Avinash Tiwari Thesis

THESIS
SUBMITTED TO THE
UNIVERSITY OF LUCKNOW
FOR THE DEGREE OF
Doctor of Philosophy
IN
CHEMISTRY
By
Avinash Tiwari
M.Sc. (Chemistry)
DEPARTMENT OF CHEMISTRY
UNIVERSITY OF LUCKNOW
LUCKNOW, (INDIA)
2014
Dedicated
To my loving Family
LUCKNOW UNIVERSITY
Dr. R.N. Pathak
Prof. & Head
Department of Chemistry
Lucknow University,
Lucknow-226007
India
Ref:
Res. CM-19, Sector-B Aliganj,

Lucknow-226024.
Res. 0522-233-1447
Mobile. 9415402641
Date:..
This is to certify that the work embodied in this thesis entitled DESIGN, SYNTHESIS
AND BIOEVALUATION OF NOVEL HETEROCYCLES AS ANTILEISHMANIAL
AGENTS has been carried out by Mr. Avinash Tiwari under the supervision of Prof. Padam
Kant and Dr. S.N. Suryawanshi (co-supervisor). He has fulfilled all the requirement of Lucknow
University, Lucknow for the degree of Doctor of Philosophy.
(Prof. R. N. Pathak)
LUCKNOW UNIVERSITY
Prof. Padam Kant
Department of Chemistry
Lucknow University,
Lucknow-226007
India
Res. 8/345, Vikas Nagar,

Lucknow-226007.
Res. 0522-233-0460
Mobile. 9450362878
Dated:........................
This is to certify that the work embodied in this thesis entitled DESIGN, SYNTHESIS
AND BIOEVALUATION OF NOVEL HETEROCYCLES AS ANTILEISHMANIAL
AGENTS has been carried out by Mr. Avinash Tiwari under my supervision. He has fulfilled
all the requirement of Lucknow University, Lucknow for the degree of Doctor of Philosophy.
The work presented in the thesis is original and has not been submitted so for to any other
institute/university either in part or full for any degree or diploma.
(Prof. Padam Kant)

Supervisor
Dr. S. N. Suryawanshi
Chief Scientist
Medicinal & Process Chemistry Division
CSIR-Central Drug Research Institute
Lucknow-226001
India
Dated:
This is to certify that the work embodied in this thesis entitled DESIGN,
SYNTHESIS
AND
BIOEVALUATION
OF
NOVEL
HETEROCYCLES
AS
ANTILEISHMANIAL AGENTS submitted to the Lucknow University, Lucknow, has been

performed by Mr. Avinash Tiwari, under my supervision at CSIR-Central Drug Research
Institute, Lucknow in order to fulfill the requirements for the award of the degree of Doctor of
Philosophy. The work presented here is original and has not been submitted so far, in part or
full, for any other degree or diploma of any other university/institute.
(Dr. S. N. Suryawanshi)
Co-Supervisor
ACKNOWLEDGEMENT
All praises be to ALMIGHTY, the most gracious, the most merciful, the most
peaceful, the cherisher and the sustainers of the worlds who guides us in darkness and
helps in difficulties. This piece of work would have not been accomplished without his
help.
It was a long journey to complete this thesis. The journey was truly adventurous
and challenging. I feel overwhelmingly ecstatic and fortunate enough to seize the gracious
opportunity to have worked under my supervisors Prof. Padam Kant (Department of
Chemistry, Lucknow University, Lucknow, India) and Dr. S. N. Suryawanshi, (Chief
Scientist, Medicinal and Process Chemistry Division, CSIR-Central Drug Research
Institute, Lucknow, India) for valuable guidance, suggestions, as well as constant
encouragement throughout my work.
I would like to express my deep and sincere gratitude to Dr. S. N. Suryawanshi
who gave me the opportunity to study at the CSIR-CDRI. His supervision and guidance
during the whole project, together with his enormous support, proved to be priceless. His
enthusiastic view on research has made a deep impression on me. He kept an eye on the
daily progress of my work. His suggestions will remain with me as an inexhaustible source
of scientific learning throughout my life. I am also thankful to Dr. S.B. Katti, Chief
Scientist, CSIR-CDRI, who provided me an opportunity to work in his guidance after
retirement of my supervisor.
I would like to pay my sincere thanks to the present Director of CSIR-CDRI, Dr.
S.K. Puri, and former Director Dr. T. K Chakraborty, for providing the necessary amenities
and giving me the opportunity to work in such a competitive environment. I am highly
indebted to Dr. B. Kundu, Head, Dr. A. K. Saxena, former Head, Medicinal Process
Chemistry Division, CSIR-CDRI to allow me to use the facilities of the department. I am
grateful to all the staff of SAIF, CSIR-CDRI for providing the instrumental facility.
I owe my deepest gratitude to Dr. Ram Pratap, Dr. R. P. Tripathi, Dr. Vijay
Laxmi, Dr. Kanchan Hajela, Dr. P. M. S. Chauhan, Dr. S. Batra, Dr. A. K. Shaw, Dr.
Atul Kumar, Dr. Atul Goel and Dr. Depankar Koley for their help and support.
I wish to express my warm and sincere thanks to Dr. Suman Gupta, Parasitology
Division for providing the valuable results of biological screening.
I offer deserved thankfulness to Mrs. Manju, Mr. H. R. Mishra, and Mr. N. P.
Mishra for their utmost help and co-operation in providing me technical assistance. I
would also like to thank my lab attendant Virender ji and Awadh Ram for providing
possible help. I am also thankful to all the members and staff of Medicinal and Process
Chemistry Division, E-I, E-II, Stores, Glass blowing section, Workshop and Library for
their cooperation during my research period.
Words cant suffice in paying my gratefulness for what I achieved and learnt from
my respected teachers to whom I owe utmost esteem and reverence. I am highly grateful for
their persistent encouragement and sympathetic attitude. I would like to express my deep
and sincere gratitude to Prof. R. N. Pathak (Head), Prof. Sudha Jain, Prof. V. K. Pandey,
Prof. Naveen Khare, Dr. Desh Deepak, Prof. R. M. Naik, Dr. R. K. Tiwari, Dr. Joy
Sarkar and Prof. A. K. S. Chauhan, Department of Chemistry, University of Lucknow,
Lucknow (U.P.) for their constant valuable suggestion and encouragement.
I could barely miss the memorable and invaluable company of my laboratory
colleagues Santosh Kumar, Ved ji, Krupal, Sachin, Shalini, Ankita, Neha, Neetu and
Akshmala for their kind cooperation fruitful suggestions, and keeping a very cheerful
environment in the lab. My wholehearted gratitude goes particularly to my seniors Dr.
Naveen Chandra, Dr. Susmita Pandey, Dr. Shishir Srivastava and Dr. Sunil K. Mishra
who have helped me through their sincere advice, moral support, constant encouragement
and cooperation and were very generous and kind towards me.
I am especially thankful to Rahul Shivahare who helped me in every endeavor
regarding the biological activity.
During these years, I have met a lot of good friends in CSIR--CDRI. My
acknowledge will remain incomplete without recognizing admirable and loving support
from Drs. Mukesh Kumar, Siddharth Sharma, Shubhashish, Sandeep Basu, Shahnawaz
Khan, S. P. Singh, Imran, Kamil, Vinay Kumar and Shashi Pandey. I would also like to
express my profound gratitude towards all the research fellows of our Division for their
persistent help, unconditional support and invaluable suggestions, pertinent names to
mention are Mr. Prem Chandra Verma, Nishant, Yarkali Krishna,Vikas Bajpai, Ashok
Kumar Maurya, Munna Prasad Gupta, Soumya Bhattacharyya, Mrs. Jaya Tiwari, Mrs.
Sukanya Panditi and Ms. Meena Devi.
I would like to pay my special thanks to Drs. Alok Kumar Verma, Vishwadeepak
Tripathi, Ajay Arya, Promod Kumar and Dr. Vishal M. Balaramnavar for giving me a lot
of support and encouragement when it was most required.
Friendship is a god given virtue and a valuable asset. I would cherish the company
of friends like Akhilesh, Amita I, Amita II, Amreen, Anand, Kamal, Lalit, Manish,
Nisha, Pankaj, Puneet, Rashmi, Saif, Saurabh, Shivam, Sudhir, Tripurari, Vaibhav and all
my friends who instilled in me the real virtues of friendship and acquaintanship. I would
also like to appreciate some of my old friends Sucharu, Sanjay, Divyendu, Arun and
Anurag for their moral support and kind cooperation throughout the Ph.D.
I am forever indebted to my family and would like to express my deep sense of
gratitude to my loving Parents (Sri Balkrishn Tiwari & Smt. Pushpa Tiwari), Sister
(Anupama and Anuradha) and Brother (Anurag) for their unconditional support,
encouragement and love to pursue my interest. My special appreciation goes to my Uncles,
Aunties, Brothers-in-law, Sisters-in-law, all my cousins and my sweet nephews and nieces
for giving me a lot of love and also for rejuvenating me during my entire Ph.D. programme.
Regards to those who have been close enough to be mentioned but not included by
name in this acknowledgement. I also expect their grant of forgiveness and acknowledge
their help and support.
This thesis would not have been possible without the financial support I received
from CSIR-UGC.
Last but not the least, I would like to offer my sincere gratitude and obeisance to
the invincible creator, with whose grace and kindness I stand today in achieving my
ambitions and desires.
(Avinash Tiwari)
CONTENTS
Page No.
List of Abbreviations
Preface
I-III
IV-V
CHAPTER 1: CHEMOTHERAPY OF LEISHMANIASIS SO FAR:

A REVIEW
1.1
Introduction
1.2
Types of leishmaniasis
1.2.1 Cutaneous leishmaniasis
1.2.2 Mucocutaneous leishmaniasis
1.2.3 Visceral leishmaniasis
1.3
Life cycle
1.4
Current status
1.5
Conventional drugs
1.5.1 Antimonials
1.5.2 Pentamidine
1.5.3 Amphotericin B
1.5.4 Paromomycin
1.5.5 Miltefosine
1.6
Recent advancements
1.6.1 Sitamaquine
1.6.2 Azithromycin
1.6.3 Imiquimod
1.6.4 Azoles
1.6.5 Combination therapy
1.7
Scope of natural product
1.8
Natural product lead based studies
1.8.1 Curcumin: The spice of life-unlocking the secrets of curcumin
1.8.2 Chalcone
1.9
Conclusion
1.10 References
1-38
CHAPTER 2.1: DESIGN, SYNTHESIS AND BIOEVALUATION OF

NOVEL TERPENYL HETEROCYCLES
2.1.1 Introduction
2.1.2 Basis of work
2.1.3 Chemistry
2.1.3.1 Synthesis of -ionone based 1,3,5-trisubstituted-4,5dihydropyrazoles (4a-j)
2.1.3.2 Synthesis of -ionone based 1,3,5-trisubstituted-4,5dihydropyrazoles (8a-e)
2.1.3.3 Synthesis of -ionone based 1,3,5-trisubstituted pyrazoles (9a-e)
2.1.4 Biological evaluation- material methods
2.1.4.1 Anti amastigote activity
2.1.4.2 Cytotoxicity assay
2.1.4.3 In Vivo assay
2.1.5 Result and discussion
2.1.6 Conclusion
39-69
1
2
2
3
3
5
7
7
8
9
10
11
12
12
13
13
14
14
15
15
23
23
26
32
33
39
41
44
44
46
47
47
48
48
49
50
51
2.1.7
2.1.8
2.1.9
Experimental section
Spectra of some selected compounds
References
52
65
67
CHAPTER 2.2: DESIGN, SYNTHESIS AND BIOEVALUATION OF

NOVEL TRIAZOLE INTEGRATED PHENYL
HETEROTERPENOIDS AS ANTILEISHMANIAL
AGENTS
2.2.1 Introduction
2.2.2 Basis of work
2.2.3 Chemistry
2.2.4 Biological evaluation- material methods
2.2.5 Result and discussion
2.2.6 Conclusion
2.2.7 Experimental section
2.2.8 Spectra of some selected compounds
2.2.9 References
70-85
CHAPTER 3: SYNTHESIS AND BIOEVALUATION OF NOVEL

ISOXAZOLE CONTAINING HETERORETINOID
AND ITS AMIDE DERIVATIVES
3.1 Introduction
3.2 Basis of work
3.3 Design and synthesis of heteroretinoid
3.4 Chemistry
3.5 Biological evaluation- material methods
3.6 Result and discussion
3.7 Conclusion
3.8 Experimental section
3.9 Spectra of some selected compounds
3.10 References
86-108
CHAPTER 4: DESIGN AND SYNTHESIS OF NOVEL

HETERORETINOID-BISBENZYLIDINE KETONE
HYBRIDS AS ANTILEISHMANIAL AGENTS
4.1 Introduction
4.2 Basis of work
4.3 Chemistry
4.4 Biological evaluation- material methods
4.5 Result and discussion
4.6 Conclusion
4.7 Experimental section
4.8 Spectra of some selected compounds
4.9 References
109-129
List of publications, patents and presentations
70
71
72
74
74
77
78
83
85
86
87
87
89
92
92
95
96
103
107
109
110
112
114
114
116
117
124
128
130-131
LIST OF ABBREVIATIONS
Anal.
Analysis
Aq.
Aqueous
Ar
Aryl
Ag2O
Silver oxide
Bn
Benzyl
brs
Broad singlet (in NMR)
Calcd
Calculated
CTABr
Cetyltrimethyl ammonium bromide
Chemical shift (parts per million)
Conc.
Concentrated
CL
Cutaneous leishmaniasis
CC50
Cytotoxicity concentration at 50%
Degree celsius
CDCl3
Deuterated chloroform
CH3COOH
Acetic acid
DMSO-d6
Deuterated dimethyl sulfoxide
DCM
Dichloromethane
DHFR
Dihydrofolate reductase
DMF
Dimethyl formamide
Doublet (in NMR)
dd
Double doublet (in NMR)
EI
Electron impact
ESIMS
Electron spray ionization mass spectrometry
ESMS
Electron spray mass spectrometry
ev
Electron volt
EtOH
Ethanol
FTIR
Fourier transformed-infra Red
Gram(s)
Hz
Hertz(s)
Hour(s)
HIV
Human immunodeficiency virus
HCl
Hydrochloric acid
IC50
Inhibitory concentration at 50%
IR
Infrared
i.p.
Intraperitoneal
Coupling constant (in NMR)
KBr
Potassium bromide
m/z
Mass to charge ratio (in Mass spectrometry)
MHz
Mega hertz
Mp
Melting point
MeOH
Methanol
Me
Methyl
M+
Molecular ion peak
Microgram
Microlitre(s)
Micrometer
Micromolar
mg
Milligram
mL
Millilitre(s)
mmol
Millimole
MIC
Minimum inhibitory concentration
Min
Minute
MCL
Mucocutaneous leishmaniasis
Multiplet (in NMR)
MeOD
Deuterated methanol
nm
Nanometer
NMR
Nuclear magnetic resonance
Na2SO4
Sodium sulphate
NaH
Sodium hydride
NH2OH
Hydroxyl amine
II
ppm
Parts per million
Percentage
PhNHNH2
Phenyl hydrazine
Quartet (in NMR)
Rf
Retention factor
rt
Room temperature
SI
Selectivity index
Singlet (in NMR)
SSG
Sodium stibogluconate
THF
Tetrahydrofuran
TMS
Tetramethylsilane
TLC
Thin layer chromatography
Triplet (in NMR)
UV
Ultraviolet
VL
Visceral leishmaniasis
wt.
Weight
WHO
World Health Organization
III
PREFACE
Leishmaniasis comprises a group of diseases with extensive morbidity and
mortality in most developing countries. They are caused by the species of the genus
Leishmania (Sarcomastigophora, Kinetoplastida) and ranges from self healing
cutaneous leishmaniasis (CL) to progressive mucocutaneous leishmaniasis (MCL) to
fatal disseminating visceral leishmaniasis (VL). The situation has become
complicated because of the emergence of post kala-azar dermal leishmaniasis
(PKDL), which appears in 0-6 months after the successful curing of VL. The WHO
has declared VL a neglected and emerging disease. While CL poses basically
cosmetic problems and MCL leads to painful disfiguration, social stigmatization and
often severe secondary infections. VL is generally lethal if left untreated.
According to the World Health Organisation, leishmaniasis currently affects
some 12 million people and there are 2 million new cases per year and with growing
tendency. Moreover, it is estimated that approximately 350 million people live at risk
of infection with Leishmania parasites. Leishmaniasis is a world-wide vector borne
disease, affecting 88 countries. Visceral leishmaniasis (VL) occurs in 65 countries.
CL is endemic in Iran, Saudi Arabia, Syria, Afghanistan and in some South American
countries. More than 90% of the VL cases worldwide are registered in India,
Bangladesh, Indonesia and Sudan. Leishmania/HIV co-infections have increased in
Mediterranean countries, where up to 70% of potentially fatal VL cases are associated
with HIV infection and up to 9% of AIDS cases suffer from newly acquired or
reactivated VL. The WHO has declared VL a neglected and emerging disease.
Most of the existing drugs like antimonials, amidines are quite toxic and
antibiotic like amphotericin B and paromomycin are quite expensive and are out of
reach of poor people. New introduction like miltefosine is also not free from toxicity
and show teratogenic effects in pregnant women. New antileishmanial drugs are
required in view of the shortcomings associated with the existing drugs. In view of
this there is a constant hunt for new lead molecules from natural sources.
Heterocycles are members of an extraordinarily significant class of
compounds, making up more than half of all known organic compounds. Heterocyclic
structures are an integral part of numerous drugs, vitamins, natural products, bio-
IV
molecules and other biologically active compounds. They have also been commonly
found as the key structural unit in the synthetic pharmaceuticals and agrochemicals.
The work embodied in this thesis is an attempt to synthesize novel heterocycles as
potential antileishmanial agents.
We focused our research work on design and synthesis of novel bioactive
scaffolds based on the heterocyclic core. The complete thesis work entitled Design,
Synthesis and Bioevaluation of Novel Heterocycles as Antileishmanial Agents
describes our endeavors leading to the accomplishment of newer and potential
antileishmanial agents. The thesis has been organized under four chapters as
summarized below:
First chapter presents a concise review on leishmaniasis, conventional treatment
options, recent advancements and scope of natural products and natural product based
lead molecules in chemotherapy of leishmaniasis.
Second chapter is divided into two parts. Chapter 2.1 deals with the design,
synthesis and antileishmanial activity of novel terpenyl heterocycles. Chapter 2.2
deals the synthesis and bioevaluation of triazole integrated phenyl heteroterpenoids as
antileishmanial agents.
Third chapter presents the synthesis and bioevaluation of novel isoxazole containing
heteroretinoid and its amide derivatives as antileishmanial agents. The synthesized
compounds were also checked for compliance to the Lipinski rule of five and it was
found that majority of the synthesized compounds followed the aforesaid rule.
Therefore, these compounds have a good potential for eventual development as oral
agents and can be potentially active drug candidate.
Fourth chapter illustrates the antileishmanial potential of novel heteroretinoidbisbenzylidine ketone hybrids. Encouraged by our previous work in chapter 3, we
have covalently linked heteroretinoid moiety with bisbenzylidine ketones and the
resulting chemically novel hybrid molecules were analyzed for their in vitro
antileishmanial activity. The activity results clearly indicate that newly synthetic
compounds reported in this chapter are promising one and provide useful model for
further structural and biological optimization.
Chapter 1
Chemotherapy of Leishmaniasis So Far: A

Review
Chapter 1
Chemotherapy of leishmaniasis so far: A Review
1.1 INTRODUCTION
Despite the fact that infectious diseases have been identified as the third major
cause of death in the world, many fall into the category of neglected diseases.
Neglected diseases have plagued mankind for centuries and continue to cause significant
public health problems in regions of the world least able to deal with the associated
economic burden.
Leishmaniasis is a neglected disease characterized by high morbidity, deeply linked
to malnutrition, humanitarian emergencies and environmental changes that affect vector
biology. It remains one of the major burdens on human health in developing countries,
and the WHO recently classified leishmaniasis as a category I: emerging or uncontrolled
disease. It is a vector-borne disease caused by the species of the genus Leishmania
(phylum-Sarcomastigophora, order-Kinetoplastida and family-Trypanosomatidae) and is
transmitted by phlebotomine sand flies. Clinical manifestations of leishmaniasis include
cutaneous
leishmaniasis
(CL),
muco-cutaneous
leishmaniasis
(MCL),
visceral
leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL).

Leishmaniasis is distributed in 88 countries, worldwide, and an estimated 1.52.0
million people both children and adults develop clinical leishmaniasis every year,
although many more subclinical infections go unrecorded. 75% of clinical cases affect
the skin (cutaneous leishmaniasis, or CL), and the remaining 25% represent systemic and
potentially fatal visceral leishmaniasis (VL, also known as kala-azar). 90% of VL cases
occur in India, Bangladesh, Nepal, Sudan and Brazil, where 70 000 or more deaths are
reported annually.1,2 It is widely recognized that this figure is a gross underestimate and
might represent only one-fifth of the true death toll. Among parasitic infections, only
malaria kills more people. In addition, leishmaniasis is in the top ten parasitic diseases for
its impact on socioeconomic development and has a burden of 2.4 million DALYs
(disability
adjusted
life
years;
http://www.who.int/whr/2002/en/whr02_en.pdf).
Increasing overlap with the spread of AIDS has heightened the threat of HIVLeishmania
co-infections, particularly in India and East Africa.3
Chapter 1
1.2 TYPES OF LEISHMANIASIS

1.2.1 Cutaneous leishmaniasis:
It is the most common form and survey revealed that
one person becomes infected by cutaneous leishmaniasis in
every 20 seconds. It is most frequently caused by Leishmania
major, Leishmania tropica and L. aethiopica in the Old
World (Mediterranean basin, Middle East, and Africa), and
by
Leishmania
braziliensis,
Leishmania
mexicana,
Leishmania amazonensis and related species in the New

World (Mexico, Central America, and South America).4
Cutaneous leishmaniasis is a disease with a varied spectrum
Figure 1.1: Cutaneous

leishmaniasis with a large
ulcerative lesion on the arm
of clinical manifestations, which range from small cutaneous nodules to gross mucosal
tissue destruction. The disease is endemic in more
than 70 countries worldwide, and 90% of cases occur
in Afghanistan, Algeria, Brazil, Pakistan, Peru, Saudi
Arabia, and Syria.5,6
CL by L. tropica and L. major occur in the
northwestern states of India (foci in Rajasthan and
Punjab). The most affected area in Rajasthan is
Bikaner district.7 Cases identified in other districts
usually are immigrants from Bikaner.8 Recently, CL
(12 cases with active lesions, out of 38 people
examined) and VL (2 cases) have been reported in
Figure 1.2: Geographical
distribution of CL in India
South India, Kerala, which has implications for the

existing elimination program.9,10
The disease is associated with poverty. In addition, the disease itself can have a
considerable socioeconomic impact on those who are affected, as CL can lead to
disfigurement, social stigmatization and isolation.11
Chapter 1
1.2.2 Mucocutaneous leishmaniasis:

Mucocutaneous leishmaniasis (MCL) is a severe form of CL that is mainly
characterized by lesions that often lead to extensive and disfiguring destruction of
mucous tissues of the nose, mouth and face, as well as the
arms and legs, causing serious disability. Mucocutaneous
leishmaniasis (also known as espundia) most commonly
manifests
with
respiratory
symptoms,
including
nasal
congestion that causes difficulty in breathing and epistaxis.

MCL is often classified simply as a severe form of CL and
thus most discussions of the disease include MCL in CL.
Figure 1.3: Mucoutaneous

leishmaniasis involving nose
Mucocutaneous lesions usually develop by organismal metastasis from distant

cutaneous lesions. Secondary infection plays a prominent role in the size and persistence
of these areas. The progress of the disease is slow and steady, with a high mortality rate if
untreated. Mucosal disease occurs in 1% to 5% of untreated patients, with primary
cutaneous lesions developing often years or even decades following the initial infection.
Children, therefore, are rarely affected.12 Multiple lesions above the waist may increase
the chance of developing mucosal disease.13 Mucocutaneous leishmaniasis occurs only in
the New World and is most common in Bolivia, Brazil, and Peru.
1.2.3 Visceral leishmaniasis:
This disease is the second-largest parasitic killer in the world (after malaria),
responsible for an estimated 500,000 infections each year worldwide. The parasite
migrates to the internal organs such as liver, spleen (hence
'visceral'), and bone marrow, and, if left untreated, will almost
always result in the death of the host.
There are two types of VL, which differ in their transmission
characteristics: zoonotic VL is transmitted from animal to vector to
human and anthroponotic VL is transmitted from human to vector
to human. In the former, humans are occasional hosts and animals,
mainly dogs, are the reservoir of the parasite.
14
Zoonotic VL is
Figure 1.4: A child

suffering with VL.
found in areas of L. infantum transmission whereas anthroponotic VL is found in areas of
Chapter 1
L. donovani transmission. Signs and symptoms include fever, weight loss, mucosal
ulcers, fatigue, anemia, and substantial swelling of the liver and spleen.
Figure 1.5: Geographical distribution of VL in India
VL, caused by L. donovani (in Asia and Africa) and L. infantum (in southern Europe, as
wells as South America where it used to be referred to as L. chagasi), is potentially fatal.
Over 90% of the global total of visceral leishmaniasis cases occur in five countries across
three continents: north eastern India, Bangladesh, and Nepal in the Indian subcontinent,
Sudan in Africa, and north eastern Brazil in South America.15 The situation is particularly
grave in the state of Bihar, India (Figure 1.5), known as the heartland of kala-azar.
The more complex form of VL is post-kala-azar dermal leishmaniasis (PKDL).
PKDL is characterized by a macular, maculo-papular or
nodular rash and is a complication of VL that is frequently
observed after treatment in Sudan and more rarely in other
East African countries and in the Indian subcontinent.16 It can
also occur in immunosuppressed individuals in L. infantumendemic areas. The interval between treated VL and PKDL is
06 months in Sudan and 6 months to 3 years in India. PKDL
Figure 1.6: A patient from

India with nodular post-kalaazar.
Chapter 1
cases are highly infectious because the nodular lesions contain many parasites,17 and such
cases are the putative reservoir for anthroponotic VL between epidemic cycles.
1.3 LIFE CYCLE

Leishmania is a protozoan and a compulsory intracellular parasite of mononuclear
phagocytes in the mammalian host. The pathogen requires two different hosts to
complete its biological cycle (Figure 1.7): an insect vector (sand flies of the genus
phlebotomine in the old world and Lutzomyia in the new world) and a vertebrate host
(e.g. humans, rodents, dogs). To survive successfully and multiply within these two
disparate biological environments the parasite must undergo profound biochemical and
morphological adaptations.18
The Leishmania parasite exists in two morphological forms known as promastigote
and amastigote. The promastigotes are ~ 20 m long and 1.5-3.00 m broad with a single
long flagellum and multiply by binary fission as an extra cellular parasite in the gut
lumen of female sandfly. The amastigotes are 2-5 m long intracellular non-motile,
uninucleate ovoid organism containing a rod shaped kinetoplast associated with a
flagellar rudiment and multiply repeatedly by binary fission, eventually destroying
macrophages of vertebrate host. When an amastigote is ingested by a Phlebotomine
sandfly it elongates in the flys gut and transforms into a flagellated promastigote or
leptomonad.
In first stage inoculation of parasite occurs when sand flies takes the blood meal
from human and inoculate promastigotes into the reticulo-endothelial system of human.
Then promastigotes attach themselves to macrophages and invade them by inducing
phagocytosis. Promastigotes cover themselves by phagolysosome. To survive in
macrophage, promastigotes transform into the amastigote within the macrophage and
amastigote divide and multiply by binary fission. When numbers of child amastigotes
reach to sufficient amount, cell bursts. Infected macrophages and free amastigotes enter
into the blood circulation. Free amastigotes may enter into other macrophages and infect
them and number of amastigotes increases by that way. The transmission cycle continues
Chapter 1
when a female sand fly feeds on an infected host and ingests macrophages infected with
amastigotes.
Figure 1.7: Different forms of parasites.(a) Promastigote, (b) Amastigote, (c) Life cycle of parasite
Chapter 1
1.4 CURRENT STATUS

Unfortunately, as yet no effective vaccines against leishmaniasis are available and
control of the disease relies primarily on chemotherapy. The chemotherapy currently
available for leishmaniasis is far from satisfactory and has been reviewed from time to
time.19,20,21 New drugs are necessary and this requirement has been fed in recent years by
the demonstration of acquired resistance to the pentavalent antimonial drugs, the first-line
chemotherapy.22
Why then have we not seen progress in the intervening decades toward
development of a new generation of more effective and safe anleishmanial drugs? The
answer primarily lies in economics. This disease, though globally massive in its impact,
mainly affects poor people in poor regions of the world. As such, these would never be
viewed as viable target markets for the pharmaceutical industry. But now funding
situation is being improved by players in drug research and development (R&D), for
example product development partnerships such as DNDi (Drugs for Neglected Diseases
initiative), iOWH (Institute for One World Health), CPDD (Consortium for Parasitic
Drug Development), funders such as the Bill and Melinda Gates Foundation, and the
pharmaceutical industry, for example Novartis.19c
This review will give an overview of the classical and current treatment for
leishmaniasis and highlights the recent advances and approaches for the development of
novel chemotherapies in area of synthetic and natural products to treat leishmaniasis.
1.5 CONVENTIONAL DRUGS

The drugs recommended for the treatment of leishmaniasis include the pentavalent
antimonials, amphotericin B and its Lipid formulation AmBisome, paromomycin,
pentamidine and miltefosine. The antimonials were first introduced in 1945 and remain
effective treatments for some forms of leishmaniasis, but the requirement for up to 28
days of parenteral administration, the variable efficacy against VL and CL, and the
emergence of significant resistance are all factors limiting the drugs usefulness. The
usefulness of the diamidine pentamidine as an antileishmanial drug has been limited by
its toxicity. The polyene antibiotic amphotericin B has proved to be highly effective for
Chapter 1
the treatment of antimonial-resistant L. Donovani (VL patients)23 and cases of MCL that
have not responded to antimonials, but it is an unpleasant drug because of its toxicity and
the need for slow infusion parenteral administration over four hours.
However, antileishmanial chemotherapy has benefited from the development of
lipid-associated formulations of amphotericin B, which have reduced toxicity and an
extended plasma half-life in comparison to the parent drug, for the treatment of fungal
infections. AmBisome is the best tested of these formulations, has proved to be
effective24 and has been approved by the Food and Drug Administration,25 but high cost
has limited its use.
Paromomycin (PM), an aminoglycoside antibiotic, was originally identified as an
antileishmanial in the 1960s and has been used in clinical trials for both VL and CL.
Perhaps the most significant recent advance has been the effective oral treatment of VL
by using miltefosine, an alkylphosphocholine originally developed as an anticancer
drug.26 A variety of other compounds discovered to have antileishmanial activity are at
various stages of development. The detailed information of each of the above mentioned
drug is given below.
1.5.1 Antimonials
Antimonials were first used almost a century ago. Initially, tartar emetic (1)
(trivalent antimonial, Sb (III) compound) was used for the treatment of leishmaniasis, but
this drug was found to be highly toxic as well as very unstable in tropical climate.27 This
led to the discovery of pentavalent antimonials. Urea stibamine (2) [Sb (V) compound]
synthesized by Brahmachari, emerged as an effective chemotherapeutic agent against
Indian kala-azar.28,29
The development of the less toxic pentavalent antimonials led to the synthesis of
sodium stibogluconate (3) (Pentostam) in 1945.30 Meglumine antimoniate (4)
(Glucantime) and Generic sodium stibogluconate are the other pentavalent antimonial
formulations currently being used in the clinic. These compounds are non-covalent
chelates of Sb (V) and in order to have an antileishmanial effect they have to cross the
phagolysosomal membrane and act against the intracellular form of parasite, the
amastigote. It is also highly likely that Sb (V) has to be converted to a trivalent form [Sb
Chapter 1
(III)] in order to be active. Sb (III) has been shown to inhibit trypanothione reductase,31
an enzyme responsible for protection from host reactive oxygen and nitrogen species to
parasites and glutathione reductase,32 and recent evidence suggests that antimony might
induce
apoptosis
and
kill
by
DNA
fragmentation
and
externalization
of
phosphatidylserine.33
Although antimonials have been in clinical use for a long time, their mode of action
is still not entirely known. The routes of entry of antimonials into Leishmania (or into
macrophages) are not known, although pentavalent arsenate, a metal related to Sb (V), is
known to enter via phosphate transporters. Despite having the side effects such as acute
pancreatitis and cardiac arrhythmia, and usually reversible muscle pains, renal failure,
cardiotoxicity and hepatotoxicity these drugs are the mainstays for the treatment of
leishmaniasis.
Recently, antimonials have become almost obsolete in certain areas of India

because of drug resistance22 developed due to the incomplete treatment and irregular use
but they are still useful in the rest of the world, where the introduction of generic brands
has reduced costs.
1.5.2 Pentamidine
Pentamidine (5) is an aromatic diamidine used to cure VL, CL, and DCL as a
second line drug when antimonials have proved ineffective. Its isothionate and
Chapter 1
methansulphonate salts are mainly used for the treatment of VL. Antileishmanial activity
of pentamidine is based on the inhibition of polyamine biosynthesis and the disruption of
mitochondrial membrane potential.34 Although, its precise mode of action is not known,
it is reported that the drug interferes with Leishmania DNA synthesis, modifying the
morphology of the kinetoplast, and promotes fragmentation of the mitochondrial
membrane, killing the parasite.
The drug has been shown to have severe side effects including cardiotoxicity,
hypotension (if administered too rapidly), renal impairment and hypoglycemia followed
by diabetes mellitus.35 Unfortunately, development of resistance, painful and
inconvenient route of administration and toxicity precludes widespread use of
pentamidine for the treatment of leishmaniasis.
1.5.3 Amphotericin B
The polyene antibiotic amphotericin B (6), widely used as an antifungal compound,
is a common second line therapy for leishmaniasis in case of antimonial failure,36 but in
some areas of the Bihar state of India where treatment failure rates for antimonials
reached >60%, Amphotericin B has become the first choice for the treatment of VL.
This drug acts on ergosterol present in the Leishmania membrane to form aqueous
pores in the membranes of the cells. By increasing the permeability of the cell membrane,
it promotes major constituent to efflux that leads to parasite cell lysis.
Despite its great efficiency, amphotericin B treatment leads to unwanted side
effects such as nephrotoxicity, hypokalemia and anaphylaxis as well as delivery related
rigor, fever and chills.2 Adverse effects of plain AmB have been circumvented with its
three clinical formulations in which deoxycholate have been replaced by other lipids.
These formulations are liposomal AmB (L-AmB: Ambiosome), AmB colloidal
dispersion (ABCD: Amphocil) and AmB lipid complex (ABL: Abelcit). These lipid
formulations of AmB retain their activity and show very high efficacy to cure this deadly
disease and are less toxic. In VL cases, liposomal AmB has been proved as an efficient
drug with more than 95% efficacy but high cost limits its use to common man suffering
10
Chapter 1
from this deadly disease.37 Recently new preferential pricing was agreed for certain
countries with a cost of $20 per 50 mg vial of AmBisome.38
1.5.4 Paromomycin
Paromomycin
(7)
(aminosidine)
is
an
aminoglycoside
antibiotic
with
antileishmanial activity. It cures both, VL and CL (more effectively) but poor oral
absorption has led to the development of parenteral and topical formulations.39 In a phase
III study of VL in India, this drug was found equally effective as amphotericin B with
94.6% cure rates.40 Paromomycin is economical but requires daily intramuscular
injections for 21 days.41 The safety profile seems to be excellent, however, ulceration and
localized tissue damage at the site of injection are commonly observed.
Paromomycin inhibits protein synthesis and modifies membrane fluidity and
permeability. It has also been revealed that cationic paromomycin binds to the negatively
charged leishmanial glycocalyx suggesting mitochondria as a primary target.42 Although,
resistance to amino glycosides is well recognized in bacteria, no clinical resistance has
been reported for Leishmania. The drug is presently under further investigation for its use
against VL in Africa and India, both as a monotherapy and in combination.43
NH
NH
H2N
OH
NH2
O
OH
HO
NH2
O
O
HO
OH OH
OH
H3C
O
Amphotericin B
OH
OH
O
OH
OH OH
NH2
O
H2N
O
CH3
HO
5
Pentamidine
(Pentacarinat)
H3C
NH2
O
H2N
OH
COOH
CH
OH 3
NH2
HO
OH
7
Paramomycin
11
Chapter 1
1.5.5 Miltefosine
Perhaps the most significant recent advances for
the treatment of VL are the discovery of orally
administered
drug
alkylphosphocholine
miltefosine
(8).
It
is
an
(hexadecylphosphocholine)
moiety originally developed as an oral antineoplastic

agent (for cutaneous cancers) and has subsequently
been applied to treat leishmaniasis.44 This drug was
registered in India for the treatment of visceral
leishmaniasis in 2002.45 It had a 9597% concluding
cure rate in large phase III and IV trials in
India.46However, there are major concerns about teratogenic potential and the long halflife of the drug. Its long half-life (approximately 150 hours) could encourage
development of clinical resistance.47
Although, the exact mode of antileishmanial action is still unsure but it has been
established that it causes apoptosis like processes in Leishmania donovani as observed in
amastigote but how it happens, still unknown.48 The intracellular accumulation of the
drug appears to be the critical step for its action, which is regulated by two transporters,
LdMT and its -subunit LdRos3, a P-type ATPase.49 The clinical resistance is not yet
reported but being an oral agent its improper use in endemic countries like India increases
the probability of resistance and spread of resistant parasites where prevalence of
infection is significantly high.
1.6 RECENT ADVANCEMENTS

Unfortunately, most of the conventional drugs are associated with a number of
shortcomings such as toxicity, prolonged treatment schedules, need for hospitalization,
high cost in endemic countries, resistance and a high rate of treatment failure in HIV coinfected patients. So far, the only effective treatment for leishmaniasis is the oral drug
miltefosine. In the light of above facts, we need to continue searching for more efficient,
inexpensive, nontoxic, and innovative drugs based on new molecular scaffold for the
12
Chapter 1
treatment of leishmaniasis. The following paragraphs describe some new developments at

the discovery and development stage.
1.6.1 Sitamaquine
The 4-methyl-6-methoxy-8-aminoquinoline, sitamaquine (9) might have an impact
as an alternative oral drug for the treatment of visceral leishmaniasis. Discovery of
sitamaquine as antileishmanial agent was based on widespread efforts in synthetic
chemistry at the Walter Reed Army Institute for Research (WRAIR).50 Several phase II
dose ranging studies in India and Kenya have been reported with variable efficacy;
however, further studies are required to define the optimal dose. Some adverse effects
included abdominal pain, headache, vomiting, and a severe renal event. The mechanism
of action of sitamaquine is currently not known, however, the drug causes alkalization of
acidocalcisomes and collapse in mitochondrial membrane potential. Further studies must
be conducted in order to explore its therapeutic potential against leishmaniasis.
OH CH 3
H3C
CH3
H3C
O
H 3CO
N
NH(CH)6
N
C 2H5 C2H 5
9
Sitamaquine
CH 3
O
OH
H 3C H 3C OH
CH3
NH 2
N
H 3C
OMe
OH O O
CH3
N
N
CH 3
N(CH 3)2
CH3
OH
CH 3
10
Azithromycin
CH3
11
Imiquimod
1.6.2 Azithromycin
Azithromycin (10), an azalide antibiotic has demonstrated activity against various
protozoa. Its high concentration in tissues, especially in macrophages, oral administration
and safety in children are the chief advantages for its use in leishmaniasis chemotherapy.
Its antiprotozoal action is on account of protein synthesis inhibition but stimulation of
phagocytosis, chemotaxis and fortification of immune response cannot be excluded.
13
Chapter 1
However, reports of its antileishmanial action from the Old and New World show
conflicting results, with variable cure rates.51,52
1.6.3 Imiquimod
Imiquimod (11) (Aldara) is extensively used for the treatment of human
papillomavirus (HPV) induced skin diseases, premalignant conditions and genital warts.
This imidazoquinoline amine is an immunomodulator, stimulating a local immune
response at the site of application, which in succession resolves the infection. It induces
the production of cytokines and nitric oxide in macrophages and has been shown to have
antileishmanial activity via macrophage activation in experimental models.53
The drug has also been used in combination with standard antimonials to treat
cutaneous leishmaniasis cases refractory to pentavalent antimonial treatment. These
results indicate that the combination of antimonials and immunomodulators could be an
alternative treatment for patients refractory to antimonials.
1.6.4 Azoles
The most recent example of development in search of new antileishmanial drugs is
therapeutic switching also called piggy-back therapy. Azoles, originally developed as
N
N
N
N
O
N
OH
O
N
Cl
12
14
Fluconazole
Ketoconazole
O
H
N
Cl
Cl
Cl
Cl
15
O
Itraconazole
N
Cl
Cl
N
N
N
Cl
13
Miconazole
CH3
O
O
N
N
NH 2
16
Posaconazole
14
Chapter 1
antifungal drugs, have been found to show activity against Leishmania parasite.
Presence of ergosterol as a membrane component is a common characteristic
between fungi and Leishmania. Like in fungi, azoles block ergosterol synthesis in
Leishmania by inhibiting the cytochrome 450 mediated 14-demethylation of
lanosterol.54 Several azole antifungals ketoconazole (12), miconazole (13), fluconazole
(14) and itraconazole (15) have been used to treat cutaneous and visceral leishmaniasis
with variable success rates.54,55 Promising results have been obtained with posaconazole
(16) against experimental L. amazonensis.
The orally active azoles are at different stages of development, offer potential for
leishmaniasis chemotherapy. However, Leishmania has potential to survive in altered
sterol profile, and also have ability to utilize and metabolize host sterol.56 This
consideration must be accounted during novel drug development.57
1.6.5 Combination therapy
After increasing resistance to most of the monotherapeutic regimens, the
combination therapy has set up new possibility in the cure of leishmaniasis. As in other
infectious diseases, combination therapy in leishmaniasis could reduce treatment
duration, drug doses and prevent drug resistance as well as potentially toxic side effects.
Several studies have been done or underway to identify such combinations for the
treatment of leishmaniasis. Some of these combinations are meglumine antimoniate with
allopurinol,58 sodium stibogluconate and paromomycin,59 imiquimod in combination with
meglumine antimoniate,60 and AmBisome plus miltefosine.61 Although it is difficult to
draw any clear conclusion about these clinical evidences of superiority of combination
therapy but these evidences can be a hope in leishmaniasis chemotherapy.
1.7 SCOPE OF NATURAL PRODUCT

Natural products have been used for centuries for the cure of various ailments.
According to a survey by National Cancer Institute 61% of the 877 small molecule new
entities introduced as drugs world-wide during 1981-2002 can be traced to or were
inspired by natural products.62 Plants are the vital source for drug candidates, chiefly
against parasites because of their long association with parasites. More than 100 plants
15
Chapter 1
have been reported to be active against various species of leishmania parasite.63 Amongst
the natural products, different classes of secondary metabolites have been reported for
their antileishmanial profile.64 These comprise quinones, alkaloids (quinoline,
isoquinoline, indole, and steroidal), terpenoids and phenolics (chalcones, flavonoids,
coumarins and lignans).
A number of quinone derivatives isolated from natural sources are reported to have
prominent in vitro and in vivo antileishmanial activity. Diospyrin (17), a bisnaphthoquinone, isolated from the bark of Diospyros Montana (Ebenaceae) showed in
vitro activity against promastigotes of L. donovani with an MIC of 1.0 g/mL.65
Plumbagin (18), originally isolated from Plumbago zylenica, was found to show
antileishmanial activity against amastigotes of L. donovani (IC50= 0.42 g/mL) and L.
amazonensis (IC50 = 1.1 g/mL). In vivo activity was also displayed by this metabolite
against L. amazonensis and L. Venezuelensis at concentrations 2.5 and 5 mg/kg/day,
respectively. The mechanism of the action of compounds 17 and 18 involves generation
of oxygen free radicals from which the parasites remain unable to defend.
The alkaloids represent an important class of natural products exhibiting

noteworthy antileishmanial activities. The quinoline alkaloids, 2-n-propylquinoline (19)
and chimanine-D (20), isolated from Galipea longiflora (Rutaceae), exhibited
antileishmanial activity against promastigotes of L.braziliensis with an IC90 values of 50,
and 25 g/mL, respectively. Likewise, dictylomide-A (21) and B (22) isolated from the
bark of Dictyoloma peruviana (Rutaceae), also showed total lyses of L. amazonensis
promastigotes at 100 g/mL concentrations.66
16
Chapter 1
O
N
CH 3
19
CH3
20
O
N
N
HO
21
22
CH 3
CH3
Indole
alkaloids
(23),
dihydrocorynantheine
corynantheine
(24)
and
corynantheidine (25) isolated from the bark of Corynanthe pachyceras (Rubiaceae)

displayed the antileishmanial activity (IC50 of 3 M) against L. major as respiratory chain
inhibitors.
H
C2 H 5
OCH3
N
N H
H
H
H 3COOC
H
C 2 H3
OCH 3
N
N H
H
H
H3COOC
23
24
N
CH 3
H 3CO
N
H
H3 COOC
H
H3COOC
25
N
N H
H
CH3
17
Chapter 1
Isoquinoline alkaloids liriodenine (26) and O-methylmoschatoline (27), isolated

from Annona foetida (Annonaceae), displayed in vitro activity against L. braziliensis
promastigote with an IC50 < 60 M. A berberine alkaloid (28), a main constituent in
various folk remedies shows prominent activity against cutaneous leishmaniasis, malaria
and amoebiasis.67 Berberine (28) has been used clinically for the treatment of
leishmaniasis for over 50 years and it has been demonstrated that it possesses significant
activity both in vitro and in vivo against several species of Leishmania.
Steroidal alkaloids, holamine (29), 15- hydroxyholamine, holacurtine (30) and

N-desmethylholacurtine obtained from Holarrhena curtisii (Apocynaceae), have also
been reported for their antileishmanial action. The metabolite holamine exhibited better
activity against L. donovani (1.56 > IC50 > 0.39 g/mL) as compared to holacurtine and
N-desmethyl holacurtine (6.25 > IC50 > 1.56 g/mL).68
O
O
CH3
CH3
CH 3
CH3 H
CH3 H
H
CH 3 O
H
O
H
H H
H 2N
29
CH 3
H
H 3C
H
OCH 3
NH
OH
H
30
18
Chapter 1
Some benzoquinolizidine alkaloids, klugine (31), cephaeline (32), isocephaeline

(33) and emetine (34), isolated from Psychotria klugii (Rubiaceae), demonstrated
significant leishmanicidal activities against L. donovani. Among these metabolites,
klugine, cephaline and isocephaline showed IC50 values of 0.40, 0.03 and 0.45g/mL
respectively. Emetine exhibited antileishmanial activity against L. donovani with an IC50
value 0.03 g/mL, however produces toxicity in treatment of cutaneous leishmaniasis
caused by L. major.69
R1
H3CO
H 3CO
N
H
R2
HN
H3CO
CH 3
OCH 3
HH
HN
OH
31 R 1 = OH; R 2 = OH
32 R 1 = OCH 3; R 2 = H
H
CH 3
OCH 3
R
33 R = OCH 3
34 R = OH
The beta-carbolines have been reported to possess in vitro trypanocidal activity

against Trypanosoma cruzi epimastigotes.70 Hermine (35), a beta-carboline amine
alkaloid isolated from Peganum harmala, reduced spleen parasite load by approximately
40, 60, 70 and 80% in free, liposomal, niosomal and nanoparticular forms, respectively in
mice model.71
Iridoids, a class of monoterpenoid glycosides, are well known for significant
leishmanicidal activity. The arbortristosides-A (36), B (37), C (38) and 6-hydroxyloganin (39), isolated from Nyctanthes arbortristis (Oleaceae) exhibited in vitro
activity against L. donovani amastigotes. In the in vivo test using infected hamsters with
L. donovani presented leishmanicidal activity at a concentration of 10 mg/kg-1 for 5 days
when administered intraperitoneally and at 100 mg/kg-1 for 5 days when administered
orally.72
19
Chapter 1
Espinanol (40), a monoterpenoid, isolated from the bark of Oxandra espintana

(Annonaceae), showed antileishmanial activity against promastigotes of twelve
Leishmania species. However, the metabolite 40 exhibited only a weak activity in vivo in
mice infected with L. amazonensis. Grifolin (41) and piperogalin (42) obtained from
Peperomia galoides, caused total lysis of L. braziliensis, L. donovani and L. amazonensis
promastigotes at 100 g/mL concentrations. At 10 g/mL concentration, metabolite 42
showed more than 90% lysis of the promastigotes.
OH
CH3
CH 3
OH
H3CO
CH3
H3C
OCH3
H 3C
CH3
OH
CH 3
H3C
40
CH3
41
OH
CH 3
CH3
CH3
CH 3
H3C
OH
42
20
Chapter 1
A sesquiterpene lactone, dehydrozaluzanin C (43), isolated from the leaves of

Munnozia maronii (Asteraceae), displayed antileishmanial activity at concentrations
between 2.5-10 g/mL against promastigotes of eleven Leishmania species. The in vivo
test using the metabolite 43 in BALB/c mice resulted in reduction of the lesions caused
by L. amazonensis. Kudtriol (44), a sesquiterpene alcohol isolated from the arial parts of
Jasonia glutinosa (Asteraceae), showed antiparasitic activity with some associated
toxicity against promastigotes of L. donovani at 250 g/mL concentration. SAR study
with metabolite 44 indicated that the presence of a C-5 hydroxy group in the orientation is essential for the expression of the leishmanicidal activity. The (+)curcuphenol (45), isolated from sponge Myrmekioderma styx, exhibited in vitro antileishmanial activities against L. donovani with an EC50 of 11.0 M.
H2C
H
OH
CH3
O
H2C
CH 3
OH
OH
H
CH2
H
O
OH
CH2
O
43
H3C
H3C
CH 3
44
CH3
45
Diterpenoids like jatrogrossidione (46) and jatrophone (47), isolated from

Euphorbiaceae species, have also been reported for their leishmanicidal potential. These
metabolites have been shown to possess antileishmanial activity along with some toxicity
against the promastigote forms of L. braziliensis, L. amazonensis and L. chagasi. SAR
H 3C
O
HO
O
H3 C
CH 3
CH2
H 3C
H
H2 C
46
CH 3
CH3
O
O
CH 3
CH 3
47
21
Chapter 1
studies with these metabolites revealed that 46 with IC100 value of 0.75g/mL displays
activity higher than 47 (IC100 = 5g/mL), but remains inactive in vivo.
Triterpenoids, ursolic acid (48) and betulinaldehyde (49) obtained from the bark of
Jacaranda copaia and the stem of Doliocarpus dentatus (Dilleniaceae) respectively,
showed antiparasitic activity against the amastigotes of L amazonensis. However, the
metabolite 49 exhibited toxicity to peritoneal macrophages in mice while 48 displayed
limited activity in vivo.
CH 2
CH 3
H3C
H3C
H
H3C
CO2 H
CH3
H3C
CH 3
CH3
HO
H
H 3C CH
3
CHO
CH3
HO
H
H 3C CH
3
49
48
Maesabalide III (MB-III) (50) an oleane triterpene saponin isolated from the
Vietnamese plant Maesa balansae73,74 was found to be 100% effective on a 0.8 mg/kg
dose. In a comparative study MB-III fared better than the liposomized amphoterecin B
(AmBisome).
However,
multiple
dose
pharmacological,
toxicological
and
pharmacokinetic studies are still needed before it can become a valid drug candidate for
development.
22
Chapter 1
1.8 NATURAL PRODUCT LEAD BASED STUDIES

In the ongoing search for better leishmanicidal compounds, plant derived products
are being evaluated. Among the bioactive natural products chalcones and curcumines
have been studied extensively. Several chalcones and curcumines have been subjected to
chemical transformations to study its antileishmanial activity.
1.8.1 Curcumin: The spice of life- unlocking the secrets of curcumin
The curcumin (51) [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hepta-diene-3,5dione], commonly called diferuloylmethane; is the active ingredient in the herbal remedy
and dietary spice turmeric. This vibrant yellow spice, derived from the rhizome of the
plant Curcuma longa, has a long history of use in traditional medicines of China and
India.
Curcumin has been the subject of hundreds of published papers over the past three
decades, studying its antioxidant,75 antiproliferative,76 anti-inflammatory,77 antitumor,78
antibacterial, and antimicrobial79 as well as antileishmanial80 activities. It was first
isolated in 1815 by Vogel81 and its chemical structure was confirmed by Lampe and
Milobedezka in 1910.82 It is an oil-soluble coloring compound, readily soluble in alkali,
ketone, acetic acid, and chloroform, while insoluble in water at acidic or neutral pH.
23
Chapter 1
Chemically it is a bis-,-unsaturated -diketone. As such, curcumin exists in

equilibrium with its enol tautomer. The bis-keto form predominates in acidic and neutral
aqueous solutions whereas the enolate form is found above pH 8. The extract from C.
longa, commonly called curcuminoids, was mainly composed of curcumin and together
with a small amount of demethoxycurcumin (DMC) and bisdemethoxycurcumin
(BDMC). Commercially available curcumin mixture contain 77% curcumin (51), 17%
DMC (52), and 3% BDMC (53).
In a study by Chatchawan et al these natural curcuminoids have been chemically
modified to give different curcuminoid analogs and these parent curcuminoids and their
analogs were assessed against protozoa of the Trypanosoma and Leishmania species.80
H3CO
HO
51
(i)
O
(i)
OH
(ii)
HO
OH
OH
OCH3
57
HO
OH
O
OCH3
56
54
H3CO
O
H3CO
OCH3
HO
OCH3
55
H3CO
OH
H3CO
HO
O
OCH3
OH
(iii)
H3CO
OCH3
58
HO
OH
O
H3CO
HO
OCH3
59
OH
Reagents and conditions: (i) H2/Pd-C, EtOH; (ii) p-TsOH, C6H6, reflux; (iii) DDQ, THF
The parent curcuminoids showed low antileishmanial activity (EC50 values of

compounds 51 and 52 for Leishmania mexicana amastigotes are 16 3 and 37 6 M,
respectively) while the control drug, pentamidine, displayed an EC50 of 16 2 M. The
most active curcumin analog (58) exhibited activity of 2.7 0.7 and 4.6 0.7 M against
L. major promastigotes and axenic Leishmania mexicana amastigotes respectively.
Curcumin analog (59) having fully conjugated keto system also displayed better activity
than curcumin while analog 54 and 55 having non conjugated keto system were found
24
Chapter 1
less active than curcumin. Thus it can be concluded that the conjugated keto system is
vital for a curcumin analog to exhibit high antileishmanial activity.
Some researcher studied the cytotoxicity of curcumin to L. donovani. Incubation of
Leishmania promastigotes with curcumin induced formation of reactive oxygen species
(ROS) and elevation of cytosolic calcium through the release of calcium ions from
intracellular stores as well as by influx of extracellular calcium leading to death of
parasite. Taken together, it indicates that curcumin has promising antileishmanial activity
that is mediated by programmed cell death.83
In spite of its efficacy and safety, curcumin has not yet been approved as a
therapeutic agent. Limited clinical efficacies such as poor solubility, bioavailability and
absorption as well as rapid metabolism have been major problems associated with
curcumin. Detailed pharmacological studies conducted on curcumin demonstrates that the
-diketone functionality of curcumin is a substrate for liver aldoketo reductases and this
may be one of the reasons for the rapid metabolism of curcumin in vivo.84
Counteracting the shortages of curcumin mentioned above, various curcumin

analogs/derivatives have been designed and synthesized in order to enhance metabolic
stability. The structural modification efforts are usually directed at variation of the
aromatic rings and their substituents, and/or replacing the heptadiendione bridge chain of
curcumin with other linkers for example 1,5-bis(3,4-dimethoxyphenyl)-1,4-penta-diene3-one
(BDMPP)
(60)
and
2,6-bis((3-methoxy-4-hydroxyphenyl)-methylene)-
cyclohexanone (BMHPC) (61) were synthesized by introduction of pentenone system
25
Chapter 1
instead of heptadiendione bridge chain of curcumin. These compounds exhibited high

antiproliferative activities.78
M. Vijey Aanandhi et al also synthesized some curcumin analogs (62-67) and
evaluated them in vitro against Leishmania promastigotes.85 The compounds having
methoxy substitution on the aromatic ring have shown better activity. Compound 63
having methoxy substitution on the aromatic ring showed IC50 and IC90 values as 25 1.7
and 50 3.5 g ml1, respectively. However, none of the compound tested was found
better than reference drug pentamidine (IC50 = 2.5 0.12 and IC90= 5.0 0.35 g ml1).
O
O
Conc. HCl
R2
R2
R1
R1
R2
R1
62. R1= H, R2= OH

63. R1= OCH3, R2= OH
64. R1= OC2H5, R2= OH
Conc. HCl
R2
R2
R1
R1
65. R1= H, R2= OH

66. R1= OCH3, R2= OH
67. R1= OC2H5, R2= OH
These reports provide the promise that curcumin and its analogs may become the
significant tools to combat with this fatal disease.
1.8.2 Chalcone
Chalcones (68) (1,3-diaryl-2-propen-1-ones), precursors of flavonoids and
isoflavonoids, constitute an important class of natural products. Chemically, they are
open-chained molecules in which two aromatic rings are linked by a three-carbon enone
fragment. Many of these molecules display an impressive array of pharmacological
activities including anticancer,86 antiinflamatory,87 antituberculosis,88 antifungal,89
antimalarial,90 and antileishmanial.91
26
Chapter 1
The leishmanicidal activity of several chalcones has been reported in the

literature.92,93 The most promising member to date is licochalcone A (69), an oxygenated
chalcone isolated from the roots of Chinese liquorice and presently thought to exert its
action by inhibiting fumarate reductase, a selective target present in the parasite
mitochondria.93
The antileishmanial activity of licochalcone A has stimulated interest in other

chalcones from natural and synthetic sources, and several members have been identified
for lead development. Chalcones are readily synthesized by base-catalyzed ClaisenSchmidt condensation of an aldehyde and an appropriate ketone in a polar solvent like
methanol. The method is versatile and convenient, although yields may be variable.
E. C. Torres-Santos demonstrated the in vitro activity of 2,6-Dihydroxy-4methoxychalcone (70) (DMC), a naturally occurring chalcone, against promastigotes and
intracellular amastigotes of Leishmania amazonensis, with 50% effective doses of 0.5
and 24 g ml1, respectively.94 Ultrastructural studies also showed that the mitochondria
of promastigotes were enlarged and disorganized in the presence of 70. Despite
amastigotes destruction, no disarrangement of macrophage organelles was observed, even
at 80 g of 70 per milliliter. These observations imply that DMC is selectively toxic to
the parasites.
Later P. Boeck et al. synthesized various analogues (71-78) of DMC using
xanthoxyline and some derivatives and evaluated them against promastigote and
amastigote form of Leishmania amazonensis. Three analogues containing nitro, fluorine
27
Chapter 1
or bromine group were found selectively active against the parasite as compared with
DMC.95
Aiming to develop new antileishmanial lead compounds, novel sulfonamide 4methoxychalcone derivatives (79a-79i) were synthesized and screened against
Leishmania braziliensis promastigotes and intracellular amastigotes to establish the
potential of sulfonamide and methoxy moieties as promising adding-groups to
28
Chapter 1
chalcones.96 Except compound 79e sulfonamide 4-methoxychalcone derivatives

displayed more potential inhibitory activity (IC50 = 3.5 0.6 to 8.6 0.4 M) than that of
4-methoxychalcone (79) (IC50 = 16.6 1.6 M). These compounds were more active
than pentamidine isothionate (IC50 = 19.6 M) but were less active than amphotericin B
(0.3 0.02 M). Conformational analysis of these sulfonamide 4-methoxychalcone
derivatives indicates that enhanced activity is probably due to the new interactions in a
new plane of these molecules caused by the addition of the sulfonamide group.
Foroumadi et al. prepared chromene-based chalcones and investigated them for
their antileishmanial activity against promastigotes form of Leishmania major.97 Two
types of novel chromeno chalcones were synthesized; Type A chalcone having carbonyl
group close to the chromene ring and Type B chalcone possessing carbonyl group away
from the chromene ring.
O
H3CO
COCH3
H3CO
c
OH
OH
a
OCH3
O
80a-e (Type A)
CHO b
OCH3
H3CO
H3CO
CHO
e
O
O
81a-e (Type B)
R
80,81a = H; 80,81b = 2-Cl; 80,81c = 3-Cl; 80,81d = 4-Cl; 80,81e = 2,4-Cl2
Reagents and conditions: (a) NaOH, CHCl3, H2O, reflux; (b) methyl vinyl ketone, 1,4-dioxane, K2CO3,
reflux; (c) appropriate aldehyde, NaOH, ethanol; (d) acrolein, 1,4-dioxane, K2CO3, reflux; (e) appropriate
acetophenone, NaOH, ethanol.
Chloro-substituted Type A chalcones (80ce) with IC50 values less than 1.0 M
were found to be the most potent compounds against the promastigote form of L. major.
Contrary to the previous studies that ring A (attached to the -position respect to the
carbonyl group) and its substitution pattern are generally less important for
antileishmanial activity compared to ring B (aryl moiety connected to the carbonyl
group), this report revealed that very good antileishmanial activity was obtained when
29
Chapter 1
ring B is 6-methoxy-2H-chromen-3-yl and ring A is 3- or 4-chlorophenyl moiety

compounds (80c-e).
Recently a series of novel quinolyl-thienyl chalcones with diverse substitution
pattern were tested against extracellular promastigotes of Leishmania major.98 All the
synthesized chalcone derivatives displayed significant antileishmanial activity (IC50 =
0.59 0.09 to 0.94 0.10 g/mL) as compared to the reference drug Amphotericin B
(IC50 = 0.56 0.20 g/mL). These quinoline-based chalcones were synthesized by
condensing formylquinolines with diverse acetylthiophenes.
Reagents and conditions: (i) AcOH, H3PO4, reflux, 46 h; (ii) POCl3, DMF, 800C; (iii) ArCOCH3, NaOH,
rt, 2 h.
(i)
(i)
(i)
O
O
H
(ii)
H3C
Cl
H
H3C
H
82b
(IC50 = 0.83 0.05 g/mL)
HH3C
(i)
Cl
(i)
S
H
CH3
H
82c
(IC50 = 0.74 0.31 g/mL)
CH3
H3C
Cl
(i)
H
82d
(IC50 = 0.62 0.24 g/mL)
Proposed stereo-,electronic and/or steric properties (i) electronic effect (attractive forces), (ii) steric
effect
30
Chapter 1
Structure-activity relationship among the two series of chalcone (82a-k and 83a-k)
was explained in terms of stereo- and electronic and/or steric properties. With the
decrease of steric crowding activity increased as evident by IC50 of compounds 82b, 82c
and 82d (IC50 = 0.83 0.05, 0.74 0.31 and 0.62 0.24 g/mL respectively).
R4
R3
R1
R4
KF / Al2O3
R2
Microwave
R2
R3
84
CHO
N
KF / Al2O3
Microwave
N
O
85
OHC
OH
OH
O
KF / Al2O3
Microwave
a) R1 = R2 = R4 = H, R3 = OBn
b) R1 = R2 = R4 = H, R3 = OMe
c) R1 = R4 = H, R2 = R3 = OMe
d) R1 = H, R2 = R3 = R4 = OMe
e) R1 = R2 = R4 = H, R3 = Cl
f) R1 = NO2, R2 = R3 = R4 = H
g) R1 = R3 = R4 = H, R2 = NO2
h) R1 = R2 = R4 = H, R3 = NO2
i) R1 = R2 = R4 = H, R3 = OH
O
86
Encouraged with the antileishmanial profile of several chalcone derivatives some

novel dihydro--ionone based chalcones were synthesized in our lab and all these
chalcone derivatives were evaluated for their in vitro antileishmanial activity in
promastigote and amastigote model.99
All these derivatives were synthesized under microwave irradiation (4-5 min.) in
presence of KF/Al2O3. The chalcones having p-methoxy and 3,4-dimethoxy substitution
as in 84b and 84c showed very good activity in promastigote as well as amastigote
model. Some of the compounds showed 100% inhibition at 5 and 2 g/mL
concentrations. Among pyridine and furan based chalcones, pyridine based chalcone (85)
showed very good antileishmanial profile in the promastigote as well as amastigote
model.
31
Chapter 1
Recently we have reported synthesis and antileishmanial potential of - and ionone based triazole integrated chalcones (87, 88) against intracellular amastigote form
of Leishmania donovani.100 Compound 66 and 67 have shown 100% and 98% inhibition
of parasitic growth at 40 M concentration with IC50 value of 15.3 2.2 M and 11.6
2.2 M respectively as compared to reference drugs miltefosine (IC50 = 8.6 0.4 M)
and miconazole (IC50 = 5.4 1.5 M).
1.9 CONCLUSION
Leishmaniasis is a life threatening disease that affects primarily to the people of
developing countries living below the poverty line. There is still no antileishmanial
vaccine and despite recognition of a large number of novel drug candidates none of them
currently undergoes clinical evaluation. Pharmaceutical research on natural products
represents a major strategy for discovering and developing new drugs. As a matter
of fact several compounds described in this review possess potent activity against
intracellular Leishmania and good efficacy in animal models of leishmaniasis; therefore,
new drug candidates could soon be available to fill the antileishmanial drug development
pipeline if funding permits.
32
Chapter 1
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87
Matsuda, H.; Morikawa, T.; Ando, S.; Toguchida, I.; Yoshikawa, M. Bioorg. Med.
Chem. 2003, 11, 1995.

88
Qian, Y.; Ma, G. Y.; Yang, Y.; Cheng, K.; Zheng, Q. Z.; Mao, W. J.; Shi, L.; Zhao, J.;
37
Chapter 1
Zhu, H. L. Bioorg. Med. Chem. 2010, 18, 4310.

89
Lahtchev, K. L.; Batovska, D. I.; Parushev, S. P.; Ubiyvovk, V. M.; Sibirny, A. A.
Eur. J. Med. Chem. 2008, 43, 2220.

90
Liu, M.; Wilairat, P.; Croft, S. L.; Tan, A. L. C.; Go, M. L. Bioorganic & Medicinal
Chemistry 2003, 11, 2729.

91
Nielsen, S. F.; Christensen, S. B.; Cruciani, G.; Kharazmi, A.; Liljefors, T. J. Med.
Chem.1998, 41, 4819.

92
Kayser, O.; Kiderlen, A. F. Phytother. Res. 2001, 15, 148.
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Chen, M.; Zhai, L.; Christensen, S. B.; Theander, T. G.; Kharazmi, A. Antimicrob.
Agents Chemother. 2001, 45, 2023.

94
Torres-Santos, E. C.; Rodrigues, J. M.; Moreira, D. L.; Kaplan, M. A. C.; Meirelles, M.
N.; Rossi-Bergmann, B. Antimicrob. Agents Chemother. 1999, 43, 1234.

95
Boeck, P.; Falcao, C. A. B.; Leal, P. C.; Yunes, R. A.; Filho, V. C.; Torres-Santos, E.
C.; Rossi-Bergmann, B. Bioorg. Med. Chem. 2006, 14, 1538.

96
Andrighetti-Frhner, C. R.; de Oliveira, K. N.; Gaspar-Silva, D.; Pacheco, L. K.;
Joussef, A. C.; Steindel, M.; Simes, C. M. O.; de Souza, A. M. T.; Magalhaes, U. O.;
Afonso, I. F.; Rodrigues, C. R.; Nunes, R. J.; Castro, H. C. European Journal of
Medicinal Chemistry 2009, 44, 755.
97
Foroumadi, A.; Emami, S.; Sorkhi, M.; Nakhjiri, M.; Nazarian, Z.; Heydari, S.;
Ardestani, S. K.; Poorrajab, F.; Shafiee, A. Chem Biol Drug Des 2010, 75, 590.
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Medicinal Chemistry 2008, 43, 2473.

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38
Chapter 2.1
Design, Synthesis and Bioevaluation of

Novel Terpenyl Heterocycles
Chapter 2.1
Design, Synthesis and bioevaluation of novel terpenyl heterocycles
2.1.1 INTRODUCTION
biology. It remains one of the major burdens on human health in developing countries,
and the WHO recently classified leishmaniasis as a Category I: emerging or uncontrolled
disease.
Clinical manifestations of leishmaniasis include cutaneous leishmaniasis (CL),
muco-cutaneous leishmaniasis (MCL), visceral leishmaniasis (VL) and post-kala-azar
dermal leishmaniasis (PKDL). Mucocutaneous leishmaniasis, visceral leishmaniasis, and
post-kala-azar dermal leishmaniasis are severe forms of leishmaniasis resulting from the
hosts inability to control the infection, whereas spontaneous healing often occurs in
cutaneous leishmaniasis because of the appropriate immune response.
Leishmaniasis is distributed in 88 countries, worldwide, and an estimated 1.52.0
million people both children and adults develop clinical leishmaniasis every year,
although many more subclinical infections go unrecorded. 75% of clinical cases affect
the skin (cutaneous leishmaniasis, or CL), and the remaining 25% represent systemic and
potentially fatal visceral leishmaniasis (VL, also known as kala-azar). 90% of VL cases
occur in India, Bangladesh, Nepal, Sudan and Brazil, where 70,000 or more deaths are
reported annually.1,2 It is widely recognized that this figure is a gross underestimate and
might represent only one-fifth of the true death toll. Among parasitic infections, only
malaria kills more people. In addition, leishmaniasis is in the top ten parasitic diseases for
its impact on socioeconomic development and has a burden of 2.4 million DALYs
(disability
adjusted
life
years;
http://www.who.int/whr/2002/en/whr02_en.pdf).
Increasing overlap with the spread of AIDS has heightened the threat of HIVLeishmania
co-infections, particularly in India and East Africa.3
At the turn of the nineteenth century, Cunningham, Borovsky, Leishman, Donovan,
Wright, Lindenberg and Vianna each independently identified the parasite that causes
leishmaniasis, to which Ronald Ross gave the generic name Leishmania (phylumSarcomastigophora, order-Kinetoplastida and family-Trypanosomatidae). Leishmania
parasites are dimorphic organisms, i.e., with two morphological forms in their life cycle:
39
Chapter 2.1
amastigotes in the mononuclear phagocytic system of the mammalian host, and

promastigotes in the digestive organs of the vector.4 The promastigotes are ~ 20 m long
and 1.5-3.00 m broad with a single long flagellum and multiply by binary fission as an
extra cellular parasite in the gut lumen of female sandfly. The amastigotes are 2-5 m
long intracellular non-motile, uninucleate ovoid organism containing a rod shaped
kinetoplast associated with a flagellar rudiment and multiply repeatedly by binary fission,
eventually destroying macrophages of vertebrate host. When an amastigote is ingested by
a Phlebotomine sandfly it elongates in the flys gut and transforms into a flagellated
promastigote or leptomonad. Leishmaniasis is transmitted through the bite of female
phlebotomine sandflies infected with the protozoan. The parasite is then internalized via
macrophages in the liver, spleen, and bone marrow.5
The classical treatment of leishmaniasis requires the administration of toxic and
poorly tolerated drugs. The pentavalent antimonials meglumine antimoniate
(Glucantime) and sodium stibogluconate (Pentostam) are the first-line compounds used
to treat leishmaniasis. Other drugs that may be used include pentamidine and
amphotericin B.6,7 However, parasite resistance greatly reduces the efficacy of
conventional medications.8 In the last 15 years, clinical misapplication of medications has
enabled the development of generalized resistance to these agents in Bihar, India, where
half of the global visceral leishmaniasis cases occur.9 Moreover, there are no effective
vaccines to prevent leishmaniasis.10,11
This disease, though globally massive in its impact, mainly affects poor people in
poor regions of the world. As such, these would never be viewed as viable target markets
for the pharmaceutical industry. But, in the past 10 years, major scientific breakthroughs
have been made in the treatment, diagnosis and prevention of leishmaniasis, and the
prices of several key medicines have been reduced. These developments have facilitated
implementation of sustainable national and regional control programmes; however,
functioning control programmes are still rare, and mortality and morbidity from
leishmaniasis worldwide show a worrying increasing trend.
40
Chapter 2.1
2.1.2 BASIS OF WORK

Although in the past decade the number of treatments has increased, but they have
several drawbacks such as difficulty in administration, length of treatment, toxicity, cost,
availability limited in disease endemic regions and increasing parasitic resistance. In view
of the above facts, the search for innovative drugs based on new molecular scaffolds that
target the specific metabolic pathway of the parasite should be highly prioritized, which
in turn requires new medicinal chemistry approaches to discover novel lead compounds
that might populate a pipeline of new therapeutics. Unfortunately, our limited discerning
of Leishmania biology makes difficult the rational designing of antileishmanial agents.
Currently, efforts are being made to search for new molecules from the natural
sources and in this endeavor diarylheptanoids,12 oxygenated abietanes,13 diterpene
quinines14,15 are showing promise as new lead molecules. Chalcones (1,3-diaryl-2propen-1-ones), precursors of flavonoids and isoflavonoids, constitute an important class
of natural products. Chemically, they are open-chained molecules in which two aromatic
rings are linked by a three-carbon enone fragment. Many of these molecules display an
impressive array of pharmacological activities including anticancer,16 antiinflamatory,17
antituberculosis,18 antifungal,19 antimalarial,20 and antileishmanial.21 The leishmanicidal
activity of several chalcones has been reported in the literature.22,23
The
recognized
synthetic
utility
of
chalcones
in
the
preparation
of
pharmacologically-interesting heterocyclic systems like pyrazolines/pyrazoles is of great

importance as these pyrazoles have been recognized owing to their pharmacological
activities,
which
includes
anti-tumor,24
anti-inflammatory,25
anti-parasitary,26
antimicrobial27 as well as anti-leishmanial.28

It was found that among the important pharmacophores responsible for
antileishmanial activity, the heterocyclic scaffold is still considered a viable lead structure
for the synthesis of more efficacious and broad spectrum antileishmanial agents. (Figure
2.1.1)
41
Chapter 2.1
Figure 2.1.1: Heterocyclic nuclei possessing antileishmanial activity

Rationally designed heterocyclic ionone like molecules29 and some novel terpenyl
2,4-diamino pyrimidines30 are showing promising antimicrobial and dihydrofolate
reductase inhibitory activities. 2,4-diaminopyrimidines31 and some de novo-designed
molecules32 are also giving further inputs in the leishmanial dihydrofolate reductase
activity.
Dihydrofolate reductase (DHFR) is a key enzyme in folate metabolism and,
therefore, in the production of thymidine.33,34 Its role in thymidine biosynthesis is the
reduction of dihydrofolate to tetrahydrofolate using the cofactor NADPH (Figure 2.1.2).
Following this reduction, tetrahydrofolate is methenylated to form methylenetetrahydrofolate, which then methylates deoxyuridine monophosphate (dUMP) to give
TMP in a reaction catalyzed by thymidylate synthase (TS) (Figure 2.1.2). During this
reaction, methylene-tetrahydrofolate is converted back to dihydrofolate, completing the
cycle. Therefore, inhibition of DHFR prevents biosynthesis of thymidine, and as a
42
Chapter 2.1
consequence, DNA biosynthesis. In addition, inhibition of DHFR probably leads to a

buildup in levels of dUMP and hence to a biosynthetic precursor, deoxyuridine
triphosphate.35 High levels of deoxyuridine triphosphate lead to incorporation of uracil
into DNA to levels beyond which the DNA repair enzymes (uracil-DNA-glycosylase)
can cope, leading to cell death.
CO 2H
CO 2H
O
O
O
N
H
N
HN
H 2N
N
H
N
CO 2H
O
Dihydrofolate Reductase
N
H
HN
H2N
NADPH
N
H
H
N
N
H
N
H
NADP
Dihydrofolate
Tetrahydrofolate
O
Me
NH
O
O P O
O-
O
OH
CO 2H
Thymidylate synthase
O
O P O
O-
methyene
tetrahydrofolate
dihdrofolate
dUMP
DHFR
NH
N
OH
TMP
tetrahydrofolate
glycine
serine
Figure 2.1.2: The reaction carried out by DHFR: the reduction of dihydrofolate to
tetrahydrofolate; and the role of reaction in the folate mediated production of TMP from
DUMP.
Based on above facts and in continuation of our studies on terpenyl pyrimidines as

novel antileishmanial agents,36 we designed some novel terpenyl heterocycles having
added aryl substitution and evaluated them for their in vitro and in vivo antileishmanial
activity and the results are parts of this chapter.
43
Chapter 2.1
2.1.3 CHEMISTRY
2.1.3.1 Synthesis of -ionone based 1,3,5-trisubstituted-4,5-dihydropyrazoles (4a-j)
The synthesis of -ionone based 1,3,5-trisubstituted-4,5-dihydropyrazoles (4a-j)
followed the general pathway outlined in scheme-2.1.1. They were prepared in two steps.
Firstly, the chalcones (3a-j) were obtained by direct condensation between the substituted
aromatic aldehydes (2) and -ionone (1), using phase transfer catalyzed condition.37
Cetyltrimethyl ammonium bromide (CTABr) was used as a phase transfer catalyst.
Secondly, cyclization of synthetic chalcones (3a-j) with phenyl hydrazine in refluxing
ethanol leads to the formation of dihydropyrazoles (4a-j). The substitution pattern in aryl
ring of compounds 3a-j and 4a-j is depicted in Table 2.1.1.
CHO
3'
R1
(i)
+
R4
R2
R3
2' 7'
4'
5'
6' 1'
3
5
8'
9'
R2
R 1 2'' 3'' R 3
4''
2
5''
1'' 6'' R 4
1
3a-j
R2
1
3a-j
R
4
3
5
(ii)
R3
R4
N N 1
4a-j
Scheme 2.1.1: Reagents and conditions: (i) Cetyl trimethyl ammonium bromide (CTABr),
NaOH, H2O, rt, 24 h; (ii) PhNHNH2, EtOH, reflux, 8 h.
The reaction of -ionone with substituted benzaldehydes was very facile and
furnished chalcones in good to excellent yield. The reaction of phenyl hydrazine with
chalcones (3a-j) was not only facile but it was also regiospecific in manner. The reaction
of chalcone 3a with phenyl hydrazine in ethanol furnished dihydropyrazole 4a in 40%
yield as a crystalline solid melting at 138-140C. The structure was assigned on the basis
of mass, IR, 1H and 13C NMR spectra.
44
Chapter 2.1
Table 2.1.1: Substitution pattern in aryl ring of compounds 3a-j and 4a-j.
Compound
3, 4
a
b
c
d
e
f
g
h
i
j
R1
R2
R3
R4
NO2
H
H
H
H
Cl
H
H
H
H
H
H
NO2
H
OCH3
H
H
Cl
H
H
H
NO2
H
OCH3
OCH3
H
Cl
H
F
OBn
H
H
H
OCH3
OCH3
H
H
H
H
H
The 1H NMR spectrum of 4a displayed doublet of doublets at 2.87 ppm (J = 17, 7

Hz, 1H) & 3.85 ppm (J = 17, 12 Hz, 1H) for two geminal protons of the dihydropyrazole
ring and doublet of doublets at 5.69 (J = 12, 7 Hz, 1H) for H-5 proton (vicinal to the
geminal protons) and it established the assigned structure 4a. The most active compound
4d was synthesized by refluxing the ethanolic solution of compound 3d with phenyl
hydrazine at 100C for 8 h. After completion of reaction, ethanol was removed by
distillation and residue was extracted with ethyl acetate. Combined organic extract was
washed with water, brine solution, dried (Na2SO4) and solvent was removed in vacuum.
The crude product was purified by Column chromatography (SiO2, 100-200 mesh). The
compound 4d was obtained in 19% yield as a yellow coloured solid melting at 90-93C.
The compound 4d was characterized by IR, NMR and mass spectrum. The IR
spectra showed a C=N stretching band at 1597 cm-1. The 1H NMR spectrum of 4d
displayed three doublet of doublets at 2.85 ppm (J = 17, 7 Hz, 1H) , 3.55 ppm (J = 17, 12
Hz, 1H) and 5.01 (J = 12, 7 Hz, 1H) for two geminal protons of dihydropyrazole ring and
one for the proton vicinal to geminal protons respectively. Analysis of
13
C, and mass
spectra provided the final structural elucidation of compound 4d.
45
Chapter 2.1
2.1.3.2 Synthesis of -ionone based 1,3,5-trisubstituted-4,5-dihydropyrazoles (8a-e)

The synthesis of -ionone based 1,3,5-trisubstituted-4,5-dihydropyrazoles (8a-e)
followed the general pathway outlined in scheme-2.1.2. They were also prepared in two
steps. Firstly, the chalcones (7a-e) were obtained by direct condensation between the
substituted aromatic aldehydes (6) and -ionone (5), using phase transfer catalyzed
condition. Secondly, cyclization of different chalcones with phenyl hydrazine in refluxing
ethanol leads to the formation of pyrazole derivatives (8a-e).
R2
CHO
R1
R1
(i)
+
R4
O
5
R3
R4
R2
R3
7a-e
R2
(ii)
7a-e
R1
R3
R4
2N N1
8a-e
Scheme 2.1.2: Reagents and conditions: (i) Cetyl trimethyl ammonium bromide (CTABr),
NaOH, H2O, rt, 24 h; (ii) PhNHNH2, EtOH, reflux, 8 h.
The substitution pattern in aryl ring of compounds 7a-e and 8a-e is depicted in
Table 2.1.2. The reaction of phenyl hydrazine with chalcones (7a-e) was also
regiospecific in manner. All the synthetic dihydro pyrazoles (8a-e) were characterized
using spectroscopic techniques (IR, 1H NMR, 13C NMR).
46
Chapter 2.1
Table 2.1.2: Substitution pattern in aryl ring of compounds 7a-e, 8a-e and 9a-e.
Compound
7, 8 and 9
a
b
c
d
e
R1
R2
R3
R4
NO2
H
H
H
H
H
H
H
H
OCH3
H
NO2
F
OCH3
OCH3
H
H
H
OCH3
OCH3
2.1.3.3 Synthesis of -ionone based 1,3,5-trisubstituted pyrazoles (9a-e)

Few dihydropyrazoles (8a-e) were aromatized using Ag2O in refluxing ethanol and
their structures were assigned by 1H NMR and 13C NMR spectra.
R2
R2
R1
R3
R4
2N N1
8a-e
R1
Ag2 O, EtOH
3 4 5
reflux, 18 h
2N N 1
R3
R4
9a-e
The substitution pattern in aryl ring of compounds 9a-e is depicted in Table 2.1.2.
We used the same reaction conditions, as used for compounds (8a-e), for aromatization
of compounds (4a-j) but we didnt find any aromatized product in significant amount.
Cyclization in all the synthesized heterocyclic compounds took place near the aromatic
ring rather than near the ionone ring. Continuous presence of doublet near 2.25 ( value)
in 1H NMR spectrum of compounds (9a-e) indicated that the cyclization took place near
the aromatic ring. If cyclization had taken place near the ionone ring then a singlet would
have been obtained in place of doublet in 1H NMR spectrum of compounds (9a-e).
2.1.4 BIOLOGICAL EVALUATION- MATERIAL METHODS

Antileishmanial screening of all the synthetic compounds was carried out by the of
Parasitology Division, Central Drug Research Institute, Lucknow, India, by using
following protocols.
47
Chapter 2.1
2.1.4.1 Anti amastigote activity

For assessing the activity of compounds against the amastigote stage of the
parasite, mouse macrophage cell line (J-774A.1) infected with promastigotes expressing
luciferase firefly reporter gene was used. Cells were seeded in a 96-well plate (4 x
104cell/100L/well) in RPMI-1640 containing 10% foetal calf serum and the plates were
incubated at 37C in a CO2 incubator. After 24 h, the medium was replaced with fresh
medium containing stationary phase promastigotes (4 x105/100L/well). Promastigotes
invade the macrophage and are transformed into amastigotes. The test compounds were
added at two fold dilutions up to 7 points in complete medium starting from 40 M conc.
after replacing the previous medium and the plates were incubated at 37C in a CO2
incubator for 72 h. At the end of the incubation, the supernatants were removed and 50
L PBS was added in each well and mixed with an equal volume of Steady Glo reagent.
After gentle shaking for 1-2 min, the reading was taken in a luminometer.38,39,40 The
values are expressed as relative luminescence units (RLU). IC50 of antileishmanial
activity was calculated by nonlinear regression analysis of the concentration response
curve using the four parameter Hill equations.
2.1.4.2 Cytotoxicity assay
The cell viability was determined using the MTT assay.41 Exponentially growing
cells (KB Cell line) (1105cells /100l/well) were incubated with test compounds for 72
hours. The test compounds were added at three fold dilutions up to 7 points in complete
medium starting from 400 M concentration, and were incubated at 37C in a humidified
mixture of CO2 and 95 % air in an incubator. Podophyllotoxin was used as a reference
drug and control wells containing dimethyl sulfoxide (DMSO) without compounds were
also included in the experiment. Stock solutions of compounds were initially dissolved in
DMSO and further diluted with fresh complete medium. After incubation, 25 L of MTT
reagent (5mg/ml) in PBS medium, followed by syringe filtration was added to each well
and incubated at 37C for 2 hours. At the end of the incubation period, the supernatant
were removed and 150 L of pure DMSO were added to each well. After 15 min. of
shaking the readings were recorded as absorbance at 544 nm on a micro plate reader. The
cytotoxic effect was expressed as 50% lethal dose, i.e., as the concentration of a
compound which provoked a 50% reduction in cell viability compared to cell in culture
48
Chapter 2.1
medium alone. CC50 values were estimated through the preformed template as described
by Huber and Koella.42
2.1.4.3 In Vivo assay
The in vivo leishmanicidal activity was determined in golden hamsters
(Mesocricetus auratus) infected with MHOM/IN/80/Dd8 strain of Leishmania donovani
obtained through the courtesy of P.C.C. Garnham, Imperial College, London (UK). The
method of Beveridge et al43 as modified by Bhatnagar et al44 and Gupta et al45 was used
for in vivo evaluation. Golden hamsters (Inbred strain) of either sex weighing 40-45g
were infected intracardiacally with 1x107 amastigotes per animal. The infection is well
adapted to the hamster model and establishes itself in 15-20 days. Meanwhile, hamsters
gain weight (85-95 g) and can be subjected to repeated spleen biopsies. Pre-treatment
spleen biopsy in all the animals was carried out to assess the degree of infection. The
animals with +1 infection (5-15 amastigotes/100 spleen cell nuclei) were included in the
chemotherapeutic trials. The infected animals were randomized into several groups on the
basis of their parasitic burdens. Five to six animals were used for each test sample. Drug
treatment by intraperitoneal (i.p.) route was initiated after 2 days of biopsy and continued
for 5 consecutive days. Post-treatment biopsies were done on day 7 after the last drug
administration and amastigote counts are assessed by Giemsa staining. Intensity of
infection in both, treated and untreated animals, and also the initial count in treated
animals was compared and the efficacy was expressed in terms of percentage inhibition
(PI) using the following formula:-
PI = 100- [ANAT x 100/ (INAT x TIUC)]

Where PI is Percent Inhibition of amastigotes multiplication
ANAT is Actual Number of Amastigotes in Treated animals
INAT is Initial Number of Amastigotes in Treated animals
TIUC is Time Increase of parasites in Untreated Control animals.
49
Chapter 2.1
2.1.5 RESULT AND DISCUSSION

The leishmanicidal activity of aryl substituted dihydropyrazoles/pyrazoles (4a-j),
(8a-e) and (9a-e) was evaluated against L. donovani intracellular amastigotes and results
have been presented in Table-2.1.3.
Table 2.1.3: Antileishmanial activity of dihydropyrazoles/pyrazoles against L. donovani

Compd.
No.
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
Miltefosine
In Vitro
Antiamastigote
Activity
IC50(M)
>20
>40
16.95
7.49
>20
>20
>20
>40
>40
>40
>20
>40
>40
>20
>20
>40
>40
>40
>20
>40
12.50
Cytotoxicity
CC50 (M)
Selectivity
Index (S.I.)
CC50/IC50
279.49
220.66
3.23
16.48
29.46
0.26
In vivo Activity
% Inhibition S.D.
(50mg/Kg5 days, i.p.
dose)
-
7.13 8.95
80.80 11.91
95.282.49
IC50 and CC50 values are the average of two independent experiments.
Miltefosine (30mg/kg x 5days, oral route) used as a reference drug.
S.D., standard deviation; i.p., intraperitoneal.
50
Chapter 2.1
The dihydropyrazoles (4a-j) prepared from ionone showed marginal to good in

vitro antileishmanial activity. Most of the compounds tested had no or marginal activity.
However, 4d did show promising antiamastigote activity with an IC50 of 7.5 M and a
selectivity index of 29.5. The in vitro antileishmanial response of this compound was
better than the reference drug, miltefosine (IC50 = 12.5 M, S.I. = 0.26). We found very
little correlation between type of substitution on the aromatic ring and the in vitro
biological activity. The dihydropyrazoles (8a-e) prepared from ionone and their
aromatized compounds (9a-e) showed only marginal in vitro activity.
The compound 4d was also selected for in vivo efficacy evaluation against L.
donovani/hamster model at the intraperitoneal (i.p.) dose of 50 mg kg15 days. The
compound exhibited significant in vivo response (81% inhibition in parasite

multiplication).
2.1.6 CONCLUSION
In summary, synthesis and biological evaluation of these terpenyl heterocycles led
us to discovery of compound 4d as good antileishmanial agent which is more active than
miltefosine in vitro. Selectivity index of compound 4d is 113.3 fold higher than
miltefosine. Despite the fact that this compound was better than the reference drug in
respect to IC50 and SI values, it was less active in vivo compare to standard drug. But due
to its merit of easy synthesis and efficacy, more analogues need to be prepared and
screened so as to identify a potential molecule for antileishmanial therapy. These
investigations revealed that these terpenyl heterocycles can be served as prototype for
development of more efficacious antileishmanial agents.
51
Chapter 2.1
2.1.7 EXPERIMENTAL SECTION

The reported melting points (C) are the uncorrected ones. The infrared spectra
were recorded on a Perkin-Elmer model 881 and FTIR 8210 PC, Schimadzu
spectrophotometers either on KBr discs or in neat. 1H NMR spectra and
13
C NMR (in
CDCl3) spectra (chemical shift in , ppm downfield from TMS) were recorded on Bruker
Advance DRX-300 MHz spectrometers. Electron impact (EI) mass spectra were recorded
on a JEOL JMS-D-300 spectrometer with the ionization potential 70 eV. Elemental
analysis was carried out on a Carlo-Erba EA 1108 instrument.
2.1.7.1 General procedure for synthesis of compounds 3a-j.
A mixture of -ionone (2.32 g, 2.49 ml, 12 mmol), substituted benzaldehydes (10
mmol), cetyl trimethyl ammonium bromide (0.14 g, 1 mmol), sodium hydroxide (1.0 g,
30 mmol) and water (50 ml) was stirred at room temperature for 24 hours. After
completion of reaction (TLC monitoring), it was extracted with ethyl acetate. The
combined organic extract was washed with water, brine solution, dried (Na2SO4) and
solvent was removed. Crude product was purified by column chromatography (SiO2, 60120 mesh).
2.1.7.2 (1E,4E)-1-(2-nitrophenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4-dien3-one (3a)
Yield: 62%. M.p. 117-119C. IR (KBr, cm1) 3093, 2923, 1668, 1611, 1568,
1519, 1351. 1H NMR (CDCl3, 300 MHz) 1.11 (s, 6H, CH3-8' and CH3-9'), 1.47-1.52
(m, 2H, CH2-5'), 1.61-1.67 (m, 2H, CH2-4'), 1.83 (s, 3H, CH3-7'), 2.10 (t, J = 6 Hz, 2H,
CH2-3'), 6.53 (d, J = 16 Hz, 1H, H-4), 6.84 (d, J = 16 Hz, 1H, H-2), 7.50-7.57 (m, 2H, H5 and H-5''), 7.62-7.72 (m, 2H, H-4'' and H-6''), 8.01-8.08 (m, 2H, H-1 and H-3'').
13
NMR (CDCl3, 75 MHz) 18.86, 21.92, 228.86, 33.80, 34.17, 39.83, 124.97, 128.35,
129.13, 130.18, 130.76, 131.26, 133.53, 136.49, 137.57, 137.70, 144.13, 148.47, 188.84.
ESMS m/z: 326 [M+1]+. Analysis calculated for C20H23NO3: C, 73.82; H, 7.12; N, 4.30;
Found: C, 73.86; H, 7.14; N, 4.26.
52
Chapter 2.1
2.1.7.3 (1E,4E)-1-(4-nitrophenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4-dien3-one (3b)

Yield: 26%. M.p. 124-125C. IR (KBr, cm1) 2924, 1668, 1610, 1510, 1337. 1H
NMR (CDCl3, 300 MHz) 1.14 (s, 6H, CH3-8' and CH3-9'), 1.51-1.56 (m, 2H, CH2-5'),
1.64-1.70 (m, 2H, CH2-4'), 1.87 (s, 3H, CH3-7'), 2.14 (t, J = 6 Hz, 2H, CH2-3'), 6.50 (d, J
= 16 Hz, 1H, H-4), 7.12 (d, J = 16 Hz, 1H, H-2), 7.59 (d, J = 16 Hz, 1H, H-5), 7.69 (d, J
= 16 Hz, 1H, H-1), 7.75 (d, J = 9 Hz, 2H, H-2'' and H-6''), 8.28 (d, J = 9 Hz, 2H, H-3''
and H-5'').
13
C NMR (CDCl3, 75 MHz) 18.82, 21.95, 228.87, 33.87, 34.18, 39.85,
2124.16, 2128.79, 129.13, 129.18, 136.44, 138.22, 139.42, 141.23, 144.21, 148.37,
188.28. ESMS m/z: 326 [M+1]+. Analysis calculated for C20H23NO3: C, 73.82; H, 7.12;
N, 4.30; Found: C, 73.86; H, 7.16; N, 4.28.
2.1.7.4 (1E,4E)-1-(3-nitrophenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4-dien3-one (3c)
Yield: 16%. M.p. 97-98C. IR (KBr, cm1) 3093, 2955, 2925, 1654, 1600, 1530,
(m, 2H, CH2-5'), 1.55-1.61 (m, 2H, CH2-4'), 1.77 (s, 3H, CH3-7'), 2.03-2.07 (m, 2H, CH23'), 6.41 (d, J = 16 Hz, 1H, H-4), 7.03 (d, J = 16 Hz, 1H, H-2), 7.46-7.55 (m, 2H, H-5 and
H-5''), 7.61 (d, J = 16 Hz, 1H, H-1), 7.80 (d, J = 8 Hz, 1H, H-6''), 8.13-8.18 (m, 1H, H4''), 8.38 (s, 1H, H-2'').
13
C NMR (CDCl3, 75 MHz) 18.84, 21.94, 228.88, 33.85,
34.19, 39.87, 122.34, 124.41, 128.16, 129.19, 129.96, 134.05, 136.46, 136.79, 138.00,
139.53, 144.09, 148.70, 188.33. ESMS m/z: 326 [M+1]+. Analysis calculated for
C20H23NO3: C, 73.82; H, 7.12; N, 4.30; Found: C, 73.86; H, 7.14; N, 4.26.
2.1.7.5 (1E,4E)-1-(3,4-dimethoxyphenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta1,4-dien-3-one (3d)
Yield: 38%. Oil. IR (Neat, cm1) 3020, 2936, 1642, 1603, 1514, 1218. 1H NMR
(CDCl3, 300 MHz) 1.10 (s, 6H, CH3-8' and CH3-9'), 1.46-1.52 (m, 2H, CH2-5'), 1.601.66 (m, 2H, CH2-4'), 1.82 (s, 3H, CH3-7'), 2.09 (t, J = 6 Hz, 2H, CH2-3'), 3.92 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 6.48 (d, J = 16 Hz, 1H, H-4), 6.81-6.90 (m, 2H, H-2 and H5''), 7.10-7.20 (m, 2H, H-2'' and H-6''), 7.49 (d, J = 16 Hz, 1H, H-5), 7.61 (d, J = 16 Hz,
1H, H-1).
13
C NMR (CDCl3, 75 MHz) 18.91, 21.90, 228.88, 33.69, 34.18, 39.81,
53
Chapter 2.1
55.90, 55.98, 109.78, 111.05, 123.00, 124.07, 127.87, 129.25, 129.35, 136.46, 136.57,
C22H28O3: C, 77.61; H, 8.29; Found: C, 77.64; H, 8.32.
2.1.7.6 (1E,4E)-1-(3,4,5-trimethoxyphenyl)-5-(2,6,6-trimethylcyclohex-1enyl)penta-1,4-dien-3-one (3e)
Yield: 44%. Oil. IR (Neat, cm1) 2935, 1644, 1604, 1585, 1503, 1459, 1128. 1H
1.60-1.67 (m, 2H, CH2-4'), 1.83 (s, 3H, CH3-7'), 2.09 (t, J = 6 Hz, 2H, CH2-3'), 3.88 (s,
3H, OCH3), 3.90 (s, 6H, 2OCH3), 6.49 (d, J = 16 Hz, 1H, H-4), 6.81 (s, 2H, H-2'' and
H-6''), 6.86 (d, J = 16 Hz, 1H, H-2), 7.46-7.60 (m, 2H, H-5 and H-1). 13C NMR (CDCl3,
75 MHz) 18.89, 21.89, 228.87, 33.71, 34.18, 39.83, 256.17, 60.96, 2105.48,
125.46, 129.10, 130.42, 136.56, 136.80, 140.21, 142.78, 143.13, 2153.43, 188.93.
ESMS m/z: 371 [M+1]+. Analysis calculated for C23H30O4: C, 74.56; H, 8.16; Found: C,
74.58; H, 8.20.
2.1.7.7 (1E,4E)-1-(2-chlorophenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4dien-3-one (3f)
= 16 Hz, 1H, H-4), 6.94 (d, J = 16 Hz, 1H, H-2), 7.27-7.34 (m, 2H, H-4'' and H-5''), 7.407.45 (m, 1H, H-6''), 7.51 (d, J = 16 Hz, 1H, H-5), 7.66-7.71 (m, 1H, H-3''), 8.04 (d, J = 16
Hz, 1H, H-1). 13C NMR (CDCl3, 75 MHz) 18.89, 21.92, 228.88, 33.76, 34.18, 39.82,
127.09, 127.62, 128.38, 128.98, 130.20, 130.99, 133.21, 135.25, 136.52, 137.12, 138.44,
143.61, 189.23. ESMS m/z: 315 [M+1]+, 317 [M+3]+. Analysis calculated for
C20H23ClO: C, 76.29; H, 7.36; Found: C, 76.33; H, 7.40.
2.1.7.8 (1E,4E)-1-(4-chlorophenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4dien-3-one (3g)
1448. 1H NMR (CDCl3, 300 MHz) 1.11 (s, 6H, CH3-8' and CH3-9'), 1.45-1.52 (m, 2H,
54
Chapter 2.1
CH2-5'), 1.60-1.67 (m, 2H, CH2-4'), 1.82 (s, 3H, CH3-7'), 2.10 (t, J = 6 Hz, 2H, CH2-3'),
6.45 (d, J = 16 Hz, 1H, H-4), 6.96 (d, J = 16 Hz, 1H, H-2), 7.36 (d, J = 8 Hz, 2H, H-3''
and H-5''), 7.45-7.55 (m, 3H, H-2'', H-6'' and H-5), 7.60 (d, J = 16 Hz, 1H, H-1).
13
NMR (CDCl3, 75 MHz) 18.88, 21.91, 228.87, 33.76, 34.18, 39.83, 126.05, 2129.17,
2129.39, 129.44, 133.44, 136.15, 136.49, 137.19, 141.24, 143.47, 188.92. ESMS m/z:
315 [M+1]+, 317 [M+3]+. Analysis calculated for C20H23ClO: C, 76.29; H, 7.36; Found:
C, 76.31; H, 7.35.
2.1.7.9 (1E,4E)-1-(3-chlorophenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4dien-3-one (3h)
= 16 Hz, 1H, H-4), 6.91 (d, J = 16 Hz, 1H, H-2), 7.25-7.54 (m, 6H, H-2'', H-4'', H-5'', H6'', H-5 and H-1).
13
C NMR (CDCl3, 75 MHz) 18.88, 21.90, 228.87, 33.76, 34.18,
39.85, 126.55, 126.78, 127.84, 129.43, 130.06, 130.13, 134.90, 136.49, 136.83, 137.24,
140.90, 143.54, 188.71. ESMS m/z: 315 [M+1]+, 317 [M+3]+. Analysis calculated for
C20H23ClO: C, 76.29; H, 7.36; Found: C, 76.31; H, 7.37.
2.1.7.10 (1E,4E)-1-(4-fluorophenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4dien-3-one (3i)
Yield: 39%. M.p. 65-66C. IR (neat, cm1) 2933, 1665, 1600, 1503, 1453, 1415.
1
H NMR (CDCl3, 300 MHz) 1.12 (s, 6H, CH3-8' and CH3-9'), 1.48-1.54 (m, 2H, CH2-
5'), 1.62-1.68 (m, 2H, CH2-4'), 1.84 (s, 3H, CH3-7'), 2.11 (t, J = 6 Hz, 2H, CH2-3'), 6.47
(d, J = 16 Hz, 1H, H-4), 6.93 (d, J = 16 Hz, 1H, H-2), 7.09 (t, J = 9 Hz, 2H, H-3'' and H5''), 7.52 (d, J = 16 Hz, 1H, H-5), 7.56-7.60 (m, 2H, H-2'' and H-6''), 7.64 (d, J = 16 Hz,
1H, H-1).
13
C NMR (CDCl3, 75 MHz) 18.89, 21.86, 228.86, 33.72, 34.17, 39.84,
115.89, 116.18, 125.38, 129.47, 130.08, 130.20, 131.20, 136.52, 136.90, 141.44, 143.32,
165.58, 189.01. ESMS m/z: 299 [M+1]+. Analysis calculated for C20H23FO: C, 80.50; H,
7.77; Found: C, 80.44; H, 7.75.
55
Chapter 2.1
2.1.7.11 (1E,4E)-1-(4-(benzyloxy)phenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta1,4-dien-3-one (3j)

(m, 2H, CH2-5'), 1.50-1.61 (m, 2H, CH2-4'), 1.74 (s, 3H, CH3-7'), 2.01 (t, J = 6 Hz, 2H,
CH2-3'), 5.02 (s, 2H, OCH2), 6.38 (d, J = 16 Hz, 1H, H-4), 6.80 (d, J = 16 Hz, 1H, H-2),
6.91 (d, J = 9 Hz, 2H, H-3'' and H-5''), 7.25-7.38 (m, 6H, -phenyl and H-5), 7.46 (d, J = 9
Hz, 2H, H-2'' and H-6''), 7.55 (d, J = 16 Hz, 1H, H-1).
13
C NMR (CDCl3, 75 MHz)
18.94, 21.90, 228.90, 33.68, 34.19, 39.83, 70.10, 2115.27, 123.70, 2127.49, 127.89,
128.17, 2128.68, 129.77, 2130.04, 136.30, 136.45, 136.58, 142.47, 142.70, 160.65,
189.14. ESMS m/z: 387 [M+1]+. Analysis calculated for C27H30O2: C, 83.90; H, 7.82;
Found: C, 83.95; H, 7.83.
2.1.7.12 General procedure for synthesis of compounds 4a-j.
To a solution of 3a-j (2 mmol) in ethanol (20 ml), phenyl hydrazine (0.216 g,
0.196 ml, 2 mmol) was added. It was refluxed for 8 hours. Ethanol was removed by
distillation and the residue was extracted with ethyl acetate (2x25 ml). The combined
organic extract was washed with water (2x25 ml), brine solution (25 ml), dried (Na2SO4)
and solvent was removed in vacuum. The crude product was purified by column
chromatography (SiO2, 100-200 mesh).
2.1.7.13 (E)-5-(2-nitrophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)4,5-dihydro-1H-pyrazole (4a).
Yield: 40%. M.p. 138-140oC. IR (KBr, cm-1) 3037, 2925, 1600, 1505, 1455, 1331.
1
H NMR (CDCl3, 300 MHz) 0.96 (s, 6H), 1.39 (m, 2H), 1.54 (m, 2H), 1.67 (s, 3H), 1.96
(m, 2H), 2.87 (dd, J = 17, 7 Hz, 1H), 3.85 (dd, J = 17, 12 Hz, 1H), 5.69 (dd, J = 12, 7 Hz,
1H), 6.18 (d, J = 16 Hz, 1H), 6.43 (d, J = 16 Hz, 1H), 6.70 (t, J = 7 Hz, 1H), 6.77 (d, J =
8 Hz, 2H), 7.08 (m, 2H), 7.38 (d, J = 8 Hz, 2H), 7.47 (m, 1H), 8.05 ( d, J = 8 Hz, 1H).
13
C NMR (CDCl3, 75 MHz) 19.09, 21.76, 2x28.91, 33.30, 34.13, 39.77, 41.97, 60.33,
2x112.93, 119.33, 125.24, 125.41, 128.27, 128.48, 2x129.09, 131.93, 133.18, 134.55,
136.84, 137.79, 144.00, 147.38, 149.52. ESMS m/z: 416 [M+1]+. Analysis calculated for
C26H29N3O2 : C, 75.15; H, 7.03; N, 10.11; Found: C, 75.21; H, 6.98; N, 10.07.
56
Chapter 2.1
2.1.7.14 (E)-5-(4-nitrophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)4,5-dihydro-1H-pyrazole (4b)

Yield: 52%. M.p. 170-171oC. IR (KBr, cm-1) 3031, 2927, 1597, 1506, 1341. 1H
NMR (CDCl3, 300 MHz) 0.96 (s, 6H), 1.41 (m, 2H), 1.55 (m, 2H), 1.68 (s, 3H), 1.97
(m, 2H), 2.84 (m, 1H), 3.64 (m, 1H), 5.19 (m, 1H), 6.16 (d, J = 16 Hz, 1H), 6.44 (d, J =
16 Hz, 1H), 6.72 (m, 1H), 6.84 (m, 2H), 7.09 (m, 2H), 7.41 (d, J = 7 Hz, 2H), 8.13 (d, J =
7 Hz, 2H).
13
C NMR (CDCl3, 75 MHz) 19.14, 21.74, 2x28.90, 33.30, 34.13, 39.74,
42.21, 63.44, 2x113.33, 119.62, 123.77, 2x124.53, 2x126.92, 2x129.21, 132.12, 133.17,
C26H29N3O2 : C, 75.15; H, 7.03; N, 10.11; Found : C, 75.19; H, 6.98; N, 10.08.
2.1.7.15 (E)-5-(3-nitrophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)4,5-dihydro-1H-pyrazole (4c)
Yield: 56%. M.p. 133-135oC. IR (KBr, cm-1) 3061, 2933, 1597, 1528, 1500, 1456,
1342. 1H NMR (CDCl3, 300 MHz) 0.97 (s, 6H), 1.40 (m, 2H), 1,53 (m, 2H), 1.68 (s,
3H), 1.97 (m, 2H), 2.88 (dd, J = 17, 7 Hz, 1H), 3.66 (dd, J = 17, 12 Hz, 1H), 5.20 (dd, J =
12, 7 Hz, 1H), 6.16 (d, J = 16 Hz, 1H), 6.45 (d, J = 16 Hz, 1H), 6.72 (m, 1H), 6.86 (d, J =
8 Hz, 2H), 7.09 (m, 2H), 7.42 (m, 1H), 7.56 (d, J = 8 Hz, 1H), 8.09 (m, 2H).
13
C NMR
(CDCl3, 75 MHz) 19.08, 21.75, 2x28.91, 33.32, 34.13, 39.76, 42.37, 63.40, 2x113.38,
119.62, 121.20, 122.70, 125.13, 2x129.06, 130.31, 132.06, 132.11, 133.15, 136.76,
C26H29N3O2: C, 75.15; H, 7.03; N, 10.11; Found: C, 75.21; H, 7.01; N, 10.06.
2.1.7.16 (E)-5-(3,4-dimethoxyphenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1enyl)vinyl) -4,5-dihydro-1H-pyrazole (4d)
1
H NMR (CDCl3, 300 MHz) 0.97 (s, 6H), 1.40 (m, 2H), 1.55 (m, 2H), 1.68 (s, 3H), 1.97
(m, 2H), 2.85 (dd, J = 17, 7 Hz, 1H), 3.55 (dd, J = 17, 12 Hz, 1H), 3.75 (s, 3H), 3.78 (s,
3H), 5.01 (dd, J = 12, 7 Hz, 1H), 6.15 (d, J = 16 Hz, 1H), 6.44 (d, J = 16 Hz, 1H), 6.74
(m, 4H), 6.92 (d, J = 8 Hz, 2H), 7.08 (t, J = 8 Hz, 2H).
13
19.10, 21.74, 2x28.90, 33.29, 34.13, 39.77, 42.60, 55.92, 55.94, 64.22, 108.71, 111.55,
2x113.47, 118.03, 119.09, 125.57, 2x128.82, 131.66, 132.53, 135.36, 136.89, 145.00,
57
Chapter 2.1
148.37, 149.44, 149.65. ESMS m/z: 431 [M+1]+. Analysis calculated for C28H34N2O2: C,
78.10; H, 7.96; N, 6.51; Found: C, 78.14; H, 7.91; N, 6.44.
2.1.7.17 (E)-5-(3,4,5-trimethoxyphenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1enyl)vinyl)-4,5-dihydro-1H-pyrazole (4e)
Yield: 11%. Oil. IR (neat, cm-1) 3014, 2933, 1596, 1501, 1460, 1222, 1127. 1H
(m, 2H), 2.85 (dd, J = 17, 8 Hz, 1H), 3.56 (dd, J = 17, 12 Hz, 1H), 3.57 (s, 3H), 3.72 (s,
6H), 4.95 (dd, J = 12, 8 Hz, 1H), 6.15 (d, J = 16 Hz, 1H), 6.43 (d, J = 16 Hz, 1H), 6.44 (s,
2H), 6.72 (m, 1H), 6.91 (d, J = 8 Hz, 2H), 7.09 (t, J = 8 Hz, 2H). ESMS m/z: 461
[M+1]+. Analysis calculated for C29H36N2O3: C, 75.62; H, 7.88; N, 6.08; Found: C,
75.60; H, 7.85; N, 6.11.
2.1.7.18 (E)-5-(2-chlorophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)4,5-dihydro-1H-pyrazole (4f)
(m, 2H), 2.76 (dd, J = 17, 7 Hz, 1H), 3.70 (dd, J = 17, 12 Hz, 1H), 5.46 (dd, J = 12, 7 Hz,
1H), 6.15 (d, J = 16 Hz, 1H), 6.43 (d, J = 16 Hz, 1H), 6.70 (t, J = 7 Hz, 1H), 6.83 (d, J =
8 Hz, 2H), 7.14 (m, 5H), 7.37 (d, J = 2 Hz, 1H).
13
C NMR (CDCl3, 75 MHz) 19.11,
21.76, 2x28.93, 33.29, 34.14, 39.77, 40.77, 60.92, 2x113.11, 119.11, 125.50, 127.38,
127.64, 128.70, 2x128.99, 129.82, 131.70, 131.79, 132.79, 136.89, 139.42, 144.35,
149.46. ESMS m/z: 405 [M+1]+, 407 [M+3]+. Analysis calculated for C26H29ClN2: C,
77.11; H, 7.22; N, 6.92; Found: C, 77.04; H, 7.21; N, 6.87.
2.1.7.19 (E)-5-(4-chlorophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)4,5-dihydro-1H-pyrazole (4g)
Yield: 42%. M.p. 94-96oC. IR (KBr, cm-1) 3059, 2922, 1595,1495. 1H NMR
(CDCl3, 300 MHz) 0.95 (s, 6H), 1.40 (m, 2H), 1.50 (m, 2H), 1.68 (s, 3H), 1.96 (m, 2H),
2.80 (dd, J = 17, 7 Hz, 1H), 3.57 (dd, J = 17, 12 Hz, 1H), 5.06 (dd, J = 12, 7 Hz, 1H),
6.13 (d, J = 16 Hz, 1H), 6.42 (d, J = 16 Hz, 1H), 6.68 (t, J = 7 Hz, 1H), 6.86 (d, J = 8 Hz,
2H), 7.07 (m, 2H), 7.16 (d, J = 9 Hz, 2H), 7.23 (d, J = 9 Hz, 2H). 13C NMR (CDCl3, 75
58
Chapter 2.1
MHz) 19.10, 21.79, 2x28.92, 33.31, 34.14, 39.74, 42.41, 63.44, 2x113.32, 119.22,
125.40, 2x127.33, 2x128.95, 2x129.32, 131.85, 132.72, 133.23, 136.82, 141.20, 144.50,
149.21. ESMS m/z: 405 [M+1]+, 407 [M+3]+. Analysis calculated for C26H29ClN2: C,
77.11; H, 7.22; N, 6.92; Found: C, 77.15; H, 7.19; N, 6.87.
2.1.7.20 (E)-5-(3-chlorophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)4,5-dihydro-1H-pyrazole (4h)
Yield: 62%. M.p. 92-94oC. IR (KBr, cm-1) 3059, 2922, 1595, 1495. 1H NMR
6.14 (d, J = 16 Hz, 1H), 6.43 (d, J = 16 Hz, 1H), 6.70 (t, J = 7 Hz, 1H), 6.88 (d, J = 8 Hz,
2H), 7.09 (m, 3H), 7.20 (m, 3H).
13
C NMR (CDCl3, 75 MHz) 19.10, 21.76, 2x28.92,
33.32, 34.14, 39.78, 42.44, 63.68, 2x113.35, 119.29, 124.04, 125.36, 120.08, 127.79,
2x128.96, 130.49, 131.85, 132.76, 135.00, 136.83, 144.62, 144.91, 149.19. ESMS m/z:
405 [M+1]+, 407 [M+3]+. Analysis calculated for C26H29ClN2: C, 77.11; H, 7.22; N, 6.92;
Found: C, 77.13; H, 7.29; N, 6.89.
2.1.7.21 (E)-5-(4-fluorophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)4,5-dihydro-1H-pyrazole (4i)
6.14 (d, J = 16 Hz, 1H), 6.44 (d, J = 16 Hz, 1H), 6.69 (t, J = 7 Hz, 1H), 6.89 (m, 2H),
6.96 (m, 2H), 7.08 (m, 2H), 7.21 (m, 2H).
13
C NMR (CDCl3, 75 MHz) 19.11, 21.78,
2x28.92, 33.31, 34.14, 39.76, 42.51, 63.45, 2x113.35, 115.88, 116.16, 119.15, 125.47,
127.53, 2x128.92, 131.79, 132.62, 136.84, 138.43, 144.60, 149.20, 160.46, 163.72.
ESMS m/z: 389 [M+1]+. Analysis calculated for C26H29FN2: C, 80.38; H, 7.52; N, 7.21;
Found: C, 80.41; H, 7.48; N, 7.20.
59
Chapter 2.1
2.1.7.22 (E)-5-(4-(benzyloxy)-phenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)
vinyl)-4,5-dihydro-1H-pyrazole (4j)
Yield: 40%. M.p. 120-122oC. IR (KBr, cm-1) 3031, 2925, 1599, 1503, 1457. 1H NMR
2.83 (dd, J = 17, 7 Hz, 1H), 3.54 (dd, J = 17, 12 Hz, 1H), 4.9 (s, 2H), 5.04 (dd, J = 12, 7
Hz, 1H), 6.13 (d, J = 16 Hz, 1H), 6.43 (d, J = 16 Hz, 1H), 6.7 (t, J = 7 Hz, 1H), 6.86 (d, J
= 8 Hz, 2H), 6.90 (d, J = 8 Hz, 2H), 7.07 (m, 2H), 7.15 (d, J = 8 Hz, 2H), 7.29 (m, 5H).
13
C NMR (CDCl3, 75 MHz) 18.09, 20.72, 2x27.88, 32.27, 33.10, 38.76, 41.53, 62.59,
69.05, 2x112.34, 2x114.36, 117.90, 124.61, 2x126.03, 2x126.46, 126.95, 2x127.55,

2x127.81, 130.55, 131.31, 134.08, 135.88, 135.92, 143.76, 148.21, 157.17. ESMS m/z:
477 [M+1]+. Analysis calculated for C33H36N2O: C, 83.15; H, 7.61; N, 5.88; Found: C,
83.11; H, 7.62; N, 5.86.
2.1.7.23 General procedure for the synthesis of compounds 8a-e.
To a solution of 7a-e (2 mmol) in ethanol (20 ml), phenyl hydrazine (0.216 g,
0.196 ml, 2 mmol) was added and the reaction mixture was refluxed for 8 h. After
completion of reaction (TLC monitoring) ethanol was removed by distillation. The
compound was extracted with ethyl acetate (100 ml). The combined organic extract was
washed with water (2x50 ml), brine solution (50 ml), dried (Na2SO4) and the solvent was
removed in vacuum. The crude product was purified by column chromatography (SiO2,
100-200 mesh).
2.1.7.24 (E)-5-(2-nitrophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl) vinyl)4,5-dihydro-1H-pyrazole (8a)
NMR (CDCl3, 300 MHz) 0.85 (s, 3H), 0.93 (s, 3H), 1.27 (m, 2H), 1.59 (s, 3H), 2.03 (m,
2H), 2.28 (d, J = 9 Hz, 1H), 2.90 (m, 1H), 3.88 (m, 1H), 5.45 (s, 1H), 5.68 (dd, J = 16, 9
Hz, 1H), 5.75 (m, 1H), 6.48 (d, J = 16 Hz, 1H), 6.79 (m, 1H), 6.85 (m, 2H), 7.17 (m, 2H),
7.45 (m, 2H), 7.54 (m, 1H), 8.13 (d, J = 8 Hz, 1H). 13CNMR (CDCl3, 75 MHz) 22.97,
23.04, 26.80, 27.91, 31.24, 32.53, 42.34, 54.81, 60.36, 2x112.91, 119.35, 121.64, 124.43,
125.41, 128.25, 128.47, 2x129.08, 133.20, 133.30, 134.55, 137.76, 138.30, 144.08,
149.06. ESMS m/z: 416 [M+1]+, 310 [M-phN2]+. Analysis calculated for C26H29N3O2: C,
60
Chapter 2.1
75.15; H, 7.03; N, 10.11; Found: C, 75.12; H, 7.01; N, 10.13.

2.1.7.25 (E)-5-(4-nitrophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)4,5-dihydro-1H-pyrazole (8b)
2H), 2.29 (d, J = 9 Hz, 1H), 2.87 (m, 1H), 3.67 (m, 1H), 5.25 (m, 1H), 5.46 (s, 1H), 5.64
(dd, J = 16, 9 Hz, 1H), 6.50 (d, J = 16 Hz, 1H), 6.82 (m, 1H), 6.91 (m, 2H), 7.17 (m, 2H),
7.48 (d, J = 8 Hz, 2H), 8.21 (d, J = 8 Hz, 2H).
13
C NMR (CDCl3, 75 MHz) 22.99,
23.04, 26.81, 27.89, 31.29, 32.55, 42.52, 54.76, 63.48, 113.30, 113.33, 119.64, 121.74,
124.35, 124.52, 124.54, 2x126.88, 2x129.07, 133.23, 138.27, 144.35, 147.45, 148.59,
149.93. ESMS m/z: 416 [M+1]+. Analysis calculated for C26H29N3O2: C, 75.15; H, 7.03;
N, 10.11; Found: C, 75.19; H, 7.07; N, 10.09.
2.1.7.26 (E)-5-(4-fluorophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)4,5-dihydro-1H-pyrazole (8c)
Yield: 64%. Oil. IR (neat, cm-1) 2957, 1600, 1501, 1438. 1H NMR (CDCl3, 300
MHz), 0.86 (s, 3H), 0.95 (m, 3H), 1.27 (m, 2H), 1.63 (s, 3H), 2.05 (m, 2H), 2.30 (d, J =
9 Hz, 1H), 2.88 (m, 1H), 3.61 (m, 1H), 5.15 (m, 1H), 5.48 (s, 1H), 5.65 ( dd, J = 16, 9
Hz, 1H), 6.51 (d, J = 16 Hz, 1H), 6.80 (m, 1H), 6.99 (m, 2H), 7.05 (m, 2H), 7.19 (m, 2H),
7.30 (m, 2H). ESMS m/z: 388 [M]+. Analysis calculated for C26H29FN2: C, 80.38; H,
7.52; N, 7.21; Found: C, 80.39; H, 7.52; N, 7.19.
2.1.7.27 (E)-5-(3,4-dimethoxyphenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2enyl)vinyl)-4,5-dihydro-1H-pyrazole (8d)
Yield: 34%. M.p. 110-112oC. IR (KBr, cm-1) 2957, 1596, 1495, 1450, 1232, 1025. 1H
NMR (CDCl3, 300 MHz) 0.84 (s, 3H), 0.94 (s, 3H), 0.94 (s, 3H), 1.27 (m, 2H), 1.61 (s,
3H), 2.03 (m, 2H), 2.29 (d, J = 9 Hz, 1H), 2.87 (m, 1H), 3.61 (m, 1H), 3.83 (s, 3H), 3.87
(s, 3H), 5.07 (m, 1H), 5.47 (s, 1H), 6.17 (dd, J = 16, 9 Hz, 1H), 6.49 (d, J = 16 Hz, 1H),
6.68 (s, 1H), 6.85 (m, 3H), 6.99 (d, J = 8 Hz, 2H), 7.15 (m, 2H).
13
C NMR (CDCl3, 75
MHz) 23.00, 23.04, 26.83, 27.81, 31.31, 32.52, 42.91, 54.75, 2x55.89, 64.18, 108.66,
111.53, 113.41, 117.97, 119.04, 121.54, 124.77, 124.80, 2x128.80, 133.41, 135.30,
61
Chapter 2.1
2.1.7.28 (E)-1-phenyl-5-(3,4,5-trimethoxyphenyl)-3-(2-(2,6,6-trimethylcyclohex-2enyl)vinyl)-4,5-dihydro-1H-pyrazole (8e)
Yield: 23%. Oil. IR (neat, cm-1) 3016, 2927, 1595, 1499, 1460, 1221, 1127. 1H
2H), 2.20 (d, J = 9 Hz, 1H), 2.81 (m, 1H), 3.53 (m, 1H), 3.74 (s, 9H), 4.93 (m, 1H), 5.37
(s, 1H), 5.55 (dd, J = 16, 9 Hz, 1H), 6.40 (d, J = 16 Hz, 1H), 6.44 (s, 2H), 6.71 (m, 1H),
6.91 (d, J = 8 Hz, 2H), 7.10 (m, 2H). 13C NMR (CDCl3, 75 MHz) 23.00, 23.05, 26.81,
27.88, 29.69, 32.55, 42.97, 54.75, 2x56.14, 60.82, 64.80, 2x102.37, 2x113.44, 119.24,
121.58, 124.65, 2x128.86, 133.40, 137.06, 137.81, 138.57, 145.21, 149.11, 2x153.81.
ESMS m/z: 461 [M+1]+. Analysis calculated for C29H36N2O3: C, 75.62; H, 7.88; N, 6.08;
Found: C, 75.58; H, 7.86; N, 6.11.
2.1.7.29 General procedure for the synthesis of compounds 9a-e.
To a solution of 7a-e (1 mmol) in ethanol (10 ml), phenyl hydrazine (0.108 g, 0.098 ml, 1
mmol) was added. The reaction mixture was refluxed for 8 h. Silver oxide (0.46 g, 2
mmol) was added and the reaction mixture was further refluxed for 10 h. It was filtered
through celite. It was taken up in ethyl acetate (50 ml), washed with water (2x 25 ml),
brine solution (25 ml), dried (Na2SO4) and the solvent was removed in vacuum. The
crude product was purified by chromatography (SiO2, 100-200 mesh).
2.1.7.30 (E)-5-(2-nitrophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl) vinyl)1H-pyrazole (9a)
Yield: 12%. Oil. IR (neat, cm-1) 3029, 2922, 1596, 1528, 1454, 1356. 1H NMR (CDCl3,
300 MHz) 0.84 (s, 3H), 0.88 (s, 3H), 1.18 (m, 2H), 1.59 (s, 3H), 1.97 (m, 2H), 2.24 (d, J
= 9 Hz, 1H), 5.39 (s, 1H), 6.07 (dd, J = 16, 9 Hz, 1H), 6.41 (d, J = 16 Hz, 1H), 6.46 (s,
1H), 7.16 (m, 2H), 7.37 (m, 1H), 7.48 (m, 5H), 7.82 (d, J = 7 Hz, 1H). ESMS m/z: 414
[M+1]+. Analysis calculated for C26H27N3O2: C, 75.54; H, 6.53; N, 10.16; Found: C,
75.51; H, 6.50; N, 10.20.
62
Chapter 2.1
2.1.7.31 (E)-5-(4-nitrophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)1H-pyrazole (9b)

Yield: 16%. M.p. 127-128oC. IR (KBr, cm-1) 3020, 2925, 1600, 1521, 1450, 1346. 1H
NMR (CDCl3, 300 MHz) 0.84 (s, 3H), 0.88 (s, 3H) 1.18 (m, 2H), 1.58 (s, 3H), 1.97 (m,
2H), 2.25 (d, J = 9 Hz, 1H), 5.4 (s, 1H), 6.12 (dd, J = 16, 9 Hz, 1H), 6.42 (d, J = 16 Hz,
1H), 6.65 (s, 1H), 7.19 (m, 2H), 7.30 ( m, 5H), 8.08 (d, J = 9 Hz, 2H). 13C NMR (CDCl3,
75 MHz) 22.98, 23.12, 26.94, 27.80, 31.48, 32.54, 54.75, 105.70, 121.47, 122.65,
2123.77, 2125.26, 128.00, 2129.17, 2129.27, 133.65, 135.03, 136.80, 139.53,
141.50, 147.28, 151.90. ESMS m/z: 414 [M+1]+. Analysis calculated for C26H27N3O2: C,
75.54; H, 6.53; N, 10.16; Found: C, 75.50; H, 6.49; N, 10.21.
2.1.7.32 (E)-5-(4-fluorophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)1H-pyrazole (9c)
(CDCl3, 300 MHz) 0.83 (s, 3H), 0.87 (s, 3H), 1.18 (m, 2H), 1.58 (s, 3H), 1.96 (m, 2H),
2.23 (d, J = 9 Hz, 1H), 5.38 (s, 1H), 6.08 (dd, J = 16, 9 Hz, 1H), 6.41 (d, J = 16 Hz, 1H),
6.50 (s, 1H), 6.90 (m, 2H), 7.11 (m, 2H), 7.20 (m, 5H).
13
23.03, 23.14, 26.96, 27.80, 31.49, 32.53, 54.72, 104.50, 115.42, 115.71, 121.33, 123.08,
2x125.10, 127.26, 2x128.95, 130.42, 130.53, 133.82, 134.40, 139.85, 142.89, 151.53,
160.93, 164.22. ESMS m/z: 387[M+1]+. Analysis calculated for C26H27FN2: C, 80.80; H,
7.04; N, 7.25; Found: C, 80.76; H, 7.01; N, 7.27.
2.1.7.33 (E)-5-(3,4-dimethoxyphenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2enyl)vinyl)-1H-pyrazole (9d)
(CDCl3, 300 MHz) 0.93 (s, 3H), 0.98 (s, 3H), 1.44 (m, 2H), 1.68 (s, 3H), 2.06 (m, 2H),
2.33 (d, J = 9 Hz, 1H), 3.66 (s, 3H), 3.90 (s, 3H), 5.48 (s, 1H), 6.17 (dd, J = 16, 9 Hz,
1H), 6.51 (d, J = 16 Hz, 1H), 6.59 (s, 1H), 6.68 (s, 1H), 6.85 (m, 2H), 7.32 (m, 5H). 13C
NMR (CDCl3, 75 MHz) 23.02, 23.14, 26.97, 27.80, 31.52, 32.52, 54.74, 55.64, 55.84,
103.81, 110.96, 111.86, 121.26, 121.34, 123.10, 123.25, 2x125.27, 127.22, 2x128.85,
133.90, 134.17, 140.16, 143.83, 148.56, 149.00, 151.44. ESMS m/z: 429 [M+1]+.
Analysis calculated for C28H32N2O2: C, 78.50; H, 7.47; N, 6.54; Found: C, 78.43; H,
63
Chapter 2.1
7.51; N, 6.51.
2.1.7.34 (E)-5-(3,4,5-trimethoxyphenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2enyl)vinyl)-1H-pyrazole (9e)
1
H NMR (CDCl3, 300 MHz) 0.84 (s, 3H), 0.88 (s, 3H), 1.18 (m, 2H), 1.58 (s, 3H), 1.98
(m, 2H), 2.25 (d, J = 9 Hz, 1H), 3.58 (s, 6H), 3.77 (s, 3H), 5.39 (s, 1H), 6.12 (dd, J = 16,
9 Hz, 1H), 6.34 (s, 2H), 6.44 (d, J = 16 Hz, 1H), 6.53 (s, 1H), 7.27 (m, 5H).
13
C NMR
(CDCl3, 75 MHz) 21.99, 22.13, 25.94, 26.78, 30.51, 31.51, 53.74, 2x54.93, 59.92,
102.88, 2x105.00, 120.28, 122.12, 2x124.38, 124.70, 126.38, 2x127.87, 132.85, 133.35,
137.03, 139.05, 142.85, 150.41, 2x152.05. ESMS m/z: 459 [M+1]+. Analysis calculated
for C29H34N2O3: C, 75.98; H, 7.42; N, 6.11; Found: C, 75.91; H, 7.45; N, 6.15.
64
Chapter 2.1
2.1.8 SPECTRA OF SOME SELECTED COMPOUNDS
Figure 2.1.3: 1H NMR of compound 3d at 300 MHz (CDCl3)
Figure 2.1.4: 13C NMR of compound 3d at 300 MHz (CDCl3)
65
Chapter 2.1
Figure 2.1.5: 1H NMR of compound 4d at 300 MHz (CDCl3)
Figure 2.1.6: 13C NMR of compound 4d at 300 MHz (CDCl3)
66
Chapter 2.1
2.1.9 REFERENCES
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69
Chapter 2.2
Design, Synthesis and Bioevaluation of

Novel Triazole Integrated Phenyl
Heteroterpenoids as Antileishmanial
Agents
Chapter 2.2
Design, synthesis and bioevaluation of novel triazole integrated

Phenyl heteroterpenoids as antileishmanial agents
2.2.1 INTRODUCTION
Leishmaniasis is a family of parasitic diseases with extensive morbidity and
mortality in 88 tropical and subtropical countries.1 These parasitic infections are caused
by an obligate intracellular protozoan parasite belonging to the genus Leishmania.
Leishmaniasis manifests mainly in three clinical forms: cutaneous leishmaniasis (CL),
mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL). VL is generally
lethal if left untreated. The situation has become complicated with the co-infection of
AIDS with leishmaniasis.
The leishmaniasis, traditionally considered rather exotic diseases of tropical areas
are beginning to have a major impact on human populations of the developed world and
is compounded by more ready access to international travel and the carelessness of
people, while the expansion of both the insect vector and the parasites due to global
warming is similarly crucial. According to the World Health Organisation, leishmaniasis
currently affects 12 million people worldwide and there around 2 million new cases per
year with growing tendency. Moreover, it is estimated that approximately 350 million
people live at risk of Leishmania infection.1 With no immediate prospect for vaccines,
chemotherapy is the only way to cure the patients suffering with this parasitic infection.
At present, only drugs in practice include pentavalent antimonials, paromomycin,
amphotericin-B and miltefosine. High toxicity and increasing resistance to the current
chemotherapeutic regimens have further complicated the situation in VL endemic regions
of the world.2 Since the chemotherapy against leishmaniasis is still inefficient; there is an
urgent need for development of new therapeutic agents from natural sources. Chalcones
represent an important class of naturally occurring small molecules, biologically active
against leishmaniasis.3 Their recognized synthetic utility in the preparation of
pharmacologically-interesting heterocyclic systems like pyrazolines is of great
importance as these pyrazolines have been recognized owing to their pharmacological
activities, which includes antitumor,4 antiinflammatory,5 antiinfective,6 antimicrobial7 as
well as antileishmanial activity.8
70
Chapter 2.2

2.2.2 BASIS OF WORK

Sterols are important components of the cell membrane that are vital to cellular
function and maintenance of cell structure. Unlike mammalian cells, which have
cholesterol as the major membrane sterol, trypanosomatids synthesize ergosterol and
other 24-methyl sterols that are required for their growth and viability. These sterols are
absent from the mammalian cells. Therefore, the sterol biosynthetic pathway from
Leishmania is considered to be an important drug target. Based on this sterol biosynthetic
pathway, most of the azoles (miconazole, ketoconazole, fluconazole and itraconazole)
(Figure 2.2.1) were designed as antifungal drugs and their antileishmanial potential9 was
reported in 1981 and later years.10,11 Antifungal agents containing triazole ring inhibit the
Leishmania amastigotes growth by preventing the 14-demethylation of lanosterol and
effectively block synthesis of ergosterol.12
O
N
N
N
N
N
Cl
Itraconazole
Cl
N
N
N
Cl
Cl
O
OH
N
Cl
O
O
N
N
O
F
H
N
Cl
Cl
Fluconazole
Ketoconazole
Cl
Miconazole
Figure 2.2.1: Chemical structures of azoles containing drugs.

However, despite reports of their usefulness, the antileishmanial activity was not
enough to induce clinical cure by themselves. Thus, the development of new, efficient,
and safe drugs for the treatment of this disease is imperative. Taking the above reports in
consideration and in continuation of our efforts to synthesize novel antileishmanial
71
Chapter 2.2

agents,13 we designed some novel triazole integrated phenyl heteroterpenoids and

evaluated them for their in vitro and in vivo antileishmanial activity.
2.2.3 CHEMISTRY
The synthetic routes followed for the preparation of the target compounds have
been outlined in Scheme 2.2.1. In the first step, triazole integrated chalcones 2 and 5
were synthesized by stirring commercially available and ionone respectively with 4(1H-1,2,4-triazol-1-yl)benzaldehyde
at
room
temperature
via
Claisen
Schmidt
condensation reactions catalysed by phase transfer catalyst, cetyltrimethyl ammonium

bromide (CTABr), in aqueous solution of NaOH. In the second step, chalcones (2, 5)
were refluxed with phenyl hydrazine and 4-fluorophenyl hydrazine hydrochloride in
ethanol to furnish 1,3,5-trisubstituted pyrazolines (3a, 3b, 6a and 6b) in quantitative
yield.
The structures of the synthesized compounds were determined by means of IR, 1H
NMR, 13C NMR and mass spectrometery. For example IR spectrum of triazole integrated
chalcone 2 (intermediate of the most active compound, 3a) revealed a strong band at
1643 cm-1, along with resonance in its 13C NMR at 188.6 indicating the presence of a
keto carbonyl function. Its 1H NMR revealed, besides aromatic proton resonances in the
region 7.67-8.56, two -protons of ,-unsaturated carbonyl moiety showed up as
doublets at 7.61 and 7.48 with trans olefinic-H coupling constant J = 16 Hz and two protons appeared as doublets at 6.96 and 6.42 (J = 16 Hz).
IR spectrum of the best compound of the series (3a) showed a C=N stretching
band at 1597 cm-1. In its 1H NMR spectra, three protons of the pyrazoline ring were seen
as doublet of doublets at 2.86 ppm (J = 16, 7 Hz), 3.63 ppm (J = 16, 12 Hz) and 5.16 (J =
12, 7 Hz). Two protons of the triazole ring were appeared downfield at 8.02 and 8.45.
This downfild shift is due to the presence of three electron withdrawing nitrogen atoms in
the triazole ring. Simillarly compound 6a showed a C=N stretching band at 1601 cm-1. In
its 1H NMR spectra, three protons of the pyrazoline ring were seen at 2.88 ppm (J = 17, 8
Hz), 3.64 ppm (J = 17, 12 Hz) and 5.20 (J = 12, 8 Hz) and triazole rings protons were
appeared at 8.08 and 8.51. Finally, mass spectra of compounds 3a and 6a showed also
well defined [M+1] ions.
72
Chapter 2.2

Scheme 2.2.1: Synthesis of triazole integrated phenyl heteroterpenoids.
R
7'
2'
3'
5
1'
5'
4 3 2
9'
6'
4'
1''
6''
5''
8'
N
H
2''
NH2
N
N
N
3''
2'''
1''' N
4'' N
3'''
5''
N N
R= H, F
N
4'''
(ii)
R
O
3 (a) R = H
(b) R = F
(i)
N
4-(1H-1,2,4-triazol-1-yl)
benzaldehyde
O
(i)
R
O
N
H
N
N
NH2
N
N
N
N N
R= H, F
(ii)
R
6 (a) R = H
(b) R = F
Reagents and conditions: (i) Cetyltrimethyl ammonium bromide (CTABr), NaOH, H2O, rt, 24
h; (ii) EtOH, reflux, 8 h.
73
Chapter 2.2

2.2.4 BIOLOGICAL EVALUATION- MATERIAL METHODS

Same as described in the Chapter 2.1.4
In vivo result is presented as means standard deviations (SD) for two experiments
(Figure 2.2.2), and analysis of data was carried out by one-way ANOVA using Graphpad
Prism (version 5.0).
2.2.5 RESULT AND DISCUSSION

The leishmanicidal activity of triazole integrated phenyl heteroterpenoids 2, 3a-b,
5, 6a and 6b was evaluated against L. donovani intracellular amastigotes and results have
been presented in Table 2.2.1. Almost all the synthesized compounds have shown more
than 90% inhibition at 40 M concentration against intracellular leishmania parasite in
vitro. Compound 2, ionone based triazole integrated chalcone, and compound 6b,
ionone based triazole integrated 4-fluorophenyl pyrazoline, showed marginal in vitro
activity. Compound 3b, ionone based triazole integrated 4-fluorophenyl pyrazoline, and
compound 5, ionone based triazole integrated chalcone, exhibited good antileishmanial
activity with IC50 12.4 and 11.6 M respectively. However, Compound 3a, ionone
based triazole integrated phenyl pyrazoline, and compound 6a, ionone based triazole
integrated phenyl pyrazoline, were found to show significant activity with IC50 6.4, 9.2
M and SI value 18, 32 respectively.
When compared with standard antileishmanials, miltefosine and miconazole, the
compound 3a showed comparable in vitro activity to miltefosine and miconazole. It is
interesting to note that compound 3a and 6a, synthesized using phenyl hydrazine have
shown increased activity as compared to the compound 3b and 6b, synthesized using 4flurophenyl hydrazine hydrochloride. This decrease in activity of , ionone based
triazole integrated phenyl pyrazoline (3a, 6a) on para fluoro substitution indicates that
replacement of para-hydrogen of phenyl ring with more electronegative fluorine atom
diminishes the antileishmanial activity.
74
Chapter 2.2

Table 2.2.1: In vitro antileishmanial activity of triazole integrated

heteroterpenoids against intracellular amastigotes and their cytotoxicity.
In vitro Antiamastigote activity
phenyl
% Inhibition at
40 M
IC50 (M)
Cytotoxicity
CC50 (M)
100
15.3 2.2
72.4 5.8
3a
100
6.4 1.2
112.4 10.9
18
3b
98.65
12.4 2.0
58.6 4.9
98.18
11.6 1.6
38.5 5.1
6a
98.03
9.2 1.7
296 15.9
32
6b
90.54
16.9 3.1
72.2 8.4
Miltefosine
100
8.6 0.4
54.7 6.8
Miconazole
100
5.4 1.5
37.4 4.1
Compound No.
Selectivity
Index (SI)
IC50 and CC50 values are the average (mean S.D.) of two independent experiments.
The Selectivity Index (SI) is defined as the ratio of CC50 on Vero cells to IC50 on L. donovani
intramacrophagic amastigotes.
IC50, half maximum inhibitory concentration; CC50, half maximum cytotoxic concentration; S.D., standard
deviation.
Based on the in vitro screening profile, compound 3a and 6a were selected for in
vivo trial in L. donovani/golden hamster model. These compounds were evaluated at the
dose of 50 mg/kg x 5 days by intraperitoneal (i.p.) route. Compound 6a has shown 58
08 % inhibition of parasite multiplication while compound 3a was found to show
significant parasitic inhibition at same dose regimen. The compound 3a has shown 79
11 % inhibition of parasitic growth which is more or less similar to first line drug,
Sodium stibogluconate (SSG) and inferior to miltefosine. In summary, we have found
compound 3a as a promising hit molecule for antileishmanial chemotherapy and it was
not toxic to mammalian cells at parasite inhibitory concentration.
75
Chapter 2.2

Figure 2.2.2: In vivo efficacy compound of 3a and 6a against L. donovani / hamster

model.
All the synthesized compounds were also checked for compliance to the Lipinski
rule of five, and the results are summarized in Table 2.2.2. According to this rule a
molecule likely to be developed as an orally active drug candidate should show no more
than one violation of the following four criteria: logP (octanolwater partition
coefficient) 5, molecular weight 500, number of hydrogen bond acceptors 10 and
number of hydrogen bond donors 5. Molecular properties of synthesized compounds
were calculated by www.molinspiration.com software, and it was found that almost all
the synthesized compounds followed the above criteria (Table 2.2.2). Therefore, these
triazole integrated phenyl heteroterpenoids have a good potential for eventual
development as oral agents and can be potentially active drug candidate.
76
Chapter 2.2

Table 2.2.2: Molinspiration calculation of molecular properties for the Lipinski Rule.
Compound
nViol
MW
miLogP
nON
nOHNH
natoms
nrotb
Acceptable
range
500
10
347.462
4.22
26.0
3a
437.591
6.051
33.0
3b
455.581
6.215
34.0
5b
347.462
3.9
26.0
6a
437.591
5.731
33.0
6b
455.581
5.895
34.0
nViol, no. of violations; MW, molecular weight; miLogP, molinspiration predicted Log P; nON, no. of
hydrogen bond acceptors; nOHNH, no. of hydrogen bond donors; natoms, no. of atoms; nrotb, no. of
rotatable bond.
2.2.6 CONCLUSION
In conclusion, we have synthesized a series of triazole integrated phenyl
heteroterpenoids and evaluated them for their in vitro activity against intracellular
amastigote form of Leishmania donovani. Among all tested compounds, compound 3a
was found to be the most active with IC50 6.4 M and better selectivity index (SI) 18 as
compared to reference drugs, miltefosine and miconazole. When evaluated in vivo in L.
donovani/hamster model, 3a has exhibited 79 11 % inhibition of parasite multiplication
at 50 mg kg-1 5 days on day 7 post treatments. The potent activity and simple synthesis
of these triazole integrated phenyl heteroterpenoids suggest that they are potential
candidates for the development of more efficacious antileishmanial agents.
77
Chapter 2.2

2.2.7 EXPERIMENTAL SECTION

The reported melting points (0C) are the uncorrected ones. The infrared spectra
spectrophotometers on KBr discs. 1H NMR spectra and
13
C NMR (in CDCl3) spectra
(chemical shift in , ppm downfield from TMS) were recorded on Bruker Advance DRX300 MHz spectrometers. Electron spray ionisation (ESI) mass spectra were recorded on a
Jeol JESMS-D-300 spectrometer with the ionization potential 70 eV. Elemental analysis
was carried out on a Carlo-Erba EA 1108 instrument.
2.2.7.1 Synthesis of (1E,4E)-1-(4-(1H-1,2,4-triazol-1-yl)phenyl)-5-2,6,6trimethyl
cyclohex-1-enyl) penta-1,4-dien-3-one (2)
A mixture of ionone (0.48 g, 0.507 ml, 2.5 mmol), 4-(1H-1,2,4-triazol-1-yl)
benzaldehydes (0.346 g, 2 mmol), cetyltrimethyl ammonium bromide (0.028 g, 0.20
mmol), sodium hydroxide (0.240 g, 6 mmol) and water (10 ml) was stirred at room
temperature for 24 h. Reaction progress was monitored by TLC and compound was
extracted with ethyl acetate (2 30 ml). The combined organic extract was washed with
water (2 30 ml), brine solution (25 ml), dried (Na2SO4) and the solvent was removed in
vacuum. The crude product was purified by column chromatography (SiO2, 60-120
mesh). Elution with 10 % ethyl acetate in hexane furnished a yellow coloured solid
(0.164 g, 24%).
Mp: 103-104OC; IR (KBr, cm-1): 2929, 1643 (C=O), 1590 (C=N); 1H NMR
(CDCl3, 300 MHz): 1.05 (s, 6 H, CH3-8 and CH3-9), 1.41-1.45 (m, 2H, CH2-5), 1.551.61 (m, 2H, CH2-4), 1.77 (s, 3H, CH3-7), 2.04 (t, J = 6 Hz, 2 H, H-3), 6.42 (d, J = 16
Hz, 1H, H-4), 6.96 (d, J = 16 Hz, 1H, H-2), 7.48 (d, J = 16 Hz, 1H, H-5), 7.61 (d, J = 16
Hz, 1H, H-1), 7.67 (brs, 4H, H-2, H-3, H-5 and H-6), 8.06 (s, 1H, H-3), 8.55 (s,
1H, H-5);
13
C NMR (CDCl3, 75 MHz): 18.86, 21.89, 228.87, 33.76, 34.18, 39.85,
2120.08, 126.64, 129.35, 2129.63, 134.87, 136.50, 137.26, 137.89, 140.69, 140.82,
143.54, 152.78, 188.66; ESI-MS m/z: 348 [M+1]+; Analysis calculated for C22H25N3O: C,
76.05; H, 7.25; N, 12.09; Found: C, 76.10; H, 7.19; N, 12.11.
78
Chapter 2.2

2.2.7.2 (E)-1-(4-(1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)-4,5-dihydro1H-pyrazol-5-yl)phenyl)l-1H-1,2,4-triazole (3a)

To a solution of 2 in ethanol (15 ml), phenyl hydrazine (0.162 g, 0.147 ml, 1.5
mmol) was added. The reaction mixture was refluxed for 8 h. Reaction progress was
monitored by TLC and compound was precipitated as yellow coloured solid. It was
filtered and dried (0.312 g, 48%).
Mp: 168-170 OC; IR (KBr, cm-1): 2927, 1597 (C=N), 1504 (C=C); 1H NMR
(CDCl3, 300 MHz): 0.97 (s, 6H), 1.38-1.41 (m, 2H), 1.53-1.56 (m, 2H), 1.68 (s, 3H),
1.95-1.97 (m, 2H), 2.86 (dd, J = 16, 7 Hz, 1H), 3.63 (dd, J = 16, 12 Hz, 1H), 5.16 (dd, J =
12, 7 Hz, 1H), 6.16 (d, J = 16 Hz, 1H), 6.45 (d, J = 16 Hz, 1H), 6.68-6.73 (m, 1H), 6.89
(d, J = 8 Hz, 2H), 7.08 (t, J = 8 Hz, 2H), 7.38 (d, J = 8 Hz, 2H), 7.58 (d, J = 8 Hz, 2H),
8.02 (s, 1H), 8.45 (s, 1H);
13
C NMR (CDCl3, 75 MHz): 19.08, 21.79, 228.92, 33.30,
34.13, 39.72, 42.39, 63.44, 2113.34, 119.32, 2120.91, 123.43, 125.32, 2127.41,
127.60, 2129.00, 131.94, 132.86, 136.79, 142.94, 144.42, 149.22, 153.14; ESI-MS m/z:
438 [M+1]+; Analysis calculated for C28H31N5: C, 76.85; H, 7.14; N, 16.00; Found: C,
76.89; H, 7.15; N, 15.96.
2.2.7.3 (E)-1-(4-(1-(4-fluorophenyl)-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)-4,5dihydro-1H-pyrazol-5-yl)phenyl)-1H-1,2,4-triazole (3b)
To a solution of 2 (0.200 g, 0.576 mmol) in ethanol (20 ml), 4-fluorophenyl
hydrazine hydrochloride (0.093 g, 0.576 mmol) was added. The reaction mixture was
refluxed for 8 h. Reaction progress was monitored by TLC. After completion of reaction
ethanol was distilled off. Compound was extracted with ethyl acetate (230 ml). The
combined organic extract was washed with water (225 ml), brine solution (25 ml), dried
(Na2SO4) and solvent was removed in vacuum. The crude product was purified by
column chromatography (SiO2, 60-120 mesh). Elution with 8% ethyl acetate in hexane
furnished the greenish yellow coloured solid (0.080 g, 31%).
(CDCl3, 300 MHz): 1.04 (s, 6H), 1.45-1.48 (m, 2H), 1.60-1.64 (m, 2H), 1.75 (s, 3H),
2.04 (t, J = 6 Hz, 2H), 2.94 (dd, J = 17, 8 Hz, 1H), 3.69 (dd, J = 17, 12 Hz, 1H), 5.15 (dd,
79
Chapter 2.2

J = 12, 8 Hz, 1H), 6.24 (d, J = 16 Hz, 1H), 6.50 (d, J = 16 Hz, 1H), 6.81-6.93 (m, 4H),
7.45 (d, J = 8 Hz, 2H), 7.66 (d, J = 8 Hz, 2H), 8.09 (s, 1H), 8.52 (s, 1H);
13
C NMR
(CDCl3, 75 MHz): 19.07, 21.74, 228.90, 33.29, 34.13, 39.72, 42.62, 64.19, 114.47,
114.57, 115.34, 115.64, 119.97, 2120.91, 125.20, 2127.48, 130.02, 131.96, 133.02,
136.37, 136.78, 141.24, 142.68, 149.38, 152.63; ESI-MS m/z: 456 [M+1]+; Analysis
calculated for C28H30FN5: C, 73.82; H, 6.64; N, 15.37; Found: C, 73.88; H, 6.67; N,
15.30.
2.2.7.4 (1E,4E)-1-(4-(1H-1,2,4-triazol-1-yl)phenyl)-5-(2,6,6-trimethylcyclohex-2enyl)penta-1,4-dien-3-one (5).
A mixture of ionone (1.152 g, 1.23 ml, 6 mmol), 4-(1H-1,2,4-triazol-1-yl)
benzaldehydes (0.865 g, 5 mmol), cetyltrimethyl ammonium bromide (0.070 g, 0.5
mmol), sodium hydroxide (0.600 g, 15 mmol) and water (25 ml) was stirred at room
temperature for 24 h. Reaction progress was monitored by TLC and compound was
extracted with ethyl acetate (2 50 ml). The combined organic extract was washed with
water (2 50 ml), brine solution (50 ml), dried (Na2SO4) and the solvent was removed in
vacuum. The crude product was purified by column chromatography (SiO2, 60-120
mesh). Elution with 10 % ethyl acetate in hexane furnished a pale yellow coloured solid
(0.400g, 23%).
Mp: 108-110OC; IR (KBr, cm-1): 2915, 1651 (C=O), 1598 (C=N); 1H NMR
(CDCl3, 300 MHz): 0.88 (s, 3H), 0.95 (s, 3H), 1.22-1.29 (m, 1H), 1.43-1.52 (m, 1H),
1.60 (s, 3H), 2.07 (brs, 2H), 2.36 (d, J = 9 Hz, 1H), 5.53 (brs, 1H), 6.39 (d, J = 16 Hz,
1H), 6.85 (dd, J = 16, 9 Hz, 1H), 7.01 (d, J = 16 Hz, 1H), 7.63-7.74 (m, 5H), 8.11 (s, 1H),
8.60 (s, 1H);
13
C NMR (CDCl3, 75 MHz): 22.92, 23.10, 26.90, 27.95, 31.25, 32.75,
54.67, 2120.13, 122.83, 125.72, 2129.72, 130.54, 131.93, 134.81, 137.99, 140.89,
141.16, 149.44, 152.81, 188.53; ESI-MS m/z: 348 [M+1]+; Analysis calculated for
C22H25N3O: C, 76.05; H, 7.25; N, 12.09; Found: C, 76.05; H, 7.29; N, 12.03.
80
Chapter 2.2

2.2.7.5 (E)-1-(4-(1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)-4,5-dihydro1H-pyrazol-5-yl)phenyl)-1H-1,2,4-triazole (6a)

To a solution of 5 (0.150 g, 0.431 mmol) in ethanol (10 ml), phenyl hydrazine
(0.046 g, 0.042 ml, 0.431 mmol) was added. The reaction mixture was refluxed for 8 h.
Reaction progress was monitored by TLC. After completion of reaction ethanol was
distilled off. Compound was extracted with ethyl acetate (225 ml). The combined
organic extract was washed with water (225 ml), brine solution (25 ml), dried (Na2SO4)
and solvent was removed in vacuum. The crude product was purified by column
chromatography (SiO2, 60-120 mesh). Elution with 8% ethyl acetate in hexane furnished
the cream coloured solid (0.086 g, 46%).
1.59 (s, 3H), 2.00 (brs, 2H), 2.27 (d, J = 9 Hz, 1H), 2.88 (dd, J = 17, 8 Hz, 1H), 3.64 (dd,
J = 17, 12 Hz, 1H), 5.20 (dd, J = 12, 8 Hz, 1H), 5.42 (brs, 1H), 5.62 (dd, J = 16, 9 Hz,
1H), 6.49 (d, J = 16 Hz, 1H), 6.77 (t, J = 7 Hz, 1H), 6.94 (d, J = 8 Hz, 2H), 7.15 (t, J = 8
Hz, 2H), 7.43 (d, J = 8 Hz, 2H), 7.63 (d, J = 8 Hz, 2H), 8.08 (s, 1H), 8.51 (s, 1H);
13
NMR (CDCl3, 75 MHz): 223.00, 26.81, 27.88, 31.31, 32.54, 42.70, 54.76, 63.48,
2113.34, 119.33, 2120.88, 121.62, 124.57, 2127.37, 2128.97, 129.09, 129.99,
133.32, 137.91, 142.91, 144.56, 148.68, 152.61; ESI-MS m/z: 438 [M+1]+; Analysis
calculated for C28H31N5: C, 76.85; H, 7.14; N, 16.00; Found: C, 76.89; H, 7.15; N, 15.96.
2.2.7.6 (E)-1-(4-(1-(4-fluorophenyl)-3-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)-4,5dihydro-1H-pyrazol-5-yl)phenyl)-1H-1,2,4-triazole (6b)
To a solution of 5 (0.150 g, 0.431 mmol) in ethanol (20 ml), 4-fluorophenyl
hydrazine hydrochloride (0.070 g, 0.431 mmol) was added. The reaction mixture was
refluxed for 8 h. Reaction progress was monitored by TLC. After completion of reaction
ethanol was distilled off. Compound was extracted with ethyl acetate (225 ml). The
combined organic extract was washed with water (225 ml), brine solution (25 ml), dried
(Na2SO4) and solvent was removed in vacuum. The crude product was purified by
column chromatography (SiO2, 60-120 mesh). Elution with 10% ethyl acetate in hexane
furnished the pale yellow coloured solid (0.090 g, 46%).
81
Chapter 2.2

1.59 (s, 3H), 2.00 (brs, 2H), 2.27 (d, J = 9 Hz, 1H), 2.87 (dd, J = 17, 8 Hz, 1H), 3.63 (dd,
J = 17, 12 Hz, 1H), 5.12 (dd, J = 12, 8 Hz, 1H), 5.43 (brs, 1H), 5.62 (dd, J = 16, 9 Hz,
1H), 6.46 (d, J = 16 Hz, 1H), 6.85-6.87 (m, 4H), 7.43 (d, J = 8 Hz, 2H), 7.65 (d, J = 8 Hz,
2H), 8.09 (s, 1H), 8.52 (s, 1H);
13
C NMR (CDCl3, 75 MHz): 222.96, 26.81, 27.82,
31.32, 32.54, 42.94, 54.76, 64.22, 114.49, 114.56, 115.31, 115.61, 119.96, 2120.89,
121.66, 124.46, 2127.45, 129.98, 133.27, 134.88, 138.05, 141.34, 142.64, 148.87,
152.60; ESI-MS m/z: 456 [M+1]+; Analysis calculated for C28H30FN5: C, 73.82; H, 6.64;
N, 15.37; Found: C, 73.88; H, 6.67; N, 15.30.
82
Chapter 2.2

2.2.8 SPECTRA OF SOME SELECTED COMPOUNDS
Figure 2.2.3: 1H NMR of compound 2 at 300 MHz (CDCl3)
Figure 2.2.4: 13C NMR of compound 2 at 75 MHz (CDCl3)
83
Chapter 2.2

Figure 2.2.5: 1H NMR of compound 3a at 300 MHz (CDCl3)
Figure 2.2.6: 13C NMR of compound 3a at 75 MHz (CDCl3)
84
Chapter 2.2

2.2.9 REFERENCES
1
Alvar, G.; Croft, S.; Olliaro, P. Adv. Parasitol. 2006, 61, 223.
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Chem. 1998, 41, 4819.

4
Johnson, M.; Younglove, B.; Lee, L.; LeBlanc, R.; Holt, H.; Hills, P.; Mackay, H.;
Brown, T.; Mooberry, S. L.; Lee, M. Bioorg. Med. Chem. Lett. 2007, 17, 5897.
5
Reddy, M. V. R.; Billa, V. K.; Pallela, V. R.; Mallireddigari, M. R.; Boominathan, R.;
Gabriel, J. L.; Reddy, E. P. Bioorg. Med. Chem. 2008, 16, 3907.

6
Sivakumar, P. M.; Seenivasan, S. P.; Kumar, V.; Doble, M. Bioorg. Med. Chem. Lett.
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7
Ozdemir, A.; Zitouni, G. T.; Kaplanckl, Z. A.; Revial, G.; Guven, K. Eur. J. Med.
Chem. 2007, 42, 403.

8
Dardari, Z.; Lemrani, M.; Sebban, A.; Bahloul, A.; Hassar, M.; Kitane, S.; Berrada, M.;
Boudouma, M. Arch Pharm (Weinheim) 2006, 339, 291.

9
Berman, J. D. Am. J. Trop. Med. Hyg. 1981, 30, 566.
10
Beach, D. H.; Goad, L. J.; Holz, G. G. Jr. Mol Biochem Parasitol 1988, 31, 149.
11
Marrapu, V. K.; Mittal, M.; Shivahare, R.; Gupta, S.; Bhandari, K. Eur. J. Med. Chem.
2011, 46, 1694.

12
Al-Abdely, H. M.; Graybill, J. R.; Loebenberg, D.; Melby, P. C. Antimicrob. Agents
Chemother. 1999, 43, 2910.

13
(a) Pandey, S.; Suryawanshi, S. N.; Gupta, S.; Srivastava, V. M. L.; Eur. J. Med.
Chem. 2004, 39, 969; (b) Suryawanshi, S. N.; Tiwari, A.; Chandra, N.; Ramesh; Gupta,
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85
Chapter 3
Synthesis and Bioevaluation of Novel

Isoxazole Containing Heteroretinoid and
its Amide Derivatives
Chapter 3
synthesis and bioevaluation of novel isoxazole containing

heteroretinoid and its amide derivatives
3.1 INTRODUCTION
Leishmaniasis is a family of parasitic diseases that affect about 12 million people in
tropical and subtropical areas in the form of three clinical expressions: visceral
leishmaniasis, which is fatal in the absence of treatment; muco-cutaneous leishmaniasis
and cutaneous leishmaniasis, which is often self curing. Classical drugs such as
antimonials (Pentostam and Glucantime) are toxic and drug resistance is increasing
dangerously in the field.1 A liposomal amphotericin B formulation (AmBisome) less
toxic than amphotericin B deoxycholate is gradually becoming the first-line therapy,
especially in immunocompromised patients, but this drug must be administered by a
parenteral route.2 Miltefosine (Impavido) was the first drug registered against visceral
leishmaniasis in the last decade; however, it is contraindicated in women of childbearing
age and shows severe gastrointestinal side effects.3
Improved treatment protocols, such as combination therapy, are under investigation
in an effort to optimize efficacy, reduce costs and prevent parasite resistance, but new
drugs are urgently needed to expand the treatment options available for these diseases.
Modern approaches are being employed that integrate genomic, proteomic and cellular
analyses for developing novel and effective anti-leishmanial drugs. Rational drug design
directed against parasite enzymes, such as dihydrofolate reductase, pteridine reductase or
malate dehydrogenase, essential for proliferation or survival, has identified specific
enzyme inhibitors, including trisubstituted pyrimidines, triazines and paullones.4
Alternatively, parallels between parasites and cancer cells, including unlimited
proliferation in the host, independence of exogenous growth factors and resistance to
apoptosis, may provide new insights into drug development,5 suggesting that anti-cancer
drugs and compounds originally developed for oncological indications should be
screened as potential leishmanicidal agents.5,6 Oral miltefosine, originally developed as
an anticancer drug, has been used for treatment of visceral leishmaniasis in India.7 While
such an approach led to the discovery of miltefosine, most anti-cancer drugs studied to
date show only moderate anti-parasitic activity and have low selectivity indices, a major
parameter in drug-toxicity evaluation.
86
Chapter 3

3.2 BASIS OF WORK

Currently, efforts are being made to search for new molecules from the natural
sources and in this endeavor diarylheptanoids,8 oxygenated abietanes,9 diterpene
quinones10,11 are showing promise as new lead molecules. Retinoids are natural and
synthetic analogues of retinoic acid, an active metabolite of vitamin A, and are specific
modulators of cell proliferation, differentiation, and morphogenesis in vertebrates.
Heteroretinoids are synthetic retinoids derived from trans-retinoic acid and include a
heteroatom in a five- or six-membered cyclic ring. In recent years retinoids,12 retinoic
acid analogs,13 heteroretinoids14 are under investigation as antiproliferative agents.
These small molecules exert most of their effects by binding to specific nuclear
retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each of which is
encoded by three separate genes designated , , and . These receptors form RXR-RAR
heterodimers that regulate transcription by binding to RA response elements (RAREs) in
the promoters of retinoid-responsive genes. All-trans-retinoic acid (ATRA) binds and
activates the RARs, whereas 9-cis-retinoic acid (9-cis-RA) binds and activates both
RARs and RXRs. Owing to their ability to regulate aberrant cell growth, retinoids are
currently being evaluated as preventive or therapeutic agents in a variety of human
premalignancies and cancer.15 Encouraging preliminary clinical results have also
demonstrated the importance of retinoids in combination chemotherapy of cancer.16
Indeed, retinoids may increase the activity of other biologic or chemotherapeutic agents,
thus offering new opportunities for the development of effective combination regimens.
Based on above facts the heteroretinoid and its amide derivatives were synthesized
and evaluated for their antileishmanial activity against L. donovani in hamsters and the
results are part of this chapter.
3.3 DESIGN AND SYNTHESIS OF HETERORETINOID

The structure of retinoid can be divided into three parts, i.e., a hydrophobic part, a
linking part, and a carboxylate part. Incorporation of the structural features of naturally
occurring ATRA afforded our designed heteroretinoid (4) shown in Figure 3.1.
87
Chapter 3

CO2H
Linking part
Hydrophobic part
ATRA
Carboxylate part
Carboxylate part
Introduction of ionone as
hydrophobic part
O
OH
O
Introduction of isoxazole
ring as linker
Heteroretinoid (4)
O
R
O
Amide derrivatives of 4
(5a-j)
Figure 3.1: Design and Synthesis of heteroretinoid (4).
88
Chapter 3

3.4 CHEMISTRY
The synthetic procedures adopted to obtain the target compounds are depicted in
scheme-3.1. The reaction of ionone with sodium hydride and diethyl oxalate in toluene
was carried out at reflux temperature to furnish 2 in quantitative yield (60%). The
compound 2 on treatment with hydroxylamine hydrochloride in ethanol under refluxing
conditions afforded compound 3 in 68% yield. During the cyclization of compound 2,
attack of nucleophillic nitrogen atom of NH2OH takes place on that carbonyl carbon
which is directly attached to an electron withdrawing ethyl carboxylate group to yield the
cylclized product 3. (Path II, Figure 3.2)
Figure 3.2: Possible ways to cyclization of compound 2
Compound 3 was subjected to base catalyzed hydrolysis to give compound 4 in

90% yield. Compound 4 was reacted with oxalyl chloride to furnish acid chloride in situ.
Acid chloride was next coupled with a set of different aliphatic and aromatic amines to
obtain compounds 5a-h. These aliphatic and aromatic amines were selected on the basis
of their easy availability, low cost and their presence as substituent in various other
antileishmanial agents.17 Compounds (5a-h) were prepared in good to excellent yield
(68%-96%) as shown in Table-3.1. Compounds 5i and 5j were synthesized directly from
compound 3 using hydrazine hydrate and hydroxylamine hydrochloride respectively. We
also synthesized the sodium salts of compounds 5i and 5j using an aqueous solution of
89
Chapter 3

NaOH to make them water soluble and to check the effect of this increased water
solubility on antileishmanial activity of these compounds. All the synthetic products were
characterized by the IR, 1H NMR, 13C NMR and mass spectral data.
Table-3.1: Reaction conditions and percentage yield of different amide derivatives

Substrate
Amine
Compound,%
Oxalyl chloride/DMF/DCM, rt, 2 h
5a, 78
Cl
5b, 90
OCH 3
5c, 83
5d, 70
5e, 65
5f, 96
5g, 90
5h, 93
H2N
H 2N
H 2N
H 2N
HN
HN
Reaction conditions
HN
HN
NH
NH2NH2
Ethanol, reflux, 3 h
5i, 78
NH2OH
THF, rt, 17 h
5j, 84
90
Chapter 3

Scheme-3.1: Synthesis of isoxazole containing heteroretinoid and its amide derivatives
Diethyl oxalate
OEt
NaH, toluene
O
2
NH2OH.HCl, ethanol
O
NaOH, ethanol
OH
O
OEt
N
O
4
3
Oxalyl chloride, DMF
D.C.M, amine
O
R
O
(5a) R =
H
N
(5b) R =
H
N
Cl
(5c) R =
H
N
OCH 3
(5d) R =
H
N
(5g) R =
NH
(5h) R =
(5i) R = NHNH 2
(5j) R = NHOH
(5k)R = N(Na)NH2
(5e) R =
(5f ) R =
(5l) R = Na(Na)OH
91
Chapter 3
3.5

BIOLOGICAL EVALUATION- MATERIAL METHODS

3.6 RESULT AND DISCUSSION

Isoxazole containing heteroretinoid (4) and its amide derivatives (5a-j) were
synthesized (scheme-3.1). All the synthesized compounds were screened for their
leishmanicidal activity against L. donovani in hamsters at 50 mg kg1 5 days dose and
results have been presented in Table-3.2. Among all fifteen tested compounds, five
compounds (3, 5a, 5d, 5k and 5l) have shown 70-76% inhibition of parasite growth. The
efficacy of these compounds was more or less similar to sodium stibogluconate (89%
inhibition of parasite growth) and superior to paromomycin (46% parasite inhibition).
Four compounds (4, 5b, 5c and 5j) exhibited 45-65% inhibition of parasite
multiplication. Compounds 5e and 5h were found to be inactive. Interestingly, primary
amine based amides (5a-d, 5i and 5j) have shown better activity as compared to amides
(5e and 5h) synthesized using secondary amines. The presence of N-H bond in secondary
amides seems to play an important role in the mode of action of the agent, since going
from secondary amide (5a-d, 5i and 5j) to tertiary amide (5e and 5h) leads to a complete
disappearance of antileishmanial effect. It is likely that N-H bond present in secondary
amide may form hydrogen bond with the macromolecular target in the parasite. Sodium
salt formation of compounds 5i and 5j increased the parasite inhibitory activity of these
compounds because of increased water solubility (5k and 5l, Table-3.2).
The synthesized compounds were also checked for compliance to the Lipinski
rule of five, and the results are summarized in Table-3.3. The rule states that a molecule
likely to be developed as an orally active drug candidate should show no more than one
violation of the following four criteria: log P (octanolwater partition coefficient) 5,
molecular weight 500, number of hydrogen bond acceptors 10 and number of
hydrogen bond donors 5. Molecular properties of synthesized compounds were
calculated by www.molinspiration.com software, and it was found that majority of the
synthesized compounds followed the above criteria (Table-3.3). Therefore, these
compounds have a good potential for eventual development as oral agents and can be
potentially active drug candidate.
92
Chapter 3

Table-3.2: Antileishmanial activity of synthetic compounds against Leishmania

donovani in hamsters.
Compound No.
Dose (mg/kg) 5
days
% inhibition on Day7 P.T.a
50
Inactive
50
76
50
45
5a
50
74
5b
50
63
5c
50
58
5d
50
70
5e
50
Inactive
5f
50
NDb
5g
50
ND
5h
50
Inactive
5i
50
50
5j
50
65
5k
50
70
5l
50
71
SSGc
50
89.08.31
50
46.79.82
Paromomycin
a
P.T: post treatment, ND: not done, SSG: reference drug (Sodium stibogluconate)
93
Chapter 3

Table-3.3: Molinspiration Calculation of molecular Properties for the Lipinski Rule
Compound
no.
Acceptable
range
nViol
MW
miLog P
nON
<500
<10
<5
292.375
2.431
21
289.375
4.89
21
261.321
4.254
19
5a
336.435
5.573
25
5b
370.88
6.251
26
5c
366.461
5.629
27
5d
342.483
5.781
25
5e
330.428
3.966
24
5f
328.456
5.028
24
5g
329.444
3.416
24
5h
405.542
5.709
30
5i
275.352
3.065
20
5j
276.336
3.558
20
nOHNH natoms
nrotb
Viol, no. of violations; MW, molecular weight; miLog P, molinspiration predicted Log P; nON, no. of
hydrogen bond acceptors; nOHNH, no. of hydrogen bond donors; natoms, no. of atoms; nrotb, no. of
rotatable bond.
94
Chapter 3

3.7 CONCLUSION
Novel isoxazole containing heteroretinoid (4) and its amide derivatives (5a-j) have
been synthesized and evaluated for their in vivo antileishmanial activity against
Leishmania donovani in hamsters. Compounds 3, 5a, 5d, 5k and 5l inhibited 70-76%
parasite growth at 50 mg kg-15 days. In conclusion, we have identified a promising new

hit for the treatment of leishmaniasis. The potent activity and simple synthesis of these
heteroretinoids suggest that they can be a possible lead for the development of novel drug
against Leishmania.
95
Chapter 3

3.8 EXPERIMENTAL SECTION

The reported melting points (C) are the uncorrected ones. The infrared spectra
spectrophotometers either on KBr discs or in neat. 1H NMR spectra and
13
C NMR (in
CDCl3) spectra (chemical shift in , ppm downfield from TMS) were recorded on Bruker
Advance DRX-300 and 200 MHz spectrometers. Electron impact (EI) mass spectra were
recorded on a Jeol JESMS-D-300 spectrometer with the ionization potential 70 eV.
Elemental analysis was carried out on a Carlo-Erba EA 1108 instrument.
3.8.1 Synthesis of (E)-ethyl 2,4-dioxo-6-(2,6,6-trimethylcyclohex-2-enyl)hex-5enoate (2)
NaH (1.25 g, 26 mmol) was stirred in dry hexane (25 ml) for 10 minutes and
hexane was pipetted out. Diethyl oxalate (7.59 ml, 52 mmol), ionone (5 ml, 26 mmol)
and toluene (45 ml) were added. Reaction mixture was refluxed for 2 h. After cooling the
reaction mixture, aqueous solution of HCl (13 ml HCl + 30 ml water) and ethyl acetate
(80 ml) were added and stirred for h. Organic layer was extracted and concentrated.
Crude was purified by column chromatography.
Yield: 60%; Oily; IR (Neat, cm-1): 2960, 2925, 2865, 1735, 1610, 1446, 1367,
1263, 1115; 1H NMR (CDCl3, 300 MHz): 0.86 (s, 3H), 0.93 (s, 3H), 1.20-1.34 (m, 1H),
1.38 (t, J = 7 Hz, 3H), 1.42-1.52 (m, 1H), 1.56 (s, 3H), 2.05 (brs, 2H), 2.33 (d, J = 9 Hz,
1H), 4.35 (q, J = 7 Hz, 2H), 5.52 (brs, 1H), 6.02 (d, J = 16 Hz, 1H), 6.41 (s, 1H), 6.87
(dd, J = 16, 9 Hz, 1H); 13C NMR (CDCl3, 75 MHz): 14.02, 22.80, 22.99, 26.76, 27.84,
31.07, 32.84, 54.65, 62.41, 99.82, 122.96, 127.83, 131.59, 149.72, 162.10, 173.05,
186.11; ESMS m/z: 293 [M+1]+; Analysis calculated for C17H24O4: C, 69.84; H, 8.27;
Found: C, 69.89; H, 8.24.
3.8.2 Synthesis of (E)-ethyl 5-(2,6,6-trimethylcyclohex-2-en-1yl)vinyl)isoxazole-3carboxylate (3)
To a solution of 2 (1.92 g, 5 mmol) in ethanol (10 ml), hydroxylamine
hydrochloride was added and reaction mixture was refluxed at 80 C for 2 h. Reaction
was monitored through TLC checking. After completion of reaction ethanol was removed
96
Chapter 3

and residue was taken up in ethyl acetate (20 ml). The organic extract was washed with
water (220 ml), brine solution (220 ml), dried (Na2SO4) and solvent was removed
under vacuum. The crude product thus obtained was chromatographed (SiO2, 60-120
mesh).
Yield: 68%; M.P: 55-57 C; IR (KBr, cm-1): 2957, 1740, 1642, 1573, 1448, 1240,
1023; 1H NMR (CDCl3, 300 MHz): 0.87 (s, 3H), 0.93 (s, 3H), 1.20-1.27 (m, 1H), 1.381.52 (m, 4H), 1.59 (s, 3H), 2.04 (brs, 2H), 2.30 (d, J = 9 Hz, 1H), 4.42 (q, J = 7 Hz, 2H),
5.50 (brs, 1H), 6.30 (d, J = 16 Hz, 1H), 6.40-6.50 (m, 2H); 13C NMR (CDCl3, 75 MHz):
14.11, 22.84, 22.97, 26.78, 27.73, 31.14, 32.65, 54.75, 62.01, 100.31, 116.10, 122.49,
132.18, 140.93, 156.49, 160.03, 170.27; ESMS m/z: 290 [M+1]+; Analysis calculated for
3.8.3 Synthesis of (E)-5-(2-(2,6,6-trimethylcyclohex-2-en-1-yl)vinyl)isoxazole-3carboxylic acid (4)
To a solution of ester 3 (0.578 g, 2 mmol) in ethanol (25 ml), NaOH (0.20 g) was
added and the solution was stirred under reflux for 2 h. After being cooled to room
temperature, the solvent was acidified with 1N HCl. The crude product was extracted
with ethyl acetate and washed with water (215 ml), brine (215 ml), dried (Na2SO4) and
solvent was removed under vacuum. The crude product thus obtained on crystallization
gave 4 as a white crystalline solid.
Yield: 90%; M.P: 99-100 0C; IR (KBr, cm-1): 3409, 2959, 1720, 1645, 1448,
1230; 1H NMR (CDCl3, 300 MHz): 0.87 (s, 3H), 0.93 (s, 3H), 1.17-1.25 (m, 1H), 1.411.51 (m, 1H), 1.59 (s, 3H), 2.04 (brs, 2H), 2.30 (d, J = 9 Hz, 1H), 5.50 (s, 1H), 6.30 (d, J
= 16 Hz, 1H), 6.47 (dd, J = 16, 9 Hz, 1H), 6.54 (s, 1H), 14.19 (brs, 1H);
13
C NMR
(CDCl3, 75 MHz): 22.85, 22.99, 26.80, 27.74, 31.17, 32.70, 54.81, 100.52, 115.98,
122.60, 132.12, 141.46, 156.17, 163.52, 170.77; ESMS m/z: 262 [M+1]+; Analysis
calculated for C15H19NO3: C, 68.94; H, 7.33; N, 5.36; Found: C, 68.89; H, 7.41; N, 5.30.
3.8.4 General procedure for the synthesis of title compounds 5a-h
To a solution of 4 (0.40 g, 1.50 mmol) in CH2Cl2 (10 ml) was added oxalyl
chloride (0.40 ml, 3.14 mmol) drop wise. After 5 minutes 2-3 drops of DMF were added
97
Chapter 3

and the resulting mixture was stirred at room temperature for 2 h. Excess of oxalyl
chloride was removed under vacuum. To the crude obtained was added amines (3 mmol)
in CH2Cl2 (10 ml) and the solution was stirred at room temperature for 2 h. After the
reaction was completed, solvent was removed under vacuum and the residue was taken in
CH2Cl2 (20 ml) followed by washing with H2O (215 ml), brine (215 ml), dried
(Na2SO4) and it was concentrated under vacuum. The crude product thus obtained was
column chromatographed (SiO2, 60-120 mesh).
3.8.5 (E)-N-phenyl-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole-3carboxamide (5a)
Yield: 78%; M.P: 55-56 0C; IR (KBr, cm-1): 3315, 3030, 2922, 2863, 1687, 1601,
1542, 1446, 1314; 1H NMR (CDCl3, 200 MHz): 0.89 (s, 3H), 0.95 (s, 3H), 1.20-1.30
(m, 1H), 1.40-1.50 (m, 1H), 1.62 (s, 3H), 2.06 (brs, 2H), 2.32 (d, J = 9 Hz, 1H), 5.53 (brs,
1H), 6.33 (d, J = 16 Hz, 1H), 6.49 (dd, J = 16, 9 Hz, 1H), 6.63 (s, 1H), 7.16 (t, J = 8 Hz,
1H), 7.37 (t, J = 8 Hz, 2H), 7.66 (d, J = 8 Hz, 2H), 8.52 (brs, 1H); 13C NMR (CDCl3, 50
MHz): 23.32, 23.44, 27.27, 28.14, 31.66, 33.11, 55.21, 100.15, 116.66, 2120.56,
122.98, 125.27, 2129.47, 132.62, 137.55, 141.44, 157.24, 159.51, 170.46; ESMS m/z:
337 [M+1]+; Analysis calculated for C21H24N2O2: C, 74.97; H, 7.19; N, 8.33; Found: C,
74.91; H, 7.21; N, 8.35.
3.8.6 (E)-N-(4-chlorophenyl)-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole3-carboxamide (5b)
1596, 1533, 1450, 1393, 1352; 1H NMR (CDCl3, 200 MHz): 0.89 (s, 3H), 0.95 (s, 3H),
1.09-1.30 (m, 1H), 1.41-1.51 (m, 1H), 1.65 (s, 3H), 2.05 (brs, 2H), 2.32 (d, J = 9 Hz, 1H),
5.52 (brs, 1H), 6.32 (d, J = 16 Hz, 1H), 6.49 (dd, J = 16, 9 Hz, 1H), 6.62 (s, 1H), 7.33 (d,
J = 9 Hz, 2H), 7.62 (d, J = 9 Hz, 2H), 8.52 (brs, 1H);
13
C NMR (CDCl3, 50 MHz):
23.28, 23.42, 27.24, 28.16, 31.62, 33.14, 55.25, 99.96, 116.53, 2121.63, 123.04,
2129.58, 130.37, 132.58, 136.02, 141.74, 157.16, 159.20, 171.20; ESMS m/z: 371
[M+1]+; Analysis calculated for C21H23ClN2O2: C, 68.01; H, 6.25; N, 7.55; Found: C,
67.95; H, 6.31; N, 7.60.
98
Chapter 3

3.8.7 (E)-N-(4-methoxyphenyl)-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)
isoxazole-3-carboxamide (5c)
1535, 1450, 1403, 1247; 1H NMR (CDCl3, 300 MHz): 0.89 (s, 3H), 0.95 (s, 3H), 1.201.28 (m, 1H), 1.42-1.54 (m, 1H), 1.62 (s, 3H), 2.06 (brs, 2H), 2.32 (d, J = 9 Hz, 1H), 3.81
(s, 3H), 5.52 (s, 1H), 6.33 (d, J = 16 Hz, 1H), 6.47 (dd, J = 16, 9 Hz, 1H), 6.62 (s, 1H),
6.90 (d, J = 9 Hz, 2H), 7.56 (d, J = 9 Hz, 2H), 8.44 (s, 1H); 13C NMR (CDCl3, 75 MHz):
22.91, 23.02, 26.84, 27.77, 31.20, 32.71, 54.79, 55.47, 99.68, 2114.27, 116.21,
2121.76, 122.57, 130.11, 132.22, 141.05, 156.59, 156.81, 159.09, 170.53; ESMS m/z:
367 [M+1]+; Analysis calculated for C22H26N2O3: C, 72.11; H, 7.15; N, 7.64; Found: C,
72.17; H, 7.19; N, 7.57.
3.8.8 (E)-N-cyclohexyl-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole-3carboxamide (5d)
Yield: 70%: M.P: 128-130 0C; IR (KBr, cm-1): 3330, 2930, 2858, 1645, 1554,
1443, 1351, 1231; 1H NMR (CDCl3, 200 MHz): 0.87 (s, 3H), 0.94 (s, 3H), 1.20-1.50
(m, 10H), 1.60 (s, 3H), 1.71-1.78 (m, 2H), 2.03 (brs, 2H), 2.30 (d, J = 9 Hz, 1H), 3.903.97 (m, 1H), 5.50 (brs, 1H), 6.29 (d, J = 16 Hz, 1H), 6.43 (dd, J = 16, 9 Hz, 1H), 6.54 (s,
1H), 6.66 (d, J = 7 Hz, 1H); 13C NMR (CDCl3, 50 MHz): 23.28, 23.41, 225.19, 25.85,
27.25, 28.10, 31.64, 233.08, 33.28, 48.81, 55.18, 100.03, 116.73, 122.88, 132.70,
141.04, 158.43, 159.46, 170.51; ESMS m/z: 343 [M+1]+; Analysis calculated for
3.8.9 (E)-morpholino(5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazol-3yl)methanone (5e)
1.41-1.54 (m, 1H), 1.60 (s, 3H), 2.04 (brs, 2H), 2.31 (d, J = 9 Hz, 1H), 3.68-3.75 (m, 4H),
3.84-3.90 (m, 4H), 5.51 (brs, 1H), 6.25-6.50 (m, 3H);
13
C NMR (CDCl3, 50 MHz):
23.23, 23.39, 27.24, 28.04, 31.65, 33.07, 43.36, 47.91, 53.81, 55.18, 62.63, 101.54,
116.48, 122.89, 132.65, 141.24, 158.87, 160.09, 169.49; ESMS m/z: 331 [M+1]+;
99
Chapter 3


7.99; N, 8.39.
3.8.10 (E)-piperidin-1-yl(5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazol-3-yl)
methanone (5f)
979; 1H NMR (CDCl3, 200 MHz): 0.88 (s, 3H), 0.94 (s, 3H), 1.18-1.27 (m, 1H), 1.431.54 (m, 1H), 1.60 (s, 3H), 1.70 (brs, 6H), 2.06 (brs, 2H), 2.30 (d, J = 9 Hz, 1H), 3.70 (m,
4H), 5.50 (brs, 1H), 6.20-6.50 (m, 3H); ESMS m/z: 329 [M+1]+; Analysis calculated for
3.8.11 (E)-piperazin-1-yl(5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazol-3yl)methanone (5g)
1.39-1.51 (m, 1H), 1.70 (s, 3H), 2.04 (brs, 2H), 2.31 (d, J = 9 Hz, 1H), 3.88 (m, 4H), 3.99
(m, 4H), 5.5 (brs, 1H), 6.27-6.45 (m, 3H);
13
C NMR (CDCl3, 50 MHz): 23.28, 23.42,
27.27, 28.10, 31.66, 33.11, 42.73, 43.45, 47.05, 47.78, 55.22, 101.62, 116.45, 122.96,
3.8.12 (E)-(4-phenylpiperazin-1-yl)(5-(2-(2,6,6-trimethylcyclohex-2enyl)vinyl)isoxazol-3-yl)methanone (5h)
1.42-1.50 (m, 1H), 1.61 (s, 3H), 2.06 (brs, 2H), 2.31 (d, J = 9 Hz, 1H), 3.2-3.30 (m, 4H),
3.90-4.07 (m, 4H), 5.51 (brs, 1H), 6.27-6.50 (m, 4H), 6.93 (d, J = 8 Hz, 2H), 7.24-7.32
(m, 2H); 13C NMR (CDCl3, 50 MHz): 23.28, 23.43, 27.27, 28.09, 31.67, 33.11, 42.92,
47.29, 49.84, 50.47, 55.21, 101.58, 116.54, 2117.15, 121.05, 122.91, 2129.66, 132.69,
100
Chapter 3

3.8.13 Synthesis of (E)-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole-3carbohydrazide (5i)

To a solution of 3 (0.50 g, 1.70 mmol) in ethanol, hydrazine hydrate (0.34, 6.8
mmol) was added and the reaction mixture was refluxed for 3 h. After completion of
reaction, ethanol was removed and residue was taken up in ethyl acetate (215 ml). The
organic extract was washed with water (215 ml), brine (215 ml), dried (Na2SO4). The
solvent was removed under vacuum. The crude product thus obtained was
chromatographed (SiO2, 60-120 mesh).
1370; 1H NMR (CDCl3, 200 MHz): 0.88 (s, 3H), 0.94 (s, 3H), 1.20-1.30 (m, 1H), 1.391.54 (m, 1H), 1.60 (s, 3H), 2.03 (brs, 2H), 2.31 (d, J = 9 Hz, 1H), 4.00 (brs, 2H), 5.51
(brs, 1H), 6.30 (d, J =16 Hz, 1H), 6.43 (dd, J = 16, 9 Hz, 1H), 6.54 (s, 1H), 8.05 (brs,
1H);
13
C NMR (CDCl3, 50 MHz): 23.23, 23.39, 27.22, 28.07, 31.63, 33.09, 55.19,
99.88, 116.51, 122.95, 132.92, 141.12, 158.53, 159.78, 170.62; ESMS m/z: 276 [M+1]+;
7.72; N, 15.30.
3.8.14 Synthesis of (E)-N-hydroxy-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)
isoxazole-3-carboxamide (5j)
To the suspension of hydroxylamine hydrochloride (4.65 g, 67 mmol) in methanol
(24 ml), solution of KOH (3.6 g, 0.064 mmol) in methanol (24 ml) was added. After
stirring for 15 min. at room temperature, it was filtered and filtrate was added to a cooled
solution of 3 in THF (10 ml) and stirred further at room temperature for 17 h. After
completion of reaction, the reaction mixture was adjusted to acidic by CH3COOH and the
crude product was extracted with ethyl acetate (215 ml) and washed with H2O (215
ml), brine (215 ml), dried (Na2SO4). The solvent was concentrated to dryness to give
desired product.
1488, 1444, 1394, 1228, 1115, 1035, 986; 1H NMR (CDCl3, 200 MHz): 0.88 (s, 3H),
0.94 (s, 3H), 1.24-1.33 (m, 1H), 1.37-1.50 (m, 1H), 1.60 (s, 3H), 2.04 (brs, 2H), 2.31 (d, J
101
Chapter 3

= 9 Hz, 1H), 5.51 (brs, 1H), 6.32-6.52 (m, 3H);
13
C NMR (CDCl3, 50 MHz): 23.24,
23.39, 27.22, 28.08, 31.62, 33.09, 55.21, 100.05, 116.41, 122.97, 132.57, 141.75, 157.03,
157.87, 170.62; ESMS m/z: 277 [M+1]+; Analysis calculated for C15H20N2O3: C, 65.20;
H, 7.30; N, 10.14; Found: C, 65.38; H, 7.27; N, 10.06.
3.8.15 Synthesis of Sodium (E)-1-(5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)
isoxazole-3-carbonyl)hydrazine-1-ide (5k)
To a solution of 5i (0.50 g, 1.81 mmol) in 5 ml of water, NaOH (0.10 g, 2.5 mmol)
was added. The solution was concentrated to dryness to give sodium salt of 5i. It was
soluble in water. Yield: 96%.
3.8.16 Synthesis of Sodium (E)-hydroxy(5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)
isoxazole-3-carbonyl)amide (5l)
To a solution 5j (0.50 g, 1.81 mmol) in 5 ml of water, NaOH (0.10 g, 2.5 mmol)
was added. The solution was concentrated to dryness to give sodium salt of 5j. It was
soluble in water. Yield: 91%.
102
Chapter 3

3.9 SPECTRA OF SOME SELECTED COMPOUNDS
Figure 3.3: 1H NMR of compound 2 at 300 MHz (CDCl3)
Figure 3.4: 13C NMR of compound 2 at 75 MHz (CDCl3)
103
Chapter 3

104
Chapter 3

105
Chapter 3

Figure 3.9: 1H NMR of compound 5c at 300 MHz (CDCl3)
Figure 3.10: 13C NMR of compound 5c at 75 MHz (CDCl3)
106
Chapter 3

3.10 REFERENCES
1
Frezard, F.; Demicheli, C.; Ribeiro, R. R. Molecules 2009, 14, 2317.
Moore E. M.; Lockwood, D. N. J. Global Infect. Dis. 2010, 2, 151.
Maltezou, H. C. J. Biomed. Biotechnol. 2010, 2010:617521.
(a) Chowdhury, S. F.; Villamor, V. B.; Guerrero, R. H.; Leal, I.; Brun, R.; Croft, S. L.;
Goodman, J. M.; Maes, L.; Ruiz-Perez, L. M.; Pacanowska, D. G.; Gilbert, I. H. J. Med.
Chem. 1999, 42, 4300; (b) Doerig, C. Biochim. Biophys. Acta 2004, 1697, 155; (c)
Kumar, A.; Katiyar, S. B.; Gupta, S.; Chauhan, P. M. S. Eur. J. Med. Chem. 2006, 41,
106. (d) Reichwald, C.; Shimony, O.; Dunkel, U.; Sacerdoti-Sierra, N.; Jaffe, C. L.;
Kunick, C. J. Med. Chem. 2008, 51, 659. (e) Sunduru, N.; Nishi; Palne, S.; Chauhan, P.
M. S.; Gupta, S. Eur. J. Med. Chem. 2009, 44, 2473.
5
Klinkert, M. Q.; Heussler, V. Mini-Rev. Med. Chem. 2006, 6, 131.
Fuertes, M. A.; Nguewa, P. A.; Castilla, J.; Alonso, C.; Perez, J. M. Curr. Med. Chem.
2008, 15, 433.

7
Richard, J. V.; Werbovetz, K. A. Curr Opin Chem Biol. 2010, 14, 447.
(a) Alves, L. V.; Do Canto-Cavalheiro, M. M.; Cysne-Finkelstein, L.; Leon, L. Biol.
Pharma. Bull. 2003, 26, 453; (b) Koide, T.; Nose, M.; Ogihara, Y.; Yabu, Y.; Ohta, N.
Biol. Pharma. Bull. 2002, 25, 131; (c) Ferriera Gomes, D. de C.; Alegrio, L. V.; freire
deLima, M. E.; Leon, L. L.; Araujo, C. A. C. Arzneim-Farsch Drug Res. 2002, 52, 120.
9
Tan, N.; Kaloga, M.; Radtke, O. A.; Kiderlen, A. F.; Oksuz, S.; Ulubelen, A.;
Kolodziej, H. Phytochemistry 2002, 61, 881.

10
Sairafianpour, M.; Christensen, J.; Staerk, D.; Budnik, B. A.; Kharazmi, A.;
Bagherzadeh, K.; Jaroszewski, J. W. J. Nat. Prod. 2001, 64, 1398.

11
Valderrama, A. J.; Benites, J.; Cortes, M.; Pessoa-Mahana, H.; Prina, E.; Fournet, A.
Bioorg. Med. Chem. 2003, 11, 4713.

12
Aggarwal, B. B.; Sundaram, Chitra; Prasad, Seema; Kannappan, Ramaswamy
Biochem. Pharmacology 2010, 80, 1613.

13
Sadikoglou, E.; Magoulas, G.; Theodoropoulou, C.; Athanassopoulos, C. M.;
Giannopoulou, E.; Theodorakopoulou, O.; Drainas, D.; Papaioannou, D.; Papadimitriou,

E. Eur. J. Med Chem. 2009, 44, 3175.
107
Chapter 3
14

Simoni, Daniele; Invidiata, F. P.; Rondanin, R.; Grimaudo, S.; Cannizzo, G.; Barbusca,
E.; Porretto, F.; DAlessandro, Nicola; Tolomeo, M. J. Med. Chem. 1999, 42, 4961.
15
(a) Evans, T. R. J.; Kaye, S. B. Br. J. Cancer 1999, 80, 1; (b) Kurie, J. M.; Hong, W.
K. Cancer J. 1999, 5, 150.

16
Caliaro, M. G.; Vitaux, P.; Lafon, C.; Lochon, I.; Nehme, A.; Valette, A.; Canal, P.;
Bugat, R.; Jozan, S. Br. J. Cancer 1997, 75, 333.

17
(a) Sunduru, Naresh; Agarwal, Anu; Katiyar, Sanjay Babu; Nishi; Goyal, Neena;
Gupta, Suman and Chauhan, Prem M. S. Bioorg. Med. Chem. 2006, 14, 7706; (b)
Sunduru, Naresh; Nishi; Palne, Shraddha; Chauhan, Prem M. S.; Gupta, Suman Eur. J.
Med. Chem. 2009, 44, 2473.
108
Chapter 4
Design and Synthesis of Novel

Heteroretinoid-Bisbenzylidine Ketone
Hybrids as Antileishmanial Agents
Chapter 4
Design and synthesis of novel heteroretinoid-bisbenzylidine

ketone hybrids as antileishmanial agents
4.1 INTRODUCTION
biology. Leishmaniasis is caused by several species of protozoan parasites, Leishmania,
and is transmitted to humans through the bite of infected female sandflies which are very
small insect vectors with a wide range of habitats. The disease classified in three clinical
forms: cutaneous, mucocutaneous and visceral. The first two result in severe skin or
muco-membranous lesions and high morbidity, and consequently high DALYs
(Disability Adjusted Life Years). Visceral leishmaniasis (VL), also known as Kala azar,
rarely results in long term illness; however, if left untreated, patients have a fatality rate
of 100% within two years. The situation has become complicated because of the
emergence of post kala-azar dermal leishmaniasis (PKDL), which appears in 0-6 months
after the successful curing of VL.1
According to the World Health Organization (WHO), leishmaniasis currently
affects some 12 million people in 88 countries and there are 2 million new cases per year.
Moreover, it is estimated that approximately 350 million people live at risk of infection
with Leishmania parasites.2 Visceral leishmaniasis (VL) occurs in 65 countries and more
than 90% of the VL cases worldwide are registered in India, Bangladesh, Nepal, and
Sudan. Leishmania/HIV co-infections have increased in Mediterranean countries, where
up to 70% of potentially fatal VL cases are associated with HIV infection and up to 9%
of AIDS cases suffer from newly acquired or reactivated VL.3 WHO recently classified
leishmaniasis as a category I: emerging or uncontrolled disease.4
Leishmaniasis control relies on chemotherapy since there are no licensed vaccines
available in the market. Available drugs are limited in number and suffer from several
limitations such as high cost, toxicity, parenteral administration, emergence and spread of
drug resistance. Antimonials are the first line of treatment options for VL, which were
discovered almost 70 years ago. These suffer from major side effects including cardiac
arrhythmia and pancreatitis. Besides their toxicity, treatment failure with antimonials use
has increased; sometimes, as high as 62% in some of the regions.1 Second line treatment
options for VL include pentamidine and amphotericin B but their widespread use is
109
Chapter 4

limited because of toxicity and cost. Perhaps the most significant recent advancement has
been the effective oral treatment of VL by using miltefosine. Despite its great efficacy,
miltefosine is also not free from toxicity and shows teratogenic effects in pregnant
women.5
4.2 BASIS OF WORK

New antileishmanial drugs are required in view of the shortcomings associated with
the existing drugs. Currently, efforts are being made to search new molecules from the
natural sources6 and in this endeavor diaryl heptanoids and aryl chalcones represent the
useful lead molecules in the area of anticancer and antileishmanial drug development.
Efforts are also being made to design multi-target-directed ligands to develop new
lead molecules for neglected tropical diseases.7 In this regard two or more small
molecules are being covalently linked to act on two or more different targets. These kinds
of hybrid molecules are under investigation and the results are quite promising.8,9 Navarro
and colleagues pointed out that in addition to the synergism in mechanism of actions
another favorable issue of this approach is the possible stabilization of the drug. This
feature might lead to a longer residence time of the drug in the body, allowing it to reach
the biological targets more efficiently, and may also result in a decrease in toxicity.10
With these views, they combined the sterol hydrazone ligand (A, Figure 4.1) to platinum.
Steroid compounds have been shown to inhibit sterol methyl transferase enzyme and
consequently Leishmania growth, by altering lipid composition of the parasites
mitochondrial inner membrane.11 Conversely, certain platinum complexes, such as
(2,2:62-terpyridine)platinum-(II), have produced remarkable leishmanicidal activity
against amastigote forms of L. donovani, exploiting the intercalative DNA properties of
the terpyridine ligand along with the covalent binding ability of the Pt (II) center.12
Therefore, the new platinum-sterol hydrazone complex (B) might exert a synergistic
mechanism of action by combining inhibition of the sterol biosynthesis pathway and dual
interaction with the DNA of the parasite.
When tested against L. mexicana promastigotes, B displayed better antileishmanial
activity than A (71% growth inhibition vs 39%, respectively), associated with motility
loss and swelling of parasites, vacuolation, and formation of parasite clusters. Studies for
110
Chapter 4

both the sterol profile and the interaction with DNA are in progress and may confirm the
designed multiple mechanism of action.
Figure 4.1: Platinum complex (B) of the sterol hydrazone ligand (A).
In recent years, in depth information is being generated on the biochemical targets
involved in the chemotherapy of cancer as compared to most neglected tropical diseases
like leishmaniasis, malaria, filaria, and Chagas disease. Biologically, most of the
biochemical targets involved in the proliferation mechanism and pathogenesis of cancer
and leishmaniasis have lots of similarities and as a result, clinically active anticancer drug
miltefosine is quite effective in chemotherapy of leishmaniasis. In view of this, a number
of biologically active anticancer natural products (curcumin, licochalcone etc.) are acting
as very good leads in the design and development of antileishmanial agents.
As a part of our research program, we have been designing antileishmanial agents
on the basis of anticancer natural products curcumin and licochalcone (Figure 4.2).13
Figure 4.2: Chemical structure of curcumin and licochalcone A.
111
Chapter 4

Recently we have reported the synthesis and antileishmanial activity of some novel
heteroretinoids.14 In continuation of our efforts in this context, we have covalently linked
heteroretinoid moiety with bisbenzylidine ketones and the resulting chemically novel
hybrid molecules were analyzed for their in vitro antileishmanial activity. Some of the
hybrid prototypes displayed good in vitro antileishmanial profile and the results are part
of this chapter.
4.3 CHEMISTRY
The overall strategy for the synthesis of novel heteroretinoid-bisbenzylidine ketone
hybrids is depicted in Scheme 4.1. The reaction of ionone with sodium hydride and
diethyl oxalate in toluene was carried out at reflux temperature to furnish 2 in quantitative
yield. The compound 2 on treatment with hydroxylamine hydrochloride in ethanol under
refluxing conditions afforded compound 3. During the cyclization of compound 2, attack
of nucleophillic nitrogen atom of NH2OH takes place on that carbonyl carbon which is
directly attached to an electron withdrawing ethyl carboxylate group to yield the cylclized
product 3.Compound 3 was subjected to base catalyzed hydrolysis to give compound 4.
Compound 4 was reacted with oxalyl chloride to furnish acid chloride (5) which was next
coupled with piperidone hydrochloride to give (E)-1- (5-(2-(2, 6, 6-trimethylcyclohex-2enyl) vinyl) isoxazole-3-carbonyl) piperidin-4-one (6). Finally, compound 6 was reacted
with various substituted benzaldehydes to obtain the desired compounds (7a-i) in
moderate to good yield.
The structures of all the synthetic compounds were determined on the basis of their
spectroscopic data and microanalysis. The IR spectra of compounds (7a-i) exhibited
characteristic absorption bands in the range of 1657-1634 cm-1 and 1599-1577 cm-1
displaying C=O and C=N stretching respectively. The ESI-MS (mass spectra) of the all
the synthetic compounds showed molecular ion peak at [M+1]+. The presence of two
carbonyl carbons in the synthetic hybrids can easily be detected by observing the
resonance at 186 (C=O of bisbenzylidine ketone function) and 169 (C=O of
heteroretinoid moiety) in their 13C NMR spectra.
112
Chapter 4

113
Chapter 4

4.4 BIOLOGICAL EVALUATION- MATERIAL METHODS

4.5 RESULT AND DISCUSSION

The in vitro biological activities of heteroretinoid-bisbenzylidine ketone hybrids
(7a-i) have shown encouraging results against L. donovani. Table 4.1 displays IC50
values of the synthetic hybrids against intracellular amastigotes and cytotoxicity of the
compounds on vero cell line. The IC50 values of the test derivatives against amastigotes
indicate that out of 9 synthetic compounds, 5 compounds (7c, 7d and 7f-h) exhibited high
activity against L. donovani (IC50 = 1.83-6.10 M), better than the reference drug sodium
stibogluconate (IC50 = 53.12 M) and miltefosine (IC50 = 8.10 M).
The overall activity profile of compounds (7a-i) demonstrated that there is
considerable difference in their IC50 values. Thus, the biological activity was influenced
to an extent by the type of substituent present and their position in the phenyl ring.
Compounds 7a-d, which have monomethoxy, dimethoxy and trimethoxy phenyl rings
were found to show interesting results. Compounds having monomethoxy substitutions
(7a, b) were found inactive whereas compound 7c, having 3,4-dimethoxy phenyl ring,
was found to exhibit better antileishmanial activity with an IC50 value of 3.75 M.
Although to a lesser extent but on further substitution diminution of biological activity
took place (7d, IC50 = 4.70 M).
Attachment of benzyloxy group at 4 position of phenyl ring rendered the molecule
inactive (7e, IC50 > 40 M). However, it was noted that the introduction of OMe group at
3 position together with 4-OBn greatly enhanced the activity (7f, IC50 = 5.02 M).
Similarly, among the methoxy derivatives (7a-d), compounds 7c and 7d exhibited better
antileishmanial potential as compared to monomethoxy derivatives (7a, b) because of the
presence of an additional OCH3 group at position 3. Considering these results and activity
profile of the target compounds (7a-i), we can say that OCH3 group at position 3 plays a
critical role in the antileishmanial activity of these compounds.
114
Chapter 4

Table 4.1: Antileishmanial activity and cytotoxicity of synthetic hybrids (7a-7i).
Entry
R1
R2
R3
R4
Antiamastigote
activity
(IC50 in M)
Cytotoxicity
(CC50 in M)
Selectivity
Index (SI)
7a
OMe
>40
NT
NA
7b
OMe
>20
NT
NA
7c
OMe
OMe
3.75 0.31
45.78 5.71
12.20
7d
OMe
OMe
OMe
4.70 0.48
25.32 3.40
5.38
7e
OBn
>40
NT
NA
7f
OBn
OMe
5.02 0.49
>400
>79.68
7g
OH
OMe
1.83 0.21
23.45 3.82
12.81
7h
OH
OEt
6.10 0.62
27.65 4.1
4.53
7i
Cl
>40
NT
NA
Standard
drug
Sodium stibogluconate
53.12 4.56
>400
>7.53
Standard
drug
Miltefosine
8.10 0.51
52.86 4.81
6.52
The Selectivity Index (SI) is defined as the ratio of CC50 (50% maximum cytotoxic concentration) on Vero
cells to IC50 (50% maximum inhibitory concentration) on L. donovani intramacrophagic amastigotes; NT =
Not tested; NA = Not available; IC50 and CC50 values are the average (mean S.D.) of three independent
experiments.
115
Chapter 4

Among the vanillin nucleus containing compounds (7g and 7h), methoxy vanillin
derivative (7g) having OCH3 group at position 3 was found more active than ethoxy
vanillin derivative (7h). In addition to that it was also found that by protecting the OH
group in compound 7g (IC50 = 1.83 M, SI = 12.81) with benzyl group, activity
decreased slightly but selectivity increased over 6 fold (7f, SI > 79.68). This indicates
that as the hydrophilicity decreases and hydrophobicity increases, selectivity increases
accordingly. The presence of p-chloro substituent has shown deleterious effect on the
antiamastigote activity of compound 7i (IC50 > 40 M) (Table 4.1).
4.6 CONCLUSION
Within this chapter, we present the efficient synthesis of a series of heteroretinoidbisbenzylidine ketone hybrids, which showed significant antileishmanial activity. The
activity results clearly indicate that newly synthetic compounds reported herein are
promising one and provide useful model for further structural and biological
optimization. Compound 7f displayed not only a lower IC50 value than that of reference
drugs, but also over 10- and 12- fold more selective as compared to that of standard drugs
sodium stibogluconate and miltefosine, respectively. The study opens up the possibility
of advancing this new class of compounds as novel antileishmanial agents. Further
studies on these heteroretinoid-bisbenzylidine ketone hybrids to optimize the efficacy are
in progress in our laboratory.
116
Chapter 4

4.7 EXPERIMENTAL SECTION

Melting points were recorded on a Buchi-530 capillary melting point apparatus and
are uncorrected. IR spectra were recorded on a Perkin-Elmer RX-1 spectrometer using
KBr pellets or neat. 1H NMR and 13C NMR spectra were recorded using BrukerSupercon
Magnet DRX-300 spectrometer using CDCl3 as solvent and tetramethylsilane (TMS) as
internal standard. Chemical shifts are reported in parts per million. Electrospray
ionization mass spectra (ESI-MS) were recorded on a JEOL SX 102/DA-6000. Elemental
analyses were performed on a Carlo-Erba-1108 C, H, N elemental analyzer (Italian).
Chromatography was executed with silica gel (60120 mesh) using mixtures of ethyl
acetate and hexane as eluants. Visualization was done under UV light and spraying with
10% sulfuric acid in methanol.
4.7.1
Synthesis of (E)-ethyl 2,4-dioxo-6-(2,6,6-trimethylcyclohex-2-enyl)hex-5enoate (2)

NaH (1.25 g, 26 mmol) was stirred in dry hexane (25 ml) for 10 minutes and
hexane was pipetted out. Diethyl oxalate (7.59 ml, 52 mmol), ionone (5 ml, 26 mmol)
and toluene (45 ml) were added. Reaction mixture was refluxed for 2 h. After cooling the
reaction mixture, aqueous solution of HCl (13 ml HCl + 30 ml water) and ethyl acetate
(80 ml) were added and stirred for h. Organic layer was extracted and concentrated.
Crude was purified by column chromatography.
1.38 (t, J = 7 Hz, 3H), 1.42-1.52 (m, 1H), 1.56 (s, 3H), 2.05 (brs, 2H), 2.33 (d, J = 9 Hz,
1H), 4.35 (q, J = 7 Hz, 2H), 5.52 (brs, 1H), 6.02 (d, J = 16 Hz, 1H), 6.41 (s, 1H), 6.87
(dd, J = 16, 9 Hz, 1H); 13C NMR (CDCl3, 75 MHz): 14.02, 22.80, 22.99, 26.76, 27.84,
31.07, 32.84, 54.65, 62.41, 99.82, 122.96, 127.83, 131.59, 149.72, 162.10, 173.05,
186.11; ESMS m/z: 293 [M+1]+; Analysis calculated for C17H24O4: C, 69.84; H, 8.27;
Found: C, 69.89; H, 8.24.
117
Chapter 4
4.7.2

Synthesis of (E)-ethyl 5-(2,6,6-trimethylcyclohex-2-en-1yl)vinyl)isoxazole-3carboxylate (3)

To a solution of 2 (1.92 g, 5 mmol) in ethanol (10 ml), hydroxylamine
hydrochloride was added and reaction mixture was refluxed for 2 h. Reaction was
monitored by T.L.C. After completion of reaction ethanol was removed and residue was
taken up in ethyl acetate (20 ml). The organic extract was washed with water (220 ml),
brine solution (220 ml), dried (Na2SO4) and solvent was removed under vacuum. The
crude product thus obtained was chromatographed (SiO2, 60-120 mesh).
Yield: 68%; M.P: 55-57 0C; IR (KBr, cm-1): 2957, 1740, 1642, 1573, 1448, 1240,
1023; 1H NMR (CDCl3, 300 MHz): 0.87 (s, 3H), 0.93 (s, 3H), 1.20-1.27 (m, 1H), 1.381.52 (m, 4H), 1.59 (s, 3H), 2.04 (brs, 2H), 2.30 (d, J = 9 Hz, 1H), 4.42 (q, J = 7 Hz, 2H),
5.50 (brs, 1H), 6.30 (d, J = 16 Hz, 1H), 6.40-6.50 (m, 2H); 13C NMR (CDCl3, 75 MHz):
14.11, 22.84, 22.97, 26.78, 27.73, 31.14, 32.65, 54.75, 62.01, 100.31, 116.10, 122.49,
4.7.3
Synthesis of (E)-5-(2-(2,6,6-trimethylcyclohex-2-en-1-yl)vinyl)isoxazole-3carboxylic acid (4)

To a solution of ester 3 (0.578 g, 2 mmol) in ethanol (25 ml), NaOH (0.20 g) was
added and the solution was stirred under reflux for 2 h. After being cooled to room
temperature, the mixture was acidified with 1N HCl. The crude product was extracted
with ethyl acetate and washed with water (215 ml), brine (215 ml), dried (Na2SO4) and
solvent was removed under vacuum. The crude product thus obtained on crystallization
gave 4 as a white crystalline solid.
1230; 1H NMR (CDCl3, 300 MHz): 0.87 (s, 3H), 0.93 (s, 3H), 1.17-1.25 (m, 1H), 1.411.51 (m, 1H), 1.59 (s, 3H), 2.04 (brs, 2H), 2.30 (d, J = 9 Hz, 1H), 5.50 (s, 1H), 6.30 (d, J
= 16 Hz, 1H), 6.47 (dd, J = 16, 9 Hz, 1H), 6.54 (s, 1H), 14.19 (brs, 1H);
13
C NMR
(CDCl3, 75 MHz): 22.85, 22.99, 26.80, 27.74, 31.17, 32.70, 54.81, 100.52, 115.98,
118
Chapter 4

122.60, 132.12, 141.46, 156.17, 163.52, 170.77; ESMS m/z: 262 [M+1]+; Analysis
calculated for C15H19NO3: C, 68.94; H, 7.33; N, 5.36; Found: C, 68.89; H, 7.41; N, 5.30.
4.7.4
(E)-1-(5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)
piperidin-4-one (6)
To a solution of (E)-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole-3-
carboxylic acid (4) (0.522 g, 2 mmol) in DCM (15 ml) oxalyl chloride (0.72 g, 5.73
mmol) and DMF (3 drops) was added and the reaction mixture was stirred at room
temperature for 2 hrs. It was concentrated in vacuo and to it was added DCM (20 ml),
triethylamine (0.404 g, 4 mmol) followed by 4-piperidone hydrochloride (0.336 g, 2.2
mmol) and the resulting reaction mixture was refluxed for 2.5 hrs. It was poured into
water (50 ml) and extracted with DCM (100 ml). The combined extract was washed with
water (50 ml X 3), brine solution (50 ml), dried Na2SO4. The solvent was removed invacuo. The crude product was column chromatographed (SiO2, 60-120 mesh). Elution
with 20% ethyl acetate in hexane furnished.(E)-1-(5-(2-(2,6,6-trimethylcyclohex-2enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one as a brown color liquid compound (0.42
g).
Yield: 61%; IR (KBr, cm-1): 2961, 1718, 1643, 1456, 1219; 1H NMR (CDCl3, 300
MHz): 0.87 (s, 3H), 0.94 (s, 3H), 1.20-1.27 (m, 1H), 1.42-1.50 (m, 1H), 1.60 (s, 3H),
2.04 (brs, 2H), 2.31 (d, J = 9.0 Hz, 1H), 4.03 (brs, 4H), 4.14 (brs, 4H), 5.51 (s, 1H), 6.31
(d, J = 16.1 Hz, 1H), 6.43-6.66 (m, 2H);
13
C NMR (CDCl3, 75 MHz): 22.8, 23.0, 26.8,
27.6, 31.2, 32.7, 40.6, 41.6, 41.8, 45.5, 54.8, 101.1, 116.0, 122.5, 132.2, 141.1, 158.4,
160.0, 169.3, 206.4; ESI-MS m/z: 343 [M+H]+; Anal. Calcd for C20H26N2O3: C, 70.15;
H, 7.65; N, 8.18; Found: C, 70.23; H, 6.59; N, 8.21.
4.7.5
General method for the Synthesis of compounds 7a-i

To a solution of (E)-1-(5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole-3-
carbonyl piperidin-4-one (6) (0.342 g, 1 mmol) in ethanol (20 ml), piperidine (0.340 g,
0.395 ml, 4 mmol), substituted benzaldehyde (2 mmol) and L-proline (0.011 g, 0.1
mmol) were added. Reaction mixture was refluxed for 8 h. After completion of reaction
(TLC monitoring), compounds 7a, 7c, 7e and 7f were precipitated as yellow coloured
119
Chapter 4

solids while remaining compounds were obtained by concentrating the reaction mixture
and extracting the organic compounds with ethyl acetate followed by water washing,
brine washing, drying (Na2SO4) and column chromatography (SiO2, 60-120 mesh).
4.7.6
(3E,5E)-3,5-bis(4-methoxybenzylidene)-1-(5-((E)-2-(2,6,6-trimethylcyclohex2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7a)
Yield: 32%; Mp: 118-120 C; IR (KBr, cm-1): 2950, 2842, 1653, 1599, 1508,
1448, 1260, 1166, 1027; 1H NMR (CDCl3, 300 MHz): 0.79 (s, 3H), 0.85 (s, 3H), 1.101.20 (m, 1H), 1.30-1.42 (m, 1H), 1.50 (s, 3H), 1.97 (m, 2H), 2.19 (d, J = 8.8 Hz, 1H),
3.77 (s, 3H), 3.79 (s, 3H), 4.99 (s, 2H), 5.18 (s, 2H), 5.42 (s, 1H), 6.12 (d, J = 15.9 Hz,
1H), 6.20-6.33 (m, 2H), 6.84-6.93 (m, 4H), 7.28 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz,
2H), 7.69 (s, 1H), 7.77 (s, 1H);
13
C NMR (CDCl3, 75 MHz): 22.8, 23.0, 26.8, 27.7,
31.2, 32.6, 43.8, 47.6, 54.7, 2 55.3, 101.1, 4 114.3, 116.0, 122.4, 127.1, 127.3, 129.0,
129.2, 2 132.2, 2 132.4, 2 132.6, 137.4, 138.1, 140.7, 158.4, 159.7, 160.7, 168.9,
186.3; ESI-MS m/z: 579 [M+H]+; Anal. Calcd for C36H38N2O5: C, 74.72; H, 6.62; N,
4.84; Found: C, 74.69; H, 6.67; N, 4.80.
4.7.7
(3E,5E)-3,5-bis(2-methoxybenzylidene)-1-(5-((E)-2-(2,6,6-trimethylcyclohex2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one(7b)
Yield: 35%; Mp: 78-80 C; IR (KBr, cm-1): 2959, 1637, 1484, 1400, 1216, 1116,
1027; 1H NMR (CDCl3, 300 MHz): 0.86 (s, 3H), 0.93 (s, 3H), 1.19-1.27 (m, 1H), 1.391.50 (m, 1H), 1.59 (s, 3H), 2.0 (brs, 2H), 2.26 (d, J = 8.8 Hz, 1H), 3.83 (s, 3H), 3.88 (s,
3H), 4.92 (s, 2H), 4.97 (s, 2H), 5.51 (s, 1H), 6.12-6.16 (m, 2H), 6.32 (dd, J = 15.9, 9.1
Hz, 1H), 6.87-6.99 (m, 3H), 7.01-7.10 (m, 2H), 7.28-7.37 (m, 3H), 7.97 (s, 1H), 8.10 (s,
1H);
13
C NMR (CDCl3, 75 MHz): 22.9, 23.0, 26.8, 27.7, 31.2, 32.6, 43.9, 47.3, 54.7,
255.4, 100.6, 2110.8, 116.0, 120.3, 120.4, 122.4, 123.5, 130.0, 130.3, 2130.9, 131.1,
2131.7, 2132.2, 133.7, 134.6, 140.4, 158.0, 158.4, 159.9, 168.7, 186.9; ESI-MS m/z:
579 [M+H]+; Anal. Calcd for C36H38N2O5: C, 74.72; H, 6.62; N, 4.84; Found: C, 74.70;
H, 6.65; N, 4.80.
120
Chapter 4
4.7.8

(3E,5E)-3,5-bis(3,4-dimethoxybenzylidene)-1-(5-((E)-2-(2,6,6-trimethyl
cyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7c)
1445, 1252, 1143, 1023; 1H NMR (CDCl3, 300 MHz): 0.78 (s, 3H), 0.85 (s, 3H), 1.101.20 (m, 1H), 1.30-1.42 (m, 1H), 1.50 (s, 3H), 1.97 (brs, 2H), 2.20 (d, J = 8.5 Hz, 1H),
3.87 (s, 6H), 3.88 (s, 6H), 5.01 (s, 2H), 5.31 (s, 2H), 5.43 (s, 1H), 6.15 (d, J = 16.0 Hz,
1H), 6.22-6.40 (m, 2H), 6.80-7.08 (m, 6H), 7.68 (s, 1H), 7.77 (s, 1H); 13C NMR (CDCl3,
75 MHz): 22.8, 23.0, 26.8, 27.7, 31.2, 32.6, 43.4, 47.8, 54.7, 455.9, 101.5, 111.1,
111.2, 113.2, 113.7, 115.9, 122.5, 124.2, 124.9, 2127.5, 2129.1, 132.2, 137.8, 138.2,
140.8, 2149.0, 2150.5, 158.6, 159.5, 169.0, 186.1; ESI-MS m/z: 639 [M+H]+; Anal.
Calcd for C38H42N2O7: C, 71.45; H, 6.63; N, 4.39; Found: C, 71.41; H, 6.68; N, 4.36.
4.7.9
(3E,5E)-3,5-bis(3,4,5-trimethoxybenzylidene)-1-(5-((E)-2-(2,6,6-trimethyl
cyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7d)
3.91 (s, 6H), 3.92 (s, 12H), 5.07 (s, 2H), 5.44 (s, 2H), 5.50 (s, 1H), 6.21-6.43 (m, 3H),
6.71 (s, 2H), 6.79 (s, 2H), 7.72 (s, 1H), 7.82 (s, 1H); 13C NMR (CDCl3, 75 MHz): 22.8,
23.0, 26.8, 27.7, 31.2, 32.6, 43.1, 48.0, 54.7, 256.2, 256.3, 260.9, 101.6, 2107.8,
2108.2, 115.9, 122.5, 129.8, 129.9, 2130.1, 132.1, 138.2, 138.4, 139.5, 139.7, 141.0,
2153.2, 2153.3, 158.7, 159.5, 169.1, 186.1; ESI-MS m/z: 699 [M+H]+; Anal. Calcd for
4.7.10 (3E,5E)-3,5-bis(4-(benzyloxy)benzylidene)-1-(5-((E)-2-(2,6,6-trimethyl
cyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7e)
1.30-1.42 (m, 1H), 1.50 (s, 3H), 1.96 (brs, 2H), 2.19 (d, J = 8.7 Hz, 1H), 4.98 (s, 2H),
5.03-5.05 (m, 4H), 5.19 (s, 2H), 5.41 (s, 1H), 6.13 (d, J = 16.1 Hz, 1H), 6.23-6.33 (m,
2H), 6.93-6.99 (m, 4H), 7.26-7.43 (m, 14H), 7.68 (s, 1H), 7.76 (s, 1H); 13C NMR (CDCl3,
75 MHz): 22.9, 23.0, 26.8, 27.7, 31.2, 32.6, 43.8, 47.6, 54.7, 270.1, 101.3, 4115.2,
121
Chapter 4

116.0, 122.5, 4127.4, 2128.1, 6128.6, 2129.1, 2129.3, 2132.2, 132.4, 132.6,
2136.4, 137.3, 138.1, 140.7, 158.4, 159.7, 160.0, 169.0, 186.3; ESI-MS m/z: 731
[M+H]+; Anal. Calcd for C48H46N2O5: C, 78.88; H, 6.34; N, 3.83; Found: C, 78.87; H,
6.37; N, 3.81.
4.7.11 (3E,5E)-3,5-bis(4-(benzyloxy)-3-methoxybenzylidene)-1-(5-((E)-2-(2,6,6trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7f)
3.88 (s, 6H), 4.98 (s, 2H), 5.13-5.14 (m, 4H), 5.30 (s, 2H), 5.43 (s, 1H), 6.16 (d, J = 15.3
Hz, 1H), 6.23-6.34 (m, 2H), 6.87-7.04 (m, 6H), 7.24-7.36 (m, 10H), 7.65 (s, 1H), 7.74 (s,
1H);
13
C NMR (CDCl3, 75 MHz): 22.8, 23.0, 26.8, 27.7, 31.2, 32.6, 43.5, 47.9, 54.7,
56.0, 56.1, 270.8, 101.5, 113.4, 113.5, 113.7, 114.2, 116.0, 122.5, 124.0, 124.8,
4127.2, 2127.9, 2128.0, 4128.6, 2129.2, 132.2, 136.5, 137.8, 138.2, 140.8,
3149.5, 2149.7, 158.6, 159.5, 169.0, 186.1; ESI-MS m/z: 791 [M+H]+; Anal. Calcd for
4.7.12 (3E,5E)-3,5-bis(4-hydroxy-3-methoxybenzylidene)-1-(5-((E)-2-(2,6,6trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7g)
1.40-1.45 (m, 1H), 1.55 (s, 3H), 2.01 (brs, 2H), 2.25 (d, J = 9.0 Hz, 1H), 3.93 (s, 6H),
5.04 (s, 2H), 5.30 (s, 2H), 5.48 (s, 1H), 6.19 (d, J = 15.9 Hz, 1H), 6.25-6.38 (m, 2H),
6.91-6.97 (m, 4H), 7.04 (s, 2H), 7.71 (s, 1H), 7.80 (s, 1H); 13C NMR (CDCl3, 75 MHz):
22.8, 22.9, 26.8, 27.6, 31.2, 32.6, 43.5, 47.8, 54.7, 256.0, 101.3, 112.9, 113.3, 2114.9,
115.9, 122.5, 124.9, 125.5, 2126.9, 2128.8, 132.2, 138.1, 138.6, 140.9, 2146.7,
2147.5, 158.5, 159.7, 169.1, 186.3; ESI-MS m/z: 611 [M+H]+; Anal. Calcd for
122
Chapter 4

4.7.13 (3E,5E)-3,5-bis(3-ethoxy-4-hydroxybenzylidene)-1-(5-((E)-2-(2,6,6trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7h)

4.17-4.22 (m, 4H), 5.05 (s, 2H), 5.31 (s, 2H), 5.50 (s, 1H), 6.00 (brs, 2H), 6.21 (d, J =
15.9 Hz, 1H), 6.32-6.40 (m, 2H), 6.96-7.00 (m, 4H), 7.05-7.08 (m, 2H), 7.72 (s, 1H), 7.80
(s, 1H);
13
C NMR (CDCl3, 75 MHz): 214.8, 22.8, 23.0, 26.8, 27.6, 31.2, 32.6, 43.5,
47.8, 54.7, 264.7, 101.4, 113.6, 114.0, 2114.8, 115.9, 122.5, 124.9, 125.4, 2126.9,
2128.8, 132.2, 138.0, 138.6, 140.8, 2145.9, 2147.5, 158.6, 159.6, 169.0, 186.2; ESIMS m/z: 639 [M+H]+; Anal. Calcd for C38H42N2O7: C, 71.45; H, 6.63; N, 4.39; Found: C,
71.41; H, 6.69; N, 4.51.
4.7.14 (3E,5E)-3,5-bis(4-chlorobenzylidene)-1-(5-((E)-2-(2,6,6-trimethylcyclohex-2enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7i)
1439, 1217, 769; 1H NMR (CDCl3, 300 MHz): 0.86 (s, 3H), 0.93 (s, 3H), 1.19-1.25 (m,
1H), 1.39-1.51 (m, 1H), 1.59 (s, 3H), 2.05 (brs, 2H), 2.27 (d, J = 9.2 Hz, 1H), 5.01 (s,
2H), 5.24 (s, 2H), 5.50 (s, 1H), 6.21 (d, J = 16.0 Hz, 1H), 6.31 (s, 1H), 6.37 (dd, J = 16.2,
9.3 Hz, 1H), 7.28-7.31 (m, 2H), 7.38-7.44 (m, 6H), 7.75 (s, 1H), 7.82 (s, 1H); ESI-MS
m/z: 587 [M+H]+; Anal. Calcd for C34H32Cl2N2O3: C, 69.50; H, 5.49; N, 4.77; Found: C,
69.47; H, 5.53; N, 4.75.
123
Chapter 4

4.8 SPECTRA OF SOME SELECTED COMPOUNDS
124
Chapter 4

Figure 4.5: 1H NMR of compound 7c at 300 MHz (CDCl3)
Figure 4.6: 13C NMR of compound 7c at 75 MHz (CDCl3)
125
Chapter 4

Figure 4.7: 1H NMR of compound 7f at 300 MHz (CDCl3)
Figure 4.8: 13C NMR of compound 7f at 75 MHz (CDCl3)
126
Chapter 4

Figure 4.9: 1H NMR of compound 7g at 300 MHz (CDCl3)
Figure 4.10: 13C NMR of compound 7g at 75 MHz (CDCl3)
127
Chapter 4

4.9 REFERENCES
1
Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling R. W.; Alvar, J.;
Boelaert, M. Nat. Rev. Microbiol. 2007, 5, 873.

2
Ashford, R. W.; Desjeux, P.; Deraadt, P. Parasitol. Today 1992, 8, 104.
Desjeux, P.; Clinics in Dermatology 1996, 14, 417.
Croft, S. L.; Olliaro, P. Clin. Microbiol. Infect. 2011, 17, 1478.
(a) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461; (b) Rocha, L. G.;
Almeida, J. R. G. S.; Macdo, R. O.; Barbosa-Filho, J. M. Phytomedicine , 2005, 12, 514.

7
Cavalli, A.; Bolognesi, M. L. J. Med. Chem. 2009, 52, 7339.
(a) Meunier, B. Acc. Chem. Res.; 2008, 41, 69; (b) Camps, P.; Formosa, X.; Galdeano,
C.; Gmez, T.; Muoz-Torrero, D.; Scarpellini, M.; Viayna, E.; Badia, A.; Clos, M. V.;
Camins, A.; Palls, M.; Bartolini, M.; Mancini, F.; Andrisano, V.; Estelrich, J.; Lizondo,
M.; Bidon-Chanal, A.; Luque, F. J. J. Med. Chem. 2008, 51, 3588; (c) Belluti, F.;
Fontana, G.; Bo, L. D.; Carenini, N.; Giommarelli, C.; Zunino, F. Bioorg. Med. Chem.
2010, 18, 3543.
9
(a) Das, B. C.; Mahalingam, S. M.; Panda, L.; Wang, B.; Campbell, P. D.; Evans, T.
Tet. Lett. 2010, 51, 1462; (b) Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Ynez, M.;
Fraiz, N.; Alcaide, C.; Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257.
10
Visbal, G.; Marchan, E.; Maldonado, A.; Simoni, Z.; Navarro, M. J. Inorg. Biochem.
2008, 102, 547.

11
Rodrigues, J. C.; Bernardes, C. F.; Visbal, G.; Urbina, J. A.; Vercesi, A. E.; de Souza,
W. Protist 2007, 158, 447.

12
Lowe, G.; Droz, A. S.; Vilaivan, T.; Weaver, G. W.; Tweedale, L.; Pratt, J. M.; Rock,
P.; Yardley, V.; Croft, S. L. J. Med. Chem. 1999, 42, 999.

13
(a) Suryawanshi, S. N.; Chandra, N.; Kumar, P.; Porwal, J.; Gupta, S. Eur. J. Med.
Chem. 2008, 43, 2473; (b) Kumar, S.; Tiwari, A.; Suryawanshi, S. N.; Mittal, M.;
Vishwakarma, P.; Gupta, S. Bioorg. Med. Chem. Lett. 2012, 22, 6728; (c) Suryawanshi,
S. N.; Tiwari, A.; Kumar, S.; Shivahare, R.; Mittal, M.; Kant, P.; Gupta, S. Bioorg. Med.
Chem. Lett. 2013, 23, 2925.
128
Chapter 4
14

Suryawanshi, S. N.; Tiwari, A.; Chandra, N.; Ramesh, Gupta, S. Bioorg. Med. Chem.
Lett. 2012, 22, 6559.
129
Publications
List of Publications
1. Chemotherapy of leishmaniasis part XIII: Design and synthesis of novel
heteroretinoid-bisbenzylidine ketone hybrids as antileishmanial agents. Tiwari,
A.; Kumar, S.; Shivahare, R.; Kant, P.; Gupta, S.; Suryawanshi, S. N. Bioorg.
Med. Chem. Lett. 2015, 25, 410-413.
2. Antidyslipidemic and Antioxidant Effects of Novel Lupeol-Derived Chalcones.
Srivastava, S.; Sonkar, R.; Mishra, S. K.; Tiwari, A.; Balramnavar, V.; Mir,
Snober; Bhatia, G.; Saxena, A. K.; Lakshmi, V. Lipids 2013, 48, 1017-1027.
3. Design, synthesis and biological evaluation aryl pyrimidine derivatives as
potential leishmanicidal agents. Suryawanshi, S. N.; Kumar, S.; Shivahare, R.;
Pandey, S.; Tiwari, A.; Gupta, S. Bioorg. Med. Chem. Lett. 2013, 23, 5235-5238.
4. Synthesis and biological evaluation of a novel series of aryl S,N-ketene acetals as
antileishmanial agents. Suryawanshi, S. N.; Kumar, S.; Tiwari, A.; Shivahare, R.;
Chhonker, Y. S.; Pandey, S.; Shakya, N.; Bhatta, R. S.; Gupta, S. Bioorg. Med.
Chem. Lett. 2013, 23, 3979-3982.
5. Chemotherapy of leishmaniasis. Part XII: Design, synthesis and bioevaluation of
novel triazole integrated phenyl heteroterpenoids as antileishmanial agents.
Suryawanshi, S. N.; Tiwari, A.; Kumar, S.; Shivahare, R.; Mittal, M.; Kant, P.;
Gupta, S. Bioorg. Med. Chem. Lett. 2013, 23, 29252928.
6. Chemotherapy of leishmaniasis part X: Synthesis and bioevaluation of novel
terpenyl heterocycles. Tiwari, A.; Kumar, S.; Suryawanshi, S. N.; Mittal, M.;
Vishwakarma, P.; Gupta, S. Bioorg. Med. Chem. Lett. 2012, 23, 248251.
7. Chemotherapy of leishmaniasis. Part IX: Synthesis and bioevaluation of aryl
substituted ketene dithioacetals as antileishmanial agents. Kumar, S.; Tiwari, A.;
Suryawanshi, S. N.; Mittal, M.; Vishwakarma, P.; Gupta, S. Bioorg. Med. Chem.
Lett. 2012, 22, 67286730.
8. Chemotherapy of leishmaniasis. Part XI: Synthesis and bioevaluation of novel
isoxazole containing heteroretinoid and its amide derivatives Suryawanshi, S. N.;
Tiwari, A.; Chandra, N.; Ramesh, Gupta, S. Bioorg. Med. Chem. Lett. 2012, 22,
65596562.
130
Publications
List of Patents
1. Triazole substituted terpenyl pyrazolidines and process for preparation therof.
(3493DEL2011); Inventors: Dr. S. N. Suryawanshi, Dr. Suman Gupta, Mr.
Avinash Tiwari, Shalini Singh, Monika Mittal, Mr. Rahul Shivahare.
2. (E)-5-(2-nitrophenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)-4,5dihydro-1H-pyrazoles
as
novel
antileishmanial
agents.
(2175DEL2010);
Inventors: Dr. S. N. Suryawanshi, Dr. Suman Gupta, Mr. Avinash Tiwari,

Monika Mittal, Preeti Vishwakarma.
Symposium/Conferences
1. Avinash Tiwari, S. N. Suryawanshi, Rahul Shivahare and Suman Gupta; Novel
retinoic acid prototype and bisbenzylidine ketone hybrids as antileishmanial
agents. Poster presentation at 5th NIPER (RBL)-CSIR-CDRI Symposium2013 held at CSIR-CDRI, Lucknow on 21-23 March 2013.
2. Avinash Tiwari, Naveen Chandra, S. N. Suryawanshi, Ramesh and Suman
Gupta; Novel isoxazole containing heteroretinoid and its amide derivatives as
antileishmanial agents. Poster presentation at 5th international Symposium on
Current Trends in Drug Discovery Research 2013 held at CSIR-CDRI,
Lucknow on 26-28 Feb. 2013.
3. Avinash Tiwari, Chemical Research Society of India A Mid Year Meeting
2012 held at Clark Awadh, Lucknow on 21-22 July 2012. (Participation)
4. Avinash Tiwari, National seminar on Natural Products & Organic Synthesis
Symposium-2012 held at Department of Chemistry, University of Lucknow,
Lucknow on March 28, 2012. (Participation)
131

Avinash Tiwari Thesis

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THESIS

Res. CM-19, Sector-B Aliganj,

Res. 8/345, Vikas Nagar,

(Prof. Padam Kant)

ANTILEISHMANIAL AGENTS submitted to the Lucknow University, Lucknow, has been

CHAPTER 1: CHEMOTHERAPY OF LEISHMANIASIS SO FAR:

CHAPTER 2.1: DESIGN, SYNTHESIS AND BIOEVALUATION OF

CHAPTER 2.2: DESIGN, SYNTHESIS AND BIOEVALUATION OF

CHAPTER 3: SYNTHESIS AND BIOEVALUATION OF NOVEL

CHAPTER 4: DESIGN AND SYNTHESIS OF NOVEL

List of publications, patents and presentations

Broad singlet (in NMR)

Cetyltrimethyl ammonium bromide

Chemical shift (parts per million)

Cytotoxicity concentration at 50%

Deuterated dimethyl sulfoxide

Doublet (in NMR)

Double doublet (in NMR)

Electron spray ionization mass spectrometry

Electron spray mass spectrometry

Fourier transformed-infra Red

Human immunodeficiency virus

Inhibitory concentration at 50%

Coupling constant (in NMR)

Mass to charge ratio (in Mass spectrometry)

Molecular ion peak

Minimum inhibitory concentration

Multiplet (in NMR)

Nuclear magnetic resonance

Parts per million

Quartet (in NMR)

Singlet (in NMR)

Thin layer chromatography

Triplet (in NMR)

World Health Organization

Chemotherapy of Leishmaniasis So Far: A

Chemotherapy of leishmaniasis so far: A Review

leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL).

Chemotherapy of leishmaniasis so far: A Review

1.2 TYPES OF LEISHMANIASIS

Leishmania amazonensis and related species in the New

Figure 1.1: Cutaneous

South India, Kerala, which has implications for the

Chemotherapy of leishmaniasis so far: A Review

1.2.2 Mucocutaneous leishmaniasis:

congestion that causes difficulty in breathing and epistaxis.

Figure 1.3: Mucoutaneous

Mucocutaneous lesions usually develop by organismal metastasis from distant

Figure 1.4: A child

found in areas of L. infantum transmission whereas anthroponotic VL is found in areas of

Chemotherapy of leishmaniasis so far: A Review

Figure 1.5: Geographical distribution of VL in India

Figure 1.6: A patient from

Chemotherapy of leishmaniasis so far: A Review

1.3 LIFE CYCLE

Chemotherapy of leishmaniasis so far: A Review

Chemotherapy of leishmaniasis so far: A Review

1.4 CURRENT STATUS

1.5 CONVENTIONAL DRUGS

Chemotherapy of leishmaniasis so far: A Review

Chemotherapy of leishmaniasis so far: A Review

Recently, antimonials have become almost obsolete in certain areas of India

Chemotherapy of leishmaniasis so far: A Review

Chemotherapy of leishmaniasis so far: A Review