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DEPARTMENT OF CHEMISTRYUNIVERSITY OF LUCKNOW

LUCKNOW, (INDIA)2014

By

THESIS

SUBMITTED TO THE

UNIVERSITY OF LUCKNOW

FOR THE DEGREE OF

Doctor of Philosophy

IN

CHEMISTRY

Avinash TiwariM.Sc. (Chemistry)

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 Dated:

Chief Scientist

Medicinal & Process Chemistry Division

CSIR-Central Drug Research Institute

Lucknow-226001

India

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 can’t 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 I-III

Preface IV-V

CHAPTER 1: CHEMOTHERAPY OF LEISHMANIASIS SO FAR: 1-38

A REVIEW

1.1 Introduction 1

1.2 Types of leishmaniasis 2

1.2.1 Cutaneous leishmaniasis 2

1.2.2 Mucocutaneous leishmaniasis 3

1.2.3 Visceral leishmaniasis 3

1.3 Life cycle 5

1.4 Current status 7

1.5 Conventional drugs 7

1.5.1 Antimonials 8

1.5.2 Pentamidine 9

1.5.3 Amphotericin B 10

1.5.4 Paromomycin 11

1.5.5 Miltefosine 12

1.6 Recent advancements 12

1.6.1 Sitamaquine 13

1.6.2 Azithromycin 13

1.6.3 Imiquimod 14

1.6.4 Azoles 14

1.6.5 Combination therapy 15

1.7 Scope of natural product 15

1.8 Natural product lead based studies 23

1.8.1 Curcumin: “The spice of life-unlocking the secrets of curcumin” 23

1.8.2 Chalcone 26

1.9 Conclusion 32

1.10 References 33

CHAPTER 2.1: DESIGN, SYNTHESIS AND BIOEVALUATION OF 39-69

NOVEL TERPENYL HETEROCYCLES

2.1.1 Introduction 39

2.1.2 Basis of work 41

2.1.3 Chemistry 44

2.1.3.1 Synthesis of β-ionone based 1,3,5-trisubstituted-4,5- 44 dihydropyrazoles (4a-j)

2.1.3.2 Synthesis of α-ionone based 1,3,5-trisubstituted-4,5- 46 dihydropyrazoles (8a-e)

2.1.3.3 Synthesis of α-ionone based 1,3,5-trisubstituted pyrazoles (9a-e) 47

2.1.4 Biological evaluation- material methods 47

2.1.4.1 Anti amastigote activity 48

2.1.4.2 Cytotoxicity assay 48

2.1.4.3 In Vivo assay 49

2.1.5 Result and discussion 50

2.1.6 Conclusion 51

2.1.7 Experimental section 52

2.1.8 Spectra of some selected compounds 65

2.1.9 References 67

CHAPTER 2.2: DESIGN, SYNTHESIS AND BIOEVALUATION OF 70-85

NOVEL TRIAZOLE INTEGRATED PHENYL

HETEROTERPENOIDS AS ANTILEISHMANIAL

AGENTS

2.2.1 Introduction 70

2.2.2 Basis of work 71

2.2.3 Chemistry 72

2.2.4 Biological evaluation- material methods 74

2.2.5 Result and discussion 74

2.2.6 Conclusion 77

2.2.7 Experimental section 78

2.2.8 Spectra of some selected compounds 83

2.2.9 References 85

CHAPTER 3: SYNTHESIS AND BIOEVALUATION OF NOVEL 86-108

ISOXAZOLE CONTAINING HETERORETINOID

AND ITS AMIDE DERIVATIVES 3.1 Introduction 86

3.2 Basis of work 87

3.3 Design and synthesis of heteroretinoid 87

3.4 Chemistry 89

3.5 Biological evaluation- material methods 92

3.6 Result and discussion 92

3.7 Conclusion 95

3.8 Experimental section 96

3.9 Spectra of some selected compounds 103

3.10 References 107

CHAPTER 4: DESIGN AND SYNTHESIS OF NOVEL 109-129

HETERORETINOID-BISBENZYLIDINE KETONE

HYBRIDS AS ANTILEISHMANIAL AGENTS

4.1 Introduction 109

4.2 Basis of work 110

4.3 Chemistry 112

4.4 Biological evaluation- material methods 114

4.5 Result and discussion 114

4.6 Conclusion 116

4.7 Experimental section 117

4.8 Spectra of some selected compounds 124

4.9 References 128

List of publications, patents and presentations 130-131

I

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

δ : Chemical shift (parts per million)

Conc. : Concentrated

CL : Cutaneous leishmaniasis

CC50 : Cytotoxicity concentration at 50%

0C : Degree celsius

CDCl3 : Deuterated chloroform

CH3COOH : Acetic acid

DMSO-d6 : Deuterated dimethyl sulfoxide

DCM : Dichloromethane

DHFR : Dihydrofolate reductase

DMF : Dimethyl formamide

d : 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

g : Gram(s)

Hz : Hertz(s)

II

h : Hour(s)

HIV : Human immunodeficiency virus

HCl : Hydrochloric acid

IC50 : Inhibitory concentration at 50%

IR : Infrared

i.p. : Intraperitoneal

J : 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

µg : Microgram

µL : Microlitre(s)

µm : Micrometer

µM : Micromolar

mg : Milligram

mL : Millilitre(s)

mmol : Millimole

MIC : Minimum inhibitory concentration

Min : Minute

MCL : Mucocutaneous leishmaniasis

m : Multiplet (in NMR)

MeOD : Deuterated methanol

nm : Nanometer

NMR : Nuclear magnetic resonance

Na2SO4 : Sodium sulphate

NaH : Sodium hydride

NH2OH : Hydroxyl amine

III

ppm : Parts per million

% : Percentage

PhNHNH2 : Phenyl hydrazine

q : Quartet (in NMR)

Rf : Retention factor

rt : Room temperature

SI : Selectivity index

s : Singlet (in NMR)

SSG : Sodium stibogluconate

THF : Tetrahydrofuran

TMS : Tetramethylsilane

TLC : Thin layer chromatography

t : Triplet (in NMR)

UV : Ultraviolet

VL : Visceral leishmaniasis

wt. : Weight

WHO : World Health Organization

IV

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-

V

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 heteroretinoid-

bisbenzylidine 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

1.1 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 1.5–2.0

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 HIV–Leishmania

co-infections, particularly in India and East Africa.3


2

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

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

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

Figure 1.1: Cutaneous

leishmaniasis with a large

ulcerative lesion on the arm

Figure 1.2: Geographical

distribution of CL in India


3

Figure 1.3: Mucoutaneous

leishmaniasis involving nose

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.

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

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

Figure 1.4: A child

suffering with VL.


4

Figure 1.5: Geographical distribution of VL in India

L. donovani transmission. Signs and symptoms include fever, weight loss, mucosal

ulcers, fatigue, anemia, and substantial swelling of the liver and spleen.

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. infantum-

endemic areas. The interval between treated VL and PKDL is

0–6 months in Sudan and 6 months to 3 years in India. PKDL

Figure 1.6: A patient from

India with nodular post-kala-

azar.


5

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 µm long and 1.5-3.00 µm 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 µm 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 fly’s 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


6

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


7

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 drug’s 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


8

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


9

(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


10

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


11

O

H3C

H3C

CH3

OH

HO O OH OH

OH

OHOHCOOH

H

O O CH3OH

H2NOH

OH

O

HO

O

NH2

OH NH2

NH2O

OHO

OH

O

HO

O

NH2

HO

H2N

OH

Amphotericin B

Paramomycin6

7

O O

NH

H2N

NH

NH2

Pentamidine(Pentacarinat)

5

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


12

1.5.5 Miltefosine

Perhaps the most significant recent advances for

the treatment of VL are the discovery of orally

administered drug miltefosine (8). It is an

alkylphosphocholine (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 95–97% concluding

cure rate in large phase III and IV trials in

India.46

However, there are major concerns about teratogenic potential and the long half-

life 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 β-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 co-

infected 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


13

ImiquimodSitamaquine Azithromycin

9 10 11

N

CH3

H3CO

NH(CH)6

NC2H5C2H5

NCH3

OO

O

CH3H3C

CH3

OH

OH

OH

H3C

H3C

O

H3C

H3C

O O

OMe

CH3

OH

CH3

CH3

OH

N(CH3)2

N

N

N

CH3

CH3

NH2

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.

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.


14

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

F F

O ON N

NN

N

O

NH2

CH3

N

N

N

O

N N O

O

O

N

N

Cl

Cl

H

Ketoconazole

Cl

Cl

ON

N

ClCl

Miconazole

N

N

N

OHN

N

N

F F

Fluconazole

NN

N

O

OONNN

N

N

O

Cl

Cl

H

Itraconazole

Posaconazole

12

13

14

15

16


15

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 14α-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


16

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 bis-

naphthoquinone, isolated from the bark of Diospyros Montana (Ebenaceae) showed in

vitro activity against promastigotes of L. donovani with an MIC of 1.0 µg/mL.65

Plumbagin (18), originally isolated from Plumbago zylenica, was found to show

antileishmanial activity against amastigotes of L. donovani (IC50= 0.42 µg/mL) and L.

amazonensis (IC50 = 1.1 µg/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 µg/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 µg/mL concentrations.66


17

Indole alkaloids dihydrocorynantheine (23), corynantheine (24) and

corynantheidine (25) isolated from the bark of Corynanthe pachyceras (Rubiaceae)

displayed the antileishmanial activity (IC50 of 3 µM) against L. major as respiratory chain

inhibitors.

NH

N

H

H3COOCH

OCH3

C2H5

H

H

NH

N

H

H3COOCH

OCH3

C2H3

H

H

NH

N

CH3

H3COOC

NH

N

H

H

H3COOC

H3CO

CH3

23 24

25

N

O

HO

CH3

N

O

CH3

19 20

N CH3 N CH3

O

21 22


18

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 µM. 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-α 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 µg/mL) as compared to holacurtine and

N-desmethyl holacurtine (6.25 > IC50 > 1.56 µg/mL).68

CH3

HCH3

H2N

H H H

CH3

O CH3

HCH3

H OH

CH3

O

H

HO

CH3

H

NH

H

OCH3

O

HH3C

29 30


19

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 0.45µg/mL

respectively. Emetine exhibited antileishmanial activity against L. donovani with an IC50

value 0.03 µg/mL, however produces toxicity in treatment of cutaneous leishmaniasis

caused by L. major.69

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-β-

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

N

HN

OH

OCH3

CH3

R1

H3COH

HR2

HN

HN

R

OCH3

CH3

H3CO

H3COH

HH

H

31 R1 = OH; R2 = OH

32 R1 = OCH3; R2 = H

33 R = OCH3

34 R = OH


20

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 µg/mL concentrations. At 10 µg/mL concentration, metabolite 42

showed more than 90% lysis of the promastigotes.

40

CH3

OH

OCH3H3CO

H3C CH3

OH CH3

CH3

CH3

OHH3C

CH3H3C

OH

H3C OH

CH3 CH3

CH3

CH3

41

42


21

A sesquiterpene lactone, dehydrozaluzanin C (43), isolated from the leaves of

Munnozia maronii (Asteraceae), displayed antileishmanial activity at concentrations

between 2.5-10 µg/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 µg/mL concentration. SAR study

with metabolite 44 indicated that the presence of a C-5 hydroxy group in the α-

orientation is essential for the expression of the leishmanicidal activity. The (+)-

curcuphenol (45), isolated from sponge Myrmekioderma styx, exhibited in vitro anti-

leishmanial activities against L. donovani with an EC50 of 11.0 µM.

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

43 44 45

H2C

O

H2CO CH2

O

H

H

HOH

CH3

OH

CH3

CH2

OH H3C CH3

H3C

OH CH3

O

H3C

H3C

OCH2

H

CH3H2C

HHO

O

CH3

O

O

CH3CH3

CH3

H3C

46 47


22

studies with these metabolites revealed that 46 with IC100 value of 0.75µg/mL displays

activity higher than 47 (IC100 = 5µg/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.

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.

48 49

HO

CH3

H

H CH3

CH3

H3C

CO2HCH3H3C

H3CHO

CH3

H

H CH3

CHOCH3H3C

H3C

H3C

CH2

H


23

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-3,5-

dione], 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.


24

O O

H3CO

HO

OCH3

OH

O O

H3CO

HO

OCH3

OH

O

H3CO

HO

OCH3

OH

O

H3CO

HO

OCH3

OH

O

H3CO

HO

OCH3

OH

O

H3CO

HO

OCH3

OH

O OH

H3CO

HO

OCH3

OH

(i)

(i)

(ii)

(iii)

51 54

55

56

57

58

59

Chemically it is a bis-α,β-unsaturated β-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

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 µM,

respectively) while the control drug, pentamidine, displayed an EC50 of 16 ± 2 µM. The

most active curcumin analog (58) exhibited activity of 2.7 ± 0.7 and 4.6 ± 0.7 µM 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


25

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

β-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-diene-

3-one (BDMPP) (60) and 2,6-bis((3-methoxy-4-hydroxyphenyl)-methylene)-

cyclohexanone (BMHPC) (61) were synthesized by introduction of pentenone system


26

O

R2

R1

O

R1

R2 R2

R1

O

R2

R1R1

R2

O

O

62. R1= H, R2= OH63. R1= OCH3, R2= OH64. R1= OC2H5, R2= OH

65. R1= H, R2= OH66. R1= OCH3, R2= OH67. R1= OC2H5, R2= OH

Conc. HCl

Conc. HCl

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 µg ml–1

, 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 µg ml–1

).

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


27

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 Claisen-

Schmidt 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-4’-

methoxychalcone (70) (DMC), a naturally occurring chalcone, against promastigotes and

intracellular amastigotes of Leishmania amazonensis, with 50% effective doses of 0.5

and 24 µg ml−1

, 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 µg 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


28

or bromine group were found selectively active against the parasite as compared with

DMC.95

Aiming to develop new antileishmanial lead compounds, novel sulfonamide 4-

methoxychalcone 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


29

chalcones.96

Except compound 79e sulfonamide 4-methoxychalcone derivatives

displayed more potential inhibitory activity (IC50 = 3.5 ± 0.6 to 8.6 ± 0.4 µM) than that of

4-methoxychalcone (79) (IC50 = 16.6 ± 1.6 µM). These compounds were more active

than pentamidine isothionate (IC50 = 19.6 µM) but were less active than amphotericin B

(0.3 ± 0.02 µM). 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.

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 (80c–e) with IC50 values less than 1.0 µM

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 β-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

OH

OCH3

OH

OCH3

CHO

O

H3CO CHO

O

H3CO COCH3

O

H3CO

O

O

H3CO

O

R

R

R

80,81a = H; 80,81b = 2-Cl; 80,81c = 3-Cl; 80,81d = 4-Cl; 80,81e = 2,4-Cl2

c

e

b

d

a80a-e (Type A)

81a-e (Type B)


30

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 µg/mL) as compared to the reference drug Amphotericin B

(IC50 = 0.56 ± 0.20 µg/mL). These quinoline-based chalcones were synthesized by

condensing formylquinolines with diverse acetylthiophenes.

Reagents and conditions: (i) AcOH, H3PO4, reflux, 4–6 h; (ii) POCl3, DMF, 800C; (iii) ArCOCH3, NaOH,

rt, 2 h.

N ClH3C

H

O

SH

HH3C

N ClH3C

H

O

H S

CH3

N ClH3C

H

O

H SCH3

HH

(i)

(i)

(ii)

(i)

(i)

(i)

(i)

82b 82c 82d

Proposed stereo-,electronic and/or steric properties (i) electronic effect (attractive forces), (ii) stericeffect

(IC50 = 0.83 ± 0.05 g/mL) (IC50 = 0.74 ± 0.31 g/mL) (IC50 = 0.62 ± 0.24 g/mL)


31

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 µg/mL respectively).

Encouraged with the antileishmanial profile of several chalcone derivatives some

novel dihydro-α-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 µg/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.

O

O

R3

R2

R1

R4

KF / Al2O3

Microwave

O R1

R2

R3

R4

O

O

N

CHO

OOHC OH

KF / Al2O3

Microwave

KF / Al2O3

Microwave

N

O

OH

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

84

85

86


32

Recently we have reported synthesis and antileishmanial potential of α- and β-

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 µM concentration with IC50 value of 15.3 ± 2.2 µM and 11.6 ±

2.2 µM respectively as compared to reference drugs miltefosine (IC50 = 8.6 ± 0.4 µM)

and miconazole (IC50 = 5.4 ± 1.5 µM).

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.


33

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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-Frӧhner, C. R.; de Oliveira, K. N.; Gaspar-Silva, D.; Pacheco, L. K.;

Joussef, A. C.; Steindel, M.; Simões, 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.

98 Rizvi, S. U. F.; Siddiqui, H. L.; Ahmad, M. N.; Ahmad, M.; Bukhari, M. H. Med Chem

Res 2012, 21, 1322.

99 Suryawanshi, S. N.; Chandra, N.; Kumar, P.; Porwal, J.; Gupta, S. European Journal of

Medicinal Chemistry 2008, 43, 2473.

100 Suryawanshi, S. N.; Tiwari, A.; Kumar, S.; Shivahare, R.; Mittal, M.; Kant, P.; Gupta,

S. Bioorganic & Medicinal Chemistry Letters 2013, 23, 2925.

Chapter 2.1

Design, Synthesis and Bioevaluation of

Novel Terpenyl Heterocycles

Chapter 2.1 Design, Synthesis and bioevaluation of novel terpenyl heterocycles

39

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

host’s 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 1.5–2.0

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 HIV–Leishmania

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 (phylum-

Sarcomastigophora, order-Kinetoplastida and family-Trypanosomatidae). Leishmania

parasites are dimorphic organisms, i.e., with two morphological forms in their life cycle:


40

amastigotes in the mononuclear phagocytic system of the mammalian host, and

promastigotes in the digestive organs of the vector.4 The promastigotes are ~ 20 µm long

and 1.5-3.00 µm 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 µm

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 fly’s 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.


41

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-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,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)


42

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 methylene-

tetrahydrofolate, 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


43

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.

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.

HN

N NH

N

H2N

O

NH

O

NH

CO2H

CO2H

HN

N NH

HN

H2N

O

NH

O

NH

CO2H

CO2H

Dihydrofolate Reductase

NADPH NADP

Dihydrofolate Tetrahydrofolate

O

OH

OP-O

O-

O

N

NH

O

O

methyene

tetrahydrofolatedihdrofolate

tetrahydrofolate

DHFR

glycine

serine

Thymidylate synthase

O

OH

OP-O

O-

O

N

NH

O

O

Me

dUMP TMP


44

2.1.3 CHEMISTRY

2.1.3.1 Synthesis of β-ionone based 1,3,5-trisubstituted-4,5-dihydropyrazoles (4a-j)

The synthesis of β-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 β-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.

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 β-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 138-140°C. The structure was assigned on the basis

of mass, IR, 1H and

13C NMR spectra.

O

+

CHO

R1

R2

R3

R4

O

R1

R2

R3

R4

1 2 3a-j

(i)

(ii)

1

23

4

5

1'

2'3'

4'

5' 6'

7'

8' 9'

1''

2'' 3''

4''

5''

6''

R1

R2

R3

R4

N N

4a-j

1

34

5

2

3a-j


45

Table 2.1.1: Substitution pattern in aryl ring of compounds 3a-j and 4a-j.

Compound

3, 4 R

1 R

2 R

3 R

4

a NO2 H H H

b H H NO2 H

c H NO2 H H

d H H OCH3 OCH3

e H OCH3 OCH3 OCH3

f Cl H H H

g H H Cl H

h H Cl H H

i H H F H

j H H OBn 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 100°C 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 90-93°C.

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.


46

2.1.3.2 Synthesis of α-ionone based 1,3,5-trisubstituted-4,5-dihydropyrazoles (8a-e)

The synthesis of α-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 α-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).

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).

O

+

CHO

R1

R2

R3

R4

O

R1

R2

R3

R4

5 7a-e

R1

R2

R3

R4

8a-e

N N

(i)

(ii)

12

34

5

6

7a-e


47

Table 2.1.2: Substitution pattern in aryl ring of compounds 7a-e, 8a-e and 9a-e.

Compound

7, 8 and 9 R

1 R

2 R

3 R

4

a NO 2 H H H

b H H NO 2 H

c H H F H

d H H OCH 3 OCH 3

e H OCH3 OCH 3 OCH 3

2.1.3.3 Synthesis of α-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.

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 didn’t 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 (δ 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.

R1

R2

R3

R4

8a-e

N N

R1

R2

R3

R4

9a-e

N N1 122

3 34 4

55 Ag2O, EtOH

reflux, 18 h


48

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/100µL/well) in RPMI-1640 containing 10% foetal calf serum and the plates were

incubated at 37ºC in a CO2 incubator. After 24 h, the medium was replaced with fresh

medium containing stationary phase promastigotes (4 x105/100µL/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 µM conc.

after replacing the previous medium and the plates were incubated at 37ºC in a CO2

incubator for 72 h. At the end of the incubation, the supernatants were removed and 50

µL 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) (1×105cells /100µl/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 µM concentration, and were incubated at 37ºC 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 µL of MTT

reagent (5mg/ml) in PBS medium, followed by syringe filtration was added to each well

and incubated at 37°C for 2 hours. At the end of the incubation period, the supernatant

were removed and 150 µL 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


49

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.


50

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.

In Vitro

Antiamastigote

Activity

IC50(µM)

Cytotoxicity

CC50 (µM)

Selectivity

Index (S.I.)

CC50/IC50

In vivo Activity

% Inhibition ± S.D.

(50mg/Kg×5 days, i.p.

dose)

4a >20 - - -

4b >40 - - -

4c 16.95 279.49 16.48 7.13 ± 8.95

4d 7.49 220.66 29.46 80.80± 11.91

4e >20 - - -

4f >20 - - -

4g >20 - - -

4h >40 - - -

4i >40 - - -

4j >40 - - -

8a >20 - - -

8b >40 - - -

8c >40 - - -

8d >20 - - -

8e >20 - - -

9a >40 - - -

9b >40 - - -

9c >40 - - -

9d >20 - - -

9e >40 - - -

Miltefosine 12.50 3.23 0.26 95.28±2.49

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.


51

The dihydropyrazoles (4a-j) prepared from β 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 µM 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 µM, 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 α 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 kg–1

×5 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.


52

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

13C NMR (in

CDCl3) spectra (chemical shift in δ, 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 β-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, 60-

120 mesh).

2.1.7.2 (1E,4E)-1-(2-nitrophenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4-dien-

3-one (3a)

Yield: 62%. M.p. 117-119°C. IR (KBr, cm–1

) 3093, 2923, 1668, 1611, 1568,

1519, 1351. 1H NMR (CDCl3, 300 MHz) δ 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, H-

5 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

C

NMR (CDCl3, 75 MHz) δ 18.86, 21.92, 2×28.86, 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.


53


3-one (3b)


) 2924, 1668, 1610, 1510, 1337. 1H

NMR (CDCl3, 300 MHz) δ 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) δ 18.82, 21.95, 2×28.87, 33.87, 34.18, 39.85,

2×124.16, 2×128.79, 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.


3-one (3c)

Yield: 16%. M.p. 97-98˚C. IR (KBr, cm–1

) 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, CH2-

3'), 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, H-

4''), 8.38 (s, 1H, H-2''). 13

C NMR (CDCl3, 75 MHz) δ 18.84, 21.94, 2×28.88, 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)penta-

1,4-dien-3-one (3d)

Yield: 38%. Oil. IR (Neat, cm–1

) 3020, 2936, 1642, 1603, 1514, 1218. 1H NMR

(CDCl3, 300 MHz) δ 1.10 (s, 6H, CH3-8' and CH3-9'), 1.46-1.52 (m, 2H, CH2-5'), 1.60-

1.66 (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 H-

5''), 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) δ 18.91, 21.90, 2×28.88, 33.69, 34.18, 39.81,


54

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-1-

enyl)penta-1,4-dien-3-one (3e)

Yield: 44%. Oil. IR (Neat, cm–1

) 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, 2×OCH3), 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). 13

C NMR (CDCl3,

75 MHz) δ 18.89, 21.89, 2×28.87, 33.71, 34.18, 39.83, 2×56.17, 60.96, 2×105.48,

125.46, 129.10, 130.42, 136.56, 136.80, 140.21, 142.78, 143.13, 2×153.43, 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-1,4-

dien-3-one (3f)


) 2933, 1660, 1600, 1566, 1441. 1H



= 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''), 7.40-

7.45 (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). 13

C NMR (CDCl3, 75 MHz) δ 18.89, 21.92, 2×28.88, 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.


dien-3-one (3g)


) 3036, 2960, 2929, 1654, 1597, 1491,

1448. 1H NMR (CDCl3, 300 MHz) δ 1.11 (s, 6H, CH3-8' and CH3-9'), 1.45-1.52 (m, 2H,


55

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

C

NMR (CDCl3, 75 MHz) δ 18.88, 21.91, 2×28.87, 33.76, 34.18, 39.83, 126.05, 2×129.17,

2×129.39, 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.


dien-3-one (3h)


) 3020, 2926, 1643, 1578, 1466. 1H



= 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'', H-

6'', H-5 and H-1). 13

C NMR (CDCl3, 75 MHz) δ 18.88, 21.90, 2×28.87, 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-1,4-

dien-3-one (3i)

Yield: 39%. M.p. 65-66°C. IR (neat, cm–1

) 2933, 1665, 1600, 1503, 1453, 1415.

1H NMR (CDCl3, 300 MHz) δ 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 H-

5''), 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) δ 18.89, 21.86, 2×28.86, 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.


56

2.1.7.11 (1E,4E)-1-(4-(benzyloxy)phenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-

1,4-dien-3-one (3j)


) 3038, 2923, 1662, 1603, 1567, 1509,


(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) δ

18.94, 21.90, 2×28.90, 33.68, 34.19, 39.83, 70.10, 2×115.27, 123.70, 2×127.49, 127.89,

128.17, 2×128.68, 129.77, 2×130.04, 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.

1H NMR (CDCl3, 300 MHz) δ 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).

13C NMR (CDCl3, 75 MHz) δ 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.


57


4,5-dihydro-1H-pyrazole (4b)


-1) 3031, 2927, 1597, 1506, 1341.

1H

NMR (CDCl3, 300 MHz) δ 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) δ 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.


4,5-dihydro-1H-pyrazole (4c)


-1) 3061, 2933, 1597, 1528, 1500, 1456,

1342. 1H NMR (CDCl3, 300 MHz) δ 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) δ 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-1-

enyl)vinyl) -4,5-dihydro-1H-pyrazole (4d)


-1) 2923, 1597, 1498, 1455, 1234, 1026.

1H NMR (CDCl3, 300 MHz) δ 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,


58

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-1-

enyl)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)


-1) 3054, 2930, 1596, 1502, 1463.

1H


(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) δ 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.


4,5-dihydro-1H-pyrazole (4g)


-1) 3059, 2922, 1595,1495.

1H NMR

(CDCl3, 300 MHz) δ 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). 13

C NMR (CDCl3, 75


59

MHz) δ 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.


4,5-dihydro-1H-pyrazole (4h)


-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) δ 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)


-1) 3035, 2924, 1596, 1503.

1H NMR



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) δ 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.


60

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)


-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).

13C NMR (CDCl3, 75 MHz) δ 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)


-1) 3029, 2923, 1598, 1518, 1345.

1H

NMR (CDCl3, 300 MHz) δ 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). 13

CNMR (CDCl3, 75 MHz) δ 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,


61

75.15; H, 7.03; N, 10.11; Found: C, 75.12; H, 7.01; N, 10.13.


4,5-dihydro-1H-pyrazole (8b)


-1) 3035, 2923, 1600, 1509, 1343.

1H


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) δ 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.


4,5-dihydro-1H-pyrazole (8c)


) 2957, 1600, 1501, 1438. 1H NMR (CDCl3, 300

MHz), δ 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.


enyl)vinyl)-4,5-dihydro-1H-pyrazole (8d)


-1) 2957, 1596, 1495, 1450, 1232, 1025.

1H

NMR (CDCl3, 300 MHz) δ 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) δ 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,


62



2.1.7.28 (E)-1-phenyl-5-(3,4,5-trimethoxyphenyl)-3-(2-(2,6,6-trimethylcyclohex-2-

enyl)vinyl)-4,5-dihydro-1H-pyrazole (8e)


) 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). 13

C NMR (CDCl3, 75 MHz) δ 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)


) 3029, 2922, 1596, 1528, 1454, 1356. 1H NMR (CDCl3,

300 MHz) δ 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.


63


1H-pyrazole (9b)


-1) 3020, 2925, 1600, 1521, 1450, 1346.

1H

NMR (CDCl3, 300 MHz) δ 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). 13

C NMR (CDCl3,

75 MHz) δ 22.98, 23.12, 26.94, 27.80, 31.48, 32.54, 54.75, 105.70, 121.47, 122.65,

2×123.77, 2×125.26, 128.00, 2×129.17, 2×129.27, 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.


1H-pyrazole (9c)


-1) 2928, 1592, 1501, 1434.

1H NMR

(CDCl3, 300 MHz) δ 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.


enyl)vinyl)-1H-pyrazole (9d)


-1) 2927, 1592, 1239, 1206.

1H NMR

(CDCl3, 300 MHz) δ 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). 13

C

NMR (CDCl3, 75 MHz) δ 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,


64

7.51; N, 6.51.

2.1.7.34 (E)-5-(3,4,5-trimethoxyphenyl)-1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-

enyl)vinyl)-1H-pyrazole (9e)


-1) 2932, 1590, 1500, 1461, 1241, 1128.

1H NMR (CDCl3, 300 MHz) δ 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) δ 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.


65

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: 13

C NMR of compound 3d at 300 MHz (CDCl3)


66

Figure 2.1.5: 1H NMR of compound 4d at 300 MHz (CDCl3)

Figure 2.1.6: 13

C NMR of compound 4d at 300 MHz (CDCl3)


67

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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

70

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 antimicrobial

7 as

well as antileishmanial activity.8



71

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 potential

9 was

reported in 1981 and later years.10,11

Antifungal agents containing triazole ring inhibit the

Leishmania amastigotes growth by preventing the 14α-demethylation of lanosterol and

effectively block synthesis of ergosterol.12

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

N

N

N

OHN

N

N

F F

NN

N

O

OO N N N

N

N

O

Cl

Cl

O

N N O

O

O

N

N

Cl

Cl

H

Itraconazole

H

KetoconazoleFluconazole

Cl

Cl

ON

N

ClCl

Miconazole



72

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 β and α 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, 13

C 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 13

C NMR at δ 188.6 indicating the presence of a

keto carbonyl function. Its 1H NMR revealed, besides aromatic proton resonances in the

region δ 7.67-8.56, two β-protons of α,β-unsaturated carbonyl moiety showed up as

doublets at 7.61 and 7.48 with trans olefinic-H coupling constant J = 16 Hz and two α-

protons appeared as doublets at δ 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 δ 8.02 and δ 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 ring’s protons were

appeared at δ 8.08 and 8.51. Finally, mass spectra of compounds 3a and 6a showed also

well defined [M+1] ions.



73

Scheme 2.2.1: Synthesis of triazole integrated phenyl heteroterpenoids.

O

N

N

N

O

O

O

N

N

N

O

N

N

N

6

N N

NN

N

R

(a) R = H(b) R = F

R

NH

NH2

R= H, F

3(a) R = H(b) R = F

R= H, F

R

NH

NH2

N N

NN

N

R

(i)

(i)

(ii)

(ii)

1

2

4

5

4-(1H-1,2,4-triazol-1-yl)benzaldehyde

1

23

4

5

1'2'

3'

4'

5'

6'

7'

8'

9'

1''2''

3''

4''5''

6''1'''

2'''

3'''

4'''5''

Reagents and conditions: (i) Cetyltrimethyl ammonium bromide (CTABr), NaOH, H2O, rt, 24

h; (ii) EtOH, reflux, 8 h.



74

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 µM concentration against intracellular leishmania parasite in

vitro. Compound 2, β ionone based triazole integrated chalcone, and compound 6b, α

ionone based triazole integrated 4-fluorophenyl pyrazoline, showed marginal in vitro

activity. Compound 3b, β ionone based triazole integrated 4-fluorophenyl pyrazoline, and

compound 5, α ionone based triazole integrated chalcone, exhibited good antileishmanial

activity with IC50 12.4 and 11.6 µM respectively. However, Compound 3a, β ionone

based triazole integrated phenyl pyrazoline, and compound 6a, α ionone based triazole

integrated phenyl pyrazoline, were found to show significant activity with IC50 6.4, 9.2

µM 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 4-

flurophenyl hydrazine hydrochloride. This decrease in activity of α, β 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.



75

Table 2.2.1: In vitro antileishmanial activity of triazole integrated phenyl

heteroterpenoids against intracellular amastigotes and their cytotoxicity.

Compound No.

In vitro Antiamastigote activity Cytotoxicity

CC50 (µM)

Selectivity

Index (SI) % Inhibition at

40 µM

IC50 (µM)

2 100 15.3 ± 2.2 72.4 ± 5.8 5

3a 100 6.4 ± 1.2 112.4 ± 10.9 18

3b 98.65 12.4 ± 2.0 58.6 ± 4.9 5

5 98.18 11.6 ± 1.6 38.5 ± 5.1 3

6a 98.03 9.2 ± 1.7 296 ± 15.9 32

6b 90.54 16.9 ± 3.1 72.2 ± 8.4 4

Miltefosine 100 8.6 ± 0.4 54.7 ± 6.8 6

Miconazole 100 5.4 ± 1.5 37.4 ± 4.1 7

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.



76

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 (octanol−water 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.



77

Table 2.2.2: Molinspiration calculation of molecular properties for the Lipinski Rule.

Compound nViol MW miLogP nON nOHNH natoms nrotb

Acceptable

range ≤1 ≤500 ≤5 ≤10 ≤5 ─ ─

2 0 347.462 4.22 4 0 26.0 5

3a 1 437.591 6.051 5 0 33.0 5

3b 1 455.581 6.215 5 0 34.0 5

5b 0 347.462 3.9 4 0 26.0 5

6a 1 437.591 5.731 5 0 33.0 5

6b 1 455.581 5.895 5 0 34.0 5

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 µM 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.



78

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

13C NMR (in CDCl3) spectra

(chemical shift in δ, ppm downfield from TMS) were recorded on Bruker Advance DRX-

300 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)-5-2,6,6−trimethyl

cyclohex-1-enyl) penta-1,4-dien-3-one (2)

A mixture of β 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): δ 1.05 (s, 6 H, CH3-8’ and CH3-9’), 1.41-1.45 (m, 2H, CH2-5’), 1.55-

1.61 (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): δ 18.86, 21.89, 2×28.87, 33.76, 34.18, 39.85,

2×120.08, 126.64, 129.35, 2×129.63, 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.



79

2.2.7.2 (E)-1-(4-(1-phenyl-3-(2-(2,6,6-trimethylcyclohex-1-enyl)vinyl)-4,5-dihydro-

1H-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 O

C; IR (KBr, cm-1

): 2927, 1597 (C=N), 1504 (C=C); 1H NMR

(CDCl3, 300 MHz): δ 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): δ 19.08, 21.79, 2×28.92, 33.30,

34.13, 39.72, 42.39, 63.44, 2×113.34, 119.32, 2×120.91, 123.43, 125.32, 2×127.41,

127.60, 2×129.00, 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)-4,5-

dihydro-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 (2×30 ml). The

combined organic extract was washed with water (2×25 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%).

Mp: 199-200 O

C; IR (KBr, cm-1

): 2926, 1604 (C=N), 1508 (C=C); 1H NMR

(CDCl3, 300 MHz): δ 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,



80

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): δ 19.07, 21.74, 2×28.90, 33.29, 34.13, 39.72, 42.62, 64.19, 114.47,

114.57, 115.34, 115.64, 119.97, 2×120.91, 125.20, 2×127.48, 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-2-

enyl)penta-1,4-dien-3-one (5).

A mixture of α 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): δ 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): δ 22.92, 23.10, 26.90, 27.95, 31.25, 32.75,

54.67, 2×120.13, 122.83, 125.72, 2×129.72, 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.



81

2.2.7.5 (E)-1-(4-(1-phenyl-3-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)-4,5-dihydro-

1H-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 (2×25 ml). The combined

organic extract was washed with water (2×25 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%).

Mp: 189-190 O

C; IR (KBr, cm-1

): 2919, 1601 (C=N), 1506 (C=C); 1H NMR


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

C

NMR (CDCl3, 75 MHz): δ 2×23.00, 26.81, 27.88, 31.31, 32.54, 42.70, 54.76, 63.48,

2×113.34, 119.33, 2×120.88, 121.62, 124.57, 2×127.37, 2×128.97, 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)-4,5-

dihydro-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 (2×25 ml). The

combined organic extract was washed with water (2×25 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%).



82

Mp: 210-212 O

C; IR (KBr, cm-1

): 2920, 1614 (C=N), 1515 (C=C); 1H NMR


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): δ 2×22.96, 26.81, 27.82,

31.32, 32.54, 42.94, 54.76, 64.22, 114.49, 114.56, 115.31, 115.61, 119.96, 2×120.89,

121.66, 124.46, 2×127.45, 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.



83

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: 13

C NMR of compound 2 at 75 MHz (CDCl3)



84

Figure 2.2.5: 1H NMR of compound 3a at 300 MHz (CDCl3)

Figure 2.2.6: 13

C NMR of compound 3a at 75 MHz (CDCl3)



85

2.2.9 REFERENCES

1 Alvar, G.; Croft, S.; Olliaro, P. Adv. Parasitol. 2006, 61, 223.

2 Croft, S. L., Sundar, S.; Fairlamb, A. H. Clin Microbiol Rev. 2006, 19, 111.

3 Nielsen, S. F.; Christensen, S. B.; Cruciani, G.; Kharazmi, A.; Liljefors, T. J. Med.

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.

2010, 20, 3169.

7 Ozdemir, A.; Zitouni, G. T.; Kaplancıklı, 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,

S. Bioorg. Med. Chem. Lett. 2012, 22, 6559.

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

86

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.



87

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 α, β, and γ. 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.



88

Figure 3.1: Design and Synthesis of heteroretinoid (4).

CO2H

Hydrophobic part

Linking part

Carboxylate partATRA

Introduction of ionone ashydrophobic part

Introduction of isoxazolering as linker

O N

OH

O

Heteroretinoid (4)

Amide derrivatives of 4(5a-j)

O N

R

O

Carboxylate part



89

3.4 CHEMISTRY

The synthetic procedures adopted to obtain the target compounds are depicted in

scheme-3.1. The reaction of α 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



90

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 Reaction conditions Compound,%

4 H2N

Oxalyl chloride/DMF/DCM, rt, 2 h 5a, 78

4 H2N Cl

Oxalyl chloride/DMF/DCM, rt, 2 h 5b, 90

4 H2N OCH3

Oxalyl chloride/DMF/DCM, rt, 2 h 5c, 83

4 H2N

Oxalyl chloride/DMF/DCM, rt, 2 h 5d, 70

4 HN O

Oxalyl chloride/DMF/DCM, rt, 2 h 5e, 65

4 HN

Oxalyl chloride/DMF/DCM, rt, 2 h 5f, 96

4 HN NH

Oxalyl chloride/DMF/DCM, rt, 2 h 5g, 90

4 HN N

Oxalyl chloride/DMF/DCM, rt, 2 h 5h, 93

3 NH2NH2 Ethanol, reflux, 3 h 5i, 78

3 NH2OH THF, rt, 17 h 5j, 84



91

O O O

OEt

O

O N

OEt

O

O N

OH

O

1 2

34

(5a) R =

(5b) R =

(5c) R =

(5d) R =

(5e) R =

(5f ) R =

HN

HN Cl

HN

N O

N

N NH

N N

(5i) R = NHNH2

(5j) R = NHOH

(5k)R = N(Na)NH2

(5l) R = Na(Na)OH

HN OCH3

Diethyl oxalate

NaH, toluene

NH2OH.HCl, ethanol

O N

R

O

5

Oxalyl chloride, DMFD.C.M, amine

NaOH, ethanol

(5g) R =

(5h) R =

Scheme-3.1: Synthesis of isoxazole containing heteroretinoid and its amide derivatives



92

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 kg–1

× 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 (octanol−water 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.



93

Table-3.2: Antileishmanial activity of synthetic compounds against Leishmania

donovani in hamsters.

Compound No. Dose (mg/kg) × 5

days

% inhibition on Day-

7 P.T.a

2 50 Inactive

3 50 76

4 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 89.0±8.31

Paromomycin 50 46.7±9.82

aP.T: post treatment,

bND: not done,

cSSG: reference drug (Sodium stibogluconate)



94

Table-3.3: Molinspiration Calculation of molecular Properties for the Lipinski Rule

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.

Compound

no. nViol MW miLog P nON nOHNH natoms nrotb

Acceptable

range ≤1 <500 ≤5 <10 <5 - -

2 0 292.375 2.431 4 0 21 7

3 0 289.375 4.89 4 0 21 5

4 0 261.321 4.254 4 1 19 3

5a 1 336.435 5.573 4 1 25 4

5b 1 370.88 6.251 4 1 26 4

5c 1 366.461 5.629 5 1 27 5

5d 1 342.483 5.781 4 1 25 4

5e 0 330.428 3.966 5 0 24 3

5f 1 328.456 5.028 4 0 24 3

5g 0 329.444 3.416 5 1 24 3

5h 1 405.542 5.709 5 0 30 4

5i 0 275.352 3.065 5 3 20 3

5j 0 276.336 3.558 5 2 20 3



95

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-1

×5 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.



96

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

13C NMR (in

CDCl3) spectra (chemical shift in δ, 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-5-

enoate (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), α 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 ½ 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): δ 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); 13

C NMR (CDCl3, 75 MHz): δ 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-3-

carboxylate (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



97

and residue was taken up in ethyl acetate (20 ml). The organic extract was washed with

water (2×20 ml), brine solution (2×20 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): δ 0.87 (s, 3H), 0.93 (s, 3H), 1.20-1.27 (m, 1H), 1.38-

1.52 (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); 13

C NMR (CDCl3, 75 MHz): δ

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-3-

carboxylic 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 (2×15 ml), brine (2×15 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,


1.51 (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): δ 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



98

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 (2×15 ml), brine (2×15 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-3-

carboxamide (5a)


-1): 3315, 3030, 2922, 2863, 1687, 1601,

1542, 1446, 1314; 1H NMR (CDCl3, 200 MHz): δ 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); 13

C NMR (CDCl3, 50

MHz): δ 23.32, 23.44, 27.27, 28.14, 31.66, 33.11, 55.21, 100.15, 116.66, 2×120.56,

122.98, 125.27, 2×129.47, 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)isoxazole-

3-carboxamide (5b)


-1): 3364, 3032, 2964, 2921, 1686,

1596, 1533, 1450, 1393, 1352; 1H NMR (CDCl3, 200 MHz): δ 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


23.28, 23.42, 27.24, 28.16, 31.62, 33.14, 55.25, 99.96, 116.53, 2×121.63, 123.04,

2×129.58, 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.



99

3.8.7 (E)-N-(4-methoxyphenyl)-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)

isoxazole-3-carboxamide (5c)


-1): 3350, 3109, 3033, 2962, 1668,

1535, 1450, 1403, 1247; 1H NMR (CDCl3, 300 MHz): δ 0.89 (s, 3H), 0.95 (s, 3H), 1.20-

1.28 (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); 13

C NMR (CDCl3, 75 MHz):

δ 22.91, 23.02, 26.84, 27.77, 31.20, 32.71, 54.79, 55.47, 99.68, 2×114.27, 116.21,

2×121.76, 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-3-

carboxamide (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): δ 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), 3.90-

3.97 (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); 13

C NMR (CDCl3, 50 MHz): δ 23.28, 23.41, 2×25.19, 25.85,

27.25, 28.10, 31.64, 2×33.08, 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-3-

yl)methanone (5e)


): 3010, 2953, 2865, 1738, 1630, 1484, 1392,


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


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]+;



100


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)


): 2939, 1638, 1490, 1447, 1396, 1250, 1225,


1.54 (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-3-

yl)methanone (5g)


): 3411, 3027, 2925, 1631, 1484, 1444, 1391,


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): δ 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-2-

enyl)vinyl)isoxazol-3-yl)methanone (5h)


): 3017, 2961, 2920, 2865, 1639, 1600, 1495,


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); 13

C NMR (CDCl3, 50 MHz): δ 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, 2×117.15, 121.05, 122.91, 2×129.66, 132.69,





101

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

carbohydrazide (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 (2×15 ml). The

organic extract was washed with water (2×15 ml), brine (2×15 ml), dried (Na2SO4). The

solvent was removed under vacuum. The crude product thus obtained was

chromatographed (SiO2, 60-120 mesh).


): 3315, 2958, 2920, 2865, 1674, 1534, 1444,


1.54 (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): δ 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 (2×15 ml) and washed with H2O (2×15

ml), brine (2×15 ml), dried (Na2SO4). The solvent was concentrated to dryness to give

desired product.


): 3450, 3014, 2964, 2922, 2862, 1739, 1644,

1488, 1444, 1394, 1228, 1115, 1035, 986; 1H NMR (CDCl3, 200 MHz): δ 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



102

= 9 Hz, 1H), 5.51 (brs, 1H), 6.32-6.52 (m, 3H); 13

C NMR (CDCl3, 50 MHz): δ 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%.



103

3.9 SPECTRA OF SOME SELECTED COMPOUNDS

Figure 3.3:

1H NMR of compound 2 at 300 MHz (CDCl3)

Figure 3.4: 13




104

Figure 3.5: 1H NMR of compound 3 at 300 MHz (CDCl3)

Figure 3.6: 13




105

Figure 3.7: 1H NMR of compound 4 at 300 MHz (CDCl3)

Figure 3.8: 13




106

Figure 3.9: 1H NMR of compound 5c at 300 MHz (CDCl3)

Figure 3.10: 13

C NMR of compound 5c at 75 MHz (CDCl3)



107

3.10 REFERENCES 1 Fre´zard, F.; Demicheli, C.; Ribeiro, R. R. Molecules 2009, 14, 2317.

2 Moore E. M.; Lockwood, D. N. J. Global Infect. Dis. 2010, 2, 151.

3 Maltezou, H. C. J. Biomed. Biotechnol. 2010, 2010:617521.

4 (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.

6 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.

8 (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.

10Sairafianpour, 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.



108

14

Simoni, Daniele; Invidiata, F. P.; Rondanin, R.; Grimaudo, S.; Cannizzo, G.; Barbusca,

E.; Porretto, F.; D’Alessandro, 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.

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

109

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



110

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 parasite’s

mitochondrial inner membrane.11

Conversely, certain platinum complexes, such as

(2,2’:6’2”-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



111

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.



112

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 α 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-2-

enyl) 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 δ 186 (C=O of bisbenzylidine ketone function) and 169 (C=O of

heteroretinoid moiety) in their 13

C NMR spectra.



113



114

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 µM), better than the reference drug sodium

stibogluconate (IC50 = 53.12 µM) and miltefosine (IC50 = 8.10 µM).

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 µM.

Although to a lesser extent but on further substitution diminution of biological activity

took place (7d, IC50 = 4.70 µM).

Attachment of benzyloxy group at 4 position of phenyl ring rendered the molecule

inactive (7e, IC50 > 40 µM). 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 µM).

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.



115

Table 4.1: Antileishmanial activity and cytotoxicity of synthetic hybrids (7a-7i).

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.

Entry R1 R

2 R

3 R

4

Antiamastigote

activity

(IC50 in µM)

Cytotoxicity

(CC50 in µM)

Selectivity

Index (SI)

7a H H OMe H >40 NT NA

7b OMe H H H >20 NT NA

7c H H OMe OMe 3.75 ± 0.31 45.78 ± 5.71 12.20

7d H OMe OMe OMe 4.70 ± 0.48 25.32 ± 3.40 5.38

7e H H OBn H >40 NT NA

7f H H OBn OMe 5.02 ± 0.49 >400 >79.68

7g H H OH OMe 1.83 ± 0.21 23.45 ± 3.82 12.81

7h H H OH OEt 6.10 ± 0.62 27.65 ± 4.1 4.53

7i H H Cl H >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



116

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 µM, 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 µM) (Table 4.1).

4.6 CONCLUSION

Within this chapter, we present the efficient synthesis of a series of heteroretinoid-

bisbenzylidine 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.



117

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 (60–120 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-5-

enoate (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), α 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 ½ h. Organic layer was extracted and concentrated.

Crude was purified by column chromatography.


): 2960, 2925, 2865, 1735, 1610, 1446, 1367,


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); 13

C NMR (CDCl3, 75 MHz): δ 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.



118

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

carboxylate (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 (2×20 ml),

brine solution (2×20 ml), dried (Na2SO4) and solvent was removed under vacuum. The

crude product thus obtained was chromatographed (SiO2, 60-120 mesh).


-1): 2957, 1740, 1642, 1573, 1448, 1240,


1.52 (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); 13


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-3-

carboxylic 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 (2×15 ml), brine (2×15 ml), dried (Na2SO4) and

solvent was removed under vacuum. The crude product thus obtained on crystallization

gave 4 as a white crystalline solid.


-1): 3409, 2959, 1720, 1645, 1448,


1.51 (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): δ 22.85, 22.99, 26.80, 27.74, 31.17, 32.70, 54.81, 100.52, 115.98,



119

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 in-

vacuo. 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-2-

enyl)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): δ 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): δ 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



120

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-trimethylcyclohex-

2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7a)

Yield: 32%; Mp: 118-120 °C; IR (KBr, cm-1

): 2950, 2842, 1653, 1599, 1508,


1.20 (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): δ 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-trimethylcyclohex-

2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one(7b)


): 2959, 1637, 1484, 1400, 1216, 1116,


1.50 (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): δ 22.9, 23.0, 26.8, 27.7, 31.2, 32.6, 43.9, 47.3, 54.7,

2×55.4, 100.6, 2×110.8, 116.0, 120.3, 120.4, 122.4, 123.5, 130.0, 130.3, 2×130.9, 131.1,

2×131.7, 2×132.2, 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.



121

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)


): 2930, 2846, 1647, 1594, 1514,


1.20 (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); 13

C NMR (CDCl3,

75 MHz): δ 22.8, 23.0, 26.8, 27.7, 31.2, 32.6, 43.4, 47.8, 54.7, 4×55.9, 101.5, 111.1,

111.2, 113.2, 113.7, 115.9, 122.5, 124.2, 124.9, 2×127.5, 2×129.1, 132.2, 137.8, 138.2,

140.8, 2×149.0, 2×150.5, 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)


): 2940, 2843, 1657, 1577, 1507,



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); 13

C NMR (CDCl3, 75 MHz): δ 22.8,

23.0, 26.8, 27.7, 31.2, 32.6, 43.1, 48.0, 54.7, 2×56.2, 2×56.3, 2×60.9, 101.6, 2×107.8,

2×108.2, 115.9, 122.5, 129.8, 129.9, 2×130.1, 132.1, 138.2, 138.4, 139.5, 139.7, 141.0,

2×153.2, 2×153.3, 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)


): 2918, 1640, 1596, 1505, 1291,


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); 13

C NMR (CDCl3,

75 MHz): δ 22.9, 23.0, 26.8, 27.7, 31.2, 32.6, 43.8, 47.6, 54.7, 2×70.1, 101.3, 4×115.2,



122

116.0, 122.5, 4×127.4, 2×128.1, 6×128.6, 2×129.1, 2×129.3, 2×132.2, 132.4, 132.6,

2×136.4, 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-(2,6,6-

trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7f)


): 2918, 2862, 1648, 1593, 1511,



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): δ 22.8, 23.0, 26.8, 27.7, 31.2, 32.6, 43.5, 47.9, 54.7,

56.0, 56.1, 2×70.8, 101.5, 113.4, 113.5, 113.7, 114.2, 116.0, 122.5, 124.0, 124.8,

4×127.2, 2×127.9, 2×128.0, 4×128.6, 2×129.2, 132.2, 136.5, 137.8, 138.2, 140.8,

3×149.5, 2×149.7, 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-(2,6,6-

trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7g)


): 2929, 1638, 1513, 1430, 1215,


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); 13


22.8, 22.9, 26.8, 27.6, 31.2, 32.6, 43.5, 47.8, 54.7, 2×56.0, 101.3, 112.9, 113.3, 2×114.9,

115.9, 122.5, 124.9, 125.5, 2×126.9, 2×128.8, 132.2, 138.1, 138.6, 140.9, 2×146.7,

2×147.5, 158.5, 159.7, 169.1, 186.3; ESI-MS m/z: 611 [M+H]+; Anal. Calcd for




123

4.7.13 (3E,5E)-3,5-bis(3-ethoxy-4-hydroxybenzylidene)-1-(5-((E)-2-(2,6,6-

trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7h)


): 2921, 2855, 1634, 1586, 1512,



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): δ 2×14.8, 22.8, 23.0, 26.8, 27.6, 31.2, 32.6, 43.5,

47.8, 54.7, 2×64.7, 101.4, 113.6, 114.0, 2×114.8, 115.9, 122.5, 124.9, 125.4, 2×126.9,

2×128.8, 132.2, 138.0, 138.6, 140.8, 2×145.9, 2×147.5, 158.6, 159.6, 169.0, 186.2; 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, 6.69; N, 4.51.

4.7.14 (3E,5E)-3,5-bis(4-chlorobenzylidene)-1-(5-((E)-2-(2,6,6-trimethylcyclohex-2-

enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (7i)


): 2927, 2856, 1657, 1590, 1525,

1439, 1217, 769; 1H NMR (CDCl3, 300 MHz): δ 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.



124

4.8 SPECTRA OF SOME SELECTED COMPOUNDS

Figure 4.3:

1H NMR of compound 6 at 300 MHz (CDCl3)

Figure 4.4: 13




125

Figure 4.5: 1H NMR of compound 7c at 300 MHz (CDCl3)

Figure 4.6: 13

C NMR of compound 7c at 75 MHz (CDCl3)



126

Figure 4.7: 1H NMR of compound 7f at 300 MHz (CDCl3)

Figure 4.8: 13

C NMR of compound 7f at 75 MHz (CDCl3)



127

Figure 4.9: 1H NMR of compound 7g at 300 MHz (CDCl3)

Figure 4.10: 13

C NMR of compound 7g at 75 MHz (CDCl3)



128

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.

3 Desjeux, P.; Clinics in Dermatology 1996, 14, 417.

4 Alvar, J.; Yactayo, S.; Bern, C. Trends Parasitol. 2006, 22, 552.

5 Croft, S. L.; Olliaro, P. Clin. Microbiol. Infect. 2011, 17, 1478.

6 (a) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461; (b) Rocha, L. G.;

Almeida, J. R. G. S.; Macêdo, R. O.; Barbosa-Filho, J. M. Phytomedicine , 2005, 12, 514.

7 Cavalli, A.; Bolognesi, M. L. J. Med. Chem. 2009, 52, 7339.

8 (a) Meunier, B. Acc. Chem. Res.; 2008, 41, 69; (b) Camps, P.; Formosa, X.; Galdeano,

C.; Gómez, T.; Muñoz-Torrero, D.; Scarpellini, M.; Viayna, E.; Badia, A.; Clos, M. V.;

Camins, A.; Pallàs, 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.; Yánez, 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,

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129

14

Suryawanshi, S. N.; Tiwari, A.; Chandra, N.; Ramesh, Gupta, S. Bioorg. Med. Chem.

Lett. 2012, 22, 6559.

Publications

130

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, 2925–2928.

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, 248–251.

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, 6728–6730.

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,

6559–6562.

Publications

131

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)-4,5-

dihydro-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 “5

th NIPER (RBL)-CSIR-CDRI Symposium-

2013” 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)

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