SYNTHESIS AND BIOLOGICAL EVALUATION OF PYRAZOLE … · 2016-04-22 · 2 22nd April 2016 CERTIFICATE BY THE GUIDE I certify that the thesis entitled “SYNTHESIS AND BIOLOGICAL EVALUATION

1

SYNTHESIS AND BIOLOGICAL EVALUATION OF PYRAZOLEDERIVATIVES AND SOME OTHER HETEROCYCLIC

COMPOUNDS

Thesis Submitted in Partial Fulfillment for the award of

Degree of Doctor of Philosophy in Pharmacy

ByP. KUMAR NALLASIVAN, M. Pharm

Under the guidance ofDr.B .JAYAKAR , M. Pharm, Ph.D

VINAYAKA MISSIONS UNIVERSITY

SALEM, TAMIL NADU, INDIA

APRIL 2016

2

22nd April 2016

CERTIFICATE BY THE GUIDE

I certify that the thesis entitled “SYNTHESIS AND BIOLOGICAL EVALUATION OF

PYRAZOLE DERIVATIVES AND SOME OTHER HETEROCYCLIC

COMPOUNDS” submitted for the degree of Doctor of Philosophy by Mr. P.KUMAR

NALLASIVAN, M.Pharm., is the record of research work carried out by him during the

period from 2005 to 2016 under my guidance and supervision and that this work has not

formed the basis for the award of any degree, diploma, associate-ship, fellowship or other

titles in this University or any other University or Institution of higher learning.

Place: SalemDate: 22.04.2016

Dr.B .JAYAKAR. , M. Pharm., Ph.D,Principal,

Vinayaka Missions College Of Pharmacy,Salem.

3

DECLARATION

I, P. Kumar Nallasivan, declare that the thesis entitled “Synthesis and Biological

Evaluation of Pyrazole and some other Heterocyclic Compounds” by me for the

award of Degree of Doctor of Philosophy is the record of research work carried out by me

during the period from 2005 to 2016 under the guidance of Prof. Dr. B. Jayakar, M.

Pharm., Ph.D and has not formed the basis for the award of any other degree, diploma,

associate-ship, fellowship or any other similar title in this or any other University or other

similar institutions of higher learning.

Place: Salem

Date: 22.04.2016

P.KUMAR NALLASIVAN. , M. Pharm.,

4

ACKNOWLEDGEMENT

“Develop an attitude, and give thanks for everything that happens to you, knowing that

every step forward is a step towards achieving something bigger and better than your current

situation”.

I wish to express my sincere and hearty gratitude, to my respected guide Dr. B. Jayakar,

Principal cum professor, Vinayaka Mission College of Pharmacy , Salem for his immense guidance,

help, intellectual supervision, and dedicated support for the timely completion of my work. I thank

him for the freedom of thought, trust and expression which he bestowed upon me. Last but not least,

it’s my fortune and so I am proud to have him as my guide.

My heartfelt gratitude to R.Venkata Narayanan,. Principal, RVS College of

pharmaceutical Sciences, Sulur for his constant support, his generous consideration and facilities

provided to me in completing this work successfully.

I dedicate my sincere thanks to Dr. B. Jayakar, Principal cum Professor, Vinayaka Missions

College of Pharmacy, Salem who has always extended his hearted support in carrying out my work

in time.

My heartfelt thanks to my dearest friend Mr.R.Sivakumar, for their enthusiastic support

by guiding me in a right way

.My sincere thanks to Prof. W.D. Sam Solomon., Head, Dept. of Pharmaceutical

Chemistry and to Dr. D. Benetto Johnson, Professor and Head, Dept. of Pharmacology,for their

timing help and constant support.

My special thanks to Asst.Prof.S.S.Rajendran for his valuable suggestions and encouragement

during the work.

5

A special word of thanks to the to all laboratory Assistants/Attenders.

I would like to thank my B.Pharm students M.Raj Kumar, K.Karthikeyan, C.Chandru,

M.Sankara Subramanian, Hari Prasad, Krishna Kumar, Fakir Muhaideen Salman, Anish Kumar and

Ganesh Prabhu

I would also like thank to my M.Pharm students Mr.Santhiagu and Mr.Suresh kumar who

has supported me throughout the work.

I thank the Almighty, who has given me this opportunity to extend my gratitude to

all those people who have helped me and guided me throughout my work and life.

I would like to express my heartfelt gratitude to my Parents, wife and my beloved daughter

whose full-hearted co-operation, love and moral support made me to complete this task

successfully.

Despite all this co-operation rendered generously by one and all, I am solely responsible for

any and all the errors and short comings of this dissertation.

P. KUMAR NALLASIVAN

6

I Dedicate

This Research Work

To

My Parents

7

CONTENTS

Chapter Title Page No.

1 Introduction 131.1.Pyrazolines 141.2.Quinazoline 231.3.Isoxazoles 301.4.Benzimidazole 381.5.In-silico Drug Design 44

2 Literature Review 592.1.Pyrazoline Derivatives 592.2.Quinazoline Derivatives 662.3.Isoxazole Derivatives 712.4.Benzimidazole Derivatives 76

3 Need For Study 884 Aim and Objective of the study 915 Materials and Instruments used

5.1.Synthesis 925.2.Characterization of newly synthesizedcompounds

96

5.3. In-Silico Studies 965.4 Toxicity Studies 985.5.Anti-Microbial Activity 995.6. Anti-inflammatory activity 995.7. Analgesic activity 1005.8.In-vitro Anti-Oxidant Activity 100

6 Results and Discussion 1016.1.Experiment 1016.2.Discussion 1346.3.In-Silico Studies 1776.4.Anti-Microbial Activity 1916.5. Evaluation of Anti-inflammatory activity 2096.6. Evaluation of Analgesic activity 222

8

6.7 In-vitro Anti-Oxidant Activity 2296.8.Infra red spectrum of synthesized compounds 2526.9.Nuclear Magnetic Resonance spectrum ofsynthesized compounds

269

7 Summary and Conclusion 278

8 References 2819 Published Articles 300

9

LIST OF TABLE

S.no. Tableno

List of table Pageno

1. 6.2.1 Spectral Data of synthesized derivatives 121

2. 6.2.2. Elemental Analysis Data of synthesized derivatives 132

3. 6.3.1Docking studies for anti-inflammatory activity using Autodock:Docked scores of newly designed compounds with COX-1and COX-2

178

4. 6.3.2 Anti-bacterial- Docked scores of newly designed compoundswith β-keto acyl acyl carrier protein (1hnj)

183

5. 6.3.3 Anti-fungal- Docked scores of newly designed compounds with14α-demethylase (1E9X)

186

6. 6.4.1 Anti Bacterial & Antifungal Activity Of Quinazolinone Derivatives[3(A-H), Iv(A-H), V(A-H)] At 100 Mg/Ml Vi(A-E) And 7(A-E)

206

7. 6.4.2 Anti Bacterial & Antifungal Activity Of Benzimidazole Derivatives[Vi(A-E), 7(A-E)] At 100 Mg/Ml

208

8. 6.5.1 Inhibitory effects of test compounds 3a-h, 4a-h,5a-h,6a-e and7a-e on Carrageenan-induced edema of the hind paw in rats

211

9. 6.5.2 Percentage inhibition of test compounds 6a-e, 7a-j and 8a-eCarrageenan-induced edema of the hind paw in rats

214

10. 6.5.3 Ulcerogenic activity of selected compounds in comparison withIndomethacin

221

11. 6.6.1 Analgesic activity of synthesized compound 3a-h 223



14. 6.6.4 Analgesic activity of synthesized compound 6a-e 226


16. 6.7.1 Reductive ability of synthesized compounds 231

17. 6.7.2Hydrogen peroxide scavenging activity of synthesizedcompounds and α-tocopherol - Hydrogen peroxide radical assayfor compound (3a-h)

236


239


242

20. 6.7.5Hydrogen peroxide scavenging activity of synthesizedcompounds and α-tocopherol - Hydrogen peroxide radical assayfor compound (6a-e)

245

10

21. 6.7.6Hydrogen peroxide scavenging activity of synthesizedcompounds and α-tocopherol - Hydrogen peroxide radical assayfor compound (7a-e)

247

11

LIST OF FIGURE

S.no. Figureno

List of figure Pageno

1. 6.3.1 Docking Studies For Anti-Inflammatory Activity Using AutoDock Binding Interaction With Cox-1 (1egq ):Compound-4b

181

2. 6.3.2 Docking Studies For Anti-Inflammatory Activity Using AutoDock Binding Interaction With Cox-1 (1egq ):Compound-4e

181

3. 6.3.3 Docking Studies For Anti-Inflammatory Activity Using AutoDock Binding Interaction With Cox-1 (1egq ):Indomethacin

181

4. 6.3.4 Docking Studies For Anti-Inflammatory Activity Using AutoDock Binding Interaction With Cox-2 (1cx2):Compound-4b

182

5. 6.3.5 Docking Studies For Anti-Inflammatory Activity Using AutoDock Binding Interaction With Cox-2 (1cx2):Compound-4e

182

6. 6.3.6 Docking Studies For Anti-Inflammatory Activity Using AutoDock Binding Interaction With Cox-2 (1cx2):Indomethacin

182

7. 6.3.7Docking studies for anti-fungal activity using Auto dockbinding interaction with β-keto acyl acyl carrier protein(1hnj):Compound - 4c

185

8. 6.3.8Docking studies for anti-fungal activity using Auto dockbinding interaction with β-keto acyl acyl carrier protein(1hnj):Compound - 4h

185

9. 6.3.9Docking studies for anti-fungal activity using Auto dockbinding interaction with β-keto acyl acyl carrier protein(1hnj):Ampicillin

185

10. 6.3.10 Anti-fungal- Docked scores of newly designed compoundswith 14α-demethylase (1E9X):Compound - 4h

188

11. 6.3.11 Anti-fungal- Docked scores of newly designed compoundswith 14α-demethylase (1E9X):Griseofulvin

188

12. 6.4.1 Anti bacterial activity of synthesized compounds andstandard

193

13. 6.4.2 Anti fungal activity of synthesized compounds 197

14. 6.5.1 Ulcerogenic activity of compound 4b, 4e, 4g and 4h,control, Indomethacin

220






20. 6.7.1Reducing power of synthesized compounds and ascorbicacid 3a-h

233

21. 6.7.2 Reducing power of synthesized compounds and ascorbicacid 4a-h

233

12

22. 6.7.3 Reducing power of synthesized compounds and ascorbicacid 5a-h

234

23. 6.7.4 Reducing power of synthesized compounds and ascorbicacid 6a-e

234

24. 6.7.5 Reducing power of synthesized compounds and ascorbicacid 7a-e

235

25. 6.7.6 Hydrogen peroxide scavenging activity of synthesizedcompounds and α-tocopherol 3a-h

249


249


250

28. 6.7.9 Hydrogen peroxide scavenging activity of synthesizedcompounds and α-tocopherol 6a-e

250

29. 6.7.10 Hydrogen peroxide scavenging activity of synthesizedcompounds and α-tocopherol 7a-e

251

13

1. INTRODUCTION

Medicinal chemistry or Pharmaceutical chemistry is a field at the combination of

chemistry and pharmacology involved with, synthesizing, designing and

developing drugs. It involves acknowledgment, construction and growth of new

chemical entities suitable for curative use. It incises the study of existing drugs,

their quantitative structure activity relationships (QSAR) and their properties.

Pharmaceutical chemistry is alert on quality analysis of medicines and aims to

promise strength for the purpose of medicinal products [1].

During the premature stages of medicinal chemistry expansion, scientists were

first and foremost concerned with the segregation of medicinal compounds found

in plants. Now, scientists in this field are also concerned with the creation of new

artificial compounds such as drugs. Medicinal chemistry is geared towards drug

innovation and development[2]. The move toward to the practice of medicinal

chemistry has developed from an empirical one relating organic peparation of

compounds, based on alteration of structures of known action. Today computers

are pushed into the service of chemists. Computers enhance the scientific

procedure in drug discovery by sustaining the chemist with storing, collecting,

analyzing, manipulating and viewing the data. Computers provide a link to

conjectural chemistry and graphic modeling, giving premeditated estimates of

molecular properties, models of biological sites, models of molecules, and even

models of drug-receptor interactions [3]

14

1.1.PYRAZOLINES

Electron-rich nitrogen heterocyclic plays an significant role in biological activities.

Pyrazoline is a five membered ring containing two adjoining nitrogen atoms, and a

double bond in the ring. Among various pyrazoline derivatives, ∆2-pyrazolines are

most frequently studied. Second nitrogen in the five-membered ring also

influences the activity or pharmacokinetic profile of molecule. ∆2-Pyrazoline

derivatives have also been reported in the literature to exhibit various biological

activities such as antimicrobial, analgesic, antipyretic, anti-inflammatory,

antihypertensive, antidepressant etc [4,5].

Pyrazolines are well known, reduced form of pyrazoles and important

nitrogen –containing five-membered heterocyclic compounds. Many methods

have been used for their syntheses. Synthesis and characterization of pyrazoline

derivatives is a developing field within the area of heterocyclic chemistry for the

past decades because of their ease of access through synthesis, wide range of

chemical reactivity and broad spectrum of biological activity and array of industrial

applications. Pyrazoline obtained fromcyclization of chalcones with aryl

hydrazines, can be oxidized to pyrazoles [6].

Chemistry of Pyrazolines

The oxo derivatives of pyrazolines, known as pyrazolines, are best classified as

follows: 5-pyrazoline, also called 2-pyrazolin-5-one (1); 4-pyrazoline, also called 2-

pyrazolin-4-one (2); and 3-pyrazoline, also called 3-pyrazolin-5-one (3). Within

15

each class of pyrazolines many tautomeric forms are possible; for simplicity only

one form is shown.

NH

NO

NH

N

O

NH

NO H

(1) (2) (3)

NN

R

OH

NNH

R

O

NN

R

OH

NN

R

O

Enol Keto Enol Keto

(4) (5)

Substitution at N1 decreases the possible number of tautomers: for 3-pyrazolines, two

tautomeric forms are possible, (4) and (5), which in nonpolar solvents are both present in

about the same ratio. 5-Pyrazolines exhibit similar behavior.

In 4-pyrazolines, the enol form predominates, although the keto form has also

been observed. The tautomeric nature of the pyrazolines is also shown by the

combination of yield separated after certain reactions. Therefore alkylation in general

takes place at C4, but sometimes it is accompanied by alkylation on N and O. Related

problems can occur during carbamoylation and acylation reactions, which also favor C4.

Pyrazolines react with aldehydes and ketones at C4 to form a carbon–carbon double

16

bond, eg (6). Coupling takes place when diazonium salts react with pyrazolines to

produce azo compounds, eg (7).

Compounds of type (7) are widely used in the dye industry. The Mannich reaction also

takes place at C4, as does halogenation and nitration. The important analgesic

aminoantipyrine (8) on photolysis in methanol undergoes ring fission to yield (9)[7].

NN

R

O

R

R

NN

R

O

NN

R

(6) (7)

NNO

NH2 CH3

CH3

CH3 OH

hv NH

O

OH

O

NHCH3

CH3

(8) (9)

17

Synthesis of pyrazoline derivatives

The pyrazoline-3-carboxylic acid (11) has been isolated by reaction of oxazoline

(10) with hydrazonyl chloride[8].

O

NAr2

H

OAr1

+ NH N

COOR

Ar3

Cl

(C4H9)4N+Br

-

Na2CO3

NN

Ar1

O

CH3

COORNH

Ar2

O

(10) (11)

NN

CH2

C(CH3)3(H3C)3C

+ NC

CN

Na H NN

C(CH3)3

ONC

NH2 C(CH3)3

The favored synthetic process for the title compounds utilizes the reaction of

hydrazines with bifunctional compounds, such as β-diketones and esters, and β-

keto acetylenic compounds. In another process, diazo compounds substitute

hydrazines and ring formation takes place through 1,3-dipolar cyclo addition.

Pyrazoles and pyrazolines are widely used in the pharmaceutical industry to ease

inflammation, fever, infections and pain. To a lesser degree, they are furthermore

used as herbicides and insecticides. Pyrazolines associated to azo compounds

are widely used in the dye manufacturing; some pyrazolines exhibit insecticidal

activity[9].

18

Pyrazolines with a free NH group are effortlessly nitrosated and bring about nitrosamines,

this cause tumors in the liver of test animals. The analgesics antipyrine (12) and

aminopyrine (13), if admixed with nitrites, are mutagenic when tested in vitro;

though, when tested in the absence of nitrites, negative consequences are

obtained [10].

NN C H 3O

C H 3

NN

C H 3

ON

CH 3

CH 3

C H 3

The oxo derivatives of pyrazolines, known as pyrazolines, are best classified as

follows: 5-pyrazoline, also called 2-pyrazolin-5-one (1); 4-pyrazoline, also called 2-

pyrazolin-4-one (2); and 3-pyrazoline, also called 3-pyrazolin-5-one (3). Within

each class of pyrazolines many tautomeric forms are possible; for simplicity only

one form is shown.

Substitution at N1 decreases the possible number of tautomers: for 3-pyrazolines, two

tautomeric forms are possible, (4) and (5), which in nonpolar solvents are both present in

about the same ratio. 5-Pyrazolines exhibit similar behavior.

In 4-pyrazolines, the enol form predominates, although the keto form has also

been observed. The tautomeric character of the pyrazolines is also illustrated by the

combination of products separated after certain reactions. Thus alkylation usually takes

place at C4, but on some instance it is accompanied by alkylation on N and O. Related

19

problems can come up during carbamoylation and acylation reactions, which also favor

C4. Pyrazolines react with aldehydes and ketones at C4 to form a carbon–carbon double

bond, eg (6). Coupling takes place when pyrazolines react with diazonium salts to

produce azo compounds, eg (7).

Compounds of type (7) are widely used in the dye industry. The Mannich reaction also

takes place at C4, as does halogenation and nitration. The important analgesic

aminoantipyrine (8) on photolysis in methanol undergoes ring fission to yield (9)[7].

The pyrazoline-3-carboxylic acid (11) has been isolated by reaction of oxazoline

(10) with hydrazonyl chloride[8].

The preferred synthetic method for the title compounds utilizes the reaction of

hydrazines with bifunctional compounds, such as β-diketones and esters, and β-

keto acetylenic compounds. In an alternative procedure, diazo compounds

replace hydrazines and ring formation takes place via 1,3-dipolar cycloaddition.

Pyrazoles and pyrazolines are widely used in the pharmaceutical industry to

alleviate inflammation, fever, pain, and infections. To a lesser extent, they are also

used as insecticides and herbicides. Pyrazolines linked to azo compounds are

extensively used in the dye industry; some pyrazolines display insecticidal

activity[9].

Pyrazolines with a free NH group are easily nitrosated and give rise to nitrosamines, which

cause tumors in the liver of test animals. The analgesics antipyrine (12) and aminopyrine

(13), if admixed with nitrites, are mutagenic when tested in vitro; however, when tested

in the absence of nitrites, negative results are obtained[10].

20

Pyrazoline-type drugs, such as phenylbutazone and sulfinpyrazone, are

metabolized Pyrazoline-type drugs, such as phenylbutazone and sulfinpyrazone,

are metabolized in the liver by micro-somal enzymes, forming glucuronide

metabolites that are easily excreted because of enhanced water solubility.

The pyrazoline derivatives, which include dipyrone (14), antipyrine (12),

aminopyrine (13) and propyphenazone, are widely used analgesics. Dipyrone, the

most widely used pyrazoline, has been the most studied. Dipyrone is an inhibitor

of cyclo-oxygenase but, unlike aspirin, its effect is rapidly reversible. The inhibition

of prostaglandin biosynthesis contributes to the analgesic activity of the pyrazoline

derivatives. Unlike the Non-steroidal anti-inflammatory agents (NSAIDs) generally,

the pyrazoline derivatives antipyrine, aminopyrine and propyphenazone are

minimally bound to plasma proteins. The pyrazolines undergo extensive

biotransformation, aminopyrine and dipyrone being converted to active

metabolites. The most frequently reported side effects of the pyrazoline

derivatives are skin rashes. Gastrointestinal side effects are rare.

NN

CH3

ON

CH3

CH3

NaO 3S

OH2

(14)

21

Important Pyrazoline and isoxazoline derivative in pharmaceuticals:

Some of the pharmaceuticals that incorporate the pyrazole nucleus are given below. Their

main uses are as antipyretic, anti-inflammatory, and analgesic agents. To a lesser extent,

they have shown efficacy as antibacterial/antimicrobial, antipsychotic, anti-emetic, and

diuretic agents. The analgesic aminopyrine, the antipyretic dipyrone, and the anti-

inflammatory phenylbutazone (15), though once widely prescribed, are rarely used in the

1990s on account of their tendency to cause agranulocytosis. Pyrazoline derivatives as

like benzimidazole derivatives have been found to possess some interesting

pharmacological activities. eg. Antipyrin, Ampyrone, edaravone, etc.

NNO

O(CH2)3CH3

(15)

NNO

O(CH2)3CH3

S

S

NH2O

O

NH2

OO

;

NNO

O(H2C)3

CH3

O

O

Cl

(16) butaglyon, an antidiabetic; (17) feclobuzo, an antiinflammatory;

22

NNO

OCH3

O

NNO

OS

O

(18) kebuzone, an antirheumatic; (19) sulfinpyrazone, an anti gout

NNNH2

O

CH3

Cl

Cl

NNO CH3

CH3

(20) muzolimin, a diuretic (21) phenazobz, an antiasthmatic

NN

CH3

O NN

CH3

O

NH2

CH3

(22)Edaravone (23)Amprone

23

Pyrazolines react with diazonium salts, an important process in the dye industry.

The majority of dyes are having pyrazoline nucleus with an azo linkage attached at C4, eg,

(24) and (25).

NNO

R2

R1

C H 3

R''

NN

NaO 3 S

NNO

R1NN

NN

NN

O

R 1

C l

C l

(24) (25)

The survey of the pertinent literature reveals that isoxazolines have been found to

possess a wide range of biological activity such as anti bacterial [11], anti HIV[12],

anti-inflammatory[13], anticancer[14]etc. Some isoxazole derivatives (26) have been

reported as anti-tubercular, anti bacterial and antifungal agets[15].

O

N O

R

(26)

Azopyrazoles (27) and azoisoxazoles (28) are possessing good antifungal

activity[16]. Similarly, 2-alkyl isoaxazolidine derivatives have been as antifungal

agents[17].

24

NN

N

NH

R1

NN

N

O

R1

(27) (28)

N

O

CH 3

ClNN

Cl

N

O

CH3

ClNN

Cl

(29) (30)

25

1.2.QUINAZOLINE

Quinazolines and quinazolinones are classes of fused het-erocycles that are of

considerable interest because of the diverse range of their biological properties [18].

Many substituted quinazoline and quinazolinone derivatives possess a wide range

of bioactivities such as antimalarial, anti-cancer, antimicrobial, antifungal, antiviral,

antiprotozoan, anti-inflammatory, diuretic, muscle relaxant, antitubercular,

antidepressant, anticonvulsant, acaricidal, weedicide, and many other biological

activities. Quinazoli-none and Quinazoline compounds are also used in

preparation of various functional materials for synthetic chemistry and also present

in various drugs molecules (Figure 1). This review is an attempt to expand the

huge potentiality and focused on the various biological activities of quinazolines

and quinazoli-nones[19].

Quinazolinones are classified as five types, based on the patterns of substitution

of the ring system[20]. These are 2-substituted-4(3H)-quinazolinones, 3-

substituted-4(3H)-quinazolinones, 4-substituted-quina-zolines, 2,3-disubstituted-

4(3H)-quinazolinones, and 2,4-disubstituted-4(3H)-quinazolinones. These

compounds may be classified into three types based upon the position of the keto

or oxo group[21]. Out of the three (2(1H)quina-zolinones, 2,4(1H,3H)quinazoline-

dione) and 4(3H)quinazolinones quinazolinone structures, 4(3H)-quinazolinones

are most found, as intermediates or as natural products in many biosynthetic

pathways.

This is slightly due to the derivarion of the structure from the anthranilates

26

(anthranilic acid or various esters, isatoic anhydride, anthranilamide, and

anthranilonitrile) while the 2(1H)-quinazolinone is majorly a product of

anthranilonitrile or benzamides with nitriles[22].

Chemical Properties of Quinazolines

In 1957 Williamson reviewed the chemistry of quinazoline and then Lindquist in

1959 and Armarego brought it up to date in 1963.

Quinazolines is stable in cold dilute acid and alkaline solutions, when these

solutions are boiled it is dissolved. When quinazoline is boiled with hydrochloric

acid O-Aminobenzaldehyde, ammonia, and formic acid are formed.

27

Hydrolysis, Oxidation, and Reduction. Oxidation of quinazoline in dilute

aqueous acid with two equivalents of hydrogen peroxide gave 3,4-dihydro-4-oxo

quinazoline at room temperature. In alkaline medium, the anhydrous neu-tral

species of quinazoline were predominantly undergo oxidation with KMnO4 which

gave 3,4-dihydro-6 4-oxo quinazoline.

Oxidation. Catalytic hydrogenation of quinazoline stopped after the absorption of

one molecule of hydrogen and gave 3, 4-dihydro quinazoline (Scheme 3).

Reduction. Sodium amalgam when undergoing reduction with Quinazoline gave

1, 2, 3, 4-tetrahydroquinazoline. Sodium borohydride and Lithium aluminum

hydride gave 1,2,3,4-tetrahydroquinazoline and 3,4-dihydro (see Scheme 4).

Nucleophilic and Electrophilic Substitution Reactions. The two known

nucleophilic substitution reactions of quinazoline are hydrazine and sodamide

most probably proceed through the intermediate addition products, and give 4-

hydrazine quinazoline and 4-amino (see Scheme 5

28

Methods for the Synthesis of Quinazoline and Quinazolinone Derivatives

(Benzoylene Urea)

Some methods were reported for the synthesis of quinazo-lines and

quinazolinones are as follows.

From Anthranilic Acid and Urea. The fusion of anthranilic acid with urea gave

1,2,3,4-tetrahydro-2,4-dioxo-quinazoline.

From O-Ureidobenzoic Acid. The o-ureidobenzoic acids are prepared from the

29

corresponding anthranilic acid and potassium cyanate. T he ureido acids are then

easily cyclized to the respective 4-dioxoquinazolines or 1,2,3,4-tetrahydro-2 by

heating with acid or alkali (see Scheme 7).

From O-Ethoxy Carbonylaminobenzoic Esters or Amides. When o-

ethoxycarbonylamino benzamide and its 4-methyl derivatives are heated above

their melting points, then they release water and form 1,2,3,4-tetrahydro-2,4-

dioxoquinazoline (see Scheme 8).

From Phthalic Acid Derivatives. The derivatives of phthalic acid used for the

preparation of dioxoquinazoline necessitate rearrangement is of the Hoffmann

Curties type or the Lossan type. Reaction of phthalamide and phthalimide or N-

methyl and N-ethyl phthalimide with alkali hypobromite gives the 1,2,3,4-

tetrehydro 2,4-dioxoquinazoline (see Scheme 9).

From Isatins. -Isatin oxime reorders itself to 1,2,3,4-tetrahydro-2,4-

dioxoquinazoline on heating with dilute sodium hydroxide; -imino derivatives of

isatin, whereas it requires oxidation with hydrogen peroxide in basic solution to

form dioxoquinazoline (see Scheme10

30

1.3.ISOXAZOLES

Isoxazoles are an important class of heterocycles, largely used in the area of

pharmaceuticals and therapeutics such as insecticidal, antibacterial, antibiotic,

antitumour, antifungal, antituberculosis, anticancer and ulcerogenic. Derivatives of

isoxazole are used in the market as COX-2 inhibitor and anti-inflammatory drugs.

Isoxazole derivatives such as sulfamethoxazole,

sulfisoxazole, oxacillin, cycloserine and acivicin have been in commercial use for many

years. Cycloserine, one of the best antibiotic drug that possess antitubercular and

antibacterial activities and used in treatment of leprosy. Acivicin is an drug of antitumour

and antileishmania, when isoxaflutole is herbicidal drug.

Isoxazoles have illustrious history; their chemistry is associated with Ludwig Claisen,

was the first to recognize the cyclic structure of 3-methyl -5- phenylisoxazole in 1888

and was shown to possess typical properties of an aromatic system under certain

reaction conditions; particularly in alkaline medium, it is found to be very highly labile.

Dunstan and Dymond first synthesized the isoxazole ring[23]. They separated a liquid

base by heating nitroethane with aqueous alkalies to obtain 3,4,5-trimethylisoxazole. A

very significant contribution to the development of isoxazole chemistry came between

1930–1946 from Quilico’s studies on the synthesis of ring system from nitrile oxides

and unsaturated compounds[24].

31

SYNTHESIS OF FUNCTIONALIZED ISOXAZOLES

Diverse applications associated with isoxazole moiety led the researchers to develop

various novel synthetic approaches for the synthesis of isoxazole ring systems. For

instance, recent review by Ajay Kumar et al[25-27] reports the use of nitrile oxides as

versatile intermediates in the synthesis of isoxazole derivatives. Oximes when treated

with PhI(OCOCF3)2 (hypervalent iodine) gives rapid formation of nitrile oxides which

were trapped in situ with terminal and cyclic alkynes efficiently to give 3,5-disubstituted

and 3,4,5-trisubstituted isoxazoles in more product formation (Scheme-1). This method

is experimentally convenient, avoids the isolation and handling of potentially harmful

and unstable hydroximoyl chlorides[28].

Sandeep Bhosale et al[29] synthesized isoxazoles and isoxazolines with 1,3-dipolar

cyclo addition of alkenes and alkynes with nitrile oxides generated in situ by treatment

of aldoximes with

Scheme 11

Nagatoshi Nishiwaki et al[30] reported one-step synthesis of different functionalized

isoxazoles by cycloaddition of carbamoylnitrile oxide with β-keto esters. Among several

salts, magnesium acetate was found to be the most efficient promoter affording

32

isoxazole in 80% yield (Scheme-4). Carbamoylnitrile oxide generated from

nitroisoxazolone underwent inverse electron-demand 1,3-dipolar cycloaddition with

1,3-dicarbonyl compounds in the presence of magnesium acetate that formed

magnesium enolate in situ.

MagtrieveTM (CrO2) in either toluene or MeCN at 800C (Scheme-11). They observed the

formation of minor amount of deoximation product along with isoxazoles and

isoxazolines. Their methodology has been shown to be equally versatile for

intramolecular nitrile oxide cycloaddition (INOC) reactions.

Shravankumar and co-workers reported a facile catalytic approach to synthesize

regioselectively both 3,5-di- and 3,4,5-trisubstiututed isoxazoles in high yields which

involve the nucleophilic organo-NHC-catalyzed 1,3 -dipolar cycloaddition of nitrile

oxide with alkynes. Triethylamine (Et3N) was employed as an effective base to

generate both nitrile oxide and the organo-NHC in situ (Scheme-12)[31]. The

multinucleus structures like isoindole linked disubstituted isoxazoles and sterically

crowded trisubstituted isoxazoles can be accessed easily selectively by this method,

which could be useful in biology and material science.

Scheme 12

33

Scheme 13

Scheme 14

Bhaskar Chakraborty and co-workers[32] reported an aqueous phase cycloaddition

reaction. They synthesized and studied the antibacterial activities of some novel

isoxazolidine derivatives by 1,3-dipolar cycloaddition reaction of nitrones with different

dipolarophiles in water. Significant rate acceleration and high yield of these reactions

are observed in water with remarkable changes in stereo and regioselectivity compared

to organic solvents. They have provided a green synthesis avoiding use of organic

34

solvents. Stokes and co-workers[33] reported that iron (II) catalyzes the formation of N-O

bonds to transform azides into 2,1-benzisoxazoles under markedly benign conditions

(Scheme-13).

Scheme 15

Highly substituted isoxazoles can be formed in good to excellent yields using mild

reaction conditions. For instance, 3,5-disubstituted 4-halo (seleno) isoxazoles have

been synthesized by the reaction of various 2-alkyn-1-one O-methyl oximes with ICl, I2,

Br2, or PhSeBr (Scheme-15)[34].

Scheme 16

35

Rai et al[35] reported that nitrile oxides generated in situ by the oxidative

dehydrogenation of aldoximes with chloramine-T reacted with , - unsaturated

compounds to afford ethyl 3,5-diarylisoxazole-4-carboxylates which exhibited

remarkable antimicrobial activity.

In a typical reaction an equimolar mixture of aldoxime, , - unsaturated compounds and

chloramine-T trihydrate in ethanol was refluxed on a water bath for 3 hours. After the

completion of the reaction, the unusual cycloadducts were obtained in good yield. The

products formed with unusual elimination of HCN under reaction conditions (Scheme-

16)

Scheme 18

36

N-(4-(5-Arylisoxazol-3-yl)phenyl)-benzenesulfonamides were synthesized under

conventional heating and microwave irradiation (Scheme-17)[36]. The method was found

to be fast, efficient and economical. The reaction proceeded smoothly with better yields

under microwave irradiation within 5-6 minutes; while under reflux conditions it required

6-8 hrs.

A series of thirteen cycloadducts 3-Aryl-5N-aryl-4,6-dioxo-pyrrolo[3,4-d]-7,8-

dihydroisooxazolines were synthesized by the reaction of in situ generated nitrile oxides

obtained from the catalytic dehydrogenation of aldoximes with chloramine- T on N-aryl

maleimides (Scheme-9)[37]. Later they demonstrated the use of nitrile oxide as a

dipolarophile in 1,3-dipolar cycloaddition with acetyl acetone and obtained the

substituted isoxazolines in good yield. Here the nitrile oxide

37

REACTIONS OF ISOXAZOLES

Isoxazoles, isoxazolines and isoxazolidines were considered as useful synthons in

organic synthesis. They have been efficiently transformed in to various classes of

medicinally important molecules. For instance, Anthracen-9-ylmethylene-(3,4 -

dimethylisoxazol -5-yl) amine was synthesized in high yield by reaction of anthracene-

9-carbaldehyde and 5-amino-3,4-dimethylisoxazole in ethanol (Scheme-19)[38].

Isoxazoloazepines were synthesized via Michael addition followed by reductive

cyclisation. For Michael addition, a convenient and highly efficient protocol was

developed by using p-TsOH adsorbed on KSF solid support under solvent-free

conditions with a variety of Michael donors and acceptors. p-TsOH-KSF solid support

is found to be a much better alternative to effect the Michael reaction in terms of better

yields (85%) and short reaction times (2 hr). The Michael adducts underwent

reductive cyclization on treatment with SnCl2-MeOH to afford substituted

isoxazolo[4,5-b]azepines in high yields (Scheme-20)[39] .

Isoxazoloazepines are also synthesized by conducting Michael reaction in presence

of PTSA absorbed on KSF and the resulting micheal adducts are converted to

isoxazoloazepines by reductive cyclization process with SnCl2 -MeOH in a one-pot

reaction. This procedure offers significant improvement over the existing Michael

reactions. All the reactions are clean, high yielding and the method is mild and

tolerates several substituents on aromatic ring and devoid of forming any undesired

side products (Scheme-19)[40]. 3,4-disubstituted isoxazole derivatives were

synthesized from the reductive cleavages of 4,5-dihydro-7H-pyrano[3,4-c]isoxazoles.

38

1.4.BENZIMIDAZOLE

Benzimidazole is also called as benziminazole, 3-benzodiazole, azindole,

benzoglyoxaline, 3-azaindole, 1,3-diazaindene with melting point of 170-1720C and

occurs as white crystals. It is used as muscle relaxant. A group of therapeutic agents

are based on the benzimidazole nucleus; this heterocyclic system provides a unifying

theme for the subset of anthelmintic compound. Benzimidazole derivatives are of wide

interest because of their diverse biological activity and clinical applications, they are

remarkably effective compounds both with respect to their inhibitory activity and their

favorable selectivity ratio.

Chemistry of Benzimidazoles

The benzimidazole nucleus is an important pharmacophore in medicinal chemistry.

The synthesis of novel benzimidazole derivatives remains a main focus of modern

drug discovery. The versatility of new generation benzimidazole would represent a

fruitful pharmacophore for further development of better medicinal agents. Since now,

researchers have been attracted toward designing more potent benzimidazole

derivatives having wide range of biological activity.

Several benzimidazoles are commercially available as pharmaceuticals.

Benzimidazoles are most widely studied drugs as antihelmintics. Recent studies have

established that benzimidazole carbamates such as albendazole (31), mebendazole

(32), flubendazole (33), and fenbendazole (34) inhibit the in vitro growth of

Trichomonas vaginalis[41] and G. lamblia[42,43] and have a broad antiparasitic spectrum

of activity, low toxicity and have been used successfully to treat gastrointestinal

helmintic infections.

39

NH

N

NHCOOCH3

S

CH3

Albendazole

NH

N

NHCOOCH3

O

Mebendazole

NH

N

NHCOOCH3

O

F

Flubendazole

NH

N

NHCOOCH3

S

Fenbenazole

Another area of important use of benzimidazole has recently been as proton pump

inhibitor. Omeprazole (35) is appeared for the treatment and reduction of risk of

recurrence of duodenal ulcer, gastric ulcer and pathological hypersecretory conditions.

Lansoprazole (6) is used for the treatment of duodenal ulcer, and Zollinger-Ellison

syndrome.

NH

N

S

NCH3

CH3

H3CO

CH3

O NH

N

S

NCH3

F3CH2CO

O

Lansoprazole

Omeprazole (35)

40

Synthetic routes of benzimidazole nucleus:

A variety of methods have been developed for the preparation of substituted

benzimidazoles. The traditional synthesis (36) of benzimidazoles involves the reaction

between a phenylenediamine and a carboxylic acid or its derivatives under harsh

dehydrating reaction conditions[44-47].

NH

NR

NH2

NH2

RCOOH4N HCl

(36)

Subsequently, several improved protocols have been developed for the synthesis of

benzimidazoles via the condensation of o-phenylenediamines with aldehydes in the

presence of acid catalysts under various reaction conditions.

Byeong Hyo Kim et al [48] described indium-mediated reductive inter-molecular

coupling reaction of 2-nitroaniline with aromatic aldehydes to benzimidazoles (37).

NH

NAr

NH2

NO2

BNP, InArCHO

MeOH, H2O, RT N

NAr

Ar

Major

(37)

Takashi Itoh et al [49] synthesized 2-arylbenzothiazoles and imidazoles using scandium

triflate as a catalyst for both a ring closing and an oxidation steps (38).

NH

XAr

XH

NO2

ArCHORT, under O2

Sc(OTf)3 (cat.)

X=S, NH X=S: Y.97-99%X=NH: Y.72-97%

(39)

41

Donglai Yang et al [50] reported a highly efficient and versatile method for the synthesis

of benzimidazoles in one step via the Na2S2O4 reduction of o-nitroanilines by heating

a solution of o-nitro aniline and an aldehyde in EtOH or another appropriate solvent, in

the presence of aqueous or solid Na2S2O4, provided facile access to a series of 2-

substituted benzimidazoles containing a wide range of functional groups not always

compatible with the existing synthetic methods (40).

NO2

NHR'F

NO2

1 eq. R'NH2

DMSO,1000C, 10 h

R R

N

NR"

R'

1 eq. R"CHO3 eq. Na2S2O4

EtOH/DMSO (4:1)800C, 12h

R

(40)

Khodabakhsh Niknam et al [51] developed a highly selective synthesis of 2-aryl-1-

arylmethyl-1H-1,3-benzimidazoles from the reaction of o-phenylenediamines and

aromatic aldehydes in the presence of metal hydrogen sulfates [M(HSO4)n] in water

and also under solvent-free conditions in good to excellent yields (41).

NH

NAr

NH2

NH2

M(HSO4)nArCHO

(41)

Reactions of benzimidazoles

Benzimidazoles undergoes following types of reactions:

Reactions with electrophilic reagents: Preferential position of attack by electrophil

is 5th position of unsubstituted benzimidazole, and 2nd preferential position is 6th in

absence of influence by the attached substituent but if the 5-substituent is powerfully

electron releasing the second substituent enters at 4th position [52]. While an electron

42

withdrawing substituent at 5th position directs the entering electrophils to 6th position

and to a lesser extent the 7th position. Examples of electrophilic aromatic substitution

reaction are

Nitration:

N

NH

CF3

F

N

NH

CF3

F

NO2

HNO3/H2SO4

(42)

N

NH

R

O2N N

NH

R

O2N

O2N

HNO3/H2SO4

+N

NH

R

O2N

NO2

R = H, Ar

(43)

Sulfonation:

N

NH

RN

NH

R

HO3SH2SO4

R = H, Ar (44)

Electrophilic attack at the 1-(or 3-) position: Alkylation and Related Reactions:

There are four possible mechanisms for the alkylation depending on the alkylation of

substrate i.e. whether alkylated by base, an anion or conjugate acid. Alkylation of

neutral imidazoles and benzimidazoles by alkyl halides usually occurs by SE2

mechanism in which electrophilic attack is directed at the pyridine like nitrogen. Under

43

neutral conditions, the benzimidazolium intermediate reacts with unchanged

benzimidazoles[53].

Electrophilic Attack at Side-Chain Substituents:

Reactions in this category include substitution and addition processes. Some of the

reactions in this category are shown

N

NR

O H

Ph 2 CHClN

NR

O

R= alkyl, aryl

(45)

N

NH

NH2

N

NH

N=CHR

RCHO

R= alkyl, aryl (46)

44

Reactions with Nucleophilic Reagents:

Substitution in the imidazole Ring:

In this type of reaction, the benzimidazole is heated in xylene with sodium amide. For

the compounds of this type, formation of anion of hetrocyclic prohibits the nucleophilic

attack at 2- position. Various other derivatives such as 5-alkyl and thioalkyl derivatives

are formed[54].

N

N

R

N

N

R

NH2

NaNH2/Xylene

R=alkyl, aryl

(47)

1.5.IN-SILICO DRUG DESIGN

Drug discovery and development is an essential, intense, lengthy and an

interdisciplinary endeavor. Drug discovery is mostly portrayed as a linear, consecutive

process that starts with target and lead discovery, followed by lead optimization and

pre-clinical in vitro and in vivo studies to determine if such compounds satisfy a

number of pre-set criteria for initiating clinical development.

Traditionally drugs were discovered by synthesizing compounds in a time consuming

multi-step processes against battery in-vivo biological screens and further

investigating the promising candidates for the pharmacokinetic properties, metabolism

and potential toxicity. Such a development processes has resulted in high attrition

rates with failures attributed to poor pharmacokinetics (39%), lack of efficacy (30%),

animal toxicity (11%), adverse effects in humans (10%) and various commercial and

miscellaneous factors. Today, the processing of drug discovery has been

45

revolutionized with the advent of genomics, proteomics, bioinformatics and efficient

technologies like, combinatorial chemistry, high throughput screening (HTS), virtual

screening, de novo design in vitro, in silico ADMET screening and structure- based

drug design.

Computer aided drug design is an interdisciplinary of bioinformatics, medicine and

biophysics. Bioinformatics and computational methods recently were used to design

new drug candidates that could potentially bind with target proteins, thus producing

drug molecules for many disease. They also promise to speedup drug research by

predicting potential effectiveness of designed compounds prior to experimental

studies and preclinical trials.

In-silico methods can help in identifying the drug targets via bioinformatics tools. They

can also be used to analyze the target structure for possible binding/ active sites,

generate candidate molecules, check for their drug likeness, dock these molecules

with the target, rank them according to their biding affinities, further optimize the

molecules to improve binding characteristics. The use of computers and

computational methods permeates all aspects of drug discovery today which is

essential core of structure-based drug design. The use of in-silico drug design

techniques increases the chance of success in many stages of the drug discovery

process, from the identification of novel targets and elucidation of their function to the

discovery and development of lead compounds with desired properties.

Computational tools provide the advantage of delivering the new drug candidates

more quickly and at lower cost[55].

46

RATIONAL DRUG DESIGN

In-silico techniques save great amounts of time and money in R&D projects. A good

modeling support is often what makes the difference between a successful drug

design project and one that fails. With a strong background in the fields of molecular

modeling, molecular biology and computational chemistry, we are able to offer full in-

silico support for projects of drug design, protein engineering and intermolecular

recognition. The possibility of developing software to tailor the in-silico approach to

different problems is what makes us unique.

TECHNIQUES

Molecular Docking and Virtual Screening: Docking studies are computational

techniques for the exploration of the possible binding modes of a substrate to a given

receptor, enzyme or other binding site. Docking is the process by which two

molecules fit together in 3D space. Docking studies may help to increase ligand

specificity; and also better therapeutic index can be achieved if the drug produces

undesirable side effects due to its binding with another site, the affinity for that

competing site can be diminished. Different types of docking include- flexible protein-

ligand docking, flexible protein-protein docking and hydrophobic docking. Docking

may play an important role in the QSAR studies and homology modeling very useful in

structure based drug design. Various docking programs are available DOCK, FLOG,

ADAM, and UGIN.

47

Molecular Dynamics: The prediction of the evolution of molecular systems

over time, the study of protein conformation, protein-protein interactions, the

simulation of biological membranes.

Quantum Mechanics: The study of chemical reactions, the effects of

substitutions on electronic properties and reactivity of molecules.

QSAR: Quantitative structure-activity relationship. The ability of predicting

biological properties of molecules without even the need of knowing their target.

Homology Modelling: Predicting the structures of proteins that has not been

yet crystallized.

DOCKING STUDIES:

The ability to propose reasonable binding modes of a designed structure to a known

receptor site called docking studies, which is crucial to the success of structure based

design. One approach is to dock or position ligand or receptor molecules together in

many different possible ways and then scores each orientation according to an

evaluation function of some kind. These studies can predict binding confirmations and

affinities of millions of molecules without the need of a single synthetic step. These

rational drug design methods accelerate the process by speeding up the discovery of

new chemical substances that may become a new drug.

DRUG-LIKENESS AND LEAD-LIKENESS

Christopher A. Lipinski[56] defined the Drug likeness as the compounds those have

sufficiently acceptable absorption, distribution, metabolism and elimination properties

48

to get successful entry in to human Phase 1 clinical trials. For the drug development,

drug properties are important prominent component. A chemically synthesized

compound library can contain many non-drug-like compounds. Therefore, recent

technologies helped to develop recognized drug-like compounds from a diverse

compound library[57-62]. These drug-like measuring and filtering technologies have

partly solved the screening problems. However, they have not been good enough to

completely solve these problems. It has been observed that many drug-like

compounds, which should be potential candidates; do not come up as hits when they

are screened against biological targets. Drug-likeness is the descriptors of all

important pharmacological properties such as potency, selectivity toward receptor,

absorption, distribution, metabolism and toxicity. In the past, these parameters were

optimized sequentially. Now, it is mandatory that these parameters should be

optimized simultaneously. Properties that have been associated with oral drug-

likeness include:

Oral bioavailability

Appropriate toxicity to pass phase I clinical trials.

Aqueous solubility

Synthetics accessibility

Pharmacokinetic viability

Blood-brain barrier permeability.

49

Lipophilicity is a key property for pharmacological activity in drug discovery and used

to estimate the permeability of a drug molecule in the cell membrane. It is measured

as logP value that distribution coefficient of compounds between n-octanol and water.

When logP value is very low or very high, the permeability of drug components get

dropped due to the inability of weakly lipophilic compounds to penetrate the lipid

portion of the membrane and the excessive partitioning of strongly lipophilic

compounds into the lipid portion of the membrane and their subsequent inability to

pass through the aqueous portion of the membrane.

Lipinski's rule helps to predict the poor absorption and permeability of potential drug

candidates. It will occur if,

A molecular weight less than 500.

An octanol-water partition coefficient log P of less than 5.

Molar refractivity not more than 150

Not more than 5 hydrogen bond donors (nitrogen or oxygen atoms with one or

more hydrogen atoms)

Not more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms).

NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used

therapeutics, primarily for the treatment of inflammation, especially arthritis[63]. In

addition to their anti-inflammatory effects, agents belonging to the NSAID class

possess both analgesic and antipyretic activities. Hence, NSAIDs are sometimes

referred to as non-narcotic analgesics or as aspirin-like drugs[64]. They provide

symptomatic relief from pain and swelling in chronic joint disease such as occurs in

50

osteo- and rheumatoid arthritis, and in more acute inflammatory conditions such as

sports injuries, fractures, sprains and other soft tissue injuries. They also provide relief

from postoperative, dental and menstrual pain, and from the pain of headaches and

migraine.

Pharmacological Actions:

All the NSAIDs have actions very similar to those of aspirin. The three main

therapeutic effects are an anti-inflammatory effect: modification of the inflammatory

reaction, an analgesic effect: reduction of certain types of (especially inflammatory)

pain and an antipyretic effect: lowering of body temperature when this is raised in

disease (i.e. fever).

In addition, all the NSAIDs share, to a greater or lesser degree, the same types of

mechanism-based side effects. These include:

gastric irritation, which may range from simple discomfort to ulcer formation

an effect on renal blood flow in the compromised kidney

a tendency to prolong bleeding through inhibition of platelet function.

Controversially, it is argued that they may also all-but especially COX-2 selective

drugs-increase the likelihood of thrombotic events such as myocardial infarction by

inhibiting prostaglandin (PG I2) synthesis.

A number of aryl and heteroaryl substituted compounds such as Diclofenac42 (1),

Lumiracoxib[65] (2), Lonazolac (3), Etodolac[66] (4) have been commercialized as non-

steroidal anti-inflammatory drugs (NSAIDS).

51

Important Non-Steroidal Anti-inflammatory Drugs:

COOH

C H 3

F

SCH 3 O

Diclofenac (48) Indomethacin (49) Sulindac (50)

PROFEN DERIVATIVES:

CH3

CH3

COOH

CH3

COOH

CH3

F

Ibuprofen (51) Flubiprofen (52)

COOH

CH3

O

H3CO

COOH

CH3

Ketoprofen (53) Naproxen (54)

NH

COOH

ClCl

52

OXICAMES:

NS

NH N

O

O H

OOC H 3 N

S

NH N

O

O H

OOC H 3

S

Piroxicam (55) Tenoxicam (56)

Others:

CH3

N

COOHCH3ON

O

COOH

Tolmetin (57) Ketorolac (58)

Selective COX-2 Inhibitors:

SO 2 NH 2

NN

CH 3

CF 3

SO 2 CH 3

O

O

Celecoxib (59) Rofecoxib (60)

COOH

N H

C l F

CH 3

(61) Lumiracoxib

NN

COOHC l

NH

O

C H 3

C H 3

COOH

Lonazolac (62) Etodolac (63)

53

While there are differences between individual drugs, all these effects are generally

thought to be related to the primary action of the drugs-inhibition of the fatty acid COX

enzyme, and thus inhibition of the production of prostaglandins and thromboxanes.

There are three known isoforms-COX-1, COX-2 and COX-3-as well as some non-

catalytic species. As it is not yet certain that COX-3 actually occurs in humans in a

functional form, we will confine the discussion mainly to a consideration of COX-1 and

COX-2. While they are closely related (> 60% sequence identity) and catalyse the

same reaction, it is clear that there are important differences between the expression

and role of these two isoforms. COX-1 is a constitutive enzyme expressed in most

tissues, including blood platelets. It has a 'housekeeping' role in the body, being

involved in tissue homeostasis, and is responsible for the production of prostaglandins

involved in, for example, gastric cytoprotection, platelet aggregation, renal blood flow

autoregulation and the initiation of parturition).

In contrast, COX-2 is induced in inflammatory cells when they are activated, and the

primary inflammatory cytokines-interleukin (IL)-1 and tumour necrosis factor (TNF)-α

are important in this regard. Thus the COX-2 isoform is responsible for the production

of the prostanoid mediators of inflammation, although there are some significant

exceptions. For example, there is a considerable pool of 'constitutive' COX-2 present

in the central nervous system (CNS) and some other tissues, although its function is

not yet completely clear.

Although, non-steroidal anti-inflammatory drugs (NSAIDS) have been used in the

treatment of various inflammatory diseases, their usage is limited by the side effects

54

produced by them, thereby necessitating the need for searching new molecular

entities[67].

Anti bacterial activity:

The emergence of resistance to the major classes of antibacterial agent is recognized

as a significant medical crisis and serious health concern. Particularly, the emergence

of multi drug-resistance strains of Gram-positive bacterial pathogens is a problem of

ever increasing significance. As the limited number of antimicrobial classes and the

common occurrence of resistance within and between classes, the search for

antibacterial agents with novel mechanism of actions is always remains an important

and challenging task.

The control of microorganism is critical for the prevention and treatment of disease.

Microorganisms also grow on and within other organism, and microbial colonization

can lead to disease, disability, and death. Thus the control or destruction of

microorganisms residing within the bodies of humans and other animals is great

importance.

Modern medicine is dependant on chemotherapeutic agents, chemical agents that are

used to treat disease. Chemotherapeutic agents destroy pathogenic microorganisms

or inhibit their growth at concentrations low enough to avoid undesirable damage to

the host. Most of these agents are antibiotics, microbial products or their derivatives

that can kill susceptible microorganisms or inhibit their growth. Drugs such as the

sulfonamides are sometimes called antibiotics although they are synthetic

chemotherapeutic agents, not microbially synthesized.

55

Antibiotics are chemical substances excreted by some microorganism which inhibit

the growth and development of other microbes. Some of these drugs that were

obtained naturally were put to chemical modifications in attempts to enhance

beneficial effects while minimizing the toxic effects. The resultant modified product is

termed as semi synthetic antibiotics. Most antibiotic currently used are semi synthetic.

The chemist has synthesized many drugs that have got the antibacterial property and

less toxicity. These drugs are called synthetic antibiotic drugs. Naturally occurring

antibiotic, their semisynthetic derivatives and synthetic antibiotics have got the same

target. i.e., antimicrobial action. Hence all these drugs were put together to be called

antimicrobial agents.

Drug resistance:

The emergence of drug resistance bacteria is posing a major problem in antimicrobial

therapy. The frequency varies with the organism and the antibiotic used. At first, there

is an emergence of a small number of drug resistant bacteria which sooner multiplies

selectively in the presence of the drug at the cost of sensitive bacteria.

Types of drug resistance:

Drug resistance is of two types, primary and acquired.

1. Primary resistance: some bacteria possess an innate property of resistance to certain

drug, e.g. resistance of E.coli to penicillin.

2. Acquired resistance: it results either from mutation or gene transfer.

56

Recent targets for finding antibacterial agents

Beta-Ketoacyl-acyl carrier protein (KAS) synthase III encoded by the fabH gene is

thought to catalyze the first elongation reaction of type II fatty acid synthesis in

bacteria and plant plastids. Beta-ketoacyl-acyl carrier protein synthase (KAS) I is

important enzyme system for the construction of the unsaturated fatty acid carbon

skeletons characterizing E. coli membrane lipids. Recent research reported that Type

II fatty acid synthesis (FAS II) pathway is an attractive targete for their efficacy against

infections caused by multi-resistant Gram-positive bacteria and Gram-negative

bacteria[68]. Among the related FAS II enzymes, beta ketoacyl-acyl carrier protein

synthase (KAS) is an essential target for novel antibacterial drug design[69,70].

The enzyme bacterial peptide deformylase (PDF) is another novel target for novel

antibacterial agents. The metalloproteases enzyme, Bacterial peptide deformylase

(PDF) deformylates the N-formyl methionine of newly synthesized

polypeptides through Fe2+-mediated catalytic reaction. PDF is essential in prokaryotes

and this enzyme is absent in mammalian cells and provides a unique target for

antimicrobial chemotherapy[71-74]. Thus, it may be another target for new

chemotherapeutic agents.

Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-

negative bacteria and are therefore essential for cell growth and viability. The

glycosyltransferase (GT) enzyme, heptosyltransferase WaaC involved in the synthesis

of the inner core region of lipopolysaccharides. It catalyzes the addition of the first l-

glycero-d-manno-heptose molecule to one molecule of 3-deoxy-d-manno-oct-2-

ulosonic acid (Kdo) residue of the Kdo2-lipid A molecule. These heptose is an

57

essential component of the Lipopolysaccharides core domain; its absence results in a

truncated lipopolysaccharide associated with the deep-rough phenotype causing a

greater susceptibility to antibiotic. Thus, WaaC represents a promising target in

antibacterial drug design[75].

Anti fungal activity:

The object of antifungal drug discovery has become a subject of greater challenge

due to increasing incidences of fungal drug resistance. This appears due largely to the

extensive use of antifungal agents to treat fungal infections. In the past decade,

number of patients diagnosed with fungal infections have increased drastically,

whereas, relatively very few clinically useful drugs were discovered. The azole

derivatives such as such as clotrimazole, fluconazole, itraconazole, ketoconazole, etc.

have been widely used to treat a verity of fungal infections. These azole derivatives

inhibit the fungal enzyme 14-alpha demethylase which is essential for the ergosterol

synthesis pathway leads to the depletion of this steroidal compound in the cell

membrane and accumulation of toxic intermediate sterols, leads increased membrane

permeability and inhibition of fungal growth[76-78]. But broad usage of these drugs led

to development of acquired resistance especially among Candida albicans. Thus,

searching not only improved version of existing drug but also for new drug targets has

become an urgent need[79].

Recent reports showed that 2-glutamine, D-fructose-6-phosphate aminotransferase

known as a new target for antifungals, it catalyzes a complex reaction involving

ammonia transfer from L-glutamine to fructose-6-phosphate, followed by isomerisation

of the formed fructosamine-6-phosphate to glucosamine-6-phosphate[80].

58

Antioxidant activity:

The knowledge of free radicals and reactive oxygen species is producing a revolution

in the field of medicine that promises a new age of health and disease management.

The formation and activity of a number of compounds, known as reactive oxygen

species, which have a tendency to donate oxygen to other substances are producing

various potential harmful effects. Evidences show that free radical damage contributes

to the etiology of many chronic health problems such as cardiovascular and

inflammatory disease, cataract and cancer. Antioxidants can prevent free radical

induced tissue damage by preventing the formation of radicals, scavenging them, or

by promoting their decomposition[81-83]

59

2. LITERATURE REVIEW

2.1.Pyrazoline Derivatives

Sahu SK et al [82] have been synthesized and evaluated for analgesic, anti-

inflammatory and antimicrobial activities of some novel pyrazoline derivatives. The

presence of 4-NO2, 2-OH and 4-Cl in phenyl ring at 5-position of pyrazoline ring of

synthesized compounds observed increase in analgesic, anti-inflammatory and

antimicrobial activities.

OH

NHNH

N

R

Mai E. Shoman et al [83] synthesized some new pyrazoline derivatives and screened

for anti-inflammatory activity and ulcerogenic activity. Most of the derivatives showed

significant anti-inflammatory activity and also reported that the incorporation of the

NO-donating group into the parent pyrazoline derivatives causes a non-significant

reduction in the anti-inflammatory activity while a marked decrease in gastric

ulcerations.

NN

R1

R2

NH

OCH3

NOH

Chimenti F et al [84] reported the synthesis and in vitro selective anti-helicobacter pylori

60

2.1.1Activity of pyrazoline derivatives.

R1N N

R3

R2

Moged AB and EvelinBM [85] reported the convergent synthesis and antibacterial

activity of pyrazole and pyrazoline derivatives of diazepam.

N

N

O

NN

CH3

Cl

NH2R2

R1

Sherif AF Rostom [86] has reported that some pyrazoline derivatives as potential

antitumor agents. From the study, it has been reported that few compounds were

reported as a significant broad spectrum of antitumor potential against most of the

tested subpanel tumor cell lines at the GI50 and TGI levels, together with a mild

cytotoxic (LC50) activity.

NN

N

N

NH2

Cl

OH

O N

61

Balakrishna kallurya et al. synthesized a series of some novel-1(substituted-

2pyrimidyl)-3-methyl-4-(arylhydrazono)-2-pyrazolin-5-ones (164) and evaluated their

antimicrobial activity against E.coli and Serratia marcesens and for antifungal activity

against Aspergillus niger and pencillium. Most of tested compounds showed

significant antifungal activity particularly against penicillium at 10µg/ml concentration

and comparable with that of standard drug Fluconazole[87].

NHN

N

N

CH3

NN

O

R1

R

(164)

a) R=4,6-dimethyl R’ = 4-chloro , b) R=4,6-dimethyl R’=2-chloro

c) R=4,6-dimethyl R’=4bromo, d)R=4,6-dimethyl R’=4-nitro

R.B Gaikar et al. synthesized some biologically active pyrazolones (65). These

compounds were synthesized by conventional as well as by ultrasound irradiation.

Synthesized compounds were characterized by H NMR, mass and IR spectral

techniques. They were also screened for antimicrobial activity[88].

O

R 3

R 2

R 1

O

N

NO

C H 3

N

(65)

62

R1 R2 R3 R1 R2 R3 R1 R2 R3

a) H H H, b) H H Me, c) H Me H,

d) H Cl H, e) H Cl Me.

Sarangapani et al. synthesized pharmacological evaluation of 3- methyl -4-(oxindol-3-

ylidenyl)-5-pyrazolones (66). The test compounds showed CNS depression, reduced

locomotor activity. All the compounds showed antibacterial activity against test

organisms employed at the concentration of 100 μg/ml and 300 μg/ml. Compound

with 5-methyl group on indolinone and 3- methyl group in pyrazole showed more

antibacterial activity among all the test compounds[89].

N

NO

CH3

R 1

NH

O

R

(66) R = 5-methyl, R1= 3-methyl

2.1.2. Analgesic and anti inflammatory activity:

Dabholkar et al. synthesized of some substituted pyrazolones (67). The final desired

compounds have been synthesized for analgesic and anti inflammatory activity[90].

NHN

N

NO

CH3

NHS NH2

R

(68) R=H, 4-chloro 2-nitro, 3- nitro, 4-nitro

63

Similarly, Gill et al. synthesized 3- methyl-4-[(chloromono-3-yl) methylene]-1-(4-

nitrophenyl) pyrazolin-5-(4H)-ones (69) and 3-methyl -4-[(1, 3, diphenyl-1H – pyrazol-

4-yl)methylene]-1-(4-nitro phenyl) pyrazolin 5 (4H)-ones (70) for the anti inflammatory

activity [91].

O

O

N

NO

CH3

NO2R1

R2

R3

N

NO

CH3

NO2

N

N

(71) (72)

R1 R2 R3 Ar

a) H H Cl 4-chlorophenyl

b) H Me H 4-methylphenyl

c) Me H Me 4-bromophenyl

d) H Me Cl phenyl

e) H H Br 2,4-dichloro-5-flourophenyl

f)Cl H Cl furyl

2.1.3. Antifungal activity:

Urmila gupta et al. synthesized antifungal activity of new fluorine containing 4-

(substituted phenylazo) pyrazoles (73) and isoxazoles (74). The structure of these

compounds has been confirmed on the basis of elemental analysis and spectral

studies[92].

64

N

N

R1

N

NR

R11

N

O

R1

N

NR

(73) (74)

R= 2-CF3, 2-F, 4-F, 2-Cl, 5-CF3. R’= CH3, CF3,

R’’= C6H5, -COC6H5, -2,4 di –NO2 C6H3

2.1.4. Anti-oxidant activity:

Bharathi. K et al. synthesized an antioxidant activity of substituted 1-acetyl-5-

(substituted phenyl)-3-(aminophenyl)-2-pyrazolines (75) and 5-(substituted phenyl)-3-

(aminophenyl)-2-isoxazolines (76). The synthesized compounds were tested for in

vitro antioxidant properties Viz., Nitric oxide scavenging, DPPH scavenging and

inhibition of iron induced lipid peroxidation in rat brain homogenate. Among the

pyrazoline derivative, bromo vanillinyl & 5-iodo vanillinyl derivative showed good

scavenging of DPPH free radical and inhibition of lipid peroxidation respectively and

among the isoxazoline derivatives. The 5-iodo vanillinyl derivative showed significant

scavenging of nitric oxide, DPPH and inhibition of lipid peroxidation[93].

(75) (76)

R= 5-Br , 4-OH, 3-OCH3 R= 5-I, 4-OH, 3-OCH3

N

HN

N

COCH3

R

N O

HN

R

65

2.1.5. Anti viral activity:

The pyrazolone scaffold, predicted by a computational modeling study using GS-

9137(2) as a pharmacophoric model, has shown to inhibit the integrase catalytic

activities in low micromolar range. Hadi et al. have synthesized various analogs (77

and 78) based on the pyrazolone scaffold and performed SAR studies. This work has

a showcase the up-to-date result of this scaffold as a promising HIV-1 integrase

inhibitor[94].

N

N

O

Br

CF3

O

O

F

ClN

N

O

Br

CF3

O

O

O

(77) (78)

A series of pyrazolone compounds as possible SARS-CoV 3CL protease inhibitors

were designed, synthesized, and evaluated by in vitro protease assay using

fluorogenic substrate peptide in which several showed potent inhibition against the

3CL protease. Interestingly, one of the inhibitors (79) was also active against 3C

protease from coxsackievirus B3. These inhibitors could be potentially developed into

anticoronaviral and anti-picornaviral agents[95].

N

N

OHOOC

NO2 (79)

66

2.2.Quinazoline Derivatives

S.K. Pandey et al[96]. have been reported three series of novel and new fused

heterocyclic system. All the synthesized compounds have been screened for their

antibacterial activity against Gram-negative bacteria, and Gram-positive bacteria, as

well as demonstrated significant antifungal activity against fungi. They concluded that

the presence of triazole nucleus at N-1 and C-2 of quinazolinone ring works better for

antimicrobial activity of this series of compounds than others[98].

N

N

O

N NH

N

S

R

R

N

N

O

N

N

R

R

N

N

O

N

N

N

M.M. Aly et al [97] have been reported the synthesis and charecterization of Novel

quinazolinone, and thio semicarbazone derivatives for their potential anticonvulsant,

analgesic, cytotoxic as well as their antimicrobial activities. It was found that

compound which has a thio- semicarbazone side chain at C-2 position ending with a

free amino group and fluorine atom, showed activities as anticonvulsant, analgesic

and cytotoxic.

N

N

OF

NNH

NH

S

Ar

I

67

ANTICONVULSANT AGENTS

S.K. Kashaw et al. [98] has been reported the synthesis of several new 1-(4-

substituted-phenyl)-3-(4-oxo-2-phenyl/ethyl-4H-quinazolin-3-yl)-urea for

anticonvulsant activity CNS depressant and sedative-hypnotic activity. They reported

that all the compounds were found to exhibit potent CNS depressants activity as

indicated by increased immobility time and concluded that newly synthesized

compounds possessed promising CNS activities.

N

N

O

NH

OHN

R1

V. Jatav et al. [99] have been reported the synthesis and evaluation of a series

of new quinazoline-4(3H)-ones for anticonvulsant, sedative hypnotic and CNS

depression activities. In this study out of 18 compounds only few showed

anticonvulsant activity in one or more test models. All except two compounds

exhibited significant sedative-hypnotic activity via actophotometer screen.

N

N

O

S

NN

Cl

Padam kant, and R.K. Saksena et al have been reported the synthesis and anti-

microbial activity of some new 2-phenyl-3- (2-methyl-3-aryl-4-oxathiazolin-2-yl) phenyl

quinazolin-4-ones and 2-phenyl-3-p-(I-aryl-3phthalimido-4-methylazetidin-2' one-2' -y')

phenyl-quinazolin-4-ones [100]

68

Agar plate diffusion technique was employed for the determination of

antibacterial activity of the synthesized compounds and against B. pumillis, B.

subtilllis, S. aureus, and S. lutea. The compounds were generally inactive. Most of the

compounds have shown moderate to good activity against four species of fungi A.

niger, A. terreus, p.fungiculosum, C. lunata.

A series of 1-deoxy-l-(α-substituted-3 amino/methylamino-4quinazolone)-D-

fructose have been synthesized by Nautiyal et a1 [101] and evaluated the effect of

aldose and amine linkage on central nervous systems. These compounds showed

higher anticonvulsant and analgesic activity as compared to the reference compound.

Hepato protective efficacy of synthetic quinazolines have been studied by Aruna

and co-workers [102] evaluating their inhibitory effects on CCl4 induced microsomal lipid

peroxidation and scavenging of hydroxyl radical formation and the compounds

showed comparable protective potential with standard liver protective compound

namely silymarin.

Synthesis of 2-aryl-5-[3'-(2' -methyl-6,8-dunstituted quinazolyl)-phenyl]-pyrazole

derivatives have been reported to possess antiviral activity against plant and animal

viruses by pandey and co-workers [103]. Few of the compounds showed maximum

activity against plant viruses' in-vitro and showed moderate antiviral activity against

chick embryo system. New 2, 3 disubstitued quinazolinone derivatives have been

synthesized and were evaluated for their anti-inflammatory activity by Tyagi et al [104].

2-methyl -3[5'- (3,6-dichlorophenyl)-2-triazolinyl]-4(3H)-quinazolinone showed 51%

anti inflammatory activity as compared to phenylbutazone (ED50 dose).

69

Anti -HIV activity of several 1-[2-phenyl-4 (4H)-oxo-3-quinazolinyl]-2-methyl-4-

arylidene-5-oxoimidazolines, 2-pheny-3- (arylamino)-4(4H)oxoquinazolines, and N1-

2mcthyl-4(4H)-oxo-3-quinazolinyl-N2-arylthiourcas has been reported by Desai and

co-workers [105]. Inhibition of HIV reverse transcriptase by 6-chloro-4 (s) cyclopropyl-

3,4-dihydro-4-[(2-pyridyl)ethynyl]-quinazolin-2(lH)-one have been reported by carroll

and coworkers[106].

3-substituted-4(3H)-quinazolinones were synthesized and studied for structure

activity relationship by Eartoli et a1[107]. These compounds displayed higher invitro

activities against filam entons fungi and shorter half-life.

Several quinazoline derivatives containing substituted thio semi carbazido and

S-methyl isothiosemicarbazido groups at the 2-position and at both the 2 & 4th

positions have been synthesized and evaluated for antitoxoplasmosis effect [108].

New 6--chloro-2,3-dihydro-4(lH)-quinazolinones have been synthesized and

evaluated for gastrointestinal prokinetic and anti-emetic activities in comparison with

structurally related benzamides and 6-chloro 2,3-dihydro- (lH)-l,3-benzoxazin-4-ones

[109].

2-[4-(3-Tert butyl; amino)-2-hydroxy propoxy)- phenyl-3 methyl-6 methoxy-

4(3H)-quinazolinone was evaluated as a selective β-1 adreno receptor ligand for

Positron Emission tomograph (PET) by Valette and coworkers [110]. A series of 2-alkyl

and 2-aryl substituted 8-hydroxy, 8-methoxy and 8-methyl quinazolin-4(3H)-ones have

been synthesized and evaluated for poly (ADP-ribose) polymerase (PARP) inhibitory

activity in permeabilized L 1210 murine leukemia cells 8-methoxy and 8-methyl

quinazolinones were synthesized by acetylation of 3-substituted anthranilamides with

70

appropriate acid chloride followed by base catalyzed cyclization. 2-Phenyl

Quinazolinoes were marginally less potent than the corresponding 2-methyl

quinazolinones, but the introduction of an electron withdrawing group or electron

donating group 4' -substituted on the 2-aryl ring invariably increased potency. Among

the synthesized compounds, 8-hydroxy or 8-methyl substituent enhanced inhibitory

activity in comparison to an 8-methoxy group [111].

Recently, Molnar-Kimber et a1., [112] and Duplainer et al.,[113], have reported a

quinazolinone derivative, Nitraqazone to possess potent phosphodiesterase inhibitory

activity that is potentially useful in treatment of asthma.

Raghu Ram Rao and co-workers [114] studied broncho dilatory action of 6-

arylbenzimidazo- (1,2-c-quinazoline).

Several other 2-substituted -4 (3H)-Quinazolineone derivatives reported to possess

various biological properties like calcium antagonist [115-119], poly (ADP-ribose)

synthetase inhibitor [120], immunotropic [121], non-steroidal anti-inflammatory [122,123],

antimicrobial [124,125]

71

2.3.Isoxazole Derivatives

K. Manna et al [124] synthesized benzofuran isoxazole derivatives and structures have

been established on the basis of spectral (IR, H NMR, Mass) data. The synthesized

benzofuran isoxazoles were evaluated for their antitubercular, antimicrobial & anti-

inflammatory activities. Some of the compounds have shown promising anti-tubercular

& antibacterial activity.

Prabodh chander sharma et al[125] synthesized new isoxazole derivatives. The

isoxazole have been reported to posses a variety of significant & diverse

pharmacological activities such as antifungal anti-HIV, antibacterial, caspose

inhibitory, antimuscarinic, anti-inflammatory, anticancer, antidepressant activity.

Y.Rajendra Prasad et al [126]synthesized antidepressant activity of some new 2-

isoxazoles. From the study, it was found that the compounds processing electron

releasing groups such as dimethyl amino, methoxy & hydroxyl substituent on both the

rings at position 3 and 5 of isoxazolines, considerably enhanced the antidepressant

activity when compared to the isoxazolines having no substitutions on the rings

O

N O

R

NH

N O

R

72

NO

OHR1

R2

R3

R4

Georgiev et al. synthesized antifungal agents on substituted 3,5-diphenyl-3-(1H-

imidazol-yl methyl)-2-alkylisoxazolidine derivatives (80). The compounds when tested

invitro in solid agar cultures exerted a very potent antifungal activity against a wide

variety of yeast and systemic mycoses and dermatophytes especially Trichophyton &

Microsporum sp., Epidermophtton floccosum and Candida stellatoidea. The in vitro

activity against Aspergillus fumigates and Candida albicans was moderate potent.

When tested in vivo in the at vaginal candidiasis model, derivative (80), although

showing significant antifungal activity when compared to controls, was less effective

than ketoconazole[127].

(80)

R1 R2 R3 R1 R2 R3

4-Cl H 4-Cl 4-Cl H 2-Cl

N

O

H22CR

R3

N N

R1

73

K. Manna et al. synthesized the structure of benzofuran isoxazoles (81) have been

established on the basis of spectral (IR, H1NMR, Mass) data. The benzofuran

isoxazoles were evaluated for their antitubercular, antimicrobial and anti-inflammatory

activities. Some of the compounds have shown promising anti-tubercular &

antibacterial activity[128].

(81)

a) -OH(o), b) -OCH3(o), c) -NCH3(p),d) -COOH(o), e) -NO2(m)

f) -OH(o), g) -OCH3(p), h) -OH(p), i) -Cl(p), j) –Cl(o),

k) -NO2(o), l) -OCH3(p), m) -H, n) Furan ring, o)–CH=CH-Ar

Prabodh chander sharma et al. synthesized new isoxazole derivatives (152). The

isoxazole have been reported to posses a variety of significant and diverse

pharmacological activities such as antifungal anti-HIV, antibacterial, caspose

inhibitory, antimuscarinic, anti-inflammatory, anticancer, antidepressant activity[129].

(82) R= H, -4-OH,-3-NO2, -3-OCH3, -4-N(CH3)2

O

N O

R

NH

N O

R

74

2.3.1. Antidepressant activity:

Y.Rajendra Prasad et al. synthesized antidepressant activity of some new 3-

(2’’-hydroxy naphthalien-1-yl)-5-phenyl-2-isoxazoles (83). All these compounds were

characterized by means of their IR, 1H NMR spectroscopic data and microanalyses.

The antidepressant activity of these compounds was evaluated by the porsolt

bevavioural despair test on swiss-webster mice. It was found that the compounds

processing electron releasing groups such as dimethyl amino, methoxy and hydroxyl

substituent on both the rings at position 3 and 5 of isoxazolines, considerably

enhanced the antidepressant activity when compared to the isoxazolines having no

substitutions on the rings[130].

NO

OHR 1

R 2

R 3

R 4 (83)

R1 R2 R3 R4

a ) -OCH3 -H -OCH3 -CH3

b) -Cl -H -Cl -H

c) -H -H -CH3 -H

d) -H -OCH3 -OCH3 -H

e) -H -H -N(CH3)2 -H

Pae et al. synthesized some isoxazoline and isoxazole derivatives (84) as 5-HT2A & 5-

HT2C receptor ligands[131].

75

(84)

R1=3,5 dichloro; n=(1); R= o (methyl)

2.3.2. Anti-viral activity:

Guy D. Diana synthesized structure activity studies of some disubstituted phenyl

isoxazole (85) against human picornavirus. A QSAR study revealed that the mean

MIC against 5 serotypes correlated with logP , The 2,6 dichloro analogue was highly

effective in-vitro against rhinoviruses with an MIC80 of 0.3µm as well as several

enteroviruses and also effective in preventing paralysis in mice infected with

coxsackievirus[132].

(85)

Iris H.hall et al. synthesized cytotoxic action of 3,5-isoxazolidinediones & 2-isoxazolin-

5-ones in murine and human tumors[133].

S

O

O

N N(..)

n

N

O

R1

R2

O

N

H3C

(CH2)5O

O

N

Cl

Cl

76

O

NH

O

O

R1

R1

O

NR1

R1

O

O

R2

IRIS H. HALL

(86) R1=Et, R2= 3,4,5-(MeO)3PhCO

2.4.Benzimidazole Derivatives

Benzimidazole is also called as benziminazole, 3-benzodiazole, azindole,

benzoglyoxaline, 3-azaindole, 1,3-diazaindene with melting point of 170-1720C and

occurs as white crystals. It is used as muscle relaxant[134]. A group of therapeutic

agents are based on the benzimidazole nucleus; this heterocyclic system provides a

unifying theme for the subset of anthelmintic compound[135]. Benzimidazole derivatives

are of wide interest because of their diverse biological activity and clinical

applications, they are remarkably effective compounds both with respect to their

inhibitory activity and their favorable selectivity ratio[136].

Benzimidazoles are regarded as a promising class of bioactive heterocyclic

compounds that exhibit a range of biological activities. Specifically, this nucleus is a

constituent of vitamin-B12[137]. Several benzimidazoles are commercially available as

pharmaceuticals. Benzimidazoles are most widely studied drugs as anthelmintic.

2.4.1. Benzimidazole as COX inhibitors:

A series of 2-[[(2-alkoxy-6-pentadecylphenyl) methyl] thio]-1H-

benzimidazoles/benzothiazoles and benzoxazoles (87) from anacardic acid and

investigated their ability to inhibit human cyclooxygenase-2 enzyme (COX-2). The

active compounds were screened for cyclooxygenase-1 (COX-1) inhibition.

77

Compound having R, R1 = CH3, is 384-fold and 19 is more than 470-fold selective

towards COX-2 compared to COX-1. Thus, this class of compounds may serve as

excellent candidates for selective COX-2 inhibition[138].

NH

N

S

R''

OR

H31C15

(87)

R = Me, R’ = H, R = Me, R’ = OMe,

R = Me, R’ = OCHF2, R = Me, R’ = Me,

R = Me, R’ = NO2, R = H, R’ = OMe,

R = Et, R’ = H, R = Et, R’ = OMe,

2.1.2. Antiviral activity:

A serious of 2-pyridyl-1H-benzimidazole-4-(N-carboximide) derivatives (88-89) are

reported as a antiviral activity against COX Sackie virus B3, a non-enveloped single

positive-strand RNA virus belonging to the picornaviridea family, which is the major

cause of virus-induced human myocarditis[139].

N

NN

ON

NN

O

F

(88) (89)

78

Ashish kumar Tewari et al. synthesized two series of N-substituted -2- substituted

benzimidazole derivatives , viz. 1-benzyl-2- substituted benzimidazole 90 (a-e) and 1-

(p- chlorophenyl )-2- substituted benzimidazole 90 (f-j) and tested for their anti-viral

activities. These compounds have been screened for Tobacco mosaic viruses and

Sunhemp rosette viruses and showed significant activities[140].

N

N

R

R'

(90)

75a) R= CH2-CH2COOH, R’= -CH2C6H5 75g) R= C6H4OH (O), R’= -C6H4Cl

75b) R= C6H4OH (O), R’= -CH2C6H5 75h) R= C6H4OH (O), R’= -C6H4Cl

75c) R= -CH=CH-C6H5, R’= -CH2C6H575i) R= C6H4OH (O), R’= -C6H4Cl

75d) R= CH (OH) CH (OH) COOH, R’= -CH2C6H5

75e) R= C6H4 COOH (O), R’= -CH2C6H5 75j) R= C6H4OH (O), R’= -C6H4Cl

75f) R= CH2-CH2COOH, R’= -C6H4Cl

2.4.2. Angiotensin-II Antagonist:

5-Nitro and 5-amino benzimidazole derivatives with varying substitutions at 2-position

of benzimidazole and compounds 91 (a-c) have been reported as good angiotensin II

antagonistic activity[141].

79

N

N

C 4H 9

R

HOOC

(91a R=H, 91b R= NO2 ,91c R= NH2)

Recently a series of 6-substituted amino carbonyl benzimidazole derivatives were

designed and synthesized as non-peptidic angiotensin II AT1 receptor antagonist.

From the preliminary studies, compounds (92) and (93) were selected for antagonistic

activity in isolated rabbit aortic strip, among the synthesized compounds (94) was

found to be more active as AT1 receptor antagonist with low toxicity[142].

NH

CH3

N

N

CH3

O

OCH3

HOOC

O

CH3

NH

CH3

N

N

CH3

O

HOOC

O

CH3

(95) (96)

Guo et al. synthesized a series of benzimidazole derivatives bearing a heterocyclic

ring imidazole, 5-chloroimidazole, 1,2,4-triazol, and imidazoline and evaluated for

angiotensin II antagonistic activities. Compound (79) showed moderate activity and

compound (80) was found to be almost equipotent with telmisartan in vivo biological

evaluation study[143].

80

N

N

CH3

CH3

N

N NNH

CH3 Cl

N

N

CH3

CH3

N

N NNH

CH3

(97) (98)

2.4.3. Antimicrobial and anti protozoal activity:

Eisa et al. synthesized some new 2-substituted benzimidazole derivatives and tested

them for antimicrobial activity against gram positive, gram negative bacterial strains.

From this study, the compounds were moderately active against different strains of

bacteria and fungus. Significant activity was observed in compound (99) oxadiazole

containing benzimidazole[144].

N

N

S

N N

O S

(99)

Khalafi-Nezhad et al. synthesized benzimidazole and imidazole choloroaryloxyalkyl

derivatives. The compounds were screened for antimicrobial activity against S.

aureus, S. typhi. Compound (100) showed considerable in vitro antibacterial activities

against both bacteria[145].

81

N

N

(CH 2 ) 4O

C l (100)

Andrzejewska et al. has synthesized 5-substituted 4, 6-dibromo and 4, 6-dichloro-2-

mercapto benzimidazoles 101 (a-b). All the compounds were screened for

antimicrobial activity against E.coli, Proteus vulgaris, Bordetella bronchiseptica,

pseudomonas aerginosa, Stenotrophomonas maltophilia, staphylococcus aureus,

Enterocooccus faecalis, Bacillus stearothermophilus, Bacillus substilis, and Bacillus

cereus. The results of this confirmed that gram positive bacteria were more

susceptible to all examined 4, 6-dihalogenated 6-subsitited-2-mercapto

benzimidazoles. The most active agents were 101 (a-b) when compared with

reference agent nitrofurantoin[146].

NH

N

S

R

R 1

R 3

101 (a-b)

a = R, R1= Cl, R3= CH2-CH2-(C6H4)-P-NO2

b= R, R1= Br, R3= CH2-CH2-(C6H5)

Shelar et al. synthesized some alkyl thio aryl substituted benzimidazole derivatives.

The synthesized compounds have been screened for in vitro antibacterial activity

against Klebsiella, E.coli and E.fecalis. The compounds (102) have shown varying

degree of antibacterial activity[147].

82

NH

N

S

R1

R2

R3

(a) R1=Cl, R2=H, R3=Cl

(b) R1=Cl, R2=Cl, R3=Cl

(c) R1=Cl, R2=NO2, R3=Cl

(102)

Anelia et al. synthesized some new thiazolo [3,2-a] benzimidazolone derivatives. The

effectiveness of compounds (103) and (104) in the intestinal phase of Trichinellosis

spiralis was 100% and in the muscle phase were 88% and 80% at a concentration of

100mg/kg[148].

N

N

S

O

OO

N

N

S

O

NH

(103) (104)

Ilkay Yildiz-Oren synthesized a series of multisubstituted benzoxazoles,

benzimidazoles and benzthiazoles (105-106) as non-nucleoside fused isosteric

heterocyclic compounds and tested for their anti-bacterial against various gram-

positive and gram-negative bacteria and anti-fungal activity against the fungus

83

Candida albicans. In these sets of non-nucleoside fused heterocyclic compounds

electron withdrawing groups at position 5 of the benzazoles increased the activity

against C. albicans [149].

NH

N

O

C l

NH

N

O

Cl

Cl

(105) (106)

NH

N

O

CH 3

Cl

(107)

Seckin Ozden et al. synthesized a series of benzimidazole-5-carboxylic acid alkyl

ester derivatives carrying amide or amidine substituted methyl or phenyl groups at the

position C-2 and evaluated for antibacterial and anti-fungal activities against S.aureus,

methicillin resistant S.aureus, S.faecalis , methicillin resistant S.epidermidis E.coli

and C.albicans. Aromatic amidines derivatives (108-109) exhibited the best inhibitory

activity[150].

84

N

N

NH

NH

Cl

O

H5C2O

N

N

NH

NH

Cl

O

H5C2O

Cl

Cl

(108) (109)

N

N

N H

N H

C l

O

H 5 C 2 O

C l

C l

C l

(110)

Shipra Parmar et al. synthesized 1-methyl [(N-alkyl phthalyl)-(benzimidazolo]-31-

chloro-41- substituted azetidin-2-ones (111-112) which shown promising antimicrobial

activity[151].

N

N

O

H 5C 2O

N OON

O

Cl

NO 2

N

N

O

H 5C 2O

N OON

O

Cl

NO 2

CH 3

(111) (112)

85

Siva kumar et al. synthesized some novel 2-(6-fluorochroman-2-yl)-1- alkyl / acyl

/aroyl-1H- benzimidazoles. Some compounds (113) exhibited promising antibacterial

activity against salmonella typhimurium [152].

N

N

O

O

F

(113)

Yun He et al. synthesized a series of 2-piperidin-4-yl benzimidazoles (114-115) and

evaluated for antibacterial activities against both gram positive and gram negative

bacteria of clinical importance, particularly enterococci[153] .

N

N

NH

Cl

Cl

NH2

N

N

NH

Cl

Cl

N

O

O

(114) (115)

N

N

NH

Cl

Cl

NHNH2

(116)

86

Yun He et al. synthesized a series of novel benzimidazole derivatives via parallel

solution phase chemistry. Many of these compounds (117) were found to inhibit the

growth of Staphylococcus aureus and E.coli[154].

N

N

NH

NH

O

NHNH

O

Cl

Cl

(117)

Some benzimidazole derivative containing oxadiazole like, 1-{[5-(alkyl/aryl)-1, 3, 4-

oxadiazol-2-yl] methyl}-2-alkyl-1H-benzimidazoles (118) are synthesized for their

antimicrobial activities. To evaluate the activity of synthesized compounds against

bacteria minimum inhibitory concentrations (MICs) were determined and for yeast and

fungi zone of inhibition was determined. Known antibiotics like ciprofloxacin and

ampicillin and amphotericin B were used for comparison.

N

N

R

O

NN

R'

(118)

(R=H or CH3), (R’= -CH3, -C2H5, -CH2Cl, -CH2CH2Cl, -C6H5, 2-Cl C6H4, 4-Cl

C6H4, 2-OH C6H4, 4-OH C6H4, 2-OC H3 C6H4, 4-OCH3C6H4

Antimicrobial activity of nitro- and halogeno-substituted benzimidazole derivatives

were synthesized and showed both antimicrobial and antiprotozoal activity. Sulfur

87

derivatives are more active towards protozoal and others are more active towards

microbes. The antibacterial activity of the benzimidazole derivatives was first tested by

the agar disc-diffusion method against Gram-positive and Gram-negative bacteria. For

the testing of anti protozoal activity mebendazole is taken as reference

compounds[155].

88

3.NEED FOR STUDY

The main objective of medicinal chemist is to design and discover new compounds

that are suitable for use as drugs. During the early stages of medicinal chemistry

development, scientists were primarily concerned with the isolation of medicinal

agents found in plants. Today, scientists in this field are equally concerned with the

creation of new synthetic drug/compounds. Medicinal chemistry is almost always

geared toward drug discovery and development. Typically, drug discovery involved the

individual synthesis of hundred of thousands of analogs of a weakly active lead

compound in an attempt to enhance the original activity, bioavailability and selectivity,

while at the same time decreasing in toxicity.

Pyrazoles are an important functionality with wide-ranging applications in

pharmaceutical sciences. All aspects of the chemistry of Pyrazoles as well as

medicinal application of Pyrazoles were covered in the literature and reported to

possess anti-bacterial, anti-fungal, anti-viral, anti-tumor, anti-inflammatory, anti-ulcer,

anti-oxidant and antihypertensive activities. Pyrazolones are an active moiety in the

class of NSAIDs and used in the treatment of arthritis, musculoskeletal and joint

disorder. Antipyrine, 2,3-dimethyl-1-phenyl-3-pyrazolin-5-one, was the first pyrazolone

derivative used in the management of pain and inflammation. Several analogues of

pyrazolidin-3,5-diones, pyrazolin-3-ones and pyrazolin-5-ones are also available as

NSAIDs; examples are felcobuzone, mefobutazone, morazone, famprofazone, and

ramifenazone. Besides these, many pyrazoline derivatives are also reported in

literature as having potent anti-inflammatory activity.

89

A systematic investigation of this class of heterocyclic lead revealed that

quinazolinone containing pharmacoactive agents play important role in medicinal

chemistry.. Quinazolinone and its derivatives represents an important class of

compounds not only for their theoretical interest but also for their hypoglycemic agent,

fungicide, antimicrobial and some of them have been tested as potential

cardiovascular drugs.

In other hand, isoxazole and benzimidazole derivatives have found practical medical

application. It also has been demonstrated to possess, antimicrobial, anticonvulsant,

analgesic, anti-inflammatory, anti-platelet, anti-tubercular and anti-tumoral activities.

Therefore, these pyrazoles, quinazolinone, isoxazoles and benzimidazoles possess

worthy and imperative bioactivities, which render them useful substances in drug

research.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used

therapeutics, primarily for the treatment of inflammation, especially arthritis. The

pharmacological activity of NSAIDs is related to the suppression of prostaglandin

biosynthesis from arachidonic acid by inhibiting the enzyme prostaglandin

endoperoxidase, popularly known as cyclo-oxygenase (COX). It was discovered that

COX exists in two isoforms, COX-1 and COX-2, which are regulated and expressed

differently. COX-1 provides cytoprotection in the gastrointestinal tract (GIT), whereas

inducible COX-2 selectively mediates inflammatory signals. Since most of the

currently available NSAIDs in the market show greater selectivity for COX-1 than

COX-2, chronic use of NSAIDs may elicit appreciable GI irritation, bleeding and

ulceration.

90

Mono therapy with a drug having both anti-inflammatory and antimicrobial activities is

highly desirable and more beneficial to the patients who suffer from inflammatory

condition associated with microbial infections.

Highly potent substances are produced by the immune system. These substances

include cytokines and oxidant molecules, such as hydrogen peroxide, free radicals,

and hypochlorous acid. The purpose of immune cell products is to destroy invading

organisms and damaged tissue, bringing about recovery. However, oxidants and

cytokines can damage healthy tissue. Excessive or inappropriate production of these

substances is associated with mortality and morbidity after infection and trauma, and

in inflammatory diseases. Inflammation, free radical damage and oxidative stress

have become major health issues in recent years, the subject of much research and

concern.

Inflammation, free radical damage and oxidative stress are not “diseases.” In fact,

they are often the by-product of normal cellular processes. However, they are

implicated in cancer, heart disease, strokes, MS, Alzheimer’s, Parkinson’s, premature

aging and almost any debilitating, degenerative condition. When it comes to fighting

inflammation, it seems that everyone knows by now that antioxidants are the heroic

arch enemy of those annoying inflammatory agents called free radicals.

In view of these observations, we have planned to synthesis of some new

quinazolinone containing pyrazole and isoxazole derivatives and some benzimidazole

derivatives which have been found to possess an interesting profile of anti-

inflammatory, along with significantly less ulcerogenic potential, antimicrobial and in

vitro anti-oxidant activities.

91

4. OBJECTIVES OF THE WORK

1. To synthesize novel 3-[4-(5-(3,4-disubstituted phenyl)-4,5-dihydro isoxazol-3-yl)

phenyl]-2-substutied phenyl Quinolin-4(3H)-one derivatives & 3-[4-(1-acetyl-5-

(3,4-disubstituted phenyl)-4,5-dihydro-1H-pyrazol-3-yl) phenyl]-2-substutied

phenyl Quinolin-4(3H)-one derivatives

2. To synthesis some 2-substituted benzimidazole derivatives.

3. To study the analgesic, anti-inflammatory & in-vitro antioxidant property of newly

synthesized compounds.

4. To evaluate its Microbiological activities and Minimal Inhibitory Concentration

(MIC) by various gram positive and gram negative bacteria and fungi.

5. To perform docking studies to investigate the interaction between synthesized

compounds and the amino acid residues of the selected bacterial, fungal, COX-

1 and COX-2 receptors and also to find out inhibitory constant (Ki) values using

AutoDock Software.

92

5. METHODOLOGY ADOPTED

All the chemicals and solvents required for the study were purchased form SD Fine,

Kemphasol, Ranbaxy, Hay man Ltd, fisher and S.D. Fine Chem. Ltd. All the solvents

procured were purified and dried. The solvent system used for Thin Layer

Chromatography in Benzene and acetone (9:1). Iodine chamber and UV Lamps were

used for visualization of TLC spots; Whatmann Filter Paper (No.1, England) was used

for filtration (Vacuum or ordinary). H1 NMR spectra were recorded on 300 MHz

instruments and the Mass spectra were recorded on Joel SX102/Da-600. FT-IR was

recorded in Shimadzu. Melting points were determined using Sulfuric acid bath which

was uncorrected.

5.1 SYNTHESIS:

Synthesis of 2-Substituted Phenyl-4H-Benzo-[1,3]-Oxazin-4-One 1(a-b)

To a stirred solution of anthranilic acid (0.01 mole) in pyridine (50ml),

substituted benzoyl chloride (0.01 mole) was added drop wise, maintaining the

temperature near 800 C for 2 hour. Reaction mixture was stirred for another 3 hours at

room temperature. While stirring a solid product separates out. Whole reaction

mixture was neutralized with sodium bicarbonate solution. A pale yellow solid

deposited which was filtered, washed with water and re-crystallized with sodium

bicarbonate solution.

93

Synthesis of 4-(4-Oxo-2-Substituted Phenylquinazolin-3(4H)-yl) -benzaldehyde

2(a-b)

Compound 1 (a-b) (0.01 mole) was dissolved in ethanol and 4-amino

benzaldehyde (0.01 mole) in ethanol was added to it with a catalytic amount of

pyridine. Then the reaction mixture was refluxed for 4 hours and after cooling a

crystalline product was obtained. Then it was filtered and re-crystallized from ethanol

to yield needle shaped shining white crystals.

Synthesis of Compound 2-Substituted Phenyl-3-(4-(3-(Substituted Phenyl-3-oxo

Prop-1-enyl) Phenyl Quniazoline-4-One: 3(a-h)

Equimolar quantities of compound 2 (a-b) and substituted acetophenone (0.01

mole) were dissolved in the minimum amount of alcohol. Then sodium hydroxide

solution (0.02 mole) was added slowly and the mixture stirred for 3 hours until the

entire mixture becomes very cloud and then the mixture was poured slowly in to 400

ml of water with constant stirring and kept in refrigerator for 24 hours. The precipitate

obtained was filtered, washed and re-crystallized from ethanol.

Synthesis of 3-[4-(1-Acetyl-5-(3,4-Disubstituted Phenyl)-4,5-Dihydro-1h-Pyrazol-

3-Yl) Phenyl]-2-Substutied Phenyl Quinolin-4(3H)-One Derivatives 4(a-h):

To a solution of chalcone 3 (a-h) (0.02 mole) in absolute alcohol (50ml), 99%

hydrazine hydrate (0.04 mole) was added drop by drop with constant stirring in the

presence of few drops of glacial acetic acid. The reaction mixture was refluxed for

94

12h, distilled of and cooled. The separated solid was cooled filtered, washed with

pet.ether and re-crystallized from the appropriate solvent.

Synthesis Of 3-[4-(5-(3,4-disubstituted phenyl)-4,5-dihydro isoxazol-3-yl)

phenyl]-2-substutied phenyl Quinolin-4(3H)-one derivatives: 5 (a-h):

A mixture of chalcone 3 (a-h) (0.02 mole), hydroxylamine hydrochloride (0.02

mole) and sodium acetate in ethanol (25 ml) was refluxed for 6 hr. Then the mixture

was concentrated by distilling out the solvent under reduced pressure and poured in

to ice water. The precipitate obtained was filtered, washed and re-crystallized.

NH2

COOH+

R

O Cl

O

N

O

R

NH2 CHO

N

N

O

R

CHO

R1

R2

CH3

O

N

N

O

R

O

R2

R1

N

N

O

R

NN

CH3

O

R1

R2

N

N

O

R

NO

R1

R2

NH2-NH2/CH3COOH

NH2OH/ethanol

1 (a-b)

2 (a-b)

3 (a-h)

4 (a-h)

5 (a-h)

a=R, R1, R2 - H , b= R- NO2, R1, R2 –H c= R- H, R1, -OCH3, R2 – H

d= R- NO2, R1,-OCH3, R2 -H e= R, R1,-H, R2 – Cl f= R- NO2, R1,- H, R2 – Cl

g= R, R1- H, R2 - NO2 h= R- NO2, R1-H, R2 - NO2

SCHEME - 1

95

Synthesis Of 2-Substituted Benzylideneamino) Benzoic Acid 6(a-e)

1.3 gm of anthranilic acid and 1.49 gm of substituted aldehyde was taken in a

round bottom flask and 50 ml of ethanol was added and refluxed in water bath for 2

hrs with occasional shaking and then evaporated solvent. An orange color precipitated

was filtered with suction and dried.

Synthesis Of 4-(Substituted Benzylidene)-2-(1H-Benzimidazole-2-yl)

benzenamine: 7(a-e)

To a mixture of 2.68 g (0.01 mol) of compound 6 and 1.08 g (0.01 mol) of o-

phenylenediamine in 250 ml round bottomed flask, 50 ml of 4 N HCl was added and

stirred for 4 hrs in magnetic stirrer. After the reaction stirring, the contents were

transferred to the beaker and made alkaline with ammonia. The formed precipitate

was filtered and the residue was dried and the filtrate was re-crystallized from alcohol.

SCHEME – II

NH2

COOH

+

R

CHO

COOH

N

R

N

NH

N

R

NH2

NH2

1 (a-e)

2(a-e)

a= H, b= NH2, c=(o-OH), d= para-OCH3, e=para N(CH3)2

96

5.2 CHARACTERIZATION OF NEWLY SYNTHESIZED COMPOUNDS

All the newly synthesized compounds were purified and subjected for the spectral

analysis to elucidate its structure.

IR spectrum of the synthesized compounds showed required peaks as the

structure assigned.

NMR spectra of the all the newly synthesized compounds were recoded in 300

MHz Bruker Instrument and shown the aromatic peak between 6 to 8 and

other characteristic peaks were present to confirm the structure.

Mass spectra’s shows that the mass charge ratio of newly synthesized

compounds

5.3 DOCKING STUDIES:

In silico methods can help in identifying drug targets via bioinformatics

tools. They can also be used to analyze the target structures for possible

binding/ active sites, generate candidate molecules, dock these molecules with the

target, rank them according to their binding affinities, further optimize the molecules to

improve binding characteristics.

5.3.1 TOOLS AND MATERIALS USED

By using Autodock software, anti-inflammatory, antibacterial and antifungal

activities of synthesized pyrazoline containing quinazoline-4-one, isoxazoline

containing quinazoline-4-one and 2-substituted benzimidazole derivatives were

predicted.

97

Procedure for prediction of Antibacterial activity studies using Autodock [156]:

The structure of β-ketoacyl-acyl carrier protein synthase (KAS) which is an

essential target for novel antibacterial drug design was retrieved from PDB.

The docking studies were done on ecKAS III (pdb id: 1HNJ) receptor. All these

molecules as well as the bound ligand of the protein 1HNJ were docked by

using the software Auto Dock and the score values are predicted.

All molecules were drawn using ChemDraw Ultra 8.0 tool and energy minimized

using Chem 3D Ultra 8.0 software. Automated docking was used to locate the

appropriate binding orientations and conformations of various inhibitors into the

1HNJ binding pocket.

Before docking the screened ligands in to the protein active site, the protein

was prepared by deleting the substrate cofactor as well as the

crystallographically observed water molecules and then protein was defined for

generating the grid. Grid maps were generated by AutoGrid program.

Each grid was centered at the crystal structure of the corresponding 1HNJ. The

grid dimensions were 60 A˚ X 60 A˚ X 60 A˚ with points separated by 0.375A˚.

For all ligands, random starting positions, random orientations and torsions

were used. During docking, grid parameters were specified for x, y and z axes

as 38.808, 30.946 and 42.249 respectively.

Procedure for prediction of Antifungal activity studies using Autodock [157]:

The structure of sterol 14α-demethylase (CYP51) which is an essential

target for novel antifungal drug design was retrieved from PDB (1E9X). All

water molecules and ligands were removed from the proteins for docking

98

studies and same procedure is followed as mentioned in antibacterial

studies.

Procedure for prediction of Anti-inflammatory activity studies using

Autodock:

The structure of COX-1 and COX-2 enzymes which are an essential target for anti-

inflammatory activity were retrieved from PDB (1egq and 1cx2 respectively). All

water molecules and ligands were removed from the proteins for docking studies

and same procedure is followed as mentioned in antibacterial studies.

5.4 Toxicity Study: [158]

Acute Toxicity Study: Determination of lethal dose 50 (LD50):

LD50 is stated in milligrams per kilogram (mg/kg): milligram of chemical per

kilogram of body weight. In this work the basic nucleus in each series were tested and

calculated its lethal dose to 50%. Healthy, adult, male albino Swiss mice weighing

between 20 and 25 g were used in the present investigation. Basic compound of each

series were tested from 5 to 2000 mg/kg body weight, (as suspension in 0.5% CMC)

in groups of 6 animals by intraperitoneally administration. The control groups of

animals received only the vehicle (0.5 % CMC). The animals were observed for 48 hrs

from the time of administration of test compound to record the mortality.

This procedure is conducted by following OECD guidelines

99

5.5 Anti- microbiological Activity:

All the newly synthesized compounds were screened for their antibacterial

activity against Staphylococcus aureus, bacillus subtilis and Pseudomonas

aerogenosa and Escherichia coli and antifungal activity against Aspergillus niger and

Saccharomyces cerevisiae at 100, 500 micro gram/ml using ampicillin, and

griseofulvin as standard drugs by cup-plate method. The zone of inhibition was

measured and calculated [159].

5.6 Anti-inflammatory Activity:

Anti-inflammatory activity of the test compounds were screened by carrageenan

induced rat paw edema method . Acute paw edema was produced by injecting

carragennin 1% w/v (0.1ml) into the sub plantar region of the left hind paw in the rats.

Test compounds 3(a-h), 4(a-h), 5(a-h) and 7 (a-e) 10mg/kg) and Indomethacin 10

mg/kg administered orally one hour before testing. The control group received vehicle

0.1 ml/100gm. The paw volume was measured by using plethysmometer at 0, 1, 2, 3,

and 4 hrs after carrageenin challenge. The percent increase in the edema (paw

100

volume) was calculated by comparing it with zero minute reading. The percentage

inhibition of edema was calculated at 4th hour assuming 100% inflammation in vehicle

group [160].

5.7 Analgesic Activity:

The newly synthesized compounds were screened for analgesic activity employed

Eddy’s hot plate method method for the assessment by hot plate method. [161] Albino

Swiss mice were divided to groups of 12 group I served as control (Normal saline

2ml/kg), group II served as standard (Pentazocine 5 mg /kg) and the remaining group

received at a dose of 5 mg/Kg of compounds at oral administration. The time of

reaction to pain stimulus of the mice placed on the hot plate heated at 550+0.50C was

recorded at 120 min after administration of test drug. The increase in reaction time

against control was calculated.

5.8 IN VITRO METHODS EMPLOYED IN ANTIOXIDANT STUDIES

5.8.1 Reducing power ability

Reducing power ability was measured by mixing 1.0 ml fractions of various

concentration prepared with distilled water to 2.5 ml of phosphate buffer (0.2 M, pH

6.6) and 2.5 ml of 1% potassium ferricyanide and incubated at 50C for 30 min. After

that 2.5 ml of trichloroacetic acid (10%) were added to the mixture and centrifuged for

10 min at 3000 g, 2.5 ml from the upper part were diluted with 2.5 ml water and

shaken with 0.5 ml fresh 0.1%, ferric chloride. The absorbance was measured at 700

nm using UV-spectrophotometer. The reference solution was prepared as above, but

contained water instead of the samples. Increased absorbance of the reaction

mixture indicates increased reducing power. All experiments were done in triplicate

101

using butylated hydroxyltoluene (BHT) as positive control. [162]

5.8.2 Hydrogen peroxide scavenging activity

Hydrogen peroxide solution (2 mM/L) was prepared with standard phosphate buffer

(pH, 7.4). The synthesized compounds (50-250 g/ml) in dimethyl sulfoxide were

added to hydrogen peroxide solution (0.6 ml). Absorbance of hydrogen peroxide at

230 nm was determined spectrophotometrically after 10 min against a blank solution

containing phosphate buffer without hydrogen peroxide. The percentage scavenging

of hydrogen peroxide of both plant fractions and standard compound (BHT) were

determined [163].

Statistical analysis

Results were statistically evaluated by analysis of variance (ANOVA) followed by

Dennett’s multiple comparison test, P<0.05 was considered to be statistically

significant.

102

6. RESULTS AND DISCUSSION

6.1. Experiment

Synthesis of 2-phenyl – 4h- benzo [d] [1,3] oxazin-4- one (1a)

2-Amino Benzoic acid 0.01 mol; 1.37 gms.

Benzoyl Chloride 0.01 mol; 1.40 gms.

Crystallization solvent Sodiumbicarbonate solution

Shape/ Nature of the compound Yellow coloured crystalline compound.

Melting point 2130 C

Percentage Yield 76.83 %

TLC behavior Single spot showed in Iodine Chamber

Synthesis of 2-(4-nitrophenyl)-4h benzo [d] [1,3] oxazin-4-one (1b)

2-Amino Benzoic acid 0.01 mol; 1.37 gm.

4-Nitro Benzoyl chloride 0.01 mol; 1.85 gm

Crystallization solvent Sodiumbicarbonate solution





Synthesis of 4-(4-oxo-2-phenylquinazoline-3 (4h)-yl) benzaldehyde (2a)

Compound 1a 0.01 mol; 2.23 gms.

4-Amino Benzaldehyde 0.01 mol; 1.21 gms.

Ethanol 25.0 ml

Crystallization solvent Methanol

103





Synthesis of 4-(2-(4-nitrophenyl)-4-oxoquinazolin -3 (4h)-yl) bnzaldehyde (2b)

Compound 1b 0.01 mol; 2.68 gms.

4-Amino Benzaldehyde 0.01 mol; 1.21 gms.

Ethanol - 25.0 ml






Synthesis of 3-(4-((z)-3-oxo-3-phenyl prop-1-enyl) phenyl)-2-phenyl quinazolin-

4(3h)-one (3a)

Compound 2a 0.01 mol; 3.26 gms

Acetophenone 0.01 mol; 1.20 gms

Ethanol 25.0 ml


Shape/ Nature of the compound Brown coloured crystalline compound.




104

Synthesis of 2-(4-nitrophenyl)-3-(4-((z)-3-oxo-3-phenyl prop-1-enyl) phenyl)

quinazolin-4(3h)-one (3b)

Compound 2b 0.01 mol; 3.71 gms

Acetophenone 0.01 mol; 1.20 gms

Ethanol 25.0 ml


Shape/ Nature of the compound Brownish coloured crystalline compound.




Synthesis of 3-(4-((z-3-(4-methoxyphenyl)-3-oxoprop-1-enyl) phenyl-2-phenyl

quinazolin-4(3h)-one (3c)


1-(4-methoxyphenyl) ethanone 0.01 mol; 1.50 gms

Ethanol 25.0 ml


Shape/ Nature of the compound Pale Yellow coloured crystalline

compound.




105

Synthesis of 3-(4-((z)-3-(4-methoxyphenyl)-3-oxoprop-1-enyl) phenyl)-2-(4-

nitrophenyl) quinazolin-4(3h)-one (3d)


1-(4-methoxyphenyl)ethanone 0.01 mol; 1.50 gms

Ethanol 25.0 ml






Synthesis of 3-(4-((z)-3-(3-chlorophenyl)-3-oxoprop-1-enyl) phenyl)-2-

phenylquinazolin-4(3h)-one (3e)

Compound 2a 0.01 mol; 3.26 gm

1-(3-chlorophenyl)ethanone 0.01 mol; 1.54 gms

Ethanol 25.0 ml






106

Synthesis of 3-(4-((z)-3-(3-chlorophenyl)-3-oxoprop-1-enyl) phenyl)-2-(4-

nitrophenyl) quinazolin-4(3h)-one (3f)


1-(3-chlorophenyl)ethanone 0.01 mol; 1.54 gms

Ethanol 25.0 ml


Shape/ Nature of the compound Yellowish white fine powder compound.




Synthesis of 3-(4-((-2)-3-(-nitrophenyl)-3-oxoprop-1-enyl)-2-phenylquinazolin-

4(3h-one) (3g)


1-(3-nitrophenyl)ethanone 0.01 mol; 1.65 gms

Ethanol 25.0 ml


Shape/ Nature of the compound Yellow colored crystalline compound.




107

Synthesis of 2-(4-nitrophenyl)-3-(4-((z)-3-(3-nitrophenyl)-3-oxoprop-1-enyl)

phenyl) qunazoline-4(3h)-one (3h)


1-(3-nitrophenyl)ethanone 0.01 mol; 1.65 gms

Ethanol 25.0 ml


Shape/ Nature of the compound Yellow color crystalline compound.




Synthesis of 3-(4-(1-acetyl-4, 5-dihydro-5-phenyl-1h-pyrazol-3-yl) phenyl)-2-

phenyl quinazolin-4(3h)-one (4a)


Hydrazine hydrate 0.02 mol; 1.00 gms

Glacial acetic acid 3 drops

Ethanol 50.0 ml


Shape/ Nature of the compound Brown color crystalline compound.




108

Synthesis of 3(4-(1-acetyl-4, 5-dihydro-5-phenyl-1h-pyrazol-3-yl) phenyl)-2-(4-

nitrophrnyl) quinazolin-4(3h)-one (4b)




Ethanol 50.0 ml



compound.




Synthesis of 3-(4-(1-acetyl-4,5-dihydro-5-(4-methoxyphenyl)-1h-pyrazol-3-

yl)phenyl)-2-phenyl quinazolin-4(3h)-one(4c)

Compound 3c 0.01mol; 4.58 gms



Ethanol 50.0 ml






109

Synthesis of 3-(4-(1-acetyl-4, 5-dihydro-5-(4-methoxyphenyl)-1h-pyrazol-3-yl)

phenyl)-2-(4-nitrophenyl) quinazolin-4(3h)-one (4d)

Compound 3d 0.01 mol; 5.03 gms



Ethanol 50.0 ml






Synthesis of 3-(4-(1-acetyl-5-(3-chlorophenyl)-4, 5-dihydro-1h-pyrazol-3-yl)

phenyl)-2-phenyl quinazolin-4(3h)-one (4e)

Compound 3e 0.01 mol; 4.11 gms



Ethanol 50.0 ml


Shape/ Nature of the compound Brown colored crystalline compound.




110

Synthesis of 3-(4-(1-acetyl-5-(3-chlorophenyl)-4,5-dihydro-1h-pyrazol-3-

yl)phenyl)-2-(4-nitrophenyl) quinazolin-4(3h)-one (4f)

Compound 3f 0.01 mol; 5.67 gms



Ethanol 50.0 ml






Synthesis of 3-(4-(1-acetyl-4,5-dihydro-5-(3-nitrophenyl)-1h-pyrazol-3-yl)phenyl)-

2-phenyl quinazolin-4(3h)-one (4g)

Compound 3g 0.01 mol; 4.73 gms



Ethanol 50.0 ml




Percentage Yield 74 %


111

Synthesis of 3-(4-(1-acetyl-4,5-dihydro-5-(3-nitrophenyl)-1h-pyrazol-3-yl)phenyl)-

2-(4-nitrophenyl) quinazolin-4(3h)-one (4h)

Compound 3h 0.01 mol; 5.18 gms



Ethanol 50.0 ml






Synthesis of 2-phenyl-3- (4- (5-phenyl-4, 5-dihydroisazol-3-yl) - phenyl

quinazolin-4(3h)-one (5a)


Hydroxylamine hydrochloride 0.02 mol; 1.39 gm

Sodium acetate in Ethanol 25.0 ml


Shape/ Nature of the compound Brown colored crystalline compound.




112

Synthesis of 2-(4-nitrophenyl)-3-(4-(5-phenyl-4,5-dihydroisazol-3-yl) - phenyl


Compound 3b 0.01 mol; 4.73 gm





compound.




Synthesis of 3-(4-(5-(4-methoxyphenyl)-4, 5-dihydroisazol-3-yl) - phenyl)-2-

phenyl quinazolin-4(3h)-one (5c)

Compound 3c 0.01 mol; 4.58 gms








113

Synthesis of 3-(4-(5-(4-methoxyphenyl)-4, 5-dihydroisazol-3-yl) - phenyl)-2-(4-


Compound 3d 0.01 mol; 5.03 gms








Synthesis of 3-(4-(5-(3-chloro phenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-phenyl

quinazolin-4(3h)-one (5e)

Compound 3e 0.01 mol; 4.11 gms








114

Synthesis of 3-(4-(5-(3-chloro phenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-(4-


Compound 3f 0.01 mol; 5.67 gms








Synthesis of 3-(4-(5-(3-nitrophenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-phenyl

quinazolin-4(3h)-one (5g)

Compound 3g 0.01 mol; 4.73 gms








115

Synthesis of 3-(4-(5-(3-nitrophenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-(4-

nitrophenyl) quinazolin-4(3h)-one (5h)

Compound 3h 0.01 mol; 5.18 gms








Synthesis of (e)-2-(benzylideneamino) benzoic acid (6a)

2-Aminobenzoic acid 0.01 mol; 1.37 gms.

Benzaldehyde 0.01 mol; 1.06 gms.

Ethanol 25.0 ml


Shape/ Nature of the compound Colorless crystalline compound.




116

Synthesis of (e)-2-(4-aminobenzylideneamino) benzoicacid (6b)

2-Aminobenzoic acid 0.01 mol; 1.37gms.

4-Amino benzaldehyde 0.01 mol; 1.21 gms.

Ethanol 25.0 ml






Synthesis of (e)-2-(2-hydroxybenzylideamino) benzoic acid (6c)


2-Hydroxy benzaldehyde 0.01 mol; 1.22 gms.

Ethanol 25.0 ml


Shape/ Nature of the compound Colorless crystalline compound.




117

Synthesis of (e)-2-(4-methoxy benzylideneamino) benzoic acid (6d)


4-Methoxy benzaldehyde 0.01 mol;1.36 gms.

Ethanol 25.0 ml






Synthesis of (e)-2-(4-(dimethylamino) benzylideneamino)

Benzoic acid (6e)


4-Dimethylamino benzaldehyde 0.01 mol; 1.49 gms.

Ethanol 25.0 ml


Shape/ Nature of the compound Orange colored compound.




118

Synthesis of (z)-2-(1h – benzo[d]imidazol-2-yl)-n-benzyliden-amine (7a).

Compound 6a 0.01 mol; 2.25 gms.

Benzene~1,2~diamine 0.01 mol; 1.08 gms.

Ethanol 25.0 ml


Shape/ Nature of the compound White coloured compound.




Synthesis of (z)-n-(4-aminobenzylidene(-2-(1h – benzo[d]imidazol-2-yl)-n-

benzyliden-amine (7b)


Benzene-1,2-diamine 0.01 mol; 1.08 gms.

Ethanol 25.0 ml


Shape/ Nature of the compound Brown colored amorphous compound.




119

Synthesis of 2-(z)-(2-(1h-benzo[d]imidazol-2-yl) phenylimino) methyl phenol (7c)

Compound 6c 0.01 mol; 2.41 gms.

Benzene-1,2-diamine 0.01 mol; 1.08 gms.

Ethanol 25.0 ml


Shape/ Nature of the compound Colorless crystalline compound



TLC behavior Single spot showed in Iodine chamber

Synthesis of (z)-n-(4-methoxy benzylidene)-2-(1h – benzo[d]imidazol-2-yl)-

benzamine (7d).

Compound 6d 0.01 mol; 3.02 gms.

Benzene-1,2-diamine 0.01 mol;1.08 gms.

Ethanol 25.0 ml


Shape/ Nature of the compound Yellow colored crystalline




120

Synthesis of (z)-n-(4-dimethylamino benzylidene)-2-(1h – benzo[d]imidazol-2-yl)-

benzamine (7e).

Compound 6e 0.01 mol; 2.29 gms.

Benzene-1,2-diamine 0.01 mol;1.08 gms.

Ethanol 25.0 ml


Shape/ Nature of the compound Orange colored crystalline compound




121

The spectral data of the synthesized compounds

Table 6.2.1

Compound

CodeNMR Data IR Data

1a 6.847-7.871(m,9H,Ar-H).

3085(C-H str,

Aromatic).,1690(C=O str

Aromatic).,1593(C=N

str).,1210(C-O str).

1b 7.214-8.158(m,8H,Ar-H).

3085(C-H str,


Aromatic).,1593(C=N


2a

9.780(s,11H,-CHO).,7.181-

7.976(m,Ar-12H).

3095(C-H str,

Aromatic).,2885,2821(C-H str,

Aldehyde).,1687(C=O str,

aldehyde).,1608(C=N

str).,1550(C=C

str,Aromatic).,1300,1236,(C-N


122

2b9.808(s,11H,-CHO).,7.214-

8.234(m,Ar-11H).

3107(C-H str,

Aromatic).,2883,2813(C-H str,

Aldehyde).,1681(C=O str

aldehyde).,1604(C=N

str).,1525,1340(Nitro

Aromatic).,1246(C-N str).,1161(C-

O str).

3a

6.393-6.425(d,1H,-CH=).,6.540-

6.576(d,1H,=CH-CO).,6.953-

7.523(m,18H,Ar-H)

3085(CH- str,Aromatic).,

3043(CH=CH str)., 1665(C=O str

a,b-unsaturared keto)., 1675

(C=O str cyclic keto)., 1586 (C=N

str).

3b

6.731-6.761(d,1H,-CH=).,7.234-

7.262(d,1H,=CH-CO).,7.234-

8.201(m,17H,Ar-H)




(C=O str cyclic

keto).,1523,1347(N=O str)

3c

3.176(s,3H-,-OCH3), 6.427-

6.453(d,1H,-CH=).,6.674-

6.681(d,1H,=CH-CO).,6.687-

7.574(m,17H,Ar-H).,


3043(CH=CH str).,2885(CH-str

Alkyl) 1665(C=O str a,b-

unsaturared keto)., 1675 (C=O str

cyclic keto)., 1586 (C=N

str).,1213(-OCH3).

123

3d

3.916(s,3H-,-OCH3), 6.737-

6.906(d,1H,-CH=).,7.010-

7.038(d,1H,=CH-CO).,6.935-

8.472(m,16H,Ar-H).,


3043(CH=CH str).,2885(CH-str

Alkyl) 1665(C=O str a,b-

unsaturared keto)., 1675 (C=O str

cyclic keto)., 1586 (C=N

str).,1523,1347(N=O str) 1213(-

OCH3).

3e

7.278-8.226(s,17H,Ar-H)., 7.238-

7.262(d,1H,-CH-CO)., 7.002-

7.009(d,1H,-CH=CH-)





str).,

3f

7.042-8.850(s,16H,Ar-H)., 6.765-

6.792(d,1H,-CH-CO)., 6.709-

6.739(d,1H,-CH=CH-)





str).,1523,1347(N=O str)

3g

7.469-8.224(s,17H,Ar-H).,7.432-

7.454(d,1H,-CH-CO)., 7.328-

7.361(d,1H,-CH=CH-)





str)

124

3h

7.038-8.472(s,16H,Ar-H).,6.980-

7.010(d,1H,-CH-CO)., 6.906-

6.935(d,1H,-CH=CH-)





str)., 1523,1347 (N=O str).

4a

6.292-7.864(s,18H,Ar-H).,3.829-

3.870(t,1H,-CH-CH2)., 3.360-

3.383(d,2H,-CH-CH2).,2287(s,3H,

CO-CH3)

3047(C-H- str, Aromatic).,

2887(C-H str, Alkyl)., 1670 (C=O

str, Aromatic keto)., 1649(C=O str,

Aliphatic Amide

keto).,1633,1612(C=C str,

Aromatic)., 1556 (C=N

str).,1456,1384( C-H Bending

Alkyl).,1276,1249(C-N str).,

4b

6.351-8.422(s,17H,Ar-H).,3.814-

3.858(t,1H,-CH-CH2)., 2.695-

2.718(d,2H,-CH-CH2).,

2.055(s,3H, CO-CH3)

3130(C-H- str, Aromatic).,

2874(C-H str, Alkyl)., 1666 (C=O

str, Aromatic keto)., 1649(C=O str,

Aliphatic Amide keto)., 1599 (C=N

str).,1535,1340(Aromatic

Nitro)1445,1384( C-H Bending

Alkyl).,1276,1249(C-N str).,

125

4c

7.063-7.869(s,17H,Ar-

H).,3.882(s,3H.-OCH3).,3.832-

3.877(t,1H,-CH-CH2)., 2.718-

2.740(d,2H,-CH-CH2).,

2.055(s,3H, CO-CH3)

3126(C-H- str,Aromatic)., 2966(C-

H str, Alkyl)., 2943(C-H str,

Methoxy).,1678 (C=O str,

Aromatic keto)., 1629(C=O str,


str).,1500,1448,1421( C-H

Bending, Alkyl).,1286,(C-N

str).,1222(C-O str, Methoxy).

4d

6.759-8.330(s,16H,Ar-

H).,3.886(s,3H.-OCH3).,3.896-

3.942(t,1H,-CH-CH2).,2.527-

2.548(d,2H,-CH-CH2).,

2.083(s,3H, CO-CH3)

3132(C-H- str,Aromatic).,

2999(C-H str, Methoxy)., 2877(C-

H str, Alkyl)., 1662 (C=O str,


Aliphatic Amide keto)., 1606(C=C

str, Aromatic)., 1595 (C=N


Nitro).,1417,1334( C-H Bending

Alkyl).,1294,1249(C-N

str).,1203(C-O str, Methoxy).,

126

4e

6.719-7.932(s,17H,Ar-H)., 3.896-

3.942(t,1H,-CH-CH2).,2.527-

2.548(d,2H,-CH-CH2).,

2.619(s,3H, CO-CH3).





str).,1558,1543(c=C str,

Aromatic).,1402( C-H Bending

Alkyl).,1151(C-N str).,

4f

7.056-7.950(m,17H,Ar-H).,4.430-

4.574(t,1H,-CH-CH2).,2.821-

2.843(d,2H,-CH-CH2).,

2.095(s,3H, CO-CH3).







Alkyl).,1274,1213(C-N str).,

4g

6.989-7.925(m,17H,Ar-H).,4.496-

4.549(t,1H,-CH-CH2).,2.726-

7.747(d,2H,-CH-CH2).,

2.143(s,3H, CO-CH3).






Nitro).,1242,(C-N str).,

127

4h

7.043-8.018(m,16H,Ar-H).,4.945-

4.899(t,1H,-CH-CH2).,2.335-

2.314(d,2H,-CH-CH2).,

2.055(s,3H, CO-CH3).







Alkyl).,1284,1205(C-N str).,

5a

6.964-7.899(m,18H,Ar-H),3.826-

3.871(t,1H,-CH-CH2),3.247-

3.269((d,2H,-CH-CH2),

3090(C-H- str, Aromatic)., 1675

(C=O str, Aromatic keto)., 1586

(C=N str).,2920 (CH str, Alkyl )

5b

7.379-8.750(m,17H,Ar-H),4.283-

4.330(t,1H,-CH-CH2),3.372-

3.355(d,2H,-CH-CH2).

3101(CH- str,Aromatic)., 1670

(C=O str, Aromatic keto)., 1620

(C=C str).,1560(C=N


Nitro).,1249(C-N str).,1103(C-O

str).,

5c

7.029-7.944(m,17H,Ar-H),4.328-

4.370(t,1H,-CH-CH2),3.360-

3.383(d,2H,-CH-

CH2),3.92(s,3H,CO-CH3).

3064(C-H- str,Aromatic).,2987(C-

H str Methoxy)., 1678 (C=O str,

Aromatic keto)., 1627(C=C str

Aromatic).,1568 (C=N

str).,1253,1166(C-O str).,

128

5d

7.019-7.964(m,16H,Ar-H).,4.229-

4.274(t,1H,-CH-CH2).,2.820-

2.843,(d,2H,-CH-

CH2.),4.413,(s,3H, CO-CH3).


(C=O str, Aromatic

keto).,1600(C=N str)., 1586 (C=N


Nitro).,2939 (CH str, Alkyl

).,1246(C-N str).,1182(C-O str,

Methoxy).

5e

6.709-6.806(m,17H,Ar-H).,4.687-

4.730(t,1H,-CH-CH2).,3.018-

3.041(d,2H,-CH-CH2).,

3086(C-H str,

Aromatic).,1658(C=0 str, Aromatic

Keto).,1586 (C=N str).,2920 (CH

str Alkyl )

5f

7.063-7.869(M,16H,Ar-H).,4.614-

4.6509(t,11H,-CH-CH2).,2.696-

2.718(d,2H,-CH-CH2).


(C=O str Aromatic keto)., 1586

(C=N str).,2920 (CH str Alkyl )

5g

7.031-7.977(m,17H,Ar-H).,4.879-

4.925(t,1H,-CH-CH2).,2.736-

2.758(d,2H,-CH-CH2).




5h

7.019-8.364(m,16H,Ar-H).,4.230-

4.274(t,1H,-CH-CH2).,2.674-

2.694(d,2H,-CH-CH2).




129

6a

10.085(s,1H,-

COOH).,7.884(s,1H,CH=N).,6.826-

7.838(m,9H,Ar-H).

3500-2500(OH str Caboxylic

Acid).,3085(CH str


Carboxylic Acid).,1623(C=C

str).,1590(C=N str).

6b

10.087(s,1H,-

COOH).,7.557(s,1H,CH=N).,7.187-

7.536(m,8H,Ar-

H).,3.9549(s,2H,NH2).


Acid).,3085(CH str



str).,1590(C=N str).,3400(N-H str

Amine).

6c

10.853(s,1H,-

COOH).,8.234(s,1H,CH=N).,6.947-

8.211(m,8H,Ar-H).,5.027(s,2H,-

OH).


Acid).,3085(CH str



str).,1590(C=N str).,1213(-

OCH3),1213(-OCH3).

6d

10.223(s,1H,-

COOH).,7.530(s,1H,CH=N).,7.247-

7.516(m,8H,Ar-H).,3.566(s,2H,CO-

CH3).


Acid).,3085(CH str




130

6e

10.472(s,1H,-

COOH).,7.464(s,1H,CH=N).,6.765-

7.453(m,8H,Ar-

H).,3.201(s,6H,N[CH3]2).


Acid).,3085(CH str



str).,1590(C=N str).2942(C-H str

Alkyl).

7a

8.299(s,1H,CH=N).,7.019-

7.843(m,13H,Ar-

H).,4.949(s,1H,NH).

3400(N-H str Amine).,3065(C-H

str Aromatic).,1590,1580(C=N str

Amine).

7b

7.790(s,1H,CH=N).,6.823-

7.753(m,12H,Ar-

H).,4.823(s,1H,NH2).

IR (KBr): 3177(N-H str. Amine),

3060(C-H str, Aromatic),

1640,1515(C=C str

),1565,1560(C=N str, Amine),

1430(N-H Bending)

7c

8.236(s,1H,CH=N).,6.067-

7.769(m,12H,Ar-H).,5.100(s,1H,-

OH).,4.349(s,1H,-NH).

IR (KBr): 3500-3000(OH str

Alcohol).,3367( N-H str), 1639(

C=C str Aromatic),1560,1525

(C=N str), 1450( N-H Bending),

1290( C-N str)

131

7d

8.784(s,1H,CH=N).,6.323-

7.890(m,12H,Ar-H).,3.065(s,1H,-

OCH3).,4.903(s,1H,-NH).

IR (KBr): 3375 (N-H str)., 3084

(C-H Aromatic), 2980(C-H str

Methoxy), 1584 (C=N str),

1514(C=C str Aromatic), 1464(C-

H Methoxy), 1263(C-N str),

1193(C-O str)

7e

8.018(s,IH,CH=N).,7.247-

7.930(m,12H,Ar-H).,3.566(s,1H,-

N[CH3]2).,4.533(s,1H,-NH).

IR (KBr): 3327(N-H str. Amine),

3016(C-H str, Aromatic), 2928(C-

H str,A lkyl), 1627 ( C=C str),

1568(C=N str)

132

Table 6.2.2 Elemental Analysis data of the synthesized compounds

S.No.Compound

Code

Molecular

Formula

Molecular

Weight

Melting

Point

Percentage

Yield

1 3a C29H20N202 428.48 220oC 76.23%

2 3b C29H19N304 473.48 244oC 78.63%

3 3c C30H22N203 458.51 189oC 72.23%

4 3d C29H21ClN202 464.94 218oC 74.83%

5 3e C29H21ClN202 464.94 217oC 75.93%

6 3f C29H18ClN304 507.92 233oC 76.42%

7 3g C29H19N304 473.48 252oC 78.76%

8 3h C29H18N406 518.48 247oC 77.23%

9 4a C31H24N402 484.55 147oC 75.62%

10 4b C31H23N504 529.55 143oC 73.98%

11 4c C32H26N403 514.57 163oC 77.53%

12 4d C32H25N505 559.57 156oC 74.78%

13 4e C31H23ClN402 518.99 166oC 77.19%

14 4f C31H22ClN504 563.99 176oC 78.98%

15 4g C31H23N504 529.55 172oC 74.00%

16 4h C31H22N606 574.54 192oC 79.39%

133

17 5a C29H21N302 443.5 169oC 75.17%

18 5b C29H20N404 488.49 154oC 73.94%

19 5c C30H23N303 473.52 158oC 75.48%

20 5d C30H22N405 518.52 172oC 78.68%

21 5e C29H20ClN302 477.94 155oC 74.13%

22 5f C29H19ClN404 522.94 146oC 71.19%

23 5g C29H20N404 488.49 151oC 76.42%

24 5h C29H19N506 533.49 166oC 75.29%

25 6a C14H11N02 225.24 189oC 72.59%

26 6b C14H12N202 240.26 196oC 74.58%

27 6c C14H11N03 241.24 184oC 76.74%

28 6d C15H13N03 255.27 209oC 76.18%

29 6e C16H16N202 268.31 198oC 75.44%

30 7a C20H15N3 297.35 298oC 74.25%

31 7b C20H16N4 312.37 225oC 75.93%

32 7c C20H15N30 313.35 237oC 74.39%

33 7d C21H17N30 327.38 204oC 72.23%

34 7e C22H20N4 340.42 220oC 78.10%

134

6.2 Discussion

Synthesis of 2-phenyl-4h-benzo[d][1,3]oxazin-4-one (1a)

To a stirred solution of anthranilic acid in pyridine, benzoyl chloride was added drop

wise at 80ºC for the period of 2 hrs. After complete addition, the reaction mixture was

stirred for another 3 hrs at room temperature. The reaction mixture was neutralized

with sodium bicarbonate and the solid separated, was filtered and washed thoroughly

with water and crystallized to give TLC pure yellow color crystals, m.p.: 213ºC (Yield:

76.83%). Its structure was confirmed on the basis of NMR and IR spectra.

NH2

O

OH

+

OCl

O

N

O

2'3'

4'

5'6'

5

6

78

The NMR spectrum of the compound showed three multiplets centered at δ 7.29,

δ 7.5 and δ 7.62, which could arise from the protons of the phenyl rings, H-3’, 4’, 5, H-

7, 8 and H-2’, 6’ respectively, while the proton H-5 appeared as doublet at δ 8.1.

These data are satisfactory for the cyclic structure. A further support to the above

structure was obtained by the IR spectral data. IR (KBr): 3085(C-H str,

Aromatic),1690(C=O str, Aromatic),1593(C=N str), 1210(C-O str).

135

Synthesis of 2-(4-nitrophenyl)-4h benzo [d] [1,3] oxazin-4-one (1b)

To a stirred solution of anthranilic acid in pyridine, nitro benzoyl chloride was added

drop wise at 80ºC for the period of 2 hrs. After complete addition, the reaction mixture

was stirred for another 3 hrs at room temperature. The reaction mixture was

neutralized with sodium bicarbonate and the solid separated, was filtered and washed

thoroughly with water and crystallized to give a crystalline TLC pure compound, m.p.:

192ºC (Yield: 78.23%). Its structure was confirmed on the basis of NMR and IR

spectra.

2'3'

5'6'

5

6

78

NH2

O

OH

+

OCl

NO2

O

N

O

NO2

The NMR spectrum of the compound showed three multiplets centered at δ 7.88,

δ 7.5 and δ 8.2, which could arise from the protons of the phenyl rings, H-2’,6’, H- 6,

7, 8 and H-3’, 4’ respectively, while the proton H-5 appeared as doublet at δ 8.1.

These data are satisfactory for the structure assigned to the above compound. A

further support to the above structure was obtained by the IR spectral data. IR (KBr):

3080(C-H str, Aromatic), 1685(C=O str Aromatic), 1598(C=N str), 1208(C-O str).

136

Synthesis of 4-(4-oxo-2-phenylquinazoline-3-(4h)-yl) benzaldehyde (2a)

A solution of compound 1a and 4-aminobenzaldehyde in ethanol containing a few

drops of pyridine was refluxed fro 4 hrs. On cooling a product, separated out, which

was filtered and crystallized from methanol to give a yellow colored crystalline

compound, m.p.: 189ºC (Yield: 77.39%), which was TLC pure and it was

characterized on the basis of NMR and IR Spectral data.

O

N

O

NH2 CHO

N

N

OCHO

56

2'3'

4'

5'6'

2"3"

5"6"

78

The NMR Spectrum of the compound showed a singlet at 9.78 arising from the

aldehydes (-CHO) group. In the aromatic region a multiplet located at 7.7 and 7.8

accounted for 4H of phenyl ring of benzaldehyde ring. The protons H-3’, 4’ and 5’ of

the phenyl ring appeared as triplet at 7.29, while the protons H-2’, 6’ could appear as

multiplet centered at 7.6. The protons H-6, 7, 8 appeared as multiplet centered at

7.4, while the proton H-5 could be seen as doublet located at 7.9. These data are

satisfactory for the structure assigned to the compound. This structure was further

supported by IR Spectra. IR (KBr): 3095(C-H str, Aromatic), 2885, 2821(C-H str,

Aldehyde), 1687(C=O str, aldehyde), 1608(C=N str), 1550(C=C str, Aromatic), 1300,

1236, (C-N str), 1159(C-O str).

137

Synthesis of 4-(2-(4-nitrophenyl)-4-oxoquinazolin -3 (4h)-yl) bnzaldehyde (2b)

To a solution of 1b in ethanol containing few drops of pyridine was added 4-

aminobenzaldehyde and the contents was refluxed for 3 hrs. On cooling a solid mass

separated out, which was filtered and crystallized from methanol to give yellow

colored crystalline compound, m.p.: 195ºC (Yield: 76.58%), which was TLC pure. Its

structure was established on the basis of NMR and IR spectral data.

56

2'3'

5'6'

2"3"

5"6"

78

O

N

O

NO2

NH2 CHO

N

N

OCHO

NO2

The NMR spectrum of the compound showed a singlet at 9.80, which could arise

from the –CHO group. The protons of the benaldehyde phenyl ring appeared as

multiplet centered at 7.80. The protons of the nitro phenyl ring appeared as a

multiplet at 7.88 to 8.22. The protons of H-6, 7, 8 appeared as multiplet centered

at 7.4, while the proton H-5 could be seen as doublet at 7.9. The increase in

chemical shift values of aromatic rings when compared to 2a compound indicates the

presence of nitro group in the structure. These data are satisfactory for the structure

assigned to the above compound. A further support to the above structure was

obtained by the IR spectral data. IR (KBr): 3107(C-H str, Aromatic), 2883, 2813(C-H

str, Aldehyde), 1681(C=O str aldehyde), 1604(C=N str), 1525, 1340 (Nitro Aromatic),

1246 (C-N str), 1161(C-O str).

138

Synthesis of 3-(4-((z)-3-oxo-3-phenyl prop-1-enyl) phenyl)-2-phenyl quinazolin-

4(3h)-one (3a)

To a solution of compound 2a and acetophenone in alcohol was added sodium

hydroxide solution and the mixture was stirred for 3 hrs. The reaction mixture was

kept in refrigerator for 24 hrs. A solid mass, which was separated out, was filtered and

crystallized from methanol to give TLC pure crystals, m.p.: 220ºC (Yield: 76.23%).

The structure was confirmed on the basis of spectral data.

5

62'

3'

4'5'

6'

2"3"

5"6"

78

N

N

OCHO

N

N

O

O

O

CH3A

B

C

Da

b

cd

e

The NMR spectrum of the compound two doublet δ 6.54 and δ 6.6 arising from the

ethylene CH=CH proton. The proton of phenyl ring A could be seen as multiplet

centered at δ 7.4 and a doublet at δ 8.1 of H-6, 7, 8 and H-5 respectively. The protons

of the phenyl ring B & C appear as δ 7.3 and δ 7.6. The protons of the phenyl ring D

could be seen as doublet at δ 7.81 and multiplet at δ 7.5 arising from the proton H-2, 6

and H-3, 4, 5 respectively. These data are satisfactory for the structure assigned to

the above compound. A further support to the above structure was obtained by the IR

spectral data. IR (KBr): 3085(CH- str, Aromatic), 3038(CH=CH str), 1675 (C=O str,

cyclic keto), 1665(C=O str, a,b- unsaturated keto), 1586 (C=N str).

139

Synthesis of 2-(4-nitrophenyl)-3-(4-((z)-3-oxo-3-phenylprop-1-enyl)-phenyl)-


A mixture of compound 2b and acetophenone in ethanol was added sodium hydroxide

and the reaction mixture was stirred for 3 hrs. The reaction mixture was processed as

usual to give TLC pure brown color crystalline compound, m.p.: 244ºC (Yield:

78.63%). The structure was established on the basis of NMR and IR Spectra.

56

2'3'

5'6'

2"3"

5"6"

78

A

B

C

Da

cd

e

N

N

OCHO

NO2

N

N

O

O

NO2

O

CH3

b

The NMR spectrum of the compound showed two doublets at δ 6.42 and δ 6.45

arising from the –CH=CH- protons. The protons of the phenyl ring A appears as

doublet at δ 7.2 (H-5), while the remaining proton appears as multiplet centered at δ

6.9 (H-6, 7, 8). A doublet appeared at δ 7.11 (H-a, e) and multiplet at δ 6.83 (H-b, c,

d), which could arise from the phenyl ring D. Another two sets of multiplets were seen

at δ 7.57 and δ 7.4, which could arise from the phenyl ring B and C respectively.



3076(CH- str, Aromatic), 3053(CH=CH str), 1687(C=O str, a,b-unsaturated keto),

1672 (C=O str, cyclic keto),1523,1347(N=O str).

140

Synthesis of 3-(4-((z-3-(4-methoxyphenyl)-3-oxoprop-1-enyl) phenyl-2-phenyl

quinazolin-4(3h)-one (3c)

To a solution of compound 2a in ethanol was added 1-(4-methoxy) phenyl ethanone

and the reaction conditions were maintained same as earlier and processed as usual

to give TLC pure crystals, m.p.: 189ºC (Yield: 72.23%) so obtained was characterized

on the basis of spectral data.

5

62'

3'

4'5'

6'

2"3"

5"6"

78

A

B

C

Da

b

cd

e

N

N

OCHO

N

N

O

O

OCH3

O

CH3

H3CO

The NMR spectrum of the compound showed a singlet at 3.17 arising from –OCH3

proton and two doublets at δ 6.57 and δ 6.63 arising from the ethylene –CH=CH-

proton. The proton of phenyl ring A could be seen as multiplet centered at δ 6.8 and a

doublet at δ 7.44 for H-6, 7, 8 and H-5 respectively. The protons of the phenyl ring B

& C could be seen as two multiplet centered at δ 6.74 and δ 7.04. The protons of the

acetophenone phenylring D could be seen as doublet at δ 7.25 and multiplet at δ 6.94

arising from the proton H-a, e and H-b, d. These data are satisfactory for the structure


obtained by the IR spectral data. IR (KBr): 3080(CH- str, Aromatic), 3048(CH=CH str),

2885(CH- str, Alkyl), 1682 (C=O str, cyclic keto), 1670(C=O str, a,b-unsaturated keto),

1583 (C=N str), 1213(-OCH3).

141

Synthesis of 3-(4-((z)-3-(4-methoxyphenyl)-3-oxoprop-1-enyl) phenyl)-2-(4-


To a solution of compound 2b in ethanol was added 1-(4-methoxy) phenyl ethanone


to give TLC pure compound m.p.: 218ºC (Yield:74.83 %) so obtained was

characterized on the basis of spectral data.

56

2'3'

5'6'

2"3"

5"6"

78

A

B

C

Da

d

e

b

N

N

OCHO

NO2

N

N

O

O

OCH3

NO2

O

CH3

H3CO

The NMR spectrum of the compound showed a singlet at δ 3.9, which could arise

from the –OCH3 group. The protons of the ethylene, -CH=CH- group appeared as

doublet at δ 6.7 and δ 6.9, indicated the successful formation of the expected

compound. There were two multiplets centered at δ 7.88 and δ 7.52, which could be

due to the protons of the phenyl ring B and C respectively. A doublet and another

multiplet was appeared at δ 7.7 and δ 7.2, which could arise due to the H-5 and H-6, 7

,8 protons of phenyl ring A respectively. These data are satisfactory for the structure


obtained by the IR spectral data. IR (KBr): 3089(CH- str, Aromatic), 3043(CH=CH str),

2879(CH-str, Alkyl), 1683 (C=O str, cyclic keto), 1660(C=O str, a,b-unsaturated keto),

1582 (C=N str), 1530, 1345(N=O str), 1210(-OCH3).

142

Synthesis of 3-(4-((z)-3-(3-chlorophenyl)-3-oxoprop-1-enyl) phenyl)-2-phenyl


To a solution of compound 2a in ethanol was added 1-(3-chloro) phenyl ethanone and

the reaction conditions were maintained same as earlier and processed as usual to

give TLC pure brown colored crystalline compound, m.p.: 217ºC (Yield: 75.93 %). Its

structure was characterized on the basis of spectral data.

5

62'

3'

4'5'

6'

2"3"

5"6"

78

A

B

C

Da

b

cd

e

N

N

OCHO

N

N

O

O

Cl

O

CH3

Cl

The NMR spectrum of the compound two doublets at δ 7.13 and δ 7.19 arising from

the ethylene –CH=CH- proton. The protons of the chloro phenyl ring D could be seen

as singlet δ 7.82 and multiplet centered at δ 7.5 arising from H-a, e and H-c, d

respectively. The protons of the phenyl ring B & C could be seen as two multiplet

centered at δ 7.29 and δ 7.6. The proton of phenyl ring A appears as doublet at δ 7.8

and multiplet centered at δ 7.46 accounted for H-5 and H-6, 7, 8 respectively. These

data are satisfactory for the structure assigned to the above compound. A further

support to the above structure was obtained by the IR spectral data. IR (KBr):

3096(CH- str, Aromatic), 3050(CH=CH str), 1680 (C=O str, cyclic keto), 1662(C=O str,

a,b-unsaturated keto, 1591 (C=N str).

143

Synthesis of 3-(4-((z)-3-(3-chlorophenyl)-3-oxoprop-1-enyl) phenyl)-2-(4-nitro-

phenyl) quinazolin-4(3h)-one (3f)

To a solution of compound 2b in ethanol was added 1-(3-chloro) phenyl ethanone and


give TLC pure yellowish white fine powder, m.p.: 233ºC (Yield: 76.42%) so obtained

was characterized on the basis of spectral data.

56

2'3'

5'6'

2"3"

5"6"

78

A

B

C

Da

c

d

e

N

N

OCHO

NO2

N

N

O

O

Cl

NO2

O

CH3

Cl

The NMR spectrum of the compound showed two doublets at δ 6.70 and δ 6.73

arising from the ethylene proton (-CH=CH-) which indicates the successful formation

of the expected product. A doublet were seen at δ 7.8 and multiplet at δ 7.46 arising

from the protons of the phenyl ring A H-5 and H-6, 7, 8 respectively. The protons of the

chlrophenyl ring D appears as multiplet centered at δ 7.5 and protons of ring B

appeared as another multiplet centered at δ 8.1, while the protons of ring C appears

as a multiplet at δ 7.28. These data are satisfactory for the structure assigned to the

above compound. A further support to the above structure was obtained by the IR

spectral data. IR (KBr): 3100(CH- str, Aromatic), 3045(CH=CH str),1682 (C=O str,

cyclic keto), 1663(C=O str, a,b-unsaturated keto), 1595 (C=N str), 1523, 1333(N=O

str).

144

Synthesis of 3-(4-((-2)-3-(-nitrophenyl)-3-oxoprop-1-enyl)-2-phenylquinazolin-

4(3h-one) (3g)

To a solution of compound 2a in ethanol was added 1-(3-nitro) phenyl ethanone and


give TLC pure compound, m.p.: 252ºC (Yield: 78.76 %)and its structure was

established on the basis of spectral data.

56

2'3'

4'5'

6'

2"3"

5"6"

78

A

B

C

Da

cd

e

N

N

OCHO O

CH3

O2N

N

N

O

O

O2N

The NMR spectrum of the compound showed two doublet at δ 6.98 and δ 7.14 arising

from the ethylene –CH=CH- proton. The protons of the nitro phenyl ring D appears as

multiplet centered for δ 8.50. The protons of the phenyl ring A could be seen as

doublet at δ 7.8 and a multiplet centered at δ 7.3 accounted for H-5 and H-6, 7, 8

respectively. The protons of the phenyl ring B & C could be seen as two multiplets

centered at δ 7.52 and δ 7.86. These data are satisfactory for the structure assigned

to the above compound. A further support to the above structure was obtained by the

IR spectral data. IR (KBr): 3084(CH- str, Aromatic), 3043(CH=CH str), 1688 (C=O str,

cyclic keto), 1669(C=O str, a,b-unsaturated keto), 1580 (C=N str).

145

Synthesis of 2-(4-nitrophenyl)-3-(4-((z)-3-(3-nitrophenyl)-3-oxoprop-1-enyl)-

phenyl)-qunazoline-4(3h)-one (3h)

To a solution of compound 2b and 1-(3- nitrophenyl) ethanone in alcohol was added

sodium hydroxide solution and the mixture was stirred for 3 hrs. The reaction mixture

was kept in refrigerator for 24 hrs. A solid mass, which was separated out, was

filtered and crystallized from methanol to give yellow colored crystals which as TLC

pure, m.p.: 247ºC (Yield: 77.23%). The structure was confirmed on the basis of NMR

& IR spectral data.

56

2'3'

5'6'

2"3"

5"6"

78

A

B

C

Da

c

d

e

N

N

OCHO

NO2

O

CH3

O2N

N

N

O

O

O2N

NO2

The NMR spectrum of the compound showed two doublets at δ 7.0 and δ 7.23 arising

from the ethylene proton (-CH=CH-) and the disappearance of the signals for the

aldehydes group indicates the successful formation of the expected product. The

remaining protons of the expected compounds were seen as a two multiplets centered

at δ 7.5 to δ 8.2. A further support to the above structure was obtained by the IR

spectral data. IR (KBr): 3102(CH- str, Aromatic), 3038(CH=CH str),1690 (C=O str,

cyclic keto), 1664(C=O str, a,b-unsaturated keto), 1578 (C=N str), 1518, 1350 (N=O

str).

146

Synthesis of 3-(4-(1-acetyl-4, 5-dihydro-5-phenyl-1h-pyrazol-3-yl) phenyl)-2-

phenyl quinazolin-4(3h)-one (4a)

To a solution of compound 3a in absolute alcohol was added hydrazine hydrate drop

by drop with constant stirring and refluxed for 12 hrs in presence of glacial acetic acid.

The reaction mixture was cooled and distilled off; the solid separated was filtered and

washed thoroughly with petroleum ether and crystallized from methanol to give TLC

brown color crystalline compound, m. p.: 147ºC (Yield: 75.62%). Its structure was

established on the basis of NMR and IR Spectra.

N

N

O

O

NH2 NH2

O

OH

CH3

N

N

O NN

O

CH3

A

B

C

D

56

78

4' 5'

3'

The NMR spectrum of the compound showed a singlet at δ 2.16 arising from the

methoxy proton (-OCH3). In the aliphatic region, -CH-CH2- protons of the pyrazole

ring appears as a doublet at δ 2.8 and a triplet at δ 4.60 accounted for H-4 and H-5

respectively, indicates the successful formation of the expected compound. The

protons of the phenyl ring “A” appeared as doublet at δ 7.89 and a multiplet centered

at δ 7.3 arises from H-5 and H-6, 7, 8 respectively. Two multiplets were seen at δ 7.2

and δ 7.0, which could arise from the protons of the phenyl ring “B” & “C”. The

147

protons of the phenyl ring “D” in the pyrazole nucleus appears as multiplet at δ 6.9.



3047(C-H- str, Aromatic), 2887(C-H str, Alkyl), 1670 (C=O str, Aromatic keto),

1649(C=O str, Aliphatic Amide keto), 1633, 1612(C=C str, Aromatic), 1556 (C=N str),

1456, 1384( C-H Bending Alkyl), 1274(C-N str).

Synthesis of 3(4-(1-acetyl-4, 5-dihydro-5-phenyl-1h-pyrazol-3-yl) phenyl)-2-(4-

nitrophenyl) quinazolin-4(3h)-one (4b)

To a solution of compound 3b in absolute alcohol was added hydrazine hydrate drop

by drop with constant stirring and the reaction conditions were maintained same as

earlier and processed as usual to give TLC pure compound, m.p.: 143ºC (Yield:

73.98%). The structure of the compound was characterized on the basis of spectral

data.

A

B

C

D

56

78

4' 5'

3'

2"

3"5"

6"

N

N

O

O

NO2

NH2 NH2

O

OH

CH3

N

N

O NN

O

CH3

NO2

4"


from the –OCH3 proton. The protons of the pyrazole nucleus appears as triplet at δ

148

4.9 (H-5’) and doublet at δ 2.4 (H-4’) in the aliphatic region. It indicates the scucessfull

formation of the expected compound. The protons of the benzene quinazolinone ring

“A” appears as multiplet and a doublet at δ 7.5 and δ 7.9, which could arise from the

proton H-6, 7, 8 and H-5 respectively. The protons of the nitrophenyl ring “B” appears

as multiplet centered at δ 7.8, while the protons of the amino phenyl ring “C”

and phenyl ring “D” appears as multiplet at δ 7.6 and δ 7.1 respectively. These data

are satisfactory for the structure assigned to the above compound. A further support to

the above structure was obtained by the IR spectral data. IR (KBr): 3132(C-H- str,

Aromatic), 2879(C-H str, Alkyl), 1666 (C=O str, Aromatic keto), 1649(C=O str, Aliphatic

Amide keto), 1599 (C=N str), 1535, 1340(Aromatic Nitro), 1446, 1384 (C-H Bending

Alkyl), 1276, 1249(C-N str).,

Synthesis of 3-(4-(1-acetyl-4, 5-dihydro-5-(4-methoxyphenyl)-1h-pyrazol-3-yl)-

phenyl)-2-phenylquinazolin-4(3h)-one (4c)

To a solution of compound 3c in absolute alcohol was added hydrazine hydrate drop

by drop with constant stirring and refluxed for 12 hrs in presence of glacial acetic acid.

The reaction mixture was processed as usual to give TLC pure compound,

m.p.: 163ºC so obtained was characterized on the basis of spectral data.

149

A

B

C

D

56

78

4' 5'

3'

N

N

O

O

OCH3

NH2 NH2

O

OH

CH3

N

N

O NN

O

CH3

OCH3

2"

3"5"

6"

The NMR spectrum of the compound showed two singlet at δ 3.88 and δ 2.2 arising

from the two methoxy protons (-OCH3) in the structure. The protons of -CH-CH2-

group in the pyrazole ring appears as triplet and doublet at δ 4.61 (H-5’) and δ 2.74

(H-4’) respectively indicates the successful formation of the expected product. The

protons of the phenyl ring “B” & “C” appears as multiplet centered at δ 7.6, while the

protons of the ring “D” appeared as doublets at δ 7.0 (H-3”, 5”) and δ 7.2 (H-2”, 6”)

respectively. The protons of benzene ring of quinazolinone nucleus (“A”) appears as a

doublet at δ 7.86 (H-5) and a multiplet centered at δ 7.4 (H-6, 7, 8). These data are

satisfactory for the structure assigned to the above compound. A further support to the

above structure was obtained by the IR spectral data. IR (KBr): 3026(C-H- str,

Aromatic)., 2966 (C-H str, Alkyl), 2943(C-H str, Methoxy),1678 (C=O str, Aromatic

keto), 1629 (C=O str, Aliphatic Amide keto), 1573 (C=N str), 1500, 1448, 1421( C-H

Bending, Alkyl),1286, (C-N str), 1222(C-O str, Methoxy).

150

Synthesis of 3-(4-(1-acetyl-4, 5-dihydro-5-(4-methoxyphenyl)-1h-pyrazol-3-yl)-

phenyl)-2-(4-nitrophenyl) quinazolin-4(3h)- one (4d)

To a solution of compound 3d in absolute alcohol was added hydrazine hydrate drop


earlier and processed as usual to give TLC pure brown colour crystalline compound,

m.p.: 156ºC (Yield: 74.78%). The structure of the compound was characterized on the

basis of spectral data.

A

B

C

D

56

78

4' 5'

3'

2"

3"5"

6"

N

N

O

O

OCH3

NO2

NH2 NH2

O

OH

CH3

N

N

O NN

O

CH3

OCH3

NO2

The NMR spectrum of the compound showed a triplet at 4.63 and a doublet at 2.5 in

the aliphatic region, which could arise from the –CH2- and –CH- proton of the

pyrazole ring respectively. It indicates the successful formation of the expected

compound. The protons of the two methoxy (-OCH3) group appears as two singlet at

3.8 and 2.0. Two sets of multiplets centered at 8.2 and 7.7 could be seen arising from

the protons of the nitro phenyl ring “B” and amino phenyl ring “C”. The protons of the

ring “A” appears as doublet at 7.9 (H-5) and multiplet centered at 7.5 (H-6, 7, 8). The

protons of the methoxy phenyl ring “D” appears as two doublets at 6.7 and 7.01 for

151

the proton H-3, 5 and H-2, 6 respectively. These data are satisfactory for the structure


obtained by the IR spectral data. IR (KBr): 3132(C-H- str, Aromatic), 2999(C-H str,

Methoxy), 2877(C-H str, Alkyl), 1662 (C=O str, Aromatic keto), 1641(C=O str, Aliphatic

Amide keto), 1606(C=C str, Aromatic), 1595 (C=N str),1533,1340(Aromatic

Nitro),1417,1340( C-H Bending Alkyl),1294,1249(C-N str),1203(C-O str, Methoxy).

Synthesis of 3-(4-(1-acetyl-5-(3-chlorophenyl)-4, 5-dihydro-1h-pyrazol-3-yl)-

phenyl)-2-phenylquinazolin-4(3h)-one (4e)

To a solution of compound 3e in absolute alcohol was added hydrazine hydrate drop




data.

A

B

C

D

56

78

4' 5'

3'

2"

5"

6"

N

N

O

O

Cl

NH2 NH2

O

OH

CH3

N

N

O NN

O

CH3

Cl

4"

The NMR spectrum of the compound showed a singlet at δ 2.2 arising from –OCH3

proton and –CH2- proton (H-5’) appear as triplet at δ 4.57 and -CH- proton (H-4’)

152

appears as doublet at δ 2.74 of pyrazole ring. It indicates that the expected compound

was successfully formed. The protons of the phenyl ring “B” & “C” appears as multiplet

centered at δ 7.3 and δ 7.6 respectively, while the chloro phenyl protons i.e., ring “D”

appears as doublet at δ 7.0 (H-6”) and a multiplet centered at δ 7.1 (H-2”, 3”,5”). The

protons of phenyl ring “A” appear as doublet and a multiplet centered at δ 7.9 and δ

7.4 of H-5 and H-6, 7, 8 respectively).



3126(C-H- str, Aromatic), 2991(C-H str, Alkyl), 1761 (C=O str, Aromatic keto),

1639(C=O str, Aliphatic Amide keto), 1579 (C=N str), 1558, 1543(c=C str, Aromatic),

1402 (C-H Bending Alkyl), 1151(C-N str).

Synthesis of 3-(4-(1-acetyl-5-(3-chlorophenyl)-4, 5-dihydro-1h-pyrazol-3-yl)-

phenyl)-2-(4-nitrophenyl) quinazolin-4(3h)-one (4f)

To a solution of compound 3f in absolute alcohol was added hydrazine hydrate drop




data.

153

A

B

C

D

56

78

4' 5'

3'

2"

5"

6"

4"

N

N

O

O

Cl

NO2

NH2 NH2

O

OH

CH3

N

N

O NN

O

CH3

Cl

NO2


from the -OCH3 proton. In the aliphatic region, a triplet at δ 4.9 (H-5’) and doublet at δ

2.6 (H-4’) from the pyrazole ring which indicates the successful formation of the

expected compound. The protons of the ring “A” appears as doublet at δ 7.8 (H-5)

and multiplet centered at δ 7.45 (H-6, 7, 8) respectively. The protons of the nitro

phenyl ring “B” appear as multiplet centered at δ 8.2, while the protons of the amino

phenyl ring “C” appears as multiplet centered at δ 7.7. The protons of the chloro

phenyl ring “D” appear as doublet at δ 7.0 (H-6”) and a multiplet centered at δ 7.1 (H-

2”, 4”, 5”) These data are satisfactory for the structure assigned to the above

compound. A further support to the above structure was obtained by the IR spectral

data. IR (KBr): 3097(C-H- str, Aromatic), 2985 (C-H str, Alkyl), 1670 (C=O str, Aromatic

keto), 1604 (C=N str), 1539, 1342(Aromatic Nitro), 1477, 1381( C-H Bending Alkyl),

1274, 1213(C-N str).

154

Synthesis of 3-(4-(1-acetyl-4, 5-dihydro-5-(3-nitrophenyl)-1h-pyrazol-3-yl) -

phenyl)-2-phenyl quinazolin-4(3h)-one (4g)

To a solution of compound 3g in absolute alcohol was added hydrazine hydrate drop


earlier and processed as usual to give TLC pure yellow color crystals, m.p.: 172ºC

(Yield: 74 %). The structure of the compound was characterized on the basis of

spectral data.

A

B

C

D

56

78

4' 5'

3'

2"

5"

6"

4"

N

N

O

O

O2N

NH2 NH2

O

OH

CH3

N

N

O NN

O

CH3

NO2

The NMR spectrum of the compound showed a singlet at aliphatic region at δ 2.2

(-OCH3) arising from the methoxy proton and triplet at δ 4.54 and doublet δ 2.74,

which could arise from the –CH2-CH- proton of pyrazole nucleus. It indicates the

successful formation of pyrazole nucleus. The protons of the phenyl rings “B” & “C”

appears as multiplet centered at δ 7.3 and δ 7.6, while the protons of the nitro phenyl

ring “D” appears as multiplet centered at δ 7.8. The protons of the benzene ring of

quinazolinone nucleus appears as doublet at δ 7.9 (H-5) and a multiplet centered at

δ 7.45 (H-6, 7, 8). These data are satisfactory for the structure assigned to the above

155


data. IR (KBr); 3130 (C-H- str, Aromatic), 2874 (C-H str, Alkyl), 1651 (C=O str,

Aromatic keto), 1624 (C=O str, Aliphatic Amide keto), 1556 (C=N str), 1527, 1402

(Aromatic Nitro), 1242, (C-N str).

Synthesis of 3-(4-(1-acetyl-4, 5-dihydro-5-(3-nitrophenyl)-1h pyrazol-3-yl)

phenyl)-2-(4-nitrophenyl) quinazolin-4(3h)-one (4h)

To a solution of compound 3h in absolute alcohol was added hydrazine hydrate drop


earlier and processed as usual to give TLC pure compound, m.p.: 192ºC (Yield: 79.39

%). The structure of the compound was characterized on the basis of spectral data.

A

B

C

D

56

78

4' 5'

3'

2"

5"

6"

4"

N

N

O

O

O2N

NO2

NH2 NH2

O

OH

CH3

N

N

O NN

O

CH3

NO2

NO2

The NMR spectrum of the compound showed a triplet at δ 4.54 and a doublet at δ 2.4

arising from the –CH2- and –CH- proton of the pyrazole ring, which indicates the

successful formation of the expected product. The protons of the nitrophenyl rings “B”

156

& “D” appear as multiplet centered at δ 7.5 and δ 8.1 respectively. The protons of the

amino phenyl ring “C” appear as multiplet centered at δ 7.7, while the protons ring “A”

appear as multiplet centered at δ 7.3 (H-6, 7, 8) and a doublet at δ 7.9 (H-5). These


support to the above structure was obtained by the IR spectral data. IR (KBr): 3057

(C-H- str, Aromatic), 2978(C-H str, Alkyl), 1639(C=O str, Aliphatic Amide keto), 1589

(C=N str), 1531, 1325(Aromatic Nitro), 1460, 1381( C-H Bending Alkyl), 1284, 1205(C-

N str). Refer Page 259.

Synthesis of 2-phenyl-3- (4- (5-phenyl-4, 5-dihydroisazol-3-yl) - phenyl

quinazolin-4(3h)-one (5a)

A mixture of compound 3a, hydroxylamine hydrochloride and sodium acetate in

ethanol was refluxed for 6 hr. The solvent was concentrated off under reduced

pressure and poured onto crushed ice, the solid mass separated was filtered and

crystallized from methanol to give brown colored crystalline compound Va as TLC

pure, m.p.169 º C (Yield: 75.17%). The structure was established on the basis of

spectral data.

N

N

O

O

NH2OH C2H5OHN

N

O NO

56

78

2'3'

4'5'

6'

2"

3"

5"6"

4'" 5'"

A

B

D

C

157

The NMR spectrum of the compound showed a triplet and double doublet at δ 4.18

and δ 2.81, which could arise from H-5”’ and H-4”’ of isoxazole ring. It indicates

the successful formation of the expected compound. The protons of ring ‘D’ appears

as singlet at δ 7.027, while the protons of the phenyl ring ‘B’ & ‘C’ appears as two

multiplet at δ 7.29 (H-3’, 4’, 5’) and 7 δ.8 (2’, 6’, 2”, 3”, 5”, 6”) respectively. The phenyl

ring ‘A’ protons appears as multiplet centered at δ 7.7 (H-6, 7, 8) and a doublet at δ

8.2 (H-5) respectively. These data are satisfactory for the structure assigned to the


spectral data. IR (KBr): 3092(C-H- str, Aromatic), 2939 (CH str, Alkyl), 1678 (C=O str,

Aromatic keto), 1586 (C=N str).

Synthesis of 2-(4-nitrophenyl)-3-(4-(5-phenyl-4,5-dihydroisazol-3-yl) - phenyl


To a solution of compound 3b in ethanol, hydroxylamine hydrochloride, sodium

acetated was added and the reaction mixture was refluxed for 6 hrs. The reaction

conditions and processed as described in earlier cases. A solid mass obtained was

crystallized to give a TLC pure pale yellow colored compound, m.p.: 157ºC (Yield:

73.94%). Its structure was established on the basis of NMR and IR data.

158

56

78

2'3'

5'6'

2"

3"

5"6"

4'" 5'"

A

B

D

C

N

N

O

O

NO2

NH2OH C2H5OHN

N

O NO

NO2

The NMR spectrum of the compound showed a triplet at δ 4.23 and a doublet at δ

2.85, which could arise from –CH- (H-5’”) and –CH2- (H-4’”) proton of the isoxazole

ring. It indicates the successful formation of the expected product. The protons of the

phenyl ring ‘D’ appears as singlet at δ 6.7. The proton H-3’, 5’ and H-2’, 6’ of phenyl

ring ‘B’ appear as multiplets at δ 8.2 and δ 8.0 respectively. The protons of phenyl

ring ‘A’ in quinazolinone nucleus appears as doublet and multiplet at δ 7.7 (H-6, 7, 8)

and δ 8.01 (H-5). The protons of ring ‘C’ appears as multiplet centered at δ 7.6.



3101(CH- str, Aromatic), 1670 (C=O str, Aromatic keto), 1620 (C=C str), 1560(C=N

str), 1506, 1344(Aromatic Nitro), 1249(C-N str), 1103(C-O str).

Synthesis of 3-(4-(5-(4-methoxyphenyl)-4, 5-dihydroisazol-3-yl) - phenyl)-2-

phenyl quinazolin-4(3h)-one (5c)

To a solution of Compound 3c in ethanol, hydroxylamine and sodium acetate was

added and the reaction mixture was refluxed for 6 hrs. The reaction mixture was

159

processed as usual to give TLC pure brownish colored crystalline compound, m.p.:

158 °C (Yield: 75.48%) so obtained were characterized on the basis of spectral data.

56

78

2'3'

4'5'

6'

2"

3"

5"6"

4'" 5'"

A

B

D

C

a

b

d

e

N

N

O

O

OCH3

NH2OH C2H5OHN

N

O NO

OCH3

The NMR spectrum of the compound showed a triplet and double doublets at δ 4.25

and δ 2.86 which could arise from the –CH- (H-5’”) and –CH2- (H-4’”) of the isoxazole

ring. The methoxy proton (-OCH3) appeared as singlet at δ 4.0, while the protons of

the methoxy phenyl ring appears as two doublets at δ 7.02 and δ 7.26 from H-b, d and

H-a, e respectively. Two multiplet were seen in aromatic region at δ 7.22 and δ 7.26,

which could arise from phenyl ring ‘B’ & ‘C’ of H-3’, 4’, 5’ and H-2’, 6’, 2”, 3”, 5”, 6”

respectively. The protons of ring ‘A’ appeared as multiplet centered at δ 7.5 (H-6, 7, 8)

and a doublet at δ 7.9 (H-5). These data are satisfactory for the structure assigned to


spectral data. IR (KBr): 3064(C-H- str, Aromatic), 2987(C-H str Methoxy), 1678 (C=O

str, Aromatic keto), 1627(C=C str Aromatic), 1568 (C=N str), 1253, 1166(C-O str).

160

Synthesis of 3-(4-(5-(4-methoxyphenyl)-4, 5-dihydroisazol-3-yl) - phenyl)-2-(4-


A mixture of compound 3d, hydroxylamine hydrochloride and sodium acetate in

ethanol was refluxed for 6 hr. The solvent was concentrated off under reduced

pressure and poured onto crushed ice, the solid mass separated was filtered and

crystallized from methanol to give yellow color crystalline compound Va as TLC pure,

m.p.: 172º C (Yield: 78.68 %). The structure was established on the basis of spectral

data.

56

78

2'3'

5'6'

2"

3"

5"6"

4'" 5'"

A

B

D

C

a

b

d

e

N

N

O

O

OCH3

NO2

NH2OH C2H5OHN

N

O NO

OCH3

NO2

The NMR spectrum of the compound showed a double doublet at δ 2.84 and a triplet

at δ 4.25, which could arise from the proton of isoxazole ring H-4’” and H-5’”

respectively. The protons of the methoxy (-OCH3) group appears as singlet at δ 4.13

and the remaining protons of the methoxy phenyl ring ‘D’ appears as two doublet at δ

7.1 and δ 7.42, which could accounted for the proton H-b, d and H-a, e respectively.

The protons of phenyl ring ‘C’ and ‘B’ appears as multiplets centered at δ 7.6 and δ

7.9 respectively. The protons of phenyl ring ‘A’ appears as multiplet at δ 7.6 (H-6, 7,

161

8) and doublet at δ 7.8 (H-5). These data are satisfactory for the structure assigned to


spectral data. IR (KBr0: 3090(CH- str, Aromatic), 2939 (CH str, Alkyl), 1678 (C=O str,

Aromatic keto), 1600(C=N str), 1586 (C=N str), 1521, 1304(Aromatic Nitro), 1246(C-N

str), 1182(C-O str, Methoxy).

Synthesis of 3-(4-(5-(3-chloro phenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-phenyl


To a solution of Compound 3e in ethanol, hydroxylamine and sodium acetate was


processed as usual to give TLC pure brown color crystalline compound, m.p.: 155 °C

(Yield: 74.13%) so obtained were characterized on the basis of spectral data.

56

78

2'3'

4'5'

6'

2"

3"

5"6"

4'" 5'"

A

B

D

C

N

N

O

O

Cl

NH2OH C2H5OHN

N

O NO

Cl

The NMR of the compound showed a triplet and a double doublet at δ 4.38 and δ

2.88, which could arise from the proton H-5’” and H-4’” of isoxazole ring. It indicates

the successful formation of the expected cyclized product. The protons of the chloro

phenyl ring appears as multiplet centered at δ 6.8, while the protons of ring B and C

162

appears at two sets of multiplet centered at δ 7.32 (H-3’, 4’, 5’) and δ 8.4 (H-2’, 6’, 2”,

3”, 5”, 6”) respectively. The protons of ring appears as multiplet at δ 7.5 (H-6, 7, 8)

and a doublet at δ 8.2 (H-5). These data are satisfactory for the structure assigned to


spectral data. IR (KBr): 3036(C-H str, Aromatic), 2922 (CH str, Alkyl), 1658(C=0 str,

Aromatic Keto), 1591 (C=N str).

Synthesis of 3-(4-(5-(3-chloro phenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-(4-


To a solution of compound 3f in ethanol, hydroxylamine hydrochloride, sodium

acetated was added and the reaction mixture was refluxed for 6 hrs. The reaction

conditions and processed as described in earlier cases. A solid mass obtained was

crystallized to give a TLC pure yellow colored crystalline compound, m.p.: 146ºC

(Yield: 71.19%). Its structure was established on the basis of NMR and IR data.

56

78

2'3'

5'6'

2"

3"

5"6"

4'" 5'"

A

B

D

C

a

b

d

c

N

N

O

O

Cl

NO2

NH2OH C2H5OHN

N

O NO

Cl

NO2

The NMR spectrum of the compound showed a triplet at δ 4.35 (H-5’”) and double

doublet at δ 2.91 (H-4’”), which indiates the successful formation of the expected

163

cyclized product. The protons of the nitro phenyl ring ‘B’ appears as multiplet

centered at δ 7.86 and chloro phenyl ring ‘D’ appears as two multiplets at δ 7.20 (H-a,

b) and δ 7.08 (H- c, d). There was another multiplet centered at δ 7.5, which could be

due to the protons of the phenyl ring ‘C’, while the protons of the phenyl ring ‘A’

appears as doublet at δ 7.72 (H-5) and a multiplet at δ 7.3 (H-6, 7, 8) respectively.



3085(CH- str, Aromatic), 2920 (CH str Alkyl), 1675 (C=O str Aromatic keto), 1586

(C=N str).

Synthesis of 3-(4-(5-(3-nitrophenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-phenyl

quinazolin-4(3h)-one (5g)

To a solution of Compound 3g in ethanol, hydroxylamine and sodium acetate was


processed as usual to give TLC pure yellow colored crystalline compound, m.p.: 151

°C (Yield: 76.42%) so obtained were characterized on the basis of spectral data.

56

78

2'3'

4'5'

6'

2"

3"

5"6"

4'" 5'"

A

B

D

C

N

N

O

O

O2N

NH2OH C2H5OHN

N

O NO

NO2

164

The NMR spectrum of the compound showed a triplet and double doublets at δ 4.925

and δ 2.758, which could arise from the protons of –HC- and –CH2- respectively of

isoxazole ring. It indicates the successful formation of the expected compound. The

protons of the nitro phenyl ring appears as multiplet centered at 8.00 and phenyl ring

‘B’ &’C’ protons appears as multiplet centered at δ 7.3 (H-3’, 4’, 5’) and δ 7.8 (H-2’, 6’,

2”, 3”, 5”, 6”) respectively.. The phenyl ring ‘A’ appears as multiplet and doublets at δ

7.5 and δ 7.9 of H-6, 7, 8 and H-5 respectively. These data are satisfactory for the

structure assigned to the above compound. A further support to the above structure

was obtained by the IR spectral data. IR ( KBr): 3085(CH- str, Aromatic), 2920 (CH str

Alkyl), 1675 (C=O str Aromatic keto), 1586 (C=N str).

Synthesis of 3-(4-(5-(3-nitrophenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-(4-

nitrophenyl) quinazolin-4(3h)-one (5h)

A mixture of compound 3h, hydroxylamine hydrochloride, sodium acetated in ethanol

was refluxed for 6 hrs. The reaction conditions and processed as described in earlier

cases. A solid mass obtained was crystallized to give a TLC pure yellow colored

crystalline compound, m.p.: 166ºC (Yield: 75.29%). Its structure was established on

the basis of NMR and IR data.

165

56

78

4'" 5'"

A

B

D

C

N

N

O

O

O2N

NO2

NH2OH C2H5OHN

N

O NO

NO2

NO2

The NMR spectrum of the compound showed a double doublet and triplet at δ 2.69

and δ 4.27, which could arise from –CH2- and –CH- of isoxazole ring respectively.

It indicates the successful formation of the expected compound. In the aromatic

region a broad multiplet centered at δ 8.2 could be seen, which arises from the

protons of ring ‘B’ & ‘D’. Another sets of multiplets were also seen at δ 7.4 and δ 7.01,

which could be accounted for the protons of H-6, 7, 8 of ring ‘A’ and protons of the

phenyl ring ‘C’. A doublet appears in the aromatic region at δ 7.7, which could arise

from the proton H-5 of ring A. These data are satisfactory for the structure assigned to


spectral data. IR (KBr): 3091(CH- str, Aromatic), 2924 (CH str Alkyl), 1661 (C=O str

Aromatic keto), 1541 (C=N str).

Synthesis of (e)-2-(benzylideneamino) benzoic acid (6a)

To a solution of anthranilic acid in ethanol was added benzaldehyde and the content

was refluxed for 2 hrs in water bath with occasional shaking. The solvent was

166

evaporated off and the residue was crystallized from the methanol to give colorless

crystalline compound VIa as TLC pure, m.p.189º C (Yield: 72.59%). The structure

was established on the basis of spectral data.

NH2

O

OH

+

CHO

N

O

OH

34

56

2'

3'4'

5'6'A

B


proton of carboxylic acid (-COOH) and the aldamine (-N=CH-) proton appear as

singlet at δ 7.86, which indicates the successful condensation of the reaction. The

protons of the phenyl ring A appears as doublet at δ 7.7 (H-6) and a triplet at δ 7.8 (H-

4), while the remaining proton appears as multiplet at δ 7.5 (H-3, 5). The protons of

phenyl ring B appears as multiplet centered at δ 6.8 and δ 7.1 of H-3’, 4’, 5’ and H-2’,

6’ respectively. A further support to the above structure was obtained by the IR

spectral data.IR (KBr): 3500-2600(OH str, Carboxylic Acid).,3117(CH str,

Aromatic).,1687(C=O str, Carboxylic Acid).,1608(C=C str).,1516(C=N str).,1309(C-O

str).,1192(C-N str)., 678(OH Bending of Carboxylic Acid). These data are satisfactory

for the structure assigned to the above compound.

Synthesis of (e)-2-(4-aminobenzylideneamino) benzoic acid (6b)

To a solution of anthranilic acid in ethanol was added 4-amino benzaldehyde and the

reaction conditions were maintained same as earlier and processed as usual to give

167

TLC pure brown colour crystalline powder m.p. 196ºC (Yeild: 74.58%) so obtained

was characterized on the basis of spectral data.

3

4

56

2'

3'

5'6'A

BNH2

O

OH

+

CHO

NH2

N

O

OH

NH2


aldamine (-N=CH-) proton and the carboxylic acid (-COOH) proton appear as singlet

at δ 10.08. The amino (-NH2-) proton appear as singlet in the aliphatic region at δ

3.95, while the protons of the amino phenyl ring B appears as two doublets at δ 7.18

and δ 7.3 arising from H-3’ , 5’ and H-2’, 6’ respectively. The protons of the phenyl ring

A appears as doublet at δ 7.4 (H-6), a triplet at δ 7.5 (H-4) and a multiplet centered at

δ 7.2 (H-3, 5). These data are satisfactory for the structure assigned to the above


data.IR (KBr): 3500-2700(OH str, Carboxylic Acid),.3360,3228(N-H str,

Amine).,3078(C-H str, Aromatic).,1689(C=O str, Carboxylic Acid).,1595(C=N

str).,1301(C-N str).,1109(C-O str).

Synthesis of (e)-2-(2-hydroxybenzylideamino) benzoicacid (6c)

To a solution of anthranilic acid in ethanol, 2-hydroxy benzaldehyde was added and

the reaction mixture was refluxed for 2 hrs in the water bath. The reaction conditions

168

and processed as described in earlier cases. A solid mass obtained was crystallized to

give a TLC colorless crystalline compound m. p. 184 °C (Yield: 76.74%). Its structure

was established on the basis of NMR and IR data.

34

56

3'4'

5'

6'A

BNH2

O

OH

+

CHO

OH N

O

OH

OH


aldamine proton and a singlet at δ 10.15 arising from the carboxylic acid proton. The

protons of the ring A appears as triplet at δ 8.0 (H-4) and doublet at δ 7.8 (H-6) while

the remaining proton appears as multiplet at δ 7.3 (H-3,5). The hydroxyl proton (-OH)

appear as singlet at δ 5.02 and the protons of ring B appear as doublet at δ 6.9 (H-3’,

5’) and triplet at δ 7.12 (H-4’) while H-5’ proton appeared as triplet at δ 7.4. These


support to the above structure was obtained by the IR spectral data. IR (KBr): 3500-

2500(OH str Carboxylic Acid).,3085(CH str Aromatic).,1680(C=O str Carboxylic

Acid).,1623(C=C str).,1590(C=N str).,1213(-OCH3).

Synthesis of (e)-2-(4-methoxy benzylideneamino) benzoic acid (6d)

To a solution of anthranilic acid in ethanol, 4-methoxy benzaldehyde was added and

the reaction mixture was refluxed for 2 hrs in the water bath. The reaction conditions

and processed as described in earlier cases. A solid mass obtained was crystallized to

169

give a TLC pure yellow color crystalline compound VId, m. p. 209 °C (Yeild: 76.81%) .

Its structure was established on the basis of NMR and IR data.

34

56

2'

3'

5'6'A

BNH2

O

OH

+

CHO

OCH3

N

O

OH

OCH3

The NMR spectrum showed a singlet at δ 3.56 arising from the –OCH3 proton and the

aldamine proton appears as singlet at δ 8.01. In the carboxylic acid region, a singlet

could be seen at δ 10.22 shows the presence of the carboxylic acid proton. The

protons of the ring B appears as doublet at δ 7.2 (H-3’, 5’) and a triplet at δ 7.5 (H-2’,

6’). A triplet at δ 7.93 (H-6) and a multiplet δ 7.62 (H-3, 4, 5) could be seen in the

aromatic region and accounted for the protons of the ring A. These data are


above structure was obtained by the IR spectral data.IR (KBr): 3500-2500(OH str

Carboxylic Acid).,3085(CH str Aromatic),1680(C=O str Carboxylic Acid).,1623(C=C


170

Synthesis of (e)-2-(4-(dimethylamino)-benzylideneamino)

Benzoic acid (6e)

To a solution of anthranilic acid in ethanol was added 4-dimethylamino benzaldehyde


to give TLC pure orange colored crystalline compound VIe, m.p. 198ºC (Yeild: 75.44)

so obtained was characterized on the basis of spectral data.

3

4

56

2'

3'

4'

5'6'A

BNH2

O

OH

+

CHO

NCH3 CH3

N

O

OH

NCH3

CH3


aldamine proton indicates the successful formation of the expected compound. A

singlet could be seen at δ 10.17 arising from the carboxylic acid proton. The protons

of dimethyl amino group appear as singlet at δ 3.2 and the protons of ring B appears

as doublet at δ 6.76 (H-3’, 5’) and a multiplet centered at δ 7.01 (H-2’, 6’). The

protons of phenyl ring A appears as triplet at δ 7.3 (H-6), while the remaining proton

appear as multiplet centered at δ 7.2 (H-3, 4, 5). A further support to the above

structure was obtained by the IR spectral data.IR (KBr): 3500-2500(OH str Carboxylic

Acid).,3085(CH str Aromatic),1680(C=O str Carboxylic Acid).,1623(C=C

str).,1590(C=N str).2942(C-H str Alkyl). These data are satisfactory for the structure

assigned to the above compound.

171

Synthesis of (z)-2-(1h – benzo[d]imidazol-2-yl)-n-benzyliden-amine (7a).

To a mixture of Compound 6a and o-phenylenediamine, 4N HCl was added and

stirred for 4 hours. The reaction mixture was made alkaline with ammonia and the

solid mass separated was filtered and washed thorough with ice cold water and

crystallized from methanol to give TLC pure white color compound VIIa; m.p.: 208º

(Yield: 74.25% ). Its structure was established on the basis of NMR and IR data.

4

5

6

3'

4'

5'6'

A B

2" 3"

4"

5"6"

C

N

O

OH

NH2

NH2N

NH

N

7


aldamine proton and the amino (-NH) proton of the imidazole nucleus appear as a

singlet at δ 4.74. It indicates the successful formation of the expected compound. The

protons of the phenyl ring appear as multiplet centered at δ 7.1, δ 7.5 and δ 7.7 of ring

B, C and A respectively. These data are satisfactory for the structure assigned to the


spectral data.IR (KBr): 3327(N-H str. Amine), 3032(C-H str, Aromatic), 1626,

1529(C=C str), 1573, 1566 (C=N str, Amine), 1446 (N-H Bending).

172

Synthesis of (z)-n-(4-aminobenzylidene(-2-(1h – benzo[d]imidazol-2-yl)-n-

benzyliden-amine (7b)

To a mixture of Compound 6b and o-phenylenediamine, 4N HCl was added and the


TLC pure brown colored amorphous powder VIIb, m.p.: 225 ºC (Yield: 75.93%) so

obtained was characterized on the basis of spectral data.

45

6

3'

4'

5'6'

A B

2" 3"

5"6"

C

7

N

O

OH

NH2NH2

NH2N

NH

N

NH2


aldamine proton which indicates the successful formation of the expected product.

The amino proton (-NH-) of the benzmidazole ring appears as singlet at δ 4.83 and

another singlet appear at aliphatic region at δ 4.0 could arise from the free amino

group (-NH2). The protons of the amino phenyl ring C appears as doublet at δ 6.32 (H-

3,5) while the remaining protons of the same phenyl ring and the phenyl ring A and B

appears as multiplet centered at δ 7.3, δ 7.8 and δ 7.5 respectively. These data are


above structure was obtained by the IR spectral data.IR (KBr): 3177(N-H str. Amine),

3060(C-H str, Aromatic), 1640,1515(C=C str ),1565,1560(C=N str, Amine), 1430(N-H

Bending).

173

Synthesis of 2-(z)-(2-(1h-benzo[d]imidazol-2-yl) phenylimino) methyl phenol (7c)

To a mixture of Compound 6c and o-phenylenediamine, 4N HCl was added and the


TLC pure colorless crystalline compound VIIc; m.p.: 237ºC (Yield: 74.39%) so

obtained was characterized on the basis of spectral data.

45

6

3'

4'

5'6'

A B

3"

4"

5"6"

C

7

N

O

OH

OH NH2

NH2N

NH

N

OH


aldamine proton and –NH proton of the benzimidazole ring appears as singlet at δ

4.34. It indicates the successful formation of the expectation product. The hydroxyl (-

OH) proton appears a singlet at δ 5.11, while the protons of the phenyl ring appears

as multiplet in the aromatic region δ 6.32 and δ 7.89. These data are satisfactory for

the structure assigned to the above compound. A further support to the above

structure was obtained by the IR spectral data. IR (KBr): 3500-3000(OH str

Alcohol).,3367( N-H str), 1639( C=C str Aromatic),1560,1525 (C=N str), 1450( N-H

Bending), 1290( C-N str).

174

Synthesis of (z)-n-(4-methoxy benzylidene)-2-(1h – benzo[d]imidazol-2-yl)-

benzamine (7d).

To a mixture of Compound 6d and o-phenylenediamine, 4N HCl was added and



crystallized from ethanol to give TLC pure yellow colored crystalline compound VIId

m.p.: 204ºC (Yield: 72.23%). Its structure was established on the basis of NMR and

IR data.

45

6

3'

4'

5'6'

A B

2" 3"

5"6"

C

7

N

O

OH

OCH3NH2

NH2N

NH

N

OCH3

The NMR of the spectrum showed a singlet at δ 8.4 arising from the aldamine proton.

The methoxyl (-OCH3) proton could be seen as singlet at δ 3.06 and the proton of the

amino group (-NH) of imidazole ring appears as singlet at δ 4.90. The protons of the

methoxy phenyl ring C appears as doublet at δ 6.3 (H-3”, 5”) and a multiplet at δ 7.38

(H-2”, 6”). The protons of the phenyl ring A & B appears as multiplet centered at δ 6.8

(H-3’, 4’, 5’, 5, 6), doublet at δ 7.1 (H-6’) and another doublet at δ 7.8 (H-4, 7)

respectively.These data are satisfactory for the structure assigned to the above


175

data. IR (KBr): 3375 (N-H str)., 3084 (C-H Aromatic), 2980(C-H str Methoxy), 1584

(C=N str), 1514(C=C str Aromatic), 1464(C-H Methoxy), 1263(C-N str), 1193(C-O str).

Synthesis of (z)-n-(4-dimethylamino benzylidene)-2-(1h – benzo[d]imidazol-2-yl)-

benzamine (7e).

To a mixture of Compound 6e and o-phenylenediamine, 4N HCl was added and



crystallized from methanol to give TLC pure orange colored crystalline compound 7e

m.p.: 220ºC (Yield: 78.10%). Its structure was established on the basis of NMR and

IR data.

45

6

3'

4'

5'6'

A B

2" 3"

5"6"

C

7

N

O

OH

NCH3

CH3NH2

NH2N

NH

N

N

CH3

CH3


aldamine proton and a singlet at δ 3.5 could arise from the two methyl group (-CH3).

There was another singlet at δ 4.5, which could arise from the NH of the imidazole

ring. The protons of the dimethyl aminophenyl ring C appears as doublet at δ 7.2 (H-

3”, 5”) and a multiplet at δ 7.4 (H-2”, 6”). The protons of the phenyl ring A and B

appears as multiplet centered at δ 7.3 (H-3’, 4’, 5, 5 & 6) and a doublet at δ 7.7 (H-6’)

176

while the remaining proton appears as doublet at δ 7.9 (H-4, 7). These data are


above structure was obtained by the IR spectral data. IR (KBr): 3327(N-H str. Amine),

3016(C-H str, Aromatic), 2928(C-H str,A lkyl), 1627 ( C=C str), 1568(C=N str).

177

6.3.In-silico studies

6.3.1. Docking studies using Auto Dock

Tools and materials used

Auto Dock

Auto Dock is an automated docking tool. It is designed to predict how small

molecules, such as substrates, bind to a receptor of known 3D structures. Auto Dock

actually consists of two main programs: one performs the docking of the ligand to a

set of grids describing the target protein; and the other Auto Grid pre-calculates these

grids. In addition to using them for docking, the atomic affinity grids can be visualized.

A graphical user interface called Auto Dock Tools or ADT was utilized to generate

grids, calculate dock score and evaluate the conformers.

Materials and Methods

The structure of β-ketoacyl-acyl carrier protein synthase (1HNJ) and 14α-demethylase

(1E9X) is an essential target for novel antibacterial and antifungal drug design

respectively, COX-1 (1egq ) and COX-2 (1cx2) receptors were retrieved from Protein

Data Bank (PDB). All the synthesized molecules were docked by using the software

Auto Dock and the score values are predicted. The protein ligand interactions were

also studied. All molecules were drawn using ChemDraw Ultra 8.0 tool and energy

minimized using Chem 3D Ultra 8.0 software.

178

Docking studies for anti-inflammatory activity using Auto dock:

Docked scores of newly designed compounds with COX-1 and COX-2

Table. No.6.3.1

Comp

ound

Auto Dock

Score

(Kcal/mol)K1 (micro M)

Interacting Aminoacid

Residues

COX-

1

COX-

2

COX-

1

COX-

2COX-1 C0X-2

3a. -4.08 -8.13 -2.54 -7.18 Arg69 Gly89

3b. -1.42 -7.65 -3.10 -6.40 lle44, Gly301,Ser46

3c. -3.72 -6.18 -2.58 -7.91 --- Arg298,Asn319

3d. -1.63 -6.92 -2.05 -7.53 Arg 97 Arg69

3e. -2.48 -7.43 -2.64 -6.99 lle44, Gly89

3f. -2.00 -6.81 -1.50 -6.23 lle44, Arg 97

3g. -1.14 -6.36 -2.32 -6.87 Arg69 Gly301,Ser46

3h. -1.78 -7.92 -1.99 -7.03 Arg 97 ---

4a. -1.16 -6.84 -2.39 -6.98Gly301,

Ser46---

4b. -4.80 -9.87 0.354 0.057 Arg120 His90

4c. -4.15 -7.56 -2.78 -7.96 Gly89 Arg69

179

4d. -2.56 -6.34 -2.14 8.00 lle44, Gly89

4e. -5.38 -8.93 0.718 0.284 Arg69 His90, Arg120

4f. -1.41 -7.63 -2.56 -8.45 Arg 97 Gly89

4g. -2.16 -7.34 -2.42 -7.86 lle44, ---

4h. -1.70 -8.23 -1.91 -6.34 Gly89 Arg 97

5a. -1.01 -6.20 -1.34 -7.45 Arg69 ---

5b. -1.26 -7.38 -2.76 -6.90 --- ---

5c. -1.38 -6.60 -1.79 -6.45. Gly89 Arg 97

5d. -1.63 -7.11 -1.80 -6.77 --- lle44,

5e. -3.78 -8.19 -1.50 -7.43 Arg69 ---

5f. -2.65 -7.80 -1.38 -7.99Gly301,

Ser46Arg 97

5g. -4.76 -7.23 -3.33 -8.01 Arg69 Gly89

5h. -4.09 -7.73 -2.89 -7.86 --- Gly89

6a. -2.46 -7.35 -2.25 -6.32 Arg69 ---

6b. -4.12 -6.57 -1.38 -7.29 Gly89 lle44,

6c. -1.37 -6.90 -2.58 -7.18 Arg 97 Arg69

6d. -1.65 -7.59 -1.50 -7.28Gly301,

Ser46---

6e. -3.87 -6.56 -2.22 -7.95Gly301,

Ser46Gly89

7a. -3.98 -7.75 -4.87 -8.51 Arg 97 lle44,

180

7b. -1.48 -6.42 -4.00 -7.06 Gly89 ---

7c. -3.68 -7.01 -2.68 -6.05 Arg69 lle44,

7d. -4.76 -6.10 -1.30 -7.85 --- Arg 97

7e. -3.64 -8.97 -3.15 -6.48 Gly89 ---

Indome

thacin-6.75 -7.4 1.91 6.34

Arg120

Tyr355

Arg120

His90, His95,

His90, Arg120

181

Docking studies for anti-inflammatory activity using Auto dock binding

interaction with COX-1 (1egq ):

Compound-4b (Figure – 6.3.1) Compound -4e (Figure – 6.3.2)

Indomethacin (Figure – 6.3.3)

Green dots with mesh - H-Bond interaction

Yellow wire cylinder - Pi-Pi interaction

Yellow wire cone - Pi-cation interaction

182

Docking studies for anti-inflammatory activity using Auto dock binding

interaction with COX-2 (1cx2):

Compound-4b (Figure – 6.3.4) Compound - 4e (Figure – 6.3.5)

Indomethacin (Figure – 6.3.6)




183

Anti-bacterial- Docked scores of newly designed compounds with β-keto acyl

acyl carrier protein (1hnj)

Table. No.6.3.2

S.No.Comp

ound

Auto dock

score

(Kcal/mol)

K1

(micro

M)

No.of H-

Bonds

Interacting amino

acid residues

1. 3a. -2.03 78.77 1 Phe 304

2. 3b. -1.76 63.23 0 ---

3. 3c. -2.45 47.61 3 Cys112,Phe304,Gly306

4. 3d. -1.55 85.12 2 Ans274,Gly306

5. 3e. -1.80 72.67 0 ---

6. 3f. -3.02 45.42 1 Phe 304

7. 3g. -2.17 43.99 1 Gly306

8. 3h. -1.63 56.11 0 ---

9. 4a. -6.47 33.75 1 Ans274,

10. 4b. -4.86 82.54 0 ---

11. 4c. -5.89 487.13 0 ---

12. 4d. -6.06 47.03 1 Gly306

13. 4e. -4.51 44.18 1 Gly306

14. 4f. -7.19 5.38 2 Gly306

15. 4g. -5.21 60.22 1 Ans274,

16. 4h. -8.09 1.18 3 ys112,Phe304,Gly306

184

17. 5a. -4.87 70.36 2 Gly306

18. 5b. -5.42 48.13 1 Ans274,

19. 5c. -3.72 186.23 1 Ans274,

20. 5d. -5.45 59.16 0 ---

21. 5e. -4.14 88.10 3 Cys112,Phe304,Gly306

22. 5f. -3.26 47.28 0 ---

23. 5g. -3.71 72.37 1 Gly306

24 5h. -4.82 65.08 1 Ans274,

32. 7c. -4.59 49.85 1 Ans274,

33. 7d. -5.26 88.45 1 Gly306

34. 7e. -3.65 78.12 2 Ans274,Gly306

185

Docking studies for anti-fungal activity using Auto dock binding interaction with

β-keto acyl acyl carrier protein (1hnj)

Compound - 4c(Figure – 6.3.7) Compound - 4h(Figure – 6.3.8)

Ampicillin (Figure – 6.3.9)




186

Anti-fungal- Docked scores of newly designed compounds with 14α-

demethylase (1E9X)

Table. No.6.3.3

S.

No.

Compou

nd

Auto dock

score

(Kcal/mol)

K1

(micro

M)

No.of H-

Bonds

Interacting amino acid

residues

1. 3a. -2.03 78.77 1 Phe 304

2. 3b. -1.76 63.23 0 ---

3. 3c. -2.45 47.61 3 Cys112,Phe304,Gly306

4. 3d. -1.55 85.12 2 Ans274,Gly306

5. 3e. -1.80 72.67 0 ---

6. 3f. -3.02 45.42 1 Phe 304

7. 3g. -2.17 43.99 1 Gly306

8. 3h. -1.63 56.11 0 ---

9. 4a. -6.47 33.75 1 Ans274,

10. 4b. -7.19 5.38 2 Gly306

11. 4c. -5.89 487.13 0 ---

12. 4d. -6.06 47.03 1 Gly306

13. 4e. -8.09 1.18 3 Cys112,Phe304,Gly306

14. 4f. -4.86 82.54 0 ---

187

15. 4g. -5.21 60.22 1 Ans274,

16. 4h. -4.51 44.18 1 Gly306

17. 5a. -4.87 70.36 2 Gly306

18. 5b. -7.42 48.13 1 Ans274,

19. 5c. -7.12 186.23 1 Ans274,

20. 5d. -5.45 59.16 0 ---

21. 5e. -4.14 88.10 3 Cys112,Phe304,Gly306

22. 5f. -3.26 47.28 0 ---

23. 5g. -3.71 72.37 1 Gly306

24 5h. -4.82 65.08 1 Ans274,

32. 7c. -4.59 49.85 1 Ans274,

33. 7d. -5.26 88.45 1 Gly306

34. 7e. -3.65 78.12 2 Ans274,Gly306

188

Anti-fungal- Docked scores of newly designed compounds with 14α-

demethylase (1E9X)

Compound - 4h (Figure – 6.3.10) Griseofulvin (Figure – 6.3.11)




189

Discussion of Docking Results.

Docking allows the scientist to virtually screen a data base of compounds and predict

the strongest binders based on various scoring functions. It explores ways in which

two molecules such as drugs and a receptor, fit together and dock to each other well.

The molecules binding to a receptor inhibit its function and thus act as drug. The

collection of drug and receptor complex was identified via docking and their relative

stabilities were evaluated using molecular dynamics and their binding affinities, using

free energy simulation.

.3.2.Discussion for anti inflammatory studies using Auto Dock

The Auto dock results for COX inhibitions revealed that the majority of the compounds

docked in to the active sites of the COX-2 receptor and exhibited H-bonding via O or –

NH group. Interestingly, these compounds were having less binding energy value

towards COX-1 receptor which shows that these compounds have more affinity

towards COX-2 receptor. These observations together with experimental results

provide a good explanation for the potent and selective inhibitory activity of 4b, 4e, 4g

and 4h. In conclusion, compound 4b had good inhibitory activity than other

compounds and can be act as an inhibitor of COX-2 enzymes. The significant anti-

inflammatory activity by carragenin method proved this point in practice.

190

6.3.3..Discussion for anti microbial studies using Auto Dock

The structure of COX-1 (PDB ID: 1EQG) and COX-2 (PDB ID: 1CX2) receptors were

retrieved from Protein Data Bank (PDB). All molecules were drawn using ChemDraw

Ultra 8.0 tool and energy minimized using Chem 3D Ultra 8.0 software. . A Lamarckian

genetic algorithm method, implemented in the program AutoDock 4.0.1, was

employed. Binding affinity was evaluated by the binding free energies (∆ G, Kcal/mol),

inhibition constant (Ki) and Hydrogen bonding.

After obtaining the PDB ID, the possible binding sites of receptors were searched

using Computed Atlas of Surface Topography of Proteins (CASTp) (Figure 6.3.1to

6.3.6). These include pockets located on protein surfaces and voids buried in the

interior of proteins. CASTp includes a graphical user interface, flexible interactive

visualization, as well as on-the-fly calculation for user uploaded structures.

The Auto dock result showed that all the designed molecules have similar orientation

in the binding pocket of selected antibacterial targets. The binding models of the

active compounds bond to active site of β-ketoacyl-acyl carrier protein synthase,

receptor is shown in Fig. No(Figure 6.3.7to 6.3.9).. From the binding model, we can

see that compound 4f and 4h is bound in to β-ketoacyl-acyl carrier protein synthase,

via hydrophilic binding by hydrogen bond they posses the highest potential binding

affinities in to the binding affinity in to the binding site of the 3D macro molecules.

Similarly, compound 5f and 5h were showed best binding score with all the

antibacterial receptors. In in-vitro studies (Table-6.3.2 and 6.3.3) also compound 4f,

191

4h,5f and 5h have emerged as active against all tested microorganism. So, it can be

predicted as the activity may be due to inhibition of either one of these targets.

In anti-fungal activity, among the 34 molecules, docking 1E9X with 4h and 5b revealed

that as good inhibitor of fungal enzyme. The binding models of the active compounds

bond to active site of β-ketoacyl-acyl carrier protein synthase, receptor is shown in

Fig. No(Figure 6.3.10to 6.3.11).. In in-vitro studies also 4h and 5b has emerged as

active against all tested micro organisms, so it can be predicted as the activity may be

due to inhibition of selected fungal targets.

6.4. Antimicrobial activity:

6.4.1 Anti Bacterial Activity:

All the newly synthesized compounds were screened for antibacterial activity against

two Gram-positive organisms, Bacillus subtilis (ATCC 6633) and Staphylococcus

aureus, (ATCC 25923) and two Gram-negative organisms, Escherichia coli (ATCC

25922) and Pseudomonas aeruginosa (ATCC 27853)) by cup-plate method172.

Antimicrobial activity is measured in vitro in order to determine a) the potency of an

antibacterial agent in solution b) the sensitivity of a given microorganism to know

concentrations of the synthesized drug.

A suspension of the test organism was well mixed with 25 ml of sterile liquid

nutrient agar media, at a temperature between 40-500 C and poured immediately in to

a pre-sterilized petri-dishes. The plates were left at room temperature to allow the

solidification. In each plate four cups of 10 mm diameter were made with a sterile

borer. Solutions of the test compounds were prepared by dissolving 10 mg of each in

192

100 ml dimethyl sulphoxide (AR grade) to get final concentration of 100 g/ml. A

reference standard for gram-positive and gram-negative bacteria was made by

dissolving accurately weighed quantity of Amphicillin in DMSO solution. Then, 100

g/ml of test solution was added to the cups, aseptically and labeled accordingly. The

plates were kept undisturbed for at least 2 hrs at room temperature to allow diffusion

of the solution properly into nutrient agar medium. After incubation of the plates at 37

1º C for 24 hr the diameter of the zone of inhibition surrounding each of the cups was

measured with the help of an antibiotic zone reader. All the experiments were carried

out in triplicate. Simultaneously controls were maintaining employing 0.1 ml of

dimethyl sulphoxide (DMSO) to observe the solvent effects and the results were

shown in Table no 6.3.1.

193

Figure 6.4.1

Anti bacterial activity of synthesized compounds

194

195

196

6.4.2 Anti-Fungal Activity:

All the compounds screened were also tested for their antifungal activity against the

organism Aspergillus niger and Saccharomyces cerevisiae by cup-plate method.

The test organisms were sub-cultured using potato dextrose agar medium. The tubes

containing sterilized medium were inoculated with test fungi and after incubation at

250C for 48 hr they were stored 4 in refrigeration. The inoculum was prepared by

taking a loopful of stock culture to about 100 ml of nutrient broth, in 250 ml clean and

sterilized flasks. The flasks were incubated at 250C for 24 hr before use.

The solutions of test substances were prepared by similar procedure described

under the antibacterial activity. A reference standard (0.1 mg/ml conc) was prepared

by dissolving 10 mg of Griseofulvin in 100 ml of DMSO to obtain a solution of 100

g/ml concentration.

The potato dextrose agar medium was sterilized by autoclaving at 121ºC (15

lb/sq. inch) for 15 minutes. The petri plates, tubes and flask plugged with cotton plugs

were sterilized in hot air oven at 150ºC for an hour. Into each sterilized Petri-plate

about 30 ml of each of molten potato dextrose agar medium inoculated with

respective fungus (6ml of inoculums to 300 ml of potato dextrose agar medium) was

transferred, aseptically. After solidification of the medium at room temperature four

cups of 10 mm diameter were made in each plate with an sterile borer. Accurately 0.1

ml (100 g/ml conc.) of test solution was transferred to the cups, aseptically and

labeled, accordingly. The reference standard 0.1 ml (100 g/ml conc.) was also

added to the cups in each plate. The plates were kept undisturbed for at least two

hours at room temperature to allow diffusion of the solution properly, into potato

197

dextrose agar medium. Then the plates were incubated at 25ºC for 48 hr. The

diameter of the zone of inhibition was read with help of an antibiotic zone reader. The

experiments were performed in triplicate in order to minimize the errors.

Figure 6.4.2

Anti-fungal activity of synthesized compounds

198

Result and Discussion

Antimicrobial activity

All the newly synthesized 3-(4-(substituted phenyl)-3-oxoprop-1-enyl) phenyl- 2-

(substituted phenyl) quniazoline-4-one 3(a-h), 3-[4-(1-acetyl-4,5-dihydro-5-substituted

phenyl-1H-pyrazol-3-yl) phenyl]-2-substutied phenyl quinazolin-4(3H)-one derivatives 4(a-h),

3-[4-(5-(substituted phenyl)-4,5-dihydro isoxazol-3-yl) phenyl]-2-substutied phenyl

quinazolin-4(3H)-one derivatives V(a-h), 2-(substituted benzylideneamino) benzoic acid VI(a-

e), and (Z)-N-4-(substituted benzylidene)-2-(1H-benzio[d]imidazole-2-yl) benzenamine 7(a-e)

were screened for their antibacterial activity against B. subtilis and S. aureus (Gram +ve),

E. coli and P. aeruginosa (Gram -ve) and antifungal activity against A. niger and C.

albicans by cup-plate method at a concentration of 100 µg / ml and measured the zone of

inhibition in mm and the results were tabulated in Table 6.3.1 & 6.4.1. The reference drug

used was Amoxycillin and Griseofulvin at a concentration of 100 µg/ml for antibacterial and

antifungal activity respectively.

The sensitivity of microorganisms to the tested compounds is identified in the

following manner:

Highly sensitive = Inhibition zone 30–40 mm

Sensitive = Inhibition zone: 20–30 mm

Slightly sensitive = Inhibition zone: 10–20 mm

Not sensitive = Inhibition zone: below 10 mm

199

Antibacterial Activity

3-(4-(substituted phenyl)-3-oxoprop-1-enyl) phenyl- 2-(substituted phenyl)

quniazoline-4-one 3(a-h)

Among the test compounds, 3-(4-((Z)-3-(3-chlorophenyl)-3-oxoprop-1-enyl) phenyl)-2-

phenyl quinazolin-4(3h)-one 3 e was found to be sensitive against gram negative

bacterial E. coli with zone inhibition of 16 mm and it is not sensitive to P. aeruginosa

where as it showed maximum zone of inhibition of 13 mm and 11 mm against gram

positive bacteria and considered to be slightly sensitive against B. subtilis and S.

aureus respectively. Compound 3 c showed slightly sensitive against only gram

positive S. aureus with zone of inhibition of 12 mm whereas remaining all compounds

did not showed any antibacterial activity against both gram positive and gram negative

bacteria. These results showed that most of these intermediate chalcone

quinazolinones were found to be least active against both gram positive and negative

bacteria.

3-[4-(1-acetyl-4,5-dihydro-5-substituted phenyl-1H-pyrazol-3-yl) phenyl]-2-

substutied phenyl quinazolin-4(3H)-one derivatives 4(a-h)

All the synthesized 3-[4-(1-acetyl-4,5-dihydro-5-substituted phenyl-1H-pyrazol-3-yl)

phenyl]-2-substutied phenyl quinazolin-4(3H)-one derivatives 4(a-h) derivatives were

sensitive to the gram positive and gram negative bacteria at concentration of 100

µg/ml. Out of 8 newly synthesized compounds, compound 4h i.e, 3-(4-(1-acetyl-4, 5-

dihydro-5-(3-nitrophenyl)-1h pyrazol-3-yl) phenyl)-2-(4-nitrophenyl) quinazolin-4(3H)-

one was found to be quite superior in its antibacterial action and showed zone of

200

inhibition of 22mm, 23mm, 20mm and 22mm against S. aureu, B. subtilis, E. coli and

P. aeruginosa respectively followed by compound 4c with zone of inhibition of 20mm,

21mm, 23mm and 22mm respectively. Their antibacterial effect was in comparison

with the effect of reference drug, amoxicillin against gram positive and gram negative

bacteria. From the results, it was found that compound 4b i.e., 3(4-(1-acetyl-4, 5-

dihydro-5-phenyl-1h-pyrazol-3-yl) phenyl)-2-(4-nitrophenyl) quinazolin-4(3H)-one

showed good activity against only gram positive organism with zone of inhibition of

20mm and 21mm as equal to Compound IVc. All other compounds in this series

showed moderate and slightly active against the gram positive and gram negative

organisms.

3-[4-(5-(Substituted phenyl)-4,5-dihydro isoxazol-3-yl) phenyl]-2-substutied

phenyl quinazolin-4(3H)-one derivatives 5(a-h)

In general all the synthesized 3-[4-(5-(Substituted phenyl)-4,5-dihydro isoxazol-3-yl)

phenyl]-2-substutied phenyl quinazolin-4(3H)-one derivatives 5(a-h) showed better

antibacterial activity against gram positive and gram negative bacteria with zone of

inhibition of 12 – 23 mm and 12-22 mm respectively in comparison to pyrazole

substituted quinazolinone derivatives. Among the synthesized compounds in this

series, compounds 5h and 5f i.e, 3-(4-(5-(3-nitrophenyl)-4, 5-dihydroisazol-3-yl)-

phenyl)-2-(4-nitrophenyl) quinazolin-4(3H)-one and 3-(4-(5-(3-chloro phenyl)-4, 5-

dihydroisazol-3-yl)- phenyl)-2-(4-nitrophenyl) quinazolin-4(3H)-one found to be quite

superior in antibacterial action against all the organism employed for the study and

followed by compound 5c, 5d, 5g and 5a showed good activity against gram positive

201

and gram negative bacteria. All other compound showed moderate activity against the

tested organism.

2-(Substituted benzylideneamino) benzoic acid 6(a-e)

In this series, most of the compounds did not show any activity against gram positive

and gram negative organism. Only the compound VIb showed moderate activity

against S. aureu, B. subtilis, E. coli and P. aeruginosa with zone of inhibition of 16

mm, 13 mm, 18mm and 15 mm respectively. Remaining all compound showed very

less sensitivity towards all the organisms with the zone of inhibition of 06–13 mm.

(Z)-N-4-(substitutedbenzylidene)-2-(1H-benzio[d]imidazole-2-yl)benzenamine7(a-

e)

The synthesized (Z)-N-4-(substituted benzylidene)-2-(1H-benzio[d]imidazole-2-yl)

benzenamine 7(a-e) derivatives showed moderate activity against gram positive and

gram negative organisms. Among the synthesized compounds, compound 7b i.e., (Z)-

N-(4-aminobenzylidene(-2-(1h – benzo[d]imidazol-2-yl)-n-benzyliden-amine showed a

better zone of inhibition of 20, 24 mm and 21, 22 mm against gram positive and gram

negative respectively when compared to the other compounds. Remaining all

compounds in this series showed slightly sensitive to all organisms.

From the results of antibacterial study of the synthesized quinazolinone derivatives

and benzimidazole dereivatives, it was concluded as follows:

Chalchone compounds 3 (a-h) intermediates did not show activity against both the

gram positive and gram negative strains employed in the study.

202

The substituted quizazolinone derivates showed good activity against the bacterial

strains when compared to its starting material chalcones.

Among the synthesized quinazolinone derivatives, isoxazole substituted

derivatives showed a better antibacterial action than pyrazole substituted

derivatives.

Over all, compound with nitro substitution i.e., 3-(4-(5-(3-nitrophenyl)-4, 5-

dihydroisazol-3-yl)- phenyl)-2-(4-nitrophenyl) quinazolin-4(3H)-one (Vh) & 3-(4-(1-

acetyl-4,5-dihydro-5-(3-nitrophenyl)-1H-pyrazol-3-yl)phenyl)-2-(4-nitro phenyl)

quinazolin-4(3H)-one (4h) is found to be quite superior in its antibacterial action and

also comparable with the reference drug.

In the pyrazole substituted quinazoline derivatives, 4-methoxy phenyl substitution

i.e, 3-(4-(1-acetyl-4,5-dihydro-5-(4-methoxyphenyl)-1h-pyrazol-3-yl)phenyl)-2-

phenyl quinazolin-4(3H)-one (4c) found to possess good antibacterial activity next

to the nitro substitution.

In isoxazole series compound with chloro substitution i.e., 3-(4-(5-(3-chloro

phenyl)-4, 5-dihydroisazol-3-yl)- phenyl)-2-(4-nitrophenyl) quinazolin-4(3h)-one (vf)

was found to be good antibacterial activity.

2-Substituted Benzylideneamino) Benzoic Acid intermediates VI (a-e) were did

show any notable anti bacterial activity.

On cyclization of benzoic acid derivatives increases the antibacterial activity.

In benzimidazole derivatives, compound 7b with amino substitution i.e., (Z)-N-(4-

aminobenzylidene-2-(1H – benzo[d]imidazol-2-yl)-N-benzylidenamine showed

good antibacterial activity against both gram positive and gram negative bacteria.

203

Antifungal Activity

3-(4-(substituted phenyl)-3-oxoprop-1-enyl) phenyl- 2-(substituted phenyl)

quniazoline-4-one 3(a-h)

Out of 8 newly synthesized 3-(4-(substituted phenyl)-3-oxoprop-1-enyl) phenyl- 2-

(substituted phenyl) quniazoline-4-one 3(a-h) derivatives, compound 3g was found to be

sensitive against both the fungal strains A. niger and S. cerviesiae with zone of

inhibition 24mm and 21mm followed by compound and 3h . It showed zone of

inhibition 22mm and 19 mm against A. niger and S. cerviesiae. Compound 3d was

found to be sensitive against A. niger (zone of inhibition 20 mm) but devoid of activity

against S. cerevisiae. Remaining all compounds showed lower degree of antifungal

action. None of the synthesized compounds were having good activity in comparison

with the standard drug griseofulvin, which showed a maximum zone of inhibition of 34

mm and 35 mm against A. niger and S. cerviesia at same concentration 100 g/ml.

3-[4-(1-acetyl-4,5-dihydro-5-substituted phenyl-1H-pyrazol-3-yl) phenyl]-2-

substutied phenyl quinazolin-4(3H)-one derivatives 4(a-h)

All the test compounds were active against A. niger except 4a at a concentration of

100 µg/ml and compound 4h , 4g and 4f was found to have good antifungal property

and showed zone of inhibition of 26, 24 and 22mm against A. niger and 28, 18 and

24mm zone of inhibition against S. cerviesiae. Rest of all the compounds in this

pyrazole substituted quinazolinone derivatives are sensitive against to both the fungal

strains A. niger and S. cerviesia. None of the synthesized compounds activity was in

comparison with the standard drug, griseofulvin.

204

3-[4-(5-(Substituted phenyl)-4,5-dihydro isoxazol-3-yl) phenyl]-2-substutied

phenyl quinazolin-4(3H)-one derivatives 5(a-h)

The newly synthesized 3-[4-(5-(Substituted phenyl)-4,5-dihydro isoxazol-3-yl) phenyl]-

2-substutied phenyl quinazolin-4(3H)-one derivatives 5(a-h) showed very good

antifungal activity among the synthesized compounds in the present study. From the

results, it was found that compound 5b i.e., 2-(4-nitrophenyl)-3-(4-(5-phenyl-4,5-

dihydroisazol-3-yl) - phenyl quinazolin-4(3H)-one quite superior in its action and also

the activity was comparable to the standard drug griseofulvin. Compounds 5b , 5e , 5a ,

5g, 5c showed 30,29, 25, 25,and 22mm zone of inhibition respectively against

Asperigillus niger and compounds 5d , 5c , 5b , 5g, and 5h showed 27, 26, 23, 22 and

22mm zone of inhibition respectively against S.cerviesiae. Remaining all other

compounds showed 17 – 10mm zone of inhibition only against both the organisms.

2-(Substituted benzylideneamino) benzoic acid 6(a-e)

None of the compounds were sensitive against A. niger and S. cerviesiae.

Compounds VIc and VIa showed slight sensitive against A. niger and S. cerviesiae

with the zone of inhibition of 15 mm & 19 mm and 14 mm & 8 mm respectively.

(Z)-N-4-(substitutedbenzylidene)-2-(1H-benzio[d]imidazole-2-yl)benzenamine7(a-

e)

Newly synthesized 4-(Substituted Benzylidene)-2-(1H-Benzimidazole-2-yl)

benzenamine 7 (a-e) showed moderate antifungal activity against A. niger and

S. cerviesiae when compare to their intermediate VI (a-e). Among these derivatives

205

comounds 7d showed superior activity and considered sensitive against A. niger with

the zone of inhibition of 29 mm followed by compound 7e and 7a which showed 22-21

mm zone of inhibition at a concentration of 100 µg/ml respectively. Similarly

compound 7d, 7c, 7e, showed 25, 24, 23 mm zone of inhibition against S.cerviesiae

and the standard compound griseofluvin showed 34-35 mm of zone of inhibition

against A. niger and S. cerviesiae respectively

From the results of antifungal study of the synthesized quinazolinone derivatives and

benzimidazole dereivatives, it was concluded as follows:

Chalchone compounds 3 (a-h) intermediates did not show activity against both the

fungal strains employed in the study.

The substituted quizazolinone derivates showed good activity against the fungal

strains when compared to its starting material chalcones.

Among the synthesized quinazolinone derivatives, isoxazole substituted

derivatives showed a better activity against Aspergilus niger while the pyrazole

substituted derivatives showed better activity against Saccharomyces verevisiae.

Compound with nitro substitution i.e., 2-(4-nitrophenyl)-3-(4-(5-phenyl-4,5-

dihydroisazol-3-yl) - phenyl quinazolin-4(3H)-one (Vb) & 3-(4-(1-acetyl-4,5-dihydro-

5-(3-nitrophenyl)-1H-pyrazol-3-yl)phenyl)-2-(4-nitrophenyl) quinazolin-4(3H)-one

(IVh) is found to be quite superior in its antifungal activity and also comparable with

the reference drug.


show any notable antifungal activity.

On cyclization of benzoic acid derivatives increases the antifungal activity.

206

Table 6.4.1

Anti Bacterial & Antifungal Activity Of Quinazolinone Derivatives [3(a-h), 4(a-h),

5(a-h)] at 100 g/ml 6(a-e) and 7(a-e)

Cpds

Zone of Inhibition (mm)

Gram Positive Gram Negative Fungal strains

B.subtilis S.aureus E.coli P.aeruginosa A. niger S. cerevisiae

3a. 5 6 --- --- --- 16

3b. 6 7 6 --- 16 14

3c. 5 12 5 4 8 18

3d. 7 --- 8 --- 20 ---

3e. 13 11 16 6 14 14

3f. 10 7 4 --- 12 16

3g. 7 7 6 10 24 21

3h. 6 6 6 --- 22 19

4a. 12 14 12 15 --- 17

4b. 20 21 11 13 19 24

4c. 20 21 23 22 12 18

4d. 14 12 11 11 17 14

4e. 13 15 10 13 19 17

4f. 19 21 10 12 22 24

4g 15 15 13 11 24 18

4h. 22 23 20 22 26 28

207

Standard Drug used:

Antibacterial Activity - Amoxicillin

Antifungal Activity - Griseofulvin

5a. 14 15 13 12 25 10

5b. 15 --- 14 ---- 30 23

5c. 20 18 16 18 22 26

5d. 16 12 14 12 14 27

5e. 18 21 12 --- 29 ---

5f. 23 22 21 18 15 18

5g. 14 15 14 15 25 22

5h. 23 26 21 22 17 22

Std* 27 30 32 28 34 35

208

Table No. 6.4.2

Anti Bacterial & Antifungal Activity Of Benzimidazole Derivatives [6(a-e), 7(a-e)]

at 100 mcg/ml

Standard Drug Used:

Antibacterial Activity – Amoxicillin

Antifungal Activity - Griseofulvin

Cpds

Zone of Inhibition (mm)

Gram Positive Gram Negative Fungal strains

B.

subtilis

S.

aureus

E.

coli

P.

aeruginosa

A.

niger

S.

cerevisiae

6a. 7 10 6 9 8 9

6b. 16 13 18 15 -- 8

6c. 5 7 6 11 15 19

6d. 9 4 9 7 11 16

6e. 8 9 7 10 14 8

7a. 8 11 10 12 21 19

7b. 20 24 21 22 -- 16

7c. 9 11 9 7 17 24

7d. 11 9 11 8 29 25

7e. 10 11 9 10 22 23

Std* 27 30 32 28 34 35

209

6.5. Evaluation of Anti-inflammatory activity

6.5.1 Acute toxicity study:

From the preliminary toxicity studies, it was observed that, all the test compounds

have revealed good safety profile till the lower most dose (5 mg/kg). No mortality of

animals observed even after 24 hrs but there were few changes in the behavioral

response like alertness, touch response and restlessness. Therefore, tolerated dose

that is 5 mg/kg b.w. was chosen for the pharmacological evaluations.

compounds 5mg/Kg 50mg/Kg 300mg/Kg LD 503a 0 1 3 50mg/kg3b 0 1 3 50mg/kg3c 0 1 3 50mg/kg3d 0 1 3 50mg/kg3e 0 1 3 50mg/kg3f 0 1 3 50mg/kg3g 0 1 3 50mg/kg3h 0 1 3 50mg/kg

6.5.2 Anti-inflammatory activity by rat paw edema method:

Inflammation is a tissue reaction to infection, irritation or foreign substance. The

inflammatory reaction is readily produced in rats in the form of paw edema with the

help of irritants. Substances such as carrageenan, formalin, bradykinin, histamine, 5-

hydroxy tryptamine, mustard or egg white, when injected in the dorsum of the foot or

rats produced acute paw edema within few minutes of the injection. Carrageenan

induced paw edema is most commonly used method in experimental pharmacology.

Anti-inflammatory activity was screened by carrageenan-induced acute inflammation

model. All the newly synthesized 3-(4-(substituted phenyl)-3-oxoprop-1-enyl) phenyl-

210

2-(substituted phenyl) quniazoline-4-one 3(a-h), 3-[4-(1-acetyl-4,5-dihydro-5-substituted

phenyl-1H-pyrazol-3-yl) phenyl]-2-substutied phenyl quinazolin-4(3H)-one derivatives

4(a-h), 3-[4-(5-(substituted phenyl)-4,5-dihydro isoxazol-3-yl) phenyl]-2-substutied

phenyl quinazolin-4(3H)-one derivatives V(a-h), 2-(substituted benzylideneamino)

benzoic acid VI(a-e), and (Z)-N-4-(substituted benzylidene)-2-(1H-benzio[d]imidazole-2-

yl) benzenamine 7(a-e) were tested for anti-inflammatory activity at a dose of 200

mg/kg (bw) and compared with Standard drug Indomethacin [10 mg/kg (bw)].

The paw volume was measured by using plythesmograph at 60, 120, 180 and 240

min. The data of 4 hrs was subjected to statistical analysis by One-way Analysis of

Variance (ANOVA) followed by Dunnet’s test. All compounds showed significant

difference when compared with control in all dose levels (p<0.001). A pValue of

<0.05, <0.01, < 0.001 were considered to be statistically significant, slightly significant

and highly significant respectively. The results are tabulated in Table 6.1 and 6.2.

211

Inhibitory effects of test compounds 3a-h, 4a-h,5a-h,6a-e and 7a-e on

Carrageenan-induced edema of the hind paw in rats

Table no :6.5.1

S.

No.Group

Treatment

(p.o)

Swelling volume (mL)

1hr 2hr 3hr 4hr

1. Control 1mL/kg 0.42±0.07 1.21±0.02 2.18±0.03 3.93±0.07

2.Indom

ethacin10mg/kg 0.43±0.05 0.74±0.01 1.07±0.02 1.67±0.013***

3. 3a 5mg/kg 0.45±0.21 1.16±0.01 1.02±0.02*** 2.37±0.008**

4. 3b 5mg/kg 0.42±0.03 0.93±0.04 1.24±0.01 2.10±0.036*

5. 3c 5mg/kg 0.46±0.03 1.04±0.02 1.17±0.02** 2.32±0.048

6. 3d 5mg/kg 0.44±0.02 0.95±0.03 1.08±0.01 2.04±0.027

7. 3e 5mg/kg 0.41±0.03 0.96±0.01 1.09±0.03 2.11±0.032

8. 3f 5mg/kg 0.46±0.03 0.95±0.03 1.07±0.02* 2.14±0.021

9. 3g 5mg/kg 0.45±0.02 0.93±0.03 1.06±0.01* 2.13±0.022

10. 3h 5mg/kg 0.43±0.01 0.90±0.03 1.24±0.01 2.15±0.039*

11. 4a 5mg/kg 0.41±0.18 0.68±0.18 1.36±0.17** 1.78±0.005*

12. 4b 5mg/kg 0.46±0.03 0.75±0.02 1.08±0.03** 1.49±0.040*

13. 4c 5mg/kg 0.42±0.02 0.86±0.02 1.13±0.01*** 1.79±0.014**

14. 4d 5mg/kg 0.46±0.01 0.81±0.03 1.08±0.02** 1.67±0.032**

212

15. 4e 5mg/kg 0.44±0.03 0.68±0.02 1.04±0.02*** 1.50±0.016**

16. 4f 5mg/kg 0.48±0.77 0.84±0.03 1.08±0.01 1.60±0.041**

17. 4g 5mg/kg 0.46±0.02 1.14±0.03 1.34±0.02 1.59±0.015

18. 44h 5mg/kg 0.41±0.03 0.98±0.02 1.18±0.02 1.75±0.025

19. 5a 5mg/kg 0.38±0.03 0.93±0.03 1.15±0.01 1.99±0.025*

20. 5b 5mg/kg 0.47±0.03 0.88±0.02 0.84±0.02* 1.66±0.046

21. 5c 5mg/kg 0.46±0.03 1.05±0.02 1.24±0.01* 1.98±0.007*

22. 5d 5mg/kg 0.44±0.01 0.93±0.01 1.14±0.02*** 1.90±0.013**

23. 5e 5mg/kg 0.41±0.02 0.86±0.02 1.05±0.01 1.50±0.015

24. 5f 5mg/kg 0.42±0.03 0.88±0.01 1.02±0.03** 1.99±0.012

25. 5g 5mg/kg 0.37±0.04 0.93±0.03 1.14±0.02 1.97±0.026*

26. 5h 5mg/kg 0.47±0.03 0.89±0.03 0.84±0.03* 1.98±0.046

27. 6a 5mg/kg 0.45±0.22 1.19±0.01 1.03±0.04*** 2.37±0.008**

28. 6b 5mg/kg 0.47±0.22 1.19±0.02 1.02±0.03*** 2.11±0.007**

29. 6c 5mg/kg 0.45±0.22 1.19±0.03 1.03±0.02*** 2.32±0.007**

30. 6d 5mg/kg 0.44±0.23 1.17±0.03 1.02±0.03*** 2.05±0.005**

31. 6e 5mg/kg 0.47±0.20 1.16±0.03 1.02±0.02*** 2.13±0.008**

32. 7a 5mg/kg 0.39±0.40 0.92±0.04 1.14±0.01*** 1.98±0.026**

33. 7b 5mg/kg 0.46±0.40 0.89±0.03 0.84±0.02*** 1.81±0.045**

213

Values are expressed in terms of mean ± S.E.M (n=6), Values are expressed in

terms of mean ± S.E.M, Significance was calculated by using one way ANOVA

with Dunnet’s t-test. The difference in results was considered significant when p

< 0.05. * p < 0.05 Vs control at 200 mg/kg b.w. ** p < 0.01 Vs control at 200 mg/kg

b.w, *** p < 0.001 Vs control at 200 mg/kg b.w

34. 7c 5mg/kg 0.48±0.01 1.06±0.02 1.24±0.01*** 1.97±0.006**

35. 7d 5mg/kg 0.41±0.20 0.94±0.02 1.14±0.02*** 1.91±0.014**

36. 7e 5mg/kg 0.37±0.30 0.88±0.02 1.05±0.01*** 1.78±0.015**

214

Percentage inhibition of test compounds 6a-e, 7a-j and 8a-e

Carrageenan-induced edema of the hind paw in rats

Table No. 6.5.2

S.

No.Group

Treatment

(p.o)

Paw

volume(ml)

as measured

by mercury

displacement

at 4 hr

Perce

ntage

inhibiti

on of

paw

edema

at 4 hr

1 Control 1mL/kg 3.93±0.07 -

2 Indomethacin 10mg/kg 1.50±0.013*** 61.80

3 3a 5mg/kg 2.37±0.008** 39.69

4 3b 5mg/kg 2.10±0.036* 46.56

5 3c 5mg/kg 2.32±0.048 40.96

6 3d 5mg/kg 2.04±0.027 48.09

7 3e 5mg/kg 2.11±0.032 46.31

8 3f 5mg/kg 2.14±0.021 45.54

9 3g 5mg/kg 2.13±0.022 45.80

10 3h 5mg/kg 2.15±0.039* 45.29

11 4a 5mg/kg 1.78±0.005* 54.70

12 4b 5mg/kg 1.66±0.040* 57.58

215

13 4c 5mg/kg 1.79±0.014** 54.45

14 4d 5mg/kg 1.97±0.032** 48.50

15 4e 5mg/kg 1.67±0.016** 58.02

16 4f 5mg/kg 1.72±0.041** 56.28

17 4g 5mg/kg 1.73±0.015 56.54

18 4h 5mg/kg 1.75±0.025 55.47

19 5a 5mg/kg 1.99±0.025* 49.36

20 5b 5mg/kg 1.80±0.046 57.76

21 5c 5mg/kg 1.98±0.007* 49.61

22 5d 5mg/kg 1.90±0.013** 51.65

23 5e 5mg/kg 1.79±0.015 54.45

24 5f 5mg/kg 1.99±0.012 49.36

25 5g 5mg/kg 1.97±0.026* 49.87

26 5h 5mg/kg 1.98±0.046 49.61

27 6a 5mg/kg 2.37±0.008** 39.69

28 6b 5mg/kg 2.11±0.007** 46.31

29 6c 5mg/kg 2.32±0.007** 40.96

30 6d 5mg/kg 2.05±0.005** 47.83

31 6e 5mg/kg 2.13±0.008** 45.80

32 7a 5mg/kg 1.98±0.026** 49.61

33 7b 5mg/kg 1.81±0.045** 53.94

34 7c 5mg/kg 1.97±0.006** 49.87

216

Values are expressed in terms of mean ± S.E.M, Significance was calculated by using

one way ANOVA with Dunnet’s t-test. The difference in results was considered

significant when p < 0.05.

* p < 0.05 Vs control at 200 mg/kg b.w. ** p < 0.01 Vs control at 200 mg/kg b.w, *** p <

0.001 Vs control at 200 mg/kg b.w

The percentage inhibition of edema was calculated at 4th hour assuming 100%

Inflammation in vehicle group.

% Anti-inflammatory activity = (Vc-Vt / Vc) x 100

Where, Vt- mean increase in paw edema volume in the drug treated group,

VC- mean increase in paw edema volume in control group.

35 7d 5mg/kg 1.91±0.014** 51.39

36 7e 5mg/kg 1.78±0.015** 54.70

217

Discussion of Anti-Inflammatory activity

From the results of antibacterial study of the synthesized quinazolinone derivatives

and benzimidazole dereivatives, it was concluded as follows:

Chalchone compounds 3 (a-h) intermediates show very less anti inflammatory

activity when compared with standard employed in the study.

The substituted quizazolinone derivates showed good anti inflammatory activity

when compared to its starting material chalcones.

Among the synthesized quinazolinone derivatives pyrazole substituted derivatives

showed a better good antiinfammatory activity action than iso oxazole substituted

derivatives.

Standard drug Indomethacin showed 61.80 % of inhibition after 4 hr from the time

of administration. Among the tested pyrazole derivativescompounds compound 4b

and 4e were more active at 4 hours (57.58% and58.02.% inhibition) of paw edema

which is superior to the standard Indomethacin and compounds 4f, and 4g and

showed 56.28, 56.54% inhibition of edema respectively after 4 hr with a p-Value of

less than 0.001 (p<0.0001) indicating that these four compounds are statistically

highly significant from the standard drug Indomethacin.

Of iso oxazole derivatives compounds 5b, and 5e showed 57.76.and 54.45 %

inhibition of edema respectively after 4 hr with a p-Value of less than 0.001

(p<0.0001) indicating that these four compounds are statistically highly significant

from the standard drug Indomethacin.

In the pyrazole substituted quinazoline derivatives, 4-methoxy phenyl substitution

i.e, 3(4-(1-acetyl-4, 5-dihydro-5-phenyl-1h-pyrazol-3-yl) phenyl)-2-(4-nitrophrnyl)

218

quinazolin-4(3h)-one (4b) and 3-(4-(1-acetyl-5-(3-chlorophenyl)-4, 5-dihydro-1h-

pyrazol-3-yl) phenyl)-2-phenyl quinazolin-4(3h)-one (4e) found to possess good

antiinfammatory activity next to the nitro substitution.

In isoxazole series compound similar to pyrazole derivatives nitro substitution

i.e., 2-(4-nitrophenyl)-3-(4-(5-phenyl-4,5-dihydroisazol-3-yl) - phenyl quinazolin-

4(3h)-one (5b) and chloro substitution of 3-(4-(5-(4-methoxyphenyl)-4, 5-

dihydroisazol-3-yl) - phenyl)-2-phenyl quinazolin-4(3h)-one (5c) was found to be

good antibacterial activity. Antiinfammatory activity


show any notable anti inflammatory activity.

On cyclization of benzoic acid derivatives increases the antiinfammatory activity.

In benzimidazole derivatives, compound 7a with amino substitution i.e., (Z)-N-(4-

aminobenzylidene-2-(1H – benzo[d]imidazol-2-yl)-N-benzylidenamine showed

good antibacterial activity against both gram positive and gram negative bacteria.

antiinfammatory activity

Ulcerogenic Activity:

Albino rats of either sex were divided into control, standard and different test groups of

six animals each group (170–250 g). They were starved for 48 h (water ad libitum)

prior to drug administration. Control group received only 0.5% sodium CMC solution,

standard group was orally administered with Indomethacin in sodium CMC solution

and test compounds 4b, 4e, 4g and 4h were administered orally at the dose of 200

219

mg/kg and 200 mg/kg, respectively. Six hours later the animals were sacrificed using

excess ether anesthesia. The stomach was excised carefully, opened along the

greater curvature; the luminal contents were removed. The mucosa was flushed with

saline and the stomach pinned on a frog board.

The ulcer index was calculated according to the method163. The lesions were counted

with the aid of hand lens (10X) and each given a severity rating as follows

Mean ulcer score for each animal will be expressed as ulcer index.

Ulcer Score Descriptive Observation

0 Normal

0.5 red coloration

1 spot ulcer

1.5 hemorrhagic streak

2 ulcers

3 perforation

220

Compound-4b (5mg/kg. (p.o)) Compound-4e(5mg/kg. (p.o))

Compound-4g(5mg/kg. (p.o)) Normal Control

Ulcer control ( Indomethacin - 10mg/kg. (p.o))

Fig. No. 6.5.1.

Ulcerogenic activity of compound 4b, 4e, 4g and 4h ,control, Indomethacin

221

Table 6.5.3.

Ulcerogenic activity of selected compounds in comparison with Indomethacin

The results are expressed as mean ± SEM (n=6). Data analyzed by one-way

ANOVA followed by Dunnett’s t-test. *p< 0.05 significant from control; **p<0.01

significant from control.

6.5.3. Ulcerogenic activity:

The gastric ulcer formation is the most common side effect with NSAIDs. The

ulcerogenic effect of compounds 4b, 4e and 4g (selected based on anti-inflammatory

profile) was evaluated in rat stress model at the therapeutic dose. The gastric

ulcerogenic effect was evaluated by calculating the ulcer index in treated animals.

Results are given in Table 6.2 that indicates these four compounds cause less gastric

ulceration at the oral dose of 200 mg/kg b.w. when compared to indomethacin which

showed ulcer index of 28.28. Hence gastrointestinal tolerance to these compounds

was better than that of standard drug.

S.No Compound Dose (p.o) Ulcer index (±SEM)

1 Control ------ 0

2 4b 5mg/kg 7.34 ± 0.35**

3 4e 5mg/kg 10.61 ± 0.14*

4 4g 5mg/kg 14.82 ± 0.18*

5 Indomethacin 10mg/kg 28.28 ± 0.48**

222

6.6.Analgesic Activity

The newly synthesized compounds were screened for analgesic activity employed by

Eddy’s hot plate method. [32] Albino Swiss mice were divided to groups of 12 group I

served as control (Normal saline 2ml/kg), group II served as standard (Pentazocine

5mg /kg) and the remaining group received at a dose of 10 mg/Kg of compounds at

oral administration. The time of reaction to pain stimulus of the mice placed on the hot

plate heated at 550+0.50C was recorded at 120 min after administration of test drug.

The increase in reaction time against control was calculated.

223

Table no. 6.6.1 Analgesic activity of synthesized compound 3a-h

Fig. No. 6.6.1. Analgesic activity of synthesized compound 3a-h

0

147.38

36.12

71.72 79.3260.21

79.31

48.43 54.71 51.57

0

50

100

150

200

control Aspirin 3a 3b 3c 3d 3e 3f 3g 3h

% Inhibition

% Inhibition

Compound Dosemg/kg

(p.o)

Reaction time in sec(Mean± SD)

% increase in painThreshold

control ----------- 3.82±0.159 ------------

Pentazocine 5mg/kg 9.45±0.245 147.38

3a 5mg/kg 5.20±0.877 36.12

3b 5mg/kg 6.56±0.978 71.72

3c 5mg/kg 6.85±0.754 79.32

3d 5mg/kg 6.12±0.823 60.21

3e 5mg/kg 6.85±0.675 79.31

3f 5mg/kg 5.67±0.711 48.43

3g 5mg/kg 5.91±0.508 54.71

3h 5mg/kg 5.79±0.654 51.57

224

Table no. 6.6.2Analgesic activity of synthesized compound 4a-h

Compound Dosemg/kg(p.o)

Reaction time insec (Mean± SD)


control --------- 3.82±0.159 ------------


4a 5mg/kg 7.80±0.837 104.19

4b 5mg/kg 9.29±0.678 143.1

4c 5mg/kg 9.05±0.154 136.91

4d 5mg/kg 7.12±0.863 86.38

4e 5mg/kg 9.32±0.695 143.9

4f 5mg/kg 9.27±0.711 142.67

4g 5mg/kg 9.01±0.568 135.86

4h 5mg/kg 9.29±0.650 143.19


0

147.38

104.19

145.1

136.91

86.38

143.9

142.67135.86

143.19

0

20

40

60

80

100

120

140

160

180

controlAspirin 4a 4b 4c 4d 4e 4f 4g 4h

% Inhibition

% Inhibition

225


Compound Dosemg/kg(p.o)

Reaction time insec (Mean± SD)


control ----------- 3.82±0.159 ---------------


5a 5mg/kg 7.80±0.837 104.19

5b 5mg/kg 8.86±0.638 131.94

5c 5mg/kg 8.05±0.134 110.73

5d 5mg/kg 7.12±0.323 86.39

5e 5mg/kg 8.65±0.605 126.44

5f 5mg/kg 8.27±0.731 116.50

5g 5mg/kg 8.11±0.528 112.30

5h 5mg/kg 8.29±0.670 117.01


0

147.38

104.19

131.94

110.73

86.39

126.44116.5 112.3 117.01

0

20

40

60

80

100

120

140

160

control Aspirin 5a 5b 5c 5d 5e 5f 5g 5h

% inhibition

% inhibition

226


Compound Dose

mg/kg

(p.o)

Reaction time in

sec (Mean ±SD)

% increase in pain

Threshold

control --------- 3.82±0.159 -----------


6a 5mg/kg 5.80±0.437 51.83

6b 5mg/kg 5.86±0.658 53.40

6c 5mg/kg 6.05±0.194 58.38

6d 5mg/kg 5.12±0.123 34.03

6e 5mg/kg 5.65±0.205 47.90


0

147.38

51.83 53.4 58.38

34.0347.9

0

20

40

60

80

100

120

140

160

control Aspirin 6a 6b 6c 6d 6e

% inhibition

% inhibition

227


Compound Dose

mg/kg

Reaction time in

sec (Mean ±SD)

% increase in pain

Threshold

control ---------- 3.82±0.159 ---------------


7a 5mg/kg 4.80±0.547 25.65

7b 5mg/kg 4.06±0.118 6.28

7c 5mg/kg 4.99±0.694 30.62

7d 5mg/kg 4.12±0.923 7.85

7e 5mg/kg 3.99±0.295 4.45


0

20

40

60

80

100

120

140

160

Drug control Aspirin 7a 7b 7c 7d 7e

Column1

228

DISCUSSION

From the results of analgesic study of the synthesized quinazolinone derivatives and

benzimidazole dereivatives, it was concluded as follows:

chalchone compounds 3 (a-h) intermediates show less signifiacant ana;gesic

activity (30-80% increase in pain threshold ) where as standard Pentazocine

shows 147.38% increase in pain threshold. the substituted quizazolinone derivates

showed good activity against the bacterial strains when compared to its starting

material chalcones.

among the synthesized quinazolinone derivatives, pyrazole substituted derivatives

showed a highly significant analgesic activity(85-160% increase in pain threshold ).

in the pyrazole substituted quinazoline derivatives, nitro i.e, 3(4-(1-acetyl-4, 5-

dihydro-5-phenyl-1h-pyrazol-3-yl)phenyl)-2-(4-nitrophrnyl) quinazolin-4(3h)-one (4b)

shows 143.12% increase in pain threshold and 3-(4-(1-acetyl-5-(3-chlorophenyl)-

4,5-dihydro-1h-pyrazol-3-yl)phenyl)-2-phenylquinazolin-4(3h)-one (4e) shows

143.90% increase in pain threshold found to possess good analgesic activity .

in isoxazole series compound similar to pyrazole derivatives nitro substitution

i.e., 2-(4-nitrophenyl)-3-(4-(5-phenyl-4,5-dihydroisazol-3-yl) - phenyl quinazolin-

4(3h)-one (5b) and chloro substitution of 3-(4-(5-(4-methoxyphenyl)-4, 5-

dihydroisazol-3-yl) - phenyl)-2-phenyl quinazolin-4(3h)-one (5c) was found to be

significant activity but comparatively less activity than pyrazole derivatives.

2-substituted benzylideneamino) benzoic acid intermediates vi (a-e) were did show

any notable analgesic activity.{5 to30%)increase in pain threshold.

229

on cyclization of benzoic acid derivatives increases the analgesic activity.{30

to50%)increase in pain threshold

6.7.In-vitro Anti-Oxidant Activity

Free radicals are known to play a definite role in a wide variety of pathological

manifestation. Antioxidants fight against by free radicals and protecting us from

various diseases and scavenge of reactive oxygen radicals or protect the antioxidant

defence mechanism. Reactive oxygen species (ROS) are capable of damaging

biological macromolecules such as DNA, carbohydrates and proteins. Reactive

oxygen species (ROS) is a collective term, which includes not only oxygen radicals

(O2., and OH.) but also some non-radical derivatives of oxygen like H2O2, HOCl, and

ozone (O3). If human disease is believed to be due to the imbalance between

oxidative stress and anti oxidative defence, it is possible to limit oxidative tissue

damage and hence prevent disease progression by antioxidant defence supplements.

It has been reported that dietary antioxidants may offer effective protection from

peroxidative damage in living systems and may play an important role in prevention of

carcinogenesis and in extending the life span of animals. In addition, antioxidant

activity may be regarded as a fundamental property important for life.

230

Reducing power of synthesized compounds and ascorbic acid

The reductive capabilities of synthesized compounds were compared with ascorbic

acid (Table 6.5.1). For the measurements of the reductive ability, we investigated the

Fe3+-Fe2+ transformation in the presence of the compounds. The reducing capacity of

a compound may serve as a significant indicator of its potential antioxidant activity.

However, the antioxidant activity of antioxidants have been attributed to various

mechanism, among which are prevention of chain initiation, binding of transition metal

ion catalysts, decomposition of peroxides, prevention of continued hydrogen

abstraction, reductive capacity and radical scavenging antioxidant activity or the

reducing power of synthesized compounds increased with increasing amount of

sample.

231

Table No. 6.7.1. Reductive ability of synthesized compounds

CompConcentration in μg/ml

50 100 150 200 250

3a 0.212±0.05 0.232±0.00 0.245±0.01 0.564±0.01 1.002±0.01

3b 0.227±0.00 0.235±0.00 0.249±0.00 1.012±0.00 1.078±0.00

3c 0.242±0.00 0.263±0.00 0.282±0.00 1.068±0.00 1.114±0.00

3d 0.251±0.00 0.249±0.00 0.258±0.00 1.114±0.00 1.135±0.00

3e 0.294±0.00 0.307±0.00 0.317±0.00 1.203±0.00 1.249±0.00

3f 0.281±0.01 0.292±0.00 0.231±0.00 0.576±0.00 1.312±0.00

3g 0.242±0.01 0.238±0.00 0.316±0.00 1.257±0.00 1.397±0.00

3h 0.292±0.00 0.280±0.00 0.411±0.00 1.002±0.00 1.479±0.00

4a 0.301±0.00 0.327±0.00 0.350±0.00 1.091±0.00 1.364±0.00

4b 0.322±0.00 0.350±0.00 1.008±0.00 1.156±0.00 1.201±0.00

4c 0.427±0.00 0.615±0.00 0.849±0.00 1.432±0.00 1.878±0.00

4d 0.442±0.00 0.793±0.00 0.952±0.00 1.268±0.00 1.734±0.00

4e 0.277±0.002 0.396±0.003 0.622±0.001 0.807±0.002 0.953±0.003

4f 0.394±0.00 0.687±0.00 0.857±0.00 1.353±0.00 1.839±0.00

4g 0.591±0.00 0.797±0.00 0.940±0.00 1.091±0.00 1.114±0.00

4h 0.281±0.002 0.290±0.001 0.297±0.004 1.083±0.003 1.119±0.002

5a 0.334±0.002 0.411±0.002 0.514±0.003 1.063±0.002 1.099±0.001

5b 0.347±0.004 0.483±0.003 0.657±0.002 1.026±0.002 1.009±0.002

5c 0.358±0.001 0.422±0.001 0.533±0.004 1.232±0.002 1.479±0.003

232

5d 0.389±0.003 0.426±0.004 0.584±0.002 0.855±0.006 0.983±0.004

5e 0.362±0.05 0.432±0.00 0.545±0.01 0.864±0.01 1.002±0.01

5f 0.377±0.00 0.435±0.00 0.549±0.00 1.020±0.00 1.180±0.00

5g 0.382±0.00 0.463±0.00 0.582±0.00 1.008±0.00 1.142±0.00

5h 0.391±0.00 0.449±0.00 0.558±0.00 1.014±0.00 1.005±0.00

6a 0.214±0.00 0.317±0.00 0.337±0.00 1.003±0.00 1.059±0.00

6b 0.291±0.01 0.302±0.00 0.331±0.00 0.476±0.00 1.052±0.00

6c 0.252±0.01 0.268±0.00 0.326±0.00 1.267±0.00 1.297±0.00

6d 0.272±0.00 0.289±0.00 0.451±0.00 1.012±0.00 1.379±0.00

6e 0.261±0.00 0.337±0.00 0.340±0.00 1.061±0.00 1.304±0.00

7a 0.282±0.00 0.350±0.00 1.018±0.00 1.136±0.00 1.211±0.00

7b 0.227±0.00 0.315±0.00 0.449±0.00 1.025±0.00 1.500±0.00

7c 0.577±0.00 0.783±0.00 0.957±0.00 1.486±0.00 1.852±0.00

7d 0.287±0.002 0.306±0.003 0.422±0.001 0.817±0.002 0.903±0.003

7e 0.354±0.00 0.607±0.00 0.757±0.00 1.053±0.00 1.190±0.00

STD 0.601±0.003 0.713±0.005 0.839±0.003 0.911±0.008 1.190±0.11

233

Fig. No. 6.7.1:Reducing power of synthesized compounds and ascorbic

acid 3a-h

Fig. No. 6.7.2: Reducing power of synthesized compounds and ascorbic acid

4a-h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

50 100 150 200 250

3

STANDARD 3a 3b 3c 3d 3e 3f 3g 3h

0

10

20

30

40

50

60

70

80

25 50 100 150 200 250

4


234

Fig. No. 6.7.3:Reducing power of synthesized compounds and ascorbic

acid 5a-h

Fig.No.6.7.4:Reducing power of synthesized compounds and ascorbic acid

6a-h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

50 100 150 200 250

5


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

50 100 150 200 250

6

STANDARD 6a 6b 6c 6d 6e

235

Fig. No. 6.7.5:Reducing power of synthesized compounds and ascorbic acid 7a-

h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

50 100 150 200 250

7


236

Table 6.7.2- Hydrogen peroxide radical assay for compound (3a-h)

Hydrogen peroxide scavenging activity of synthesized compounds and α-

tocopherol

CompoundConcentration

(µg/ml)

Absorbance at

230 nm% inhibition IC 50 µg/ml

Control 0.735

3a

25

50

100

200

400

0.620±0.002

0.592±0.003

0.503±0.002

0.436±0.003

0.362±±0.002

15.26±0.008

19.45±0.009

31.56±0.004

40.68±0.006

50.74±0.003

312.52

3b

25

50

100

200

400

0.618±0.004

0.560±0.002

0.498±0.002

0.420±0.004

0.351±0.002

15.91±0.007

23.80±0.006

32.24±0.005

46.53±0.006

52.92±0.004

321.17

3c

25

50

100

200

400

0.659±0.002

0.572±0.003

0.476±0.001

0.393±0.003

0.346±0.003

10.34±0.006

22.17±0.005

35.23±0.004

46.53±0.007

52.92±0.006

322.33

237

3d

25

50

100

200

400

0.660±0.003

0.583±0.001

0.498±0.003

0.402±0.002

0.373±0.002

10.20±0.005

20.68±0.006

32.24±0.007

45.30±0.005

49.25±0.006

362.12

3e

25

50

100

200

400

0.650±0.002

0.601±0.003

0.540±0.001

0.488±0.002

0.364±0.002

11.56±0.005

18.23±0.006

26.53±0.004

33.60±0.006

50.47±0.006

308.11

3f

25

50

100

200

400

0.643±0.003

0.546±0.002

0.469±0.002

0.399±0.003

0.357±0.001

12.51±0.006

25.71±0.004

36.19±0.006

45.71±0.003

51.42±0.009

289.33

3g

25

50

100

200

400

0.614±0.002

0.533±0.003

0.451±0.002

0.397±0.003

0.330±0.002

16.46±0.006

27.48±0.007

38.63±0.005

45.98±0.004

55.10±0.006

354.25

238

Values shown are mean ± SEM for four test p<0.01, as compared to control

3h

25

50

100

200

400

0.630±0.002

0.580±0.004

0.469±0.002

0.403±0.003

0.344±0.001

14.28±0.006

21.10±0.007

36.19±0.003

45.17±0.005

53.19±0.004

356.27

239

Hydrogen peroxide radical assay for compound (4a-h)

Table 6.7.3

Compound Concentration

(µg/ml)

Absorbance

at 230 nm

% inhibition IC 50 µg/ml

Control 0.735

4a 25

50

100

200

400

0.500±0.003

0.439±0.001

0.376±0.003

0.303±0.002

0.253±0.002

31.19±0.007

40.27±0.006

48.84±0.005

58.77±0.006

65.57±0.006

220.12

4b 25

50

100

200

400

0.510±0.002

0.445±0.003

0.380±0.003

0.292±0.002

0.242±0.003

30.61±0.005

39.45±0.006

48.29±0.008

60.27±0.003

67.07±0.005

195.25

4c 25

50

100

200

400

0.228±0.003

0.183±0.002

0.136±0.002

0.091±0.003

0.056±0.001

68.97±0.005

75.10±0.003

81.49±0.006

87.61±0.006

92.38±0.004

55.37

240

4d 25

50

100

200

400

0.222±0.003

0.194±0.001

0.140±0.002

0.100±0.003

0.040±0.002

69.79±0.004

73.60±0.006

80.95±0.004

86.39±0.005

94.55±0.007

62.12

4e 25

50

100

200

400

0.508±0.002

0.463±0.001

0.396±0.001

0.332±0.004

0.263±0.002

30.88±0.006

37.00±0.005

46.12±0.008

54.82±0.006

64.21±0.005

167.28

4f 25

50

100

200

400

0.507±0.003

0.496±0.002

0.363±0.004

0.298±0.003

0.247±0.001

31.02±0.008

32.51±0.004

50.61±0.007

59.45±0.007

0.247±0.004

189.12

4g 25

50

100

200

400

0.516±0.002

0.478±0.003

0.418±0.001

0.356±0.003

0.238±0.002

29.79±0.004

34.96±0.005

43.12±0.009

51.15±0.006

67.61±0.005

226.12

241

4h 25

50

100

200

400

0.507±0.002

0.462±0.003

0.388±0.001

0.322±0.004

0.259±0.002

31.02±0.009

37.14±0.005

47.21±0.006

56.19±0.004

64.76±0.008

280.12


242


Table 6.7.4


(µg/ml)

Absorbance

at 230 nm

%

inhibition

IC 50 µg/ml

Control 0.735

5a 25

50

100

200

400

0.512±0.002

0.472±0.003

0.390±0.002

0.312±0.03

0.254±0.002

30.34±0.006

35.78±0.004

46.93±0.007

57.55±0.005

65.44±0.006

242.12

5b 25

50

100

200

400

0.510±0.002

0.451±0.004

0.383±0.001

0.309±0.004

0.248±0.003

30.61±0.007

38.63±0.006

47.89±0.004

57.95±0.008

66.25±0.004

242.17

5c 25

50

100

200

400

0.228±0.003

0.165±0.002

0.128±0.003

0.099±0.002

0.058±0.001

68.97±0.004

77.55±0.007

82.58±0.006

86.53±0.009

92.10±0.005

.69.34

243

5d 25

50

100

200

400

0.213±0.004

0.179±0.003

0.134±0.001

0.086±0.004

0.044±0.002

71.02±0.005

75.64±0.006

81.76±0.007

88.29±0.006

94.01±0.007

71.21

5e 25

50

100

200

400

0.506±0.002

0.447±0.004

0.382±0.001

0.290±0.003

0.249±0.002

31.15±0.008

39.18±0.007

48.02±0.005

60.54±0.007

66.12±0.004

153.37

5f 25

50

100

200

400

0.510±0.003

0.439±0.003

0.386±0.001

0.319±0.002

0.282±0.003

30.61±0.009

40.27±0.004

47.48±0.006

56.59±0.008

61.63±0.005

280.56

5g 25

50

100

200

400

0.506±0.003

0.488±0.001

0.397±0.004

0.358±0.002

0.257±0.003

31.15±0.007

33.60±0.006

45.98±0.008

51.29±0.004

65.03±0.006

200.13

244

5h 25

50

100

200

400

0.512±0.001

0.436±0.004

0.380±0.002

0.313±0.004

0.243±0.003

30.34±0.007

40.68±0.009

48.29±0.005

57.41±0.005

66.93±0.004

208.55


245

Hydrogen peroxide radical assay for compound (6a-e)

Table 6.7.5


(µg/ml)

Absorbance

at 230 nm

%

inhibition

IC 50 µg/ml

Control 0.735

6a 25

50

100

200

400

0.670±0.002

0.646±0.004

0.530±0.002

0.476±0.001

0.328±0.001

0.80±0.004

12.10±0.003

27.89±0.009

35.23±0.005

55.37±0.007

295.12

6b 25

50

100

200

400

0.650±0.003

0.597±0.002

0.515±0.002

0.467±0.004

0.372±0.002

11.56±0.008

18.77±0.006

29.03±0.004

36.46±0.007

49.38±0.006

342.12

6c 25

50

100

200

400

0.659±0.002

0.583±0.003

0.412±0.001

0.349±0.002

0.320±0.004

10.34±0.011

20.68±0.004

43.94±0.008

52.51±0.005

56.46±0.008

312.77

246

6d 25

50

100

200

400

0.666±0.004

0.613±0.002

0.530±0.004

0.460±0.002

0.369±0.003

9.38±0.007

16.59±0.007

27.89±0.006

37.41±0.005

49.79±0.006

271.12

6e 25

50

100

200

400

0.654±0.002

0.632±0.001

0.528±0.004

0.471±0.002

0.380±0.004

11.02±0.007

14.01±0.006

29.25±0.006

35.91±0.004

48.29±0.007

329.26


247


Table 6.7.6


(µg/ml)

Absorbance at

230 nm

% inhibition IC 50 µg/ml

7a 25

50

100

200

400

0.650±0.002

0.592±0.004

0.523±0.004

0.440±0.001

0.357±0.003

11.56±0.005

19.45±0.004

28.84±0.005

40.13±0.008

51.42±0.006

320.55

7b 25

50

100

200

400

0.662±0.002

0.578±0.003

0.510±0.002

0.470±0.004

0.360±0.004

90.93±0.004

21.36±0.009

30.61±0.005

36.05±0.006

51.02±0.007

361.17

7c 25

50

100

200

400

0.219±0.002

0.172±0.003

0.102±0.004

0.091±0.003

0.032±0.004

70.20±0.006

76.59±0.004

86.12±0.009

87.61±0.007

95.64±0.005

61.12

248

7d 25

50

100

200

400

0.647±0.002

0.555±0.003

0.479±0.003

0.359±0.002

0.331±0.003

11.97±0.005

24.48±0.006

34.82±0.004

51.15±0.007

54.96±0.005

334.12

7e 25

50

100

200

400

0.639±0.002

0.541±0.004

0.472±0.001

0.417±0.003

0.349±0.003

13.06±0.007

26.39±0.006

35.78±0.004

43.26±0.009

52.51±0.007

312.55

α-tocopherol

(standard)

25

50

100

200

400

0.232 ± 0.708

0.197 ± 0.008

0.176 ± 0.001

0.091 ± 0.002

0.071 ± 0.007

68.43 ± 0.10

73.46 ± 0.15

76.05 ± 0.06

87.61 ± 0.10

90.34 ± 0.05

42.12


249

Fig. No. 6.7.6. Hydrogen peroxide scavenging activity of synthesized

compounds and α-tocopherol 3a-h

Fig. No. 6.7.7.: Hydrogen peroxide scavenging activity of synthesized


0

10

20

30

40

50

60

70

80

90

100

25 50 100 200 400

3


0

10

20

30

40

50

60

70

80

90

100

25 50 100 200 400

4


250




compounds and α-tocopherol 6a-e

0

10

20

30

40

50

60

70

80

90

100

25 50 100 200 400

5


0

10

20

30

40

50

60

70

80

90

100

25 50 100 200 400

6


251


compounds and α-tocopherol 7a-e

Hydrogen peroxide converts into the singlet oxygen (O2) and hydroxy radicals, and

become powerful oxidizing agent. Thus the removal of H2O2 is very important for

antioxidant defence in cell or food systems. H2O2 can cross membranes and may

oxidize a number of compounds. All the compounds were capable of scavenging

hydrogen peroxide in an amount dependent manner, which could be seen by its

graded increase in percentage of inhibition.

Compound 4c and4d exhibited greater hydrogen peroxide scavenging activity (55.3

and 62.1 µg/ml respectively), when compared to the other compounds. The positive

control (α-tocopherol) had an IC50 value of 42.12 µg//ml. Thus from the present

investigation it can be said that the among synthesized pyrazole derivatives 4c,4d,

among isooxazole derivatibves 5c, 5d and among benzimidazole derivatives 7c

exhibited remarkable antioxidant property in various in vitro assay systems.

0

20

40

60

80

100

120

25 50 100 200 400

7


252

IR Graph of 3(4-(1-ACETYL-4,5-DIHYDRO-5-PHENYL-1H-PYRAZOL-3-YL)PHENYL)-2-(4-NITROPHRNYL)QUINAZOLIN-4(3H)-ONE(IV-b)

253

IR Graph of 3-(4-(1-ACETYL-4,5-DIHYDRO-5-(4-METHOXYPHENYL)-1H-PYRAZOL-3-YL)PHENYL)-2-PHENYLQUINAZOLIN-4(3H)-ONE(IV-c)

254

IR Graph of 3-(4-(1-ACETYL-4,5-DIHYDRO-5-(4-METHOXYPHENYL)-1H-PYRAZOL-3-YL)PHENYL)-2-(4-NITROPHENYL)QUINAZOLIN-4(3H)- ONE(IV-d)

255

IR Graph of 3-(4-(1-ACETYL-5-(3-CHLOROPHENYL)-4,5-DIHYDRO-1H-PYRAZOL-3-YL)PHENYL)-2-PHENYLQUINAZOLIN-4(3H)-ONE(IV-e)

256

IR Graph of 3-(4-(1-ACETYL-5-(3-CHLOROPHENYL)-4,5-DIHYDRO-1H-PYRAZOL-3-YL)PHENYL)-2-(4-NITROPHENYL)QUINAZOLIN-4(3H)-ONE(IV-f)

257

IR Graph of 3-(4-(1-ACETYL-4,5-DIHYDRO-5-(3-NITROPHENYL)-1H-PYRAZOL-3-YL)PHENYL)-2-PHENYLQUINAZOLIN-4(3H)-ONE(IV-g)

258

IR Graph of 3-(4-(1-ACETYL-4,5-DIHYDRO-5-(3-NITROPHENYL)-1H PYRAZOL-3-YL)PHENYL)-2-(4-NITROPHENYL)QUINAZOLIN-4(3H)-ONE(IV-h)

259

IR Graph of 3-(4-(4,5-DIHYDRO-5-PHENYLISOXAZOL-3-YL)PHENYL)-2-(4-NITROPHENYL)QUINAZOLIN-4(3H)-ONE(V-b)

260

IR Graph of 3-(4-(4,5-DIHYDRO-5-(4-METHOXYPHENYL)ISOXAZOL-3-YL)PHENYL)-2-PHENYKLQUINAZOLIN-4(3H)-ONE(V-c)

261

IR Graph of 3-(4-(5-(3-CHLOROPHENYL)-4,5-DIHYDROISOXAZOL-3-YL)PHENYL)-2-PHENYLQUINAZOLIN-4(3H)-ONE(V-e)

262

IR Graph of 3-(4-(4,5-DIHYDRO-5-(3-NITROPHENYL)ISOXAZOL-3-YL)PHENYL)-2-(4-NITROPHENYL)QUINAZOLIN-4(3H)-ONE(V-h)

263

IR Graph of (E)-2-(BENZYLIDENEAMINO) BENZOIC ACID(VI-a)

264

IR Graph of (E)-2-(4-AMINOBENZYLIDENEAMINO) BENZOIC ACID (VI-b)

265

IR Graph of (Z)-2-(1H-BENZO[d]IMIDAZOL-2-YL)-N-BENZYLIDENEAMINE(VII-a)

266

IR Graph of 2-((Z)-(2-(1H-BENZO[d] IMIDAZOL-2-YL) PHENYLIMINO) METHYL)PHENOL (VII-c)

267

IR Graph of (Z)-N-(2,4-DINITROBENZYLIDENE)-2-(1H-BENZO[d]IMIDAZOL-2-YL)BENZAMINE(VII-d)

268

IR Graph of (Z)-N-(4-(DIMETHYLAMINO)BENZYLIDENE)-2-(1H-BENZO[d]IMIDAZOL-2-YL)BENZENAMIANE(VII-e)

269

NMR Graph of 2-(4-NITROPHENYL)-3-(4-((Z)-3-OXO-3-PHENYLPROP-1-ENYL)PHENYL)QUINAZOLIN-4(3H)-ONE (III-b)

270

NMRGRAPH of 2-(4-NITROPHENYL)-3-(4-((Z)-3-(3-NITROPHENYL)-3-OXOPROP-1-ENYL)PHENYL)QUNAZOLINE-4(3H)-ONE (III-h)

271

NMR Graph of 3-(4-(1-ACETYL-4,5-DIHYDRO-5-PHENYL-1H-PYRAZOL-3-YL)PHENYL)-2-PHENYLQUINAZOLIN-4(3H)-ONE (IV-a)

272

NMR Graph of 3-(4-(1-ACETYL-5-(3-CHLOROPHENYL)-4,5-DIHYDRO-1H-PYRAZOL-3-YL)PHENYL)-2-PHENYLQUINAZOLIN-4(3H)-ONE(IV-E)

273

NMR Graph of 3-(4-(1-ACETYL-5-(3-CHLOROPHENYL)-4,5-DIHYDRO-1H-PYRAZOL-3-YL)PHENYL)-2-(4-NITROPHENYL)QUINAZOLIN-4(3H)-ONE(IV-F)

274

NMR Graph of 3-(4-(4,5-DIHYDRO-5-(4-METHOXYPHENYL)ISOXAZOL-3-YL)PHENYL)-2-(4-

NITROPHENYL)QUINAZOLIN-4(3H)-ONE(V-d)

275

NMR Graph of (E)-2-(4-(dimethylamino)benzylideneamino Benzoic acid (VI-e)

276

NMR Graph of (z)-n-(2,4-dinitrobenzylidene)-2-(1h-benzo[d]imidazol-2-yl)benzamine(VII-d)

277

NMR Graph of (z)-n-(4-(dimethylamino)benzylidene)-2-(1h-benzo[d]imidazol-2-yl)benzenamiane(VII-e)

278

7.SUMMARY AND CONCLUSION

All the newly synthesized 3-(4-(substituted phenyl)-3-oxoprop-1-enyl) phenyl- 2-

(substituted phenyl) quniazoline-4-one 3(a-h), 3-[4-(1-acetyl-4,5-dihydro-5-substituted

phenyl-1H-pyrazol-3-yl) phenyl]-2-substutied phenyl quinazolin-4(3H)-one derivatives

4(a-h), 3-[4-(5-(substituted phenyl)-4,5-dihydro isoxazol-3-yl) phenyl]-2-substutied

phenyl quinazolin-4(3H)-one derivatives V(a-h), 2-(substituted benzylideneamino)

benzoic acid VI(a-e), and (Z)-N-4-(substituted benzylidene)-2-(1H-benzio[d]imidazole-2-

yl) benzenamine 7(a-e) were characterized by TLC, UV, IR, 1H NMR and mass spectral

analysis. Compounds were evaluated for anti-inflammatory as well as gastric

ulcerogenic effects, antimicrobial properties and in vitro anti-oxidant effects.

In the acute toxicity studies, the examined compounds did not show toxic effects at

doses up to 1000 mg/kg b.w. Therefore, 1/10th of the tolerated dose was chosen for

the pharmacological evaluation.

pyrazoline derivatives 4b and 4e showed highly significant anti-inflammatory activity

and other compounds 4f and 4g exhibited promising activity in carrageenan-induced

rat paw edema in rat, 4b,4e and 4g show low ulcerogenicity compared with the

standard drug Indomethacin. Since GI problems due to NSAID continue to be the

major impediment to their use in therapeutics, GI protection of the new anti-

inflammatory derivatives proves them useful lead molecules for the development of

better NSAID with greatly improved therapeutic index. Molecular docking studies auto

279

dock of these derivatives with COX-2 and COX-1 receptors are also showed minimum

binding energy for invitro active compounds and may be considered as good

inhibitors of COX-2. A Lamarckian genetic algorithm method implemented in the

program Auto dock 4.0, was employed.

The newly synthesized compounds were also screened for their antibacterial activity

and antifungal activity against gram positive bacteria (B. subtilis and S. aureus), gram

negative bacteria (E. coli and P. aeruginosa) and A. niger and S. cerevisiae fungal

strains respectively by cup plate method. Among the test compounds, the compounds

4h, 4c, 5fand 7b have emerged as active against all tested microorganisms and all

other compounds showed moderate activity whereas chalcone derivatives show very

lessactivity. Further, the anti fungal evaluation result of the synthesized compounds

indicated that compounds 4h and 5b have the potential to be selective as lead

compounds.

With in vitro anti microbial results in hand it is thought worth-while to do in silico

studies to support the in vitro activities .Theoretically all the compounds showed very

good binding energy. So, it can be predicted as the activity may be due to inhibition of

enzyme β-keto acyl acyl carrier protein in case of anti bacterial activities and 14-α

demethylase enzyme for antifungal activity to confirm these studies. Further enzyme

assays are required to confirm these studies.

280

Hence, this study widened the scope of developing these quinazolinone containing

pyrazole and quinazolinone containing isooxazole and as promising anti-bacterial and

anti-fungal agents.

In in-vitro anti-oxidant activities, most of the compounds assayed showed excellent

reducing power and free radical scavenging activities. Compounds 4c, 4d, and 7c are

the most active among the series showing both high reducing power and hydrogen

peroxide scavenging activities.

These results and previous experimental and docking studies strongly suggest that

most of pyrazole molecules synthesized in this study may indeed be promising drug

candidates with interesting pharmacological profile and most of these derivatives

could be a fruitful pharmacophore for further development of better anti inflammatory,

antimicrobial and anti-oxidant agents.

281

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