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PHYTOCHEMICAL AND PHARMACOLOGICAL INVESTIGATION

OF MEDICINAL PLANTS AND SYNTHESIS OF SOME BIOACTIVE

MOLECULES

THESIS

SUBMITTED FOR THE AWARD OF THE DEGREE OF

Doctor of Philosophy

IN

Chemistry

BY

SHAISTA AZAZ

Under Supervision of

Dr. Mehtab Parveen, Professor

DEPARTMENT OF CHEMISTRY

ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2017

ACKNOWLEDGEMENTS

This is probably the most difficult part of this work to write, because printed

words fail to express the depth of feelings hidden behind them and convey the

actual extent of influence others cast on one’s work or life. First and foremost,

I would like to express my deepest faith in Almighty ALLAH, the

Omnipotent, the Omnipresent for it is indeed His blessings which provided

me enough zeal to complete this work.

A debt of gratitude to my supervisor, Prof. Mehtab Parveen,

Department of Chemistry, Aligarh Muslim University, Aligarh, for her

generosity, faith, encouragement and excellent guidance, that has been

instrumental in completion of this thesis. The discussions with her were

always a lifeline for new thoughts. Her valuable suggestions added to the

output of the research, inspired me to have a better grip over my research

field.

I am thankful to Prof. M. Shakir, Chairman, Department of

Chemistry, Aligarh Muslim University, Aligarh, for providing the necessary

research facilities.

It is a matter of great contentment for me to recompense my sincere and

deep sense of appreciation to Prof. M. Mushfiq (Ex. Chairman),

Department of Chemistry, A.M.U., Aligarh for his momentous advices and

suggestions during my research. I cordially express my gratefulness to Dr.

Mahboob Alam, Division of Bioscience, Dongguk University, South Korea,

for his persistent encouragement and support.

I gratefully acknowledge the financial help from UGC in the form of

Maulana Azad National fellowship.

Thanks are due to USIF, Aligarh Muslim University, Aligarh for SEM

analysis and SAIF Punjab University, Chandigarh, for providing XRD, NMR

and Mass spectral facilities. Thanks also to CEMDRX Physics Department,

University of Coimbra, Portugal, for carrying out X-ray analysis.

I render my special thanks to my lab colleagues Dr. Ali M. Malla,

Dr. Faheem Ahmad and Afroz Aslam for their help, and coordination

extended by them.

Thanks to all my dear friends Zeba Nasir, Shama Yasmeen,

Nausheen Bano, Mumtaz Riyasat, Saira Khatoon, Shahnaz Rahman, Saba Samreen Ansari, Mohd. Danish, Mohd. Asif,

Khairu, Abad Ali, and my seniors, Dr. Saima Tarannum, Dr.

Nayeem Ahmad, and all those who have always stood by me.

Putting into words is rather restrictive, still I would like to owe my deep

sense of gratitude to my respected and beloved father Late Azaj Ahmad

and mother Aquila Khatoon with whom my learning process started and

who encouraged me to proceed along a path where untiring determination

stretches its arms towards perfection. I would like to express my profound

gratitude to all the family members especially my sisters and brother,

Shagufta Azaz, Shaheen Azaz, Shadab Ali and my brother-in-law

Dr. Mohd. Khalid who have always been a source of inspiration and

encouragement for me. A Special thanks to my cousin, Shahid jamal who

has been played a role of elder brother for me during this journey.

Shaista Azaz

CANDIDATE’S DECLARATION

I, Shaista Azaz, Department of Chemistry certify that the work embodied in

this Ph.D thesis is my own bonafide work carried out by me under the supervision of

Prof. Mehtab Parveen at Aligarh Muslim University, Aligarh. The matter embodied

in this Ph.D. thesis has not been submitted for the award of any other degree.

I declare that I have faithfully acknowledged, given credit to and referred to

the research workers wherever their works have been cited in the text and the body of

the thesis. I further certify that I have not willfully lifted up some other’s work, para,

text, data, result, etc. reported in the journals, books, magazines, reports, dissertations,

thesis, etc., or available at web-sites and included them in this Ph.D. thesis and cited

as my own work.

Date: (Signature of the candidate)

SHAISTA AZAZ (Name of the candidate)

Certificate from the Supervisor

This is to certify that the above statement made by the candidate is correct to the best of our knowledge.

Signature of the Supervisor:

Name & Designation: Dr. Mehtab Parveen, Professor Department: CHEMISTRY

(Signature of the Chairman of the Department with seal)

COURSE/ COMPREHENSIVE EXAMINATION/ PRE-SUBMISSION SEMINAR COMPLETION CERTIFICATE

This is to certify that Miss. Shaista Azaz, Department of Chemistry has

satisfactorily completed the course work/comprehensive examination and pre-

submission seminar requirement which is part of her Ph.D. programme.

Date: ……………. (Signature of the Chairman of the Department)

COPYRIGHT TRANSFER CERTIFICATE

Title of the Thesis : PHYTOCHEMICAL AND PHARMACOLOGICAL

INVESTIGATION OF MEDICINAL PLANTS AND

SYNTHESIS OF SOME BIOACTIVE MOLECULES

Candidate’s Name: SHAISTA AZAZ

Copyright Transfer

The undersigned hereby assigns to the Aligarh Muslim University, Aligarh,

copyright that may exist in and for the above thesis submitted for the award of

Ph.D. degree.

(Signature of the Candidate)

Note: However, the author may reproduce or authorize others to reproduce material

extracted verbatim from the thesis or derivative of the thesis for author’s

personal use provided that the source and the university’s copyright notice are

indicated

Abstract

1

The present thesis entitled “Phytochemical and Pharmacological Investigation of

Medicinal Plants and Synthesis of Some Bioactive Molecules” has been categorized

into six chapters. The first chapter includes a critical review of literature about natural

product and green chemistry. The second and third chapter comprises of

phytochemical investigation of two plants i.e. Iphiona scabra and Garcinia nervosa

and their biological studies of isolated compounds and cytotoxicity of crude aqueous

and ethanolic extracts of their leaves. The fourth, fifth and sixth chapter deals with the

catalyst promoted synthesis of a series of biological active compounds viz. hydrazone,

pyrazolone and acrylonitrile derivatives, respectively.

Chapter-1: General Introduction

Plants have been an integral part of the ancient culture of India, China and Egypt as

medicine. Medicinal plants have been a valuable source of therapeutic agents, and

also used as folk medicines for various human ailments. The therapeutic properties of

plants have attracted considerable attention of chemists to explore the chemical

behaviour of the medicinal plants, which led to the new branch of chemistry known as

Phytochemistry, deals with extraction, isolation, purification and identification of

compounds from plants sources.

Over the past decade, green chemistry has demonstrated how fundamental scientific

methodologies can protect human health and the environment in an economically

beneficial manner. Significant progress is being made in several key research areas,

such as catalysis, the design of safer chemicals and environmentally benign solvents,

and the development of renewable feed stocks. The area of catalysis is sometimes

referred to as a “foundational pillar” of green chemistry. Catalytic reactions often

reduce energy requirements and decrease separations due to increased selectivity.

This chapter highlights the importance of natural product with special reference to

flavonoids, alkaloids, terpenoids and coumarins as well as green chemistry. It also

embodies the recent literature of isolation of novel compounds from the plant sources,

general introduction about spectroscopic techniques used for the characterization of

compounds and biological assay techniques used in the thesis.

Abstract

2

Chapter-2: Phytochemical Constituents of Iphiona scabra Leaves and Their

Biological Studies

This chapter deals with the phytochemical investigation of the leaves of Iphiona

scabra (Family: Compositae) i.e. extraction, isolation and characterization of isolated

compounds. Herein, five compounds have been isolated from this plant by column

chromatography followed by fractional crystallization. Among the isolated

compounds, compound 2 is novel and has not been reported from any natural sources

and also has no report of its chemical synthesis so far, while compound 5 has been

isolated first time from natural source. The structures of all the isolated compounds

have been elucidated on the basis of chemical and physical evidences (elemental

analysis, UV, 1H NMR,

13C NMR and MS). Molecular structure of compound 2 and 3

is further authenticated by X-ray crystallographic studies. Interaction of compounds 3-

5 with DNA is extensively studied using various biophysical techniques. The multi-

technique approach employed in the present study reveals that compounds 3-5 are

found to interact with DNA via non-intercalation mode. Moreover, aqueous and

ethanolic extracts are screened for in vivo cytotoxicity against brine shrimp nauplii by

lethality bioassay and exhibit potential cytotoxic activity. Following are the five

isolated compounds.

Is-1: n-tetracosan-1-ol (1)

Petroleum ether-benzene (9:1) fraction yielded compound 1, white solid

Yield 120 mg

Molecular formula C24H50O

Melting point 78 °C

Molecular mass 354

Is-2: 6-hydroxy-8,11,11-trimethylbicyclo[7.2.0]undec-4-ene-4-carboxylic acid (2)

Petroleum ether-benzene (8:2) fraction yielded compound 2, white shining crystals

Yield 35 mg

Molecular formula C15H24O3

Melting point 131-132 °C

Molecular mass 252

Abstract

3

Is-3: 4,6-Diacetylresorcinol (3)

Petroleum ether-benzene (6:4) fraction yielded compound 3, colourless shining

crystals

Yield 40 mg



Molecular mass 194

Is-4: 4-Acetylresorcinol (4)

Blank benzene fraction yielded compound 4, yellowish brown solid

Yield 60 mg



Molecular mass 152

Is-5: 1,2-bis(o-tolyloxy)ethane (5)

Benzene-ethyl acetate (8:2) fraction yielded compound 5, bright yellow solid

Yield 60 mg



Molecular mass 242

Journal of Asian Natural Products Research, 2014, 16, 406-411

Abstract

4

Chapter-3: Phytochemical Constituents of Garcinia nervosa Leaves and Their

Biological Studies

This chapter reports the isolation and structure elucidation of compounds isolated

from the leaves of Garcinia nervosa (Family: Guttiferae). Herein, five compounds

have been isolated from this plant by column chromatography followed by

crystallization. The structures of all the isolated compounds have been elucidated on

the basis of chemical and physical evidences (elemental analysis, UV, IR, 1H NMR,

13C NMR and MS) and comparison of their spectral data with literature compounds.

Molecular structure of compound 1 and 2 is further authenticated unambiguously by

X-ray crystallographic analysis. Among the isolated compounds, compound 1 is novel

and has not been reported from any natural source. Interaction study of compound 1

and 2 with DNA has also been carried out using different biophysical techniques.

Further all the isolated compounds along with extracts were also test for antioxidant

study. The ethanolic and aqueous extracts of leaves were screened for in vivo

cytotoxicity assay and shows significant cytotoxic activity. Following are the five

isolated compounds.

Gn-1: 5,7-dihydroxy-3-(3'-hydroxy-4',5'-dimethoxyphenyl)-6-methoxy-4H-

chromen-4-one (1)

Petroleum ether-benzene (6:4) fraction furnishes compound 1, dark yellowish crystals

Yield 50 mg



Molecular mass 361

Gn-2: 1-(2,5-dioxoimidazolidin-4-yl)urea (DL-allantoin) (2)

Petroleum ether-benzene (6:4) fraction furnishes compound 2, white solid

Yield 30 mg

Molecular formula C4H6N4O3


Molecular mass 158

Abstract

5

Gn-3: 4-methoxychalcone (3)

Benzene-ethyl acetate (8:2) fraction furnishes compound 3, cream coloured crystals

Yield 60 mg



Molecular mass 238

Gn-4: 2',4',4-trihydroxychalcone (4)

Benzene-ethyl acetate (6:4) afforded compound 4, light yellow crystals

Yield 85 mg



Molecular mass 256

Gn-5: 5,7-dihydroxy-3-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one (5)


Yield 70 mg



Molecular mass 300

Journal of Photochemistry and Photobiology, B: Biology, 2017, 167, 176-188

Abstract

6

Chapter-4: [Et3NH][HSO4] catalyzed facile synthesis of Hydrazone derivatives

In the present chapter, a series of hydrazone analogues 3(a-o) were synthesized, via a

solvent-free facile nucleophilic addition between hydrazine hydrate 2 and

appropriately substituted aromatic aldehydes 1(a-o) (Scheme 1). The molecular

structure of compound (3f) was well supported by single crystal X-ray

crystallographic analysis and also verified by DFT calculations. This new synthetic,

eco-friendly, sustainable protocol resulted in a remarkable improvement in the

synthetic efficiency (90-98%) yield, high purity, using [Et3NH][HSO4] as a catalyst

and an environmentally benign solvent eliminating the need for a volatile organic

solvent and additional catalyst. This ionic liquid is air and water stable and easy to

prepare from cheap amine and acid. The present methodology is a green protocol

offering several advantages such as, excellent yield of products, minimizing

production of chemical wastes, shorter reaction profile, mild reaction conditions,

simple operational procedure, easy preparation of catalyst and its recyclability up to

five cycles without any appreciable loss in catalytic activity. The synthesized

compounds were also tested for antioxidant activity.

Scheme 1 Synthetic pathway for the synthesis of hydrazone derivatives 3(a-o)

New Journal of Chemistry, 2015, 39, 469- 481

Abstract

7

Chapter-5: SiO2/ZnBr2 mediated synthesis of Pyrazolone derivatives in water

under Microwave irradiation

In the present chapter, a library of 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one

derivatives 4(a-s) have been synthesized (Scheme 1), via a facile environmentally

benign cyclization reaction, involving various aromatic aldehydes with

ethylacetoacetate and phenylhydrazine/2,4 dinitrophenylhydrazine (2,4-DNP) to yield

target 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one derivatives in excellent yields (94-

98%) with high purity, employing SiO2/ZnBr2 as a recyclable Lewis acid catalyst in

water under microwave heating. The molecular structure of compounds 4a and 4d

were well supported by single crystal X-ray crystallographic analysis. The present

protocol is more practical, efficient, eco-friendly and compatible as compared to

existing methods. We have tested our synthesized compounds for antioxidant studies.

Scheme 2 Synthetic scheme for the synthesis of pyrazolone derivatives 4(a-s)

RSC Advances, 2016, 6, 148- 162

Abstract

8

Chapter-6: Silica bonded N-(propylcarbamoyl)sulfamic acid (SBPCSA)

mediated synthesis of Acrylonitrile derivatives

In the present chapter, a library of acrylonitrile derivatives 3(a-o) have been

synthesized using an environmentally benign heterogenous catalyst silica bonded N-

(propylcarbamoyl)sulfamic acid (SBPCSA) under solvent free conditions. Herein,

each series was typically accessed via Knoevenagel condensation between 2-

thiopheneacetinitrile (2) and appropriately substituted heterocyclic/aromatic

aldehydes 1(a-o) to yield target acrylonitrile derivatives in excellent yields (92-98%)

with high purity. It possesses environmentally benign properties such as non-toxicity,

biocompatibility, recyclability, physiological inertness, inexpensiveness and thermal

stability. The structure and morphology of the catalyst was established on the basis of

FT-IR, powder X-ray diffraction (XRD), scanning electron microscopy (SEM) and

energy-dispersive X-ray spectroscopy (EDX). The synthesized compounds have been

characterized by FT-IR, 1H NMR,

13C NMR, MS and elemental analysis and were

also screened for antioxidant studies.

Scheme 3 Synthetic scheme for the synthesis of acrylonitrile derivatives 3(a-o)

Catalysis Letters, 2016, 146, 1687-1705

Abbreviations

AcOH Acetic acid Aq. Aqueous FT-IR Fourier transform infrared CDCl3 Deuterated chloroform CHCl3 Chloroform DCM Dichloromethane DFT Density functional theory DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide EtOH Ethanol MeOH Methanol g Gram h Hours Hz Hertz IL Ionic liquid mg Milligram mL Millilitre mmol. Millimole meq. Milliequivalent M.p. Melting point min Minute µg Microgram Fig. Figure NMR Nuclear magnetic resonance MW Microwave M.W Molecular weight RT Room temperature THF Tetrahydrofuran TLC Thin layer chromatography

Summary

Summary

i

The present thesis entitled “Phytochemical and Pharmacological Investigation of

Medicinal Plants and Synthesis of Some Bioactive Molecules” has been categorized

into six chapters. The first chapter includes a critical review of literature about natural

product and green chemistry. The second and third chapter comprises of

phytochemical investigation of two plants i.e. Iphiona scabra and Garcinia nervosa

and their biological studies of isolated compounds and cytotoxicity of crude aqueous

and ethanolic extracts of their leaves. The fourth, fifth and sixth chapter deals with the

catalyst promoted synthesis of a series of biological active compounds viz. hydrazone,

pyrazolone and acrylonitrile derivatives, respectively.

Chapter-1: General Introduction

Plants have been an integral part of the ancient culture of India, China and Egypt as

medicine. Medicinal plants have been a valuable source of therapeutic agents, and

also used as folk medicines for various human ailments. The therapeutic properties of

plants have attracted considerable attention of chemists to explore the chemical

behaviour of the medicinal plants, which led to the new branch of chemistry known as

Phytochemistry, deals with extraction, isolation, purification and identification of

compounds from plants sources.

Over the past decade, green chemistry has demonstrated how fundamental scientific

methodologies can protect human health and the environment in an economically

beneficial manner. Significant progress is being made in several key research areas,

such as catalysis, the design of safer chemicals and environmentally benign solvents,

and the development of renewable feed stocks. The area of catalysis is sometimes

referred to as a “foundational pillar” of green chemistry. Catalytic reactions often

reduce energy requirements and decrease separations due to increased selectivity.

This chapter highlights the importance of natural product with special reference to

flavonoids, alkaloids, terpenoids and coumarins as well as green chemistry. It also

embodies the recent literature of isolation of novel compounds from the plant sources,

general introduction about spectroscopic techniques used for the characterization of

compounds and biological assay techniques used in the thesis.

Summary

ii

Chapter-2: Phytochemical Constituents of Iphiona scabra Leaves and Their

Biological Studies


scabra (Family: Compositae) i.e. extraction, isolation and characterization of isolated

compounds. Herein, five compounds have been isolated from this plant by column

chromatography followed by fractional crystallization. Among the isolated

compounds, compound 2 is novel and has not been reported from any natural sources

and also has no report of its chemical synthesis so far, while compound 5 has been

isolated first time from natural source. The structures of all the isolated compounds

have been elucidated on the basis of chemical and physical evidences (elemental

analysis, UV, 1H NMR, 13C NMR and MS). Molecular structure of compound 2 and 3

is further authenticated by X-ray crystallographic studies. Interaction of compounds 3-

5 with DNA is extensively studied using various biophysical techniques. The multi-

technique approach employed in the present study reveals that compounds 3-5 are

found to interact with DNA via non-intercalation mode. Moreover, aqueous and

ethanolic extracts are screened for in vivo cytotoxicity against brine shrimp nauplii by

lethality bioassay and exhibit potential cytotoxic activity. Following are the five

isolated compounds.

Is-1: n-tetracosan-1-ol (1)

Petroleum ether-benzene (9:1) fraction yielded compound 1, white solid

Yield 120 mg

Molecular formula C24H50O


Molecular mass 354

Is-2: 6-hydroxy-8,11,11-trimethylbicyclo[7.2.0]undec-4-ene-4-carboxylic acid (2)

Petroleum ether-benzene (8:2) fraction yielded compound 2, white shining crystals

Yield 35 mg



Molecular mass 252

OH

O OH

OH

Summary

iii

Is-3: 4,6-Diacetylresorcinol (3)

Petroleum ether-benzene (6:4) fraction yielded compound 3, colourless shining

crystals

Yield 40 mg



Molecular mass 194

Is-4: 4-Acetylresorcinol (4)

Blank benzene fraction yielded compound 4, yellowish brown solid

Yield 60 mg



Molecular mass 152

Is-5: 1,2-bis(o-tolyloxy)ethane (5)

Benzene-ethyl acetate (8:2) fraction yielded compound 5, bright yellow solid

Yield 60 mg



Molecular mass 242

Journal of Asian Natural Products Research, 2014, 16, 406-411

OO

OHHO

O

OH

OH

CH3O O

CH3

Summary

iv

Chapter-3: Phytochemical Constituents of Garcinia nervosa Leaves and Their

Biological Studies

This chapter reports the isolation and structure elucidation of compounds isolated

from the leaves of Garcinia nervosa (Family: Guttiferae). Herein, five compounds

have been isolated from this plant by column chromatography followed by

crystallization. The structures of all the isolated compounds have been elucidated on

the basis of chemical and physical evidences (elemental analysis, UV, IR, 1H NMR, 13C NMR and MS) and comparison of their spectral data with literature compounds.

Molecular structure of compound 1 and 2 is further authenticated unambiguously by

X-ray crystallographic analysis. Among the isolated compounds, compound 1 is novel

and has not been reported from any natural source. Interaction study of compound 1

and 2 with DNA has also been carried out using different biophysical techniques.

Further all the isolated compounds along with extracts were also test for antioxidant

study. The ethanolic and aqueous extracts of leaves were screened for in vivo

cytotoxicity assay and shows significant cytotoxic activity. Following are the five

isolated compounds.

Gn-1: 5,7-dihydroxy-3-(3'-hydroxy-4',5'-dimethoxyphenyl)-6-methoxy-4H-

chromen-4-one (1)

Petroleum ether-benzene (6:4) fraction furnishes compound 1, dark yellowish crystals

Yield 50 mg



Molecular mass 361

Gn-2: 1-(2,5-dioxoimidazolidin-4-yl)urea (DL-allantoin) (2)

Petroleum ether-benzene (6:4) fraction furnishes compound 2, white solid

Yield 30 mg

Molecular formula C4H6N4O3


Molecular mass 158

O

OOHH3CO

HO

OH

OCH3OCH3

HN

NH

O

NH

ONH2

O

Summary

v

Gn-3: 4-methoxychalcone (3)

Benzene-ethyl acetate (8:2) fraction furnishes compound 3, cream coloured crystals

Yield 60 mg



Molecular mass 238

Gn-4: 2',4',4-trihydroxychalcone (4)


Yield 85 mg



Molecular mass 256

Gn-5: 5,7-dihydroxy-3-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one (5)


Yield 70 mg



Molecular mass 300

Journal of Photochemistry and Photobiology, B: Biology, 2017, 167, 176-188

O

OCH3

O

HO

OH

OH

OHO

OH O OH

H3CO

Summary

vi

Chapter-4: [Et3NH][HSO4] catalyzed facile synthesis of Hydrazone

derivatives

In the present chapter, a series of hydrazone analogues 3(a-o) were synthesized, via a

solvent-free facile nucleophilic addition between hydrazine hydrate 2 and

appropriately substituted aromatic aldehydes 1(a-o) (Scheme 1). The molecular

structure of compound (3f) was well supported by single crystal X-ray

crystallographic analysis and also verified by DFT calculations. This new synthetic,

eco-friendly, sustainable protocol resulted in a remarkable improvement in the

synthetic efficiency (90-98%) yield, high purity, using [Et3NH][HSO4] as a catalyst

and an environmentally benign solvent eliminating the need for a volatile organic

solvent and additional catalyst. This ionic liquid is air and water stable and easy to

prepare from cheap amine and acid. The present methodology is a green protocol

offering several advantages such as, excellent yield of products, minimizing

production of chemical wastes, shorter reaction profile, mild reaction conditions,

simple operational procedure, easy preparation of catalyst and its recyclability up to

five cycles without any appreciable loss in catalytic activity. The synthesized

compounds were also tested for antioxidant activity.


N

HN

HAr

ArNH2NH2.H2O [Et3NH][HSO4] (20 mol%)

Solvent Free, 120 oCH

O

Ar

1(a-o) 2 3(a-o)

N FNO2

O2NOCH3

H3CO

OCH3

H3CO

H3CO

OHH3CO OH

OH

HO

HO

OH

O

O

O

OF

O

OH3C

O

OBr

O

O

NH2

H3C

CH31a 1b 1c 1d 1e

1f 1g 1h 1i 1j

1k 1l 1m 1n 1o

Summary

vii

New Journal of Chemistry, 2015, 39, 469- 481

Chapter-5: SiO2/ZnBr2 mediated synthesis of Pyrazolone derivatives in water

under Microwave irradiation


derivatives 4(a-s) have been synthesized (Scheme 1), via a facile environmentally

benign cyclization reaction, involving various aromatic aldehydes with

ethylacetoacetate and phenylhydrazine/2,4 dinitrophenylhydrazine (2,4-DNP) to yield

target 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one derivatives in excellent yields (94-

98%) with high purity, employing SiO2/ZnBr2 as a recyclable Lewis acid catalyst in

water under microwave heating. The molecular structure of compounds 4a and 4d

were well supported by single crystal X-ray crystallographic analysis. The present

protocol is more practical, efficient, eco-friendly and compatible as compared to

existing methods. We have tested our synthesized compounds for antioxidant studies.


NN

O

HHN NH2 H3C

O

OC2H5

O SiO2/ZnBr2 (0.1 g)

MW, 60oC(10-15min)

H

O

Ar Ar1(a-n) 2(a) 3 4(a-n)

Water (10 mL)

NN

O

HHN NH2 H3C

O

OC2H5


MW, 60oC(10-15min)

H

O

Ar ArWater (10 mL)

NO2

O2N

NO2

O2N1(o-s) 2(b) 3 4(o-s)

NH

NH

NH

HO H3C

O

O

O

O

O

O

O

O

H3C F

Br

OCH3

H3CO O2NNO2

F

H3CO

NH3C

CH3 NO2 OCH3

H3CO

O

O

NH2

FNH3C

CH3

OCH3

H3CO

1a 1b 1c 1d 1e 1f

1g 1h 1i 1j 1k 1l

1m 1n 1o 1p 1q 1r 1s

Summary

viii

RSC Advances, 2016, 6, 148- 162

Chapter-6: Silica bonded N-(propylcarbamoyl)sulfamic acid (SBPCSA)

mediated synthesis of Acrylonitrile derivatives



(propylcarbamoyl)sulfamic acid (SBPCSA) under solvent free conditions. Herein,

each series was typically accessed via Knoevenagel condensation between 2-

thiopheneacetinitrile (2) and appropriately substituted heterocyclic/aromatic

aldehydes 1(a-o) to yield target acrylonitrile derivatives in excellent yields (92-98%)

with high purity. It possesses environmentally benign properties such as non-toxicity,

biocompatibility, recyclability, physiological inertness, inexpensiveness and thermal

stability. The structure and morphology of the catalyst was established on the basis of

FT-IR, powder X-ray diffraction (XRD), scanning electron microscopy (SEM) and

energy-dispersive X-ray spectroscopy (EDX). The synthesized compounds have been

characterized by FT-IR, 1H NMR, 13C NMR, MS and elemental analysis and were

also screened for antioxidant studies.

Scheme 3 Synthetic scheme for the synthesis of acrylonitrile derivatives 3(a-o)

SCN

CN

S

Solvent f ree,

SBPCSA (80 mg)

80 oC

H

20-30 min

O

HAr Ar

1 (a-o) 3 (a-o)2

O

O

O

OH3C

O

OF

O

OBr

O

O

NH2

NNO2

O2N H3C

CH3 OCH3

H3CO

H3CO

OCH3

H3CO

HO

HOOH

OHNH N

HNH

H3C HO

1a 1b 1c 1d 1e

1f 1g 1h 1i 1j

1k 1l 1m 1n 1o

Summary

ix

Catalysis Letters, 2016, 146, 1687-1705

CONTENTS

CHAPTER-1: General Introduction 1-41 1.1. Natural products 1-16 1.2. Green chemistry 16-20 1.3. Methods of chemical analysis 20-28 1.4. Biological studies 28-33 1.5. References 34-41 CHAPTER-2: Phytochemical constituents of Iphiona scabra leaves and their biological studies 42-74 2.1. Introduction 42-43 2.2. Results and discussion 43-65 2.3. Experimental 66-71 2.4. Conclusion 72 2.5. References 73-74 CHAPTER-3: Phytochemical constituents of Garcinia nervosa leaves and their biological studies 75-111 3.1. Introduction 75-76 3.2. Results and discussion 76-101 3.3. Experimental 101-108 3.4. Conclusion 108 3.5. References 109-111 CHAPTER-4: [Et3NH][HSO4] catalysed facile synthesis of Hydrazone derivatives 112-147 4.1. Introduction 112-118 4.2. Results and discussion 118-136 4.3. Experimental 136-144 4.4. Conclusion 145 4.5. References 146-147

CHAPTER-5: SiO2/ZnBr2 mediated synthesis of Pyrazolone derivatives in water under Microwave irradiation 148-184 5.1. Introduction 148-153 5.2. Results and discussion 154-173 5.3. Experimental 173-181 5.4. Conclusion 181 5.5. References 182-184 CHAPTER-6: Silica bonded N-(propylcarbamoyl) sulfamic acid (SBPCSA) mediated synthesis of Acrylonitrile derivatives 185-221 6.1. Introduction 185-189 6.2. Results and discussion 189-210 6.3. Experimental 210-217 6.4. Conclusion 217 6.5. References 218-221

Chapter-1

1

1.1. Natural Products Nature is an ancient pharmacy that used to be the solitary source of therapeutics for

the early eras. Ancient civilizations of both China and India have provided a wealth of

knowledge on the use of traditional medicines. At the beginning of the nineteenth

century, the era of “modern” drugs began. In the 1800s, the most widely used drug in

the world was synthesized by Felix Hoffmann, known as aspirin. In 1805, the first

pharmacologically active compound morphine was isolated by Friedrich Serturner,

from the opium plant.1,2 Subsequently, countless active compounds have been

separated from natural sources. The World Health Organization (WHO) estimated

that 80% of the earth inhabitants mainly depend on traditional medicines for their

health care.3 Medicines, such as anticancer, antihypertensive, and antimigraine have

benefited greatly from natural products.1,4

1.1.1. Sources of natural products

The anecdote of bioactive natural products started more than 100 years ago. Their

usual definition in the widest sense is chemical compounds isolated/derived from the

nature i.e. living organisms such as plants, animals, marines and microorganisms.

These compounds may be derived from primary or rather secondary metabolism of

this organisms.5 Chemistry of natural products is related to the isolation, biosynthesis

and structure elucidation of new products that led to new biological agents. On

account of their chemical diversity and various activities against diseases, they have

been playing an important role in pharmaceutical and agricultural research.6

Approximately half of all new drugs in the time frame reported are of natural origin or

designed on the basis of natural product structure7 and nearly half of the 20 best-

selling non protein drugs are related to natural products.8 The paclitaxel (Taxol), most

widely used breast cancer drug have been isolated from the bark of Taxus brevifolia

(Pacific Yew).9 In 1992, FDA approved Taxol for various uses.10 Arteether,

introduced in 2000, as Artemotil is derived from artemisinin which was first isolated

from the plant Artemisia annua and are both approved antimalarial drugs.11

Grandisines A and B, indole alkaloids were isolated from the leaves of Elaeocarpus

grandis, exhibit binding affinity for the human δ-opioid receptor and are potential

leads for analgesic agents.12 Apomorphine is a derivative of morphine isolated from

the poppy (Papaver somniferum), used to treat Parkinson’s disease.13 Tubocaurarine,

used as a muscle relaxant in surgical operations was isolated from Chondrodendron

Chapter-1

2

tomentosum.14 Beside this, several other plant-derived compounds presently used in

clinical trials are; Digitoxin, Podophyllotoxin, Compothecin,

Vincristine, Vinblastine, Epipodophyllotoxin, Bruceatin, Flavopiridol etc.15-17

1.1.2. Classes of natural products

Plants produce an enormous variety of natural products with highly diverse structures.

The compounds are classified into four different groups according to their

biosynthetic origin: flavonoids, alkaloids, terpenoids and coumarins.

Flavonoids are polyphenolic compounds, present ubiquitously throughout the

plant kingdom. They embrace a wide range of substances, which possess 2-phenyl-

benzyl-γ-pyrone, in their structural framework. Their biosynthesis pathway (part of

the phenylpropanoid pathway) begins with the condensation of one p-coumaroyl-CoA

molecule with three molecules of malonyl-CoA to yield chalcone, catalyzed by

chalcone synthase (CHS). The next step is isomerization of chalcone to flavanone by

chalcone isomerase (CHI).14 From this step onwards, the pathway branches to

different flavonoid classes, including aurones, dihydrochalcones, flavanonols

(dihydroflavonols), isoflavones, flavones, flavonols, leucoanthocyanidins,

anthocyanins and proanthocyanidins.

Alkaloids are defined as heterocyclic nitrogen compounds, biosynthesized from

amino acids. Many other substances, however, that do not exactly match this rule are

classified as alkaloids, either for historical reasons or due to their bioactivities. With

currently more than 12,000 known structures, alkaloids represent one of the biggest

group of natural products.18 Due to this large number and the high structural diversity,

it is impossible to give a comprehensive summary of all different types of alkaloids.

Terpenoids, also named isoprenoids, are the largest class of natural products in

plants and comprise more than 40,000 different structures. They are derived from

five-carbon isoprene units, and according to the number of isoprene molecules

incorporated, they can be classified into hemiterpenes (C5), monoterpenes (C10),

sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40), and

polyterpenes.14 In plants, terpenoids originate from two different biosynthetic

routes19: the cytosolic mevalonic acid (MVA) pathway and the plastid-located

desoxyxylulose phosphate (DXP) pathway (also called methylerythritol phosphate or

MEP pathway). Both biosynthetic routes yield the activated isoprene units

dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), which are

Chapter-1

3

joined by head-to-tail or tail-to-tail linkage and subsequently can undergo cyclization

and other modifications e.g., oxidations or rearrangements. While hemiterpenes,

monoterpenes, diterpenes, and tetraterpenes are derived from the DXP pathway,

triterpenes, steroids, and certain sesquiterpenes originate from mevalonic acid.

Literature survey reveals that the natural products possess inherent wide range

of pharmacological activities such as anticancer, antimicrobial, antioxidant and

antiviral activities. Some of the examples have been summarized in Table (1-4).

Table 1 Biological activities of naturally occurring Flavonoids

Name Structure Source

Anticancer

Baicalin O

OOHHO

O

OHHO

O

OH

OH

Scutellaria

baicalensis20

Eryvarin

O

O

HO

OHO OH

Erythrina

mildbraedii21

Antioxidant

Lanneaflavonol

OO

OOH

OHOCH3

OH

OH

Lannea alata22

Chapter-1

4

Rutin

OHO

OH O

OHOH

OO O

HOOHOH

O

OH

OHOH

Hedysarum

carnosum23

Antimicrobial

Papyriflavonol A

OHO

OH O

OH

OH

OH

Broussnetia

papyrifera24

Sophoraisoflavanone

A

OHO

OH O

OCH3

OH

Echinosophora

koreensis24

Antiviral

Leachianone G OHO

OH O

OH

OH

Morus alba25

Vogelin J OHO

O

OH

O

Ficus virens26

Chapter-1

5

Table 2 Biological activities of naturally occurring Alkaloids

Anticancer

Khasuanine A

NH

O

OH

N O

Melodinus

khasianus27

Sanguinarine NO

O

O

O

Sanguinaria

canadensis28

Antimicrobial

Sampangine N N

O

OCH3

Cananga

odorata29

Isocorydine N

H3CO

H3COHO

H3CO

CH3H

Berberis

microphylla30

Antioxidant

Vindolicine

NMe

N

N

N

Me

MeO

MeO

H

H

H

H COOMe

COOMeOAc

OAcH

HO

Catharanthus

roseus31

Chapter-1

6

Bicculline O

ON

O

O OO

Fumaria

officinalis32

Antiviral

Lanuginosine

NO

O

CH3

O

Magnolia

grandiflora33

Isatibisindosulfonic

acid B NH

NH

O SO3H

Isatis indigotica34

Table 3 Biological activities of naturally occurring Terpenoids

Antimicrobial

Oleananoic acid

acetate

O

OH

O

O

Bougainvillea

glabra35

Rediocide C

OHO

HOCH2OH

O

O

HO

O

OH

H

OO

C6H5OCOH

H H

Trigonostemon

reidioides36

Chapter-1

7

Antiviral

Biperovskatone

H

OO

O HOO

HO

H

Perovskia

atriplicifolia37

Chlorajaponilide A

O O

OH

H

HOH

O

OO

O

O

OO

O

O

Chloranthus

japonicus38

Antioxidant

Artepestrin F O

OHO

OHO

HH

H

Artemisia

rupestris39

Garcinielliptone Q

HO

H

HO

H

Garcinia subellipti

ca40

Anticancer

Helioscopinolide E

OO

CH3

OH

H

Euphorbia

tuckeyana41

Chapter-1

8

Istanbulin A OO

O

OHH

Chloranthus

multistachys42

Table 4: Biological activities of naturally occurring Coumarins

Anticancer

Imperatorin OO OO

Angelica

dahurica43

Osthole OH3CO O

Ferulago

campestris44

Antimicrobial

Anthogenol

O OO

HOH3C

H3CHO

Aegle marmelos45

Agasyllin

O OO

O

OCH3

CH3

Ferulago

campestris46

Antiviral

Inophyllum A O O

O

OOH

H

Calophyllum

inophyllum47

Chapter-1

9

Pseudocordatolide C

O O

O

O

CH3

CH3H3C

HO

Calophyllum

lanigerum48

Antioxidant

Fraxin O OHO

O

H3CO

OOH

HO

HO

HO

Actinidia

deliciosa49

Esculin

O OHO

OOOH

OH

HOHO

Actinidia

chinensis49

A large number of naturally occurring novel compounds of therapeutic importance

have been isolated to every year. Although it is not possible to cover all the literature

related to the isolation of natural products from plant sources, some important, recent

and relevant naturally occurring compounds are as follows:

Flavonoids

Liu et al.50 have isolated three new carboxylated flavonoids, uncinatic acids A-

C (1-3), from the herb of Selaginella uncinata. In addition, the isolates were tested

for their cytotoxicity against A549 and BGC-823 cell lines in vitro.

OO

OOH

HO OO

OOH

HO

O

OHOH

O

O

OHOH

O

1 2

Chapter-1

10

O

OOH

HO

OH

OH

O

3

Zou et al.51 and co-workers isolated six new flavonoids, unciflavones A-F (4-9), from medicinal plant Selaginella uncinata.

O

OOH

HOOH

O

O

OH

O

OOH

HOOH

O

O

OH

4 5

O

OOH

HOOH

O

OH

O

OOH

HOOH

HOOH

O

6 7

O

OOH

HOOH OH

O

O

OOH

HOO OH

O

HO

8 9

Long et al.52 have isolated six new flavonoids, involvenflavones A-F (10-15),

from Selaginella involven. These compounds also exhibited a potent effect against

the injury of human umbilical vein endothelial cell (HUVECs) induced by high

concentrations of glucose in vitro.

Chapter-1

11

O

O

OCH3

H3CO

OH

OH

O

OHO

HOOH

OH

O

O

OCH3

H3CO

OH

OH

O

OHO

HOOH

OH 10 11

O

O

OCH3

H3CO

OH

OH

O

OHO

HOOH

OH

O

O

OH

H3CO

OH

OH

O

OHO

HOOH

OH 12 13

O

O

OH

H3CO

OH

OH

O

OHO

HOOH

OH

O

O

OH

HO

OHOH

14 15

Zou and co-workers53 reported the isolation of six new flavonoids,

seladoeflavones A-F (16-21), from the herbs of Selaginella doederleinii, along with

one known flavonoid (22). In addition, bioassay of the isolates revealed that 20-

22 exhibited moderate cytotoxicity against three human cancer cell lines NCI-H460,

A549, and K562 in vitro with IC50 values ranging from 8.17 to 18.66 μM.

O

O

HO

OH

OH

R

OH

O

O

HO

OH

OH

R

OH

16 R= CH=CHCOCH3; 2,3-dihydro 18 R= CHO; 2,3-dihydro 17 R= CH=CHCOCH3 19 R= CHO 21 R= H; 2,3-dihydro 20 R= COOH; 2,3-dihydro 22 R= H; 2,3-dihydro

Chapter-1

12

Xu et al.54 have isolated two new flavonoids, saniculamins A and B (23 and 24),

together with three known flavonoid derivatives brosimacutin, 6-flavonol, and

eucomol from the whole plants of Sanicula lamelligera.

O

O

HO

OH

OH

O

O

HO

OHOH

23 24

Alkaloids

Sun et al.55 reported the isolation of four new dimeric bromopyrrole alkaloids (26-

29), including hexazosceptrin (25).

NHN

OHNH

H2N NH

NH2

HN

HN

O

O

NH

NH

Br

Br

HN

HN

O

O

NH

NH

Br

Br

O

OCH3

O

OR

25 26 R = H; 27 R = CH3

HN

HN

O

O

NH

NH

Br

Br O

OH

NHN

NH2

HN

HN

O

O

NH

NH

Br

Br

NHN

NH2

N

NHNH2

28 29

Jin and co-woorkers56 have isolated two new imidazole alkaloids, lepidiline C

and D (30 and 31) along with two known imidazole alkaloids (lepidiline A and B),

from the root of Lepidium meyenii.

N N

H

H3C CH3

Cl

H3CO

N N

CH3

H3C CH3

Cl

H3CO

30 31

Chapter-1

13

Chen et al.57 isolated two new alkaloids, nigellisoquinomine (32) and

nigellapyrrolidine (33) along with two known alkaloids, agrocybenine and 4,6,6-

trimethyl-3,4-epoxypiperidin-2-one, from the seeds of Nigella glandulifera Freyn.

Compound 32 exhibited potent protein tyrosine phosphatase 1B (PTP1B) inhibitory

activity with an IC50 value of 3.65±0.08 mM.

NN

OO O

OHOHHO

HO NH O

32 33 Dong and co-workers58 have reported the isolation of two new alkaloids,

dehydrostenine A (34) and B (35), from the roots of Stemona sessilifolia.

N

O

H

H

H

O

N

O

H

H

H

O 34 35

Yang et al.59 have isolated four tropane alkaloids, a new compound triuniamine

A (38) along with previously reported known compounds darlingine (36), 10-

hydroxydarlingine (37) and 2,3-dihydrodarlingine (39) from the stems of Triunia

montana.

O

ON

H

R

CF3CO2H2N O

OHO

CF3CO2O

ON

HCF3CO2

36 R= H; 37 R= OH 38 39

Coumarins

Aminudin et al.60 have reported the isolation four new 4-substituted coumarins,

incrassamarin A (40), B (41), C (42) and D (43) with (7S,8S)-7,8-dihydro-5-hydroxy-

7,8-dimethyl-4-propyl-2H,6H-benzo[1,2-b;5,4-b']dipyran-2,6-dione (44), friedelin,

carpachromene, amentoflavone, epiafzelechin and L-quercitrin from the barks and

leaves of Calophyllum incrassatum. Compound (40) displayed cytotoxic activity

Chapter-1

14

against A-549 cell lines with IC50 87.71 mg/mL and showed inhibition towards a-

glucosidase enzymatic activity with IC50 93.25 mM.

O O O

OO

O O O

OHO

O O O

OHO

40 41 42

O O O

OHO

O O O

OHO

43 44

Wanga et al.61 have been isolated two new dicoumarins, chimsalicifoliusins A

(45) and B (46), a new tricoumarin, chimsalicifoliusin C (47), and nine known

coumarin from Chimonanthus salicifolius. Compounds 1-3 showed modest

cytotoxicity against Hela and HL-60 cell lines, with IC50 values ranging from 14.2 to

29.6 mM, while only chimsalicifoliusin C (47) had the cytotoxicity against PC-3 cell

line.

O

H3CO

HOOCH3

O

O O

H3CO

O

O

HO

OCH3

OOO

OH

OCH3

45 46

O

H3CO

HO O

O O

O O

OCH3

H3CO

O

H3CO

O

47

Bashir et al.62 reported the isolation of two new sesquiterpene coumarins,

fnarthexone (48) and fnarthexol (49), along with three known coumarin derivatives,

Chapter-1

15

conferol, conferone and umbelliferone from the plant Ferula narthex Boiss. Conferol

was found to be the most potent with IC50 value of 11.51, 0.09 mg/mL in vitro

leishmanicidal activity.

OO O

O H

OO OH

HHO

48 49

Dastan and co-workers63 isolated a new disesquiterpene and five sesquiterpene

coumarins from the roots of Ferula pseudalliacea. Compound 50, 54 and 55

displayed the highest potency against HeLa cells with IC50 of 2.2, 6.7, and 4.9 µM,

respectively.

O

OH

H

OO O

O

OO

O O

50 51

O

OO

O O O

OO O

O

52 53

O O

HO

OO O OH

OO

O 54 55

Liu et al.64 isolated two new hexahydrobenzo[c]phenanthridine alkaloids,

ambiguanine H (56) and ambiguanine I (57) together with six known alkaloids (58-

63), from the Corydais ambigua var. amurensis leaves. All the compounds except

Chapter-1

16

compound 61 showed the remarkable protective effect on myocardium ischemia-

hypoxia cells and compound 59 was the most active compound with cell viability of

49.4, 53.3, 68.2 and 84.5%, which were stronger than that of salvia acid B.

N

R2

R1

CH3

R4

OO

H

R3

N

OCH3

OCH3

CH3

H3CO

OO

H

HO

OH 56-62 63

56 R1=R3=R4= OH, R2= OCH3; 57 R1= OCH3, R2=R3=R4= O; 58 R1=R2=R4= OCH3, R3= OAc; 59 R1=R2=R4= OCH3, R3= OH; 60 R1=R2= OCH3, R3=R4=OH;

61 R1=R3= OH, R2=R3= OCH3, 62 R1=R2= OCH3; R3=R4= OH

Sakunpak and co-workers65 isolated two new monoterpene coumarins,

minutin A (64) and minutin B (65), from Micromelum minutum leaves along with

four known coumarins.

OOHOO

O

OH

OO

O

OH

OOH

64 65

1.2. Green Chemistry

The world-wide synthetic community has been already aware about the development

of green chemistry processes, where non-toxic substances can be used and the

generation of waste can be avoided. Green synthesis protocols not only provide

essential atom-economy, energy savings, waste reduction, and easy workup but also

avoid hazardous chemicals.66 Greener aspects in molecular design now invariably

include the use of bio-renewable raw materials in benign reaction media and

recyclable nano-catalysts in atom-economical synthesis as thrust areas.67 This may

encompass an unconventional reaction activation methodology, such as mechano-

chemical mixing, catalysis, solvent less reactions and microwave and ultrasonic

irradiation. The object of green chemistry is not only the development of novel

methods but is to develop alternative sustainable variants to existing ones. Thus, in

order to design new reaction systems satisfying the green chemistry principles, we

have carried out the synthesis of bioactive compounds viz. hydrazones, pyrazolones

Chapter-1

17

and acrylonitriles by employing ionic liquids, microwave and silica supported

catalysts.

1.2.1. Ionic liquids

Ionic liquids (ILs), are ionic salts that are liquids at ambient or below ambient

temperatures have been widely utilized as promising alternatives to hazardous, toxic,

volatile and highly flammable organic solvents. In fact, various attractive and unique

physicochemical properties of ILs such as extremely low vapor pressures, high

salvation interactions with inorganic and organic compounds, excellent thermal and

chemical stabilities, good ionic conductivities and broad electrochemical windows

make ILs attractive candidates for the replacement of volatile organic compounds.

The combination of all these unique properties opens new avenues to an extensive

range of applications, including, organic synthesis and catalysis, extraction, inorganic

synthesis, nanomaterial synthesis, separation, biocatalysis, pharmaceuticals and

polysaccharide dissolution.68

Hu et al.69 reported condensation of Meldrum’s acid with aromatic aldehydes

proceeded efficiently in a reusable ionic liquid, ethylammonium nitrate (EAN), at

room temperature in the absence of any catalyst with high yields.

O

O Ar

H

O

OO

OO

O

Ar H

O EAN, RT

0.5-2 h

66 67 68

Ar = C6H5, p-Me2NC6H4, p-MeOC6H4, p-OHC6H4, 3,4-(OCH2)C6H3, p-ClC6H4, p-NO2C6H4, o-NO2C6H4, 2-Furyl, C6H5CH=CH

Xu et al.70 have introduced, 1-methyl-3-butylimidazolium hydroxide

([bmim]OH) as a novel basic ionic liquid catalyst for the Markovnikov addition of N-

heterocycles to vinyl esters under mild conditions.

N

NH

R2

R1O R3

ON

N

R2

R1

O R3

O[bmim]OH

50 °C, 2-12 h

69 70 71 R1= H, CH3; R2=NO2, H, CH3

R3=CH3, CH3(CH2)2, (CH3)2CH, CH3(CH2)3, CH3(CH2)4, Ph

Chapter-1

18

1.2.2. Microwave assisted synthesis

Among the different aspects of green synthetic methods, synthesis involving

microwave irradiation has gained more popularity as a powerful tool for rapid and

efficient synthesis due to selective and efficient absorption of microwave (MW)

energy by polar molecules.71 Microwave synthesis has considered as a green

technology because it allows solvent-free reactions and low energy consumption

compared to traditional methods. The short reaction time and expanded reaction range

offered by microwave-assisted organic synthesis are suited to the increased demands

in industry. As, there is a requirement in the pharmaceutical industry for a higher

number of novel chemical entities to be produced, which requires chemists to employ

a number of resources to reduce the time for the production of compounds.72

Tu et al.73 synthesize a series of furo[3′,4′ 5,6]pyrido[2,3-d]pyrimidine

derivatives by three-component reactions between an aldehyde, 2,6-

diaminopyrimidine-4(3H)-one, and tetronic acid/indane-1,3-dione, without using any

catalyst in MW.

H

O

HN

N

O

NH2H2N O O

OH2OMW HN

N NH

O

O

H2N

O

R

R

72 73 74 75

R= H, 4-Cl, 4-Br, 4-F, 4-Me, 2-Cl, 2-NO2

Taran et al.74 have reported the use of Cu (I)-species anchored to functionalized

chitosan microspheres to obtain 1,4-substitued triazoles at 150 °C in 15 min.

OON3

OO NNN

Cu (I)MW, 150 oC

76 77 78

Singh et al.75 outlined an efficient one-pot synthesis of substituted pyridines in

high yields using a multicomponent reaction of aromatic aldehydes, malononitrile,

and thiophenol in ethanol using KF/Alumina in MW at 80 oC.

Chapter-1

19

CHO

R3

R2

R1 CN

CN

SH

R4

N CN

CN

NH2

S

R4

R3

R2R1

KF, AluminaMW, 80 oC

79 80 81 82

R1=R3= H, OCH3; R2= H, OCH3, CH3, F, Cl, Br, OH, NO2; R4= H, OCH3, CH3

1.2.3. Silica supported catalysts

Heterogeneous supported catalysts have been gained much attention in recent years,

as they possess a number of advantages.76-78 Immobilization of catalysts on solid

support improves the available active site, stability, hygroscopic properties, handling

and reusability of catalysts79 which all factors are important in industry. Therefore,

use of supported and recoverable catalysts in organic transformations has economical

and environmental benefits. Lewis acid immobilized on solid surface have gained

much attention in organic synthesis, e.g. hydroxyapatite supported Lewis acid catalyst

has been developed for the transformation of trioses in alcohols,86 Nb2O5·nH2O has

been described as heterogeneous catalyst with water tolerant Lewis acid sites,87 silica

gel supported aluminium chloride has been reported for the solvent-free synthesis of

bis-indolylmethanes,88 polystyrene supported Al(OTf)3 was used for the synthesis of

acylals from aldehydes.89

Jetti et al.90 reported silica-bonded N-propyl sulfamic acid (SBNPSA) catalyzed

one-pot three component biginelli condensation of different substituted aromatic

aldehydes with ethyl acetoacetate and urea/thoiurea to the respective 3,4-

dihydropyrimidin-2-(1H)-ones and thiones.

O O

OEt Ar

O

H H2N

X

NH2

SBPSANH

NH

Ar

X

O

EtO

MeEthanol, ref lux

83 84 85 86 SBPSA= silica bonded propyl sulfamic acid

Ar= C6H5, 4-NO2-C6H4, 3-Cl-C6H4, 4-Cl-C6H4, 3-Br-C6H4, 4-OCH3-C6H4, 2,4-Cl-C6H3, 3-NO2-C6H4, 4-F-C6H4; X= O, S

Li et al.91 used silica-supported aluminum chloride for one-pot mannich-type

reactions of acetophenone with aromatic aldehydes and aromatic amines.

Chapter-1

20

O

CH3

O

H NH2

R1R2

SiO2-OAlCl2RT, EtOH

O

NH

R1 R2

87 88 89 90

R1= H, 4-CH3O, 4-CH3, 4-Br, 4-Cl, 4-OH, 4-N(CH3)2; R2= H, 4-CH3, 3-NO2, 4-NO2, 4-Cl, 3-Br, 3-COOH, 4-COOH, 2-Cl, 2-NO2

Bigdeli and coworkers92 successfully synthesized xanthane derivatives using

silica supported perchloric acid (HClO4-SiO2) as a catalyst.

R H

O OH HClO4. SiO2

ClCH2CH2ClO

R2

91 92 93 R= C6H5, 4-BrC6H5, 4-MeOC6H5, 2-MeOC6H5, 4-MeC6H5, 4-ClC6H5, 2-ClC6H5,

4-NO2C6H5, 3-NO2C6H5, 4-FC6H5, C6H5CH2, CH3CH2, (CH3)2CH, CH3CH2CH2

1.3. Methods of Chemical Analysis In recent years, immense development in the field of natural products chemistry and

synthetic organic chemistry has taken place due to the availability of powerful

analytical techniques. The spectral techniques provide substantial information

regarding the structure of individual compounds. The spectroscopic techniques like

infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (1H NMR

and 13C NMR) and mass spectrometry (MS) have been used for the structure

elucidation and identification of the synthesized and natural compounds. A brief

review on spectroscopic techniques like UV, IR, 1H NMR, 13C NMR and MS has

been discussed here in the present study by taking a relevant example.

1.3.1. Ultraviolet Spectroscopy

Ultraviolet spectroscopy has become a major technique for the structure elucidation of

natural compounds especially flavonoids.93 The UV spectra of flavones in methanol

typically exhibit two major absorption peaks in the region 240-400 nm. Band I at

around 300-380 due to cinnamoyl moeity and Band II at around 240-240 nm due to

benzoyl moiety. On increasing oxygenation of the B-ring in flavones, a bathochromic

Chapter-1

21

shift in Band I occurs, while Band II remains unaffected. Further on increasing

hydroxylation of the A-ring in flavones produces a bathochromic shift in Band II with

a smaller effect on Band I. Isoflavones can easily distinguished from flavones by their

UV spectra. The UV spectra of isoflavones typically exhibited an intense Band II

adsorption with only a shoulder or low intensity peak representing Band I. The Band

II adsorption of isoflavones usually occurs at around 245-270 nm. The Band II of

isoflavones is unaffected by the increased hydroxylation of B-ring, however, Band II

shifted bathochromically by increasing oxygenation in the A ring.94 In case of

chalcones, the major absorption band (Band I) usually occurs in the range of 340-390

nm.

O

O

A

B

Benzoyl Cinnamoyl Band II Band I

O

O

A

Band II Band I

B O

A

B

Band II Band I

(Flavone) (Isoflavone) (Chalcone)

The structural information obtained from the UV spectrum could be further

confirmed by the use of specific shift reagents such as sodium methoxide (NaOMe),

sodium acetate (NaOAc), aluminium chloride (AlCl3) and aluminium chloride/

hydrochloric acid (AlCl3/HCl) sodium acetate/ boric acid (NaOAc/H3BO3).95 The

addition of these shift reagents separately to an alcoholic solution of the flavonoids

lead to a significant shifts in the UV absorption bands and thus provides sufficient

information about the orientation of the various hydroxyl groups present in the

flavonoid nucleus.

The UV spectra of Isoflavones containing A-ring hydroxyl groups usually show

bathochromic shift in the presence of NaOMe94 for both Band I and Band II. NaOAc,

being a weaker base than NaOMe, hence it ionizes the more acidic 7-hydroxyl group

in isoflavone causing a bathochromic shift of 6-20 nm in Band II. NaOAc/H3BO3

mixture is used for the detection of A-ring ortho-dihydroxyl groups in isoflavones.

However, the ortho-dihydroxyl groups in B-ring are not detectable by this shift

reagent because the B-ring lacks effective conjugation with the major chromophore.

On addition of NaOAc/H3BO3,94 a bathochromic shift by 10-15 nm in Band I is

Chapter-1

22

observed in presence of 6,7-dihydroxyl group in the A-ring of isoflavones. Further In

presence of AlCl3, the ortho-dihydroxyl groups in B-ring are not detectable due to

less or no conjugation with the major chromophore, however in absence of C-5

hydrogen bonded hydroxyl group, the ortho- dihydroxyl groups in ring-A can easily

detected by the use of AlCl3. The 6,7- or 7,8-dihydroxyl isoflavones exhibit

bathochromic shift for both Band I and Band II using AlCl3/HCl. The UV spectra of

all 5-hydroxyisoflavones undergo a consistent 10-14 nm bathochromic shift in Band

II in the presence of AlCl3/HC1. However, the spectra of isoflavones lacking a free 5-

hydroxyl group are unaffected by this reagent.94

The UV spectra of chalcones in the presence of NaOMe causes a bathochromic

shift of 60-100 nm of Band I for 4-hydroxyl, 2-hydroxyl and 4'-hydroxyl group. There

is an increase in the peak intensity along with bathochromic shift in case of 4-

hydroxyl group while the other two do not affect the intensity. The ortho-dihydroxyl

groups in B-ring of chalcones are readily detected by the 28-36 nm bathochromic shift

observed in Band I, while A-ring ortho-dihydroxyl groups show smaller shift in UV

spectra of chalcones on addition of NaOAc/H3BO3.94 The ortho-dihydroxyl groups in

B-ring of chalcones can also be detected by a 40-70 nm bathochromic shift of Band I

(relative to the Band I position in the AlCl3/HCl UV spectrum) on the addition of

AlCl3. A-ring ortho-dihydroxyl groups can also be detected by this procedure with

smaller shift in UV spectra. Band I in the UV spectra of 2'-hydroxylchalcones usually

undergoes a large bathochromic shift of 48-64 nm in the presence of AlCl3/HCl,

however, in the spectra of 2',3',4'-trihydroxylchalcones and its derivatives, the shift is

only about 40 nm. Lower wavelength bands also appear to shift bathochromatically,

but since these bands are often poorly defined, the shifts are difficult to determine.

1.3.2. Infra-red Spectroscopy

Infrared Spectroscopy is the analysis of interaction of infrared light with a molecule.

The IR spectroscopy is based on the absorption of the infrared radiation by molecule

which causes an excitation of molecule from a lower to the higher energy levels. It is

a simple and reliable technique used for the identification of the natural and

synthesized compounds. IR spectrum usually extends from radiation around 4000 cm-

1to 400 cm-1. It is divided into two regions, the functional group region and the

fingerprint region. The fingerprint region is different for each molecule just like a

fingerprint is different for each person. Two different molecules may have similar

Chapter-1

23

functional group regions because they have similar functional groups, but they will

always have a different fingerprint region. The fingerprint region extends from about

1450 cm-1 to 400 cm-1 and it has many absorption bands which makes it quite

complex. The IR spectra of all the flavonoids and isoflavonoids show absorption

bands in the region 1500-1600 cm-1 due to aromatic rings, along with a carbonyl band

at 1620-1670 cm-1.96 The carbonyl absorption does not appear in flavanoids,

isoflavanoids pterocarpanoids and chalcanoids. The presence of hydroxyl groups in

hydroxyflavonoids is evidenced by absorption in the region 3300-3450 cm-1.97

Absorption at 925 cm-1 is indicative of a methylenedioxy group and the presence of

gem dimethyl group is indicated by the appearance of a band at 1400 cm-1.98 The

glycosidic nature of a flavonoid is reflected by broad bands at 3250 and 1650 cm-1.99

However, although these absorption bands are present in most flavonoid glycoside,

they may also occur in the spectra of polyhydroxyflavonoids.

1.3.3. Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance (1H and 13C NMR) spectroscopy is well-resolved

method of analysis for organic compounds, because in many cases it provides a way

to determine an entire structure using one set of analytical tests. NMR spectroscopy is

used by chemists and biochemists to investigate the properties of organic molecules

(natural as well as synthetic), although it is applicable to any kind of sample that

contains nuclei possessing spin. The intramolecular magnetic field around an atom in

a molecule changes the resonance frequency, thus giving access to details of the

electronic structure of a molecule. The use of NMR spectroscopy on the sciences has

been substantial because of the range of information and the diversity of samples,

including solutions and solids. Different functional groups are obviously

distinguishable, and identical functional groups with differing neighboring substituent

still give distinguishable signals.

In 1964-65, two groups of workers, Waiss et.al.100 and Mabry et.al.101

independently investigated the usefulness of trimethylsilyl ether derivatives for

obtaining NMR spectra of flavonoids which were insoluble in CDCl3. The most

detailed and systematic studies of the 1H NMR spectra of flavonoids are documented

by Mabry94,102 Batterham and Highet,103 Okigawa et al.,104 Lewis et al.,105 Massicot

and Marathe,106 Miura et al.,107 and Pelter et al.,108 These studies have simplified the

task of determination of the substitution pattern of flavonoids with the help of NMR

Chapter-1

24

spectroscopy. 1H NMR signals in trimethyl silylated flavonoids178 normally occur

between 0 and 9 ppm. Most of the trimethylsilyl protons signal of flavonoid TMS

ethers occurs between 0.1-0.5 ppm. The chemical shift of the protons of ring A and B

prove to be independent of each other but are affected by the nature of ring C. This

technique is also helpful to distinguish flavones, isoflavones, flavanones, flavonols

and chalcone.94 13C NMR spectral data furnish key information about the carbon

backbone of the compound while 1H NMR gives information about the structural

environment of each proton. It helps in determining the total number of carbons and

the number of oxygenated carbons in the skeleton. It also helps in establishing the

nature of carbon either it is primary, secondary or tertiary. 13C NMR occurs over a

range of 0-200 ppm downfiled from TMS compared with a range of only 0-10 ppm

for 1H resonance. In proton decoupled spectra each carbon atom is represented by one

line and its chemical shift is determined primarily by the electron density at that

carbon atom. The electron resonances at lowest field are generally those of carbonyl

carbons and oxygenated aromatic carbons, whereas those at highest field will

represent non-oxygenated aliphatic carbons. In case of natural products especially

flavonoids it may be of use in special situations. The different types of aglycone are

not distinguishable on the basis of aromatic carbon resonances but the chemical shifts

of the central three carbon unit are often quite distinctive. The chemical shift values

of 13C NMR for ring C in flavonoids109 are given in Table 5.

Table 5 Chemical shift of 13C NMR for ring C in flavonoids (δ in ppm)

Flavonoids C-2 C-3 C=O

Flavones 160.5-165 103-111.8 176.3-184

Isoflavones 149.8-155.4 122.3-125.9 174.5-181

Flavonols 145-150 136-139 172-177

Flavanones 75-80.3 42.8-44.6 189.5-196.5

Chalcones 136.9-145.4* 116.6-128.1* 188.6-194.6

Aurones 146.1-147.7 111.6-111.9 182.5-182.7

* For chalcone C-2 and C-3 represents C-β and C-α, respectively.

The chemical shift values 1H and 13C NMR (δ, ppm) of a isoflanonoid (5,6,7-

trimethoxy-3-(3',4',5'-trimethoxyphenyl)-4H-chromen-4-one) and chalcone

(isoliquiritigenin-4,4'-dimethyl ether) nucleus of our interest in the context of the

Chapter-1

25

compounds isolated in the present work are shown in the following table (Table 6

and 7).

O

OHH3CO

OCH3O

O

HO

OH

OCH3

OHOCH3

H3CO

94 95

Table 6 1H NMR spectral data of representative isoflavone and chalcone (δ in ppm)

Assignment of protons

Isoflavone Assignment of protons

Chalcone

H-2 7.86 (s) H-2,6 7.61 (d, J=9 Hz)

H-6 6.62 (d, J=2.5 Hz) H-3,5 6.95 (d, J=9 Hz)

H-8 6.66 (d, J=2.5 Hz) H-3' 6.45 (d, J=2.5Hz)

H-5' 6.79 (s) H-5' 6.51 (dd,J=2.5,9Hz)

3×OCH3 3.84, 3.85, 4.01 (s) H-6' 7.81 (d, J=9Hz)

5-OH 13.5 (s) H-α 7.49 (d, J=16Hz)

7-OH 10.51 (s) H-β 7.89 (d, J=16Hz)

4'-OH 9.41 (s) 2×OCH3 3.88 (s)

2'-OH 13.56 (s)

Table 7 13C NMR spectral data of representative isoflavone and Chalcone (δ in ppm)

Assignments of Carbons

Isoflavone Assignments of Carbons

Chalcone

2 153.1 1 127.2

3 127.0 2 130.1

4 177.3 3 114.2

5 159.2 4 161.5

6 100.0 5 114.2

7 162.1 6 130.1

8 94.0 α 117.4

4a 156.6 β 143.9

8a 105.0 CO 191.4

1' 122.5 1' 114.0

2' 146.0 2' 166.3

3' 148.8 3' 101.0

Chapter-1

26

4' 148.5 4' 165.7

5' 118.1 5' 107.2

6' 148.2 6' 131.0

OCH3 55.8, 55.7, 55.2 OCH3 55.8

1.3.4. Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that ionizes chemical species and

sorts the ions based on their mass to charge ratio. It proves to be essential tool for the

identification and quantification of natural products and synthesized compounds,

primarily because of its speed, sensitivity, selectivity and its versatility in analyzing

solids, liquids and gases.110,111 It has become an indispensable apparatus to the modern

organic chemists by providing investigators with molecular formulae, isotopic profiles

and fragmentation data that are especially useful for structure elucidation. Mass

spectroscopy has been successfully employed for the structure determination of

flavonoids. The mass spectrum consists of a series of signals each of which represents

a charged fragment of the parent flavonoid produced by electron impact within the

spectrophotometer. The molecular ion normally appears as a major peak in the MS of

aglycones and must be an even mass number due to the presence of only oxygen,

carbon, and hydrogen atoms. Characteristics fragments in the MS originate by fission

of the M+ ion into A and B ring derived fragments. These fragmentations usually

involve one of the two competing pathways, I is Retro-Diels Alder and II one is

normal. The dominant pathway is determined by the aglycone type, although on

occasion neither pathway produces significant fragments. The common fragmentation

process of isoflavone is shown in Scheme 1 using 5,6,7-trimethoxy-3-(3',4',5'-

trimethoxyphenyl)-4H-chromen-4-one (96)112 as a typical example. It showed M+· at

m/z 360. The fragment ions at m/z 345, 330 and 315 were due to the successive loss

of three methyl groups. The fragment ions at m/z 332 and 329 are due to the loss of

CO and OCH3, respectively. The Retro-Diels-Alder (RDA) cleavage representing

ring-A at m/z 153, 152 and ring-B at m/z 208, 207 and 178 suggested the presence of

two hydroxyl group in ring-A and one hydroxyl and three methoxy group in ring-B.

Further the common fragmentation process of chalcone have shown in Scheme 2

using isoliquiritigenin-4,4'-dimethyl ether (97)94 as a typical example.

Chapter-1

27

O

OOCH3

H3CO

H3CO

OCH3

OCH3OCH3

O

OCH3

H3CO

H3CO

OCH3

OCH3OCH3

- CO- CH3 m/z 388

m/z 373

m/z 358

m/z 374

C

OH3CO

H3COOCH3

O

OCH3

OCH3OCH3

HC

M+H = m/z 403

m/z 210 m/z 192

- CH3

- CH3

96

Scheme 1 Mass fragmentation pattern of representative isoflavone

OCH3

m/z 269

m/z 254

- CH3

- CH3

OH

H3COm/z 256

OH

O

H3CO

OCH3

m/z 284

O

O

H3CO

OCH3

m/z 284

OH

C O

CH

CH

COO

C O

HCH2CHCHC

m/z 151 m/z 161

H3COOCH3

OCH3

H3CO

OCH3

O

OH

H3CO

m/z 133

m/z 149

m/z 126

m/z 165

- CO

97

Scheme 2 Mass fragmentation pattern of representative chalcone

Chapter-1

28

It showed M+· at m/z 284. The fragment ions at m/z 269 and 254 are corresponding to

the loss of methyl group from molecular ion peak and from the fragment m/z 269. The

fragment ion at m/z 256 showed the loss of carbonyl group from molecular ion peak.

Other fragmentation peaks have shown in scheme.

1.4. Biological Studies 1.4.1. DNA binding studies

Binding studies of chemical entities with DNA are important in the development of

molecular probes and new therapeutic reagents.113 These interactions play a central

role in rational drug designing and simultaneously DNA sequence recognition by

these drugs has been of great interest. Studies have demonstrated that DNA is the

primary target of anticancer drugs.114-116 Molecules can bind to DNA through a series

of interactions like coordination by the DNA bases, intercalation and non-covalent

interaction including hydrogen bonding between the coordinated ligands and

phosphate oxygen atoms of the sugar-phosphate group.117,118

1.4.2. Structural features of duplex DNA

Double-helical DNA consists of two complementary, anti-parallel, sugar-phosphate

poly-deoxyribonucleotide strands which are associated with specific hydrogen-

bonding between nucleotide bases.119 The backbone of these paired strands called the

helical grooves, within which the edges of the heterocyclic bases are exposed. B-form

of DNA duplex is biologically important and it is characterized by a shallow-wide

major groove and a deep-narrow minor groove.

1.4.3. Interaction of DNA-duplex with small organic molecules

1.4.3a. Covalent interaction of duplex-DNA with small organic molecules

Cancer chemotherapy was found as agents that interact with DNA or alkylate it, and

such compounds continue to be clinically important today. Alkylating agents are

involved in reaction with the preferential N-7 position of guanine and N-3 of adenine

in DNA this leads to interference in DNA replication. In the first mechanism, an

alkylating agent attaches alkyl groups to DNA bases. This alteration results in the

DNA being fragmented by repair enzymes in their attempts to replace the alkylated

bases. A second mechanism by which alkylating agents cause DNA damage is the

formation of cross-bridges, bonds between two stands of DNA. In this process, two

Chapter-1

29

bases are linked together by an alkylating agent that has two DNA-binding sites.

Cross-linking prevents DNA from being separated for synthesis or transcription. The

third mechanism of action of alkylating agents causes mis-pairing of the nucleotides

leading to mutations. Another well known covalent DNA binder used as an anticancer

drug is cis-platin (cis-diammine-dichloroplatinum), which makes an intra/ interstrand

cross-link with the nitrogens on the DNA bases and used extensively in testicular,

ovarian, head, and neck cancers.120 The early success of cis-platin as an anticancer

drug has led to the development of other less toxic derivatives such as carboplatin.

However, these agents are mostly non-specific in their action.121

1.4.3b. Non-covalent interactions of small molecules with duplex-DNA

(i) Duplex-DNA intercalators:

Molecules that bind to double-stranded DNA by intercalative mode have been

significantly used as drugs. The binding of these molecules to DNA is characterized

by insertion of planar aromatic rings between the DNA base pairs. This interaction

can be quite strong and the stability of intercalation complexes is governed by van der

Waals, hydrophobic and electrostatic forces. The two major types of intercalation-

binding modes are: (1) classical intercalation and (2) threading intercalation. Binding

by the classical mode is studied by DNA staining dye ethidium bromide and

antimalarial drug quinacrine.122 An important contributor to the binding affinity of

ethidium bromide and quinacrine to DNA is the stacking interaction of the respective

heteroaromatic rings with the DNA base pairs. Intercalation preferentially occurs at

G/C-rich sequences (CpG sites), because these sequences get unstacked easily.

Intercalators generally cause more significant distortion of the native conformations

of DNA, a factor that contributes to the disruption of protein binding. As threading

intercalators typically have two side chains on opposite sides of a planar aromatic ring

system, the process of complex formation with DNA is more complicated. In such

cases, one of the side-chains must slide through the intercalation cavity in order to

form the complex. Favourable interactions of the side-chains with both the major and

minor grooves contribute to the complex stability of the threading intercalators.

(ii) Duplex-DNA groove-binding molecules:

Groove binders are another major class of small molecules that bind to DNA and play

an important role in drug development. In this case molecules can bind to both the

major or minor groove of DNA. Due to the dimensional difference, the major grooves

Chapter-1

30

are the site for binding of many DNA interacting proteins.123 Proteins can recognize

and bind to DNA by reading the sequence information in either groove, but most

often by major groove recognition. However, nonpeptidyl compounds show a reverse

preference, they bind with the minor groove, thus potentially allowing simultaneous

major-groove recognition by proteins.124 Duplexes that are made up of polypurine-

polypyrimidine sequences can be read by oligomers that bind in the major groove and

form hydrogen bond with bases of the purine strand. The amenability of the minor-

groove to bind with small molecules has led many investigators to focus on this

aspect.125-127 It has been speculated that the evolution of antibiotic minor-groove

binders that target the DNA of competing organisms is related to the more attractive

dimensions of the minor groove for small molecules.128 Minor-groove binding usually

involves greater binding affinity and higher sequence specificity than that of

intercalator binding. The forces that dominate small molecule-minor-groove binding

interactions are electrostatic, van der Waals, hydrophobic and hydrogenbonding. A

number of crystal structure analysis and NMR studies of Hoechst 33258 complex to

various oligonucleotide duplexes containing stretches of AT base pair have been

reported.129,130 Design of low molecular mass compounds, which bind with high

affinity and specificity to pre-determined DNA sequences that are 10-16 base pairs

long, is a key issue in chemical biology.131 Hence the first strategy is to generate new

DNA targetable compounds, e.g. the groove-binding agents.

1.4.2. Molecular docking studies

Molecular docking is a key tool in structural molecular biology and computer-assisted

drug design. The motive of ligand-protein docking is to predict the predominant

binding mode (s) of a ligand with a protein of known three-dimensional

structure.132 Docking methods typically use an energy-based scoring function to

identify the energetically most favorable ligand conformation when bound to the

target.133 Successful docking methods explore high-dimensional spaces effectively

and use a scoring function that correctly ranks candidate dockings. Docking can be

used to perform virtual screening on large libraries of compounds, rank the results,

and propose structural hypothesis of how the ligands inhibit the target, which is

invaluable in lead optimization. The general hypothesis is that lower energy scores

represent better protein-ligand bindings compared to higher energy values. Therefore,

molecular docking can be formulated as an optimization problem, where the task is to

Chapter-1

31

find the ligand-binding mode with the lowest energy.133 In our experiment, rigid

molecular docking studies were performed to predict the binding modes of

compounds with DNA. The target protein/DNA used in the study has been

downloaded from protein data bank (PDB ID: 1BNA). Mol files were converted into

PDB format using Avogadro 1.0.1. Energy minimization and molecular optimization

of structures were done using Arguslab 4.0.1. Geometry optimization was carried

using AM1 (Austin Model 1), semiempirical quantum mechanics force field in

Arguslab 4.0.1. and Discovery studio 4.0 software have also been used to predict the

possible binding orientations of compounds with target DNA or protein.

1.4.3. Antioxidant studies

Antioxidants play an important role as health protecting factor. Scientific evidence

suggests that antioxidants reduce the risk for chronic diseases including cancer and

heart disease. Primary sources of naturally occurring antioxidants are whole grains,

fruits and vegetables. Plant sourced antioxidants like vitamin C, vitamin E, carotenes,

phenolic acids etc. have been recognized as having the potential to reduce disease

risk. Most of the antioxidant compounds in a typical diet are derived from plant

sources and belong to various classes of compounds with a wide variety of physical

and chemical properties. Free radicals are an atom or molecule that bears an unpaired

electron and is extremely reactive, capable of engaging in rapid change reaction that

destabilize other molecules and generate many more free radicals. In plants and

animals these free radicals are deactivated by antioxidants. These antioxidants act as

an inhibitor of the process of oxidation, even at relatively small concentration and

thus have diverse physiological role in the body. The harmful free radicals such as

hydroxyl, peroxyl and the superoxide anion are constantly being produced as a result

of metabolic reactions in living systems. Several diseases caused by free radicals have

been reported such as atherosclerosis, cancer, liver cirrhosis, diabetes, etc.134 and the

scavenging effects of these free radicals by the chemical compounds have great

potential in ameliorating these disease progresses.

A simple, rapid and inexpensive method to measure antioxidant capacity of any

compounds involves the use of the free radical, 2,2-Diphenyl-1-picrylhydrazyl

(DPPH) which is widely used to test the ability of compounds to act as free radical

scavengers or hydrogen donors and to evaluate antioxidant activity.136 The DPPH

assay method is based on the reduction of DPPH, a stable free radical.137 DPPH free

Chapter-1

32

radical method is an antioxidant assay based on electron-transfer that produces a

violet solution in ethanol.138 This free radical is stable at room temperature and it

reduced in the presence of an antioxidant molecule, giving rise to colorless ethanol

solution. The use of the DPPH assay provides an easy and rapid way to evaluate

antioxidants by spectrophotometry,138 so it can be useful to assess various products at

a time. The free radical DPPH with an odd electron gives a maximum absorption at

517 nm (purple colour). When Antioxidants react with DPPH, it reduced to the

DPPH-H and as consequence the absorbance decreased from the DPPH.139 Radical to

the DPPH-H form, results in decolorization (yellow colour) with respect to the

number of electrons captured.140 More the decolorization, more is the reducing ability.

This test has been the most accepted model for evaluating the free radical scavenging

activity of any new drug.141 When a solution of DPPH is mixed with that of a

substance that can donate a hydrogen atom, then this gives rise to the reduced form

(Diphenylpicrylhydrazine; non radical) with the loss of this violet colour (although

there would be expected to be a residual pale yellow colour from the picryl group still

present).142

1.4.4. Cytotoxicity

Plants are used as food sources, some of them may have mutagenic or genotoxic

potential.143 Numerous research studies have recently focused on both pharmacology

and toxicity of medicinal plants used by humans.144 The toxicity of the plants may

originate from different contaminants or from plant chemical compounds that are part

of the plant. Around half of the anticancer drugs currently used in clinical trials are of

natural origin, and it has been estimated that about 60% of new chemical entities

(NCEs) introduced in the recent years were natural products or were derived from a

natural lead compound. Therefore, the screening of traditional medicinal plants has

great importance to identify new medicinal plants and to isolate new cytotoxic

compounds for life threatening diseases like cancer. Various assays are used for the

detection of toxicity of herbal extracts based on different biological models, such as in

vivo assays on laboratory animals. However, recent studies employed efforts for

alternative biological assays that include species of Artemia (A. salina, A. franciscana

and A. urmiana. These toxicity tests are considered a useful tool for preliminary

assessment of toxicity.145 Brine shrimp (A. salina) is most extensively studied of the

Artemia species, estimated to represent over 90% of the studies in which Artemia is

Chapter-1

33

used as an experimental test organism. The Brine Shrimp Toxicity Assay was

proposed and developed by Michael et al.146 and later adapted by Vanhaecke et al.,147

Meyer et al.,148 and Sleet and Brendel.149 Brine Shrimp Lethality Assay (BSLA) has

been applied as an alternative bioassay technique to screen the toxicity of plant

extracts148,150 toxicity of heavy metals,151 toxicity of cyanobacteria152 and algae,153

toxicity of nanoparticles,154 as well as screening of marine natural products.155 It is

capable of detecting various bioactivities present in crude extracts of medicinal plants

and has been used as an indicator for general toxicity and as a guide for the detection

of antitumor and pesticidal compounds.156

Chapter-1

34

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Chapter-2

42

2.1. INTRODUCTION

The genus Iphiona is a small genus of the family Compositae belongs to the major

group Angiosperms (Flowering plants). It is chiefly distributed in North Eastern

Africa to Central Asia.1 It is characterized with ligulate female florets and sweeping

hairs in the upper part. The spine tips of the pollen grains are distinctly set off from

their bases and the vascular thickening mostly has a dome-like arrangement.2 Previous

investigation revealed that polysulphated flavonoids were the major constituents of

this genera. Fourteen flavonoids were isolated from the aerial parts of I. mucronata

out of which seven were sulphated and seven were non-sulphated.3 In addition, aerial

parts of I. mucronata afforded six sesquiterpene xylosides. Further, a non-toxic

pyrrolizidine alkaloid and two diterpene glycosides (atractyloside and

carboxyatractyloside) were isolated from I. aucheri and this plant is also responsible

for poisoning racing camels (Camels dromedarius) in the United Arab Emirates U. A.

E.4

A number of compounds have been isolated from I. scabra which includes

flavonoids3: the 3-sulphate, 3,4′-disulphate and 3,7,4′-trisulphate of isorhamnetin; the

3,7-disulphate and 3,7,4′-trisulphate of quercetin; and the 7-sulphate of hispidulin; the

3-glucosides and 3-galactosides of isorhamnetin and quercetin; and artemetin,

salvigenin and 5-hydroxy-3,6,4′-tetramethoxyflavone; sesquiterpene xylosides1:

seven eudesmane derivatives, a secoeudesmane and two new sesquiterpene glycosides

named iphionane and isoiphionane; 10-Epi-cubebolxyloside5: α-Elemol-[α-

xylopyranoside-triacetate] and l0-epi-cubebol-[α-xylopyranoside-triacetate]. The

remarkable role of ethanolic extract of Iphiona scabra in medicinal chemistry such as

anti-inflammatory, antiplatelet aggregation, ovipositive deterrents, maximum

inhibition of egg hatchability and hypotensive effects in albino rats6 draw the attention

of phytochemist towards this genus.

Its biological importance and scanty of work on this plant accelerated our

interest to carry out comprehensive investigation of leaves of I. scabra. In this chapter

we report the isolation and characterization of novel 6-hydroxy-8,11,11-trimethyl-

bicyclo[7.2.0]undec-4-ene-4-carboxylic acid (2), along with four known compounds

n-tetracosan-1-ol (1), 4,6-Diacetylresorcinol (3) 4-Acetylresorcinol (4) and 1,2-bis(o-

tolyloxy)ethane (5). Compound 2 is a novel compound. To the best of our knowledge,

compound (2) has neither been isolated from any natural source nor has its synthesis

Chapter-2

43

been reported so far. While, compound 5 has been isolated first time from natural

source. The structure of all the isolated phyto-constituents was established on the

basis of IR, 1H NMR, 13C NMR, Mass spectral analysis. The structure of the

compound 2 and 3 were further confirmed by single X-ray crystallographic study.

DNA binding study of compound 3-5 were also carried out to predict binding mode of

compounds with ctDNA. Further antioxidant activity of isolated compounds and

cytotoxicity of plant extracts were also tested.

2.2. RESULTS AND DISCUSSION


scabra i.e. extraction, isolation and characterization of its chemical constituents,

screening of antioxidant activity, cytotoxicity of aqueous and ethanolic extracts of

leaves and DNA binding study of isolated compounds 3-5. The air dried powdered

leaves of Iphiona scabra (2 kg) were extracted with 95% ethanol and concentrated to

dark gummy mass under reduced pressure. It was then fractionated successively with

petroleum ether, benzene, ethylacetate, acetone and methanol. Petroleum ether and

benzene fraction, ethylacetate and acetone fractions exhibited similar behavior on

thin-layer chromatography (TLC) examination with different solvents system eg.

Benzene-chloroform (9:1-1:1), benzene-pyridine-formic acid (BPF-36:9:5), toluene-

ethylformate-formic acid (TEF-5:4:1), hence were mixed together and

chromatographed over silica gel column. Then each column was successively eluted

with a gradient increase in polarity of solvent to give different fractions. Further these

fractions were purified by repeated column chromatography followed by fractional

crystallization afforded pure compounds.

The following five compounds (1-5) have been isolated from the Iphiona scabra

leaves.

1. n-tetracosan-1-ol (Is-1)

2. 6-hydroxy-8,11,11-trimethylbicyclo[7.2.0]undec-4-ene-4-carboxylicacid(Novel)

(Is-2)

3. 4,6-Diacetylresorcinol (Is-3)

4. 4-Acetylresorcinol (Is-4)

5. 1,2-bis(o-tolyloxy)ethane (Is-5)

Chapter-2

44

2.2.1. Characterization of compound 1 (Is-1)

Compound 1 was isolated by the elution of column with petroleum ether-benzene

(9:1) as a white solid (120 mg), m.p. 78 oC.7 Elemental analysis and molecular ion

peak at m/z 354 was in agreement with molecular formula C24H50O. The IR spectrum

displayed characteristic peaks at 3460 and 1611 cm-1 which were ascribed to alcoholic

OH group and CH2 stretching. In 1H NMR spectrum a singlet at δ 3.63 integrating for

one proton has been ascribed for hydroxyl group. A triplet for three protons at δ 0.88

ascribed for CH3 group. Another triplet appears at δ 3.50 for two protons have been

assigned to H-1 protons. A multiplet at δ 1.56 integrating for four protons has been

assigned to H-2 and H-23 protons. Another multiplet δ 1.25 for fourty protons was

assigned (H-3 to H-22). 13C NMR spectrum showed peak at δ 63.11 (C-1), δ 31.94 (C-

2), δ 25.75 (C-3) other carbons resonating at δ 29.38-29.71 for (C-4 to C-21), δ 32.82

(C-22), δ 22.70 (C-23) δ 14.13 (CH3). Further, Co-TLC with the authentic sample and

finally comparison of melting point and spectral data with the literature7 confirmed

the structure of compound 1.

In the light of above discussion, compound 1 was identified as n-tetracosan-1-

ol.

OH


Compound 2 was isolated from the column with petroleum ether-benzene (8:2)

mixture and crystallizes with CHCl3-MeOH as white shinning crystals (35 mg), m.p.

131-132 oC. Elemental analysis and molecular ion peak at m/z 252 was in agreement

with molecular formula C15H24O3. The IR spectrum (Fig. 1) displayed characteristic

peaks at 3550 and 3500 cm-1 ascribed to hydroxyl groups. Another band at 1690 cm-1

corresponded to carboxylic group which was further confirmed by the appearance of

effervescences with NaHCO3. A characteristic band at 1630 cm-1 exhibited the

presence of double bond. In 1H NMR spectrum (Fig. 2) an independent doublet

integrating for one proton at δ 6.81 (J = 4.0 Hz) assigned to H-5 proton. A double

doublet signal integrating for one proton at δ 4.98 (J = 6.8, 4.0 Hz) was ascribed to H-

Chapter-2

45

6 proton. Multiplet at δ 1.38 and 1.25 for one proton each has been assigned to H-1

and H-9 protons, respectively. A singlet for six protons at δ 0.95 was ascribed to the

methyl group attached to C-11. Another singlet integration for three protons at δ 0.98

was attributed to methyl group attached to C-8 carbon. A pair of singlet integrating

for one proton each at δ 10.20 and 9.50 was assigned to O-H proton of carboxylic

group and 6-OH proton, respectively. Moreover, eight methylene protons have been

resonated at δ 2.81 (H-3a), 2.34 (H-3b), 1.83 (H-7), 1.54 (H-2), and 1.11 (H-10). 13C

NMR spectrum (Fig. 3) exhibited the presence of 15 carbons. The signal at δ 166.1

was ascribed to the carbonyl group. The vinylic carbon were resonated at δ 140.5 (C-

5) and 134.3 (C-4). The germinal methyl groups were found to appear at δ 25.5 and

25.8 while the other methyl group was resonating at δ 18.0. A number of signals were

ascribed at δ 48.9 (C-1), 27.3 (C-2), 25.2 (C-3), 78.0 (C-6), 42.5 (C-7), 29.5 (C-8),

38.2 (C-9), 40.2 (C-10) and 34.3 (C-11) of the two cyclic ring. Mass spectrum showed

molecular ion peak at m/z 252 (Fig. 4). Further single-crystal X-ray diffraction

analysis confirms the molecular structure of compound 2.

Fig. 1 FT-IR spectrum of compound 2

Chapter-2

46

Fig. 2 1H NMR spectrum of compound 2

Fig. 3 13C NMR spectrum of compound 2

Chapter-2

47

Fig. 4 Mass spectrum of compound 2

In the light of above discussion, compound 2 was identified as 6-hydroxy-

8,11,11-trimethyl-bicyclo[7.2.0]undec-4-ene-4-carboxylic acid, which is being

reported for the first time from natural source and has not been synthesized so far.

O OH

OH

1 2 3 45

67891011


Elution of column with petroleum-ether and benzene (6:4) afforded compound 3 as a

white solid which was crystallized with chloroform-benzene as colorless shining

crystals, m.p. 178-180 °C.8 Elemental analysis and molecular ion peak at m/z 194 was

in agreement with molecular formula C10H10O4. The UV spectrum of the compound

exhibited λmax at 212, 275 and 312 nm. The IR spectrum revealed characteristic

absorption bands for hydroxyl group (3430 cm-1), phenyl ring (1589 and 1490 cm-1)

m/z 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

%

0

100 433 252.7

202.2 415

401 224.2

250.1 119 578.1

95 253.9

85

Chapter-2

48

and for carbonyl group (1658 cm-1). Positive alcoholic ferric chloride test confirmed

the presence of phenolic OH group. The compound responded positively to haloform

test indicating the presence of methyl ketone. Its 1H NMR spectrum exhibited two

sharp singlets at δ 6.38 and 8.18 integrating for one proton each were ascribed to H-3

and H-6 protons, respectively. An independent sharp singlet at δ 12.91 of two protons

was assigned to two hydroxyl groups. Moreover, sharp singlet at δ 2.61 for six

protons indicating the presence of two methyl groups. 13C NMR spectrum indicated

peak at δ 26.12 and 202.52 which show the presence of methyl carbon and carbonyl

carbon, respectively. Further, peaks at δ 113.66 (C-1 and C-5), 168.93 (C-2 and C-4),

104.99 (C-3) and 136.31 (C-6) showed the presence of aromatic carbons. Finally

comparison of melting point, spectral data with the literature8 and single X-ray

crystallographic studies confirmed the structure of the compound 3.

In the light of above discussion, compound 3 was identified as 4,6-

Diacetylresorcinol.

OO

OHHO


Elution of the column with blank benzene gave compound 4 which on crystallization

with benzene and ether yielded yellowish brown solid (60 mg), m.p. 144-145 °C.9

Elemental analysis and the molecular ion peak at m/z 152 [M+•] is in agreement with

the molecular formula C8H8O3. The UV spectrum of the compound exhibited λmax at

212, 275 and 310 nm. It responded positively to alcoholic ferric chloride test pointing

out the presence of phenolic hydroxyl group which was further confirmed by the

presence of a characteristic band at 3301 cm-1 in its IR spectrum. The peaks

resonating at 1631, 1520 and 1440 cm-1 have been assigned to carbonyl group and

phenyl ring, respectively. The compound gave a positive haloform test indicating the

presence of methyl ketone. Its 1H NMR spectrum showed a pair of doublet integrating

for one proton each at δ 6.25 (J = 4.0 Hz) and 7.63 (J = 8.0 Hz) have been assigned to

H-3 and H-6 protons, respectively. A double doublet integrating for one proton at δ

Chapter-2

49

6.34 (J = 8.8, 2.4 Hz) has been attributed to H-5 proton. Similarly a pair of singlets

corresponding to one proton each at δ 12.62 and 10.45 has been ascribed to C-2 and

C-4 hydroxyl protons, respectively. Further a sharp singlet for three protons

resonating at δ 2.50 has been assigned to methyl protons. The 13C NMR displayed

characteristic peaks at δ 189.40, 164.84 and 164.42 were assigned to carbonyl carbon

and hydroxylated carbons (C-2 and C-4). The signals resonating at δ 132.96 (C-6),

112.63 (C-1), 107.96 (C-5) and 102.36 (C-3) have been attributed to aromatic ring

carbons, respectively. Similarly a peak at δ 25.86 was assigned to methyl carbon.

Moreover Co-TLC with the authentic sample and finally comparison of melting point

and spectral data with the literature9 confirmed the structure of the compound 4.

In the light of above discussion, compound 4 was identified as 4-

Acetylresorcinol.

O

OH

OH


Elution of the column with benzene-ethylacetate (8:2) gave compound 5 which on

crystallization with chloroform and acetone yielded bright-yellow solid (60 mg), m.p.

87 °C.10 Elemental analysis and molecular ion peak at m/z 242 [M+•] was in good

agreement with the molecular formula C16H18O2. IR spectrum displayed

characteristics peaks at 1624 and 1500 cm-1 have been assigned (C=C)str of phenyl

ring. Peaks at 1283 and 1146 cm-1 have been assigned to asymmetric and symmetric

C-O-C stretching, respectively. Further peak at 1442 has been attributed to CH2

bending and peaks at 1389, 1317 have been assigned to CH3 bending. In 1H NMR

spectrum a pair of multiplets at δ 7.18-7.15 and 6.91-6.98 corresponding to four

protons each, has been ascribed to the phenyl ring protons. Further two singlets at δ

4.36 and 4.37 have been attributed to four methylene protons. A sharp singlet

integrating for six protons resonating at δ 2.34 has been assigned to methyl protons.

The 13C NMR displayed characteristic peaks at δ 23.37 and 62.84 for methyl and

methylene carbon, respectively. Peaks at δ 122.25 (C-6' and C-6"), 128.73 (C-5' and

Chapter-2

50

C-5"), 128.85 (C-4' and C-4"), 129.26 (C-3' and C-3"), 144.26 (C-1' and C-1") and

164.16 (C-2' and C-2") have been attributed to aromatic ring carbons. Moreover

comparison of melting point and spectral data with the literature10 confirmed the

structure of the compound 5 which is being reported for the first time from natural

source.

In the light of above discussion, compound 5 was identified as 1,2-bis(o-

tolyloxy)ethane.

CH3O O

CH3

2.2.6. X-ray crystallographic analysis of compounds 2 and 3

X-ray structure of compound 2 and 3 have been shown in Fig. 5 and Fig. 7.

The asymmetric unit of compound 2, crystallizes in the non-centrosymmetric and

chiral space group P21 (Fig. 5). Crystallographic data and structure refinement has

been shown in Table 1. There are two symmetry independent molecules in the

asymmetric unit. The torsion angle C3-C4-C5-C6 of the double bond in the nine-

membered ring is 23.6 (3)° for molecule A and 3.4 (3)° for molecule B. These values

are quite different from those of related compounds. For example, for pestalotiopsin A

this value is 152°11 and for (E)-15-norcaryophyll-4-en-8b-yl tosylate it is 158°12.

These differences may be due to the presence of different substituents at positions 4

and 6, in pestalotiopsin A, a methyl group and a methoxy group, respectively, and in

(E)-15-norcaryophyll-4-en-8b-yl tosylate, a methyl group at position 4 and no

substituent at position 6. The crystal structure is stabilized by O-H···O hydrogen

bonds between the hydroxyl and carboxyl groups (Fig. 6, Table 2).

Chapter-2

51

Fig. 5 Asymmetric unit of compound 2 with the ellipsoids drawn at the 50%

probability level, with the atomic labeling scheme

Fig. 6 Packing diagram of compound 2 viewed down the b axis with the H-bonds

show as dashed lines

Chapter-2

52

Fig. 7 Asymmetric unit of compound 3 with the ellipsoids drawn at the 50%

probability level, with the atomic labeling scheme

The symmetry equivalent molecules are linked in zigzag chains running parallel to the

b axis with a periodicity of seven atoms, graph-set symbol C(7) according to Etter’s

graph-set theory.10 There are also H-bonds between the symmetry nonequivalent

molecules with graph-set descriptor D1,1(2). The combination of the four H-bonds

generates rings with descriptor R4,4(12).

The single crystal X-ray diffraction analysis of compounds 3 was in good agreement

with the previous report.8 Compound 3 crystallizes in the monoclinic crystal system

with the space group P21/c (Fig. 7). Crystallographic data and structure refinement

has been shown in Table 1.

Chapter-2

53

Table 1 Crystal data and structure refinements of compounds 2 and 3

Empirical formula Compound (2) C15H22O3

Compound (3) C10H10O4

Formula weight 250.33 194.18 Temperature (K) 293(2) 293(2) Wavelength (Å) 0.71073 0.71073 Crystal system Monoclinic Monoclinic Space group P21 P21/c

a (Å) 12.3489(8) 7.0908(4) B (Å) 9.9037(6) 11.3687(6) c (Å) 12.9732(7) 11.6475(7)

α (°) 90 90.00

β (°) 112.783(4) 100.444(3)

γ (°) 90 90.00

Volume (Å3) 1462.83(15) 923.39(9) Z 4 4

Calculated density (g/cm-3) 1.137 1.397 Absorption coefficient (mm-1) 0.078 0.109

F(000) 544 408 Crystal size (mm) 0.41 × 0.16 × 0.12 0.59×0.40×0.33

θ range for data collection (°) 2.91-31.99 3.43-27.23 Index ranges -16<h<18, -14<k<13,

19<l<17 -9<h<9; -14<k<14;

-14<l<14 Reflections collected 21996 17124

Independent reflections 4661 12348 Completeness to 2θ=50º 99.6% 99.9%

Refinement method Full-matrix LS on F2 Full-matrix LS on F2 Data/restrains/parameters 4661/1/333 2044/0/136

Goodness-of-fit on F2 1.055 1.083 Final Rindices [I>2σ(I)] R1=0.0520; wR2=0.1272 R=0.0464; wR=0.1508

R indices (all data) R1=0.0730; wR2=0.1422 R=0.0521; wR=0.1572

Largest diff. peak/hole (e Å-3) 0.197/-0.231 0.246/-0.214

Chapter-2

54

Table 2 H-bond geometry (Å, º) of compound 2.

D-H H...A D...A D-H...A

O2A—H2A...O3Bi 0.82 1.86 2.666(2) 167

O3A—H3A...O1Aii 0.82 1.99 2.752(2) 155

O2B—H2B...O3Aiii 0.82 1.88 2.659(2) 158

O3B—H3B...O1Biv 0.82 1.94 2.751(2) 171

(symmetry codes i: 1-x, y-1/2, 2-z; ii: -x, y-1/2, 2-z; iii: -x, y+1/2, 1-z; iv: 1-x, y+1/2, 1-z)

2.2.7. DNA binding studies

2.2.7a. UV-Vis spectroscopy

The UV-Vis spectroscopy is widely used to examine the binding mode of drug with

DNA.13 Drugs can bind to DNA via both covalent and non-covalent interactions.14

We have exploited this technique to study the interaction of compounds (3-5) with

ctDNA. The absorption spectra were recorded for a fixed concentration of ctDNA

with the increasing concentration of compounds (0-30 μM) (Fig. 8). With the increase

in concentration of compounds 3-5, a “hyperchromism” was observed with no

apparent shift in the absorption band of DNA. Hyperchromism has been attributed to

the presence of non-covalent interactions: external contact (electrostatic binding) and

groove surface (major and minor) along outside the DNA helix. As there is no change

in the position of absorption bands (bathochromic or hypsochromic shift), it can be

inferred that compounds 3-5 exhibit groove binding interactions to ctDNA.

Chapter-2

55

Fig. 8 UV-Visible absorption spectra of (a) compound 3 (0-30μM) (b) compound 4

(0-30μM) (c) compound 5 (0-30μM) in the presence of constant concentrations of

ctDNA in Tris-HCl buffer (pH 7.4)

(a)

(b)

(c)

Chapter-2

56

2.2.7b. Steady state fluorescence measurements

Fluorescence quenching experiments were used to investigate the interaction of

isolated compounds (3-5) with DNA. Quenching can arise by two different

mechanisms such as dynamic and static quenching. As seen in Fig. 9, with the

increasing concentration of compounds 3-5, there is decrease in the fluorescence

intensity, which validates the interaction of compounds with ctDNA.

The fluorescence quenching data were analyzed by Stern-Volmer equation. (Table 3)

F₀/F = Ksv+1 = Kqτ₀ [Q] +1 (1)

where Fo and F denote the steady-state fluorescence intensities in the absence and in

the presence of quencher, respectively, Ksv is the Stern-Volmer quenching constant

(which is a measure of quenching efficiency), [Q] is the concentration of the

quencher. Kq is the bimolecular quenching rate constant and τ₀ is the life time of

biomolecules without the quencher. Analysis of quenching mechanism was confirmed

from the values of biomolecular quenching rate constants, which are evaluated by

using the Eq. (2) (Table 3)

Ksv= Kqτ0 (2)

The bimolecular quenching rate constant for compounds 3-5 were calculated to be

7.60×1011, 5.26×1011, 7.89×1011 M-1s-1, respectively. These values are greater than

maximum collision quenching rate constant 2×1010 M1s-1 which depicts that the

mechanism of quenching is static. Furthermore, for static quenching, the relationship

between fluorescence intensity and concentration of a quencher can be described by

the following equation. Eq (3)

Log (F₀/F -1) = logKb + n log [Q] (3)

In the above equation F0 and F are the steady state fluorescence intensities in the

absence and presence of quencher, respectively. Kb is binding constant, n is the

number of binding sites and Q is the concentration of quencher. Kb and n values of

the compounds 3-5 can be obtained from the intercept and slope of the corresponding

plot of log (F0/F-1) vs log [Q] (Fig. 10). The binding constant (Kb) for compounds 3-

5 were calculated to be 2.83×103, 2.54×103, 4.26×103, respectively and the number of

binding sites (n) for all the compounds 3-5 were found to be 1. The data shown in

(Table 3) also suggests that the interaction of the compounds 3-5 with DNA follows

the order: 5>3>4.

Chapter-2

57

Fig. 9 Fluorescence emission spectra of (a) compound 3 (0-70μM) (b) compound 4

(0-70μM) (c) compound 5 (0-70μM) in the presence of constant concentrations of

ctDNA (12µM)

(a)

(b)

(c)

Chapter-2

58

Fig. 10 Stern-Volmer plot of compounds 3-5. F0 and F are the fluorescence intensity

of ctDNA in the absence and presence of compounds (3-5). Plot of log(F0/F-1) versus

log[Compounds] to calculate the binding constant

Chapter-2

59

Table 3 Binding parameters obtained from the fluorescence quenching method.

Compounds Ksv (M-1) Kq (M-1s-1) Kb (M-1) n ΔG° (kJ mol-

1)

R2

3 7.60×103 7.60×10

11 2.83×10

3 1.02 -19.68 .99

4 5.26×103 5.26×10

11 2.54×10

3 1.02 -19.40 .99

5 7.89×103 7.89×10

11 4.26×10

3 1.08 -20.71 .99

2.2.7c. Circular dichroism study

CD spectroscopy is useful technique to understand the structural changes of DNA in

presence of interacting drug molecules and to investigate the mode of interactions

with these drugs. Classical intercalative molecules are known to perturb the CD

spectra of DNA significantly due to strong base stacking interactions and stable DNA

conformations, while simple groove binding and electrostatic interactions has less or

negligible effects on the CD spectra of DNA.15 The CD spectra of DNA in the

presence of the compounds (3-5) were carried out to study the change in secondary

structure of ctDNA. As seen in Fig. 11(a-c), with the increasing concentration of

compounds 3-5, there was slight increase in the intensity of the positive and negative

peaks of the DNA was observed. This change in CD spectra of DNA might be due to

the unwinding of DNA helix and transformation from B form of DNA to A form

structure as UV-Vis spectroscopic study suggests the non-intercalative interaction of

compounds with DNA.

Chapter-2

60

Fig. 11 CD spectra of ctDNA in the presence of (a) compound 3 (0-30μM) (b)

compound 4 (0-30μM) (c) compound 5 (0-30μM)

(a)

(b)

(c)

Chapter-2

61


Molecular docking studies have played a very significant role in understanding the

mechanistic pathway of DNA-drug interactions.16 In order to study the exact

interaction, compounds 3-5 were successively docked within the DNA duplex of

sequence d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) (Fig. 12). The

resulting docking experiments informed that compounds 3-5 were interact with B-

DNA (PDB ID: 1BNA) at the G-C rich intercalation site of the minor groove with a

patchdock scores of 2630, 2404 and 3850 having atomic contact energy of -175.85, -

171.79 and -249.44 kJ mol-1, respectively. The results obtained by PatchDock were

further confirmed by HEX 8.0 software and binding energy was found to be: -183.32,

-146.00 and -188.21 kJ mol-1 for compounds 3-5, respectively. The outcomes strongly

aided the previous results with a good binding efficiency between receptor and

ligands with the calculated PatchDock score of 3850 (Hex; -188.21, compound 5),

2630 (Hex; -183.32, compound 3) and 2404 (Hex; -146.00, compound 4) for DA18,

DG22, DG4, DC11, DG16 and DA17 respectively, These binding modes are further

stabilized by van der Waals and hydrophobic contacts. On the basis of obtained

results from molecular docking studies, the greater binding affinity of compounds

(5>3>4) is in good agreement with the experimental results acquired from electronic

and fluorescence studies.

Chapter-2

62

Fig. 12 Docking model of compounds 3-5 with ctDNA.

Chapter-2

63

2.2.9. Antioxidant activity (DPPH assay)

The antioxidant activity was measured by DPPH assay, is a primary tool to investigate

free radical scavenging activity of the compounds. The statistically analyzed results of

scavenging activity of DPPH radicals of isolated compounds (1-5), ethanolic extract

and aqueous extract are shown in Table 4. It can be inferred from the data that

isolated compounds displayed better antioxidant activity in comparison to both the

extracts. As can be seen from Table 4, IC50 values of the extracts and compounds

were found in the order; compound 3 > 4 > 2 > 1> 5 > ethanolic extract > aqueous

extract. Compound 3 exhibited more scavenging activity with (IC50= 22.15 μg/mL)

than compound 4 it might be due to different substitution patterns of two hydroxyl

groups on phenyl ring.17 The ethanolic extract of plant show good antioxidant activity

than the aqueous extract. The remarkable differences in radical scavenging activities

may be due to the presence of various active compounds in the extract.

Table 4 Antioxidant activity of compounds (1-5) and plant extracts (ethanolic and aqueous).

Compounds and extracts IC50 value (μg/mL)a

1

2

37.67

28

3 22.15

4 26.42

5 38.73

Ethanolic extract 70

Aqueous extract 82

Standard (Ascorbic acid) 15 aIC50 value represents the concentration of three experiments required to exhibit 50 % antioxidant activity.

2.2.10. In vivo cytotoxicity assay

The in vivo cytotoxicity activity was carried out on ethanolic and aqueous extract of

Iphiona scabra leaves using the brine shrimp (Artemia salina) lethality bioassay

method (Fig. 13). In this experiment, Vincristine sulfate was used as standard. This

bio-screening assay is considered as a very useful method for the assessment of the

toxic potential of various plant extracts.18 It is one of the most convenient and simple

bioassay for testing the cytotoxicity of plant extracts. This bioassay has good

Chapter-2

64

correlation with cytotoxic activity in some human solid tumors, hence it is useful to

discover new class of natural active antitumor agents.19 It has also been used for the

detection of fungal toxins.20 Rapidness, simplicity and low requirements are the

advantages of this method. This method is based on the survival of number of brine

shrimps after 24 h of incubation as described by Meyer et al.21 This procedure

determines LD50 value in μg/mL of extracts in the brine medium (Table 5). In the

present study, each of the test samples display different mortality rates at different

concentrations, the percentage mortality increases with an increase in concentration.

Based on the results, the brine shrimp lethality of the plant extracts were found to be

concentration-dependent. The calculated LD50 value of the ethanolic and aqueous

extract of leaves are found to be 10.89 μg/mL and 16.71 μg/mL, respectively, as

compared to the standard drug Vincristine sulfate whose LD50 is 8.70 μg/mL. These

results clearly indicate significant cytotoxicity of the crude extracts of I. scabra. The

variation in results may be due to different type of compounds present in the extracts.

Polyphenolic compounds that are ubiquitously found in plants have ability to prevent

cancer and tumor growth.22 This significant lethality of the crude plant extratcts to

brine shrimp give an indication about the plants ability as an antitumor agent.

Table 5 Cytotoxicity assay of plant extracts (ethanolic and aqueous).

Extracts LD50 value (μg/mL)

Ethanolic extract 10.89±0.20

Aqueous extract 16.71±1.42

Standard (Bleomycin) 8.70±0.03 aLD50 value represents the concentration of three experiments required to exhibit 50 % cytotoxicity.

Chapter-2

65

Fig. 13 Determination of LD50 values for ethanolic and aqueous extract of Iphiona scabra leaves from linear correlation between logarithms of concentration versus

percentage of mortality.

Chapter-2

66

2.3. EXPERIMENTAL

2.3.1. Materials and methods

All the solvents, chemicals and Calf Thymus DNA were purchased from commercial

sources (Sigma-Aldrich) used without further purification. Melting points were

determined on a Kofler apparatus and are uncorrected. Elemental analysis (C, H, N)

was conducted using Carlo Erba analyzer model 1108. The IR spectra were recorded

with a Shimadzu IR-408 Perkin-Elmer1800 instrument (FTIR) and the values are

given in cm-1. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance-II

400 MHz and 100 MHz instrument in DMSO-d6/CDCl3 solvent instrument with TMS

as an internal standard and J values were measured in Hertz (Hz). Chemical shifts are

reported in ppm (δ) relative to TMS. Mass spectra were recorded on a JEOL D-300

mass spectrometer. Thin layer chromatography (TLC) glass plates (20×5 cm) were

coated with silica gel G (Merck) and exposed to iodine vapors to check the

homogeneity as well as the progress of the reaction.

2.3.2. Extraction and Isolation

The leaves of Iphiona scabra were collected from Yemen in summer season and

identified by Prof. Abdulnasser Al-Gifri, Professor of Plant Taxonomy, University of

Aden, Yemen. A voucher specimen bearing number (6036, 12 May 2006) was

deposited at the University of Aden, Yemen. The leaves were dried under shade and

crushed to make powder. The air dried powdered leaves of I. scabra (2 kg) were

extracted with 95% ethanol three times under reflux temperature. The solvent was

removed by steam distillation and the extract was concentrated to dark gummy mass

under reduced pressure. The dark gummy mass was fractionated successively with

petroleum ether, benzene, ethylacetate, acetone and methanol. The petroleum ether

and benzene fraction, ethyl acetate and acetone fraction showed similar behavior on

TLC examinations in different solvent systems like petroleum ether-benzene (1:1),

benzene-chloroform (9:1-1:1), benzene-pyridine-formic acid (BPF-36:9:5), toluene-

ethylformate-formic acid (TEF-5:4:1), hence they were mixed together and

chromatographed over silica gel column. Elution of the column with a gradient of

increasing solvent system like petroleum ether, petroleum ether-benzene (9:1-1:1),

benzene, benzene-ethylacetate (9:1-1:1), ethylacetate, ethylacetate-methanol (9:1-1:1)

successively and finally with blank methanol, yielded different fractions. All those

fractions which exhibited similar behavior on TLC examination were pooled together.

Chapter-2

67

Repeated column chromatography of the pooled fractions followed by fractional

crystallization afforded five compounds marked as: Is-1, Is-2, Is-3, Is-4, and Is-5.

2.3.3. Spectral characterization of isolated compounds (1-5)

n-tertacosanenol (1)

White solid (120 mg), m.p. 78 oC, Anal. Calc. for C24H50O: C, 81.28; H, 14.21;

found: C, 81.25; H, 14.24.

IR (KBr, νmax cm-1): 1611 (C-H), 3460 (OH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 0.88 (t, 3H, CH3), 1.25 (m, 4H, H-3 to H-

22), 1.56 (m, 4H, H-2 and H-23), 3.50 (t, 2H, H-1), 3.63 (s, 1H, OH), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 14.13 (CH3), 22.70 (C-23), 25.75 (C-3),

29.38-29.71 (C-4 to C-21), 31.94 (C-2), 32.82 (C-22), 63.11 (C-1).

MS (ESI) (m/z): 354.39 [M+•] (C24H50O).

6-hydroxy-8,11,11-trimethyl-bicyclo [7.2.0] undec-4-ene-4-carboxylic acid (2)

White shinning crystals (35 mg), m.p. 131-132 oC. Anal. Calc. for C15H24O3; C,

71.39; H, 9.59; found: C, 71.10; H, 9.53.

IR (KBr, νmax cm-1): 1630 (C=C), 1690 (C=O), 3550, 3500 (OH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 0.95 (s, 6H, 2×CH3), 0.98 (s, 3H, CH3),

1.11 (m, 2H, H-10), 1.25 (m, H-9), 1.38 (m, H-1), 1.54 (m, 2H, H-2), 1.83 (m, 2H,

H-7), 2.34 (dd, 1H, H-3b, J = 12.0, 8.0 Hz), 2.81 (dd, 1H, H-3a, J = 12.0, 8.0 Hz),

4.98 (dd, 1H, H-6, J = 6.8, 4.0 Hz), 6.81 (d, 1H, H-5, J = 4.0 Hz), 9.50 (s, 1H, 6-

OH), 10.20 (s, 1H, COOH). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 18.0, 25.5, 25.8 (CH3), 25.2 (C-3), 27.3

(C-2), 29.5 (C-8), 34.3 (C-11), 38.2 (C-9), 40.2 (C-10), 42.5 (C-7), 48.9 (C-1), 78.0

(C-6), 134.3 (C-4), 140.5 (C-5), 166.1 (COOH).

MS (ESI) (m/z): 252 [M+•] (C15H24O3).

4,6-Diacetylresorcinol (3)

Colourless shining crystals (40 mg), m.p. 178-180 °C. Anal. Calc. for C10H10O4; C,

61.85; H, 5.19; found: C, 61.83; H, 5.21. UV (MeOH) λmax: 212, 275 and 312 nm.

IR (KBr, νmax cm-1): 1589, 1490 (C=C), 1658 (C=O), 3430 (OH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.61 (s, 6H, 2×CH3), 6.38 (s, 1H, H-3),

8.18 (s, 1H, H-6), 12.91 (s, 2H, 2×OH). 13C NMR (100 MHz, DMSO-d6, δ, ppm):, 26.12 (CH3), 104.99 (C-3), 113.66 (C-1,

Chapter-2

68

C-5), 136.31 (C-6), 168.93 (C-2, C-4), 202.52 (C=O),

MS (ESI) (m/z): 194.06 [M+•] (C10H10O4).

1-(2,4-dihydroxyphenyl)ethanone (4-Acetylresorcinol) (4)

Yellowish brown solid (60 mg), m.p. 144-145 °C. Anal. Calc. for C8H8O3; C, 63.15;

H, 5.30; found: C, 63.14; H, 5.33. UV (MeOH) λmax: 215, 275 and 310 nm.

IR (KBr) ν cm-1: 1520, 1440 (C=C), 1631 (C=O), 2925, 3301 (OH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.53 (s, 3H, CH3), 6.25-6.26 (d, 1H, H-3, J

= 4.0 Hz), 6.34-6.37 (dd,1H, H-5, J = 8.8, 2.4 Hz), 7.63-7.65 (d, 1H, H-6, J = 8.0

Hz), 10.45 (s, 1H, H-4, OH), 12.62 (s, 1H, 2-OH). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 25.86 (CH3), 112.63 (C-1), 102.36 (C-3),

107.96 (C-5), 132.96 (C-6), 164.42 (C-4), 164.84 (C-2), 189.40 (C=O),

MS (ESI) (m/z): 152 [M+•] (C8H8O3).

1,2-bis(o-tolyloxy)ethane (5)

Yellow solid (60 mg). m.p. 89 °C. Anal. Calc. for C16H18O2; C, 79.31; H, 7.49;

found C, 79.30; H, 7.47.

IR (KBr) ν cm-1: 1146, 1283 (C-O-C), 1317, 1389 (CH3), 1442 (CH2), 1500, 1624

(C=C). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.34 (s, 6H, 2×CH3), 4.36 (s, 2H, H-1),

4.37 (s, 2H, H-2), 6.91-6.98 (m, 4H, phenyl ring), 7.18-7.15 (m, 4H, phenyl ring). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 23.37 (CH3), 62.84 (C-1 and C-2), 122.25

(C-6' and C-6"), 128.73 (C-5' and C-5"), 128.85 (C-4' and C-4"), 129.26 (C-3' and

C-3"), 144.26 (C-1' and C-1"), 164.16 (C-2' and C-2").

MS (ESI) (m/z): 242 [M+•] (C16H18O2).

2.3.4. Single crystal X-ray crystallography studies of compounds 2 and 3

The crystal structure of the compound 2 and 3 were determined by X-ray diffraction

experiments performed on a Bruker Apex II diffractometer. The diffraction data was

collected at room temperature 293(2) K using graphite monochromated Mo Kα (λ=

0.71073 Å). Data reduction was performed with APEX II.23 Lorentz and polarization

corrections were applied. Absorption corrections were applied using SADABS.24 The

crystallographic structures were solved using direct methods (SHELXS-97).24 The

structure refinements were carried out with SHELXL-97 software.25 The refinements

were made by full-matrix least-squares on F2, with anisotropic displacement

Chapter-2

69

parameters for all non-hydrogen atoms. All the hydrogen atoms were located in a

difference Fourier synthesis, placed at calculated positions and then, included in the

structure factor calculation in a riding model using SHELXL-97 defaults with the

exception of the hydrogen atoms bonded to hetero atoms, which were refined freely.

MERCURY 3.326 was used for figure plotting. PLATON27 was used for data analysis.

Additional information to the structures determination is given in Table 1. Atomic

coordinates, thermal parameters and bond lengths and angles have been deposited at

the Cambridge Crystallographic Data Centre with reference numbers CCDC-934640

(compound 2), 1451250 (compound 3).


2.3.5a. Sample preparation

1 mg of calf thymus DNA was dissolved in 1 ml 20 mM phosphate buffer (pH = 7.4)

at 298 K with occasional stirring up to 24 h to ensure the formation of a homogenous

solution. The concentration of the DNA was determined spectrophotometrically using

260 = 6600 M-1 cm-1 as reported earlier.28 The stock solution of the compounds (3-5)

were first dissolved in DMSO and then final stock solution was prepared in the same

buffer (volume of DMSO does not exceed 5 % by V/V). All experiments were carried

out at 298 K in 2 mM phosphate buffer pH (7.4) unless otherwise mentioned.

2.3.5b. pH determination

pH measurements were carried out on Mettler Toledo pH meter (Seven Easy S20–K)

using Expert “Pro3 in 1” type electrode. The least count of the pH meter was 0.01 pH

unit.

2.3.5c. UV-Visible spectroscopy

The UV-measurements of calf thymus DNA were recorded on a UV-1800 Shimadzu

spectrophotometer by using a cuvette of 1 cm path length. The absorbance values of

DNA were recorded in absence and presence of compounds (3-5) in the range of 240-

300 nm. Appropriate blanks corresponding to the DNA solution and buffer were

subtracted to correct the base line.

Chapter-2

70

2.3.5d. Fluorescence spectroscopy

Fluorescence measurements were performed on a Shimadzu spectrofluorimeter,

model RF-5301 equipped with PC. The fluorescence spectra were measured at 25 ±

0.1 °C with a 1 cm path length cell. Both excitation and emission slits were set at 5

nm. Intrinsic fluorescence was measured by exciting the ctDNA solution at 440 nm

and emission spectra was recorded in the range of 550-700 nm. ctDNA concentration

was 12µM and the concentrations of compounds (3-5) were 0-70 µM.

2.3.5e. Circular dichroism measurements

To monitor the secondary structural changes in ctDNA at different concentrations

compounds (3-5) was carried out on JASCO-J 813 spcetropolarimeter equipped with

a Peltier-type temperature controller at 25 degree using a quartz cell a path length of

0.1 cm. Two scans were accumulated at a scan speed of 100 nm min-1, with data

being collected in the range of 220-320. CD spectra of ctDNA in absence and

presence of compounds (3-5) were recorded. In addition respective blanks were

subtracted.


The rigid and symmetric molecular docking studies of compounds 3-5 have been

carried out using PatchDock site having default setting of Clustering RMSD 4.0. The

visualization of the docked systems has been performed using Discovery Studio 4.0

software. The crystal structure of B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB

ID: 1BNA) retrieved from protein data bank. The energy optimized structure of

compounds 4,5 and Crystallographic Information File (cif) of compound 3 were first

saved to the Protein Data Bank (PDB) format and then were used as a ligand for

docking study. The initial study of the DNA-drug interaction was carried out by

docking of compounds 3-5 with DNA (PDB ID: 1BNA). For this, the whole DNA

crystal structure was considered as an active site to avoid bias towards either DNA

minor or major grooves during the docking study.

2.3.7. Antioxidant assay

Isolated compounds 2-5 and plant extracts (aqueous and ethanolic) were tested for

their antioxidant property by 1,1-diphenylpicrylhydrazyl (DPPH) method.29 In this

procedure, drug stock solution (1 mg/mL) was diluted to final concentration of 25, 50,

75 and 100 μg/mL in methanol. Methanolic DPPH solution (1 mL, 0.3 mmol) was

Chapter-2

71

added to 3.0 mL of drug solution of different concentrations. The tube was kept at an

ambient temperature for 30 min and the absorbance was measured at 517 nm in UV

VIS-1800 spectrophotometer. The scavenging activity was calculated by following

formula:

[% inhibition = [(Acontrol - Asample/Acontrol) × 100]

Where Acontrol is the absorbance of the L-ascorbic acid (standard) and Asample is the

absorbance of different samples. The methanolic DPPH solution (1 mL, 0.3 mM) was

used as control. The inhibitory concentration (IC50) value represents the concentration

required to exhibit 50 % antiradical activity. The IC50 values were calculated by the

linear regression analysis of dose-response curve plotted between % inhibition and

concentration. The experiments are run in triplicates.

2.3.8. In vivo cytotoxicity assay of plant extracts

Brine shrimp lethality bioassay is commonly used in the bioassay for the bioactive

compounds.30 The in vivo cytotoxicity assay is performed on brine shrimp nauplii

(Artemia salina) in accordance with the Meyer method.21 The egg of brine shrimp are

hatched in a tank filled with artificially prepared sea water (brine, 3.8 % NaCl)

exposed to incandescent light. Air is supplied at the bottom of the tank with the help

of air supplier fitted with tube to keep the shrimps in uniform motion. The assay is

performed 24 h after hatching and no food supplement is given during the hatching

and experimental periods. A test sample (5.0 mg) is dissolved in 1 mL DMSO to

obtain stock solution of 5 mg/mL. Different concentrations of test samples are

obtained from stock solution and placed in separate vials, and the volume of each vial

is made up to 5 mL with brine to obtain the desired final concentrations (10, 20, 40,

60 and 80 μg/mL). The negative control is prepared in the same manner without the

samples. Vincristine sulphate is used as a standard anti-cancer drug. Thirty brine

shrimp nauplii are then placed in each vial. After 24 h of incubation, the vials are

observed using a magnifying glass, and the number of survivors in each vial are

counted and noted. The LD50 values are calculated using the plot of percentage of

mortality versus logarithm of concentration. The percentage mortality (%M) was also

calculated by dividing the number of dead nauplii by the total number, and then

multiplied by 100 %. This is to ensure that the death (mortality) of the nauplii is

attributed to the bioactive compounds present in the plant extracts. All tests are

performed in triplicate and expressed as mean.

Chapter-2

72

2.4. CONCLUSION

In the present chapter, we report the isolation of a novel 6-hydroxy-8,11,11-trimethyl-

bicyclo [7.2.0] undec-4-ene-4-carboxylic acid (2) along with four known compounds

n-tertacosanol (1), 4,6-Diacetylresorcinol (3), 4-Acetylresorcinol (4) and 1,2-bis(o-

tolyloxy)ethane (5) from the leaves of Iphiona scabra. Compound 5 has been isolated

first time from natural source. Interaction of isolated compounds (3-5) with ctDNA

was studied. Antioxidant activity along with plant extract (aqueous and ethanolic) was

extensively studied. It found that all the compounds interact via non-intercalation

mode (groove binding). Compound 5 showed greater binding affinity while

compound 3 showed greater antioxidant activity. The cytotoxicity activity show that

the extracts of I. scabra have promising antitumor agent based on the LC50 values of

both the extracts.

Chapter-2

73

2.5. REFERENCES

1. M. G. El-Ghazouly, N. A. El-Sebakhy, A. A. Seif El-Din, C. Zderoa, F.

Bohlmann, Phytochemistry, 1987, 26, 439.

2. H. Merxmuller, P. Leins, and H. Roessler, edited by V. H. Heywood, J. B.

Harborne, B. L. Turner, Academic Press, London, 1977, 590.

3. A. A. Ahmed, T. J. Mabry, Phytochemistry, 1987, 26, 1517.

4. E. Roeder, T. Bourauel, U. Meier, H. Wiedenfeld, Phytpchemistry, 1995, 37,

353.

5. M. A. Mogib, J. Jakupovic, A. M. Dawidar, Phytochemistry, 1989, 28, 2202.

6. A. Sharaby, H. A. Rahman, S. Moawad, Saudi. J. Biol. Sci., 2009, 16, 1.

8. M. K. Kokila, K. Nirmala, A. Puttaraja, N. Shamala, Acta Cryst. C, 1992, 48,

1133.

9. Y. Sun, Z. Liu, J. Wang, L. X. L. Zhu, Chromatographia, 2009, 70, 1.

10. W. Zhao, J. Wu, W. Liu, P. Wang, Supramol. Chem., 2013, 25, 69.

11. M. Pulici, F. Sugawara, H. Koshino, J. Uzawa, S. Yoshida, E. Lobkovsky, and

J. Clardy, J. Org. Chem., 1996, 61, 2111.

12. S. Shankar, R. M. Coates, J. Org. Chem., 1998, 63, 9177.

13. F. A. Tanious, D. Ding, D. A. Patrick, C. Bailly, R. R. Tidwell, W. D. Wilson,

Biochemistry., 2000, 39, 12091.

14. Q. L. Zhang, J. G. Liu, H. Chao, G. Q. Xue, L. N. Ji, J. Inorg. Biochem., 2001,

83, 49.

15. M. Parveen, F. Ahmad, A. M. Malla, M. S. Khan, S. U. Rehman, M. Tabish,

M. R. Silva, P. S. P. Silva, J. Photochem. Photobiol. B, 2016, 159, 218.

16. X. Y. Meng, H. X. Zhang, M. Mezei, M. Cui, Curr. Comput. Aided Drug Des.,

2011, 7, 146.

17. E. Bendary, R. R. Francis, H. M. G. Ali, M. I. Sarwat, S. E. Hady, Annals of

Agricultural Science, 2013, 58, 173.

18. (a) S. A. Gadir, J. Chem. Pharm. Res. 2012, 4, 5145; (b) J.R. Naidu, R. Ismail,

S. Sasidharan, Trop. J. Pharm. Res. 2014, 13, 101.

19. J. L. McLaughlin, L. L. Rogers, J. E. Anderson, Drug Inf. J. 1998, 32, 513.

20. T. R. Prashith Kekuda, K. S. Vinayaka, K. V. Soumya, S. K. Ashwini, R.

Kiran, Int. J. Toxicol. Pharmacol. Res., 2010, 2, 26.

Chapter-2

74

21. B. N. Mayer, N. R. Ferrigni, J. E. Putnam, L. B. Jacobsen, D. E. Nichols, J. L.

McLaughlin, Planta Med., 1982, 45, 31.

22. W. Ren, Z. Qiao, H. Wang, L. Zhu, L. Zhang, Med. Res. Rev., 2003, 23, 519.

23. Bruker APEX2, SAINT Bruker AXS Inc. Madison,Wisconsin, USA, 2003.

24. G. M. Sheldrick, SADABS, University of Gottingen, Germany, 2003.

25. G. M. Sheldrick, Acta Crystallogr. A, 2008, 64, 112.

26. C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P. Shields, R.

Taylor, M. Towler, J. V. Streek, J. Appl. Crystallogr., 2006, 39, 453.

27. A. L. Spek, Acta Crystallogr. D, 2009, 65, 148.

28. C. V. Kumar, E. H. Asuncion, J. Am. Chem. Soc., 1993, 115, 8547.

29. K. Kato, S. Terao, N. Shimamoto, M. Hirata, J. Med. Chem., 1988, 31, 793.

30. G. X. Zhao, Y. H. Hui, J. K. Rupprecht, J. L. McLaughlin, K.V. Wood, J. Nat.

Prod., 1992, 55, 347.

Chapter-3

75

3.1. INTRODUCTION

The Genus Garcinia, belonging to the family Guttiferae (Clausiaceae) comprises

about 300 species. These species are native to Asia, Africa, South America and

Polynesia. The plants are small to medium sized evergreen trees or shrubs which may

grow up to 30 m in height and are widely distributed in the tropical and temperate

regions of the world.1 Most of the species produce edible fruits in which the most

famous species is G. mangostana or mangosteen (locally known as ‘manggis’).

Garcinia species is commonly used as folklore medicine for the treatment of

diarrhoea, itches, earaches, wounds, ulcers and fevers.2 The literature survey revealed

that isolated compounds from Garcinia species possess promising pharmacological

properties such as anticancer, anti-inflammatory, antibacterial, antiviral, antifungal,

anti-HIV, antidepressant and antioxidant.3-7 This genus has been a major source of

prenylated xanthones, benzophenones, triterpenoids and biflavonoids mainly with a

3/8 linkage.8-11 Previous phytochemical investigations on Garcinia nervosa revealed

the presence of xanthones (narvosaxanthone)12, biflavanoids (flavanoylflavone)2,13,

isoflavones (nervosin, irigenin and 7-methyltectorigenin)14 and chalcone (5′-bromo-

2′-hydroxy-4,4′,6′-trimethoxy chalcone, 2′-hydroxy-4,4′-dimethoxy chalcone and 2′-

hydroxy-3,4,4′,6′-tetramethoxy dihydrochalcone15. The methanolic extract of G.

nervosa exhibited positive response against the three cancer cell lines (HeLa, MCF-7,

HT-29) along with high anti-oxidative and anti-inflammation properties.5 Further the

methanolic extract of the leaves of G. nervosa var. pubescens King showed strong

inhibitory effects on platelet-activating factor (PAF) receptor binding.2 Although a

large no. of compounds have been isolated from G. nervosa in the last 30 years but

medicinal importance and scanty work on this plant accelerates our interest to carry

out the comprehensive investigations of the plant G. nervosa. Further intensive

studies and proper modern analysis of medicinal properties of the isolated compounds

is still missing. Thus it would be an interesting study not only the purpose of

expanding knowledge, but also as an alternative for medicinal purpose.

As part of our research work to explore the phytochemical and biological profile

of plants,16,17 we have investigated the chemical constituents of the leaves of Garcinia

nervosa, which led to the isolation of five compounds in which compound 1 is a novel

isoflavone. To the best of our knowledge, compound (1) has neither been isolated

from any natural source. Their structures have been elucidated on the basis of

Chapter-3

76

chemical and physical evidences (elemental analysis, UV, IR, 1H NMR, 13C NMR,

MS and X-ray crystallographic studies) and comparison of their spectral data with

literature compounds. DNA binding study of compound 1 and 2 were carried out to

probe the binding mode and extent of interaction of these compounds with calf

thymus DNA (ct-DNA). Moreover, antioxidant activity and cytotoxicity of plant

extracts were also tested.


This chapter deals with the phytochemical investigation of the leaves of Garcinia

nervosa i.e. extraction, isolation and characterization of its chemical constituents,

screening of antioxidant activity, cytotoxicity of extracts (aqueous and ethanolic) and

DNA binding study of isolated compounds 1 and 2. The air-dried leaves of G. nervosa

were extracted with 95% ethanol for three times under reflux temperature and filtered

it to yield a filtrate. The solvent was evaporated under reduced pressure and the

gummy mass was obtained fractionated successively with petroleum ether, benzene,

ethyl acetate, acetone and methanol. The benzene and ethyl acetate fractions showed

similar behavior on thin-layer chromatography (TLC) examination with different

solvents system eg. Benzene-chloroform (9:1-1:1), benzene-pyridine-formic acid

(BPF-36:9:5), toluene-ethylformate-formic acid (TEF-5:4:1), hence they were mixed

together. The mixed benzene and ethyl acetate extracts was chromatographed over a

silica gel column, eluting stepwise with gradient increase in polarity of solvents.

Further repeated column chromatography followed by fractional crystallization of

various fractions afforded pure compounds. The following five compounds (1-5) have

been isolated from the G. nervosa leaves.

1. 5,7-dihydroxy-3-(3'-hydroxy-4',5'-dimethoxyphenyl)-6-methoxy-4H-

chromen-4-one (Novel) (Gn-1)

2. 1-(2,5-dioxoimidazolidin-4-yl)urea (DL-allantoin) (Gn-2)

3. 4-methoxychalcone (Gn-3)

4. 2',4',4-trihydroxychalcone (Gn-4)

5. 5,7-dihydroxy-3-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one (Gn-5)

Chapter-3

77

3.2.1. Characterization of compound 1 (Gn-1)

Elution of column with petroleum ether-benzene (6:4) gave compound 1 which on

crystallization with CH3Cl-MeOH gave dark yellowish crystals, m.p. 260-262 °C.

Elemental analysis and molecular ion peak at m/z 361.29 [M+H]+• were in good

agreement with the molecular formula C18H16O8. The UV spectrum of compound (1)

exhibited characteristic absorption bands at 262 and 329 nm, suggesting its isoflavone

nature which was further confirmed by a positive ferric chloride test and a pink color

with Na-Hg and HCl suggesting that it was a hydroxylated isoflavone derivative. The

Micro-Ziesel determination showed the presence of three methoxyl groups. The IR

spectrum (Fig. 1) showed absorption bands at 3384, 1667, 1622, 1460-1583,

indicating the presence of hydroxyl, α,β-unsaturated carbonyl (C=O), C=Cγ-pyron and

C=Caromatic functional groups in the molecule, respectively. In the 1H NMR spectrum

(Fig. 2), the isoflavone nucleus was evidenced by the presence of a sharp singlet at δ

8.19 for H-2 proton. Further an independent sharp singlet integrating for one proton at

δ 6.46 ascribed to H-8 proton. A pair of meta-coupled doublets at δ 6.63 (J = 2.5 Hz)

and 6.71 (J = 2.5 Hz) each integrating for one proton were attributed to H-2' and H-6'

protons, respectively. Two broad singlets and one sharp singlet at δ 9.03, 10.50 and

13.01 integrating for 3 hydroxyl protons, respectively. The presence of three methoxy

groups was established by the appearance of three singlets of three protons each at δ

3.76, 3.81 and 3.83 respectively. The 13C NMR spectrum (Fig. 3) showed signals at δ

180.25, 153.87 and 121.97 were attributed to C-4, C-2 and C-3, characteristic of

flavone nucleus. Further signals at δ 152.72 (C-5), 131.32 (C-6), 157.34 (C-7), 93.76

(C-8), 104.93 (C-4a), 152.67 (C-8a), 126.00 (C-1'), 110.17 (C-2'), 150.20 (C-3'),

136.27 (C-4'), 153.30 (C-5'), 104.24 (C-6') have been assigned to aromatic carbons.

The three methoxy group were resonating at δ 59.84 (6-OCH3), 59.79 (4'-OCH3),

55.61 (5'-OCH3). The above assigned structure was further supported by the mass

spectrum (Fig. 4). It showed [M+•] at m/z 360. The fragment ions at m/z 345, 330 and

315 were due to [M-15], [M-30] and [M-45], corresponding to the successive loss of

three methyl groups. The RDA cleavage represents ring-A at m/z 153, 152 and ring-B

at m/z 208, 207. The ion at m/z 332 was due to the loss of CO unit from the flavones

unit (Scheme 1). The structure of compound (1) was further confirmed by single-

crystal X-ray diffraction analysis and DFT calculations.

Chapter-3

78

Fig. 1 FT-IR spectrum of compound 1

Fig. 2 1H NMR spectrum of compound 1

Chapter-3

79

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

38.94

39.15

39.36

39.57

39.78

39.99

40.20

55.61

59.79

59.84

78.21

78.53

78.73

78.86

93.76

104.24

104.93

110.17

121.97

126.00

131.32

136.27

150.20

152.67

152.72

153.30

153.87

157.34

180.25

Current Data ParametersNAME Sep02-2015EXPNO 181PROCNO 1

F2 - Acquisition ParametersDate_ 20150902Time 20.08INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT DMSONS 1024DS 4SWH 29761.904 HzFIDRES 0.454131 HzAQ 1.1010548 secRG 812DW 16.800 usecDE 6.00 usecTE 299.2 KD1 2.00000000 secd11 0.03000000 secDELTA 1.89999998 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 9.60 usecPL1 -2.00 dBSFO1 100.6228298 MHz

======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 -3.00 dBPL12 14.31 dBPL13 18.00 dBSFO2 400.1316005 MHz

F2 - Processing parametersSI 32768SF 100.6128193 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40

GN-12 BRUKERAVANCE II 400 NMRSpectrometerSAIFPanjab UniversityChandigarh

[email protected] Fig. 3 13C NMR spectrum of compound 1

WATERS, Q-TOF MICROMASS (ESI-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

%

0

100SHAISTA GN-12 13 (0.259) Cm (4:20-22:30) TOF MS ES+

4.10e4361.2940980

346.302260

383.3032272

384.336000

759.612348

743.631497

Fig. 4 Mass spectrum of compound 1

Chapter-3

80

Scheme 1 Mass fragmentation pattern of compound 1

In the light of above discussion, compound 1 was identified as 5,7-dihydroxy-3-

(3'-hydroxy-4',5'-dimethoxyphenyl)-6-methoxy-4H-chromen-4-one, it is a novel

compound.

O

OOHH3CO

HO

OH

OCH3OCH3

12

345

6

7 8 8a

4a1' 2' 3'

4'5'

6'

O

OOHH3CO

HO

OH

OCH3OCH3

O

OHH3CO

HO

OH

OCH3OCH3

O

OHH3CO

HO OH

OCH3OCH3

CO

m/z 360

m/z 152 m/z 207

m/z 345

m/z 330

m/z 315

m/z 332

- CO

- CH3

- CH3

- CH3RDA

Chapter-3

81


Elution of column with petroleum ether-benzene (6:4) gave white solid which on

crystallization with CHCl3-MeOH gave colorless crystals with m.p. 230 °C.18

Elemental analysis and molecular ion peak at m/z 158.04 [M+•] were found in good

agreement with the molecular formula C4H6N4O3. The IR spectrum of the compound

showed absorption bands at 1781, 1717 and 1660 cm-1 (C=O), 3439, 3345 cm-1

(NH2), 3219, 3062 cm-1 (N-H)str, 1531 cm-1 (N-H)bend indicating the presence of

C=O, NH2 and CONH functional groups in the molecule. In the 1H NMR spectrum,

the signals for H-3 and H-6 appeared as doublets at δ 6.9 (J = 8.1 Hz) and 5.3 (J = 8.1

Hz), respectively. The remaining protons appeared as singlets at δ 10.51, 8.09 and

5.81 were assigned to H-1, H-4 and H-8, respectively. In 13C NMR spectrum, the two

carbonyl group present in the ring appeared very close to each other at δ 157.40 and

157.80 for C-2 and C-7, respectively while the third carbonyl present outside of the

ring appeared at δ 174.10. The signal resonating at δ 62.01 was assigned to C-4

carbon of the ring. The structure of compound (2) was further confirmed by single-

crystal X-ray diffraction analysis and comparison of melting point, spectral data with

literature.18

In the light of above discussion, compound 2 was identified as 1-(2,5-

dioxoimidazolidin-4-yl)urea, known as DL-Allantoin.

HN

NH

O

NH

ONH2

O1

23

4

5

67

8


Compound 3 was isolated from the column with benzene-ethylacetate (8:2) and

crystallized with CHCl3-MeOH as light cream colored crystals, m.p. 76-78 °C.19

Elemental analysis and molecular ion peak at m/z 238, agreed with molecular formula

C16H14O2. A red color with conc. H2SO4 and orange to red color with aq. NaOH

suggested it was chalcone. The IR spectrum displayed the characteristics bands at

1661 cm-1 (C=O) and 1475 cm-1 (C=C). Its UV spectrum showed the maximum

absorption at 345 nm and minimum absorption at 245 nm. The 1H NMR spectrum

Chapter-3

82

showed a singlet of three protons at δ 3.76 assigned to a methoxy group. A pair of

ortho-coupled doublets at δ 6.89 (J = 8.7 Hz) and 7.48 (J = 8.7 Hz) integrating for two

protons each were attributed to (H-3, H-5) and (H-2, H-6) protons, respectively.

Another pair of doublets at δ 7.41 (J = 15.0 Hz) and 7.79 (J = 15.0 Hz) were ascribed

to α and β protons of chalcone. An ortho-coupled doublets at δ 7.87 (J = 8.4 Hz) for

two protons were attributed to 2', 6' protons. A multiplet at δ 7.50 resonating for three

protons was assigned to 3', 4', and 5' protons. 13C NMR spectrum showed peaks at δ

119.04 and 145.2 for α, β unsaturated carbon, δ 190.1 for C=O, characteristics of

chalcone nucleus. A peak at δ 55.3 was attributed to methoxy carbon. Further signals

at δ 137.50 (C-1'), 128.3 (C-2' and C-6'), 130.2 (C-3' and C-5'), 134.05 (C-4'), 127.1

(C-1), 132.3 (C-2 and C-6), 114.02 (C-3 and C-5) and 159.6 (C-4) were ascribed to

aromatic carbons. Finally comparison of melting point and spectral data with

literature19 confirm the structure of compound 3.

On the basis of above results compound 3 was identified as 4-

methoxychalcone.

O

OCH3



crystallized as yellow needles from CHCI3-MeOH m.p. 240 °C.20 Elemental analysis

and molecular ion peak at m/z 256 was in agreement with molecular formula

C15H12O4. A red color with conc. H2SO4 and orange to red color with aq. NaOH

suggested it was chalcone. Its IR spectrum showed the characteristic bands at 3324,

1665 and 1465 cm-1 for hydroxyl, α,β unsaturated ketone and C=C, respectively. In

UV spectrum it exhibited the absorption maxima at 236 and 368 nm. The 1H NMR

spectrum exhibited a meta coupled doublet at δ 6.20 (J = 2.4 Hz) for one proton was

attributed to H-3' proton, while a double doublet at δ 6.40 (J = 8.9, 2.4 Hz) integrating

for one proton was correspond to H-5' proton. One ortho-coupled doublet at δ 8.10 (J

= 8.9 Hz) for one proton was attributed to H-6' proton. Three singlet at δ 13.00 (5-

Chapter-3

83

OH), 10.20 (7-OH) and 9.31 (4'-OH) for one proton each were attributed to phenolic

OH. A pair of ortho-coupled doublets at δ 7.59 (J = 8.6 Hz) and 6.83 (J = 8.6 Hz)

integrating for two protons each were assigned to (H-2, H-6) and (H-3, H-5) protons,

respectively. α and β-protons of chalcone appeared as doublets at δ 7.74 (J = 15.4 Hz)

and 7.58 (J = l5.4 Hz). 13C NMR spectrum showed signals at δ 118.24, 146.07 and

190.35 were attributed to C-α, C-β and unsaturated ketone, respectively. Other peaks

at δ 127.8 (C-1), 130.7 (C-2 and C-6), 116.9 (C-3 and C-5), 160.8 (C-4), 113.7 (C-1'),

167.0 (C-2'), 105.1 (C-3'), 168.2 (C-4'), 110.1 (C-5'), 131.6 (C-6') have been assigned

to aromatic carbons. Finally comparison of melting point and spectral data with


On the basis of above results compound 4 was identified as 2',4',4-

trihydroxychalcone, known as isoliquiritigenin.

O

HO

OH

OH



crystallized as yellow needles from CHCI3-MeOH m.p. 223 °C.21 Elemental analysis

and molecular ion peak at m/z 300 was in agreement with molecular formula

C16H12O6. Its UV spectrum showed maximum absorption at 262 nm and an inflection

at 329 nm. The Micro-Ziesel determination showed the presence of three methoxyl

groups. The IR spectrum showed the characteristic bands at 3314, 1667 and 1632 cm-1

indicating the presence of hydroxy, C=O and C=C, respectively. In the 1H NMR

spectrum, the isoflavone nucleus was evidenced by the presence of a sharp singlet at δ

7.90 for H-2 proton. Further an independent sharp singlet integrating for one proton at

δ 6.50 ascribed to H-8 proton. The two ortho-coupled doublets at δ 6.95 (J = 8.9 Hz)

and 7.48 (J = 8.9 Hz) for two proton each were assigned to (H-2', H-6') and (H-3' and

H-5'), respectively. The three singlet in the offset region at δ 13.30 (5-OH), 10.53 (7-

OH) and 9.38 (4'-OH) were attributed to phenolic OH. Another singlet at δ 3.82

Chapter-3

84

integrating for three protons were indicated the presence of methoxy group. 13C NMR

spectrum, showed signals at δ 153.56, 125.12 and 178.03 were attributed to C-2, C-3

and C-4, characteristic of flavone nucleus. Further signals at δ 153.02 (C-5), 131.58

(C-6), 157.45 (C-7), 95.15 (C-8), 157.10 (C-9), 108.01 (C-10), 124.58 (C-1'), 135.94

(C-2' and C-6'), 118.20 (C-3' and C-5') and 157.05 (C-4') have been assigned to

aromatic carbons. Further comparison of melting point and spectral data with


In light of above discussion, compound 5 was identified as 5,7-dihydroxy-3-(4-

hydroxyphenyl)-6-methoxy-4H-chromen-4-one, known as Tectorigenin.

OHO

OH O OH

H3CO

3.2.6. X-ray crystallographic analysis and DFT study of compound 1 and 2

X-ray structure of compound 1 and 2 have been given in Fig. 5. Compound 5,7-

dihydroxy-3-(3'-hydroxy-4',5'-dimethoxyphenyl)-6-methoxy-4H-chromen -4-one (1)

crystallizes in the monoclinic system with the non-centrosymmetric and chiral space

group C2. Crystallographic data and structure refinement is shown in Table 1. It is an

isoflavone, composed of a benzopyrone moiety, a phenyl moiety, three methoxy

groups and three hydroxyl groups (Fig. 5). The atoms of the benzopyrone moiety are

nearly coplanar, with a dihedral angle between ring A (C3–C8) and ring C (C1–

C3/C8/O1/C9) of 3.8(1)º. To avoid steric hindrance, the phenyl ring and the

benzopyrone moiety of the title compound are rotated by 44.0(1)º, with respect to

each other but this is a common feature of biphenyl type structures. The methoxy

groups are all oriented out of the benzene rings (Table 2). The methoxy group O8–

C18 has positional disorder of the methyl group. This disorder was modeled over two

positions (labeled A and B) with an occupancy ratio of 0.514 (15):0.486 (15). The

molecules are linked by O—H…O hydrogen bonds forming zigzag chains along the b

axis (Table 3, Fig. 6)

Chapter-3

85

Fig. 5 Asymmetric unit of compound 1 and 2 with the ellipsoids drawn at the 50%

probability level, with the atomic labelling scheme

(1)

(2)

Chapter-3

86

Fig. 6 Packing diagram for compound (1) viewed down the c axis. Hydrogen bonds

are shown as dashed lines (Mercury, version 3.1 [12]). For clarity, only the major

disorder component and the strongest hydrogen bonds are displayed

with descriptor C(6) and C(12) according to Etter’s graph-set theory.22 There are also

one strong intramolecular O—H …O hydrogen bond with descriptor S(6) forming a

6-membered ring (Table 3). The crystal structure is also stabilized by several C—

H…O hydrogen bond. There are π–π stacking interactions between adjacent rings of

the isoflavone skeletons. Ring C stacks with ring C at (1-x,y,1-z) with a centroid-

centroid distance of 3.52Å and with ring C at (1-x,y,2-z) with a centroid-centroid

distance of 3.65Å. The other important stacking interactions occur between ring A

and ring C at (1-x,y,1-z) with a centroid-centroid distance of 3.76Å and between ring

A and ring C at (1-x,y,1-z) with a centroid-centroid distance of 3.86Å. These π–π

interactions organize the molecules in columns along the c axis. Aromatic π–π

stacking interactions are common in flavonoid crystal structures.23,24

Compound 2 crystallizes in monoclinic structure with space group P21/c (Fig. 5,

Table 1) The single crystal X-ray analysis of compound (2) is in good agreement with

the previous reports.25

Chapter-3

87

Table 1 Crystallographic data and structure refinement of compounds (1) and (2).

Empirical formula Compound (1) C18H16O8

Compound (2) C4H6N4O3

Formula weight 360.31 158.13

Temperature (K) 293(2) 293(2)

Wavelength (Å) 0.71073 0.71073

Crystal system Monoclinic Monoclinic

Space group C2 P21/c

a (Å) 27.4955(13) 8.0205(2)

b (Å) 8.4744(4) 5.14940(10)

c (Å) 7.0859(3) 14.7807(4)

α (°) 90 90.00

β (°) 102.915(2) 93.0687(14)

γ (°) 90 90.00

Volume (Å3) 1609.30(13) 609.58(3)

Z 4 4

Calculated density (g/cm3) 1.487 1.723

Absorption coefficient (mm-1) 0.119 0.148

F(000) 752 328

Crystal size (mm3) 0.60×0.40×0.28 0.53×0.37×0.31

θ range for data collection (°) 3.00-27.19 3.65-26.16

Index ranges -34<h<34; -10<k<10;

-9<l<9

-9<h<9; -6<k<6;

-18<l<18

Reflections collected/unique 16619/3509 11295/1223

Completeness to θ = 25.00º 99.8% 99.8%

Refinement method Full matrix LS on F2 Full matrix LS on F2

Data/restraints/parameters 3509/3/250 1223/0/115

Goodness–of–fit on F2 1.066 1.042

Final R indices [I> 2σ(I)] R1=0.0338;wR2=0.0902 R=0.0295;wR=0.0758

R indices (all data) R1=0.0357;wR2=0.0919 R=0.0329;wR=0.0788

Largest diff. peak and hole (e Å-3) 0.218 and -0.317 0.209 and -0.195

Chapter-3

88

Table 2 Comparison of selected geometrical parameters for compound (1) as determined by X-Ray diffraction and from DFT calculation (Å,°).

Experimental DFT

O1—C8 1.371(2) 1.370

O1—C9 1.347(2) 1.348

C1—C9 1.347(3) 1.355

C1—C10 1.482(3) 1.484

C2—O2 1.241(2) 1.245

C4—O3 1.337(2) 1.340

C5—O4 1.379(2) 1.379

O4—C16 1.425(3) 1.441

C6—O5 1.346(2) 1.349

C12—O6 1.367(2) 1.360

C13—O7 1.373(3) 1.383

O7—C17 1.431(3) 1.443

C14—O8 1.386(2) 1.377

O8—C18 A 1.417(1)/B 1.420(1) 1.436

C8—O1—C9 118.85(15) 119.53

C5—O4—C16 113.01(17) 116.35

C13—O7—C17 115.06(19) 114.71

C14—O8—C18 A 114.0(3)/B 120.0(3) 114.69

C4—C5—O4—C16 89.1(2) 60.11

C14—C13—O7—C17 78.0(3) 66.89

C13—C14—O8—C18 A 89.2(6)/B 138.6(7) 70.71

C9—C1—C10—C15 140.8(2) 137.74

Table 3 H-bond geometry (Å, º) of compound (1)

D—H...A D—H H...A D...A ∠(D—H...A)

O3—H3...O2 (intra) 0.82 1.85 2.580(2) 147.4

O5—H5...O6i 0.82 2.11 2.830(2) 145.8

O6—H6...O8 ii 0.82 2.01 2.775(2) 154.5

C9—H9...O3 iii 0.93 2.57 3.483(3) 165.8

C11—H11...O4 vi 0.93 2.52 3.364(2) 151.0

C17—H17C...O2 ii 0.96 2.56 3.410(3) 147.5

(symmetry codesi:1-x,-1+y,1-z; ii:3/2-x,1/2+y,2-z; iii: x,1+y,z; iv: 1-x,1+y,1-z)

Chapter-3

89

Table 4 Comparison of selected geometrical parameters for compound (2) as determined by X-Ray diffraction and from the DFT geometry optimization (Å,°).

Experimental DFT

O1—C1 1.2221(16) 1.2031

O2—C2 1.2148(16) 1.2019

O3—C4 1.2438(15) 1.2159

N1—C1 1.3371(16) 1.3774

N1—C3 1.4537(16) 1.4542

N2—C1 1.3910(16) 1.4109

N2—C2 1.3531(16) 1.3731

N3—C3 1.4241(15) 1.4405

N3—C4 1.3595(15) 1.3844

N4—C4 1.3302(16) 1.3844

C2—C3 1.5368(16) 1.5587

C1—N1—C3 112.66(10) 112.97

C1—N2—C2 111.96(10) 113.38

C3—N3—C4 120.57(10) 121.54

O1—C1—N1 127.89(12) 128.34

N1—C1—N2 107.68(10) 105.60

N2—C2—C3 106.62(10) 105.38

N1—C3—C2 100.74(9) 101.74

N3—C4—N4 116.99(11) 114.28

C4—N3—C3—N1 70.21(15) 67.05

C3—N3—C4—N4 177.82(11) 179.45

To check the influence of the intermolecular interactions on the molecular geometry,

we have performed a DFT calculation of the equilibrium geometry of the free

molecule starting from the experimental X-ray geometry. The DFT calculation of

compound (1) reproduce well the observed experimental bond lengths and valence

angles of the molecule but some of the calculated dihedral angles in the free molecule

differ significantly from those of the molecule in the crystal (Table 2, Fig. 7). The

methoxy group O6—C18 of the optimized geometry is closer to the position of the

major disorder component (Fig. 7). In case of compound (2) the agreement between

the experimental and calculated geometries is good (Table 4, Fig. 8).

Chapter-3

90

Fig. 7 Comparison of the molecular conformation of compound 1, as established from the X-ray study (red-major disorder component, yellow-minor disorder component)

with the optimized geometry (blue). (Software used for visualization: VMD, version 1.9.1, January 29, 2012).

Fig. 8 Comparison of the molecular conformation of compound 2, as established from the X-ray study (red) with the optimized geometry (blue) (Software used for

visualization: VMD, version 1.9.1, January 29, 2012).


3.2.7a. UV-Vis spectroscopy

UV-visible spectroscopy has been widely used to study the interaction between small

molecules and DNA. Results showed that compound (1) exhibits maximum

absorption at ~ 268 nm and ~ 334 nm. On addition of increasing concentrations of

ctDNA, a hyperchromism was observed at 268 nm while the peak at 334 remains

unchanged (Fig. 9a). On the other hand, compound (2) exhibits maximum absorbance

at ~ 225 nm. With addition of increasing DNA concentrations, hyperchromism was

observed at 225 nm (Fig. 9b). It is well established that observation of

Chapter-3

91

hyperchromism effect in complex formation suggests that small molecules binds to

DNA by external contact via non-covalent interactions.26 Hence, absorbance spectral

studies provide an evidence of non-intercalative binding pattern of compounds (1) and

(2) with DNA. To assess the binding ability of these compounds with DNA, intrinsic

binding constant (Kb) was calculated by the ratio of intercept to slope of plot from

Benesi-Hildebrand equation.27 The double reciprocal plot of 1/A-Ao vs 1/CDNA was

linear (Fig. 9c and d). The estimated values of binding constant (Kb) of compound

(1) and (2) was found to be of order of 104 M-1 (Fig. 9c and d).

(a) (b)

(c) (d)

Fig. 9 Interaction of compound with ctDNA, UV-Visible absorption spectra of (a) compound 1 (5 μM) (b) compound 2 (5μM) in the presence of increasing

concentrations of ctDNA (0-35 µM) in Tris-HCl buffer (pH 7.2) (c) and (d) represents the inverse plot of compound 1 and 2, respectively.

3.2.7b. Fluorescence spectral study

To further determine the interaction of compounds (1) and (2) with DNA,

fluorescence spectroscopy experiments were performed. As evident from results,

Chapter-3

92

quenching was observed in the fluorescence intensity of compound (1) and (2) and no

apparent shift in the emission maxima on addition of increasing concentrations of

ctDNA (Fig. 10a and b). This quenching suggests the interaction of compounds (1)

and (2) with DNA. Further, we also determined the quenching constant of these

compounds by plotting the ratio of fluorescence intensity (F0/F) in the presence and

absence of ctDNA as a function of increasing concentrations of DNA (Fig. 10c and

d).

Fig. 10 Interaction of compound with ctDNA, Fluorescence emission spectra of (a) compound 1 (10 μM) (b) compound 2 (10 μM) in the presence of increasing conc. of

ctDNA (0-50μM) Stern-Volmer plot of (c) compound 1 (d) compound 2. Stern-Volmer quenching constant (Ksv) was calculated from the slope and was found

to be 0.64 ×104 M-1 and 0.31 × 104 M-1 for compounds (1) and (2), respectively.

Quenching constant values for both these compounds was lower than classical

intercalators,28,29 hence indicating non-intercalative interaction with ctDNA.

Fluorescence spectral studies data was also used to calculate binding stoichiometry

(n) and binding constant (K) of compound-DNA complex using following equation.30

log [(F0-F)/F] = log K + n log[Q]

(a) (b)

(c) (d)

Chapter-3

93

where, F0 and F are fluorescence intensity in absence and presence of DNA

(quencher), respectively. Results obtained via a plot of log [(F0-F)/F] vs log [DNA]

are presented in Table 5. Binding constants of compounds (1) and (2) were found to

be 3.9 × 104 M-1 and 1.44 × 104 M-1, respectively.

Table 5 Parameters obtained using fluorescence studies.

Compounds Ksv (×104)(M-1) K (×104)(M-1) n R2

1 0.64 × 104 3.9 × 104 1.193 0.998

2 0.31 × 104 1.44 × 104 1.155 0.963

3.2.7c. Competitive displacement assay study

In order to decipher the binding modes of compound (1) and (2) with ctDNA,

competitive displacement assay was performed. Such an assay involves the use of

DNA binding dyes such as EtBr (intercalator) or Hoechst 33258 (groove binder). Any

small molecule/drug that displaces the bound dye from ctDNA is expected to bind in a

similar fashion as bound dye.31-34 Therefore, changes in fluorescence intensity of dye-

DNA complex on addition of small molecule help predict the binding mode of such

molecules. In case of EtBr displacement assay, with increasing concentrations of

compounds (1) and (2), there was no change in fluorescence intensity of EtBr-DNA

complex (Fig. 11a and b). This suggests that compound (1) and (2) follow the non-

intercalative mode of binding. To further explore the exact binding mode of the

compound (1) and (2), Hoechst 33258 dye was used in dye displacement assay.

Hoechst dye binds in the minor groove of DNA and shows enhancement in

fluorescence intensity on binding to DNA.34 As evident from results, increase in

concentrations of compound (1) and (2) decreases the fluorescence of Hoechst-DNA

system (Fig. 12a and b). This confirms the binding mode of compound (1) and (2) is

groove binding rather that intercalation.

Chapter-3

94

Fig. 11 Ethidium bromide (EB) displacement assay. Fluorescence emission spectra of DNA-EBcomplex in presence of increasing concentration of (a) compound 1 (0-

50μM) (b) compound 2 (0-50μM)

Fig. 12 Hoechst 33258 (HO) displacement assay. Fluorescence emission spectra of DNA-HO complex in presence of increasing concentration of (a) compound 1 (0-

50μM) (b) compound 2 (0-50μM) 3.2.7d. KI quenching study

Iodide quenching studies help to determine the binding mode of drug with DNA

molecules. Such an assay depends on the fact that when small molecules are

intercalated in DNA, iodide ions are repelled by negatively charged phosphate groups

of DNA and fluorescence of such molecules remains unaffected in presence of DNA.

However, molecules which are present as groove binders (exposed to external

surface) are easily approachable for quenchers even in the presence of DNA.35 The

quenching constant of anionic quenchers in absence and presence of DNA is

calculated via Stern-Volmer equation:

F0/F = 1 + Ksv [Q]

where F0 and F are fluorescence intensity in the absence and presence of anionic

quencher. Ksv is the quenching constant obtained from slope of F0/F vs [Q] plot (Fig.

(a) (b)

(a) (b)

Chapter-3

95

13a and b). Ksv values determine the type of binding of molecules with DNA

molecule. Decrease in Ksv occurs in intercalation and it remains unchanged when

interaction is electrostatic or groove binding.36,37 As evident from results, no

significant differences in Ksv values are observed in absence and presence of DNA for

compounds (1) and (2). Therefore, it can be concluded that these compounds exhibit

non-intercalative binding mode with ctDNA.

Fig. 13 KI quenching studies. The quenching of compound 1 and 2 fluorescence was recorded in (a) absence and (b) presence of ctDNA. Stern-Volmer plot was used to

calculate the Ksv in the absence and presence of DNA.

3.2.7e. Circular dichroism study

CD spectroscopy technique is a sensitive method to detect changes in secondary

structure of DNA molecule.38 Changes in the intrinsic CD spectra of DNA backbone

depends on the non-covalent interaction of molecules with DNA.39

Fig. 14 CD spectra of ctDNA in the presence of (A) compound 1 and (B) compound 2

was obtained at 25 °C with increasing concentration of compounds 1 and 2 (0-100μM)

(a) (b)

Chapter-3

96

As seen from Fig. 14, the CD spectrum of free DNA exhibits a positive peak at ~ 276

nm and a negative peak at ~ 244 nm. The positive band at ~ 276 nm is due to base

stacking and negative band at ~ 244 nm correspond to helicity of right-handed B-form

of DNA.40,41 Further, it is important to note that both these bands (peaks) are very

sensitive to the interaction of small molecules with DNA.42 Groove binding molecules

cause less or no perturbation on the positive and negative bands of CD spectra of

ctDNA, whereas intercalating molecules are known to produce significant effect on

intensities of both bands.43,44 Binding mode of compounds (1) and (2) with ctDNA

was studied using CD spectroscopy. Fig. 14 shows that no detectable change in CD

spectrum was recorded upon addition of compounds (1) and (2) to ctDNA solution.

These results confirm the non-intercalative mode of binding of compounds (1) and (2)

with ctDNA.


In order to study the exact interaction of the compounds (1) and (2) docking studies

were carried out. As evident from results (Table 6, Fig. 15), compound 1 exhibits

strong binding affinity with B-DNA with binding energy -6.82 kcal/mol and forms

seven hydrogen bonds with guanine (G-8 and G-9) and thymine (T-10). On the other

hand, compound 2 also exhibits strong binding affinity to B-DNA but lesser than

compound 1 with binding energy -5.93 kcal/mol and forms 5 hydrogen bonds with

adenosine (A-6), cytosine (C-7), guanine (G-5) and thymine (T-6). Due to higher H-

bonding of compound 1 than compound 2 with DNA, therefore, it can be concluded

that relative DNA binding affinity of compounds follow the order compound 1 >

compound 2.

Table 6 AutoDock results (Binding energy, Inhibition constant and no. of hydrogen bonds) of docked ligands in B-DNA

Ligands AutoDock Binding Energy (Kcal/mol)

AutoDock Inhibition Energy

(µM)

No. of Hydrogen bonds

Compound 1 -6.82 10.1 7

Compound 2 -5.93 46.03 5

Chapter-3

97

Fig. 15 Docking model of compounds 1 and 2 with ctDNA.

3.2.9. Antiproliferative activity

The ability of compounds (1) and (2) to inhibit the growth of cancer cells was

evaluated using well-characterized human cancer cell lines, MCF-7 and MDA-MB

231. MTT assay was used to determine cell viability (Fig. 16). As evident from Fig.

16, dose-dependent inhibition of cell proliferation was observed in compounds (1) and

(2) treated breast cancer cells. Next, we determined the IC50 value of compounds (1)

and (2) for MCF-7 and MDA-MB 231 cells. IC50 value for compound (1) for MCF-7

and MDA-MB 231 cell line was found to be 8.44±3.5 μM and 6.94±2.6 μM,

respectively. On the other hand, compound (2) exhibited IC50 for MCF-7 and MDA-

MB 231 cell lines, 14.2±4.6 μM and 20.0±3.1 μM respectively. IC50 values show high

potential of compound (1) as anticancer drug candidate. These results further suggest

that compounds (1) and (2) exhibit anticancer activity and hence can be used as

potential anticancer drugs.

Chapter-3

98

Fig. 16 % cell viability or proliferation of MFC and MDA-MB 231 cell lines with different concentration of compounds 1 and 2


The antioxidant activities of all the isolated compounds along with ethanolic and

aqueous extract were investigated by DPPH scavenging activity. It is based on the

reduction of DPPH, a stable free radical.45 The free radical DPPH gives a maximum

absorption at 517 nm (purple colour). Antioxidants react with DPPH and reduced it to

a stable form by donating hydrogen free radical. When a solution of DPPH is mixed

with a substance that can donate a hydrogen atom, then this gives rise to the reduced

form of DPPH with the loss of this violet color. The reduction capability of DPPH

radicals was determined by a decrease in their absorbance at 517 nm induced by

antioxidants using ascorbic acid as a reference. The potencies for the antioxidant

activity of five isolated compounds along with ethanolic and aqueous extract to the

reference compound are shown in Table 7. Almost all the tested compounds (1-5)

possessed strong scavenging activity against the DPPH with IC50 values (8.29-27.80

µg/mL). Compound 1, 4 and 5 showed highest antioxidant activity with IC50 8.29,

Chapter-3

99

12.08 and 10.95 µg/mL, respectively than the standard drug (Ascorbic acid IC50 =

5.48 µg/mL). It is due to presence of 5,7-dihydroxyl group in ring A and OH/OCH3

group in ring B.46 Further 4'-OCH3 derivatives of chalcone (3) showed the moderate

inhibition (IC50= 16.55 µg/mL). The ethanolic extract showed good inhibitory activity

while aqueous extract showed moderate inhibition.

Table 7 Antioxidant activity of compounds (1-5) and plant extracts.

Compounds and extracts IC50 value (µg/mL)a

1 8.29±0.3

2 16.55±0.8

3 18.79±0.4

4 12.08±0.9

5 10.95±0.9


Aqueous extract

Ascorbis Acid (Standard)

27.80±0.2

5.48±0.3 aIC50 value represents the concentration of three experiments required to exhibit 50 % antioxidant activity.

3.2.11. Cytotoxicity study of plant extracts

The in vivo cytotoxicity of ethanolic and aqueous extract of Garcinia nervosa leaves

were evaluated to brine shrimp nauplii (Artemia salina) using Vincristine sulfate as

standard (Fig. 17). Brine Shrimp Lethality Assay is the most convenient system for

monitoring toxicity of various plant species. This method is very useful for

preliminary assessment of toxicity of the plant extracts. This bioassay has good

correlation with cytotoxic activity in some human solid tumors, and has led to the

discovery of new class of natural active antitumor agents.47 Rapidness, simplicity and

low requirements are several advantages of this assay. This method is based on the

survival of number of brine shrimps after 24 h of incubation as described by Meyer et

al.48 The LD50 value of extracts (ethanolic and aqueous) and standard (Vincristine

sulfate) is reported in μg/mL (Table 8). In the present study, each of the test samples

display different mortality rates at different concentrations, the percentage mortality

increases with an increase in concentration. The calculated LD50 value of the

ethanolic and aqueous extract of leaves are found to be 11.06 μg/mL and 23.74

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100

μg/mL, respectively, as compared to the standard drug Vincristine sulfate whose LD50

is 8.70 μg/mL. These data clearly indicates significant cytotoxicity of the crude

extracts of Garcinia nervosa. The variation in results may be due presence of different

types of compounds in the extracts. Polyphenolic compounds especially flavonoids

that are found in plants have demonstrated to be very effective antitumor agents.49,50

Fig. 17 Determination of LD50 values for ethanolic and aqueous extract of Garcinia nervosa leaves from linear correlation between logarithms of concentration versus

percentage of mortality

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101

Table 8 Cytotoxicity assay of plant extracts (ethanolic and aqueous).

Extracts LD50 value (μg/mL)a


Aqueous extract 23.74±1.74

Standard (Bleomycin) 1.50±0.03

aLC50 value represents the concentration of three experiments required to exhibit 50 % cytotoxicity.

3.3. EXPERIMENTAL

3.3.1. Materials and Methods

Chemicals were purchased from Merck and Sigma-Aldrich as ‘synthesis grade’ and

used without further purification. Melting points were determined on a Kofler

apparatus and are uncorrected. The instrumentation detail of elemental analysis (C, H,

N), IR, NMR and Mass have been discussed in chapter 2. The UV spectra were

recorded with (UV-1800, Shimadzu Corp., Tokyo, Japan). Fluorescence emission

spectra were recorded using RF- 5301PC spectrofluorometer (Shimadzu 360 Corp.,

Tokyo, Japan). Thin layer chromatography (TLC) glass plates (20×5 & 50×10) were

coated with silica gel G254 (E-Merck) and exposed to iodine vapors to check the

purity of collected fractions and compounds.

3.3.2. Extraction and Isolation

The leaves of G. nervosa were collected by the former colleagues from Zaria, Nigeria.

It was identified by Prof. Wazahat Husain, Department of Botany, AMU, Aligarh. A

voucher specimen has also been deposited at the herbarium of department of botany

AMU Aligarh. The air-dried leaves of G. nervosa were crushed to make powder (2.0

kg) and extracted exhaustively with 95% ethanol for three times under reflux

temperature and filtered to yield a filtrate. The solvent was evaporated under reduced

pressure to afford a crude extract and fractionated successively with petroleum ether,

benzene, ethyl acetate acetone and methanol. The benzene and ethyl acetate fractions

show similar behavior on thin-layer chromatography (TLC) examination in different

solvent systems like petroleum ether-benzene (9:1-1:1), benzene-chloroform (9:1-

1:1), benzene-acetone (9:1- 1:1), benzene-pyridine-formic acid (BPF-36:9:5), toluene-

ethylformate-formic acid (TEF-5:4:1), and also respond positively to ferric chloride

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102

test for phenolic OH group and Shinoda’s test for flavonoids, hence they were mixed

together. The mixed benzene and ethyl acetate extracts was chromatographed on a

silica gel column, eluting stepwise with blank petroleum ether then with petroleum

ether-benzene mixture in different ratios (9:1-1:1), benzene, benzene-ethyl acetate

(9:1-1:1), ethyl acetate, ethyl acetate-methanol (9:1-1:1) and finally with blank

methanol to obtain different fractions. All those fractions which show similar

behavior on TLC examination were pooled together. Further repeated column

chromatography of pooled fractions followed by crystallization afforded five

compounds, labeled as Gn-1, Gn-2, Gn-3, Gn-4, and Gn-5.

3.3.3. Spectral characterization of isolated compounds (1-5)

5,7-dihydroxy-3-(3'-hydroxy-4',5'-dimethoxyphenyl)-6-methoxy-4H-chromen-4-

one (1)

Light yellowish crystals (50 mg), m.p. 260-262 °C, Anal. Calc. for C18H16O8; C,

50.97; H, 4.28; found: C, 50.95; H, 4.27. UV λmax (MeOH, nm): 262, 329

IR (KBr) ν cm-1: 1460, 1583 (C=C), 1622 (C=Cγ-prone), 1667 (C=O), 3384 (OH). 1H NMR (400 MHz, CDCl3, δ): 3.76 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.83 (s,

3H, OCH3). 6.63 (d, 1H, H-2', J = 2.5 Hz), 6.46 (s, 1H, H-8), 6.71 (d, 1H, H-6', J =

2.5 Hz), 8.19 (s, 1H, H-2), 9.03 (brs, 1H, 3'-OH), 10.50 (brs, 1H, 7-OH), 13.01 (s,

1H, 5-OH), 13C NMR (100 MHz, CDCl3, δ): 55.61 (5'-OCH3), 59.79 (4'-OCH3), 59.84 (6-

OCH3), 93.76 (C-8), 104.24 (C-6'), 104.93 (C-4a), 110.17 (C-2'), 121.97 (C-3),

126.00 (C-1'), 131.32 (C-6), 136.27 (C-4'), 150.20 (C-3'), 152.67 (C-8a), 152.72 (C-

5), 153.30 (C-5'), 153.87 (C-2), 157.34 (C-7), 180.25 (C-4).

MS (ESI) (m/z): 361.29 [M+H]+• (C18H16O8).

1-(2,5-dioxoimidazolidin-4-yl)urea (2)

Colorless crystals (30 mg), m.p. 230 °C, reported 230-232 °C, Anal. Calc. for

C4H6N4O3; C, 30.38; H, 3.82; found: C, 30.36; H, 3.85.

IR (KBr) ν cm-1: 1781, 1717, 1660 (C=O), 1531, 3062, 3219 (NH), 3345, 3439

(NH2). 1H NMR (400 MHz, CDCl3, δ): 5.3 (d, 1H, H-6, J = 8.1), 5.81 (s, 2H, H-8), 6.9 (d,

1H, H-3, J = 8.1), 8.09 (s, 1H, H-4), 10.51 (brs, 1H, H-1). 13C NMR (100 MHz, CDCl3, δ): 62.01 (C-4), 157.40 (C-2), 157.80 (C-7), 174.10

(C-5).

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103

MS (ESI) (m/z): 158.04 [M+•] (C4H6N4O3).

4-methoxychalcone (3)

Light cream colored crystals (60 mg), m.p. 76-78 °C. Anal. Calc. for C16H14O2; C,

80.65; H, 5.92; found: C, 80.61; H, 5.94. UV λmax (MeOH, nm): 345, 245

IR (KBr) ν cm-1: 1475, 1641 (C=C), 1661 (C=O). 1H NMR (400 MHz, CDCl3, δ): 3.76 (s, 3H, OCH3), 6.89 (d, 2H, H-3 and H-5, J =

8.7 Hz), 7.41 (d, 1H, α-H, J = 15.0 Hz), 7.48 (d, 2H, H-2 and H-6, J = 8.7 Hz), 7.79

(d, 1H, β-H, J = 15.0 Hz), 7.50-7.65 (m, 3H, H-3', H-4', H-5'), 7.87 (d, 2H, H-2' and

H-6', J = 8.4 Hz),

13C NMR (100 MHz, CDCl3, δ): 55.3 (OCH3), 114.02 (C-3 and C-5), 119.04 (C-α),

127.1 (C-1), 128.3 (C-2' and C-6'), 130.2 (C-3' and C-5'), 132.3 (C-2 and C-6),

134.05 (C-4'), 137.50 (C-1'), 145.2 (C-β), 159.6 (C-4), 190.1 (C=O).

MS (ESI) (m/z): 238 [M+•] (C16H14O2).

2',4',4-trihydroxychalcone (4)

Yellow crystals (85 mg), m.p. 240 °C, Anal. Calc. for C15H12O4; C, 70.31; H, 4.72;

found: C, 70.30; H, 4.73. UV λmax (MeOH, nm): 236, 368

IR (KBr) ν cm-1: 1465, 1649 (C=C), 1665 (C=O), 3324 (OH). 1H NMR (400 MHz, CDCl3, δ): 6.20 (d, 1H, H-3', J = 2.4 Hz), 6.40 (dd, 1H, H-5', J

= 8.9 2.4, Hz), 6.83 (d, 2H, H-3 and H-5, J = 8.6 Hz), 7.74 (d, 1H, α-H, J = 15.4 Hz),

7.58 (d, 1H, β-H, J = 15.4 Hz), 7.59 (d, 2H, H-2 and H-6, J = 8.6 Hz), 8.10 (d, 1H,

H-6', J = 8.9 Hz), 9.31 (4'-OH), 10.20 (7-OH), 13.00 (5-OH), 13C NMR (100 MHz, CDCl3, δ): 105.1 (C-3'), 110.1 (C-5'), 113.7 (C-1'), 116.9 (C-3

and C-5), 118.24 (C-α), 127.8 (C-1), 130.7 (C-2 and C-6), 131.6 (C-6'), 146.07 (C-

β), 160.8 (C-4), 167.0 (C-2'), 168.2 (C-4'), 190.35 (C=O).

MS (ESI) (m/z): 256 [M+•] (C15H12O4).

5,7-dihydroxy-3-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one (5)

Yellow crystals (70 mg), m.p. 240 °C. Anal. Calc. for C16H12O6; C, 64.03; H, 4.00;

found: C, 64.00; H, 4.00. UV λmax (MeOH, nm): 262, 329

IR (KBr) ν cm-1: 1468, 1632 (C=C), 1667 (C=O), 3314 (OH). 1H NMR (400 MHz, CDCl3, δ): 3.82 (s, 3H, OCH3), 6.50 (s, 1H, H-8), 6.95 (d, 2H,

H-2' and H-6', J = 8.9 Hz), 7.48 (d, 2H, H-3' and H-5', J = 8.9 Hz), 7.90 (s, 1H, H-

2), 9.38 (4'-OH), 10.53 (7-OH), 13.3 (5-OH), 13C NMR (100 MHz, CDCl3, δ): 95.15 (C-8), 108.01 (C-10), 118.20 (C-3' and C-

Chapter-3

104

5'), 124.58 (C-1'), 125.12 (C-3), 131.58 (C-6), 135.94 (C-2' and C-6'), 153.02 (C-5),

153.56 (C-2), 157.05 (C-4'). 157.10 (C-9), 157.45 (C-7), 178.03 (C-4),

MS (ESI) (m/z): 300 [M+•] (C16H12O6).

3.3.4. Crystal Structure Determination

The crystal structure of compound 5,7-dihydroxy-3-(3ʹ-hydroxy-4ʹ,5ʹ-

dimethoxyphenyl)-6-methoxy-4H-chromen-4-one (1) and 1-(2,5-dioxoimidazolidin-4-

yl)urea (2) was determined in a single crystal X-ray diffraction experiment performed

on a Bruker Apex II diffractometer. The instrumentation detail and procedure have

been discussed in chapter 2. We have chosen to model the disordered methyl group

over two positions, restraining the O-CH3 bond length to 1.420(1) Å with the DFIX

instruction from SHELXL-97. Additional information to the structure determination is

given in Table 1. The crystal data have been deposited at the Cambridge

Crystallographic Data Centre (compound (1) CCDC No. 1479579, compound (2)

1451249).

3.3.5. DFT calculations

The geometry optimization was performed using the GAMESS package,51 starting

with the experimental X-ray geometry but using an average position of the disordered

methyl group i.e., with no splitting of the original position. The calculations were

performed within density functional theory (DFT) using B3LYP (Becke three-

parameter Lee-Yang-Parr) for exchange and correlation, which combines the hybrid

exchange functional of Becke52,53 with the correlation functional of Lee, Yang and

Parr.54 The calculations were performed with an extended 6-311G(d,p) basis set. We

imposed tight conditions for convergence of both the self consistent field cycles and

the maximum density and energy gradient variations (10-5 atomic units). At the end of

the geometry optimization we conducted a Hessian calculation to guarantee that the

final structure corresponds to a true minimum, using the same level of theory as in the

geometry optimization.

3.3.6. Sample preparation for DNA binding study

ctDNA was dissolved in 10 mM Tris-HCl buffer (pH 7.2) at 4 °C with occasional

stirring to make a homogenous solution. In the next stage, purity of DNA solution was

analyzed by recording the absorbance ratio (A260nm / A280nm). The absorbance

ratio was between 1.8 and 1.9 and hence no further purification was needed. DNA

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105

concentrations used in different experiments were determined using average molar

extinction coefficient value of 6600 M-1 cm-1 of a single nucleotide at 260 nm.55 Stock

solution (3 mM) of compounds (1) and (2) was prepared in DMSO.

3.3.6a. UV-Visible spectroscopy

UV-visible spectral studies of compounds (1) and (2) were carried out using UV-VIS

spectrophotometer (UV-1800, Shimadzu Corp., Tokyo, Japan). Absorbance spectra of

isolated compounds were recorded in respective wavelengths in absence and presence

of increasing concentrations of ctDNA. Briefly, in the reaction mixture, fixed

concentration of synthesized compounds (1) and (2) (5 μM) was titrated with

increasing concentrations of ct-DNA (0-35 μM) in 10 mM Tris-HCl (pH 7.2).

3.3.6b. Steady state fluorescence spectroscopy

Fluorescence emission spectra of compounds (1) and (2) were recorded using RF-

5301PC spectrofluorometer (Shimadzu 360 Corp., Tokyo, Japan). Compounds (1) and

(2) were excited at 268 nm and 225 nm, respectively and emission spectra were

recorded in the respective wavelength range after setting the widths of excitation slit

at 5 nm and emission slit at 10 nm. To a 1 mL reaction mixture, fixed concentration of

compounds (1) and (2) (10 μM) was used and titrated with increasing concentrations

of ctDNA (0-50 μM). All fluorescence spectroscopy experiments were carried out in

10 mM Tris-HCl (pH 7.2).

3.3.6c. Competitive displacement assays

DNA binding dyes such as ethidium bromide (EtBr) and Hoechst 33258 (HO) are

used to decipher the binding modes of drug-DNA interaction. In case of EtBr

displacement assay, ctDNA (20 μM) and EtBr (2.5 μM) were dissolved in 10 mM

Tris-HCl (pH 7.2). Later, increasing concentrations of compounds (1) and (2) (0-50)

were added to EtBr-DNA solution. Solution was excited at 475 nm and emission

spectra were recorded in range 500-700 nm. Groove binding dye, HO (2 μg/ml) and

ctDNA (20 μM) were dissolved in 10 mM Tris-HCl (pH 7.2) and titrated with

increasing concentrations of compounds (1) and (2) (0-50 μM). HO DNA complex

was excited at 343 nm and emission was recorded from 400-600 nm.

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106

3.3.6d. KI quenching study

Iodide quenching experiments were performed in the presence and absence of ctDNA.

Briefly, compounds (1) and (2) (20 μM) were dissolved in 10 mM Tris-HCl (pH 7.2)

and titrated with increasing concentrations of KI (0-6 mM). Compounds (1) and (2)

were excited at 268 nm and 225 nm, respectively and emission spectra were recorded

in the wavelength range 300-400 nm. In a different experiment, compounds (1) and

(2) (20 μM) and ctDNA (20 μM) were taken and then an increasing concentration of

KI (0-6 mM) was added. Quenching constant values (Ksv) in the presence and

absence of DNA was calculated via Stern-Volmer equation.

3.3.6e. Circular dichroism (CD) spectral study

CD measurements of ctDNA (50 μM) alone and in the presence of increasing

concentrations of compounds (1) and (2) were carried out using JASCO-J-720 CD

spectropolarimeter, equipped with a temperature controller to keep the temperature of

the sample constant. CD experiments were carried out at 25 ˚C. All the CD spectra

were recorded in a range from 225-320 nm with a scan speed 200 nm/min with

spectral bandwidth of 1.0 nm. Each spectrum was the average of three scans.

Background spectrum of buffer (10 mM Tris-HCl, pH 7.2) was subtracted from the

spectra of DNA and compounds (1) or (2)-DNA complex. The results were expressed

as ellipticity (mdeg).


The chemical structure of compounds (1) and (2) was drawn using ChemDraw 12.0

and the structures were saved in *.mol format. Mol files were converted into PDB

format using Avogadro 1.0.1. Energy minimization and molecular optimization of

structures were done using Arguslab 4.0.1.56 Geometry optimization was carried using

AM1 (Austin Model 1), semiempirical quantum mechanics force field in Arguslab

4.0.1. The best conformer thus obtained was based on energy minimization and

geometry optimization. The final structures exhibiting lowest energy were saved in

*.pdb for input in docking protocol. Docking studies between double strand DNA

with a sequence of d(CGCGAATTCGCG)2 dodecamer (PDBID: 1BNA) and

compounds (1) and (2) were performed with the standard AutoDock (v4.2) suit using

Lamarckian Genetic Algorithm.57,58 Before starting the docking protocol, the target

receptor (PDBID: 1BNA) and individual ligands were prepared using standard

docking protocol and saved into ‘PDBQT’ format. In docking calculations, the target-

Chapter-3

107

ligand poses so obtained are ranked using an energy based scoring function. To

determine the most favorable binding sites of ligands in DNA as target, blind docking

was performed. The input ‘grid parameter’ files were modified and the grid size was

adjusted to X=60, Y=60 and Z=110 with 0.375 nm grid spacing. All docking

parameters were set to default values. After docking, the top pose conformation of

each docked ligand was visualized via PyMOL software (Molecular Graphics System,

version 1.5.0.1, Schrodinger.LLC), to identify the possible interactions between

ligand and DNA.59

3.3.8. Cell culture and antibodies

Human breast cancer MCF-7 and MDA-MB 231 cell lines were purchased from

American Type Culture Collection (ATCC, Manassas, VA). MCF-7 and MDA-MB

231 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-

Aldrich, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum

(FBS, Sigma-Aldrich, St. Louis, MO) and 1% penicillin and streptomycin. Cells were

grown in tissue culture flasks at 37 ˚C in an atmosphere of 5% CO2 incubator and

were checked for mycoplasma contamination by mycoplasma PCR detection kit

(Sigma-Aldrich, St. Louis, MO).

3.3.8a. Cell proliferation and viability assay

Cytotoxic effect of compounds (1) and (2) on breast cancer MCF-7 and MDA-MB

231 cells was determined using colorimetric MTT reduction assay.60 Briefly, breast

cancer cells (2×104 cells/ml) were seeded on 96-well culture plate for overnight

adherence. After complete adherence, cells were treated with increasing

concentrations of compounds (1) and (2) for 24h at 37 ˚C in 5% CO2. Then, 20 μL of

MTT (5 mg/ml) was added each well and re-incubated for additional 3 h. Formazan

blue crystals formed were dissolved in 100 μl of DMSO. Absorbance was read at 570

nm using ELISA plate reader (Bio Tek Instruments Inc., USA). Viability or

proliferation of treated cells was expressed as a percentage of untreated cells (100%).

IC50 value for compounds (1) and (2) was calculated by determining the concentration

that causes 50% inhibition of cell growth of MCF-7 and MDA-MB 231 cells. All

experiments were performed in triplicates.

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108

3.3.8b. Statistical analysis

Experimental values were expressed as mean ± SEM of three independent

experiments. Data was analyzed by one way- analysis of variance (ANOVA) using

GraphPad Prism 5.01 (California, USA) to examine statistically significant

differences. P-values < 0.05 were considered statistically significant.


All the synthesized compounds were tested for their antioxidant property by 1,1-

diphenylpicrylhydrazyl (DPPH) method.61 The procedure follow for this assay has

been discussed in chapter 2. In this procedure, drug stock solution (1 mg/mL) was

diluted to final concentration of 2, 4, 6, and 8µg/mL in methanol.

3.3.10. In vivo cytotoxicity assay of plant extracts

Brine shrimp lethality bioassay is commonly used in the bioassay for the bioactive

compounds.62 The in vivo cytotoxicity assay is performed on brine shrimp nauplii

(Artemia salina) in accordance with the Meyer method.48 The procedure follow for

this assay have been discussed in detail in chapter 2.

3.4. CONCLUSION

In the present chapter, we have reported the isolation of a novel isoflavone, 5,7-

dihydroxy-3-(3'-hydroxy-4',5'-dimethoxyphenyl)-6-methoxy-4H-chromen-4-one (1)

from the leaves of Garcinia nervosa along with 1-(2,5-dioxoimidazolidin-4-yl)urea

(DL-allantoin), 4-methoxychalcone (3), 2',4',4-trihydroxychalcone (4), 5,7-dihydroxy-

3-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one (5). Molecular structure of

compound 1 and 2 was further authenticated by X-ray crystallography and density

functional theory (DFT) studies. Interaction of compounds 1 and 2 with DNA is

extensively studied using various biophysical techniques. DNA binding studies

revealed that groove binding is the effective interaction between isolated compounds

and ctDNA. It was also supported by molecular docking results which showed that

compound 1 and 2 binds with -6.82 and -5.93 kcal/mol, respectively. Further the

antiproliferative activity against MCF-7 and MDA-MB 231 cell lines suggested that

compound 1 is more potent anticancer agent. Compound 1, 4 and 5 showed highest

antioxidant activity with IC50 = 8.29, 12.08 and 10.95 µg/mL. The ethanolic and

aqueous extract of leaves possess potential cytotoxic activity.

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109

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Chapter-4

112

4.1. INTRODUCTION

Hydrazones are a special group of the Schiff base compounds possessing an

azomethine -NHN=CH- proton in their structural frame work. Hydrazone and their

derivatives constitute an important class of compounds in organic chemistry due to

their promising biological activities.1 It has been documented in the literature that

hydrazone derivatives exhibit a wide spectrum of biological properties such as anti-

inflammatory,2 analgesic,3 antipyretic4 as well as chelating properties towards various

metal ions.5 Savini et al. have reported anticancer, anti-HIV and antimicrobial

activity6 of heterocyclic hydrazones. Further, Melnyk et al. reported hydrazone

derivatives as antimalaria drugs.7 The synthetic efforts for this class of compounds are

very well studied and generally entail the reaction of carbonyl compounds with

hydrazine hydrate in organic solvents8 like ethanol, methanol, tetrahydrofuran,

butanol, glacial acetic acid, ethanol-glacial acetic acid. Another synthetic route for the

synthesis of hydrazones is the coupling of aryldiazonium salts with active hydrogen

compounds. Hydrazide-hydrazones compounds are not only intermediates but they

are also very effective organic compounds in their own right. When they are used as

intermediates, coupling products can be synthesized by using the active hydrogen

component of –CONHN=CH- azometine group.

In addition, Pasternak9 reported the synthesis of hydrazone derivative of 14-

hydroxydihydromorphinone 2(a-c) from noloxone, oxymorphone and naltrexone 1(a-

c) by reacting with anhydrous hydrazine in absolute ethanol at room temperature.

Compounds b and c show high affinity for opiate binding sites in vitro.

O

N

R1

HO

OHO O

NR1

HO

NHO NH2

anhy. hydrazine, absolute ethanol

Stirring, RT

1(a-c) 2(a-c)

(a) R1= CH2-CH=CH2 (b) R1= CH3 (c) R1= CH2-C3H5

Kaplancikli et al.10 outlined the synthesis of hydrazone derivatives 3(a-j) by

the reaction of 3-cyclohexylpropionic acid hydrazide (1) with various benzaldehydes

Chapter-4

113

2(a-j). The synthesized compounds were also evaluated for anti-inflammatory activity

and cytotoxicity.

NHNH2O HNO

CHO

R

N

R

1 2(a-j) 3(a-j)

Absolute ethanolReflux, 3-5 h

R= (a) H; (b) NO2; (c) CH3; (d) Br; (e) F; (f) OH; (g) OCH3; (h) Cl; (i) CH(CH3)2;

(j) N(CH3)2

Belkheiri et al.11 synthesize a novel series of hydrazones 3(a-j) by heating

syringaldehyde (1) with hydrazine derivatives 2(a-j) in absolute EtOH under reflux

for 6-12 h and tested for their antioxidant as well as carbonyl scavenging activity.

CHO

H3COOCH3

OCH3

C

H3COOCH3

OCH3

NHHN R

EtOHreflux, 6-12 h

RNHNH2

1 2(a-j) 3(a-j)

S

N F

F

CF3ClClN

N

(a) (b) (c) (d) (e)

NN

OO O

ONH

H2NN

NH

CH3

O

(f) (g) (h) (i) (j)

El-Sayed et al.12 illustrated the synthesis of new arylhydrazone derivatives 3(a-

f) by the reaction of 1-(4-chlorophenyl)-4,4,4-trifuorobutane-1,3-dione (1) with

diazonium salt to form 1-(4-chlorophenyl)-2-(2-arylhydrazone)-4,4,4-trifuorobutane-

1,3-dione 2(a-f). Further they react 1-(4-chlorophenyl)-2-(2-arylhydrazone)-4,4,4-

trifuorobutane-1,3-dione with hydrazine hydrate in ethanol to yield 3-(4-

Chapter-4

114

chlorophenyl)-4-(2-arylhydrazono)-5-trifuoromethyl)-4H-pyrazoles 3(a-f). The newly

synthesized compounds were investigated in vivo for their anti-inflammatory

activities they were also tested for their in vitro inhibitory activity against ovine COX-

1 and COX-2.

Cl

O

O

FF F

N2Cl

Stirring, 30 minNH

NO

F

R3

R2R1

R

OF F

Cl

NNHO

F

R3

R2R1

R

OF F

ClN

N

R3R2

R1

R

NNF

FCl FNH2NH2, EtOH

Reflux, 4h

4(a-f) 3(a-f)

CH3COONa, EtOHR

R1R2

R3

1 2(a-f) 3(a-f)

(a) R= Cl, R1=R2=R3= H; (b) R2= Cl, R=R1=R3= H; (c) R2= Br, R=R1=R3= H; (d) R2= NO2, R=R1=R3= H; (e) R2= OCH3, R=R1=R3= H; (f) R=R2= H, R1=R3= CF3

Ivanov and co-workers13 carried out the reaction of substituted aromatic

aldehydes 1(a-h) with 4-hydrazino-N-hexyl-1,8-naphthalimide (2) in ethanol in

presence of acetic acid to yield substituted arylhydrazones of N-hexyl-1,8-

naphthalimide 3(a-h).

O

XY

N

O

O

C6H13

NH

H2N EtOH, AcOHReflux, 5-15 min

N

O

O

C6H13

NH

N

X

Y2'3'

5'6'

78

9

54

1(a-h) 2 3(a-h)

Chapter-4

115

(a) X= N(CH3)2, Y= H; (b) X= OCH3, Y= H; (c) X= CH3, Y= H; (d) X=Y= H; (e)

X= Cl, Y= H; (f) X= CN, Y= H; (g) X= NO2, Y= H; (h) X= OCH3, Y= OCH3

Kumar and Chauhan14 synthesized a series of novel flavones hydrazide

derivatives 5(a-j) by incorporating the substituted flavone moiety 4(a-j) to phenoxy

benzoic acid (1). They were also screened for anticonvulsant activity and compound

5g was found to be most active against maximal electroshock seizure (MES) than

standard drug (phenytoin).

O

OH

O O

OCH3

O

O

NHNH2

O

Conc. H2SO4

CH3OH

O

O

R1

R2R3

O

N

R1

R2R3

NH

O

OCH3OH, Glacial AA

1 2

5(a-j) 3

4(a-j)

Reflux, 30-40 h

NH2NH2.H2OReflux, 22 h

(a) R1=R2= H, R3= Cl; (b) R1= Cl, R2=R3= H; (c) R1= NO2, R2=R3= H; (d) R1=R3= H, R2= NO2; (e) R1=R2= H, R3= NO2; (f) R1=R2= H, R3= OH; (g) R1=R2= H; R3= N(CH3)2; (h) R1=R2=R3= H; (i) R1= H, R2= OCH3, R3= OH; (j) R1= OH, R2=R3=

H

Saeed and co-workers15 reported the synthesis of 2-(phenyl-hydrazono)-

succinic acid dimethyl ester by reacting phenylhydrazine with dimethylacetylene

dicarboxylate at room temperature.

Chapter-4

116

NH

NH2 H3CO

O

O

OCH3 HN NOCH3

OH3CO O

Toluene-DCM (1:1)Stirring, 2h

A part from these conventional methods a wide range of methods for

synthesizing hydrazones in the presence of Ultrasound, Microwave and catalysts are

available in literature. Leite et al.16 reported the reaction of aromatic

aldehydes/ketones 1 with hydrazides 2 (semicarbazide, thiosemicarbazide and

aminoguanidine) in aqueous medium (acid conditions) under ultrasound irradiation to

yield aryl-hydrazones 3.

Ar R

O

H2N NH

NH

XH Ultrasound, (20-30) min Ar

R

N NH

N

X

H

H

1 2 3H2O, AA (0.4 mL)

Ar/R= p-OMePh/H; C4H4O/H; 3,4-ClPh/H; p-BrPh/H; PhCH=CH/H; p-OHPh/H;

Arylthioacetaldehyde/H; ph/CH3; p-ClPh/CH3

X= S, O, NH

Ajani et al.17 synthesize a series of 2-quinoxalinone-3-hydrazone derivatives

3(a-g) from 3-hydrazinoquinoxalin-2(1H)-one (1) using microwave irradiation at 140

°C for 1-3 min and evaluated for their antimicrobial activities.

N

HN O

NH

NH2 N

HN O

NH

NX

X= 2(a-g)

1 3(a-g)

Microwave, 140 oC1-3 min

X= (a) Acyclic ketone (b) 6-substituted-3-acetylcoumarin (c) cyclopentanone (d) 5-

substituted isatin (e) anthrone (f) camphor (g) 2-substituted cyclohexanone

Jeselnik et al.18 reported the reaction of 5 or 8-oxobenzopyran-2-ones (1-3)

with a variety of aromatic and heteroaromatic hydrazines (4-5) using microwave

under catalyst free condition at 150 °C for the synthesis of hydrazone derivatives.

Chapter-4

117

O O

NHCOPhO

RR O O

NHCOPhN

RR

MW, 150 oC, 10 min

O O

NHCOPh

O

MW, 150 oC, 10 minO O

NHCOPh

N

R2HNR2HN

R2NHNH2 (5)

1-2 6(a-g)

3 7(a-e)

R1NHNH2 (4)

R= H/Me; R1= Ph; 2,5-F2C6H3; 3-CF3C6H4; 6-chloropyridazin-3-yl; R2= Ph; 3-CF3C6H4; pyridin-2-yl; 4-NO2C6H4; 2,5-F2C6H3

Polshettiwar and Varma19 developed a new protocol for the synthesis of

heterocyclic hydrazones using polystyrene sulfonic acid (PSSA) as a catalyst in water

under microwave irradiation at 100 °C.

R

NHH2N

PSSA/H2O100 oC, MW

OO

H

O

O

NH

R

N

O

OH

N NH

R

1(a-c)

3(a-c)

5(a-c)

3

4

R= H, CH3, Cl

Sadjadi et al.20 synthesized benzoyl hydrazones derivatives 1 from reaction of

benzoic acid hydrazide and aldehyde/ketone derivatives. Further they also reported

synthesis of N-acyl-benzoyl derivatives 3(a-h) by the reaction of benzoyl hydrazones

derivatives 1 with acylcholorides (2) in presence of heteropolyacids.

Chapter-4

118

NH

ON R2

R1R

HPA, CH3CNReflux

N

ON R2

R1R R3OR3 Cl

O

1 2 3(a-h)

HPA= H6[PMo9V3O40], H5[PMo10V2O40], H4[PMo11VO40], H3[PMo12O40] (a) R= H, R1= C6H5, R2= H, R3= Me; (b) R= H, R1= 4-OHC6H5, R2= H, R3= Me;

(c) R= H, R1= 2-OHC6H5, R2= H, R3= Me; (d) R= H, R1= 4-OMeC6H5, R2= H, R3= Me; (e) R= H, R1= 3-Br,5-OH-C6H3, R2= H, R3= Me; (f) R= Cl, R1= C6H5, R2= H, R3= Me; (g) R= Cl, R1= C6H5, R2=Me, R3= Me; (h) R= Cl, R1= Me, R2= Me, R3=

Me

Wang et al.21 synthesized various aromatic hydrazones by the reaction of

aromatic ketone with hydrazine hydrate in ionic 1iquid at 100 °C with good yields.

O

R1R2

NNH2

R1R2

NH2NH2.H2OIonic liquid

1(a-g) 2(a-g)

100 oC, 4h

Ionic liquids = [Bmim] BF4, [Bmim] PF6, [Bmim] CH3COO (a) R1= H, R2= H; (b) R1= 4-Cl, R2= NO2; (c) R1= 4-Br, R2= NO2; (d) R1= 4-

CH(CH3)2, R2= NO2; (e) R1= 4-Cl, R2= 4-NO2; (f) R1= 4-Cl, R2= 4-NH2; (g) R1= 4-Br, R2= 4-NH2

Motivated by these finding and promising biological profile of hydrazone

derivatives and in continuation of our on-going efforts endowed with the finding of

new synthetic protocols under the principles of green chemistry, this chapter report the

development of an efficient Bronsted acid ionic liquid (BAIL) [Et3NH][HSO4]

promoted synthesis of hydrazone derivatives. The ionic liquid [Et3NH][HSO4]

employed in the present study has been characterized on the basis of 1H NMR and 13C

NMR spectral analysis. This ionic liquid is air and water stable and easy to prepare

from cheap amine and acid.


In the present chapter, a series of hydrazone analogues 3(a-o) have been synthesized

via solvent-free facile nucleophilic addition between hydrazine hydrate and

Chapter-4

119

appropriately substituted aromatic aldehydes 1(a-o). The molecular structure of

compound (3f) was well supported by single crystal X-ray crystallographic analysis

and also verified by DFT calculations. This new synthetic, eco-friendly, sustainable

protocol resulted in a remarkable improvement in the synthetic efficiency (90-98%

yield) with high purity, using [Et3NH][HSO4] as a catalyst as well as an

environmentally benign solvent which eliminate the need of a volatile organic solvent

and additional catalyst. The present methodology is a green protocol offering several

advantages such as, excellent yield of products, minimizing production of chemical

wastes, shorter reaction profile, mild reaction conditions, simple operational

procedure, easy preparation of catalyst and its recyclability up to five cycles without

any appreciable loss in catalytic activity. The synthesized compounds have been

screened for antioxidant activity and the results obtained were promising.

4.2.1. Characterization of the catalyst [Et3NH][HSO4]

The catalyst [Et3NH][HSO4] used in the study has been characterized on the basis

of 1H NMR and 13C NMR spectral analysis. 1H NMR spectrum displayed a triplet at

around δ 1.20 integrating for nine protons has been assigned to methyl group (3×CH3)

protons. Similarly a multiplet resonating at δ 3.15 corresponding to six protons has

been attributed to methylene (3×CH2) protons. A sharp singlet at around δ 8.85 for

one proton has been assigned to -NH (D2O-exchangeable) proton (Fig. 1). 13C NMR

spectrum showed a pair of resonance signals at around δ 10.23 and 52.01 assigned to

methyl (CH3) and methylene (CH2) carbons, respectively (Fig. 2).

Chapter-4

120

Fig. 1 1H NMR spectrum of [Et3NH][HSO4]

Fig. 2 13C NMR spectrum of [Et3NH][HSO4]

The synthetic pathways of a series of hydrazone derivatives 3(a-o) have been

shown in Scheme 1. Herein, the series was typically accessed via a nucleophilic

Chapter-4

121

addition between appropriately substituted aromatic/heterocyclic aldehydes 1(a-o)

and hydrazine hydrate (2) to yield target hydrazone derivatives 3(a-o). All the

compounds were obtained in excellent yields (90-98%) with high purity.


4.2.2. Chemistry

The structural elucidation of the synthesized compounds 3(a-o) was established on the

basis of elemental analysis, IR, 1H NMR, 13C NMR and mass spectral analysis. The

analytical results for C, H and N were within ±0.4% of the theoretical values. IR

spectrum of all the synthesized compounds, showing the absence of absorption signal

for carbonyl moiety, authenticates the reaction at carbonyl group. Moreover, all the

synthesized compounds exhibited a characteristic peak at around (1583-1595),

assigned to C=N group. Other diagnostic peaks for functional groups such as OH,

NO2, C=Oγ pyrone have been discussed in the experimental section. In the 1H NMR

spectra, each compound displayed a sharp singlet at around δ 8.65-8.94 ascribed to

N

HN

HAr

ArNH2NH2.H2O [Et3NH][HSO4] (20 mol%)

Solvent Free, 120 oCH

O

Ar

1(a-o) 2 3(a-o)

N FNO2

O2NOCH3

H3CO

OCH3

H3CO

H3CO

OHH3CO OH

OHHO

HO

OH

O

O

O

OF

O

OH3C

O

OBr

O

O

NH2

H3C

CH31a 1b 1c 1d 1e

1f 1g 1h 1i 1j

1k 1l 1m 1n 1o

Chapter-4

122

the -CH=N proton. Similarly sharp singlets resonating at around δ 7.23, 7.21, 7.29 and

7.24 each integrating for two protons, has been attributed to H-2 and H-2' protons of

γ-pyrone ring in compounds 3k, 3l, 3m and 3n, respectively. 13C NMR spectra,

showed peaks resonating at around δ 160.09-164.40 corresponds to –C=N moiety and

the signals at δ 178.50, 178.42, 178.49, 177.42 and 179.50 have been attributed to

carbonyl group (C=Oγ-pyrone) of compounds 3k, 3l, 3m, 3n, and 3o, respectively. The

mass spectral analysis of the synthesized compounds was also in good conformity

with the proposed structures.

The FT-IR spectrum of compound 3a (Fig. 3) displayed characteristics peak at

1594 cm-1 for C=N group. Other peaks resonating at 1456 and 2909 cm-1 attributed to

C=C and =C-H stretching, respectively. The 1H NMR spectrum of compound 3a (Fig.

4) displayed two sharp singlet at around δ 8.65 and 8.68 for two protons ascribed to

the -CH=N proton. Similarly, a sharp singlet at δ 3.10 for twelve protons attributed to

CH3 protons. A double doublet at δ 6.76 for four protons have been ascribed to (H-3,

H-6) and (H-3', H-6') protons of benzene ring. Similarly, another double doublet at δ

7.20 for four protons have been ascribed to (H-2, H-6) and (H2', H-6') protons

respectively. 13C NMR spectrum of compound 3a (Fig. 5) displayed characteristics

absorption bands resonating at around δ 160.54 and 42.10 for C=N and CH3,

respectively. Further a series of signals resonating at around δ 111.12-152.43 have

been assigned to aromatic carbons. The detailed spectral characterization of all the

synthesized compounds has been discussed in experimental section.

Fig. 3 FT-IR spectrum of compound 3a

Chapter-4

123

Fig. 4 1H NMR spectrum of compound 3a

Fig. 5 13C NMR spectrum of compound 3a

Chapter-4

124

The configuration around C=N was authenticated by single crystal X-ray

crystallographic analysis of compound 3f (Fig. 6), the crystallographic data of

compound 3f has been presented in Table 1. In compound 3f both C=N were found to

have E,E-geometry. Among the three possible geometrical isomers (E,E/Z,Z/E,Z),

(E,E)-isomers were obtained as the exclusive product (Fig. 6), which has been well

established further on the basis of density functional theory (DFT) calculations (Fig.

7). This E,E-selectivity can be interpreted as a way to minimize steric interactions

among various substituents. To compare the relative stability of the three possible

isomers E,E, Z,Z and E,Z we have performed the calculation of the vacuum single-

point energies of the optimized geometries (Fig. 8) to obtain the energy differences. It

was found that the E,E-isomer is stabilized by 5.68 and 9.24 kcal mol-1 more than the

E,Z and Z,Z-isomers, respectively (Table 2). This difference in energy is the reason

that during the crystallization process, the E,E-isomer gets exclusively crystallized

out. The rotations about single bonds (intramolecular torsions) are worth 1-3 kcal mol-

1 but can be as high as 10 kcal mol-1 due to steric factors or restricted rotations so this

can elucidate the calculated energy differences.22 The single crystal X-ray diffraction

analysis of compound (3f) was found to be in good agreement with the previous

report.23

Fig. 6 X-ray molecular structure of compound (3f). Displacement ellipsoids are

plotted at the 50% probability level.

Chapter-4

125

Table 1 Crystallographic data and structure refinement of compound (3f)

Empirical formula C20H24N2O6

Formula weight 388.41

Temperature (K) 293(2)

Wavelength (Å) 0.71073

Crystal system Monoclinic

Space group C 2/c

a (Å) 29.7753(9)

b (Å) 4.8973(2)

c (Å) 13.9889(4)

α (deg.) 90

β (deg.) 103.7400(10)

γ (deg.) 90

Volume (Å3) 1981.47(12)

Z 4

Calculated density (g/cm3) 1.302

Absorption coefficient (mm–1) 0.097

F(000) 824

Crystal size (mm) 0.55 × 0.33 × 0.31

θ range for data collection (deg.) 3.00 –27.58

Index ranges –38<h<38, –6<k<6, –18<l<18

Reflections collected/unique 17606 /2300 [R(int) = 0.0421]

Completeness to θ = 25.00º 99.8 %

Refinement method Full–matrix least–squares on F2

Data/restraints/parameters 2300/0/130

Goodness–of–fit on F2 1.064

Final R indices [I> 2σ(I)] R1 =0.0396 wR2 = 0.1091

R indices (all data) R1 = 0.0489 wR2 = 0.1214

Largest diff. peak and hole (e Å – 3) 0.170 and –0.209

Chapter-4

126

Fig. 7 Comparison of the molecular conformation of compound (3f), as established

from the X-ray study (red) with the optimized geometry (blue)

Fig. 8 Optimized structures of different isomers of compound (3f) (a) E,E-isomer (b) Z,Z-isomer and (c) E,Z-isomer

(a) (b)

(c)

Chapter-4

127

Table 2 Single-point energy calculations, with energies in hartree, kcal mol-1 and relative conformational energies (kcal mol-1) between different isomers and the E,E-isomer.

Isomers Energy (hartree)a

Energy (kcal mol-1)

E(X)-E(E,E)b

(kcal mol-1) E,E-isomer -1337.65585 -839342.0864438645 −

E,Z-isomer -1337.64679 -839336.4015441923 5.68

Z,Z-isomer -1337.64112 -839332.8437758544 9.24 a1 hartree = 627.47237 kcal mol-1. bEnergy difference between different isomers and

E,E-isomer.

4.2.3. Results of density functional theory (DFT) calculations

In order to gain some insight into the influence of the intermolecular interactions on

the molecular geometry, DFT calculation of the equilibrium geometry of the free

molecule starting from the experimental X-ray geometry has been performed. The

DFT calculations closely reproduce the solid state geometry of the molecule (3f). The

corroboration between the experimental and calculated bond lengths and angles was

found to be very good, with the differences being smaller than 0.02 Å and 1.651,

respectively, but the calculated torsion angle of one of the methoxy group deviates

appreciably by 24.71 from the experimental value (Table 3). Overall, our data

suggests that the supramolecular aggregation has some significance in the

stabilization of the observed geometry of compound (3f), in spite of all the

interactions being weak.

Table 3 Comparison of selected geometrical parameters of compound (3f) as determined by X-Ray diffraction and from DFT calculation (Å,°).

Experimental [1] Experimental (This study)

DFT study

O1-C4 1.365(2) 1.3585(14) 1.361

O2-C5 1.374(2) 1.3709(12) 1.365

O3-C6 1.370(2) 1.3607(14) 1.361

N1-N1i 1.419(2) 1.4102(18) 1.389

N1-C1-C2 123.00(19) 123.03(12) 122.58

C1-N1-N1 112.25(17) 112.03(13) 112.66

N1i-N1-C1-C2 179.37(18) 179.01(13) 180.00

Chapter-4

128

C3-C4-O1-C8 -0.7(3) -0.68(18) 2.29

C4-C5-O2-C9 -105.8(2) -105.69(13) -81.04

C7-C6-O3-C10 2.8(3) 3.0(2) -2.13

Symmetry code: (i) -x,1-y, 1-z.

4.2.4. Optimization of reaction conditions

In order to develop an eco-friendly approach for the synthesis of biologically active

hydrazone derivatives, the optimum reaction conditions regarding the choice of

solvent, temperature of reaction and amount of catalyst on the model reaction using p-

dimethylaminobenzaldehyde (1a) and hydrazine hydrate 2 was investigated to

establish best reaction conditions.

To achieve the optimum concentration of catalyst, the model reaction was

investigated for different concentrations 5, 10, 15, 20 and 25 mol% (Table 4, entries

1-5) of [Et3NH][HSO4] at 120 °C under solvent-free condition. It is obvious from

(Table 4, entry 4) that 20 mol% of the catalyst is satisfactory to gain the optimum

yield in the shortest reaction time. Using less than 20 mol% of catalyst, moderate

yields of the product (76-89%) were obtained with extended reaction times, while

with an excess mol% of catalyst (25 mol%) there was no further increase in the yield

of the product, probably due to the saturation of the catalytic sites of the catalyst.

Table 4 Effect of catalyst loading on the yield and time period of model reaction

(3a)a.

Entry Catalyst (mol%) Time(min)b Yield (%)c

1 5 120 76 2 10 70 82 3 15 55 89 4 20 30 96 5 25 30 96

aReaction conditions: p-dimethylaminobenzaldehyde (1a, 2 mmol), hydrazine hydrate (2, 1 mmol), solvent free, 120 oC; bReaction progress monitored by TLC; cIsolated yield of products.

In order to study the solvent effect, the model reaction was carried out in

different solvent systems. Initially, the reaction was investigated in MeOH and EtOH

(Table 5, entries 1 and 2) the reaction took a longer time (6-8 h) with moderate yields

of 64% and 60%, respectively, whereas in water (Table 5, entry 3), the product was

Chapter-4

129

obtained in better yield (68%) after refluxing for 5 h. In CH2Cl2 and DMF, moderate

yields of the product were obtained after a stretched reaction periods (Table 5, entries

5-6), whereas in acetic acid, the reaction period was reduced to 4 h (Table 5, entry 4)

and there was an enhancement in the yield by 5% in comparison to CH2Cl2, probably

due to an electromeric effect offered by acetic acid activating the carbonyl group of

the reactants, decreasing the activation energy, thereby rendering it more reactive

towards nucleophilic attack. Furthermore, when the model reaction was carried out

under solvent-free condition, there was a noteworthy increase in the yield of the

product in a shorter time period (Table 5, entry 7). In view of the above results, it was

concluded that solvent-free is the best reaction condition for the synthesis of present

hydrazone derivatives in excellent yields. To optimize the reaction temperature, the

model reaction was carried out at different temperatures under solvent free condition.

It was observed that the increase in temperature from 25 °C to 120 °C, has a

significant effect on the reaction in terms of the yield and reaction time. The yield of

the product increased from 84-98% during the course of reaction (Table 5, entries 7-

11). However, no increase in the yield of product was observed when the reaction

temperature was raised from 120 °C to 140 °C (Table 5, entry 11).

Table 5 Effect of various solvents and temperature on the model reactiona

Entry

Solvent Temp (oC) Time (h)b Yield (%)c

1 MeOH Reflux 6 64 2 EtOH Reflux 8 60 3 Water Reflux 5 68 4 CH3COOH Reflux 4 76 5 CH2Cl2 Reflux 10 71 6 DMF Reflux 12 68 7 Solvent free Room temp 2.5 84 8 Solvent free 60 2.0 89 9 Solvent free 80 1.5 92 10 Solvent free 120 30d 98 11 Solvent free 140 30d 98

aReaction conditions: p-dimethylamino benzaldehyde (1a, 2 mmol), hydrazine hydrate (2, 1 mmol), Different solvents (20 mL, entry 1-6, refluxing temperature), Solvent free (entry 7-11, temperature 25-140 oC), Catalyst (20 mol%); bReaction progress monitored by TLC (entry 1-9, hrs); cIsolated yield of products; dReaction progress monitored by TLC (entry 10-11, min).

Chapter-4

130

A comparative study of a variety of other Bronsted acid ionic liquid catalysts

was conducted to investigate the superiority of [Et3NH][HSO4]. It is obvious from

(Table 6) that the catalytic activity was strongly affected by the anionic part of the

ionic liquids. In case of [HSO4] anion, higher yields were obtained (Table 6, entries

1-3). However, when [H2PO4] and [CH3COO] anions were probed for their

efficiency, lower yields (81-90%) were obtained as compared to [HSO4] anion (92-

98%), probably due to the weaker acidity of the phosphate and acetate anions than

[HSO4]. These results suggest that [Et3NH][HSO4] is the best ionic liquid catalyst for

the synthesis of present hydrazone derivatives.

Table 6 Comparison of the efficiency of [Et3NH] [HSO4] for the synthesis of (3a)a

Entry

Catalyst Time (min)b Yield (%)c

1 [Et3NH][HSO4] 30 98 2 [Me3NH][HSO4] 45 92 3 [Et2NH2][HSO4] 40 96 4 [Et3NH][H2PO4] 48 90 5 [Me3NH][H2PO4] 52 86 6 [Et2NH2][H2PO4] 46 89 7 [Et3NH][CH3COO] 57 84 8 [Me3NH][CH3COO] 62 81

aReaction conditions: p-dimethylamino benzaldehyde (1a, 2 mmol), hydrazine hydrate (2, 1 mmol), Solvent free, 120 oC, Different catalysts (20 mol%); bReaction progress monitored by TLC; cIsolated yield of products.

Keeping in view the above optimize conditions, reactions were carried out at

120 °C in the presence of 20 mol% of [Et3NH][HSO4] under solvent-free conditions

the efficacy of this approach was explored for the synthesis of hydrazone derivatives.

A comparative study has also been done in ethanol in absence of the catalyst, the

reaction took a prolonged time period (6-8 hours) for completion with a moderate

yield (60-72%) of the products (Table 7).

Chapter-4

131

Table 7 [Et3NH][HSO4] catalyzed synthesis of hydrazone derivatives 3(a-o).

S. No.

Products

Reaction in

absence of catalysta

Time Yield (h)c (%)d

Reaction in presence of

catalystb

Time Yield (min)c (%)d

3a

3b

3c

3d

3e

3f

N N

N

NCH3

CH3

CH3

H3C

11'

2 2'

3

3'

4

4'

5

5'

66'

NN

F

F

N N

NO2

NO2

N N

O2N

NO2

N NH3CO

OCH3

H3CO

OCH3

NN

H3CO

H3CO

OCH3

OCH3

OCH3

OCH3

6.0 69

7.5 72

6.0 70

7.0 70

6.5 68

6.0 72

30 98

42 96

37 94

40 98

35 90

30 97

Chapter-4

132

3g

3h

3i

3j

3k

3l

3m

3n

N NHO OH

OCH3

H3CO

NN

OH

HO

NN

HOOH

HO

OHOH

OH

11'

2

2'

3

3'

4

4'

55'

6

6'

7

7'

8

8'

9

9'10

10'

OH

N N

HO

O

O

NN

O

O

1

1'

2

2'

3 3'44'

5

5'6 6'

7

7'

8

8'

4a 4a'

8a

8a'

O

O

NN

O

OFF

O

O

NN

O

OCH3H3C

O

O

NN

O

OBrBr

8.0 65

7.5 67

8.0 63

7.0 60

6.5 70

6.0 68

7.0 64

6.5 66

45 92

42 94

35 97

40 96

35 98

30 96

40 98

37 94

Chapter-4

133

3o

O

O

NN

O

ONH2

H2N

8.0 62

45 92

aReaction conditions: p-dimethylamino benzaldehyde (1a, 2 mmol), hydrazine hydrate (2, 1 mmol), ethanol bReaction conditions: p-dimethylamino benzaldehyde (1a, 2 mmol), hydrazine hydrate (2, 1 mmol), Solvent free, 120 oC, Catalyst (20 mol%); bIsolated yield of products cReaction progress monitored by TLC, dIsolated yield of products

4.2.5. Reaction Mechanism

A plausible mechanistic pathway is proposed to illustrate the synthesis of hydrazone

derivatives catalyzed by [Et3NH][HSO4] (Scheme 2). The initial step involves the

protonation of formyl group (-CHO) of differently substituted aromatic aldehydes and

3-formylchromones (I) by protic ionic liquid catalyst [Et3NH][HSO4] to form

intermediate (II), which facilitates the nucleophilic attack of hydrazine hydrate to

promote the formation of C-N bond to yield intermediate (III). The subsequent

elimination of water molecule from intermediate (III) enhanced by catalyst

[Et3NH][HSO4] eventually yield compound (IV) followed by regeneration of the

catalyst. The repetition of catalytic loop for compound (IV) with another molecule (I)

finally ends up with target hydrazone products 3(a-o).

Chapter-4

134

Scheme 2 Plausible mechanistic pathway for the synthesis of target hydrazone derivatives 3(a-o)

4.2.6. Reusability of catalyst

The reusability of the catalyst was also explored for the selected model reaction. The

catalyst was reused five times and the results demonstrate that the catalyst can be

reused without a significant reduction in the yield (Table 8). After the completion of

the reaction, cold water was added to the reaction mixture and the products were

isolated by filtration. The ionic liquid was recovered from the filtrate by removing the

water under reduced pressure.

H

O

H

OSO

OOO N

Et

EtEtHH

H

OH SO

OOO N

Et

EtEtH

H

OH

NH-NH2. H2O

HO

N-NH2

H

-H2O

H

SO

OOO N

Et

EtEtH

H

N-NH2 SO

OOO N

Et

EtEtHH

Repeatation of catalytic cycle with another molecule of (I)

N NH

HFinal Product

(I)

(II)

HH

H

NH-NH2. H2OH

Catalytic loop

H

O

(III)

(IV)Ar

Ar

Ar

Ar

Ar

Ar

ArAr

Ar

Chapter-4

135

Table 8 Reusability of [Et3NH][HSO4] in the synthesis of (3a)a

Entry Reaction cycle Isolated yield (%)b

1 1st (fresh run) 90

2 2nd cycle 89

3 3rd cycle 87

4 4th cycle 82

5 5th cycle 81 aReaction conditions: p-dimethylamino benzaldehyde (1a, 2 mmol), hydrazine hydrate (2, 1 mmol), Solvent free, 120 oC, Catalyst (20 mol%); bIsolated yield of products.


The antioxidant activities of all the synthesized compounds 3(a-o) were investigated

by DPPH scavenging activity. The reduction capability of DPPH radicals was

determined by a decrease in their absorbance at 517 nm induced by antioxidants using

ascorbic acid as a reference. The potencies for the antioxidant activity of compounds

3(a-o) to the reference compound are shown in Table 9. Almost all the tested

compounds possessed strong scavenging activity against the DPPH with IC50 values

(5.57-8.37 µg/mL). 4-Flouro derivative 3b (IC50=7.33 µg/mL) showed less activity

than methoxy derivatives 3(e-g) while 4-Dimethyamino derivative 3a (IC50=6.57

µg/mL) showed significant activity. Other compounds having OH group on benzene

ring were found to be possessing strong antioxidant activity. Further formyl

derivatives having flouro (3l) and bromo (3n) substitution on 6th position showed the

lowest scavenging activity (IC50=8.24 and 8.37 µg/mL) whereas methyl substitution

(3m) does not affect on activity. Amino derivative of formylchromone (3o) showed

the best inhibitory activity with IC50=5.57 µg/mL. Rest of the compounds showed

significantly good activity against DPPH.

Chapter-4

136

Table 9 Antioxidant activity of compounds 3(a-o) by DPPH assay

% inhibition (absorbance at 517 nm)

Compounds 2 μg/mL 4 μg/mL 6 μg/mL 8 μg/mL IC50(μg/mL)a

3a 10.71±0.4 30.51±0.4 40.23±0.7 64.56±0.5 6.57

3b 11.68±0.3 17.54±0.4 40.30±0.3 56.73±0.5 7.33

3c 9.35±0.7 17.21±0.3 32.57±0.5 53.48±0.4 7.95

3d 10.27±0.3 18.50±0.3 30.24±0.4 57.52±0.5 7.71

3e 17.30±0.5 26.23±0.4 35.48±0.4 70.41±0.7 6.50

3f 16.70±0.4 22.30±0.3 32.48±0.3 77.70±0.3 6.31

3g 10.57±0.4 28.90±0.5 43.39±0.7 67.30±0.6 6.34

3h 18.5±0.3 32.43±0.3 61.95±0.5 67.30±0.4 5.56

3i 11.80±0.6 21.46±0.7 63.87±0.4 67.30±0.3 5.85

3j 30.11±0.3 47.05±0.5 51.33±0.5 59.05±0.3 5.68

3k 7.60±0.4 41.33±0.7 54.12±0.6 71.70±0.3 5.61

3l 9.41±0.5 12.52±0.4 30.54±0.7 52.32±0.5 8.24

3m 12.46±0.4 20.53±0.3 37.38±0.4 64.12±0.4 6.90

3n 8.64±0.5 13.98±0.5 28.55±0.3 51.76±0.7 8.37

3o 14.5±0.3 30.24±0.5 63.02±0.4 69.56±0.4 5.57

Control - - - - -

Standard Ascorbic acid

16.50±0.3 44.35±0.3 57.22±0.4 66.25±0.3 5.48

aIC50 value represents the concentration of three experiments required to exhibit 50 % antioxidant activity.

4.3. EXPERIMENTAL





N), IR, NMR and Mass have been discussed in chapter 2. Thin layer chromatography

(TLC) glass plates (20×5 cm) were coated with silica gel G (Merck) and exposed to

iodine vapor to check the homogeneity as well as the progress of the reaction.

Chapter-4

137

4.3.2. Synthesis of ionic liquids

The simple ammonium ionic liquids of general type [amine][HSO4] were synthesized

by the known standard literature methods24 in the following way.

Triethylammonium sulfate [Et3NH][HSO4]

The synthesis of ionic liquid was carried out in a 250 mL round bottomed flask, which

was immersed in a recirculating heated water-bath and fitted with a reflux condenser.

Sulfuric acid (49 g, 0.5 mol) 98% solution in water was added drop wise into

triethylamine (50.5 g, 0.5 mol) at 60 °C for 1 hour. After the addition, the reaction

mixture was stirred for an additional period of 1 hour at 70 °C to ensure the reaction

had proceeded to completion. Then the traces of water were removed by heating the

residue at 80 °C in high vacuum (5 mm Hg) until the weight of the residue remained

constant. The yield of [Et3NH][HSO4] was 98%. 1H NMR (400 MHz, DMSO-d6, δ,

ppm): 1.20 (t, 9H), 3.15 (m, 6H), 8.85 (s, 1H, D2O exchangeable). 13C NMR (100

MHz, DMSO-d6, δ, ppm): 10.23 (CH3), 52.01 (CH2).

The following ionic liquids were synthesized by the same procedure.24

Trimethylammonium sulfate [Me3NH][HSO4]. 1H NMR (400 MHz, DMSO-d6, δ,

ppm): 2.57 (s, 9H), 2.79 (s, 1H, D2O exchangeable).

Diethylammonium sulfate [Et2NH2][HSO4]. 1H NMR (400 MHz, DMSO-d6, δ,

ppm): 1.18 (t, 6H), 2.95 (m, 4H), 8.20 (s, 2H, D2O exchangeable).

Triethylammonium dihydrogen phosphate [Et3NH][H2PO4]. 1H NMR (400 MHz,

DMSO-d6, δ, ppm): 1.16 (t, 9H), 3.25 (m, 6H), 8.79 (s, 1H, D2O exchangeable).

Trimethylammonium dihydrogen phosphate [Me3NH][H2PO4]. 1H NMR (400

MHz, DMSO-d6, δ, ppm): 2.50 (s, 9H), 2.79 (s, 1H, D2O exchangeable).

Diethylammonium dihydrogen phosphate [Et2NH2][H2PO4]. 1H NMR (400 MHz,

DMSO-d6, δ, ppm): 1.18 (t, 6H), 2.95 (m, 3H), 8.11 (s, 2H, D2O exchangeable).

Triethylammonium acetate [Et3NH][CH3COO]. 1H NMR (400 MHz, DMSO-d6, δ,

ppm): 1.15 (t, 9H), 2.12 (s, 3H), 3.14 (m, 6H), 8.82 (s, 1H, D2O exchangeable).

Trimethylammonium acetate [Me3NH][CH3COO]. 1H NMR (400 MHz, DMSO-d6,

δ, ppm): 2.54 (s, 9H), 2.15 (s, 3H), 2.73 (s, 1H, D2O exchangeable).

Chapter-4

138

4.3.3. General procedure for the synthesis of hydrazone derivatives 3(a-o)

To a mixture of an aromatic/heterocyclic aldehyde 1(a-o) (0.02 mol) and hydrazine

hydrate 2 (0.01 mol), 20 mol% of [Et3NH][HSO4] was added and the reaction

mixture was heated on an oil bath at 120 °C for (20-30 min) with stirring. During the

reaction process, the reaction mixture spontaneously solidified. After completion of

the reaction as evident from thin layer chromatography (TLC), the reaction mixture

was allowed to cool at room temperature. Water was added and the reaction mixture

was further stirred for 5 min. The solid obtained was removed by filtration, washed

with appropriate solvents and then recrystallized from methanol. The water was

removed from filtrate under reduced pressure to recover [Et3NH][HSO4], which was

then reused in subsequent cycles.

4.3.4. Spectral characterization of synthesized compounds 3(a-o)

4,4'-[(1E,1'E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(N,N-

dimethylaniline) (3a)

Brown crystalline solid; yield 98%; m.p. 213-214 oC,25 reported 215 oC; Analytical

cal. C18H22N4: C, 73.44; H, 7.53; N, 19.03; found: C, 73.42; H, 7.53; N, 19.05.

IR (KBr, νmax cm-1): 1157, 1456 (C=C), 1594 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.10 (s, 12H, 4×CH3), 6.76 (dd, 2H, H-3

and H-5), 6.78 (dd, 2H, H-3ʹand H-5ʹ), 7.20 (dd, 2H, H-2 and H-6), 7.21 (dd, 2H, H-

2ʹ and H-6ʹ), 8.65 (s, 1H, -CH=Nazine), 8.68 (s, 1H, -CH=Nazine).

13C NMR (100 MHz, DMSO-d6, δ, ppm): 42.10 (CH3), 111.12 (C-3 and C-5),

111.13 (C-3ʹand C-5ʹ), 122.50 (C-1ʹ), 122.52 (C-1), 133.21 (C-2 and C-6), 133.24

(C-2ʹand C-6ʹ), 152.41 (C-4ʹ), 152.43 (C-4), 160.54 (C=N).

MS (ESI) (m/z): 294.18 [M+•].

(1E,2E)-1,2-bis(4-fluorobenzylidene)hydrazine (3b)

Yellow crystalline solid; yield 96%; m.p. 75-78 oC. Analytical cal. C14H10F2N2: C,

68.85; H, 4.13; N, 11.47; found: C, 68.83; H, 4.14; N, 11.48.

IR (KBr, νmax cm-1): 1154, 1453 (C=C), 1583 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.93 (dd, 2H, H-2ʹ and H-6ʹ), 7.95 (dd,

2H, H-2 and H-5), 8.31 (dd, 2H, H-3ʹ and H-5ʹ), 8.32 (dd, 2H, H-3 and H-5), 8.67 (s,

1H, -CH=Nazine), 8.70 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 115.11 (C-3 and C-5), 115.14 (C-3ʹ and

Chapter-4

139

C-5ʹ), 130.75 (C-2 and C-6), 130.78 (C-2ʹ and C-6ʹ), 131.20 (C-1), 131.21 (C-1ʹ),

162.54 (C=N), 164.23 (C-4), 164.24 (C-4ʹ).

MS (ESI) (m/z): 244.08 [M+•].

(1E,2E)-1,2-bis(3-nitrobenzylidene)hydrazine (3c)

Yellow solid; yield 94%; m.p. 198 oC, reported 196-197 oC;26 Analytical cal.

C14H10N4O4: C, 56.38; H, 3.38; N, 18.78; found: C, 56.35; H, 3.39; N, 18.80.

IR (KBr, νmax cm-1): 1461, 1560 (C=C), 1334, 1510 (NO2), 1586 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.72 (m, 1H, H-5), 7.74 (m, 1H, H-5ʹ),

8.10 (d, 1H, H-4), 8.13 (d, 1H, H-4ʹ), 8.62 (s, 1H, H-2), 8.63 (s, 1H, H-2ʹ), 8.69 (s,

1H, -CH=Nazine), 8.73 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 120.32 (C-2), 120.35 (C-2ʹ), 125.45 (C-4),

125.47 (C-4ʹ), 129.56 (C-5), 129.59 (C-5ʹ), 132.12 (C-1), 132.15 (C-1ʹ), 134.97 (C-

6), 134.98 (C-6ʹ), 150.10 (C-3), 150.11 (C-3ʹ), 162.44 (C=N).

MS (ESI) (m/z): 298.07 [M+•].

(1E,2E)-1,2-bis(4-nitrobenzylidene)hydrazine (3d)

Yellow solid; yield 98%; m.p. 296 oC, reported 297-298 oC;26 Analytical cal.


IR (KBr, νmax cm-1): 1152, 1458 (C=C), 1338, 1512 (NO2), 1585 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.92 (dd, 2H, H-2 and H-6), 7.94 (dd, 2H,

H-2ʹ and H-6ʹ), 8.12 (dd, 2H, H-3 and H-5), 8.16 (dd, 2H, H-3ʹ and H-5ʹ), 8.79 (s,

1H, -CH=Nazine), 8.82 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 125.68 (C-2 and C-4), 125.69 (C-2ʹ and

C-4ʹ), 140.15 (C-1), 140.18 (C-1ʹ), 150.01 (C-6), 150.02 (C-6ʹ), 162.13 (C=N).

MS (ESI) (m/z): 298.07 [M+•].

(1E,2E)-1,2-bis(3,4-dimethoxybenzylidene)hydrazine (3e)

Yellow crystalline solid; yield 90%; m.p. 191-193 oC, reported 193 oC;27 Analytical

cal. C18H20N2O4: C, 65.84; H, 6.14; N, 8.53; found: C, 65.81; H, 6.16; N, 8.54.

IR (KBr, νmax cm-1): 1461, 1562 (C=C), 1591 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.79 (s, 12H, 4×CH3), 6.98 (d, 1H, H-5),

6.99 (d, 1H, H-5ʹ), 7.42 (d, 1H, H-6), 7.43 (d, 1H, H-6ʹ), 7.69 (s, 1H, H-2), 7.70 (s,

1H, H-2ʹ), 8.85 (s, 1H, -CH=Nazine), 8.89 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 57.65 (CH3), 108.89 (C-5), 108.90 (C-5ʹ),

111.56 (C-2), 111.57 (C-2ʹ), 123.23 (C-6), 123.25 (C-6ʹ), 127.87 (C-1), 127.89 (C-

Chapter-4

140

1ʹ), 148.78 (C-3), 148.79 (C-3ʹ), 152.66 (C-4), 152.68 (C-4ʹ), 160.80 (C=N).

MS (ESI) (m/z): 328.14 [M+•].

(1E,2E)-1,2-bis(3,4,5-trimethoxybenzylidene)hydrazine (3f)

Yellow crystalline solid; yield 97%; m.p. 194 oC, reported 192-194 oC;23 Analytical


IR (KBr, νmax cm-1): 1460, 1558 (C=C), 1595 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.89 (s, 18H, 6×CH3), 7.18 (dd, 2H, H-2

and H-6), 7.20 (dd, 2H, H-2ʹ and H-6ʹ), 8.90 (s, 1H, -CH=Nazine), 8.92 (s, 1H, -

CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 58.45 (-OCH3), 106.11 (C-2

and C-6), 106.13 (C-2ʹ and C-6ʹ), 131.20 (C-1), 131.22 (C-1ʹ), 142.32 (C-6), 142.34

(C-6ʹ), 152.32 (C-3 and C-5), 152.35 (C-3ʹ and C-5ʹ), 160.09 (C=N).

MS (ESI) (m/z): 388.16 [M+•].

4,4'-[(1E,1'E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(2-methoxyphenol)

(3g)

Light yellow solid; yield 92%; m.p. 211-213 oC; Analytical cal. C16H16N2O4: C,

63.99; H, 5.37; N, 9.33; found: C, 63.97; H, 5.38; N, 9.34.

IR (KBr, νmax cm-1): 1458, 1559 (C=C), 1617 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.73 (s, 6H, 2×CH3), 5.76 (s, 1H, -OH),

6.87 (d, 1H, H-5), 6.89 (d, 1H, H-5ʹ), 7.42 (s, 1H, H-2), 7.44 (s, 1H, H-2ʹ), 7.45 (d,

1H, H-6), 7.46 (d, 1H, H-6ʹ), 8.84 (s, 1H, -CH=Nazine), 8.87 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 56.12 (-OCH3), 114.02 (C-2), 114.04 (C-

2ʹ), 118.97 (C-5), 118.99 (C-5ʹ), 123.42 (C-6), 123.44 (C-6ʹ), 129.12 (C-1), 129.13

(C-1ʹ), 150.12 (C-3), 150.14 (C-3ʹ), 152.43 (C-4), 152.47 (C-4ʹ), 161.44 (C=N).

MS (ESI) (m/z): 300.11 [M+•]

2,2'-[(1E,1'E)-hydrazine-1,2-diylidenebis(methanylylidene)]diphenol (3h)

Yellow solid; yield 94%; m.p. 211-213 oC, reported 213 oC;28 Analytical cal.


IR (KBr, νmax cm-1): 1458, 1556 (C=C), 1588 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 5.40 (s, 2H, -OH), 7.01 (m, 1H, H-5), 7.02

(m, 1H, H-5ʹ), 7.08 ( d, 1H, H-3), 7.09 (d, 1H, H-3ʹ), 7.59 (m, 1H, H-4), 7.60 (m, 1H,

H-4ʹ), 7.88 (d, 1H, H-6), 7.89 (d, 1H, H-6ʹ), 8.91 (s, 1H, CH=Nazine). 8.94 (s, 1H, -

CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 117.67 (C-3), 117.69 (C-3ʹ), 118.23 (C-1),

Chapter-4

141

118.25 (C-1ʹ), 125.50 (C-5), 125.51 (C-5ʹ), 132.21 (C-6), 132.23 (C-6ʹ), 134.56 (C-

4), 134.70 (C-4ʹ), 162.34 (C-2). 162.35 (C-2ʹ), 163.08 (C=N).

MS (ESI) (m/z): 240.09 [M+•].

5,5'-[(1E,1'E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(benzene-1,2,3-

triol) (3i)

Light brown solid; yield 97%; m.p. 198-200 oC; Analytical cal. C14H12N2O6: C,

55.27; H, 3.98; N, 9.21; found: C, 55.31; H, 3.99; N, 9.24.

IR (KBr, νmax cm-1): 1460, 1554 (C=C), 1592 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 5.42 (s, 6H, -OH), 6.92 (s, 2H, H-2 and H-

6), 6.93 (s, 2H, H-2ʹ and H-6ʹ), 8.67 (s, 1H, -CH=Nazine), 8.69 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 107.94 (C-2 and C-6), 107.96 (C-2ʹ and

C-6ʹ), 132.98 (C-1), 132.99 (C-1ʹ), 137.33 (C-4), 137.35 (C-4ʹ). 147.01 (C-3 and C-

5), 147.02 (C-3ʹ and C-5ʹ), 162.21 (C=N).

MS (ESI) (m/z): 304.07 [M+•]

1,1'-[(1E,1'E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(naphthalen-2-ol)

(3j)

Yellow solid; yield 96%; m.p. > 300 oC, reported 308 oC;29 Analytical cal.


IR (KBr, νmax cm-1): 1456, 1562 (C=C), 1586 (C=N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 5.45 (s, 2H, -OH), 7.53 (m, 1H, H-6), 7.55

(m, 1H, H-6ʹ), 7.62 (m, 1H, H-5), 7.63 (m, 1H, H-5ʹ), 7.69 (d, 1H, H-3), 7.70 (d, 1H,

H-3ʹ), 7.90 (d, 1H, H-4), 7.93 (d, 1H, H-4ʹ), 7.95 (m, 1H, H-7), 7.96 (m, 1H, H-7ʹ),

8.40 (d, 1H, H-8), 8.42 (d, 1H, H-8ʹ), 8.72 (s, 1H, -CH=Nazine), 8.75 (s, 1H, -

CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 107.50 (C-1), 107.52 (C-1ʹ) 110.89 (C-3),

110.90 (C-3ʹ), 115.56 (C-8), 115.57 (C-8ʹ), 123.90 (C-6), 123.91 (C-6ʹ), 124.96 (C-

9), 124.92 (C-9ʹ), 127.01 (C-4), 127.03 (C-4ʹ), 127.22 (C-5), 127.24 (C-5ʹ), 128.21

(C-7), 128.23 (C-7ʹ), 141.11 (C-10), 141.12 (C-10ʹ), 158.02 (C-2), 158.03 (C-2ʹ),

163.22 (C=N),

MS (ESI) (m/z): 340.12 [M+•]

3,3'-[(1E,1'E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(4H-chromen-4-

one) (3k)

Yellow crystalline solid; yield 98%; m.p. 290-292 oC. Analytical cal. for

Chapter-4

142


IR (KBr, νmax cm-1): 1454, 1574 (C=C), 1594 (C=N), 1612 (C=Cγ-pyrone), 1652

(C=Oγ-pyrone). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.23 (s, 2Hγ-pyrone ring, H-2 and H-2ʹ), 7.42

(m, 4H, H-6, H-6ʹ, H-7 and H-7ʹ), 7.81 (dd, 2H, H-5 and H-5ʹ), 7.82 (dd, 2H, H-8

and H-8ʹ), 8.74 (s, 1H, -CH=Nazine), 8.78 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 110.74 (C-3), 110.79 (C-3ʹ), 121.26 (C-8),

121.27 (C-8ʹ), 123.90 (C-4a), 123.97 (C-4aʹ), 124.23 (C-6), 124.25 (C-6ʹ), 127.85

(C-5), 127.82 (C-5ʹ), 133.50 (C-7), 133.59 (C-7ʹ), 151.62 (C-8a), 151.65 (C-8aʹ),

163.15 (C=N), 163.23 (C-2ʹ), 163.25 (C-2), 178.50 (C-4) (C=Oγ-pyrone).

MS (ESI) (m/z): 344.08 [M+•]

3,3'-[(1E,1'E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(6-fluoro-4H-

chromen-4-one) (3l)

Yellow solid; yield 96%; m.p. 294 oC; Analytical cal. C20H10F2N2O4: C, 63.16; H,

2.65; N, 7.37; found: C, 63.12; H, 2.66; N, 7.40.


(C=Oγ-pyrone ). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.21 (s, 2Hγ-pyrone ring, H-2 and H-2ʹ), 7.30

(dd, 2H, H-7 and H-7ʹ), 7.32 (dd, 2H, H-8 and H-8ʹ), 7.78 (s, 2H, H-5 and H-5ʹ),

8.81 (s, 1H, -CH=Nazine), 8.83 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 110.71 (C-3), 110.73 (C-3ʹ), 121.12 (C-5),

121.20 (C-8), 121.15 (C-5ʹ), 122.25 (C-8ʹ), 124.50 (C-4a), 124.56 (C-4aʹ), 128.50

(C-7), 128.46 (C-7ʹ), 135.23 (C-6), 135.21 (C-6ʹ), 152.80 (C-8a), 152.82 (C-8aʹ),

162.95 (C-2), 162.99 (C-2ʹ), 164.40 (C=N), 178.42 (C-4) (C=Oγ-pyrone).

MS (ESI) (m/z): 380.06 [M+•]

3,3'-[(1E,1’E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(6-methyl-4H-

chromen-4-one) (3m)

Yellow crystalline solid; yield 98%; m.p. 293-295 oC; Analytical cal. C22H16N2O4:

C, 70.96; H, 4.33; N, 7.52; found: C, 70.95; H, 4.30; N, 7.56.


(C=Oγ-pyrone). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 1.83 (s, 6H, 2×CH3), 7.29 (s, 2Hγ-pyrone ring,

H-2 and H-2ʹ), 7.31 (dd, 2H, H-7 and H-7ʹ), 7.35 (dd, 2H, H-8 and H-8ʹ), 7.75 (s,

Chapter-4

143

2H, H-5 and H-5ʹ), 8.92 (s, 1H, -CH=Nazine), 8.94 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 29.02 (-CH3), 110.72 (C-3), 110.78 (C-

3ʹ), 121.24 (C-8), 121.22 (C-8ʹ), 123.91 (C-4a), 123.95 (C-4aʹ), 129.15 (C-5), 129.19

(C-5ʹ), 133.52 (C-7), 133.51 (C-7ʹ), 135.12 (C-6), 135.10 (C-6ʹ), 151.63 (C-8a),

151.65 (C-8aʹ), 163.13 (C=N), 163.21 (C-2), 163.23 (C-2ʹ), 178.49 (C-4) (C=Oγ-

pyrone).

MS (ESI) (m/z): 372.11 [M+•]

3,3'-[(1E,1’E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(6-bromo-4H-

chromen-4-one) (3n)

Yellow solid; yield 94%; m.p. 297-299 oC; Analytical cal. C20H10Br2N2O4: C,

47.84; H, 2.01; N, 5.58; found: C, 47.82; H, 2.04; N, 5.57.


(C=Oγ-pyrone). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.24 (s, 2Hγ-pyrone ring, H-2ʹ and H-2), 7.31

(dd, 2H, H-8 and H-8ʹ), 7.40 (dd, 2H, H-7 and H-7ʹ), 7.77 (s, 2H, H-5 and H-5ʹ), 8.66

(s, 1H, -CH=Nazine), 8.69 (s, 1H, -CH=Nazine). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 110.12 (C-3), 110.13 (C-3ʹ), 120.98 (C-8),

120.99 (C-8ʹ), 123.54 (C-4a), 123.59 (C-4aʹ), 124.17 (C-5), 124.15 (C-5ʹ), 128.35

(C-6), 128.38 (C-6ʹ), 133.72 (C-7), 133.76 (C-7ʹ), 151.67 (C-8a), 151.68 (C-8aʹ),

162.21 (C-2), 162.26 (C-2ʹ), 163.89 (C=N), 177.42 (C-4) (C=Oγ-pyrone).

MS (ESI) (m/z): 501.90 [M+•]

3,3'-[(1E,1'E)-hydrazine-1,2-diylidenebis(methanylylidene)]bis(2-amino-4H-

chromen-4-one) (3o)

Yellow solid; yield 92%; m.p. > 300 oC; Analytical cal. C20H14N4O4: C, 64.17; H,

3.77; N, 14.97; found: C, 64.18; H, 3.78; N, 14.99.


(C=Oγ-pyrone). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.23 (s, 2Hγ-pyrone ring, H-2ʹ and H-2), 7.39

(m, 4H, H-6, H-6ʹ, H-7 and H-7ʹ), 7.80 (dd, 2H, H-8 and H-8ʹ), 7.90 (dd, 2H, H-5

and H-5ʹ), 8.68 (s, 1H, -CH=Nazine), 8.71 (s, 1H, -CH=Nazine), 9.02 (brs, 4H, D2O

exchangeable). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 112.40 (C-3), 112.42 (C-3ʹ), 121.45 (C-8),

121.49 (C-8ʹ), 123.42 (C-4a), 123.44 (C-4aʹ), 124.21 (C-6), 124.23 (C-6ʹ), 125.63

Chapter-4

144

(C-5), 125.65 (C-5ʹ), 133.43 (C-7), 133.45 (C-7ʹ), 152.10 (C-8a), 152.12 (C-8aʹ),

163.21 (C=N), 166.80 (C-2), 166.82 (C-2ʹ), 179.50 (C=Oγ-pyrone).

MS (ESI) (m/z): 374.10 [M+•].

4.3.5. X-ray analysis and DFT studies of compound (3f)

The crystal structure of compound 3f was determined by Bruker Apex II

diffractometer. The instrumentation detail and procedure have been discussed in

chapter 2. Pertinent crystallographic data for compound (3f) is summarized in Table

1, while as selected structural parameters has been presented in Table 3. The

geometry optimization of the compound (3f) was performed using the PC

GAMESS/Firefly QC package,30 which is partially based on the GAMESS (US)

source code,31 starting from the experimental X-ray geometry (E,E-isomer). The

calculation was performed within density functional theory (DFT) using B3LYP

(Becke three-parameter Lee-Yang-Parr) for exchange and correlation, which

combines the hybrid exchange functional of Becke32,33 with the correlation functional

of Lee, Yang and Parr.34 The calculation was performed with an extended 6-

311G(d,p) basis set. Tight conditions for convergence of both the self-consistent field

cycles and the maximum density and energy gradient variations were imposed (10-5

atomic units). At the end of this geometry optimization, we conducted a Hessian

calculation to guarantee that the final structure corresponds to a true minimum, using

the same level of theory as in the geometry optimization. The geometries of the Z,Z

and Z,E isomers were also optimized with the same level of theory and obtained from

the optimized geometry of the E,E-isomer, performing the necessary rotations of

bond/torsion angles using the UCSF Chimera software package version 1.8.35 For the

optimized geometries of the E,E, Z,Z and E,Z-isomers, we performed single point

energy calculations with the conditions mentioned above (DFT: B3LYP functional

and 6-311G(d,p) basis set).



diphenylpicrylhydrazyl (DPPH) method.36 The procedure follow for this assay has

been discussed in detail in chapter 2. In this procedure, drug stock solution (1 mg/mL)

was diluted to final concentration of 2, 4, 6, and 8µg/mL in methanol.

Chapter-4

145

4.4. CONCLUSION

The present protocol reports the convenient and eco-friendly approach for the

synthesis of hydrazone derivatives 3(a-o) in excellent yields (90-98%) by employing

[Et3NH][HSO4] as a catalyst. This solvent-free, green synthetic procedure eliminates

the use of toxic solvents and thus makes it attractive one in organic synthesis. The

notable features of this protocol are shorter reaction time, high purity, mild reaction

conditions, operational simplicity, cleaner reaction profile, enhanced reaction rates

and easy workup. The ionic liquid [Et3NH][HSO4] used in the present study exhibits

stability towards air and water and is easy to prepare from cheap amine and acid. The

recyclability study of the catalyst demonstrates that it can be used up to five cycles

without any appreciable loss in catalytic activity. All the synthesized compounds

showed promising antioxidant activity. Thus, present synthetic approach provides

better scope for the synthesis of hydrazone derivatives and will be more practical than

the existing methods.

Chapter-4

146

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Salvayre, M. Baltas, Eur. J. Med. Chem., 2010, 45, 3019.

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148

5.1. INTRODUCTION

Heterocyclic compounds are acquiring more importance in recent years because of

their pharmacological activities. Pyrazolones have a particular value due to their

broad spectrum of biological activity and their wide ranging utility as synthetic tools

to design the various bioactive molecules. Pyrazolone derivatives are an important

class of heterocyclic compounds as they play a vital role both in medicinal chemistry

and in organic synthesis.1 Pyrazolone, is a derivative of pyrazole that has an

additional keto (C=O) group. There are three possible isomers: 3-pyrazolone, 4-

pyrazolone, and 5-pyrazolone. Pyrazolones are traditionally synthesized by treatment

of β-keto esters with hydrazine substrates under acidic conditions.2 In the field of

organic synthesis, pyrazolones are important intermediates for the synthesis of

dihydro[2,3-c]pyrazoles,3-6 phenylpyrazoles,7 and chromeno[2,3-c]pyrazol-4(1H)-

ones.8 In addition, They also serve as precursors in agrochemichal industries for the

preparation of herbicides, fungicides and insecticides,9 liquid crystals10, dyes11 and

thermally stable polymers.12 pyrazolone derivatives are useful for the extraction and

separation of various metal ions.13 Chemical oxidation of pyrazolones to azo

dienophiles provides suitable substrates for hetero Diels-Alder cycloadditions.14 They

have been found useful as solvent extraction reagents in both acidic and non- acidic

media.15,16

The chemistry of pyrazolone has gained increasing attention due to its diverse

pharmacological properties such as analgesic, anti-inflammatory, antimicrobial,

anticancer, antitubercular, antioxidant and antitumor activities.17-22 Antipyrine,

synthesized in 1883 was the first pyrazolone derivative that was used for clinical

trials. Antipyrine was used as the first agent to reduce fever and also for arthritis.

Nowadays a large no. of drugs such as Edaravone, Phenazone, Propyphenazone,

ARONIS023059, Metamizole sodium, TELIN and many others are available in the

market possess pyrazolone moiety in their structural framework. The biological

activities of pyrazol-5-ones depend on the nature of the substituents present on

pyrazolone ring.23 Compounds like 3-alkyl-4-aryl methyl pyrazol-5-ones are reported

to exhibit potent antihyperglycemic activity, while 1-phenyl-3-

tetrafluoroethylpyrazol-5-one is an anxiolytic. 3-methyl-1-phenyl-2-pyrazolin-5-one

(edaravone) a strong novel free radical scavenger is used for the treatment of patients

with acute brain infarction.24 A new derivative of edaravone, 4,4-dichloro-1-(2,4-

Chapter-5

149

dichlorophenyl)-3-methylpyrazol-5-one was identified as a potent blocker of human

telomerase and is considered to be avaluable substance for medical treatment of

cancer and related diseases.25 Thiadiazole substituted pyrazol-5-ones derivatives were

found to be potent KDR/VEGFR-2 kinase inhibitors in regulating angiogenesis which

is crucial for the proliferation of tumor cells.26 The diverse pharmacological properties

have encouraged the chemists to developed new synthetic methodologies to

synthesize a large number of novel therapeutic agents. Several sophisticated

methodologies have been postulated in the literature for the synthesis of a pyrazolone

moiety.

Vijesh et al.27 Reported two step synthesis of novel pyrazolone derivatives 3(a-

f). First they react phenyl hydrazine/2,4 dinitrophenyl hydrazine with

ethylacetoacetate in ethanol under reflux condition to form 2-Aryl-5-methyl-2,4-

dihydro-3H-pyrazol-3-one (1). Further the targeted pyrazolone derivatives 3(a-f) were

obtained in excellent yields by refluxing 2-Aryl-5-methyl-2,4-dihydro-3H-pyrazol-3-

one (1) with various substituted heterocyclic aldehydes 2(a-f) in presence of

anhydrous sodium acetate in acetic acid (AA) for 8h. The synthesized compounds

were also evaluated for antimicrobial activity.

NN O

H3CAbsolute

alcohol 30 mL

Reflux, 12h NNH

CHOAr NN O

H3CNHN

Ar

AA, NaOAc

NHNH2R

R

R

R

R

R

O

OO

Reflux, 8h

C2H5

1 2(a-f) 3(a-f)

R=H, NO2

Ar= 2,4-dichlorophenyl, thioanisyl, 2,5-diclorothiophene, biphenyl, 4-anisyl, 4-

chlorophenyl

Mariappan et al.28 carried out the synthesis of 3-methyl pyrazol-5-one

derivatives 5(a-j) by reacting ethylacetoacetate and hydrazine hydrate at 60 °C for 1h

to yield 3-methyl pyrazol-5-one (3). Further in presence of 20% alcoholic solution of

sodium hydroxide, 3-methyl pyrazol-5-one reacts with aromatic and aliphatic

aldehydes 4(a-j) to yield 3-methyl pyrazol-5-one derivatives 5(a-j).

Chapter-5

150

NH2NH2

EthanolN N

H3C

H

O

N N

H3C

H

O

HCRCHO

R

N N

H3C

H

O

CHCH3

alc. NaOH (20%)

CH3CHO

4(a-i)

(4j)

5(a-i)

(5j)

Stirring, 60oC, 1h Stirring, 30 min

O O

OC2H51

2 3

R= N(CH3)2, C6H5CH=CH, 2,3-OCH3, 3NO2, 3-Cl, H, 4-OCH3, 3-OCH3, OH

Verma and co-workers29 synthesized a series of 4-arylidene-3-methyl-1-phenyl-

5-pyrazolone derivatives 5(a-j) by reacting various substituted aromatic aldehydes

4(a-j) with 3-methyl-1-phenyl-5-pyrazolone (3) via Knoevenagel condensation by

conventional as well as by exposure to microwave irradiations. All the synthesized

compounds were also tested for anti-inflammatory as well as antimicrobial activity.

H3CO

OO

C2H5

NHNH2 NN

H3C

O NN

H3C

ORCHO

AA, heat

Stirring, 45 min

150-160 ºC, 3-4 h or

MW 2-4 minR

1 2 3 4(a-j) 5(a-j)

R= 4-H, 4-Cl, 4-OH, 4-F, 4-OCH3, N(CH3)2, 3-OCH3, 3-Cl, 2-CH3, 3-OC2H5

Rasapalli et al.30 developed a facile one pot approach for synthesis of new

pyrazolones derivatives 4(a-j) from the reaction of dimethyl 3-oxopentanedioate (1),

phenylhydrazine (2) and aromatic aldehydes 3(a-j) in presence of acetic acid and

NH4OAc (20% mmol).

HO

O O

OH

O

NH

NH2 NH4OAc (20%mmol)N

N

ROOC

Ar

OAA, Stirring, 15 min

1 2 3(a-j) 4(a-j)

Ar H

OH

Ar= 3-OMe-4-OEtC6H3, 4-OHC6H4, 3,4,5-(OMe)3C6H2, 4-ClC6H4, indole, furan, 4-

N(CH3)2C6H5, thiophene, 2,4-(OMe)2C6H3, 2-BrC6H5

Chapter-5

151

Atudosie et al.31 documented one-pot approach for the synthesis of new

pyrazolones derivatives containing a phenothiazine unit 4(a-d) using 4Å molecular

sieves as catalyst in DMA at 60 °C.

S

N

OCl

R2

O

OEt

O

S

N

ON

N O

R2

R1NH2NH2. H2O

R1

4 Å molecular sieves powderedDMA, 60 oC, 24 h

1 2 3 4(a-d)

R1=H, CH3, CF3; R2= Me, Ph

Mosaddegh et al.32 effectively synthesize a series of 4,4'-(arylmethylene) bis(3-

methyl-1-phenyl-1H-pyrazol-5-ol) derivatives 3(a-j) by refluxing aromatic aldehydes

(2) with 1-phenyl-3-methyl-5-pyrazolone (1) using Ce(SO4)2.4H2O as reusable,

environmentally friendly catalyst in water/ethanol solution for 5-25 min.

NN OPh

Ar H

O Ce(SO4)2.4H2O

EtOH/H2O, Reflux

Ar

HNN N

NOPh PhHO

1 2 3(a-j)

Ar= C6H5, 4-ClC6H4, 2,4-Cl2C6H3, 3-NO2C6H4, 4-BrC6H4, 4-CH3C6H4, 3-CH3C6H5, 4-OCH3C6H5, 3-OHC6H4, 2,4-(OCH3)2C6H4

Yang et al.33 successfully synthesized 4-amino-5-pyrazolone derivatives (3)

bearing a chiral quaternary centre by the reaction of 4-substituted pyrazolones (1)

with azodicarboxylates (2) employing N,N'-dioxide gadolinium(III) complex as the

catalyst.

NN

O R1

R2PhNN

R3OOC

COOR3N

N

O

R2Ph

NR1

HN COOR3

COOR3

L1-Gd-(OTf3) (0.05 or 1 mol%)4 A MS, -20 oC, CH2Cl2

1 2 3

R1= Bn, 2-MePhCH2, 3-MePhCH2, 4-MePhCH2, 2-OMePhCH2, 3-OMePhCH2, 4-

OMePhCH2, 2-ClPhCH2, 3-ClPhCH2, 4-ClPhCH2, 2-BrPhCH2, 2,4-Cl2PhCH2, 2-

Chapter-5

152

furanylmethyl, 2-thienylmethyl, 1-naphthylmethyl, 2-naphthylmethyl, Me, Et, n-

propyl, allyl, (CH2)4; R2=Me, Ph; R3= Et, i-Pr, Bu

Zang and co-workers34 described a new methodology for the synthesis of 4-[(5-

hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-phenyl-methyl]-5-methyl-2-phen-yl-

1,2-dihydro-pyrazol-3-ones 3(a-m) using ionic liquid [HMIM][HSO4] as a catalyst in

ultrasound irradiation at room temperature.

NN OPh

Ar H

O

EtOH, Ultrsound, RT

Ar

HNN N

NOPh PhHO

[HMIM][HSO4] (10 mol%)

1 2 3(a-m)

Ar= C6H5, 4-ClC6H4, 2-ClC6H4, 2,4-Cl2C6H3, 2-BrC6H4, 4-NO2C6H4, 3-

NO2C6H4, 2-NO2C6H4, 2-OHC6H4, 4-OHC6H4, 2-OCH3C6H4, 4-OCH3C6H4, 4-

N(CH3)2C6H4

Ziarati et al.35 reported simple and green four-component reaction of

phenylhydrazine, ethylacetoacetate, aromatic aldehydes 4(a-o) and β-naphthol in

water under ultrasound irradiation using CuI nanoparticles as a catalyst for synthesis

of 2-aryl-5-methyl-2,3-dihydro-1H-3-pyrazolones 5(a-o).

HN NH2R1 Me

O

OEt

O

OHCHO

R2

CuI nanoparticles, H2OUltrasound, RT, 30-40 min HN

N OHO

1 2

3 4(a-o) 5(a-o)

R2

R1

R1=H, 4-Cl; R2=H, 4-Cl, 4-NO2, 4-Me, 2-Me, 2-Cl, 4-Br, 2-NO2, 4-OMe

Gunasekaran et al.36 developed new protocol for the synthesis of a series of

novel 2-aryl-5-methyl-2,3-dihydro-1H-3-pyrazolones 5(a-o) through one-pot four-

component reaction between phenylhydrazine (1), methyl acetoacetate (2), β-naphthol

(3) and aromatic aldehydes (4) in the presence of p-toluenesulphonic acid in water.

Chapter-5

153

These 2-aryl-5-methyl-2,3-dihydro-1H-3-pyrazolones were also screened for in-vitro

antimycobacterial activity against mycobacterium tuberculosis H37Rv (MTB).

OHO

OMeORNHNH2

p-TSA, H2OReflux, 4-12h

ArCHOAr

HNN O

R HO 1 2 3 4 5(a-o)

R= C6H5, 4-FC6H4, 4-ClC6H4

Ar= 4-NO2C6H4, 2-NO2C6H4, 4-ClC6H4, C6H5, 4-PriC6H4, 4-MeOC6H4, 4-

MeC6H4, 4-ClC6H4, 4-NO2C6H4, 2,4-Cl2C6H3, 3-NO2C6H4, 4-FC6H4

Sujatha et al.37 carried out efficient synthesis of 4,4'-(arylmethylene) bis

(1H-pyrazol-5-ols) 3(a-j) by ceric ammonium nitrate (CAN) catalysed tandem

Knoevenagel-Michael reaction of two equivalents of 5-methyl-2- phenyl-2,4-dihydro-

3H-pyrazol-3-one (1) with various aromatic aldehydes (2) in water.

NN O

Ar H

O Ar

HNN N

NOHO

CAN (5 mol%), H2OStirring, RT

1 2 3(a-j)

Ar= C6H5, 3-CH3C6H4, 4-CH3C6H4, 4-OCH3C6H4, 4-FC6H4, 4-NO2C6H4, 3,4-(OCH3)2C6H3, 3-OCH3-4-OHC6H3, 2-Furfuryl, 2-Pyridyl

In light of the above observations, a series of biologically active pyrazolone

analogues have been synthesized via one pot synthesis of aromatic/heterocyclic

aldehydes, ethylacetoacetate and phenylhydrazine/2,4 dinitrophenylhydrazine (2,4-

DNP) in water under microwave heating using SiO2/ZnBr2 as a catalyst. This new

synthetic eco-friendly approach resulted in a remarkable improvement in synthetic

efficiency (94-98%), high purity, minimizing the production of chemical wastes

without using highly toxic reagents for the synthesis. The synthesized compounds

have been screened for antioxidant study.

Chapter-5

154



derivatives 4(a-s) have been synthesized using SiO2/ZnBr2 as a recyclable Lewis acid

catalyst in water under microwave heating. The molecular structure of compounds 4a

and 4d were well supported by single crystal X-ray crystallographic analysis. The

present protocol bears a wide substrate tolerance and is believed to be more practical,

efficient, eco-friendly and compatible as compared to existing methods. Further all

synthesized compounds were tested for antioxidant activity and results obtained were

promising.

5.2.1. Characterization of catalyst (SiO2/ZnBr2)

The catalyst was prepared by employing standard procedures depicted in the

literature.38 The formation of SiO2/ZnBr2 system was evaluated by FT-IR, powder

XRD and SEM-EDX analysis. The stability of the catalyst was shown by TGA/DTA

analysis. The FT-IR spectrum of the catalyst (SiO2/ZnBr2) is depicted in Fig. 1.

Fig. 1 FT-IR Spectrum of catalyst (SiO2/ZnBr2)

The FT-IR spectrum of the catalytic system displayed a symmetrical stretching band

at 3483 cm-1 for hydroxyl group and the band resonating at 1632 cm-1 was attributed

to the bending vibration of adsorbed water.39 Moreover, asymmetric and symmetric

stretching vibration band for Si-O-Si appeared at 1093 cm-1 and 797 cm-1,

respectively. In addition the peak resonating at 910 and 467 cm-1 has been assigned

Chapter-5

155

for the stretching vibration of Si-OH39 and Zn-Br, respectively. Thus, FT-IR spectrum

of the catalytic system authenticates the coating of ZnBr2 on the SiO2 surface as all

the characteristic peaks related to silica and ZnBr2 has been present in the spectrum of

SiO2/ZnBr2.

Formation of the catalytic system (SiO2/ZnBr2) was further confirmed by

powder XRD analysis (Fig. 2). X-ray diffractograms (XRD) of the catalyst were

recorded in the 2θ range of 20-80°. A single broad peak in the range of 2θ=20-30°

ascribed to the amorphous nature of silica. The characteristic diffraction peaks of pure

ZnBr2 were reported to appear at 13.7°, 21.1°, 27.5°, 46.1°and 53.4°.40 The XRD

analysis of SiO2/ZnBr2 exhibited diffraction peak for ZnBr2 only at 46.2° and 53.4°.

However, the other characteristic peaks (21.1° and 27.5°) were merged with the broad

peak of SiO2 (2θ=20-30°). The appearance of these characteristics peaks indicating

the dispersion of ZnBr2 on the silica material and thus confirming the formation of

SiO2/ZnBr2 matrix.

Fig. 2 Powder XRD pattern of catalyst (SiO2/ZnBr2)

SEM analysis was employed to study the surface morphology of the catalytic

system (Fig. 3). SEM micrographs of the catalyst showed that the particles of ZnBr2

were well dispersed on silica surface. The successful incorporation of zinc bromide

was also confirmed by EDX analysis (Fig. 4). EDX spectrum showed the presence of

Zn and Br in addition to O and Si elements.

The thermal stability of the catalyst was determined by TGA analysis (Fig. 5).

The only weight loss of 16.94% in the range of 40-120 °C was attributed to loss of

physically adsorbed water molecules in the silica gel framework. TGA is further

20 30 40 50 60 70

50

100

150

200

250

300

350

Inten

sity (

a.u.)

2θ (degree)

Chapter-5

156

supported by DTA analysis in which a prominent peak at 93.04 °C showed

endothermic reaction which help in the removal of water molecule (Fig. 5). Further

there is no weight loss upto 800 °C. Therefore it can be concluded that physiosorbed

and chemisorbed ZnBr2 on silica surfaces is stable upto 800 °C.41

(a) (b)

Fig. 3 SEM micrograph of (a) pure SiO2 (b) SiO2/ZnBr2 catalyst.

Fig. 4 EDX analysis of the catalyst (SiO2/ZnBr2)

Chapter-5

157

Fig. 5 TGA/DTA of catalyst (SiO2/ZnBr2)

5.2.2. Chemistry


derivatives 4(a-s) have been synthesized (Scheme 1), via the reaction between

aromatic/heterocyclic aldehydes 1(a-s) with phenylhydrazine / 2,4

dinitrophenylhydrazine (2,4-DNP) 2(a-b) and ethylacetoacetate (3) employing

SiO2/ZnBr2 as a catalyst in water under microwave heating.

Chapter-5

158

NN

O

HHN NH2 H3C

O

OC2H5


MW, 60oC(10-15min)

H

O

Ar Ar

1(a-s) 2a 3 4(a-n)

Water (10 mL)

NN

O

HHN NH2 H3C

O

OC2H5


MW, 60oC(10-15min)

H

O

Ar Ar

1(o-s) 2b 3 4(o-s)

Water (10 mL)

NO2

O2N

NO2

O2N

NH

NH

NH

HO H3C

O

O

O

O

O

O

O

O

H3C

F Br

OCH3

H3CO O2NNO2

F

H3CO

NH3C

CH3

NO2 OCH3

H3CO

O

O

NH2

FNH3C

CH3

OCH3

H3CO

1a 1b 1c 1d 1e

1f 1g 1h 1i

1j 1k 1l 1m 1n

1o 1p 1q 1r 1s


The structural elucidation of the synthesized compounds 4(a-s) was established

on the basis of elemental analysis, IR, 1H NMR, 13C NMR and mass spectral studies.

The analytical results for C, H and N were within ±0.3% of the theoretical values. The

absence of peak for aldehydic carbonyl in IR spectrum, confirmed the reaction at the

carbonyl moiety. Moreover, all the compounds displayed a characteristic peak for

C=N and C=O groups, resonating at around 1578-1603 cm-1 and 1680-1700 cm-1,

respectively, which signifies the formation of a pyrazolone ring. Characteristic peaks

Chapter-5

159

for the different functional groups such as methoxy, nitro and hydroxyl etc. have been

discussed in experimental section. In 1H NMR spectrum, each compound displayed a

sharp singlet at around δ 7.32-7.99 ascribed to the olefinic proton, a broad singlet at

around δ 12.02-12.46 (D2O exchangeable) has been ascribed to -NH proton of indole

ring. Similarly, a sharp singlet at around δ 9.32-9.82 corresponds to the H-2 proton of

indole ring (4a-4c). Furthermore, sharp singlets resonating at around δ 10.64, 10.04,

10.12, 10.10 each integrating for one proton, has been attributed to H-2 proton of γ-

pyrone ring of compounds 4d, 4e, 4f and 4g, respectively. 13C NMR spectra, showed

peaks resonating at around δ 137.31-153.80 and δ 162.77-170.16 corresponds to -

C=N and -C=O moiety of pyrazolone ring, respectively. Similarly signals at δ 174.19-

174.69 have been attributed to carbonyl group (C=Oγ-pyrone) of compounds (4d-4h).

The mass spectral analysis of the synthesized compounds was also in good conformity

with the proposed structures.

The FT-IR spectrum of compound 4a (Fig. 6) displayed characteristics peaks at

1594 and 1680 cm-1 attributed to C=N and C=O respectively, while the peak

resonating at 3251 cm-1 attributed to –NH stretching of the indole ring. Other peaks

resonating at 1157, 1456 cm-1 for C=C stretching, 2928, 3064 cm-1 for –C-H and =C-

H stretching, respectively. The 1H NMR spectrum of compound 4a (Fig. 7) displayed

a sharp singlet at δ 7.91 for one protons ascribed to the =C-H proton. Multiplets at

around δ 7.52-8.02 for five protons have been attributed to phenyl ring. A broad

singlet at δ 12.46 for one proton has been ascribed to -NH proton of the indole ring. A

sharp singlet at δ 9.82 for one proton has been ascribed to H-2 proton of the indole

ring. Another sharp singlet at δ 2.39 for three protons and multiplet at δ 7.14-7.67 for

four protons were attributed to CH3 protons and indole ring respectively.

Chapter-5

160

Fig. 6 FT-IR spectrum of compound 4a

Fig. 7 1H NMR spectrum of compound 4a

Chapter-5

161

13C NMR spectrum of compound 4a (Fig. 8) displayed characteristics absorption

bands resonating at around δ 162.77, 150.44, 136.40 and 138.90 for C=O, C=N, C-1'

and C-2', respectively. Further a series of signals resonating at around δ 118.40-

138.19 and δ 112.17-136.56 have been assigned to phenyl ring and indole ring,

respectively. The detailed spectral characterizations of all the synthesized compounds

have been discussed in experimental section.

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

12.93

38.93

39.13

39.34

39.55

39.76

39.97

40.18

62.90

78.32

78.65

78.98

112.17

112.71

118.05

118.12

118.40

121.91

123.23

123.66

128.06

128.37

136.40

136.56

138.19

138.90

150.44

162.77

Current Data ParametersNAME May14-2015EXPNO 12PROCNO 1

F2 - Acquisition ParametersDate_ 20150515Time 1.37INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT DMSONS 1024DS 4SWH 29761.904 HzFIDRES 0.454131 HzAQ 1.1010548 secRG 32DW 16.800 usecDE 6.00 usecTE 295.7 KD1 2.00000000 secd11 0.03000000 secDELTA 1.89999998 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 9.60 usecPL1 -2.00 dBSFO1 100.6228298 MHz

======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 -3.00 dBPL12 14.31 dBPL13 18.00 dBSFO2 400.1316005 MHz

F2 - Processing parametersSI 32768SF 100.6128193 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40

I-P BRUKERAVANCE II 400 NMRSpectrometerSAIFPanjab UniversityChandigarh

[email protected] Fig. 8 13C NMR spectrum of compound 4a

The selective Z-geometry across C=C was authenticated by single crystal X-ray

crystallographic analysis of compound 4a and 4d (Fig. 9), which was found to be

stabilized by an intricate array of H-bonding (Fig. 10) and π.....π interactions (Fig.

11). The crystallographic data of compound 4a and 4d have been presented in Table

1.

Chapter-5

162

Fig. 9 Asymmetric unit showing thermal ellipsoids (50% probability level) of (a) compound 4a (b) compound 4d.

(a)

(b)

Chapter-5

163

(a)

(b)

Fig. 10 2D view showing intricate H-bonding interactions in (a) compound 4a; (b)

compound 4d.

Chapter-5

164

(a)

(b)

Fig. 11 Diagrammatic representation of π...π interactions in (a) compound 4a, π...π and -CH ...π interactions in (b) compound 4d

Chapter-5

165

Table 1 Crystallographic data and structure refinement of compounds 4a and 4d

Parameters Compound 4a Compound 4d

Empirical formula C19H15N3O C20H14N2O3

Formula wt. 301.34 330.33

Crystal system Monoclinic Triclinic

Space group P21/n P-1

a, Å 5.810(5) 7.870(5)

b, Å 9.256(5) 8.298(3)

c, Å 26.893(5) 11.843(5)

α (°) 90 85.806(4)

β (°) 94.997(5) 80.900(5)

γ (°) 90 89.987(5)

U, Å3 1440.7(15) 761.6(7)

Z 4 2

ρcalc Mg/m3 1.389 1.441

μ, mm-1 0.089 0.099

Temperature (K) 100 100

θ max 25.50 25.50

F(000) 632 344

Refl. collected 11010 9548

Independent refl. 2087 2314

GOFa 1.038 1.037

Final Rb indices [I>2σ(I)] R1 = 0.0501; wR2 =

0.1268

R1 = 0.0402; wR2 =

0.0970

R indices (all data) R1 = 0.0675; wR2 =

0.1380

R1 = 0.0524; wR2 =

0.1038


To optimize the best reaction conditions for synthesis of biologically active

pyrazolone derivatives, effect of solvent, catalyst loading, effect of temperature have

been investigated on the model reaction. Initially, indole-3-carbaldehyde (1a, 2

mmol), phenylhydrazine (2a, 2 mmol) and ethylacetoacetate (3, 2 mmol) were

Chapter-5

166

refluxed in water (10 mL) at 60 °C without catalyst. The reaction took a longer time

period of 24 h to complete and afforded desired product 4a in less yield (Table 2,

entry 1), signifying the need of a catalyst. The reaction was then studied in the

presence of different catalysts such as AlCl3, ZnBr2, FeCl3, SiO2-Cl, SiO2/ZnBr2.

Our analysis revealed that the catalytic activity of various catalysts in water at 60 °C

was found to be in the order of SiO2/ZnBr2 > AlCl3 > FeCl3 > ZnBr2 > SiO2-Cl

(Table 2, entries 2-6). To compare the efficiency as well as competence of the

reactions under aqueous condition, the model reaction was also examined in the

presence of SiO2/ZnBr2 in different solvents like MeOH, EtOH, CH3COOH, CH2Cl2,

DMF and THF. The use of relatively less polar aprotic solvents CH2Cl2, DMF and

THF yielded the product 4a in moderate yield (58-62%), after extended reaction time

(Table 2, entries 10-12). However, in polar protic solvents MeOH, EtOH, and AcOH

relatively high yield (65-70%) of the product 4a was obtained with dip in reaction

time (Table 2, entries 7-9), whereas when reaction was performed in water in the

presence of SiO2/ZnBr2, there was remarkable increase in the yield (86%) of the

product 4a with prominent fall in reaction time (Table 2, entry 6). In order to further

improve the protocol to make it more energy efficient microwaves was introduced.

The use of microwaves (Anton Paar, Monowave 300) enhanced the protocol

remarkably with high yield of the product 4a (98%) and short reaction period (10

min) (Table 2, entry 13).

Table 2 Effect of different reaction media on model reaction (4a)a

S.

No.

Solvent Condition Time (h)b Yield (%)c

1 Water 60 °C, without catalyst 24h 46

2 Water 60 °C, AlCl3 8h 56

3 Water 60 °C, ZnBr2 10h 52

4 Water 60 °C, FeCl3 12h 53

5 Water 60 °C, SiO2-Cl 16h 51

6 Water 60 °C, SiO2/ZnBr2 4h 86

7 MeOH 60 °C, SiO2/ZnBr2 6h 65

8 EtOH 60 °C, SiO2/ZnBr2 8h 70

9 CH3COOH 60 °C, SiO2/ZnBr2 6h 68

10 CH2Cl2 60 °C, SiO2/ZnBr2 10h 62

Chapter-5

167

11 DMF 60 °C, SiO2/ZnBr2 14h 60

12 THF 60 °C, SiO2/ZnBr2 18h 58

13 Water 60 °C, SiO2/ZnBr2, MW 10 min 98 aReaction condition: indole-3-carbaldehyde (1a, 2 mmol), phenylhydrazine (2a, 2 mmol), and ethylacetoacetate (3, 2 mmol), different solvent (10 mL), different catalyst (0.10 g) bReaction progress monitored by TLC; cIsolated yield of products

To achieve the optimum concentration of the catalyst, the model reaction (4a)

was investigated at different concentrations 0.02-0.12 g (Table 3, entries 1-6) of the

catalyst SiO2/ZnBr2 at 60 °C in water under MW. The best results were obtained with

the use of 0.10 g of catalyst. Using less than 0.10 g of catalyst, moderate yields of the

product 4a (66-83%) were obtained with extended reaction times, while increasing

catalyst amount 0.10-0.12 g, there was no further increase in the yield of the product

4a, possibly due to the saturation of the catalyst. The above results signify that 0.10 g

of SiO2/ZnBr2 is optimum dose in terms of efficient yield and reduced reaction time.

Table 3 Effect of catalyst loading on the yield and reaction time of model reaction

(4a)a

Entry Catalyst (g) Time (min)b Yield (%)c

1 0.02 35 60 2 0.04 30 68 3 0.06 28 75 4 0.08 25 83 5 0.10 10 98 6 0.12 10 98

aReaction condition: indole-3-carbaldehyde (1a, 2 mmol), phenylhydrazine (2a, 2 mmol), and ethylacetoacetate (3, 2 mmol), water (10 mL), SiO2/ZnBr2 (0.02-0.12 g), MW-60 °C bReaction progress monitored by TLC; cIsolated yield of products

To optimize the reaction temperature, the model reaction was carried out at

different temperatures in water under microwave heating (Table 4, entries 1-7). It

was observed that increase in temperature from 25 °C to 60 °C, has a noteworthy

effect on the model reaction in terms of yield and reaction time (Table 4, entries 1-6).

However, no further enhancement in the yield of product 4a was observed when the

reaction temperature was raised from 60 °C to 65 °C (Table 4, entry 7).

Chapter-5

168

Table 4 Effect of reaction temperature on the model reaction (4a)a

Entry Temperature (°C) Time (min)b Yield (%)c

1 25 75 72

2 40 50 74

3 45 35 78

4 50 30 84

5 55 25 88

6 60 10 98

7 65 10 98 aReaction condition: indole-3-carbaldehyde (1a, 2 mmol), phenylhydrazine (2a, 2 mmol), and ethylacetoacetate (3, 2 mmol), water (10 mL), SiO2/ZnBr2 (0.10 g), MW, different temperature (25-65 °C) bReaction progress monitored by TLC cIsolated yield of products After optimization of the reaction conditions, the catalyst SiO2/ZnBr2 was

examined under the optimized reaction conditions using both conventional and

microwave heating. A wide range of aromatic/heterocyclic aldehydes was reacted

with ethylacetoacetate and phenylhydrazine/2,4-dinitrophenylhydrazine (2,4-DNP) to

afford the target pyrazolone in excellent yields. The catalyst showed good efficiency

under conventional heating giving the products in 4-6 h. However, microwave

induction produced excellent yields (94-98%) of products in 10-15 min. The above

results demonstrate that SiO2/ ZnBr2 is an efficient catalyst for the synthesis of wide

range of pyrazolones in high yields under mild aqueous conditions.

Chapter-5

169

Table 5 Synthesis of pyrazolones derivatives 4(a-s)

S. No.

Products

Conventional Methoda

Microwave irradiationb

Time (h)c

Yield (%)d

Time (min)c

Yield (%)d

4a

NN

O

H

NH

123

45

677a

3a

1'2'

3"4"

5"6"

1"2"

4

81

10

98

4b NN

O

H

NH

OH

4.5

77

10

95

4c NN

O

H

NH

CH3

4.5

80

15

98

4d

NN

O

H

O

O

123 4

5

6

788a

4a

1' 2'

1"2"

3"4"

5"6"

4

84

10

98

4e NN

O

H

O

O

CH3

5

78

15

95

4f

NN

O

H

O

O

F

4.5

82

15

96

Chapter-5

170

4g NN

O

H

O

O

Br

6

79

15

94

4h NN

O

H

O

O

H2N

5.0

76

15

95

4i NN

O

H

OCH3

OCH3

1 23

45

6

6"

1"2"

3"4"

5"1' 2'

5.5

81

10

97

4j NN

O

H

OCH3

OCH3H3CO

6.0

74

15

94

4k

NN

O

H

NO2

4.0

83

10

97

4l N

N

O

H

NO2

4.5

75

10

94

4m NN

O

H

NH3C CH3

5.5

84

10

98

Chapter-5

171

4n NN

O

H

F

5

76

15

96

4o NN

O

H

O2N

NO2

6

74

15

94

4p NN

O

H

O2N NO2

NO2

4.5

78

10

97

4q NN

O

H

O2N OCH3

OCH3

NO2

5

76

15

95

4r NN

O

H

NH3C CH3

O2N

NO2

4

82

10

98

4s NN

O

H

F

NO2

O2N

5.5

75

15

94

aReaction conditions: indole-3-carbaldehyde (1a, 2 mmol), phenylhydrazine (2a, 2 mmol), and ethylacetoacetate (3, 2 mmol), water (10 mL), SiO2/ZnBr2 (0.10 g), 60 °C bReaction conditions: indole-3-carbaldehyde (1a, 2 mmol), phenylhydrazine (2a, 2 mmol), and ethylacetoacetate (3, 2 mmol), water (10 mL), SiO2/ZnBr2 (0.10 g), MW-60 °C cReaction progress monitored by TLC. dIsolated yield of the products

Chapter-5

172

5.2.4. Reusability of the catalyst

The reusability of the catalyst (SiO2/ZnBr2) was also explored for the selected model

reaction in order to reduce the cost of the process (Fig. 12). After the first fresh run

with 98% yield, the catalyst was removed by simple filtration, washed with

ethylacetate and dried at 160 °C for 10 h. The recovered catalyst was further tested up

to five more reaction cycles. The results revealed that there is little drop in the yield of

the product after every successive run of the catalyst. This little drop in the catalytic

activity is believed to be due to the leaching of ZnBr2. SEM and EDX analysis of the

recovered catalyst was also performed to ascertain its morphology and composition

(Fig. 13). It was observed that the composition of the catalytic system was almost

consistent with the fresh catalyst (Fig. 3) there have been no significant changes in the

morphology of the catalyst.

Fig. 12 Recycling data of the catalyst (SiO2/ZnBr2) for the model reaction

Fig. 13 (a) SEM micrograph (b) EDX spectrum of the recovered catalyst

(SiO2/ZnBr2)

(a) (b)

Chapter-5

173


The antioxidant activities of all the synthesized pyrazolone derivatives 4(a-s) were

investigated by DPPH scavenging activity. The reduction capability of DPPH radicals

was determined by a decrease in their absorbance at 517 nm induced by antioxidants

using ascorbic acid as a reference. The potencies for the antioxidant activity of

compounds 4(a-s) to the reference compound are shown in Table 6.

Table 6 Antioxidant activity of compounds 4(a-s) by DPPH assay



4a 43.94±0.3 47.80±0.4 49.73±0.1 51.44±0.5 6.45

4b 32.84±0.4 52.19±0.2 59.69±0.3 63.87±0.5 4.57

4c 36.22±0.2 38.90±0.6 52.84±0.7 56.37±0.2 6.05

4d 33.44±0.3 37.62±0.3 39.22±0.4 49.30±0.2 9.10

4e 26.79±0.5 32.58±0.1 39.11±0.4 44.37±0.7 9.82

4f 41.26±0.4 42.97±0.5 47.90±0.7 49.62±0.6 8.04

4g 36.58±0.4 39.92±0.5 47.40±0.4 54.96±0.2 6.68

4h 38.26±0.3 45.58±0.3 50.78±0.5 58.38±0.4 5.53

4i 21.46±0.6 25.83±0.7 29.36±0.4 31.40±0.3 18.79

4j 32.26±0.4 35.09±0.5 36.87±0.4 38.47±0.3 19.04

4k 33.54±0.4 38.69±0.2 44.15±0.6 49.30±0.2 8.25

4l 25.18±0.3 28.18±0.6 33.65±0.5 37.72±0.1 13.73

4m 23.68±0.4 29.15±0.2 35.15±0.5 41.58±0.2 10.90

4n 33.11±0.4 37.19±0.1 43.08±0.6 45.76±0.3 9.66

4o 26.36±0.2 35.47±0.7 39.65±0.1 42.06±0.3 10.50

4p 25.18±0.3 28.18±0.6 33.65±0.4 37.72±0.1 13.73

4q 24.65±0.5 25.83±0.2 32.04±0.6 34.51±0.1 16.59

4r 21.11±0.4 33.33±0.2 30.86±0.5 39.97±0.2 11.30

4s 38.15±0.4 41.80±0.7 43.62±0.2 47.37±0.3 9.93

Control - - - - -

(Ascorbic acid) 16.50±0.2 44.35±0.3 57.22±0.4 66.25±0.3 5.48 aIC50 value represents the concentration of three experiments required to exhibit 50 % antioxidant activity.

Chapter-5

174

Almost all the tested compounds possessed strong scavenging activity against the

DPPH with IC50 values (4.57-19.04µg/mL). Compounds having indole ring 4(a-c)

showed potant antioxidant activity (4.57-6.45 µg/mL). Compound 4b

(IC50=4.57µg/mL) showed the highest antioxidant activity due to existence of –NH

and OH group. Further formyl derivatives having NH2 group on 2th position showed

the high scavenging activity (IC50=5.53µg/mL) after compound 4b whereas methyl

substitution on 6th position does not affect on activity. N-N'dimethyl derivatives 4m

and 4r showed good inhibitory activity with IC50=10.90 and 11.30µg/mL where as

methoxy derivatives showed the less inhibitory activity than other (IC50=16.59-

19.04µg/mL). Rest of the compounds showed significantly good activity against

DPPH.

5.3. EXPERIMENTAL

5.3.1. Materials and methods All the chemicals and reagents were purchased from Merck and Sigma-Aldrich

(India) as ‘synthesis grade’ and used without further purification. The microwave

synthesis was performed in Anton Paar, Monowave 300 microwave synthesizer.

Melting points were determined on a Kofler apparatus and are uncorrected. The

instrumental detail of elemental analysis (C, H, N), IR, NMR and Mass have been

discussed in chapter 2. X-ray diffractograms (XRD) of the catalyst were recorded in

the 2θ range of 20-80° with a scan rate of 41 min-1 on a Shimadzu-6100 X-ray

diffractometer with Ni-filtered Cu Ka radiation at a wavelength of 1.54060 A. The

scanning electron microscope (SEM-EDX) analysis was obtained using a JEOL

(JSM-6510) equipped with an energy dispersive X-ray spectrometer at different

magnification. TGA has been carried out with DTG-60H (Simultaneous DTA-TG

Apparatus), Shimadzu instrument. The progress of the reaction homogeneity of

reaction mixture was monitored by Thin layer chromatography (TLC) glass plates

120×5 cm with silica gel G (Merck) using benzene:acetone (8:2) as mobile phase.

5.3.2. Preparation of the silica-supported zinc bromide (SiO2/ZnBr2) catalyst

Silica gel (70-230 mesh) (10 g) was added to a solution of ZnBr2 (12 mmol, 2.7 g) in

EtOH (50 mL), and the mixture was heated at reflux for 1 h. The solvent was removed

using rotary evaporator, and the product was dried under vacuum at 160 °C for 10 h.38

Chapter-5

175

The other catalyst i.e. SiO2-Cl used for the comparative study has been synthesized

according to the previously published standard procedures.42

5.3.3. General procedure for the synthesis of pyrazolones under microwave

irradiation

A mixture of substituted aromatic/heterocyclic aldehyde 1(a-s) (2 mmol),

phenylhydrazine/2,4-dinitrophenylhydrazine 2(a-b) (2 mmol), ethylacetoacetate (3, 2

mmol), and SiO2/ZnBr2 (0.10 g) in 10 mL water was taken in a G30 vial and

irradiated using microwaves with continuous stirring at 60 °C for 10-15 min. After

completion of reaction (monitored by TLC), the reaction mixture was allowed to cool

at room temperature and diluted with cold water (5 mL). The catalyst was separated

by filtration and the resulting solution was extracted with ethyl acetate (3×10 mL).

The combined organic layer was washed with water, dried over anhydrous Na2SO4

and evaporated under reduced pressure. The crude product was crystallized with

chloroform-methanol to afford the pure product. The recovered catalyst was reused

for subsequent cycles without a significant loss in yield.

5.3.4. Spectral characterization of synthesized compounds 4(a-s)

(Z)-4-((1H-Indol-3-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (4a)

Orange crystalline solid; yield 98%; m.p. 245-250 °C; Analytical cal. C19H15N3O: C,

75.73; H, 5.02; N, 13.94; found: C, 75.70; H, 5.03; N, 13.96.

IR (KBr, νmax cm-1): 1157, 1456 (C=C), 1594 (C=N), 1680 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.39 (s, 3H, -CH3), 7.26-7.39 (m, 4H,

indole ring), 7.52-8.02 (m, 5H, phenyl ring), 7.91 (s, 1H, -CH=C), 9.82 (s, 1H, H-

2indole ring), 12.46 (brs, 1H, -NH, D2O exchangeable), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 12.93 (CH3), 112.17-136.56 (indole ring),

118.40-138.19 (phenyl ring), 136.40 (C-1'), 138.90 (C-2'), 150.44 (-C=N), 162.77

(C=O), MS (ESI) (m/z): 301.12 [M+•].

(Z)-4-((5-Hydroxy-1H-indol-3-yl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-

one (4b)

Yellow crystalline solid; yield 95%; m.p. 256 °C; Analytical cal. C19H15N3O2: C,

71.91; H, 4.76; N, 13.24; found: C, 71.90; H, 4.79; N, 13.22.


indole ring), 7.23-7.98 (m, 5H, phenyl ring), 7.83 (s, 1H, -CH=C), 9.70 (s, 1H, H-

Chapter-5

176

2indole ring), 10.20 (s, 1H, -OH, D2O exchangeable), 11.22 (brs, 1H, -NH, D2O

exchangeable), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 13.15 (CH3), 111.64-137.65 (indole ring),

118.20-140.51 (phenyl ring), 133.44 (C-1'), 143.23 (C-2'), 147.89 (-C=N), 165.63

(C=O),

MS (ESI) (m/z): 317.12 [M+•].

(Z)-3-Methyl-4-((5-methyl-1H-indol-3-yl)methylene)-1-phenyl-1H-pyrazol-5(4H)-

one (4c)

Yellow solid; yield 98%; m.p. 248 °C; Analytical cal. C20H17N3O: C, 76.17; H, 5.43;

N, 13.32; found: C, 76.18; H, 5.40; N, 13.34.


phenyl ring), 6.89-7.27 (m, 3H, indole ring), 7.71 (s, 1H, -CH=C), 9.32 (s, 1H, H-

2indole ring). 12.02 (brs, 1H, -NH, D2O exchangeable), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 14.23 (CH3), 112.64-135.65 (indole ring),

116.24-142.54 (phenyl ring), 133.04 (C-1'), 142.13 (C-2'), 146.87 (-C=N), 164.55

(C=O),

MS (ESI) (m/z): 317.12 [M+•].

(Z)-3-Methyl-4-((4-oxo-4H-chromen-3-yl)methylene)-1-phenyl-1H-pyrazol-5(4H)-

one (4d)

Red crystalline solid; yield 98%; m.p. 222 °C. Analytical cal. C20H14N2O3: C, 72.72;

H, 4.27; N, 8.48; found: C, 72.70; H, 4.30; N, 8.47.

IR (KBr, νmax cm-1): 1153, 1452 (C=C), 1600 (C=N), 1692 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.33 (s, 1H, -CH3). 7.12-7.34 (m, 5H,

phenyl ring), 7.99 (s, 1H, -CH=C), 8.19-7.27 (m, 4H, chromone ring), 10.64 (s, 1H,

H-2γ-pyrone ring), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 14.23 (CH3), 117.59 (C-8), 118.38-142.54

(phenyl ring), 118.54 (C-3), 123.10 (C-4a), 124.56 (C-6), 125.75 (C-5), 134.67 (C-1'),

135.92 (C-7), 137.81 (-C=N), 150.67 (C-2, γ-pyrone ring), 155.47 (C-2'), 161.83 (C-

8b), 163.35 (C=O), 174.29 (C-4, C=Oγ-pyrone ring). MS (ESI) (m/z): 330.10 [M+•].

(Z)-3-Methyl-4-((6-methyl-4-oxo-4H-chromen-3-yl)methylene)-1-phenyl-1H-

pyrazol 5(4H)-one (4e)

Orange solid; yield 95%; m.p. 230 °C; Analytical cal. C21H16N2O3: C, 73.24; H,

Chapter-5

177

4.68; N, 8.13; found: C, 73.26; H, 4.69; N, 8.10.






8b), 163.05 (C=O), 174.19 (C-4, C=Oγ-pyrone ring).

MS (ESI) (m/z): 344.12 [M+•].

(Z)-4-((6-Fluoro-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-1-phenyl-1H-

pyrazol-5(4H)-one (4f)

Red solid; yield 96%; m.p. 214 °C; Analytical cal. C20H13FN2O3: C, 68.96; H, 3.76;

N, 8.04; found: C, 68.97; H, 3.78; N, 8.01.






8b). 163.25 (C=O), 174.69 (C-4, C=Oγ-pyrone ring).

MS (ESI) (m/z): 348.09 [M+•].

(Z)-4-((6-Bromo-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-1-phenyl-1H-

pyrazol-5(4H)-one (4g)

Brown solid; yield 94%; m.p. 248 °C; Analytical cal. C20H13BrN2O3: C, 58.70; H,

3.20; N, 6.85; found: C, 58.71; H, 3.22; N, 6.82.




(phenyl ring), 118.64 (C-3), 120.41(C-6), 123.34 (C-4a), 125.39 (C-5), 133.19 (C-1'),

Chapter-5

178


8b), 163.23 (C=O), 174.47 (C-4, C=Oγ-pyrone ring).

MS (ESI) (m/z): 408.01 [M+•].

(Z)-4-((2-Amino-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-1-phenyl-1H-

pyrazol-5(4H)-one (4h)

Brown solid; yield 95%; m.p. 240 °C; Analytical cal. C20H15N3O3: C, 69.56; H, 4.38;

N, 12.17; found: C, 69.53; H, 4.39; N, 12.19.


phenyl ring), 7.72 (s, 1H, -CH=C), 8.08-7.27 (m, 4H, chromone ring), 8.57 (s, 2H, -

NH, D2O exchangeable), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 14.22 (CH3), 117.26 (C-8), 118.25-142.37

(phenyl ring), 118.61 (C-3), 123.28 (C-4a), 123.41(C-6), 125.33 (C-5), 133.29 (C-1'),

135.16 (C-7), 137.38 (-C=N), 155.34 (C-2'), 161.66 (C-8b), 163.28 (C=O), 170.24 (C-

2, γ-pyrone ring), 174.41 (C-4, C=Oγ-pyrone ring). MS (ESI) (m/z): 345.11 [M+•].

(Z)-4-(3,4-Dimethoxybenzylidene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (4i)

Yellow solid; yield 97%; m.p. 208 °C; Analytical cal. C19H18N2O3: C, 70.79; H,

5.63; N, 8.69; found: C, 70.71; H, 5.60; N, 8.71.

IR (KBr, νmax cm-1): 1157, 1450 (C=C), 1578 (C=N), 1700 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.31 (s, 1H, -CH3), 3.38 (s, 6H, 2×-OCH3),

7.19-7.97 (m, 5H, phenyl ring), 7.28-7.32 (m, 3H, phenyl ring), 7.32 (s, 1H, -CH=C). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 14.62 (CH3), 56.06 (O-CH3), 111.74 (C-

5), 115.23 (C-2), 118.21-142.39 (phenyl ring), 122.25 (C-6), 128.24 (C-1), 133.22 (C-

1'), 137.32 (-C=N), 148.61 (C-3), 149.41 (C-4), 155.14 (C-2'), 163.26 (C=O).

MS (ESI) (m/z): 322.13 [M+•].

(Z)-3-Methyl-1-phenyl-4-(3,4,5-trimethoxybenzylidene)-1Hpyrazol-5(4H)-one (4j)

Yellow crystalline solid; yield 94%; m.p. 214 °C; Analytical cal. C20H20N2O4: C,

68.17; H, 5.72; N, 7.95; found: C, 68.20; H, 5.70; N, 7.94.

IR (KBr, νmax cm-1): 1150, 1456 (C=C), 1600 (C=N), 1691 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.32 (s, 1H, -CH3), 3.34 (s, 9H, 3×-OCH3),

7.12 (s, 2H, phenyl ring), 7.19-7.97 (m, 5H, phenyl ring), 7.35 (s, 1H, -CH=C). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 14.89 (CH3), 56.36 (OCH3), 105.13 (C-2

and C-6), 118.52-142.29 (phenyl ring), 129.24 (C-1), 133.29 (C-1'), 134.34 (C-5),

Chapter-5

179

137.38 (-C=N), 154.11 (C-3 and C-4), 155.39 (C-2'), 163.28 (C=O).

MS (ESI) (m/z): 352.14 [M+•].

(Z)-3-Methyl-4-(4-nitrobenzylidene)-1-phenyl-1H-pyrazol-5(4H)-one (4k)

Orange crystalline solid; yield 97%; m.p. 210 °C; Analytical cal. C17H13N3O3: C,

66.44; H, 4.26; N, 13.67; found: C, 66.41; H, 4.28; N, 13.68.

IR (KBr, νmax cm-1): 1157, 1456 (C=C), 1578 (C=N), 1687 (C=O), 1522, 1365 (NO2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.36 (s, 1H, -CH3), 7.20-7.99 (m, 5H,

phenyl ring), 7.49 (s, 1H, -CH=C), 8.12 (d, 2H, H-2 and H-6), 8.18 (d, 2H, H-3 and

H-5), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 15.09 (CH3), 118.12-142.22 (phenyl ring),

124.18 (C-3 and C-4), 128.21 (C-1'), 132.17 (C-2 and C-6), 139.24 (C-1), 145.69 (C-

2'), 145.88 (-C=N), 148.14 (C-5), 163.25 (C=O).

MS (ESI) (m/z): 307.10 [M+•].

(Z)-3-Methyl-4-(3-nitrobenzylidene)-1-phenyl-1H-pyrazol-5(4H)-one (4l)

Yellow solid; yield 94%; m.p. 202 °C; Analytical cal. C17H13N3O3: C, 66.44; H,

4.26; N, 13.67; found: C, C, 66.41; H, 4.27; N, 13.69.

IR (KBr, νmax cm-1): 1152, 1454 (C=C), 1579 (C=N), 1693 (C=O), 1520, 1360 (NO2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.32 (s, 1H, -CH3), 7.22-7.95 (m, 5H,

phenyl ring), 7.47 (s, 1H, -CH=C), 7.67-8.42 (m, 4H, phenyl ring), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 15.19 (CH3), 118.15-142.32 (phenyl ring),

123.08 (C-4), 123.36 (C-6), 125.17 (C-2), 127.23 (C-1'), 128.18 (C-5), 133.34 (C-1),

144.63 (C-2'), 146.98 (-C=N), 147.08 (C-3), 163.29 (C=O).

MS (ESI) (m/z): 307.10 [M+•].

(Z)-4-(4-(Dimethylamino)benzylidene)-3-methyl-1-phenyl-1Hpyrazol-5(4H)-one

(4m)

Orange crystalline solid; yield 98%; m.p. 194 °C, reported 188-192 °C;43 Analytical

cal. C19H19N3O: C, 74.73; H, 6.27; N, 13.76; found: C, 74.70; H, 6.28; N, 13.78

IR (KBr, νmax cm-1): 1154, 1452 (C=C), 1591 (C=N), 1689 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.30 (s, 1H, -CH3), 3.13 (s, 1H, 2×CH3),

7.13-7.97 (m, 5H, phenyl ring), 6.84-8.66 (m, 4H, phenyl ring), 7.58 (s, 1H, -CH=C). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 15.19 (CH3), 41.28 (N-CH3), 111.32 (C-3

and C-5), 118.09-138.84 (phenyl ring), 123.92 (C-1), 128.69 (C-1'), 137.43 (C-2 and

C-6), 148.18 (C-4), 151.56 (C-2'), 153.80 (-C=N), 170.16 (C=O).

Chapter-5

180

MS (ESI) (m/z): 305.15 [M+•].

(Z)-4-(4-Fluorobenzylidene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (4n)

Yellow solid; yield 96%; m.p. 104 °C, reported 98-102 °C;43 Analytical cal.

C17H13FN2O: C, 72.85; H, 4.67; N, 9.99; found: C, 72.82; H, 4.69; N, 10.00.


phenyl ring), 7.19-7.97 (m, 5H, phenyl ring), 7.42 (s, 1H, -CH=C). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 15.02 (CH3), 115.36 (C-3 and C-5),

118.02-139.81 (phenyl ring), 127.60 (C-1'), 128.91 (C-1), 132.03 (C-2 and C-6),

145.50 (C-2'), 148.85. (-C=N), 162.18 (C-4), 168.12 (C=O). MS (ESI) (m/z): 280.10

[M+•].

(Z)-4-Benzylidene-1-(2,4-dinitrophenyl)-3-methyl-1H-pyrazol-5(4H)-one (4o)

Yellow solid; yield 94%; m.p. 194 °C;44 Analytical cal. C17H12N4O5: C, 57.96; H,

3.43; N, 15.90; found: C, 57.93; H, 3.45; N, 15.91.

IR (KBr, νmax cm-1): 1152, 1450 (C=C), 1528, 1368 (NO2), 1597 (C=N), 1686 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.36 (s, 1H, -CH3), 7.40-7.66 (m, 5H,

phenyl ring), 7.42 (s, 1H, -CH=C), 8.20-9.08 (m, 3H, phenyl ring). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 15.20 (CH3), 111.48 (C-3 and C-5),

120.14-144.09 (phenyl ring), 123.02 (C-1), 126.69 (C-1'), 136.43 (C-2 and C-6),

148.88 (C-4), 150.56 (C-2'), 150.80 (-C=N), 170.12 (C=O).

MS (ESI) (m/z): 352.08 [M+•].

(Z)-1-(2,4-Dinitrophenyl)-3-methyl-4-(3-nitrobenzylidene)-1H-pyrazol-5(4H)-one

(4p)

Yellow crystalline solid; yield 95%; m.p. 274 °C, reported 272-274 °C;45 Analytical


IR (KBr, νmax cm-1): 1152, 1454 (C=C), 1520, 1360 (NO2), 1593 (C=N), 1700 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.34 (s, 1H, -CH3), 7.54 (s, 1H, -CH=C),

7.68-8.30 (m, 4H, phenyl ring), 8.22-9.02 (m, 3H, phenyl ring). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 15.20 (CH3), 120.15-145.32 (phenyl ring),

122.06 (C-6), 123.28 (C-4), 124.17 (C-2), 127.10 (C-1'), 128.28 (C-5), 133.04 (C-1),

144.03 (C-2'), 146.99 (-C=N), 147.88 (C-3), 164.20 (C=O).

MS (ESI) (m/z): 397.30 [M+•].

(Z)-4-(3,4-Dimethoxybenzylidene)-1-(2,4-dinitrophenyl)-3-methyl-1H-pyrazol-

Chapter-5

181

5(4H) one (4q)

Yellow solid; yield 97%; m.p. 224 °C;44 Analytical cal. C19H16N4O7: C, 55.34; H,

3.91; N, 13.59; found: C, 55.31; H, 3.93; N, 13.60.

IR (KBr, νmax cm-1): 1157, 1456 (C=C), 1522, 1362 (NO2), 1594 (C=N), 1688 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.32 (s, 1H, -CH3), 3.36 (s, 6H, 2×-OCH3),

7.20-7.31 (m, 3H, phenyl ring), 7.46 (s, 1H, -CH=C), 8.20-9.04 (m, 3H, phenyl ring). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 14.88 (CH3), 56.02 (OCH3), 111.72 (C-5),

115.53 (C-2), 120.21-144.39 (phenyl ring), 122.25 (C-6), 128.14 (C-1), 130.22 (C-1'),

139.32 (-C=N), 148.68 (C-3), 149.45 (C-4), 150.14 (C-2'), 164.26 (C=O).

MS (ESI) (m/z): 412.10 [M+•].

(Z)-4-(4-(Dimethylamino)benzylidene)-1-(2,4-dinitrophenyl)-3-methyl-1H-pyrazol-

5(4H)-one (4r)

Orange crystalline solid; yield 98%; m.p. 240 °C;44 Analytical cal. C19H17N5O5: C,

57.72; H, 4.33; N, 17.71; found: C, 57.73; H, 4.30; N, 17.73.

IR (KBr, νmax cm-1): 1156, 1452 (C=C), 1526, 1361 (NO2), 1593 (C=N), 1691 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.32 (s, 1H, -CH3), 3.10 (s, 1H, 2×CH3),

6.80-8.46 (m, 4H, phenyl ring), 7.49 (s, 1H, -CH=C), 8.28-9.01 (m, 3H, phenyl ring). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 15.20 (CH3), 41.31 (N-CH3), 111.48 (C-3

and C-5), 120.14-144.09 (phenyl ring), 123.02 (C-1), 126.69 (C-1'), 136.43 (C-2 and

C-6), 148.88 (C-4), 150.56 (C-2'), 150.80 (-C=N), 170.12 (C=O).

MS (ESI) (m/z): 395.12 [M+•].

(Z)-1-(2,4-Dinitrophenyl)-4-(4-fluorobenzylidene)-3-methyl-1H-pyrazol-5(4H)-one

(4s)

Yellow solid; yield 94%; m.p. 162 °C, reported 160-163 °C;45 Analytical cal.

C17H11FN4O5: C, 55.14; H, 2.99; N, 15.13; found: C, 55.15; H, 2.96; N, 15.15.

IR (KBr, νmax cm-1): 1159, 1453 (C=C), 1526, 1360 (NO2), 1598 (C=N), 1698 (C=O). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.36 (s, 1H, -CH3), 7.18-7.80 (m, 4H,

phenyl ring), 7.42 (s, 1H, -CH=C), 7.96-7.98 (m, 3H, phenyl ring). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 15.01 (CH3), 115.31 (C-3 and C-5),

120.23-145.84 (phenyl ring), 127.60 (C-1), 128.91 (C-1'), 132.00 (C-2 and C-6),

145.50 (C-2'), 148.85 (-C=N), 162.18 (C-4), 169.10 (C=O).

MS (ESI) (m/z): 370.07 [M+•].

5.3.5. Single crystal X-ray crystallographic studies of compound 4a and 4d

Chapter-5

182

The crystal structure of compounds 4a and 4d were determined by X-ray diffraction

experiments performed on a Bruker Apex II diffractometer. The instrumentation

detail and procedure have been discussed in chapter 2. Pertinent crystallographic data

for compounds 4a and 4d have been summarized in Table 1. The crystal data have

been deposited at the Cambridge Crystallographic Data Centre (CCDC) with

reference number, compound 4a with CCDC 1409997 and compound 4d with CCDC

1432605.



diphenylpicrylhydrazyl (DPPH) method.46 The general procedure for this assay is

discussed in chapter 2. In this procedure, drug stock solution (1 mg/mL) was diluted

to final concentration of 2, 4, 6, and 8µg/mL in methanol.

5.4. CONCLUSION

This chapter reports a convenient, eco-friendly and sustainable approach for the one-

pot synthesis of a series of pyrazolone derivatives 4(a-s) in excellent yields (94-98%)

by employing recyclable and reusable SiO2/ZnBr2 Lewis acid catalyst in water under

microwave heating. The scheme not only offers use of microwave at low temperature

and substantial yield of products but also affords mild reaction conditions, water as a

green solvent, shorter reaction times, high purity, operational simplicity and easy

workup. This green synthetic procedure eliminates the use of toxic solvents and thus

makes it attractive one in organic synthesis. All the compounds displayed moderate to

good antioxidant activity. The results validate that the nature of substituent attached to

the heterocyclic moieties is important for biological activity. Thus, present synthetic

approach provides a better scope for the synthesis of pyrazolone analogues and will

be a more practical alternative to the other existing methods.

Chapter-5

183

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Chapter-6

185

6.1. INTRODUCTION

Carbon-carbon (C-C) bond formation reactions are of significant importance in

organic synthesis as they unlock new pathways to new chemical entities.1-3 The

Knoevenagel condensation between aldehydes and ketones with activated methylene

compounds is one of the reactions which facilitates C-C bond formation4,5 and has

been exploited in the synthesis of some vital drugs such as entacapone6 pioglitazone7

and lumefantrine.8 The use of ammonia, amines, pyridine, piperidine and their salts as

basic catalysts in Knoevenagel condensation restricts their applications due to their

hazardous environmental concerns, carcinogenic nature, problem in product

separation and reusability.9 Therefore there is a need for exploring the cheap and

easily available catalysts for Knoevenagel condensation. Over the past few decades

there has been an upsurge in the number of biologically active compounds that

contain an acrylonitrile moiety. Acrylonitrile derivatives constitute an important class

of compounds in organic chemistry due to their promising biological activities such as

antiproliferative,10 antifungal,11 antitumor,12 antibacterial,13 antitubercular,14

antioxidative,15,16 tuberculostatic,17 antitrichomonal18 and antiparasitic.19 Various

medicinally important drugs also possess acrylonitrile moiety in their structural

framework viz. DG172,20 Rilpivirine (Edurant),21 Entacapone,6 Cyenopyrafen22 and

Dynole 34-2.23

The synthesis of acrylonitrile compounds have been previously achieved by the

use of Wittig reactions,24 McMurry coupling25 and the Heck reaction.26 It is pertinent

to mention here that Knoevenagel condensation is a facile and versatile method for

the formation of acrylonitrile derivatives.27-29 In the quest to achieve higher efficiency

several catalysts have been employed viz Bronsted acid catalysts,30 Lewis acids such

as MgBr2·OEt2,31 SnCl2,32 Al2O3

33 ionic liquids [C6-mim] PF6,34

[Bmim]Cl·xAlCl3,35 [Bpy]Cl·xAlCl3,35 [Bmim]BF4,

36 ethylammonium nitrate

(EAN)37 and silica supported acid catalyst.38,39

Several sophisticated methodologies have been postulated in the literature for

the synthesis of acrylonitrile moiety. However, in recent years organic chemists have

been developed various new methodologies for the efficient synthesis of acrylonitrile

derivatives. Some of them have been discussed below.

Trilleras et al.40 developed new protocol for the synthesis of a series of (E)-2-

(benzo[d]thiazol-2-yl)-3-arylacrylonitriles 3(a-j) by microwave assisted Knoevenagel

Chapter-6

186

condensation under solvent-free conditions from the corresponding 2-

(benzo[d]thiazol-2-yl)acetonitrile (1) and aromatic aldehydes 2(a-j).

S

N CN

Ar H

O

S

N CN

ArMW, 150 W

Solvent-free20-30 min

1 2(a-j) 3(a-j)

Ar= C6H5; 4-CH3C6H5; 4-NO2C6H5; 4-ClC6H5; 4-BrC6H5; 4-FC6H5; 4-CF3C6H5;

4-OCH3C6H5; 3,4,5(OCH3)3C6H2; 3,4-OCH2OC6H3

Quiroga et al.41 outlined the synthesis of two different thiophene substituted

arylacrylonitriles derivatives 3(a-k) and 6(a-e) by the reaction of 2-thienylacetonitrile

(1) and 3-thienylacetonitrile (4) with aromatic aldehydes 2(a-k) and 5(a-e)

respectively, in the presence of potassium tert-butoxide in ethanol.

SCN

CHO

R S

NC

H

R

1 2(a-k) 3(a-k)

potassium tert-butoxideEtOH, Stirring, RT

R= 4-Cl, H, 4-F, 4-OCH3, 4-Br, 4-CH3, 4-CF3, 3,4-OCH2O, 2-F, 3,4,5-(OCH3)3, 2-

CF3

S

CHO

RS

NC

H

R

CN

4 5(a-e) 6(a-e)

potassium tert-butoxide

EtOH, Stirring, RT

R= 4-Cl, H, 4-F, 4-OCH3, 4-Br

Khan et al.42 reported an efficient synthesis of acrylonitrile derivative in

presence of hydrotalcite catalyst in ionic liquid medium at room temperature.

Chapter-6

187

Ar H

O CN

Y

CN

YH

ArWith or without catalyst

Ionic liquid, RT

Catalyst= Mg:Al (3:1) hydrotalcites; Ionic liquid= [bmim]BF4, [bmim]PF6

Y= CN, COOEt

Ar= Ph; 4-NO2C6H4; 4-OHC6H4; 3-BrC6H4; 3,4,5-(OMe)3C6H2; 4-ClC6H4; 4-

PhC6H4; C6H13; Me

Parveen et al.43 reported stereoselective synthesis of Z-acrylonitrile derivatives

using SiO2-Cl as catalyst via a facile Knoevenagel condensation between p-

nitrophenylacetonitrile and appropriately substituted aromatic aldehydes 1(a-i) and 3-

formyl chromones 1(a-e).

Ar H

OCN

CN

H

Ar

NO2

NO2

SiO2-Cl (2.5 mol%)EtOH, Stirring(RT)

1(a-i) 2 3(a-i)

Ar= 3,4,5-(OMe)3C6H2; 2,3-(OMe)2C6H3; 3,5-(OMe)2C6H3; 3,4-(OMe)2C6H3; 3-

NO2C6H4; 4-N(CH3)2C5H4; 3-OMe,4-OHC6H3; 3,4,5-(OMe)3C6H2; 3,5-

(OH)2C6H3

CN

NO2

SiO2-Cl (2.5 mol%)EtOH, Stirring(RT)O

OCHO

R2

R1

O

O

R2

R1

H

CN

NO2

1(a-e) 2 3(a-e)R1=R2= H; R1= H, R2= Br; R1= NH2, R2= H

Hranjec et al.44 synthesize a series of novel 2-benzimidazolyl substituted

acrylonitriles by condensation of 2-cyanomethylbenzimidazole (1) with

corresponding aromatic/heteroaromatic aldehydes 2(a-m) in absolute ethanol by

adding a few drops of piperidine.

N

HN CN

Ar H

O

N

HN CN

ArH

Piperidine, EtOHReflux, 2-4h

1 2(a-m) 3(a-m)

Chapter-6

188

Ar= 4-CNC6H4; 4-CH3C6H4; 4-FC6H4; 4-BrC6H4; 3,5-(OMe)2C6H3; 4-NHCOCH3;

C6H4N; 4-N(CH3)2; 3-OH,4-N(CH2CH3)2C6H3; C4H3O; C4H3S; C4H3N-CH3;

C3H3N2

Torre et al.45 reported the synthesis of a new series of (E)-2-(benzo[d]thiazole-

2-yl)-3-heteroarylacrylonitriles 3(a-k) by the reaction between 2-(benzo[d]thiazol-2-

yl)acetonitrile (1) and different substituted heteroaromatic aldehydes 2(a-k) using

ethanol as solvent and catalytic amounts of triethylamine (TEA).

S

N CN

Ar H

O

S

N CN

HAr

TEA, EtOHStirring, RT

1 2(a-k) 3(a-k)

ONH3C O

NO

NF

NO

NNH

NO

Cl

NN CH3

Cl

O N

2a 2b 2c 2d 2e

2f 2g 2h 2i 2j 2k

Mogilaiah et al.46 developed an efficient and simple Knoevenagel condensation

catalyzed by NH2SO3NH4 in solvent free condition under microwave irradiation for

the synthesis of acrylonitrile derivatives in excellent yield.

Ar H

O CN

RNH2SO3NH4

MW, 400W H

Ar CN

R1 2 3

R= CN, COOC2H5, CONH2

Ar= C6H5, 4-CH3C6H4, 4-OCH3C6H4, 4-ClC6H4, 4-NO2C6H4, 4-OHC6H5, 4-OH,3-

OCH3C6H3, 3,4-(OCH3)2C6H3, 3,4-(O-CH2-O)C6H3, 3-NO2C6H4, C5H3O

Chapter-6

189

Ali et al.47 outlined the synthesis of (Z)-2-phenyl-3-(1H-pyrrol-2-

yl)acrylonitriles derivatives (3) by the reaction of various 1H-pyrrole-2-

carboxaldehydes (1) with phenylacetonitrile derivatives (2) in either ethanol/water or

in presence of 18-crown-6 in toluene and KOH using benzyltrimethylammonium

hydroxide as a catalyst.

NH

H

O

CNNH

NCR1

R2

R1

R2

PhCH2NMe3(OH)EtOH/H2O

or18-Crown-6

KOH, PhCH31 2 3

R1= H, 4-Br, 4,5-Cl2

R2= H, 2-F, 2-Cl, 2-Br, 2-CF3, 2-Me, 2-Ph, 2-OMe, 2-CN, 3-F, 3-Cl, 3-Br, 3-CF3, 3-

Me, 3-OPh, 4-F, 4-Cl, 4-Br, 4-I, 4-CF3, 4-Me, 4-Et, 4-OMe, 4-CN, 4-NO2, 4-i-Pr, 4-t-

Bu, 4-Ph, 4-Cl, 2,4-Cl2, 3,4-Cl2, 2,4-F2, 3,5-(CF3)2, 2,4-Me2

In recent years, immobilization of catalysts on solid support, leads to clean

chemical synthesis for environmental as well as economical point of view.48,49

Heterogeneous catalysts have gained much attraction of chemist due to economic and

environmental importance.50 Due to the favorable chemical and physical properties of

silica surfaces, it is possible to impart any reactive functional group e.g., sulfonic,

amine, carboxyl and thiol etc. on silica surfaces through well-known silane

chemistry.51,52 Therefore, use of supported and recoverable catalysts in organic

transformations has economical and environmental benefits.53 In this regards, carbon

based solid acid catalysts have attracted much more attention due to more activity and

selectivity.54-56 These sulfonated silica/carbon hybrids have been prepared from the

sulfonation of carbonized biomaterials have been extensively used in organic

synthesis.57-59 In light of these observations, A novel silica bonded N-

(propylcarbamoyl)sulfamic acid (SBPCSA) has been synthesized as a green, efficient

and recyclable catalyst for the synthesis of acrylonitrile derivatives 3(a-o).




Chapter-6

190

(propylcarbamoyl)sulfamic acid (SBPCSA) under solvent free conditions. Further all

synthesized compounds were tested for antioxidant activity and results obtained were

promising.

6.2.1. Characterization of the Catalyst

The catalyst SBPCSA was prepared by the concise route outlined in Scheme 1. The

characterization and morphology of the catalyst was established on the basis of FT-

IR, powder X-ray diffraction (XRD), scanning electron microscopy (SEM) and

energy-dispersive X-ray spectroscopy (EDX). The stability of catalyst was shown by

TGA-DTA analysis.

OHOH

OH

OH

OH

(MeO)3Si ClToluene, Reflux, 48 h

OO

O

OH

Si Cl OO

O

OH

Si NH

NH2

O

OO

O

OH

Si NH

NH

O

S

O

OHO

Dry CH2Cl2

ClSO3H

Room temp., 2 h

SiO2

SiO2 SiO2

SiO2

SBPCSA

NH2CONH2

Ethanol, Reflux, 8h

Scheme 1 Synthetic scheme for the formation of catalyst SBPCSA

The FT-IR spectrum of the catalyst (SBPCSA) is depicted in Fig. 1. The FT-IR

spectrum displayed peaks at 3152 cm-1 attributed to the presence of the N-H bond

stretching frequency of sulphonamide groups, which has been overlapped with O-H

stretching frequency of silanol group or the adsorbed water molecules.60 The

Chapter-6

191

corresponding C=O stretching and bending frequency of N-H were observed at 1717

and 1575 cm-1, respectively. The peak at 1401.96 cm-1 is assigned for C-H bending.

Further, the peak at about 797 and 1112 cm-1 is attributed to the typical symmetric

stretching vibration of Si-O-Si and S=O bond of supported SO3H group, respectively.

Moreover stretching vibration of S-N bond in the –HN-SO3H is observed at 767 cm-

1.61

Fig. 1 FT-IR spectrum of the catalyst SBPCSA

To study the surface morphology of the catalyst, SEM micrographs of the

catalyst was employed (Fig. 2). The SEM result showed the adsorption of N-

(propylcarbamoyl) sulfamic acid on silica surfaces. The composite is a fine

homogeneous powder in which particles were of uneven size and shape, well

dispersed on the surface of the silica gel. The successful incorporation of urea and

SO3H groups was also confirmed by EDX analysis (Fig. 3) of the catalyst, which

showed the presence of N, S, O and Si elements signifying the formation of expected

catalytic system. In the fresh catalyst S is present by weight% 13.38 and atomic%

6.79 while N is present by weight% 22.37 and atomic% 26.00. Further elemental

mapping of the catalyst showed the uniform distribution of elements present in the

catalyst (Fig. 4).

Chapter-6

192

(a)

(b) Fig. 2 SEM analysis of (a) SiO2 (b) catalyst SBPCSA

Chapter-6

193

Fig. 3 EDX analysis of catalyst SBPCSA

(a) (b)

(c) (d)

(e)

Fig. 4 Elemental mapping of catalyst SBPCSA (a) carbon (b) nitrogen (c) oxygen (d)

silicon (e) sulfur showing uniform distribution of catalyst surface

The chemical modification of silica support with N-(propylcarbamoyl)sulfamic

acid, was further explored by powder XRD analysis (Fig. 5). The XRD pattern of the

catalyst was recorded in the 2θ range of 10°-80°. The XRD of the catalyst showed

characteristic single broad peak of silica in the range of 2θ=18°-25° displaying

Chapter-6

194

amorphous nature of silica. However, the XRD of SPBCSA also showed crystallinity

with the characteristic peaks of urea.62

Fig. 5 XRD analysis of catalyst SBPCSA

The thermal stability of the catalyst was evaluated by TG analysis (Fig. 6). The

TG curve showed first weight loss of 62.84% around 336 °C which can be attributed

to the decomposition of amide chain along with SO3H group and physically adsorbed

water molecules. Further another weight loss of 9.1% at 442 °C can be attributed to

the loss of Si group covalently bonded to silica surface. Hence it can be concluded

that catalyst is stable up to 336 °C. TGA is further supported by DT analysis (Fig. 6)

in which a prominent peak at 297 and 365 °C showed endothermic reaction which

help in the removal of water, SO3H group, amide moiety, Si group.

10 20 30 40 50 60 70 800

500

1000

1500

2000

2500

3000

Inten

sity (

a.u.)

2θ (degree)

Chapter-6

195

Fig. 6 TGA/DTA analysis of catalyst SBPCSA

The optimum concentration of H+ ion was determined by acid-base titration of

the aqueous suspension of the weighed amount of thoroughly washed catalyst with

standard NaOH (0.01 N) solution and was found to be 0.49 meq/g of the support. The

concentration of the residual H+ ion of the recovered catalyst was also measured and

found to be small loss of H+ ion. It signified that SO3H moiety was tightly anchored

with SPBCSA, possibly due to a covalent linkage.

6.2.2. Chemistry

The synthetic pathways of a series of new acrylonitrile derivatives 3(a-o) have been

shown in Scheme 2. Herein, each series was typically accessed via Knoevenagel

condensation between 2-thiopheneacetinitrile (2) and appropriately substituted

heterocyclic/aromatic aldehydes 1(a-o) to yield target acrylonitrile derivatives in

excellent yields (92-98%) with high purity.

SCN

CN

S

Solvent f ree,

SBPCSA (80 mg)

80 oC

H

20-30 min

O

HAr Ar

1 (a-o) 3 (a-o)2

O

O

O

OH3C

O

OF

O

OBr

O

O

NH2

NNO2

O2NH3C

CH3 OCH3

H3CO

H3CO

OCH3

H3CO

HO

HOOH

OH NH N

HNH

H3C HO

1a 1b 1c 1d 1e

1f 1g 1h 1j 1j

1k 1l 1m 1n 1o

Scheme 2 Synthetic scheme for the formation of acrylonitrile derivatives

The structural elucidation of the synthesized compounds 3(a-o) have been established

on the basis of elemental analysis (C, H, N), IR, 1H NMR, 13C NMR and mass

Chapter-6

196

spectral analysis. The results for C, H and N analysis were within ±0.3% of the

theoretical values. The spectral analysis has been in good corroboration with the

expected structural framework of the synthesized compounds. All the synthesized

compounds showed the absence of the carbonyl peak in the IR spectra, which

confirmed the reaction at the carbonyl moiety. Moreover, all compounds exhibited a

characteristic peak for cyano group resonating at around 2212-2220 cm-1. Other

characteristic peaks for the different functional groups such as C=Oγ-pyrone, OH and

NO2 have been discussed in experimental section. The 1H NMR spectra of each

compound displayed a sharp singlet at around δ 7.40-8.21 ascribed to the olefinic

proton (H-1''). Similarly, sharp singlets resonating at around δ 7.25, 7.01, 7.28, 7.23

each integrating for one proton has been attributed to H-2 protons of γ-pyrone ring of

chromones 3a, 3b, 3c and 3d, respectively. Doublet at around δ 7.12-7.39 and 7.26-

7.78 have been attributed to H-3' and H-5' protons of thiophene ring and a multiplet at

around δ 7.13-7.39 has been attributed to H-4' proton of thiophene ring. In 13C NMR

spectra, peaks resonating at around δ 126.00-137.73 have been ascribed to carbons of

thiophene ring. Similarly, peaks resonating at around δ 117.38-119.70 corresponds to

-C≡N moiety and the signals at δ 178.50, 177.82, 177.09, 177.87 and 178.10 have

been attributed to carbonyl group (C=Oγ-pyrone) of compounds 3a, 3b, 3c, 3d and 3e,

respectively. Furthermore peaks at δ 107.20-115.49 and 152.12-153.90 corresponds to

C-1" and C-2", respectively. The mass spectral analysis of the synthesized compounds

was also in good conformity with the proposed structures.

The FT-IR spectrum of compound 3f (Fig. 7) displayed characteristics peaks at

2218 cm-1 attributed to C≡N. Peak resonating at 1518, 1320 cm-1 attributed to NO2.

Other peaks resonating at 1600, 1562 and 1440 cm-1 attributed to C=C. The 1H NMR

spectrum of compound 3f (Fig. 8) displayed a sharp singlet at δ 7.67 for one protons

ascribed to the =C-H proton. Two doublets at around δ 7.47 and 7.48 for one proton

each have been attributed to H-4 and H-6, respectively. A sharp singlet at δ 8.56 for

one proton has been ascribed to H-2 proton. Further doublet at around δ 7.12 and 7.26

have been attributed to H-3' and H-5' protons of thiophene ring and triplet at around δ

7.39 has been attributed to H-4' proton of thiophene ring. 13C NMR spectrum of

compound 3f (Fig. 9) showed a series of signals resonating at around δ 136.51,

122.12, 147.35, 124.82, 129.26 and 136.11 have been assigned to aromatic carbons.

Similarly, peaks resonating at around δ 135.21, 126.72, 128.90, and 130.18 have been

ascribed to carbons of thiophene ring. Furthermore, peak resonating at around δ

Chapter-6

197

118.34 corresponds to -C≡N moiety, peak at δ 107.68 and 152.44 corresponds to C-1"

and C-2", respectively.

Fig. 7 FT-IR spectrum of compound 3f

Fig. 8 1H NMR spectrum of compound 3f

Chapter-6

198

Fig. 9 13C NMR spectrum of compound 3f

In the present study, it was not possible to confirm geometry across C=C on

the basis of 1H NMR analysis. In order to gain some insight into the influence of the

electronic interactions on the molecular geometry, quantum mechanical calculations

of the equilibrium geometry of the free molecule have been performed. For this

purpose we choose compound 3f for the DFT study. Of the two possible geometrical

isomers (E/Z) of the compound 3f, E-isomer was obtained as the sole product. To

probe the relative stability of the two possible isomers, MM2 energy-minimization

calculations were performed. It was found that the E-isomer is stabilized by 12.53

kcal/mol of energy than Z-isomer and this energy difference is satisfactory enough to

suggest that during the crystallization process, the E-isomer gets exclusively

crystallized out from the solution. The ground state optimized structure of E-isomer

interpreted by DFT is shown in Fig. 10, wherein the thiophene part of the molecule is

planar with the acrylic moiety, while the nitrophenyl group is pushed out of the plane,

probably by the electronic interactions between the two lone pairs of sulfur atom of

thiophene ring and hydrogen atom (H23) of nitrophenyl ring (:S:–H23). The

optimized geometry exhibits the E-configuration of the thiophene and nitrophenyl

group about the acrylic double bond (Fig. 10).

Chapter-6

199

Fig. 10 Ground state optimized structure of (a) (E)-isomer and (b) (Z)-isomer of compound 3f

The energies of highest occupied molecular orbital (EHOMO) and lowest

unoccupied molecular orbital (ELUMO) and their distributions using DFT-B3LYP/6-

311G were calculated using grid based density functional theory (DFT) at the

B3LYP/6-311G basis set level. The calculated energies of HOMO and LUMO were

found to be -11.89 and -4.36 eV, respectively, for the optimized E-isomer. The

energies of HOMO and LUMO, including neighboring orbitals, were all negative,

indicating that E-isomer is stable. It was observed that the highest occupied molecular

orbitals in E-isomer is located in the thiophene ring and acrylic moiety while lowest

unoccupied molecular orbitals is mainly localized on the nitro phenyl ring (Fig. 11).

Fig. 11 Electron density distribution in (a) HOMO and (b) LUMO of (E)-isomer of

compound 3f

E-isomer selectivity in the present protocol was further verified on the basis of

dreiding energy concept calculated by using ChemAxon, E and Z-isomers were found

to have 63.70 and 92.21 kcal/- mol of dreiding energy, respectively. This energy

difference 29.15 kcal/mol is favorable for the selective formation of E-isomer (Fig.

(a) (b)

(a) (b)

Chapter-6

200

12). On the basis of these results it was believed that all the synthesized compounds

possess E configuration.

Fig. 12 Dreiding models of the E and Z-geometrical isomers of the compound 3f


To obtain the best reaction conditions for the present protocol effect of solvent,

amount of catalyst and temperature of reaction have been investigated on the model

reaction using 3-nitrobenzaldehyde (1f) and 2-thiopheneacetonitrile (2) in the

presence of SBPCSA to establish the best possible reaction conditions for the

synthesis of acrylonitrile 3f. Initially, the model reaction was performed in organic

solvents like MeOH and EtOH (Table 1, entries 1-2) a moderate yield of the product

70 and 68%, respectively, was obtained after a stretched time period (6-8h). This

moderate yield can be attributed to the nucleophilic nature of these solvents, which

may force the reaction to undergo nucleophilic competition between these solvents

(MeOH, EtOH) and active methylene (2-thiopheneacetonitrile) for electrophilic

carbon of carbonyl group that will eventually results in lower yield of the desired

product (3f), whereas in water (Table 1, entry 3) the product was obtained in 66%

yield after refluxing for 3 h. This apparent drop in reaction time in water is believed to

be due to the hydrogen bonding between the water and carbonyl group of the

substrate, which may stabilizes the intermediate structure during the formation of

product. In CH3COOH, the yield of the desired product increased significantly (75%)

with reduced reaction time (Table 1, entry 4). This enhancement in yield in

CH3COOH is probably due to its ability to trigger the carbonyl group of the reactants

by electromeric effect, thereby enhancing the electrophilicity of carbon, rendering it

Chapter-6

201

more feasible for nucleophilic attack by the active methylene 2-thiopheneacetonitrile.

However, when the reaction was carried out in non-coordinating solvents like CH2Cl2

and DMF no further significant increase in the yield of the product was observed in

comparison to CH3COOH after a stretched reaction time (Table 1, entries 5 and 6).

Moreover, when the model reaction was explored in solvent-free condition, there was

remarkable increase in the yield of the product (98%) with prominent fall in reaction

time (20 min) (Table 1, entry 7). It is evident from the result that nature of solvent

plays a crucial role in the degree of selectivity of the desired product (3f). In view of

condition is the best condition for the synthesis of acrylonitrile derivatives.

Table 1 Effect of different solvents on model reaction

Entry Solvent Temp (oC) Time (hrs)a Yield (%)b 1 MeOH Reflux 6 70 2 EtOH Reflux 8 68 3 Water Reflux 3 66 4 CH3COOH Reflux 3 75 5 CH2Cl2 Reflux 6.5 65 6 DMF Reflux 7 63 7 Solvent-free Reflux 20c 98

Reaction condition: 3-nitrobenzaldehyde (1f, 2 mmol), 2-thiopheneacetonitrile (2, 2 mmol), different solvents (20 mL), SBPCSA (80 mg), 80 oC. aReaction progress monitored by TLC; bIsolated yield of products. cReaction progress monitored by TLC(entry 7, min).

To achieve the optimum concentration of the catalyst for the present protocol,

the model reaction was investigated at different concentrations i.e., 10, 20, 40, 60, 80

and 100 mg (Table 2) of the catalyst SBPCSA at 80 °C under solvent-free condition

and the results were noted in terms of reaction time and yield. It was inferred from

Table 2 that with every subsequent increase in concentration of catalyst from 10-80

mg, there has been a noteworthy improvement in the yield from 65 to 98%, with

prominent drop in reaction time from 120-20 min (entries 1-5, Table 2). However,

further increase in the concentration of the catalyst (80 mg) did not have any

significant effect on the reaction. Thus, it can be concluded from the above results that

80 mg of the catalyst is adequate to gain the optimum yield in the shortest reaction

time under neat conditions at 80 °C, therefore 80 mg of the catalyst was selected for

further studies.

Chapter-6

202

Table 2 Effect of catalyst loading on model reaction

Entry Catalyst (mg) Time (min)a Yield (%)b

1 10 120 65 2 20 80 72 3 40 55 79 4 60 40 84 5 80 20 98 6 100 20 98

Reaction condition: 3-nitrobenzaldehyde (1f, 2 mmol), 2-thiopheneacetonitrile (2, 2 mmol), solvent free, SBPCSA (10-100 mg), 80 oC. aReaction progress monitored by TLC, bIsolated yield of products.

To optimize the reaction temperature, the model reaction was carried out at

different temperatures in presence of catalyst SBPCSA (Table 3). The reaction was

initially tested at room temperature and does not give satisfactory yield of desired

product 3f (Table 3, entry 1). It was observed that the increase in temperature from

25 to 80 °C, has a noteworthy effect on the model reaction with increase in the yield

of desired product 3f (66-98%) with the prominent decrease in the reaction time

(Table 3, entry 1-5). However, a further increase in the temperature from 80 to 100

°C did not show any further increase in the yield of the product 3f (Table 3, entry 6).

Thus, keeping in view the above optimized reaction conditions; 80 °C was preferred

as the optimal temperature for all the reactions in the presence of 80 mg of SBPCSA

under solvent-free conditions.

Table 3 Effect of temperature on model reaction Entry Temperature (oC) Time (hrs)a Yield (%)b

1 Room Temp 3.0 66 2 40 2.5 72 3 55 2.0 85 4 70 1.0 92 5 80 20c 98 6 100 20c 98

Reaction condition: 3-nitrobenzaldehyde (1f, 2 mmol), 2-thiopheneacetonitrile (2, 2 mmol), solvent free, SBPCSA (80 mg), 25-100 oC. aReaction progress monitored by TLC; bIsolated yield of products. cReaction progress monitored by TLC (entry 5-6, minutes).

Chapter-6

203

A comparative study of a variety of catalysts was conducted to probe the

superiority of SBPCSA for the present protocol (Table 4). The model reaction was

first investigated without catalyst and it was found that the reaction took prolonged

reaction time with trace of the yield. Further the reaction was tested with blank-SiO2,

SiO2-NH4SO4 and SiO2-Cl, moderate yield of products were obtain with prolonged

time period (Table 4, entries 2-4). When the reaction was carried out in presence of

SiO2-H2SO4 and SiO2-HClO4 there was an increase in the yield of product with

decrease in the reaction time (Table 4, entries 5, 6). To obtain better yield in less

reaction time we further use sulfamic acid and SBNPU to catalyze the reaction and

found better results in term of yield and time (Table 4, entries 7, 8). Moreover, when

the model reaction was probed with SBPCSA the yield of product (3f) increased

exponentially (98%) with a prominent dip in reaction time (20 min).

Table 4 Comparison of the efficiency of SBPCSA with different catalysts on the model reaction

Entry Catalyst Time h/(min)a Yield (%)b 1 --- 300 traces 2 SiO2 240 40 3 SiO2-NH4SO4 80 74 4 SiO2-Cl 100 70 5 SiO2-H2SO4 65 76 6 SiO2-HClO4 48 81 7 NH2SO3H 55 89 8 SBNPU 60 83 9 SBPCSA 20 98

Reaction condition: 3-nitrobenzaldehyde (1f, 2 mmol), 2-thiopheneacetonitrile (2, 2 mmol), solvent free, different catalysts (80 mg), 80oC. aReaction progress monitored by TLC, bIsolated yield of products. Using these optimized reaction conditions as discussed above, the efficacy of this

approach was explored for a wide variety of heterocyclic/aromatic aldehydes

possessing electron-withdrawing and electron-donating groups for the synthesis of

acrylonitrile derivatives in excellent yields (92-98%) (Table 5). In the present study a

comparative study has also been carried out for the present protocol with the

conventional Knoevenagel condensation by carrying out the reaction of aldehydes

with 2-thiopheneacetonitrile under reflux in ethanol in the presence of 5 mol% of

piperidine. It was observed that the reaction took prolonged reaction time (6-8h) for

completion with moderate yield (60-72%). The results revealed that employing

Chapter-6

204

SBPCSA in the present protocol enhances selectivity and product conversion thus,

proves beneficial over the conventional method for the synthesis of acrylonitrile in

terms of yield and reaction time.

Table 5 Synthesis of acrylonitrile derivatives 3(a-o)

Product

Structure

Reaction in presence of piperidinea

Time Yield (hrs) (%)

Reaction in presence of

catalystb Time Yield (min) (%)

3a O

O H

CN

S

12

345

6

78

1''

1'2'

3'4'

5'

2''4a

8a

6.0 69

20 96

3b

O

O H

CN

SH3C

7.5 72

24 94

3c

O

O H

CN

SF

6.0 70

20 98

3d

O

O H

CN

SBr

7.0 70

22 96

3e

O

O H

CN

S

NH2

7.5 68

25 94

3f

H

CN

S

NO2

1

23

4

56

1'

2'3'

4'

5'

1'' 2''

6.5 70

20 98

3g

H

CN

S

O2N

6.0 72

20 96

Chapter-6

205

3h NH3C

CH3

H

CN

S

7.5 67

25 98

3i

H

CN

S

H3COOCH3

8.0 60

26 98

3j

H

CN

S

OCH3

H3CO

H3CO

8.0 68

30 95

3k

H

CN

S

OHHO

HO

8.0 62

26 92

3l

H

CN

S

OH

7.0 60

30 94

3m

NH

HCN

S

1 23

456

1'

2'3'

4'5'

1''2''

7 7a

3a

6.0 65

20 98

3n

NH

H3C

HCN

S

6.5 62

22 94

Chapter-6

206

3o

NH

HO

HCN

S

7.0 68

24 95

aReaction conditions: Differently substituted heterocyclic/aromatic aldehydes 1(a-o) with 2-thiopheneacetonitrile (2) under reflux in ethanol in the presence of piperidine (5 mol%) bReaction conditions: Differently substituted heterocyclic/aromatic aldehydes 1(a-o) with 2-thiopheneacetonitrile (2) in presence of SBPCSA (80 mg) at 80 °C 6.2.4. Recyclability of the Catalyst

The reusability of the catalyst was also explored for the selected model reaction in

order to reduce the cost of the process. After completion of the reaction, catalyst was

removed by simple filtration, washed with ethanol and dried under vacuum at 80 °C

for 6h and was further tested up to four more reaction cycles (Fig. 13). Recycling and

reuse of the catalyst showed minimal decreases in yields. Although the reaction rate

get decreased gradually with repetition of the reaction cycle, however we succeeded

in obtaining the desired product 3f in satisfactory yield (85%) even after 5th repetition

of the model reaction without any addition of the fresh catalyst. To ascertain the

variation in morphological features of the recovered catalyst, we carry out its SEM-

EDX analysis (Fig. 14). It was observed that the composition of the catalytic system

was almost consistent with the fresh catalyst and also there was no significant change

observed in the morphology of the catalyst as compared to the fresh catalyst.

Chapter-6

207

Fig. 13 Recyclability of the catalyst SBPCSA for the model reaction

Fig. 14 SEM and EDX analysis of recovered catalyst SBPCSA

(a)

(b)

Chapter-6

208

6.2.5. Reaction Mechanism

A plausible mechanistic pathway is proposed in Scheme 3, to illustrate the synthesis

of acrylonitrile derivatives catalyzed by SBPCSA. The initial step is assumed to be

the protonation of formyl group (-CHO) of substrates (I) by protic SBPCSA catalyst

to form intermediate (II), which facilitates the nucleophilic attack of 2-

thiopheneacetonitrile to promote the formation of C-C bond to yield intermediate

(III). The subsequent elimination of H2O molecule from intermediate (III) promoted

by catalyst SBPCSA eventually yielded target compound followed by regeneration of

the catalyst.

H

O

S

CN

SH

H2O

Catalytic Cycle

SC

N

(I)

H

OH

C

H

N H

OH

H

SH

CN

H

OH

(II)(III)

Ar

ArAr

Ar

Acrylonitrile

Condensation Ar

SO

OOHSiO2

SBPCSA

O SO

OSiO2

SO

OOSiO2

OO

O

OH

Si NH

NH

OSO

OHO

SiO2

O SO

OSiO2

HH

Scheme 3 Plausible mechanistic pathway for the synthesis of acrylonitrile derivatives

Chapter-6

209


The antioxidant activities of all the synthesized acrylonitrile derivatives 3(a-o) were

investigated by DPPH scavenging activity. The reduction capability of DPPH radicals

was determined by a decrease in their absorbance at 517 nm induced by antioxidants

using ascorbic acid as a reference. The potencies for the antioxidant activity of

compounds 3(a-o) to the reference compound are shown in Table 6.

Table 6 Antioxidant activity of compounds 3(a-o) by DPPH assay



3a 22.72±0.3 30.86±0.1 40.72±0.3 48.01±0.5 8.36

3b 19.82±0.2 21.54±0.4 37.19±0.4 43.94±0.1 9.40

3c 14.57±0.2 21.97±0.4 31.08±0.1 45.12±0.1 9.33

3d 25.08±0.3 33.70±0.5 37.72±0.4 46.08±0.2 9.28

3e 33.22±0.2 41.80±0.6 45.76±0.1 48.23±0.3 8.16

3f 18.32±0.4 24.00±0.5 28.29±0.7 31.88±0.2 15.48

3g 18.32±0.1 22.72±0.5 28.72±0.3 37.51±0.2 12.29

3h 21.46±0.5 25.83±0.6 29.36±0.4 38.37±0.3 11.14

3i 20.79±0.6 24.43±0.2 27.54±0.4 31.29±0.3 18.86

3j 22.61±0.1 25.08±0.5 28.08±0.4 32.26±0.2 19.09

3k 36.76±0.2 39.65±0.2 49.62±0.6 56.05±0.3 6.32

3l 41.26±0.5 45.23±0.1 46.90±0.4 57.34±0.7 5.93

3m 35.58±0.4 29.90±0.2 43.08±0.5 48.23±0.2 8.46

3n 28.51±0.4 31.51±0.1 37.72±0.2 49.41±0.3 8.83

3o 20.68±0.1 26.90±0.2 37.08±0.6 55.84±0.2 7.57

Control - - - - -

Standard (Ascorbic acid)

16.50±0.2

44.35±0.3 57.22±0.4 66.25±0.3 5.48

aIC50 value represents the concentration of three experiments required to exhibit 50%

antioxidant activity.

Almost all the tested compounds possessed strong scavenging activity against the

DPPH with IC50 values (5.93-19.39µg/mL). Compounds 3k, 3l and 3o showed

highest antioxidant activity with IC50 6.32, 5.93 and 7.57µg/mL, respectively than the

standard drug (Ascorbic acid IC50=5.48 µg/mL). It is due to presence of -OH group in

Chapter-6

210

the structure. Compound having NH2 group showed the greater scavenging activity

(IC50=8.16µg/mL). Further formyl and indole derivatives of acrylonitrile derivatives

showed moderate to good antioxidant activity where as methoxy derivatives of

aromatic aldehyde showed the less inhibitory activity than other (IC50=18.86-

19.09µg/mL).

6.3. EXPERIMENTAL





N), IR, NMR and Mass have been discussed in chapter 2. XRD, SEM and TGA-DTA,

have been discussed in chapter 5. Homogeneity as well as progress of reaction was

checked with thin layer chromatography (TLC) glass plates (20×9×5 cm) coated with

silica gel G (Merck) using benzene-acetone (8:2) mixture as mobile phase and

exposed to iodine vapors.

6.3.2. Synthesis of Catalyst

6.3.2a. Preparation of 3-Chloropropylsilica63

In a typical procedure 5 mL (25 mmol) of (3-chloropropyl)-trimethoxysilane was

dissolved in 100 mL of dried toluene. 5 g of SiO2 was added to this mixture and the

solution was stirred for 18 h at 60 °C. Then the solid residue was filtered, washed with

toluene and dried in vacuum.

6.3.2b. Preparation of urea functionalized propylsilica

The synthesized chloropropyl silica (3 g) was added to a solution of urea (1.2 g, 20

mmol) in ethanol (50 mL) in a round-bottom flask and the mixture was stirred under

reflux condition for 8 h. The obtained solid was then filtered and washed with ethanol

followed by drying at 80 °C for 12 h.

6.3.2c. Preparation of Silica Bonded N-(Propycarbamoyl)sulfamic acid (SBPCSA)64

To a mixture of urea functionalized propyl silica (2.5 g) in CH2Cl2 (20 mL),

chlorosulphonic acid (1.2 mL) was added drop wise at room temperature over a

period of 30 min. After addition was completed, the mixture was further stirred for 90

min and HCl gas evolution was monitored with a pH paper indicator. The mixture

Chapter-6

211

was filtered and washed with CH2Cl2 (50 mL) and dried in vacuum at 80 °C to afford

SBPCSA.

6.3.3. General procedure for the synthesis of acrylonitrile derivatives

To a mixture of substituted aromatic aldehydes 1(a-o) (2 mmol) and 2-

thiopheneacetonitrile 2 (2 mmol), 80 mg of SBPCSA was added and the reaction

mixture was heated on an oil bath at 80 °C for (20-30 min) with stirring. After

completion of the reaction as evident from thin layer chromatography (TLC), the

reaction mixture was diluted with ethanol and filtered off to recover the catalyst for

further use in catalytic cycles. The filtrate was evaporated under reduced pressure to

obtain the product. The crude product was further purified by crystallization from

appropriate solvent to afford the pure product 3(a-o).

6.3.4. Spectral characterization of synthesized compounds 3(a-o)

(E)-3-(4-Oxo-4H-chromen-3-yl)-2-(thiophen-2-yl)acrylonitrile (3a)

Yellow crystalline solid, yield 96%, m.p. 213-214 °C, Analytical cal. C16H9NO2S: C,

68.80; H, 3.25; N, 5.01; found: C, 68.77; H, 3.26; N, 5.03.

IR (KBr, νmax cm-1): 1612, 1560, 1462 (C=C), 1652 (C=Oγ-pyrone), 2219 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.20 (m, 1H, H-4'), 7.25 (s, 1H, H-2), 7.38

(d, 1H, H-3'), 7.40 (s, 1H, =CHolifinic), 7.45 (m, 1H, H-6), 7.58 (m, 1H, H-7), 7.59 (d,

1H, H-8), 7.70 (d, 1H, H-5'), 8.01 (d, 1H, H-5), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 110.45 (C-1"), 116.82 (C-8), 117.91

(C≡N), 118.50 (C-3), 123.83 (C-6), 124.82 (C-5), 125.54 (C-4a), 127.15 (C-4'),

127.42 (C-3'), 131.51 (C-5'), 135.51 (C-7), 136.05 (C-2'), 150.12 (C-2), 152.44 (C-

8a), 152.84 (C-2"), 178.45 (C-4), 178.50 (C=Oγ-pyrone).

MS (ESI) (m/z): 279.04 [M+•].

(E)-3-(6-Methyl-4-oxo-4H-chromen-3-yl)-2-(thiophen-2-yl)acrylonitrile (3b)

Yellow solid, yield 94%, m.p. 198-200 °C, Analytical cal. C17H11NO2S: C, 69.61; H,

3.78; N, 4.77; found: C, 69.60; H, 3.79; N, 4.74.

IR (KBr, νmax cm-1): 1658 (C=Oγ-pyrone), 1618, 1558, 1461 (C=C), 2215 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.38 (s, 3H, CH3), 7.01 (s, 1H, H-2), 7.22

(m, 1H, H-4'), 7.58 (d, 1H, H-8), 7.39 (d, 1H, H-3'), 7.45 (s, 1H, =CHolifinic), 7.75 (d,

1H, H-5'), 7.80 (s, 1H, H-5), 7.84 (m, 1H, H-7). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 21.81 (CH3), 113.12 (C-8), 115.49 (C-1"),

Chapter-6

212

117.38 (C≡N), 118.96 (C-3), 123.87 (C-5), 125.77 (C-4a), 127.01 (C-4'), 127.53 (C-

3'), 131.84 (C-5'), 133.92 (C-6), 134.89 (C-2'), 137.43 (C-7), 150.56 (C-2), 152.32

(C-8a), 154.80 (C-2"), 177.81 (C-4), 177.82 (C=Oγ-pyrone).

MS (ESI) (m/z): 293.05 [M+•].

(E)-3-(6-Fluoro-4-oxo-4H-chromen-3-Yl)-2-(thiophen-2-Yl)acrylonitrile (3c)

Yellow crystalline solid, yield 98%, m.p. 211 °C, Analytical cal. C16H8FNO2S: C,

64.64; H, 2.71; N, 4.71; found: C, 64.62; H, 2.70; N, 4.74.

IR (KBr, νmax cm-1): 1620, 1559, 1464 (C=C), 1662 (C=Oγ-pyrone), 2219 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.28 (s, 1H, H-2), 7.20 (m, 1H, H-4'), 7.36

(d, 1H, H-3'), 7.48 (s, 1H, =CHolifinic), 7.50 (d, 1H, H-8), 7.68 (d, 1H, H-7), 7.73 (d,

1H, H-5'), 7.80 (s, 1H, H-5). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 109.81 (C-5), 112.22 (C-1"), 118.02 (C-3),

118.13 (C≡N), 121.73 (C-8), 123.52 (C-7), 125.22 (C-4a), 127.26 (C-3'), 128.84 (C-

4'), 131.03 (C-5'), 136.73 (C-2'), 149.61 (C-2), 152.12 (C-2"), 152.91 (C-8a), 161.84

(C-6), 177.00 (C-4), 177.09 (C=Oγ-pyrone).

MS (ESI) (m/z): 297.03 [M+•].

(E)-3-(6-Bromo-4-oxo-4H-chromen-3-yl)-2-(thiophen-2-yl)acrylonitrile (3d)

Yellow solid, yield 96%, m.p. 220 °C, Analytical cal. C16H8BrNO2S: C, 53.65; H,

2.25; N, 3.91; found: C, 53.63; H, 2.26; N, 3.93.

IR (KBr, νmax cm-1): 1627, 1554, 1467 (C=C), 1665 (C=Oγ-pyrone), 2214 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.10 (d, 1H, H-8), 7.20 (m, 1H, H-4'), 7.23

(s, 1H, H-2), 7.38 (d, 1H, H-3'), 7.63 (d, 1H, H-5'), 7.78 (d, 1H, H-7), 7.94 (s, 1H, H-

5), 8.09 (s, 1H, =CHolifinic). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 107.20 (C-1"), 118.70 (C-8), 118.90

(C≡N), 119.08 (C-3), 124.8 (C-6), 127.25 (C-4a), 128.26 (C-3'), 129.81 (C-4'), 131.82

(C-5'), 135.82 (C-5), 136.90 (C-2'), 143.51 (C-7), 149.90 (C-2), 153.15 (C-2"), 157.91

(C-8a), 177.81 (C-4), 177.87 (C=Oγ-pyrone).

MS (ESI) (m/z): 358.94 [M+•].

(E)-3-(2-Amino-4-oxo-4H-chromen-3-yl)-2-(thiophen-2-yl)acrylonitrile (3e)

Yellow solid, yield 94%, m.p. 230-232 °C, Analytical cal. C16H10N2O2S: C, 65.29;

H, 3.42; N, 9.52; found: C, 65.26; H, 3.44; N, 9.53.

IR (KBr, νmax cm-1): 1614, 1565, 1461 (C=C), 1651 (C=Oγ-pyrone), 2220 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.18 (m, 1H, H-4'), 7.36 (d, 1H, H-3'), 7.48

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(m, 1H, H-6), 7.56 (d, 1H, H-8), 7.58 (m, 1H, H-7), 7.70 (d, 1H, H-5'), 7.75 (d, 1H,

H-5), 8.5 (s, 2H, N-H, D2O exchangeable), 8.12 (s, 1H, =CHolifinic). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 106.88 (C-3), 114.28 (C-1"), 117.02 (C-8),

118.06 (C≡N), 124.51 (C-4a), 124.81 (C-6), 125.80 (C-5), 126.42 (C-3'), 127.71 (C-

4'), 131.73 (C-5'), 135.85 (C-7), 136.33 (C-2'), 153.56 (C-2"), 158.45 (C-8a), 168.12

(C-2), 178.14 (C-4), 178.10 (C=Oγ-pyrone).

MS (ESI) (m/z): 294.05 [M+•].

(E)-3-(3-Nitrophenyl)-2-(thiophen-2-yl)acrylonitrile (3f)

Yellow crystalline solid, yield 98%, m.p. 150 °C, Analytical cal. C13H8N2O2S: C,

60.93; H, 3.15; N, 10.93; found: C, 60.90; H, 3.16; N, 10.95.

IR (KBr, νmax cm-1): 1518, 1320 (NO2), 1600, 1562, 1440 (C=C), 2218 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.12 (d, 1H, H-3'), 7.26 (d, 1H, H-5'), 7.39

(t, 1H, H-4'), 7.47 (d, 1H, H-6), 7.48 (d, 1H, H-4), 7.67 (s, 1H, =CHolifinic), 8.26-8.31

(t, 1H, H-5), 8.50 (s, 1H, H-2). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 107.68 (C-1"), 118.34 (C≡N), 122.12 (C-

2), 124.82 (C-4), 126.72 (C-3'), 128.90 (C-4'), 129.26 (C-5), 130.18 (C-5'), 135.21

(C-2'), 136.11 (C-6), 136.51 (C-1), 147.35 (C-3), 152.44 (C-2").

MS (ESI) (m/z): 256.03 [M+•].

(E)-3-(4-Nitrophenyl)-2-(thiophen-2-yl)acrylonitrile (3g)

Yellow crystalline solid, yield 96%, m.p. 125 °C, Analytical cal. C13H8N2O2S: C,

60.93; H, 3.15; N, 10.93; found: C, 60.90; H, 3.17; N, 10.94.

IR (KBr, νmax cm-1): 1512, 1322 (NO2), 1612, 1560, 1445 (C=C), 2214 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.17 (m, 1H, H-4'), 7.20 (d, 1H, H-3'), 7.41

(s, 1H, =CHolifinic), 7.60 (d, 1H, H-5'), 8.0 (dd, 2H, H-2 and H-6), 8.15 (dd, 2H, H-4

and H-6), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 108.26 (C-1"), 119.70 (C≡N), 122.80 (C-3

and C-5), 126.62 (C-3'), 128.34 (C-4'), 128.51 (C-2 and C-6), 131.52 (C-5'), 136.61

(C-2'),

141.04 (C-1), 153.18 (C-2"), 157.81 (C-4).

MS (ESI) (m/z): 256.03 [M+•].

(E)-3-(4-(Dimethylamino)phenyl)-2-(thiophen-2-yl)acrylonitrile (3h)

Brown crystalline solid, yield 98%, m.p. 132 °C, Analytical cal. C15H14N2S: C,

70.83; H, 5.55; N, 11.01; found: C, 70.80; H, 5.56; N, 11.03;

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IR (KBr, νmax cm-1): 1614, 1567, 1445 (C=C), 2218 (C≡N), 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.11 (s, 6H, 2×CH3), 6.82 (dd, H-3 and H-

5), 7.18 (m, 1H, H-4'), 7.24 (d, 1H, H-3'), 7.54 (d, 1H, H-5'), 7.62 (dd, 2H, H-2 and

H-6), 8.02 (s, 1H, =CHolifinic). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 42.15 (CH3), 108.10 (C-1"), 112.32 (C-3

and C-5), 118.42 (C≡N), 124.01 (C-1), 126.00 (C-3'), 127.31 (C-4'), 128.50 (C-2 and

C-6), 137.73 (C-2'), 137.79 (C-5'), 151.85 (C-4), 153.80 (C-2").

MS (ESI) (m/z): 254.09 [M+•].

(E)-3-(3,4-Dimethoxyphenyl)-2-(thiophen-2-yl)acrylonitrile (3i)

Pale yellow solid, yield 98 %, m.p. 136 °C, Analytical cal. C15H13NO2S: C, 66.40; H,

4.83; N, 5.16; found: C, 66.41; H, 4.80; N, 5.18.

IR (KBr, νmax cm-1): 1445, 1560, 1592 (C=C), 2215 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.86 (s, 6H, OCH3×2). 7.1 (d, 1H, H-5),

7.20 (m, 1H, H-4'), 7.39 (d, 1H, H-3'), 7.45 (s, 1H, =CHolifinic), 7.68 (d, 1H, H-5'),

7.76 (d, 1H, H-6), 7.82 (s, 1H, H-2). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 56.21 (CH3), 108.08 (C-1"), 112.61 (C-2),

117.35 (C-5), 118.20 (C≡N), 123.80 (C-6), 129.21 (C-1), 127.91 (C-3'), 128.73 (C-

4'), 132.94 (C-5'), 135.05 (C-2'), 148.84 (C-4), 149.83 (C-3), 153.42 (C-2").

MS (ESI) (m/z): 271.07 [M+•].

(E)-2-(Thiophen-2-yl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (3j)

Yellow crystalline solid, yield 95%, m.p. 149 °C, Analytical cal. C16H15NO3S: C,

63.77; H, 5.02; N, 4.65; found: C, 63.75; H, 5.05; N, 4.64.

IR (KBr, νmax cm-1): 1449, 1562, 1605 (C=C), 2212 (C≡N). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 3.89 (s, 9H, 3×OCH3), 7.12 (s, 2H, H-2 and

H-6), 7.21 (m, 1H, H-4'), 7.33 (d, 1H, H-3'), 7.78 (d, 1H, H-5'), 7.97 (s, 1H,

=CHolifinic), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 56.51 (CH3), 107.62 (C-2 and C-6), 108.22

(C-1"), 117.90 (C≡N), 127.02 (C-3'), 128.21 (C-1), 128.90 (C-4'), 131.91 (C-5'),

135.94 (C-2'), 138.86 (C-4), 152.12 (C-2"), 154.84 (C-3 and C-5).

MS (ESI) (m/z): 301.08 [M+•].

(E)-2-(Thiophen-2-yl)-3-(3,4,5-trihydroxyphenyl)acrylonitrile (3k)

Yellow solid, yield 92 %, m.p. 158 °C, Analytical cal. C13H9NO3S: C, 60.22; H,

3.50; N, 5.40; found: C, 60.20; H, 3.53; N, 5.39.

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IR (KBr, νmax cm-1): 1448, 1556, 1610 (C=C), 2214 (C≡N), 3448 (OH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 5.35 (s, 3H, 3×OH, D2O-exchangeable).

7.13 (m, 1H, H-4'), 7.32 (d, 1H, H-3'), 7.42 (s, 2H, H-2 and H-6), 7.54 (d, 1H, H-5'),

8.00 (s, 1H, =CHolifinic), 13C NMR (100 MHz, DMSO-d6, δ, ppm): 106.86 (C-6), 107.41 (C-2), 108.45 (C-1"),

118.22 (C≡N), 127.85 (C-3'), 127.90 (C-4'), 131.08 (C-5'), 131.24 (C-1), 134.54 (C-

4), 135.81 (C-2'), 146.32 (C-5), 148.80 (C-3), 152.44 (C-2").

MS (ESI) (m/z): 259.03 [M+•].

(E)-3-(2-Hydroxynaphthalen-1-yl)-2-(thiophen-2-yl)acrylonitrile (3l)

Bright yellow solid, yield 94%, m.p. 132 °C, Analytical cal. C17H11NOS: C, 73.62; H,

4.00; N, 5.05; found: C, 73.62; H, 4.03; N, 5.02.

IR (KBr, νmax cm-1): 1448, 1565, 1602 (C=C), 2212 (C≡N), 3425 (OH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 5.35 (s, 1H, -OH, D2Oexchangeable), 7.20

(m, 1H, H-4'), 7.53 (m, 1H, H-6), 7.38 (d, 1H, H-3'), 7.62 (m, 1H, H-5), 7.68 (d, 1H,

H-5'), 7.69 (d, 1H, H-3), 7.90 (d, 1H, H-4), 7.95 (m, 1H, H-7), 8.21 (s, 1H,

=CHolifinic), 8.40 (d, 1H, H-8). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 107.50 (C-1), 108.08 (C-1"), 110.89 (C-3),

115.56 (C-8), 118.20 (C≡N), 123.90 (C-6), 124.96 (C-9), 126.41 (C-3'), 127.01 (C-4),

127.22 (C-5), 127.90 (C-4'), 128.21 (C-7), 131.18 (C-5'), 135.91 (C-2'), 141.11 (C-

10), 153.41 (C-2"), 158.02 (C-2).

MS (ESI) (m/z): 227.04 [M+•].

(E)-3-(1H-Indol-3-yl)-2-(thiophen-2-yl)acrylonitrile(3m)

Yellow crystalline solid, yield 98%, m.p. 195 °C, Analytical cal. C15H10N2S: C,

71.97; H, 4.03; N, 11.19; found: C, 71.97; H, 4.03; N, 11.19.

IR (KBr, νmax cm-1): 1610, 1550, 1445 (C=C), 2212 (C≡N), 3324 (NH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.13-7.15 (m, 1H, H-4'), 7.39 (d, 1H, H-3'),

7.58 (d, 1H, H-5'), 7.79 (s, 1H, H-2), 7.94-7.97 (m, 2H, H-5 and H-6), 8.09 (s, 1H,

=CHolifinic), 8.13 (d, 1H, H-4), 8.28 (d, 1H, H-7), 10.17 (s, 1H, -NH, D2O-

exchangeable). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 110.16 (C-3), 111.32 (C-7), 114.49 (C-1"),

118.02 (C-4), 118.09 (C≡N), 119.36 (C-5), 122.92 (C-6), 126.27 (C-3'), 127.50 (C-2),

128.19 (C-3a), 129.05 (C-4'), 131.56 (C-5'), 136.84 (C-2'), 137.43 (C-7a), 153.90 (C-

2")

Chapter-6

216

MS (ESI) (m/z): 250.06 [M+•].

(E)-3-(5-Methyl-1H-indol-3-yl)-2-(thiophen-2-yl)acrylonitrile (3n)

Yellow solid, yield 94%, m.p. 196 °C, Analytical cal. C16H12N2S: C, 72.70; H, 4.58;

N, 10.60; found: C, 72.70; H, 4.55; N, 10.63.

IR (KBr, νmax cm-1): 1604, 1560, 1445 (C=C), 2217 (C≡N), 3324 (NH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 2.56 (s, 3H, -CH3), 7.08 (d, 1H, H-6), 7.09

(d, 1H, H-7), 7.15 (m, 1H, H-4'), 7.37 (d, 1H, H-3'), 7.42 (s, 1H, H-4), 7.68 (d, 1H, H-

5'), 7.79 (s, 1H, H-2), 7.99 (s, 1H, =CHolifinic), 10.02 (s, 1H, -NH, D2Oexchangeable). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 21.30 (CH3), 110.42 (C-3), 111.90 (C-7),

114.81 (C-1"), 118.16 (C≡N), 120.58 (C-4), 121.82 (C-6), 126.62 (C-3a), 126.91 (C-

3'), 127.81 (C-2), 127.90 (C-4'), 129.34 (C-5), 131.16 (C-5'), 134.22 (C-7a), 136.74

(C-2'), 153.32 (C-2").

MS (ESI) (m/z): 264.07 [M+•].

(E)-3-(5-Hydroxy-1H-indol-3-yl)-2-(thiophen-2-yl)acrylonitrile (3o)

Light yellow solid, yield 95%, m.p. 202 °C, Analytical cal. C15H10N2OS: C, 67.65;

H, 3.78; N, 10.52; found: C, 67.66; H, 3.75; N, 10.54.

IR (KBr, νmax cm-1): 1612, 1562, 1448 (C=C), 2219 (C≡N), 3324 (NH), 3435 (OH). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 5.35 (s, 1H, -OH, D2O-exchangeable), 7.40

(s, 1H, H-4), 7.18 (d, 1H, H-6), 7.19 (m, 1H, H-4'), 7.29 (d, 1H, H-7), 7.32 (d, 1H, H-

3'), 7.60 (d, 1H, H-5'), 7.78 (s, 1H, H-2), 8.07 (s, 1H, =CHolifinic), 10.06 (s, 1H, -NH,

D2O-exchangeable). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 110.42 (C-3), 112.56 (C-7), 114.83 (C-1"),

118.71 (C≡N), 120.50 (C-4), 121.84 (C-6), 125.62 (C-3a), 126.90 (C-3'), 127.20 (C-

2), 127.91 (C-4'), 129.32 (C-5), 131.15 (C-5'), 134.07 (C-7a), 135.71 (C-2'), 152.32

(C-2"). MS (ESI) (m/z): 277.06 [M+•].

6.3.5. Density functional theory (DFT) calculations

The density functional theory (DFT) calculations were conducted with a hybrid

functional B3LYP (Becke’s three parameters nonlocal exchange function with the

Lee- Yang-Parr correlation function)65,66 at the 6-311G basis set using the GAMESS

interface in ChemBio3D ultra ver. 14.0 (PerkinElmer, MA, USA).

Chapter-6

217



diphenylpicrylhydrazyl (DPPH) method.67 The general procedure for this assay have

been discussed in chapter 2. In this procedure, drug stock solution (1 mg/mL) was

diluted to final concentration of 2, 4, 6, and 8µg/mL in methanol.

6.4. CONCLUSION

This chapter reports a convenient and eco-friendly protocol for the synthesis of

acrylonitrile derivatives 3(a-o) in excellent yield (92-98%) by employing silica

bonded N-(propylcarbamoyl)sulfamic acid (SBPCSA) under solvent free conditions.

This protocol offers several advantages over other existing synthetic methodologies in

terms of yield and purity of products, simple procedures, reaction times, catalyst

stability and recyclability. It possesses environmentally benign properties such as

non-toxicity, biocompatibility, recyclability, physiological inertness, inexpensiveness

and thermal stability. This synthetic scheme possesses diverse applicability and is

compatible to a range of functional groups (electron donating/electron withdrawing).

All the compounds displayed moderate to good antioxidant activity. Thus, present

synthetic approach provides a better scope for the synthesis of acrylonitrile analogues

and will be a more practical alternative to the other existing methods.

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218

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in chemistryir.amu.ac.in/11865/1/t10239.pdfreduce energy requirements and decrease separations due...

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