PHENOL
OH
OCH3
N
NH
O2N NO2
111
Table No.6 Structure and nomenclature of Vanillin derivatives (Compound VI - VIII)
S. No Compound Mol.formula Chemical name Structure
6 VI C9H11N3O3
N-[(Z)-(4-HYDROXY-3-ETHYLPHENYL)
METHYLIDENE]HYDRA
ZINECARBOXAMIDE OCH3
OH
N NH
O
NH2
7 VII C22H20N2O4
4,4'-{BENZENE-1,2-DIYLBIS[NITRILO(E)
METHYLYLIDENE]}BIS (2-METHOXY
PHENOL)
N N
OH
O
CH3
OH
O
CH3
8 VIII C16H14O3
(2E)-3-(3-HYDROXY-2-
METHYLPHENYL)-1-PHENYLPROP-2-
EN-1-ONEO
O
OH CH3
112
Table No.7 Structure and nomenclature of Vanillin derivatives (Compound IX - XI)
S. No Compound Mol.formula Chemical name Structure
9 IX C12H14O3
(1Z)-1-(4-HYDROXY-3-
METHOXYPHENYL)PENT-1-EN-3-ONE
H
O
CH3OH
O
CH3
10 X C21H18O3
{3-[(Z)-(3-HYDROXY-2-
METHOXYCYCLOHEXA-2,4-DIEN-1-YL
IDENE) METHY PHENYL}(PHENYL)
METH ANONE OH
O
CH3
O
11 XI C13H16N2O3
4-[(Z)-2-{[(2E,3Z)-3-
(HYDROXYIMINO)BUTAN-2-YLIDENE]
AMINO}ETHENYL]-2-METHOXYPHENOL
H
O
CH3OH
N
CH3 N
CH3
OH
113
Table No.8 Structure and nomenclature of Carvone derivatives (Compound XII -XIV )
S. No Compound Mol.formula Chemical name Structure
12 XII C16H18N4O4
(2E)-1-(2,4-DINITROPHENYL)-2-[2-
METHYL-5-(PROP-1-EN-2-YL) CYCLOHEX-
2-EN-1-YLIDENE]HYDRAZINE
CH3
CH3
CH2
N
NH
O2N
NO2
13 XIII C10H16N2
(1E)-[2-METHYL-5-(PROP-1-EN-2-
YL)CYCLOHEX-2-EN-1-
YLIDENE]HYDRAZINE
CH3
CH3 CH2
N NH2
14 XIV C11H17N3O
(2E)-2-[2-METHYL-5-(PROP-1-EN-2-
YL)CYCLOHEX-2-EN-1-
YLIDENE]HYDRAZINE CARBOXAMIDE
N
CH3 CH2
CH3
NH NH2
O
114
Table No.9 Structure and nomenclature of Carvone derivatives (Compound XV - XVI)
S. No Compound Mol.formula Chemical name Structure
15 XV C25H30N2
N,N’-BIS[(1E)-2-METHYL-5-(PROP-1-EN-2-
YL)CYCLOHEX-2-EN-1-
YLIDINE]BENZENE-1,2-DIAMINE
N NCH3
CH3 CH2 CH3CH2
16 XVI C10H15NO
(1E)-N-HYDROXY-2-METHYL-5-(PROP-1-
EN-2-YL) CYCLO HEX -2-EN-1-IMINE
CH3
N
CH3 CH2
OH
115
Table No.10 Structure and nomenclature of Camphor derivatives (Compound XVII - XIX)
S. No Compound Mol.formula Chemical name Structure
17 XVII C26H40N2
N,N’-BIS-[(2E)-1,7,7-
TRIMETHYLBICYCLO[2.2.1]HEPT-2-
YLIDENE]BENZENE-1,2-DIAMINE-
ETHANE(1:1)
CH3
N
N
CH3
CH3CH3
CH3CH3
18 XVIII C24H30O2
2-BENZYL-1,7,7-
TRIMETHYLBICYCLO[2.2.1]HEPT-2-
BENZOATE
CH3O
O
CH3 CH3
19 XIX C16H23N
N-[(2E)-1,7,7-
TRIMETHYLBICYCLO[2.2.1]HEPT-2-
YLIDENE]ANILINE
CH3
N
CH3 CH3
116
Fig.No.19. FT-IR Spectrum of Compound
Fig. No.18. FT-IR of compound I
117
Fig.No.19. HNMR Spectrum of Compound I
CH3
CH3CH3
N NH C
O
NH2
118
Fig.No.21. MASS Spectrum of Compound I
Fig.No.20. MASS spectrum of compound I
CH3
CH3CH3
N NH C
O
NH2
119
Fig.No.21. FT-IR Spectrum of Compound II
120
Fig.No.22. HNMR Spectrum of Compound II
N
CH3
CH3
CH3
O
121
Fig.No.23. MASS Spectrum of Compound II
N
CH3
CH3
CH3
O
122
Fig.No.24. FT-IR Spectrum of Compound III
123
Fig.No.25. HNMR Spectrum of Compound III
OH
OCH3
N
NH2
124
Fig.No.26. MASS Spectrum of Compound III
OH
OCH3
N
NH2
125
Fig.No.27. FT-IR Spectrum of Compound IV
126
Fig.No.28. HNMR Spectrum of Compound IV
OH
OCH3
N
NH
127
Fig.No.29. MASS Spectrum of Compound IV
OH
OCH3
N
NH
128
Fig.No.30. FT-IR Spectrum of Compound V
129
Fig.No.31. HNMR Spectrum of Compound V
OH
OCH3
N
NH
O2N NO2
130
Fig.No.32. MASS Spectrum of Compound V
OH
OCH3
N
NH
O2N NO2
131
Fig.No.33. FT-IR Spectrum of Compound VI
132
Fig.No.34. HNMR Spectrum of Compound VI
OCH3
OH
N NH
O
NH2
133
Fig.No.35. MASS Spectrum of Compound VI
OCH3
OH
N NH
O
NH2
134
Fig.No.36.FT-IR Spectrum of Compound VII
135
Fig.No.37. HNMR Spectrum of Compound VII
N N
OH
O
CH3
OH
O
CH3
136
Fig.No.38. MASS Spectrum of Compound VII
N N
OH
O
CH3
OH
O
CH3
137
Fig.No.39. FT-IR Spectrum of Compound VIII
138
Fig.No.40. HNMR Spectrum of Compound VIII
O
O
OH CH3
139
Fig.No.41. MASS Spectrum of Compound VIII
O
O
OH CH3
140
Fig.No.42. FT-IR Spectrum of Compound IX
141
Fig.No.43. HNMR Spectrum of Compound IX
H
O
CH3OH
O
CH3
142
Fig.No.44. MASS Spectrum of Compound IX
H
O
CH3OH
O
CH3
143
Fig.No.45. FT-IR Spectrum of Compound X
144
Fig.No.46. HNMR Spectrum of Compound X
OH
O
CH3
O
145
Fig.No.47. MASS Spectrum of Compound X
OH
O
CH3
O
146
Fig.No.48. FTIR Spectrum of Compound XI
147
Fig.No.49. HNMR Spectrum of Compound XI
H
O
CH3OH
N
CH3 N
CH3
OH
148
Fig.No.50. MASS Spectrum of Compound XI
H
O
CH3OH
N
CH3 N
CH3
OH
149
Fig.No.51. FT-IR Spectrum of Compound XII
150
Fig.No.52. HNMR Spectrum of Compound XII
CH3
CH3
CH2
N
NH
O2N
NO2
151
Fig.No.53. MASS Spectrum of Compound XII
CH3
CH3
CH2
N
NH
O2N
NO2
152
Fig.No.54. FT-IR Spectrum of Compound XIII
153
Fig.No.55. HNMR Spectrum of Compound XIII
CH3
CH3 CH2
N NH2
154
Fig.No.56. MASS Spectrum of Compound XIII
CH3
CH3 CH2
N NH2
155
Fig.No.57. FT-IR Spectrum of Compound XIV
156
Fig.No.58. HNMR Spectrum of Compound XIV
N
CH3 CH2
CH3
NH NH2
O
157
Fig.No.59. MASS Spectrum of Compound XIV
N
CH3 CH2
CH3
NH NH2
O
158
Fig.No.60. FT-IR Spectrum of Compound XV
159
Fig.No.61. HNMR Spectrum of Compound XV
N NCH3
CH3 CH2 CH3CH2
160
Fig.No.62. MASS Spectrum of Compound XV
N NCH3
CH3 CH2 CH3CH2
161
Fig.No.63. FT-IR Spectrum of Compound XVI
162
Fig.No.64. HNMR Spectrum of Compound XVI
CH3
N
CH3 CH2
OH
163
Fig.No.65. MASS Spectrum of Compound XVI
CH3
N
CH3 CH2
OH
164
Fig.No.66. FT-IR Spectrum of Compound XVII
CH3
N
N
CH3
CH3CH3
CH3CH3
165
Fig.No.67. HNMR Spectrum of Compound VII
CH3
N
N
CH3
CH3CH3
CH3CH3
166
Fig.No.68. MASS Spectrum of Compound XVII
CH3
N
N
CH3
CH3CH3
CH3CH3
167
Fig.No.69. FT-IR Spectrum of Compound XVIII
168
Fig.No.70. HNMR Spectrum of Compound XVIII
CH3O
O
CH3 CH3
169
Fig.No.71. MASS Spectrum of Compound XVIII
CH3O
O
CH3 CH3
170
Fig.No.72. FT-IR Spectrum of Compound XIX
171
Fig.No.73. HNMR Spectrum of Compound XIX
CH3
N
CH3 CH3
172
Fig.No.74. MASS Spectrum of Compound XIX
CH3
N
CH3 CH3
173
PHARMACOLOGICAL SCREENING
Acute Toxicity Studies: (Anne monks, 19991) Healthy and adult male
albino swiss mice weighing between 20-25g were used in this
investigation. Animals were fasted for 24 hours and divided into groups
of six animals each. The test compounds, suspended in sodium
carboxymethyl cellulose (CMC) solution (0.1%) were administered
intraperitoneally in doses of 5mg to 1000mg per kg (b.w.). The control
groups of animals received only the vehicle (0.1% sodium CMC). The
animals were observed for 48 hours from the time of administration of
test compound to record the mortality. It is approved by Institutional
Animal Ethical Commite (SSRCP/41/2010/CPCSEA-6755, Date: 09-11-
2010), a copy of the same is enclosed at the end of the thesis.
Anti-bacterial activity:The compounds (I-XIX) synthesized were
evaluated for antibacterial activity as per the reported methods 238.
The antibacterial activity of synthesized compounds was
performed against two gram positive bacteria viz., B.subtilis and
S.aureus and two gram negative bacteria viz., E.coli and P. vulgaris by
using cup plate method. Ampicillin sodium was employed as standard
to compare the results.
Culture medium: Nutrient broth was used for the preparation of
inoculum of the bacteria and nutrient agar was used for the screening
method.
174
Composition of Nutrient agar medium
Peptone 5.0 gmSodium chloride 5.0 gmBeef extract 1.5 gmYeast extracts 1.5 gmAgar 15.0 gmDistilled water up to 1000 mlpH 7.4 ± 0.2
The test organisms were subcultured using nutrient agar medium.
The tubes containing sterilized medium were inoculated with respective
bacterial strain. After incubation at 37 ±1oC for 24 hours, they were
stored in refrigerator. The stock cultures were maintained. Bacteria
inoculum was prepared by transferring a loopful of stock culture to
nutrient broth (100 ml) in conical flasks (250 ml). The flasks were
incubated at 37oC ±1oC for 48 hours before the experimentation.
Solution of the test compounds were prepared by dissolving 10mg each
in dimethylformamide (10 ml, AR grade). A reference standard for both
gram positive and gram negative bacteria was made by dissolving
accurately weighed quantity of ampicillin sodium in sterile distilled
water, separately.
The nutrient agar medium was sterilized by autoclaving at 121oC
(15 lb/sq. inches) for 15 min. The petriplates, tube and flasks plugged
with cotton were sterilized in hot-air oven at 160oC, for an hour. Into
each sterilized petriplate (10 cm diameter), about 27 ml of molten
nutrient agar medium was poured and inoculated with the respective
175
strain of bacteria (6 ml of inoculum to 300 ml of nutrient agar medium)
was transferred aseptically. The plates were left at room temperature to
allow the solidification. In each plate, three cups of 6 mm diameter
were made with sterile borer. Then 0.1 ml of the test solution was
added to the respective cups aseptically and labeled, accordingly. The
plates were kept undisturbed for at least 2 hours in refrigerator to allow
diffusion of the solution properly into nutrient agar medium. After
incubation of the plates at 37o ± 1oC for 24 hours, the diameter of zone
of inhibition surrounding each of the cups was measured with the help
of an antibiotic zone reader. All the experiments were carried out in
triplicate. Simultaneously, controls were maintained employing 0.1 ml
of dimethyl formamide to observe the solvent effects. The results are
presented in Tables No.16.
Antifungal Activity:
The compounds (I-XIX) synthesized using the above mentioned
procedures were evaluated for antifungal activity as per the reported
methods (British pharmacopoeia, 1953)
All those compounds screened for antibacterial activity were also tested
for their antifungal activity. The fungi employed for screening were
A.niger, A.flavus, F.oxysporum and C.verticulata. The test organisms
were sub-cultured using potato-dextrose-agar medium. The tubes
containing sterilized medium were inoculated with test fungi and after
176
incubation at 25oC for 48 hours, they were stored at 4oC in refrigerator.
The inoculum was prepared by taking a loopful of stock culture to about
100 ml of nutrient broth, in 250 ml conical flasks. The flasks were
incubated at 25oC for 24 hours before use.
The solutions of test compounds were prepared by a similar
procedure described under the antibacterial activity. Reference
standard (1mg/ml conc.) was prepared by dissolving 10 mg of
clotrimazole in 10 ml of dimethylformamide (AR grade). Further, the
dilution was made with dimethylformamide itself to obtain a solution of
100 g/ml concentration.
The potato-dextrose-agar medium was sterilized by autoclaving at
121oC (15 lb/sq. inches) for 15 minutes. The petriplates, tubes and
flasks with cotton plugs were sterilized in hot-air oven at 150oC, for an
hour. In each sterilized petriplate, about 27 ml of molten potato-
dextrose-agar medium inoculated with respective fungus (6 ml of
inoculum in 300 ml of potato-dextrose medium) was added aseptically.
After solidification of the medium at room temperature three discs of 6
mm diameter were made in each plate with a sterile borer. Accurately
0.1 ml (100 g/disc) of test solution was transferred to the discs
aseptically and labeled, accordingly. The reference standard, 0.1ml (10
g/disc) was also added to the discs in each plate. The plates were
kept undisturbed at room temperature for 2 hours, at least to allow the
177
solution to diffuse properly into the potato-dextrose-agar medium. Then
the plates were incubated at 25oC for 48 hours. The diameter of the
zone of inhibition was read with the help of an antibiotic zone reader.
The experiments were performed in triplicate in order to minimize the
errors. The results are presented in Table.No.190.
Anthelmintic activity: (Dash GK, 2003)
The synthesized compounds were screened for Anthelmintic
activity by using earth worms. Six Indian adult earth worms (Pheretima
postuma) of nearly equal size 5-8 cm in length and 0.2-0.3cm in width
were placed in standard drug solution and test compound solutions at
room temperature. Normal saline was used as control. The standard
drug and test compounds were dissolved in minimum quantity of
dimethyl formamide (DMF) and adjusted the volume up to 15ml with
normal saline solution to get the concentration of 0.1%w/v, 0.2%w/v and
0.5%w/v. Albendazole was used as standard drug. The compounds
were evaluated for the time taken for complete paralysis and death of
earthworms. The mean lethal time for each test compound was
recorded and compared with standard drug. The time taken by worms
to become motionless was noted as paralysis time.
To ascertain the death of motionless worms, they were frequently
applied with external stimuli, which stimulate and induce movement in
the worms, if alive. The mean lethal time and paralysis time of the earth
178
worms for different test compounds and standard drug were tabulated in
Tables No.21.
The results of all the above mentioned experiments are discussed
in next chapter.
In – vitro antioxidant studies:
Chemicals and Reagents:
ABTS [2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid)]
diammonium salt was obtained from Sigma Aldrich Co, St Louis,
USA.and p-nitroso dimethyl aniline (p-NDA) were obtained from Acros
Organics, New Jersey, USA. Ascorbic acid, Nitro blue tetrazolium (NBT)
were obtained from SD Fine Chemicals Ltd., Mumbai, India. 2-Deoxy –
D-ribose was obtained from Himedia Laboratories Pvt. Ltd., Mumbai,
India. All other chemicals used were of analytical grade.
The scavenging of DPPH was performed using 2, 2- Diphenyl 1-
picryl hydrazyl solution (DPPH, 100 μM): Accurately weighed 22 mg of
DPPH and dissolved in 100 ml 0f methanol. From this stock solution, 18
ml was diluted to 100 ml with methanol to obtain 100 μM DPPH
solutions.
The scavenging of nirtric oxide radicals was performed using
Sodium nitroprusside solution (10 Mm): Weighed accurately 0.30 g of
sodium nitroprusside and dissolved in dissolved in distilled water to
make up the volume to 100 ml in a volumetric flask.
179
Naphthyl ethylene diamine dihydrochloride (NEDD): Weighed
accurately 0.1 g of NEDD and dissolved in 60 ml of 50% glacial acetic
acid by heating and made up the volume to 100 ml with distilled water in
a volumetric flask.
Sulphanilic acid reagent (0.33% W/V): Weighed accurately 0.33 g
of sulphanilic acid and dissolved in 20% glacial acetic acid by heating
and made up the volume to 100 ml in a volumetric flask.
A. Scavenging of ABTS radical cation
This method involves the scavenging of ABTS [2, 2΄azino bis (3-
ethylbenzo- thiazoline – 6- sulphuric acid)] radical cation. The principle
behind the technique involves the reaction between ABTS and
potassium persulphate to produce the ABTS radical action, a blue green
chromogen. In the presence of antioxidant reductant the coloured
radical is converted back to colourless ABTS, the absorbance of which
is measured at 734 nm.
Preparation of standard solutions: Required amount of ascorbic acid
was accurately weighed and dissolved in distilled water to prepare 1
mM stock solution. Solutions of different concentrations of ascorbic
acid 10 nM, 30 nM, 100 nM, 300 nM, 1 M, 3 M, 100 M, 300 M, 1
mM were prepared from stock solution.
Preparation of test compound solutions: Required amount of test
compound was dissolved in methanol and 1mM stock solution was
180
prepared. Solutions of concentrations ranging from 100 nM to 1 mM
were prepared from the stock solution.
Preparation of ABTS solution:
ABTS (54.8 mg) was dissolved in 50 ml of distilled water to 2 mM
concentration and potassium persulphate (17 mM) 0.3 ml was added.
The reaction mixture was left to stand at room temperature overnight in
dark before usage.
Standard graph of ascorbic acid: 0.16 ml of ABTS solution and 1 ml
of DMSO was added to 2.8 ml of ascorbic acid solution in a test tube
wrapped with aluminium foil and its absorbance was read out at 517 nm
using UV-visible double beam spectrophotometer. The results were
plotted on a graph and IC50 value was determined.
ABTS Assay procedure for the test compounds:
To 0.2 ml of various concentrations of the test compounds, 1.0 ml of
distilled DMSO and 0.16 ml of ABTS solution was added to make a final
volume of 1.36 ml. Absorbance was measured spectrophotometrically,
after 20 min at 734 nm. The assay was performed in triplicates.
The IC50 values of test compounds were determined by the procedure
similar to ascorbic acid determination.
B. Scavenging of DPPH:
The DPPH free radical is reduced to a corresponding hydrazine when it
reacts with hydrogen donors. The DPPH radical is purple in colour and
181
upon reaction with hydrogen donor’s changes to yellow in colour. It is a
discoloration assay, which is evaluated by the addition of the antioxidant
to a DPPH solution in ethanol or methanol and the decrease in
absorbance was measured.
Preparation of standard ascorbic acid solutions: Required amount
of ascorbic acid was accurately weighed and dissolved in distilled water
to prepare 1 mM stock solution. Solutions of different concentrations of
ascorbic acid 10 nM, 30 nM, 100 nM, 300 nM, 1 M, 3 M, 100 M, 300
M, 1 mM were prepared from stock solution.
Preparation of test compound solutions: Required amount of test
compound was dissolved in methanol and 1mM stock solution was
prepared. Solutions of concentrations ranging from 100 nM to 1 mM
were prepared from the stock solution.
Preparation of DPPH solutions: 0.05 mM of DPPH was prepared by
dissolving 19.71 mg of DPPH in 100 ml of methanol. The solution was
protected from sunlight to prevent the oxidation of DPPH.
Standard graph of ascorbic acid: 0.2 ml of DPPH solution was added
to 2.8 ml of ascorbic acid solution in a test tube wrapped with aluminium
foil and its absorbance was read out at 517 nm using UV-visible double
beam spectrophotometer. The results were plotted on a graph and IC50
value was determined.
DPPH Assay procedure for the test compounds:
182
Aq. soln
0.2 ml of DPPH solution was added to 2.8 ml of the test compounds in a
test tube wrapped with aluminium foil and its absorbance was read out
at 517 nm using UV-visible double beam spectrophotometer. The
assay was performed in triplicates.
The IC50 values of test compounds were determined by the procedure
similar to ascorbic acid determination.
C. Scavenging of Nitric oxide radical
Sodium nitroprusside in aqueous solution at physiological pH,
spontaneously generates nitric oxide, which interacts with oxygen to
produce nitrite ions, which can be estimated by the use of modified
Griess Illosvay reaction251. In the present investigation, Griess Illovay
reagent is modified by using Naphthyl ethylene diamine dichloride
(0.1% W/V) instead of 1-naphthylamine (5%). Nitrite ions react with
Griess reagent, which forms a purple azo dye. In presence of test
components, likely to be scavengers, the amount of nitrites will
decrease. The degree of decrease in the formation purple azo dye will
reflect the extent of scavenging. The absorbance of the chromophore
formed was measured at 540 nm.
Sodium Nitroprusside NO (Nitric Oxide)
NO HNO3 + HNO2Nitric Acid Nitrous Acid
Preparation of standard ascorbic acid solutions: Required amount
of ascorbic acid was accurately weighed and dissolved in distilled water
Dissolved O2/ Water
183
to prepare 1 mM stock solution. Solutions of different concentrations of
ascorbic acid 10 nM, 30 nM, 100 nM, 300 nM, 1 M, 3 M, 100 M, 300
M, 1 mM were prepared from stock solution.
Preparation of test compound solutions: Required amount of test
compound was dissolved in methanol and 1mM stock solution was
prepared. Solutions of concentrations ranging from 100 nM to 1 mM
were prepared from the stock solution.
Standard graph of ascorbic acid: The reaction mixture (6 ml)
containing sodium nitroprusside (10 millimole, 4ml), phosphate buffer
saline (PBS, PH 7.4, 1 ml was added to 2.8 ml of the ascorbic acid
solution and incubated at 25 ºC for 15 min. After incubation, 0.5 ml of
the reaction mixture containing nitrite ion was removed, 1 ml of
sulphanilic acid reagent was added, mixed well and allowed to stand for
5 min for completion of diazotization. Then, 1 ml of NEDD was added,
mixed and allowed to stand for 30 min in diffused light. A pink coloured
chromophore was formed. The absorbance was measured at 540 nm
using UV-visible double beam spectrophotometer. The results were
plotted on a graph and IC50 value was determined.
Nitric Oxide Assay procedure for the test compounds:
The reaction mixture (6 ml) containing sodium nitroprusside (10
millimole, 4ml), phosphate buffer saline (PBS, PH 7.4, 1 ml was added
to 2.8 ml of the test compounds and incubated at 25 ºC for 15 min. After
184
incubation, 0.5 ml of the reaction mixture containing nitrite ion was
removed, 1 ml of sulphanilic acid reagent was added, mixed well and
allowed to stand for 5 min for completion of diazotization. Then, 1 ml of
NEDD was added, mixed and allowed to stand for 30 min in diffused
light. A pink coloured chromophore was formed. The absorbance was
measured at 540 nm using UV-Visible double beam spectrophotometer.
The assay was performed in triplicates.
The IC50 values of test compounds were determined by the
procedure similar to ascorbic acid determination. The datas of the
above performed antioxidant activities are represented in Table No.18.
The above mentioned methods are performed and the results are
discussed in the next chapter.
Anti-inflammatory studies:
Materials:
Polypropylene cages with paddy husk
Plethysmograph
Carrageenan
Diclofenac sodium
Test compounds
Normal saline and water
Animals:
185
All the experiments were carried out using male, Wistar rats (150-
200 g).On arrival the animals were placed at random and allocated to
treatment groups in polypropylene cages with paddy husk as bedding.
Animals were housed at a temperature of 24 ± 2oC and relative humidity
of 30 – 70 %. A 12:12 light: day cycle was followed. All animals were
allowed to free access to water and fed with standard commercial rat
chaw pallets. All the experimental procedures and protocols used in this
study were reviewed by the Institutional Animal Ethics Committee.
Drugs and Chemicals
The drugs and fine chemicals were purchased from Sigma-
Aldrich, India. All other chemicals and solvents were obtained from local
firms (India) and were of highest pure and analytical grade.
Vehicle
Test compounds and Diclofenac sodium were suspended in 0.5%
w/v CMC and administered orally to animals. Carrageenan diluted
separately in normal saline and injected.
Acute Anti-inflammatory Studies:(Saxena RS et al., 1987)
Carrageenan, induced rat paw edema model were used for
evaluating potential of test compounds on inflammation. For each
model, rats were divided in two groups (n = 6). 200-250 mg /kg of test
compound and diclofenac sodium (10 mg/kg) were administered orally
186
one hour before the sub plantar injection of edematogenic agent. The
control groups of animals were received vehicle (1 ml/kg) orally.
Plethysmograph used for measuring paw volume (mm) of rats. Edema
(T) was calculated as follows: T = Tt – T0 Where Tt is the right hind paw
volume (mm) at time‘t’, T0 is hind paw volume (mm) before subplantar
injection.
In this method, acute inflammation was produced by the
subplantar administration of 0.1 ml of 1% w/v carrageenan in the right
paw of the rat. The volume (mm) of the paw was measured immediately
and at 1, 2, 3 and 4 hr intervals after the administration of the
carrageenan. The results are presented in Table No.19..
Analgesic activity:
Materials:
Polypropylene cages with paddy husk
Eddy’s hot plate
Pentazocine lactate as standard
Test compounds
Normal saline and double distilled water
Animals:
All the experiments were carried out using male, swiss Albino
mice (25-30 g). On arrival the animals were placed at random and
allocated to treatment groups in polypropylene cages with paddy husk
187
as bedding. Animals were housed at a temperature of 24 ± 2oC and
relative humidity of 30 – 70 %. A 12:12 light: day cycle was followed. All
animals were allowed to free access to water with standard commercial
chaw pallets. All the experimental procedures and protocols used in this
study were reviewed by the Institutional Animal Ethics Committee.
Drugs and Chemicals:
The drugs and fine chemicals were purchased from Sigma-
Aldrich, India. All other chemicals and solvents were obtained from local
firms (India) and were of highest pure and analytical grade.
Hot Plate Method:
Each group of six mice’s was selected for the present study. One
group served as control and received the vehicle, and one group
received the standard drug pentazocine lactate (30 mg/kg, i.p.). The
drug concentration of 50 mg/kg suspended in acacia was administered
orally to other groups. The mice were placed on Eddy’s hot plate kept at
a temperature of 55 ± 0.5 o C for a maximum time of 15 sec. Reaction
time was recorded when the animals licked their fore-and hind paws
and jumped, at before 0 and 15, 30, 45, and 60 min after administration
of test drugs. In Statistical Analysis all the results were expressed as
mean ± standard error (SEM). Data was analyzed using one-way
ANOVA followed by Dunnett’s t-test. P-values < 0.05 were considered
188
as statistically significant. The results of analgesic activity of title
compounds are presented in Table No.20.
189
RESULTS
Table No.11. Physical properties of the synthesized compounds (I-
XIX)
Sl.No. Compound Meting
point (oC) % yield Molecularweight
Molecularformula
1 Compound No. I 112 43.06%. 209.3 C11H19N3O
2 Compound No. II 130 49.75%. 331.4 C23H25NO
3 Compound No. III 178 59% 166.1 C8H10N2O2
4 Compound No. IV 86 70.3%. 242.2 C14H14N2O2
5 Compound No. V 182 13.7% 332.2 C14H12N4O6
6 Compound No. VI 224 25 % 209.2 C9H11N3O3
7 Compound No.VII 182 14.91% 376.4 C22H20N2O4
8 Compound No.VIII 97 64.5 254.2 C16H14O3
9 Compound No. IX 130 63.4 206.2 C12H14O3
10 Compound No. X 100 55.6% 318.3 C21H18O3
11 Compound No. XI 220 69.6% 248.2 C13H16N2O3
12 Compound No.XII 195 59% 330.3 C16H18N4O4
13 Compound No.XIII 160 70.3% 330.3 C16H23N
14 Compound No.XIV 95 13.7 207.2 C11H17N3O
15 Compound No.XV 163 25 358.5 C25H30N2
16 Compound No.XVI 95 78 165.2 C10H15NO
17 Compound No.XVII 157 61.6 376.5 C26H40N2
18 CompoundNo.XVIII 79 67.7 348.4 C24H30O2
19 Compound No.XIX 176 50.6 227.3 C10H16N2
190
Table No.12. TLC Profile of the Synthesized compounds (I-XIX)
Sl.No Compound Solvent System Proportion Rf Value
1 Compound No. I Acetic Acid: N-Butanol: CCl4 4.5:0.2:0.3 0.33
2 Compound No. II N-Butanol: N-Hexane 4.5:0.5 0.66
3 Compound No. III Acetone: Carbontetrachloride 5:5 0.85
4 Compound No. IV Benzene: Chloroform 4:1 0.7
5 Compound No. V Benzene: Ethanol 2.5:2.5 0.78
6 Compound No. VI Ethanol: N-Hexane 4:1 0.48
7 Compound No. VII Benzene: Ether 4.5:0.5 0.79
8 Compound No. VIII Ethanol- N-Hexane 4.5:0.5 0.85
9 Compound No. IX Ethanol- N-Hexane 4.5:0.5 0.76
10 Compound No. X Ethanol-Water 4.5:0.5 0.63
11 Compound No. XI Ethanol- N-Hexane 4.5:1.5 0.73
12 Compound No. XII Benzene: Ethanol 4.5:0.5 0.8
13 Compound No. XIII Ether: Water :Acetic Acid 5:2.5:2.5 0.7
14 Compound No. XIV Acetone : Alcohol 2.5:2.5 0.70
15 Compound No. XV Ethanol: N-Hexane 4:1 0.48
16 Compound No. XVI Benzene: Ether 4.5:0.5 0.79
17 Compound No.XVII Ethanol: Water 1:4 0.95
18 CompoundNo.XVIII
n-Hexane:CarbonTetrachloride 4.5:0.5 0.89
19 Compound No. XIX Ether:Carbon Tetrachloride 4.5:0.5 0.96
191
Table 13: Elemental analysis of novel semisynthetic compounds (I– XIX)
Sl.No Compound Calculated FoundC% H% N% O% C% H% N% O%
1 CompoundNo. I 63.12 9.21 20.09 7.93 63.13 9.15 20.08 7.64
2 CompoundNo. II 83.32 7.56 4.21 4.12 83.34 7.60 4.23 4.83
3 CompoundNo. III 57.32 5.98 17.23 18.34 57.82 6.07 16.86 19.26
4 CompoundNo. IV 70.12 4.88 12.01 14.09 69.41 5.82 11.56 13.21
5 CompoundNo. V 51.12 4.12 17.09 27.12 50.61 3.64 16.86 28.89
6 CompoundNo. VI 51.78 5.23 20.04 22.89 51.67 5.30 20.09 22.94
7 CompoundNo. VII 70.15 5.87 7.43 17.08 70.20 5.36 7.44 17.00
8 CompoundNo. VIII 75.43 5.55 - 18.92 75.57 5.55 - 18.88
9 CompoundNo. IX 69.91 6.78 - 23.76 69.88 6.84 - 23.27
10 CompoundNo. X 79.21 5.74 - 15.04 79.22 5.70 - 15.08
11 CompoundNo. XI 63.01 6.49 - 19.23 62.89 6.50 - 19.33
12 CompoundNo. XII 58.19 5.32 16.96 19.34 58.17 5.49 16.96 19.37
13 CompoundNo. XIII 73.12 9.79 17.08 - 73.13 9.82 17.06 -
14 CompoundNo. XIV 63.75 8.54 20.19 7.79 63.74 8.27 20.27 7.72
15 CompoundNo. XV 83.78 8.45 7.82 - 83.75 8.43 7.81 -
16 CompoundNo. XVI 72.67 9.21 8.45 9.56 72.69 9.15 8.48 9.68
17 CompoundNo. XVII 82.98 8.89 7.42 - 82,93 9.64 7.44 -
18 CompoundNo.XVIII 82.88 8.03 - 9.18 82.72 8.10 - 9.18
19 CompoundNo. XIX 84.56 9.21 6.12 - 84.53 9.31 6.16 -
192
Table No.14. FT-IR Spectral Datas of the Semisynthetic Compounds (I –
XIX)
Compound Compound structure Spectralpeaks cm-1
Functionalgroups
CompoundNo. I
CH3
CH3CH3
N NH C
O
NH2
341232861686109615121379942
-NH2 stretching-NH stretching-C=O stretching-C-N stretching-C-H bending-C-C stretching-C-H bending
CompoundNo. II N
CH3
CH3
CH3
O 3248305516311076
-C=C stretching-C=C stretching-C=O stretching-C-N stretching
CompoundNo. III
OH
OCH3
N
NH2
3477.132928.551597.271507.80
- OH stretching-CH stretching-NH bending-CH stretching
CompoundNo. IV
OH
OCH3
N
NH
3493.593312.983043.562942.091599.541355.421159.941507.34
- OH stretching-CH stretching-CH stretching-C=N stretching-CH stretching-CH stretching-C-O stretching-NH bending
193
CompoundNo. V
OH
OCH3
N
NH
O2N NO2
3459.083320.613095.151582.601504.841414.121326.37
-OH stretching-CH stretching-CH stretching-C=N stretching-NH stretching-C=C stretching-NO2 (S)
Compound Compound structure Spectralpeaks cm-1
Functionalgroups
CompoundNo. VI
OCH3
OH
N NH
O
NH23515.903463.203294.841646.771605.021441.231350.76
-NH stretching-OH stretching-CH stretching-C=O stretching-C=N stretching-C=C stretching-C-H bending
CompoundNo. VII N N
OH
O
CH3
OH
O
CH3
3402.913000.582936.361599.931523.631456.901225.80
-OH stretching-CH stretching-CH stretching-C=N stretching-NH bending-C=C stretching
CompoundNo. VIII O
O
OH CH3
3256.271656.941317.10820.03
-OH stretching-C=O stretching-C-O stretching-C-H bending
194
CompoundNo. IX
H
O
CH3OH
O
CH3
3259.971656.981581.23819.48
-OH stretching-C=O stretching-C=C stretching-C-H bending
CompoundNo. X
OH
O
CH3
O
3249.781656.581580.97819.94
-OH stretching-C=O stretching-C=C stretching-C-H bending
Compound Compound structure Spectralpeaks cm-1
Functionalgroups
CompoundNo. XI
H
O
CH3OH
N
CH3 N
CH3
OH
3209.261581.031361.65755.59
-OH stretching-C=O stretching-C=C stretching-C-H bending
CompoundNo. XII
CH3
CH3
CH2
N
NH
O2N
NO2
3438.413321.522922.53
16441583.12 ,1329.20
-NH stretching-CH stretching-CH stretching-C=C stretching-N=O-N=O
CompoundNo. XIII
CH3
CH3 CH2
N NH2
3377.463101.811519.80
-NH stretching-CH stretching-C=C stretching
195
CompoundNo. XIV
N
CH3 CH2
CH3
NH NH2
O3456.583404.973206.722924.281688.851572.92
-NH stretching-CH stretching-CH bending-CH stretching-C=O stretching-C=C stretching
CompoundNo. XV
N NCH3
CH3 CH2 CH3CH2
3439.172810.011581.501497.66
-NH stretching-CH stretching-C=C stretching-C=C stretching
Compound Compound structure Spectralpeaks cm-1
Functionalgroups
CompoundNo. XVI
CH3
N
CH3 CH2
OH 3216.463080.032913.901643.65
-OH stretching-NH stretching-CH stretching-C=C stretching
CompoundNo. XVII
CH3
N
N
CH3
CH3CH3
CH3CH3
3430.631733.671497.00810.78
-NH stretching-C=O stretching-CH stretching-CH bending
196
CompoundNo.XVIII
CH3O
O
CH3 CH3
3455.123324.363085.131733.751497.27
-CH stretching-CH stretching-C=O stretching-CH stretching-C-O stretching
CompoundNo. XIX
CH3
N
CH3 CH3
3430.993236.841546.861418.20810.69
-NH stretching-CH stretching-C=O stretching-CH stretching-CH bending
197
Table No.15.1HNMR Spectral Datas of compounds (I – XIX)
Sl.No Compound1HNMRδppm Protons J Value
1 Compound No. I
7.5 s7.0 s6.0 s5.20 t2.0 d1.71 t
1H- CH1H-NH2H-NH21H-CH
4H -CH29H-CH3
7.12.51.27.16.86.4
2 Compound No.II
7.8 – 7.39m5.81s5.20s2.00t1.71
10 H – Ar-H1H - =CH1H- =CH4H-CH2
9H – CH3
5.96.96.96.86.4
3 Compound No.III
8.1s7.0m6.7 q5.0s
3.73s
2H- NH21H-Ar-H1H-Ar- H1H- OH3H-CH3
12.25.55.5
10.26.2
4 Compound No.IV
8.1s6.46-7.01m
5.0s4.0s
3.73s
1H-Ar- CH8H-Ar-H
1H- Ar- OH1H- NH
3H-OCH3
5.75.9
11.213.17.2
5 Compound No.V
8.87q8.33q8.1s7.0q
6.98q6.7q5.0s4.0s
3.73s
1H-Ar-H1H-CH
2H-Ar-H1H-Ar-H1H-Ar-H1H-Ar-H
1H- Ar- OH1H-NH3H-CH3
5.57.15.95.55.55.5
11.22.56.2
6 Compound No.VI
8.1s8.0s7.0q6.7q5.0s
3.73s2.0s
1H-CH1H-NH
2H-Ar-CH1H-Ar-CH1H-Ar-OH3H-OCH32H-NH2
7.18.55.95.5
13.18.18.6
198
7 Compound No.VII
8.39s7.3q
7.01q6.96q6.65q5.0s
3.73s
2H-CH4H-Ar-CH2H-Ar-CH2H-Ar-CH2H-Ar-CH2H-Ar-OH6H-OCH3
8.65.25.95.95.9
11.18.1`
8 Compound No.VIII
8.17t7.81m
7.45-7.54m7.39t6.75q6.60q6.50q5.0s
3.73s
1H-CH2H-Ar-CH3H-Ar-CH
1H-CH1H-Ar-CH1H-Ar-CH1H-Ar-CH1H-Ar-OH3H-OCH3
7.15.95.87.15.55.55.5
11.28.1
9 Compound No.IX
7.37t6.69q6.64q6.57q6.24t5.0s
3.73s2.98t1.11t
1H-=CH1H-Ar-CH1H-Ar-CH1H-Ar-CH1H-=CHIH-Ar-OH3H-OCH32H-CH23H-CH3
6.95.55.55.57.1
10.88.16.96.2
10 Compound No.X
13.9s7.74-7..36m
6.28q6.16t5.66t3.50s2.63d
1H-Ar-OH9H-Ar-CH1H-Ar-CH1H-=CH1H-=CH
3H-OCH32H- CH2
11.25.35.56.95.88.14.2
11 Compound No.XI
6.69q6.64q6.6t
6.57q5.2d5.0s
3.73s2.0s
1.90s
1H-Ar-CH1H-Ar-CH
1H-CH1H-Ar-CH1H-=CH
1H-Ar-OH3H-OCH31H-OH3H-CH3
5.55.57.15.55.8
11.28.19.03.9
199
12 Compound No.XII
8.87q8.33q7.0s
6.98q5.5q
4.88d4.63d2.2q
2.09-1.84m1.52-1.2m
1H-Ar-CH1H-Ar-CH
1H-NH1H-Ar-CH1H-=CH1H-=CH1H-=CH1H-CH2H-CH22H-CH2
5.55.51.45.56.96.97.17.14.24.2
13 Compound No.XIII
7.0s5.5q
4.88d4.63d2.2q
2.09-1.84m1.71d
1.5-1.2m
2H-NH21H-=CH1H-=CH1H-=CH1H-CH2H-CH26H-CH32H-CH2
1.25.85.85.86.94.26.76.8
14 Compound No.XIV
7.0s6.0s5.5d
4.88d4.63d2.2q
2.09-1.85m1.71d
1.52-1.2m
1H-NH2H-NH21H-=CH1H-=CH1H-=CH1H-CH2H-CH26H-CH32H-CH2
8.51.26.96.96.97.18.68.14.2
15 Compound No.XV
7.3q5.7q5.5q
4.88d4.63d2.2q
2.09-1.84m1.71d
1.5-1.2m
4H-Ar-CH1H-=CH1H-=CH2H-=CH2H-=CH2H-CH4H-CH29H-CH34H-CH2
12.16.96.96.86.88.66.84.26.8
16 Compound No.XVI
5.5q4.83d4.69d2.2q
2.09-1.84m1.71d
1.5-1.2m
1H-=CH1H-=CH1H-=CH1H-CH2H-CH26H-CH32H-CH2
6.96.96.97.14.24.84.2
200
17 Compound No.XVII
7.3q2.60-2.34m
1.5t1.4m
1.11m
1H-Ar-CH8H-CH22H-CH4H-CH2
18H-CH3
5.54.18.66.88.8
18 CompoundNo.XVIII
7.99d7.46d7.37d7.21d7.12d7.08d3.06m1.90q1.52q1.49q1.42d1.16s1.11t
2H-Ar-CH1H-Ar-CH2H-Ar-CH2H-Ar-CH2H-Ar-CH1H-Ar-CH2H-CH22H-CH22H-CH22H-CH21H-CH3H-CH36H-CH3
5.45.55.45.45.45.54.24.24.24.25.86.86.7
19 Compound No.XIX
7.3t1.9d1.5m1.4d1.1s
5H--Ar-CH4H-CH21H-CH2H-CH26H-CH3
13.16.85.84.26.7
201
Table No.16: Antibacterial Activity of the Compounds (I – XIX).
Standard: Ampicillin sodium
Compound Antibacterial Activity (Zone of inhibition in mm)B. Subtillis S. Aureus E. coli P. vulgaris
I 18 17 16 14
II 14 15 14 12
III 14 16 13 13
IV 13 17 13 11
V 14 14 14 12
VI 17 13 15 13
VII 15 14 14 11
VIII 16 15 15 13
IX 19 18 23 16
X 17 15 19 15
XI 14 16 20 14
XII 15 15 18 15
XIII 16 17 19 12
XIV 14 15 18 13
XV 17 12 13 11
XVI 15 11 14 10
XVII 16 12 13 11
XVIII 15 13 12 13
XIX 16 11 14 12
Standard(10 µg/cup) 22 20 18 17
*Concentration of Test Compound:100 µg/cup
202
Table No.17: Antifungal Activity of Compounds (I – XIX).
Standard: Clotrimazole
Compound Antifungal Activity (Zone of inhibition in mm)
A. niger C.verticulata
F.oxysporum A. flavus
I 19 16 12 10
II 15 13 10 08
III 16 15 09 09
IV 14 15 08 09
V 12 13 11 10
VI 13 12 10 07
VII 14 13 11 06
VIII 13 14 08 09
IX 17 16 13 11
X 14 14 06 10
XI 15 11 12 09
XII 15 12 13 10
XIII 16 13 12 08
XIV 12 14 09 09
XV 16 14 07 10
XVI 15 13 06 09
XVII 17 12 10 04
XVIII 14 13 08 09
XIX 14 12 10 03Standard
(10 µg/cup) 21 22 23 15
*Concentration of Test Compound:100 µg/cup
203
Table No.18: Antioxidant activity of compounds (I-XIX).
Standard: Ascorbic acid
CompoundIC50 Value (µM)
ABTS DPPH Nitric Oxide
Standard 4.83 5.87 4.02
I 11.93 10.44 9.45
II 8.52 8.21 8.24
III 6.98 7.78 7.01
IV 10.04 11.00 9.83
V 11.83 9.45 10.78
VI 9.73 10.45 9.68
VII 12.89 10.22 11.93
VIII 7.83 7.94 6.52
IX 6.95 7.87 5.96
X 8.73 7.45 7.04
XI 6.39 7.79 6.62
XII 8.05 7.88 8.15
XIII 6.89 7.76 6.93
XIV 9.32 7.99 8.83
XV 9.03 11.77 9.83
XVI 8.89 11.66 7.41
XVII 9.52 10.58 9.21
XVIII 8.43 10.75 9.67
XIX 9.12 9.25 9.54
204
Table No.19: Anti-inflammatory activity of the compounds (I –XIX).
*** p<0.0001, ** p<0.001, * p<0.05 compared to control at respective Time period.NA = Not Applicable. Standard = Diclofenac Sodium
Compound1hr 2hr 3hr 4hr
Mean±SD % red Mean±SD % red Mean±SD % red Mean±SD % redControl 3.32±0.18 NA 3.47±0.19 NA 3.57±0.17 NA 3.27±0.25 NA
Standard 2.33±0.16* 29.81 2.13±0.16* 38.61 2.07±0.1* 42.01 1.83±0.13 44.03I 3.17±0.15 4.51 3.02±0.13 12.96 2.93±0.1 17.92 2.78±0.11 14.98II 2.8±0.17 15.66 2.7±0.2 22.19 2.62±0.16* 26.61 2.48±0.18* 34.15III 2.73±0.16 17.77 2.63±0.15* 24.20 2.5±0.11* 29.97 2.37±0.15* 37.52IV 2.53±0.17* 23.79 2.42±0.09* 30.25 2.27±0.1* 36.41 2.12±0.09* 35.16V 2.65±0.21 20.18 2.45±0.21* 29.39 2.33±0.24* 34.73 2.2±0.25* 32.72VI 3.1±0.11 6.62 2.95±0.1 14.98 2.83±0.15 20.72 2.7±0.16 17.43VII 2.45±0.15* 26.20 2.25±0.19* 35.15 2.15±0.13* 39.77 2.02±0.04* 38.22VIII 2.87±0.16 13.55 2.73±0.10 21.32 2.83±0.11 20.72 2.52±0.09 22.93IX 2.52±0.19* 24.09 2.38±0.13* 31.41 2.22±0.11* 37.81 2.10±0.08* 35.78X 3.28±0.11 4.09 3.20±0.08 9.09 3.12±0.09 13.33 3.0±0.07 12.28XI 3.22±0.07 5.84 3.12±0.10 11.36 3.08±0.07 14.44 2.98±0.07* 32.86XII 3.15±0.10 7.89 3.07±0.12 12.78 2.93±0.08 18.61 2.9±0.11 15.20XIII 3.02±0.07 11.69 2.90±0.08 17.61 2.80±0.12 22.22 2.68±0.11 21.63XIV 2.9±0.07 15.20 2.80±0.08 20.45 2.72±0.11* 24.44 2.6±0.14* 33.97XV 3.18±0.07 7.01 3.10±0.08 11.93 3.0±0.07 16.66 2.9±0.07* 35.20XVI 2.68±0.07 21.63 2.55±0.08* 27.55 2.44±0.08* 32.22 2.3±0.08* 32.74XVII 3.10±0.16 9.35 3.0±0.12 14.77 3.0±0.11 16.66 2.92±0.11 14.61XVIII 2.77±0.05 19.00 2.62±0.07* 25.56 2.53±0.05* 29.72 2.43±0.05* 28.94XIX 2.72±0.09 20.46 2.55±0.05* 28.57 2.45±0.05* 32.50 2.25± 0.05* 35.15
205
Table No.20: Analgesic activity of the compounds (I –XIX).
*** p<0.0001, ** p<0.001, * p<0.05 compared to control at respective Time period,NA = Not Applicable, Standard = Pentazocine Lactate
Compound0.5hr 1hr 2hr
Mean ±SDTime (min) %Protection Mean ±SD %Protection Mean ±SD %Protection
Control 4.42 ± 0.16 NA 4.27± 0.19 NA 4.33 ± 0.11 NAStandard 11.1 ± 0.89* 159.25 12.1 ± 0.05* 183.37 13.2 ± 0.34* 204.89
I 4.95 ± 0.22 12 5.7 ± 0.45 33.48 6.15 ± 0.82 42.03II 5.13 ± 0.12 16 6.3 ± 0.53 47.54 7.2 ± 0.61 66.28III 5.38 ± 0.14 21.7 7.2 ± 0.12 68.61 8.72 ± 0.46* 101.38IV 7.23 ± 0.25 63.5 8.2 ± 0.23 92.03 9.12 ± 0.22* 110.62V 8.56 ± 0.09* 93.66 9.42 ± 0.22* 120.60 10.65 ± 0.43* 145.40VI 5.11 ± 0.43 15.6 5.83 ± 0.17 36.53 6.37 ± 0.51 47.11VII 9.00 ± 0.22* 103.61 10.02 ± 0.15* 134.6 11.50 ± 0.44* 165.58VIII 5.45 ± 0.51 23 6.27 ± 0.38 46.83 7.21 ± 0.21 66.51IX 7.09 ± 0.02 67.15 11.0 ± 0.51 57.6 11.80 ± 0.24* 172.5X 6.05 ± 0.15 36.87 6.17 ± 1.00 44.4 7.15 ± 0.32 65.12XI 5.01 ± 0.15 13.34 5.43 ± 0.23 27.16 6.11 ± 0.81 41.10XII 4.85 ± 0.05 9.72 5.31 ± 0.32 24.35 6.17 ± 0.28 42.49XIII 5.34 ± 0.16 20.8 5.81 ± 0.32 36.29 6.61 ± 0.51 52.65XIV 6.14 ± 0.19 38.91 6.37 ± 0.21 49.18 7.15 ± 0.43 65.12XV 5.63 ± 0.22 27.3 6.52 ± 0.11 52.69 7.91 ± 0.21 82.67XVI 7.67 ± 0.21 73.52 6.13 ± 0.79 43.55 5.91 ± 0.21 36.48XVII 5.65 ± 0.21 27.82 6.82 ± 0.23 59.7 7.75 ± 0.23 78.98XVIII 6.67 ± 0.16 50.90 7.56 ± 0.45 77.04 8.59 ± 0.34 98.38XIX 9.15 ± 0.19* 107.01 10.18 ± 0.14* 138.90 12.08 ± 0.23* 178.98
5.19 ± 0.29 17.42 6.89 ± 0.84 61.35 7.38 ± 0.21 70.43
206
Table No.21: Anthelmintic activity of compounds (I –XIX).
Compound Concentration%w/v
Time in Minutes Mean±SDFor paralysis For Death
Control 0.9% - -
Standard0.1% 49±0.56 68±0.210.2% 44±0.15 62±0.310.5% 38±0.21 53±0.24
I0.1% 60±0.18 158±0.190.2% 57±0.14 145±0.240.5% 53±0.32 139±0.34
II0.1% 49±0.26 152±0.170.2% 42±0.35 137±0.340.5% 39±0.29 128±0.21
III0.1% 57±0.34 162±0.180.2% 55±0.51 143±0.260.5% 52±0.28 135±0.27
IV0.1% 61±0.31 170±0.360.2% 58±0.25 157±0.150.5% 55±0.36 145±0.34
V0.1% 63±0.42 178±0.280.2% 59±0.25 162±0.310.5% 57±0.51 149±0.27
VI0.1% 65±0.24 182±0.540.2% 62±0.32 167±0.510.5% 60±0.29 151±0.34
VII0.1% 67±0.14 168±0.260.2% 63±0.51 149±0.190.5% 59±0.26 140±0.34
VIII0.1% 53±0.34 165±0.280.2% 51±0.31 152±0.350.5% 48±0.24 143±0.34
IX0.1% 42±0.52 142±0.210.2% 39±0.26 137±0.510.5% 30±0.41 125±0.18
X0.1% 52±0.52 167±0.340.2% 50±0.32 155±0.510.5% 47±0.42 149±0.42
207
Standard: Albendazole
XI0.1% 68±0.93 159±0.910.2% 64±0.82 152±0.880.5% 53±0.39 142±0.29
XII0.1% 71±0.72 171±0.220.2% 63±0.78 167±0.850.5% 61±0.64 157±0.87
XIII0.1% 66±0.16 162±0.980.2% 61±0.93 153±0.920.5% 53±0.99 143±0.31
XIV0.1% 59±0.03 149±0.960.2% 56±0.80 139±0.180.5% 53±0.17 127±0.61
XV0.1% 57±0.05 129±0.630.2% 51±0.09 118±0.080.5% 49±0.68 112±0.97
XVI0.1% 63±0.04 139±0.380.2% 59±0.20 124±0.290.5% 52±0.99 118±0.97
XVII0.1% 69±0.08 150±0.380.2% 59±0.96 134±0.760.5% 49±0.60 128±0.89
XVIII0.1% 69±0.09 149±0.950.2% 58±0.28 139±0.430.5% 55±0.03 129±0.92
XIX0.1% 53±0.42 134±0.630.2% 49±0.92 129±0.010.5% 43±0.97 120±0.11
208
DISCUSSIONS
Rapid development of phytochemistry and pharmacological testing methods
in recent years has introduced lot of medicinal plants and many of their active
constituents have been reported. The discovery of aspirin like semisynthetic
derivatives is playing a main role in drug dsiscovery. Based on this concept the
present study was performed to synthesize some novel semisynthetic
derivatives of some commonly used medicinal compounds isolated from the
natural origin.
The compounds selected in the present study are some essential oils with
very popular medicinal properties. The aim of the present study was to avoid the
wastage of these medicinal compounds through vaporization i.e. to convert these
medicinal compounds into their respective non-volatile derivatives.
The volatile substances used in the present study are citral, camphor,
carvone, and vanillin. All the four compounds are used in our day to day life and
were found to contain various medicinal properties, which were also proven
scientifically. In this present study the derivatives of citral, camphor, carvone, and
vanillin are synthesized and evaluated for the possible pharmacological activity.
The derivatives of the above mentioned compounds were synthesized as
per the scheme mentioned in earlier chapter. The compounds synthesized were
subjected for thin layer chromatography to identify the purity of the synthesized
compound.
209
The synthesized compound was subjected for physical analysis like
melting point, solubility, and elemental analysis; and was also subjected for
1HNMR spectroscopy, MASS spectroscopy and FT-IR spectroscopic studies for
characterizing the structure of the synthesized derivatives.
Physical, Analytical and Spectral datas of Citral derivatives
Compound I
Compound I was synthesized as per scheme I and recrystallize to obtain
pure compound. The purity was determined using TLC, the compound 1
exhibited Rf of about 0.8 using solvent system benzene: Ethanol (4.5:0.5). The
melting point was found to be 195oC. Percentage yield was found to be 59%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H Stretching at 3438.41cm-1methyl
at 3321.52 cm-1, C-H stretching salicylic at 2922.53 cm-1, conjugate C=C at
1644.cm-1,NO2 at 1583.12 and 1329.20 cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 7.5 s (1H –CH),
7.0 s (1H-NH), 6.0 s (2H -NH2), 5.20 t (1H-CH), 2.0 d (4H -CH2), 1.71 t (9H-CH3).
The mass spectrum of the compound showed its molecular ion peak (M+)
at m/z at 209. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound I
as 2-[(2Z)-3,7-dimethylocta-2,6-dien-1-ylidene]hydrazine carboxamide.
210
Compound II
Compound II was synthesized as per scheme II and recystallized to obtain
pure compound. The purity was determined using TLC; the compound exhibited
a Rf value of about 0.66 using solvent system N-butanol: n-hexane (4.5:0.5).
The melting point was found to be 195oC. Percentage yield was found to be 59%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of 3248(C=C stretching in aromatic
compound), 3055(C=C stretching in aromatic compound), 1631(C=O stretching
in amide), 1076(C-N stretching), 997.01, 918,766(C-H bending in alkenes),
488(C-C bending).
1HNMR spectrum showed characteristic signals (δ ppm) at 7.8 – 7.39m (10
H – Ar-H) 5.81s (1H - =CH), 5.20s (1H- =CH), 2.00t (4H-CH2), 1.71s (9H – CH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 331. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound II
as (2Z)-N-diphenylmethlidene -3, 7-dimethylocta-2,6-dienamide
Physical, Analytical and Spectral datas of Vanillin derivatives
Compound III
Compound III was synthesized as per scheme III and recystallized to
obtain pure compound. The purity was determined using TLC. The compound
211
showed Rf of about 0.85 using solvent system Acetone: carbon tetrachloride
(5:5). The melting point was found to be 178oC. Percentage yield was found to
be 59%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of phenolic OH(S) at 3477.13cm-1,
CH(S) at2928.55cm-1, C=N at 1597.27cm-1,NH(B) at 1507.80 cm-1,Aromatic
CH(B) deformation at 809.74&750.86cm-1
1HNMR spectrum showed characteristic signals (δ ppm) at 8.1s (2H- NH2),
7.0m (1H-Ar-H), 6.7 q (1H-Ar- H), 5.0s (1H- OH), 3.73s (3H-CH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 166. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound III
as 4[(E)-hydrazinylidenemethyl]-2-methoxyphenol.
Compound IV
Compound IV was synthesized as per scheme IV and recystallized to
obtain pure compound. The purity was determined using TLC; the compound
exhibited Rf of about 0.7 using solvent system Benzene: chloroform (4:1). The
melting point was found to be 86oC. Percentage yield was found to be 70.3%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound
212
showed characteristic absorbance peak of O-H(S) at3493.59 cm-1, C-H(S) at
3312.98 cm-1, two aromatic rings at 3043.56 cm-1& 2942.09 cm-1, C=N at
1599.54 cm-1, CH bending at 1355.42 cm-1, C-O (S) at 1159.94, NH(B) at
1507.34, Aromatic at 614.88 cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 8.1s (1H-Ar-
CH) 6.46- 7.01m (8H-Ar-H) 5.0s (1H- Ar- OH), 4.0s (1H- NH), 3.73s(3H-OCH3)
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 242. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound IV
as 2-methoxy-4-[(E)-(2-phenylhydrazinylidene) methyl] phenol.
Compound V
Compound V was synthesized as per scheme V and recrystallize to obtain
pure compound. The purity was determined using TLC; the compound exhibited
Rf of about 0.78 using solvent system Benzene: Alcohol (2.5:2.5). The melting
point was found to be 182oC. Percentage yield was found to be 65.7%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of OH(B) at 3459.08cm-1, CH(B) at
3320.61cm-1, Aromatic ring at 3095.15cm-1,C=N at 1582.60cm-1, NH(B) at
1504.84cm-1, C=C aromatic (S) at 1414.12 cm-1, NO2 (S) at 1326.37 cm-1.
213
1HNMR spectrum showed characteristic signals (δ ppm) at 8.87q (1H-Ar-
H), 8.33q (1H-CH), 8.1s (2H-Ar-H), 7.0q (1H-Ar-H), 6.98q (1H-Ar-H), 6.7q (1H-
Ar-H), 5.0s (1H- Ar- OH), 4.0s (1H-NH), 3.73s (3H-CH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 332. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound V
as4-{(E)[2-(2,4-dinitrophenyl) hydrazinylidene]methyl}-2-methoxy phenol.
Compound VI
Compound VI was synthesized as per scheme VI and recystalized to
obtain pure compound. The purity was determined using TLC; exhibited Rf of
about 0.48 using solvent system Ethanol: n-hexane (4:1). The melting point was
found to be 224 oC Percentage yield was found to be 54%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of NH(S) at 3515.90cm-1,OH(S) at
3463.20 cm-1, CH aromatic (S) at 3294.84 cm-1, C=O(S) at 1646.77 cm-1 ,C=N(S)
at 1605.02 cm-1,C=C aromatic(S) at 1441.23 cm-1 , CH(B) at 1350.76 cm-1 .
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 209. It exhibited the fragmentation pattern characteristic of the
compound.
214
1HNMR spectrum showed characteristic signals (δ ppm) at 8.1s (1H-CH),
8.0s (1H-NH), 7.0q (2H-Ar-CH), 6.7q (1H-Ar-CH), 5.0s (1H-Ar-OH), 3.73s (3H-
OCH3), 2.0s (2H-NH2).
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound VI
as N-[(Z)-(4-hydroxy-3-methyl phenyl) methylidene] hydrazinecarboxamide.
Compound VII
Compound VII was synthesized as per scheme VII and recystallized to
obtain pure compound. The purity was determined using TLC; the compound
exhibited Rf of about 0.79 using solvent system Benzene: ether (4.5:0.5). The
melting point was found to be 182oC. Percentage yield was found to be 59%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound
showed characteristic absorbance peak OH(S) at 3402.91 cm-1, CH aromatic (S)
at 3000.58 cm-1, CH alkane (S) at 2936.36 cm-1, C=N (S) at 1599.93 cm-1, NH(B)
at 1523.63 cm-1, C=C aromatic (S) at 1456.90 cm-1, C-O (S) at 1225.80 cm-1 .
1HNMR spectrum showed characteristic signals (δ ppm) at 8.39s (2H-CH),
7.3q (4H-Ar-CH), 7.01q (2H-Ar-CH), 6.96q (2H-Ar-CH), 6.65q (2H-Ar-CH), 5.0s
(2H-Ar-OH), 3.73s (6H-OCH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 376. It exhibited the fragmentation pattern characteristic of the
compound. The analytical and spectral data proves the identity and the purity of
the compound. From the spectral datas it also confirms the structure of
215
compound VII as 4,4'-{benzene-1,2-diylbis[nitrilo(e)methylylidene]}bis(2-methoxy
phenol).
Compound VIII
Compound VIII was synthesized as per schemeVIII and recystallized to
obtain pure compound. The purity was determined using TLC; exhibited Rf of
about 0.85 using solvent system, Alcohol- n-hexane (4.5:0.5). The melting point
was found to be 97oC. Percentage yield was found to be 64.5%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H stretching at 3256.27cm-1,C=0
bending at 1656.94cm1. Aromatic C-H bending at 820.03cm-1, Nitro group at
1317.10 cm-1.
HNMR spectrum showed characteristic signals (δ ppm) at 8.17t (1H-CH),
7.81m (2H-Ar-CH), 7.45-7.54m (3H-Ar-CH), 7.39t (1H-CH), 6.75q (1H-Ar-CH),
6.60q (1H-Ar-CH), 6.50q (1H-Ar-CH), 5.0s (1H-Ar-OH), 3.73s (3H-OCH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 254. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound
VIII as (2E)-3-(3-hydroxy-2-methylphenyl)-1-phenylprop-2-en-1-one.
216
Compound IX
Compound IX was synthesized as per scheme IX and recystallized to
obtain pure compound. The purity was determined using TLC; the compound
exhibited Rf of about 0.76 using solvent system, Alcohol- n-hexane (4.5:0.5).
The melting point was found to be 130oC. Percentage yield was found to
be 63.4%.
The percentage of elements (C, H, O, N) obtained from elemental analysis was
matching with the calculated percentage. FT-IR Spectrum of Compound showed
characteristic absorbance peak of N-H bending at 3259.97cm-1, C=0 stretching
at 1656.98cm-1, Aromatic C-H bending at 819.48cm-1,Nitro group at 1318.82 cm-
1. C=C stretching at1581.23cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 7.37t (1H-=CH),
6.69q (1H-Ar-CH), 6.64q (1H-Ar-CH), 6.57q (1H-Ar-CH), 6.24t (1H-=CH), 5.0s
(IH-Ar-OH), 3.73s (3H-OCH3), 2.98t (2H-CH2), 1.11t (3H-CH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 206. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound IX
as (1Z)-1-(4-hydroxy-3-methoxyphenyl) pent-1-en-3-one.
Compound X
Compound X was synthesized as per scheme X and recystallized to obtain
pure compound. The purity was determined using TLC, the compound exhibited
217
Rf of about 0.63 using solvent system, Alcohol-water (4.5:0.5). The melting point
was found to be 100oC. Percentage yield was found to be 55.6 %.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H stretching at 3249.78cm-1, C=0
bending at 1656.58cm-1, Aromatic C-H bending at 819.94cm-1,C=C at
1580.97cm-1. aromatic rings at 1460.55cm-1 .
1HNMR spectrum showed characteristic signals (δ ppm) at 13.9s (1H-Ar-
OH), 7.74-7.36m 9H-Ar-CH), 6.28q (1H-Ar-CH), 6.16t (1H-=CH), 5.66t (1H-=CH),
3.50s (3H-OCH3), 2.63d (2H- CH2).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 318. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound X
as {3-[(Z)-(3-hydroxy-2-methoxycyclohexa-2,4-dien-1-ylidene) methyl
phenyl}(phenyl) methanone.
Compound XI
Compound XI was synthesized as per scheme XI and recystallized to
obtain pure compound. The purity was determined using TLC; exhibited Rf of
about 0.73 using solvent system, Alcohol- n-hexane (4.5:1.5). The melting point
was found to be 220oC. Percentage yield was found to be 69.6%.
218
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound
showed characteristic absorbance peak of N-H bending at 3209.26cm-1, C=0
bending at 1581.03cm-1, Aromatic C-H bending at 755.59cm-1,C-H bending
acyclic at 1361.65cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 6.69q (1H-Ar-
CH), 6.64q (1H-Ar-CH), 6.6t (1H-CH), 6.57q (1H-Ar-CH), 5.2d (1H-=CH), 5.0s
(1H-Ar-OH), 3.73s (3H-OCH3), 2.0s (1H-OH), 1.90s (3H-CH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 248. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound XI
as 4-[(Z)-2-{[(2E,3Z)-3-(hydroxyimino) butan-2-ylidene]amino}ethenyl]-2-
methoxyphenol.
Physical, analytical and spectral datas of carvone derivatives
Compound XII
Compound XII was synthesized as per scheme XII and recystallized to
obtain pure compound. The purity was determined using TLC, the compound
exhibited Rf of about 0.8using solvent system, Benzene: Ethanol (4.5:0.5). The
melting point was found to be 195oC. Percentage yield was found to be 59%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
219
showed characteristic absorbance peak of N-H Stretching at 3438.41cm-1methyl
at 3321.52 cm-1, C-H stretching alicyclic at 2922.53 cm-1, conjugate C=C at
1644.cm-1,NO2 at 1583.12 and 1329.20 cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 8.87q (1H-Ar-
CH), 8.33q (1H-Ar-CH), 7.0s (1H-NH), 6.98q (1H-Ar-CH), 5.5q (1H-=CH), 4.88d
(1H-=CH), 4.63d (1H-=CH), 2.2q (1H-CH), 2.09-1.84m (2H-CH2), 1.52-1.2m (2H-
CH2).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 330. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound
XII as (2E)-1-(2, 4-dinitrophenyl)-2-[2-methyl-5-(prop-1-en-2-yl) cyclohex-2-en-1-
ylidene] hydrazine.
Compound XIII
Compound XIII was synthesized as per scheme XIII and recystallized to
obtain pure compound. The purity was determined using TLC, the exhibited Rf of
about 0.7 using solvent system; ether: water: acetic acid (5:2.5:2.5). The melting
point was found to be 160oC. Percentage yield was found to be 70.3%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H stretching at 3377.46cm-1, C-H
cyclic stretching at 3101.81 cm-1, C=C at 1519.80 cm-1.
220
1HNMR spectrum showed characteristic signals (δ ppm) at 7.0s (2H-NH2),
5.5q (1H-=CH), 4.88d (1H-=CH), 4.63d (1H-=CH), 2.2q (1H-CH), 2.09-1.84m
(2H-CH2), 1.71d (6H-CH3), 1.5-1.2m (2H-CH2).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 164. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound
XIII as (1E)-[2-methyl-5-(prop-1-en-2-yl) cyclohex-2-en-1-ylidene]hydrazine.
Compound XIV
Compound XIV was synthesized as per scheme XIV and recystallized to
obtain pure compound. The purity was determined using TLC, the compound
exhibited Rf of about 0.70 using solvent system; Acetone: Alcohol (2.5:2.5). The
melting point was found to be 95oC. Percentage yield was found to be 53.3%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound
showed characteristic absorbance peak of N-H stretching at 3456.58 cm-1, C-H
stretching at 3404.97 cm-1, C-H bending methyl at 3206.72 cm-1, CH stretching
at 2924.28 cm-1, C=0 twisting at 1688.85 cm-1, C=C ring stretching at b1572.92
cm-1, CH bending acyclic at 1379.08 cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 7.0s (1H-NH),
6.0s (2H-NH2), 5.5d (1H-=CH), 4.88d (1H-=CH), 4.63d (1H-=CH), 2.2q (1H-CH),
2.09-1.85m (2H-CH2), 1.71d (6H-CH3), 1.52-1.2m (2H-CH2).
221
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 207. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound
XIV as (2E)-2-[2-methyl-5-(prop-1-en-2-yl) cyclohex-2-en-1-ylidene] hydrazine
carboxamide.
Compound XV
Compound XV was synthesized as per scheme XV and recystallized to
obtain pure compound. The purity was determined using TLC, the compound
exhibited Rf of about 0.0.48 using solvent system; Ethanol: n-hexane :( 4:1). The
melting point was found to be 163oC. Percentage yield was found to be 57%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H stretching at 3439.17 cm-1, C-H
stretching and methyl at 2810.01 cm-1, C=C ring stretching aromatic at 1581.50
cm-1, C=C ring aromatic at 1497.66 cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 7.3q (4H-Ar-
CH), 5.7q (1H-=CH), 5.5q (1H-=CH), 4.88d (2H-=CH), 4.63d (2H-=CH), 2.2q (2H-
CH), 2.09-1.84m (4H-CH2), 1.71d (9H-CH3), 1.5-1.2m (4H-CH2).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 358. It exhibited the fragmentation pattern characteristic of the
compound.
222
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound
XV as N,N’-bis[(1E)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-ylidine]benzene-
1,2-diamine.
Compound XVI
Compound XVI was synthesized as per scheme XVI and recystallized to
obtain pure compound. The purity was determined using TLC; the compound
exhibited Rf value of about 0.8 using solvent system benzene: Ethanol (4.5:0.5).
The melting point was found to be 195oC. Percentage yield was found to be 59%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H Stretching at 3438.41cm-1methyl
at 3321.52 cm-1, C-H stretching alicyclic at 2922.53 cm-1, conjugate C=C at
1644.cm-1,NO2 at 1583.12 and 1329.20 cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 5.5q (1H-=CH),
4.83d (1H-=CH), 4.69d (1H-=CH), 2.2q (1H-CH), 2.09-1.84m (2H-CH2), 2.0s (1H-
OH), 1.71d (6H-CH3), 1.5-1.2m (2H-CH2).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 165. It exhibited the fragmentation pattern characteristic of the
compound. The analytical and spectral data proves the identity and the purity of
the compound. From the spectral datas it also confirms the structure of
compound XVI as (1E)-N-hydroxy-2-methyl-5-(prop-1-en-2-yl) cyclohex-2-en-1-
imine.
223
Physical, Analytical and Spectral datas of Camphor derivatives
Compound XVII
Compound XVII was synthesized as per scheme XVII and recystallized to
obtain pure compound. The purity was determined using TLC, the compound
exhibited Rf of about 0.79 using solvent system; Benzene: ether (4.5:0.5). The
melting point was found to be 95oC. Percentage yield was found to be 78%.
The percentage of elements (C, H, O, N) obtained from elemental analysis
was matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H stretching at 3216.46 and
3080.03 cm-1, C-H stretching at 2913.90 cm-1, C=C ring aromatic at 1437.43 cm-
1, C=O 1643.65 cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 7.3q (1H-Ar-
CH), 1.60-1.34m (8H-CH2), 1.5t (2H-CH), 1.4-1.2m (4H-CH2), 1.11m (18H-CH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 376. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound
XVII as N, N’-bis-[(2E)-1,7,7-trimethyl bicyclo[2.2.1]hept-2-ylidene]benzene-1,2-
diamine-ethane(1:1).
Compound XVIII
Compound XVIII was synthesized as per scheme XVIII and recystallized to
obtain pure compound. The purity was determined using TLC; the compound
224
exhibited Rf of about 0.89 using solvent system N-hexane: Carbon tetrachloride
(4.5:0.5).
The melting point was found to be 79oC. Percentage yield was found to be
67.7%w/w.
The percentage of elements (C, H, O, N) obtained from elemental analysis was
matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H stretching at 3455.12cm-1,
Aromatic C-H bending at 3085.13cm-1, C=0 bending at 1733.75cm-1,C-H methyl
group at 3324.36, Aromatic C-H bending at 826.26cm-1, aromatic rings at
1497.27-1 cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 7.99d (2H-Ar-
CH), 7.46d (1H-Ar-CH), 7.37d (2H-Ar-CH), 7.21d (2H-Ar-CH), 7.12d (2H-Ar-CH),
7.08d (1H-Ar-CH), 3.06m (2H-CH2), 1.90q (2H-CH2), 1.52q (2H-CH2), 1.49q
(2H-CH2), 1.42d (1H-CH), 1.16s (3H-CH3), 1.11t (6H-CH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 348. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound
XVIII as 2-benzyl-1,7,7-trimethylbicyclo [2.2.1] hept-2-benzoate.
Compound XIX
Compound XIX was synthesized as per scheme XIX and recystallized to
obtain pure compound. The purity was determined using TLC; the compound
225
exhibited Rf of about 0.96 using solvent system, Ether: Carbon tetrachloride
(4.5:0.5).
The melting point was found to be 176oC. Percentage yield was found to
be 50.6%.
The percentage of elements (C, H, O, N) obtained from elemental analysis was
matching with the calculated percentage. FT-IR Spectrum of Compound I
showed characteristic absorbance peak of N-H bending at 3441.99cm-1,
Aromatic C-H bending at 3061.84cm-1, C=0 stretching at 1733.75cm-1, Aromatic
C-H bending at 810.69cm-1,aromatic rings at 1497.12.cm-1.
1HNMR spectrum showed characteristic signals (δ ppm) at 7.3t (5H--Ar-
CH), 1.6d (4H-CH2), 1.5m (1H-CH), 1.4d (2H-CH2), 1,1s (6H-CH3).
The mass spectrum of the compound showed its molecular ion (M+) peak
at m/z at 227. It exhibited the fragmentation pattern characteristic of the
compound.
The analytical and spectral data proves the identity and the purity of the
compound. From the spectral datas it also confirms the structure of compound
XIX as N-[(2E)-1,7,7-trimethylbicyclo [2.2.1] hept-2-ylidene] aniline.
II. Pharmacological Screening.
The semisynthetic derivatives of citral, vanillin, carvone and camphor was
synthesized as per the scheme and procedures discussed in our earlier chapters.
The compounds synthesized were subjected for various pharmacological
activities and their datas were represented in the tables illustrated in previous
chapters. The pharmacological activities are acute toxicity studies, analgesic
226
activity, anti-inflammatory activity, anthelmintic activity, Antimicrobial activity,
antifungal activity, and invitro antioxidant activity.
Acute toxicity studies:
The acute toxicity studies were performed as per OECD guidelines 423. All
the compounds synthesized were administered intraperitonially for mice (20-25
gm) and monitored for the mortality rate for next 48hrs. There was no mortality
rate and there were no behavioral changes observed for 48hrs. This proved the
compounds synthesized were safe up to 2000mg/kg body weight.
Sl.No. Compounds LD50 Value (mg/kg body weight)
1 Compound No. I 2150
2 Compound No. II 2310
3 Compound No. III 2815
4 Compound No. IV 2620
5 Compound No. V 2885
6 Compound No. VI 2620
7 Compound No. VII 2410
8 Compound No. VIII 2765
9 Compound No. IX 2816
10 Compound No. X 2011
11 Compound No. XI 2620
12 Compound No. XII 928
13 Compound No. XIII 889
227
14 Compound No. XIV 1110
15 Compound No. XV 920
16 Compound No. XVI 967
17 Compound No. XVII 1240
18 Compound No.XVIII 1011
19 Compound No. XIX 1150
Antimicrobial activity:
The antibacterial activity of compounds synthesized was performed
against two gram positive bacteria viz., B.subtilis and S.aureus and two gram
negative bacteria viz., E.coli and P. vulgaris by using cup plate method.
Ampicillin sodium was used as standard. The compounds showed good to mild
antimicrobial activity.
The two citral derivatives synthesized in the study has shown antimicrobial
activity among them compound I has shown better antimicrobial activity with a
greater zone of inhibition of 18mm in comparison with 14mm of compound II. But
when compared with the standard drug Ampicillin, compound II has shown
moderate antibacterial activity. While comparing the structure of compound I and
compound II, both the compounds contain Nitrogen hetero atom where as the
compound II contains Keto group and the aromatic rings which are absent in
compoundI. This states that removal of keto group is essential for the
antimicrobial activity, attaching the aromatic ring substituted amine to the
228
aldehyde group will show better activity than attaching a nitrogen attached to a
aliphatic compounds.
Nine derivatives of vanillin have been subjected for antimicrobial activity,
all the compounds have shown mild to moderate antibacterial activity. Among
them compoundIX has shown good activity against both gram positive and gram
negative bacteria in comparison with other derivatives. But when compared with
standard except compound IX other derivatives has shown mild antibacterial
activity, compound IX has shown moderate to good antimicrobial activity against
gram positive and gram negative bacteria. The zone of inhibition of compound IX
was 19mm and 18mm against B. Subtillis and S. Aureus, whereas zone of
inhibition of Ampicillin (standard) was found to be 22 mm and 20mm respectively.
This clearly shows that compound IX has good antimicrobial activity. The
compound IX has shown excellent and better activity against gram negative
bacteria’s with zone of inhibition of 23mm and 16mm when compared with
ampicillin (standard) with zone of inhibition of 18mm and 17mm against E.Coli
and P.Vulgaris respectively. This proves compound IX is a better compound with
good and excellent antimicrobial activity against gram positive and gram negative
bacteria.
When comparing the structure of compoundIX with the other derivatives;
compound IX comes under the category of chalcones as reported earlier in the
literature chalcones has shown to have antimicrobial activity. Thus it could be
concluded that the presence of keto group in vanillin is essential for microbial
activity and also attachment of higher aromatic substances or heteroatom’s like
229
nitrogen will not increase the activity and also has not diminished the activity but
to get better activity it will be better to substitute the aldehyde group with smaller
alkyl chains to achieve more potent antimicrobial compounds.
Five derivatives of carvone were synthesized and all the compounds were
subjected for antimicrobial activity against both gram positive and gram negative
bacteria. All the five derivatives were found to posses’ mild to moderate
antimicrobial activity. In comparison with ampicillin (standard), compoundXIII was
found to have good antimicrobial activity against both gram positive and gram
negative bacteria. The zone of inhibition of compound XIII was found to be
16mm and 17mm, whereas ampicillin (Standard) showed zone of inhibition of 22
mm and 20mm against B.Subtilis and S.aureus respectively.
In Compound XIII, the oxygen of the keto group was replaced by hydrazyl
group whereas on other compounds the oxygen was replaced with other
substituents like aromatic rings and alkyl derivatives. The presence of the
hydrazyl group may be responsible for increase in the antimicrobial activity.
All the three camphor derivatives has shown mild to moderate
antimicrobial activity, but none of the derivatives showed comparatively good
antimicrobial activity in comparison with the standard drug (Ampicillin).
Antifungal activity:
Citral derivatives were subjected for antifungal activity against A.niger, C.
verticulata, F. oxysporum, A. flavus and compared with standard drug. Among
them compound I showed better activity with zone of inhibition of 19mm against
A.niger but was not comparative active against other organisms.
230
Various derivatives of vanillin were synthesized and subjected for
antifungal activity, all the derivatives showed mild antifungal activity with respect
to the standard drug. Among them compound IX was found to have a better
activity with zone of inhibition of 17mm, 16mm, 13mm and 11mm against A.niger,
C. verticulata, F. oxysporum, and A. flavus respectively
Even the carvone derivatives have not shown any comparative activity
against the organisms used. Still all the compounds were more or less with the
same potency it clearly shows that the activity of the parent compound has not
been affected much with the substitution. Thus more derivatives have to be
synthesized by modifying the structure or by substituting other substituents in
other suitable portions of the compound.
Carvone derivatives have shown mild to moderate antifungal activity.
Among the carvone derivatives compoundXV have shown moderate activity in
comparison with the standard drug. It has shown the zone of inhibition of 16mm,
14mm, 07 mm and 10mm against A.niger, C. verticulata, F. oxysporum, and A.
flavus respectively.
Camphor derivatives showed mild to moderate activity against the
organisms used for antifungal activity. CompoundXVII has shown better activity
when compared with the other derivatives of camphor. It has the zone of
inhibition of 17mm, 12mm, 10mm and 04mm against A.niger, C. verticulata, F.
oxysporum, and A. flavus respectively.
Thus it was concluded that the compounds synthesized was not very
effective against the fungus, since the parent compound has antifungal property
231
in future more works should be targeted on these derivatives to optimize the
structure and to come up with a potent and a safer drug.
Invitro antioxidant activity:
A.Scavenging of ABTS radical cation:
ABTS radical anion scavenging activity was performed for all the derivatives of
citral, vanillin, and carvone. The experiments were performed in triplicates and
their average was taken and the IC50 values were calculated. Ascorbic acid was
used as standard. The IC50 values of the synthesized compounds were compared
with IC50 of ascorbic acid.
The Ic50 values of citral are more when compared to that of ascorbic acid
which shows that both the derivatives have very less scavenging property.
Among the vanillin derivatives compounds IX and III had moderate radical
scavenging activity. They have shown IC50 of 6.39 and 6.98 respectively, which is
comparable with the IC50 value 4.83 of Ascorbic acid. Compounds (IV-VII) have
showed very poor radical scavenging activity.
The carvone derivatives subjected for ABTS radical scavenging activity showed
mild to moderate scavenging activity. Among them compounds XIII showed
moderate scavenging activity when compared with the IC50 value of ascorbic
acid. Next compound with better scavenging activity is compound XII with a IC50
value 8.05. The other compounds showed very poor or mild scavenging property.
All the camphor derivatives have shown the IC50 value around and above more
than 9 which show the derivatives have very poor radical scavenging activity.
232
B.DPPH radical scavenging activity
The compounds synthesized were subjected for DPPH radical scavenging
activity and the results were compared with Ascorbic acid. Both the citral
derivatives have shown IC50 at a higher concentration it clearly indicates that
citral derivatives have very less radical scavenging property.
Vanillin derivatives have shown mild to moderate scavenging property.
Compound III, VIII, IX, X, XI has shown IC50 values around seven which is less
than the IC50 value of standard. Thus it can be concluded that these compounds
has moderate radical scavenging activity. Whereas, other derivatives of vanillin
have shown IC50 value at a higher concentration.
Except compound XIII and XIV all other derivatives of carvone has the IC50
values at a very high concentration. Compound XIII and XIV have their IC50 of
7.76 and 7.99 respectively, which proves that both the compounds posses
moderate antioxidant property.
The results of camphor derivatives show the IC50 value at higher concentration
except compound XVII. The IC50 Compound XVII was 7.88 which can be
considered as moderate scavenging property when comparing with the IC50
value of standard.
C.Nitric oxide radical scavenging activity
The IC50 values of the derivatives were around 9μM which is high when
compared to the IC50 values of the standard drug. This proves that citral
derivatives have shown very mild antioxidant activity.
233
Vanillin derivatives showed mild to moderate antioxidant activity. Compound
III, VIII, IX and XI has shown good antioxidant activity with the IC50 values of 7.01,
6.52, 5.96 and 6.62μM respectively. Other derivatives had the IC50 values at
higher concentration i.e. above 9μM.
Compound XIII of carvone derivatives showed good nitric oxide scavenging
activity at IC50 value of about 6.93μM concentration. The other derivatives of
carvone have not shown satisfactory results in scavenging the nitric oxide
radical.
The camphor derivatives had the IC50 value above 9 μM concentration. This
proves that the camphor derivatives have very mild nitric oxide radical
scavenging activity.
Anti-inflammatory activity
The anti inflammatory activity of all the synthesized compounds was
carried out using Male, Wister rats. Anti-inflammatory activity was evaluated by
carrageenan induced paw edema model using the standard drug diclofenac
sodium (10mg/ml) and results are presented in Table NO. 19. The results
mentioned showed good significance value with P < 0.05.
As per the results represented in Table No.19., all the derivatives
synthesized showed anti-inflammatory activity. Among the two citral derivatives
compound II showed potent anti-inflammatory activity whereas compound I
showed moderate anti-inflammatory activity.
All the nine derivatives of vanillin have showed satisfactory anti-inflammatory.
Compound III, IV, V, VI, VII, VIII and IX showed good potent anti-inflammatory,
234
whereas compound X and XI showed moderate anti-inflammatory activity in
comparison with standard drug Diclofenac sodium (10μg/ml).
Among the derivatives of carvone, compoundXVI and XIV showed potent
anti-inflammatory activity in par with the standard drug. The other derivatives
showed moderate activity with 15 to 30 percentage reduction of inflammation
when compared with the standard drug.
The results of camphor derivatives states that they posses moderate to good
potent anti-inflammatory activity. Compound XIX and XVIII showed good potency
and were in par with the standard drug. The compound XVIII also showed good
moderate activity.
Analgesic Activity:
All the derivatives of citral, camphor, carvone and vanillin have been
evaluated for their analgesic activity by Eddy’s hot plate method. The results of
analgesic activity are presented in Table No.20. The data represents that none of
the tested compounds shown analgesic activity as good as the standard drug.
The citral derivatives showed less to moderate activity when compared with the
standard drug. Among the vanillin derivatives CompoundIII, IV, V, VII and IX
showed good activity when compared to the standard drug, whereas the other
vanillin derivatives i.e. compound VI, VIII, X, and XI showed less to moderate
analgesic activity in comparison with the standard drug.
The carvone derivatives (compound XII – XVI) showed moderate activity in
comparison to the standard drug. The datas prove that compound XIX of
camphor derivative showed good potent analgesic activity when compared with
235
the standard drug. The other two derivatives i.e. compound XVII and XVIII
showed moderate analgesic activity when compared with the standard drug.
Anthelmentic activity
All the compounds were screened for Anthelmintic activity by using Indian
adult earth worms (Pheretima postuma). The compounds were evaluated for the
time taken for complete paralysis and death of earthworms by taking albendazole
as the standard drug with 0.1, 0.2, and 0.5 % concentrations. The compounds
were evaluated and results are presented in Table No.21.
As per data’s recorded in Table No.20. Citral derivatives were found effective in
causing paralysis but have consumed more time to cause death. Among the two
derivatives compoundII was found to be more effective and the time consumed
was less and in par with the standard whereas it has not shown to be as effective
as the standard drug in causing death.
Vanillin derivatives also showed the same results as that of citral
derivatives; they have taken more time to cause death when compared with the
standard drug (Albendazole). Among the vanillin derivatives, compound X has
showed excellent paralytic effect on Indian earth worms but was not as effective
as standard drug in causing death. All the other derivatives showed good
paralytic effect on Indian earth worms. Carvone and camphor derivatives also
shown good paralytic effect but they consumed more time in causing death to
Indian earthworms.
236
SUMMARY AND CONCLUSION
Nature always stands as a golden mark to exemplify the outstanding
phenomenon of symbiosis. Several herbs consist of powerful ingredients, which
are helpful to cure a number of health problems. A crude (untreated) extract from
any one of these sources typically contains novel, structurally diverse chemical
compounds, which the natural environment is a rich source of Chemical diversity
in nature is based on biological and geographical diversity, so researchers travel
around the world obtaining samples to analyze and evaluate in drug discovery
screens or bioassays. As a result of rapid development of phytochemistry and
pharmacological testing methods in recent years, new plant drugs are finding
their way into medicine as purified phytochemicals, rather than in the form of
traditional galenical preparations. The earliest pure compounds discovered were
salicin, isolated from the bark of the white willow, Salix alba, in 1825-26. It was
subsequently converted to salicylic acid via hydrolysis and oxidation, and proved
as successful as an antipyretic (fever reducing) that it was actively manufactured
and used worldwide. The use of salicylic acid, however, often led to severe
gastrointestinal toxicity. This was overcome when Felix Hoffmann of Bayer
Company converted salicylic acid into acetylsalicylic acid (ASA) via acetylation.
Bayer then began marketing ASA under the trade name aspirin in 1899. Today,
aspirin is still the most widely used analgesic and antipyretic drug in the world.
Based on the above concept four pharmacologically potential compounds
were selected. Various derivatives of these compounds were synthesized using
237
simple synthetic procedures. A total of nineteen semi-synthetic derivatives were
synthesized and their structures are confirmed by Physical and spectral analysis.
The derivatives of the above mentioned compounds were synthesized as
per the scheme mentioned in earlier chapter. The compounds synthesized were
subjected for thin layer chromatography to identify the purity of the synthesized
compound.
The synthesized compound was subjected for physical analysis like
melting point, solubility, and elemental analysis; and was also subjected for
1HNMR spectroscopy, MASS spectroscopy and FT-IR spectroscopic studies for
characterizing the structure of the synthesized derivatives.
The physical, elemental and spectral datas of the above mentioned compounds
was interpreted and the structure of the synthesized derivatives was elucidated.
The list of newly synthesized derivatives is represented in the table below.
Table No.22. List of the Newly Synthesized Derivatives along with their
IUPAC Name.
Sl.NOCOMPOUND
NUMBERCOMPOUND NAME
1 Compound No. I2-[(2Z)-3,7-DIMETHYLOCTA-2,6-DIEN-1-YLIDENE]HYDRAZINE
CARBOXAMIDE
2 Compound No. II(2Z)-N-DIPHENYLMETHYLIDENE-3,7-DIMETHYLOCTA-2,6-
DIENAMIDE
3 Compound No. III 4[(E)-HYDRAZINYLIDENEMETHYL]-2-METHOXYPHENOL
4Compound No.IV
2-METHOXY-4-[(E)-(2-
PHENYLHYDRAZINYLIDENE)METHYL]PHENOL
5 Compound No. V4-{(E)[2-(2,4-DINITROPHENYL)HYDRAZINYLIDENE]METHYL}-2-
METHOXY PHENOL
238
6Compound No.VI
N-[(Z)-(4-HYDROXY-3-
METHYLPHENYL)METHYLIDENE]HYDRAZINECARBOXAMIDE
7Compound No.VII
4,4'-{BENZENE-1,2-DIYLBIS[NITRILO(E)METHYLYLIDENE]}BIS(2-
METHOXY PHENOL)
8Compound No.VIII
(2E)-3-(3-HYDROXY-2-METHYLPHENYL)-1-PHENYLPROP-2-EN-1-
ONE
9Compound No.IX
(1Z)-1-(4-HYDROXY-3-METHOXYPHENYL)PENT-1-EN-3-ONE
10 Compound No. X{3-[(Z)-(3-HYDROXY-2-METHOXYCYCLOHEXA-2,4-DIEN-1-YLIDENE)
METHY PHENYL}(PHENYL) METH ANONE
11Compound No.XI
4-[(Z)-2-{[(2E,3Z)-3-(HYDROXYIMINO)BUTAN-2-
YLIDENE]AMINO}ETHENYL]-2-METHOXYPHENOL
12Compound No.XII
(2E)-1-(2,4-DINITROPHENYL)-2-[2-METHYL-5-(PROP-1-EN-2-
YL)CYCLOHEX-2-EN-1-YLIDENE]HYDRAZINE
13Compound No.XIII
(1E)-[2-METHYL-5-(PROP-1-EN-2-YL)CYCLOHEX-2-EN-1-
YLIDENE]HYDRAZINE
14Compound No.XIV
(2E)-2-[2-METHYL-5-(PROP-1-EN-2-YL)CYCLOHEX-2-EN-1-
YLIDENE]HYDRAZINE CARBOXAMIDE
15Compound No.XV
N,N’-BIS[(1E)-2-METHYL-5-(PROP-1-EN-2-YL)CYCLOHEX-2-EN-1-
YLIDINE]BENZENE-1,2-DIAMINE
16Compound No.XVI
(1E)-N-HYDROXY-2-METHYL-5-(PROP-1-EN-2-YL)CYCLOHEX-2-EN-
1-IMINE
17Compound No.XVII
N,N’-BIS-[(2E)-1,7,7-TRIMETHYLBICYCLO[2.2.1]HEPT-2-
YLIDENE]BENZENE-1,2-DIAMINE-ETHANE(1:1)
18CompoundNo.XVIII
2-BENZYL-1,7,7-TRI METHYLBICYCLO[2.2.1]HEPT-2-BENZOATE
19Compound No.XIX
N-[(2E)-1,7,7-TRI METHYLBICYCLO[2.2.1]HEPT-2-YLIDENE]ANILINE
The above mentioned derivatives were subjected for various
pharmacological activities like Acute Toxicity Studies, Analgesic Activity, Anti-
239
Inflammatory Activity, Anthelmintic Activity, Antimicrobial Activity, Antifungal
Activity and Invitro Antioxidant Activity.
The main advantage of semi synthetic drug is they can act with higher
potency than their original natural products such as onset of action, potency, site
of action etc.
Based on the above facts Four Pharmacologically potential compounds
were selected. The various derivatives of this compound were synthesized by
using simple synthetic procedures. The total Nineteen semi synthetic derivatives
were synthsized and their structures are conformed by physical and spectral
analysis.
All the synthesized compounds were subjected for different activities, the
Anti-bacterial activity of the synthesized compounds were performed against two
Gram positive and Gram negative bacteria. The compound II, IX and XIII has
potent Anti-bacterial activity. Further more studies on these derivatives for safer
and potent Anti-bacterial drug.
The Anti-fungal activity of these derivatives compound I, IX, XV and XVII
has shown moderated activity, thus in future more works has to be carried out on
these derivatives to come up with potent moiety.
The compound III to XVIII showed good potent Anti-inflamatory activity
when compared with standard drug.
The acute toxicity studies showed that all theNineteen derivatives were
safe even up 1000mg/kg and thus a dose of 300mg/kg i.p. was used as safer
dose in experimental animals.
240
Based on the above it could be concluded that compound III, IV, IX
and XIII were found to have good potency in all activity performed. Thus structure
of these derivatives has to be optimized to explore the desired Pharmacological
activity
241
CONCLUSIONS
The conclusions drawn from the results discussed in the earlier chapters are as
follows.
1. Synthetic work performed in the present study was positive, as the
predicted compounds were obtained and confirmed using the spectral and
physical data’s.
2. Acute toxicity studies showed that all the nineteen derivatives were safe
even up to 1000 mg/kg and thus a dose of 300 mg/kg i.p. was used as
safer dose in experimental animals.
3. The antibacterial activity of the synthesized was performed against two
gram positive bacteria and negative bacteria proved that compound II,
compound IX, and compound XIII has potent antibacterial activity. This
concludes that further more studies on these derivatives could end up with
a safer and potent antibacterial drug.
4. Antifungal activity of these nineteen derivatives has not shown promising
results but compound I, Compound IX, Compound XV and compound XVII
has shown moderate activity, thus in future more works has to be carried
out on these derivatives to come up with a potent moiety.
5. Antioxidant activities carried out using radical ion scavenging property of
the molecules. Mostly all the molecules were having good activity.
Compound III, Compound IX and Compound XIII showed good radical ion
scavenging activity in all the three methods. Since antioxidant compounds
are likely to posses’ anticancer activity. Thus in future these compounds
242
should be subjected for anticancer screening to identify their anticancer
activity.
6. Compound III, IV, V, VI, VII, VIII, IX, XVI, XIV, XIX and XVIII showed good
potent anti-inflammatory when compared with the standard drug. Thus
more studies have to be carried out using other models to identify their
potency.
7. Analgesic activity results showed that Compound III, IV, V, VII IX and XIX
posses potent analgesic activity. Since these compounds has also proved
their anti inflammatory activity in our previous study. More pharmacological
studies has to be carried out and SAR studies showed be performed on
these derivatives to optimize their analgesic and anti-inflammatory activity.
8. The anthelmentic activity showed that all the derivatives have good
paralytic effect but was not so effective in causing death, thus the structure
has to be optimized in order to derive more potent and safer drug.
Overall it could be concluded that compound III, IV, IX and XIII were found
to have good potency in all the activity performed. Thus structure of these
derivatives has to be optimized to explore the desired pharmacological activity.
243
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