Chapter - 4shodhganga.inflibnet.ac.in/bitstream/10603/45072/7/chapter 4.pdf · -Bill Cosby Synthesis, characterization and evaluation of antioxidant activities of 3-chloro-1-(10-methoxy-

204

Chapter - 4

In order to succeed, Your desire for success should be greater than

Your fear of failure. -Bill Cosby

Synthesis, characterization and evaluation of antioxidant activities of 3-chloro-1-(10-methoxy- 5H-dibenz[b,f]azepine-5-yl) propan-1-one conjugated with different amino acids, aminophenols and substituted aminophenols.

Chapter 4 Introduction

Page 205

INTRODUCTION

The investigation on limitless series of

structurally related 5H-dibenz[b,f]azepines with a wide range of physical, chemical and

biological properties were discussed in chapter 1 and 2. Among the series of 5H-

dibenz[b,f]azepine analogues, 10-methoxy-5H-dibenz[b,f]azepine(IV) is one, having the

following structural formula figure 4.1.

Fig.4.1. 10-methoxy-5H-dibenz[b,f]azepine(IV)

This is used as an intermediate for the synthesis of registered anticonvulsant drug,

oxcarbazepine1 and its structure was recently been reported2.

Even though a vast versatile literature is available on the various analogues of 5H-

dibenz[b,f]azepines, the reports on 10-methoxy-5H-dibenz[b,f]azepine analogues were

less. A few reports on structural, preparative and patent work are highlighted in the

following paragraph. Structural aspects of 10-methoxy-5H-dibenz[b,f]azepine, a

iminostilbene analogues was determined by X-ray crystallography studies3. Preparation

of iminostilbene derivatives and the reaction between 10-methoxy-5H-dibenz[b,f]azepine

and bis (trichloromethyl)carbonate results in 10-methoxy-N-chlorocarbonyl-

iminostilbene with good yield were reported4.

Chapter 4 Introduction

Page 206

The invention relates to an improved process for preparation of 10-methoxy-5H-

dibenz[b,f]azepine-5-carbonyl chloride from 10-methoxy-5H-dibenz[b,f]azepine without

the use of phosgene and its further conversion to 10-oxo-10,11-dihydro-5H-

dibenz[b,f]azepine-5-carboxamide(oxcarbazepine) without the use of strong mineral

acids was recently reported5. Fuenfschilling et al developed a new industrial process for

the preparation of oxcarbazepine using 10-methoxy-5H-dibenz[b,f]azepine as

intermediate6. Till date only a few data are available on 10-methoxy-5H-

dibenz[b,f]azepine, its derivatives and their pharmacology. An attempts has been made to

synthesize, characterize and evaluate the antioxidant activities of 10-methoxy-5H-

dibenz[b,f]azepine and its analogues.

Chapter 4 Present Investigation

Page 207

Present Investigation

Survey of the literature on 10-methoxy-5H-

dibenz[b,f]azepine and its derivatives reveals that no efforts have been made towards the

study of the effect of antioxidant activity on incorporation of different amino acids,

aminophenols and substituted aminophenols. In the course of our research and

development of new antioxidants, we synthesize various analogues of 10-methoxy-5H-

dibenz[b,f]azepine. Initially we synthesized the basic molecule 10-methoxy-5H-

dibenz[b,f]azepine by literature method (discussed in chapter 2). Preliminary findings on

antioxidant studies reveals that 10-methoxy-5H-dibenz[b,f]azepine showed dominant

DPPH scavenging activity and also antioxidant properties in various in vitro assays.

These finding prompted us to synthesize the series of its analogues and to evaluate their

antioxidant properties.

Literature reveals that 10-methoxy-5H-dibenz[b,f]azepine and its various analogues

are having various pharmacological importance5 (eg., anti-epiliptical agent in the

treatment of AIDS-related neural disorders and for the treatment of Perkinson’s

diseases). 10-methoxy-5H-dibenz[b,f]azepine has been also used as an intermediate for

the synthesis of oxcarbazepine. But no significant reports on antioxidant activities were

found. The chapter is divided into three sections (section I, section II, and section III).

In section I, we have synthesized three N-acylated derivatives of 10-methoxy-5H-

dibenz[b,f]azepines and evaluated their antioxidant activities. Structures of N-acylated

derivatives of 10-methoxy-5H-dibenz[b,f]azepines were showed in the table 4.1.


Page 208

Table 4.1. List of the structures of N-acylated derivatives of 10-methoxy-5H- dibenz[b,f]azepine

Compound name

Compound number

structure

10-methoxy-5H-dibenz[b,f]azepine-5-carbonyl

chloride XXVI

2-chloro-1-(10-methoxy-5H-dibenz[b,f]azepin-5-

yl)ethanone XXVII

3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-

yl) propan-1-one XXVIII

The obtained analogues were subjected to test their capacity for antioxidant activity. All

the N-acylated analogues showed significantly less activity, but among them 3-chloro-1-

(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one(XXVIII) analogue showed

considerable activity. Thus, in order to improve the antioxidant activity of 3-chloro-1-

(10-methoxy-5H-dibenz[b,f]azepine-5yl)propan-1-one and to study the structure activity

relationships, we undertook the present investigation.

In this direction, we have synthesized a series of 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5-yl)propan-1-one containing different amino acids, aminophenols

and substituted aminophenols and their ability for antioxidant potential were evaluated

employing a wide range of well-established in vitro systems.

N

H3CO

OCl

N

H3CO

O

Cl

N

H3CO

O

Cl


Page 209

The systematic studies on the synthesis, characterization and evaluation of antioxidant

activities of newly synthesized analogues of 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5-yl)propan-1-one are presented in section II and III.

Section II reveals about the synthesis, characterization and evaluation of

antioxidant activities of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-

one conjugated with different amino acids. The amino acids used for conjugation are

glycine, alanine, threonine, cysteine, methionine, proline, phenylalanine, tyrosine,

hydroxyproline, tryptophan and histidine. Totally eleven amino acid analogues were

synthesized and their structures were listed in the table 4.2.


Page 210

Table 4.2. List of the structures of newly synthesized 3-chloro-1-(10-methoxy-5H- dibenz[b,f]azepine-5yl)propan-1-one conjugated with different amino acid.

Compound name

Compound number

Structure

2-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)-3oxopropylamino)acetic acid [Glycine analogue]

XXIX

2-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)-3 oxopropylamino)propanoicacid [Alanine analogue]

XXX

2-(3-(10-methoxy-5H-dibenz[b,f]azepine-yl)-3-oxopropyl amino)-3-hydroxy butanoic acid [Threonine analogue] XXXI

2-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)-3-oxopropylamino)-3-mercapto propanoic acid [Cysteine analogue] XXXII

2–(3–(10-methoxy-5H–dibenzo[b,f]azepin–5–yl)–3–oxopropylamino)–4–(methylthio) butanoic acid [Methionine analogue] XXXIII

N

H3CO

O

NH

O OH

N

H3CO

O

HN

OHO

N

H3CO

O

NH

HO O

OH

N

H3CO

O

NH

SH

O

OH

N

H3CO

O

NH

S

O

OH


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Compound name

Compound number

Structure

1-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)- 3-oxopropyl) pyrrolidine-2-carboxylic acid

[Proline analogue]

XXXIV

2-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)- 3 oxopropylamino)-3-Phenyl propanoicacid [Phenylalanine analogue] XXXV

3-(4-hydroxyphenyl)-2-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)-3-oxopropylamino)propanoicacid [Tyrosine analogue]

XXXVI

1-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)-3-oxopropyl)-3-hydroxy-pyrolidine-2-carboxylic acid [Hydroxyproline analogue]

XXXVII

N

H3CO

O

N

OHO

N

H3CO

O

NH

O

OH

N

H3CO

O

NH

HO

O

OH

N

H3CO

O

NHO

OHO


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Compound Name

Compound number

Structure

2-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)- 3-oxopropylamino)-3-(1H-indol-3-yl) propanoic acid

[Tryptophan analogue] XXXVIII

2-(3-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)- 3-oxopropylamino)-3-(1H-imidazol-4-yl) propanoic acid [Histidine analogue]

XXXIX

Section III reveals about the synthesis, characterization and evaluation of

antioxidant activities of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-

one conjugated with different aminophenols and substituted aminophenols. The

aminophenols used for conjugation are para aminophenol, ortho aminophenol and meta

aminophenol, The substituted aminophenols used for conjugation are 4-amino-2-

methoxyphenol and 4-amino-2-nitrophenol. Totally three aminophenol and two

substituted aminophenol analogues were synthesized and their structures were listed in

the table 4.3.

N

H3CO

O

NH NH

O

HO

N

H3CO

O

NH NH

N

O

HO


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Table 4.3. List of the structures of newly synthesized 3-chloro-1-(10-methoxy-5H- dibenz[b,f]azepine-5-yl)propan-1-one conjugated with different aminophenols and substituted aminophenols.

Compound name

Compound number

Structure

3-(4-hydroxyphenylamino)-1-(10-methoxy-5H-dibenz[b,f]azepin-5-yl)propan-1-one

[para- aminophenol analogue]

XXXX

3-(2-hydroxyphenylamino)-1-(10-methoxy-5H- dibenz[b,f]azepin-5-yl)propan-1-one [ortho- aminophenol analogue]

XXXXI

3-(3-hydroxyphenylamino)-1-(10-methoxy-5H-dibenz[b,f]azepin-5-yl)propan-1-one [meta- aminophenol analogue]

XXXXII

3-(4-hydroxy-3-nitrophenylamino)-1-(10-methoxy-5H-dibenz[b,f]azepin-5-yl)propan-1-one [4-amino-2-nitrophenol analogue]

XXXXIII

3-(4-hydroxy-3-methoxyphenylamino)-1-(10- Methoxy-5H-dibenz[b,f]azepin-5-yl)propan-1-one [4-amino-2-methoxyphenol analogue]

XXXXIV

N

H3CO

O

OH

NH

N

O

NH

OH

O2N

H3CO

N

O

NH

OH

H3CO

H3CO

N

O

NH

H3CO

OH

N

O

NH

H3CO

OH

Chapter 4 Materials and Methods

Page 214

MATERIALS AND Methods

The organic solvents such as methanol-AR grade

(E-Merck), chloroform-AR (E-Merck), n-hexane AR(E-Merck), benzene AR (Qulegens),

diethyl ether AR (Ranbaxy), ethyl acetate AR (E-Merck), acetic acid-AR (E-Merck) used

were of analytical grade. Distilled water-double distilled water by quartz distillation unit.

All the chemical reagents were obtained from the standard commercial sources unless

otherwise indicated. Phosgine, chloro acetyl chloride, chloro propionyl chloride,

triethylamine, sodium bicarbonate, anhydrous sodium sulphate (Ranbaxy), tlc aluminium

sheets-Silica gel 60 F254 were purchased from E-Merck. Amino acids like glycine, alanine,

threonine, cysteine, methionine, proline, phenylalanine, tyrosine, hydroxyproline,

tryptophan and histidine were purchased from Sigma Aldrich. Aminophenols and

substituted aminophenols like p-aminophenol, o-aminophenol, m-aminophenol, 4-amino 2-

nitrophenol, and 4-amino-2-methoxyphenol were purchased from S.d. fine chemicals. The

reagents and solvents used in the present investigation were purified and dried according to

standard procedures7-10. Anhydrous sodium sulphate was activated by heating over naked

flame for 3-4 hr, cooled in desiccators over fused calcium chloride and stored in air-tight

bottle.

Melting points of the compounds were determined using SELACO-650 and Veego

VMP-III model hot stage melting point apparatus and are uncorrected. The pH of the

solution was measured using pH meter, model APX 175 (Control Dynamics

Chapter 4 Materials and method

Page 215

Instrumentation Pvt.Ltd). The pH meter was standardized using buffer tablets of 9.2, 7.0

and 4.0 at 25 0C Identification and structure elucidation of newly

synthesized compounds under study was carried out by using various spectroscopic

techniques such as IR, 1H NMR, Mass and elemental analysis.

A Nicolet 5700 FTIR instrument was used for recording IR spectra for the

synthesized compounds. A 2.0 to 3.0 mg of compound was prepared as KBr pellet and

the IR spectra were recorded. NMR- A Bruker DRX-500MHz spectrometer operating at

500.13 MHz was used. 1H NMR spectra in CDCl3 and DMSO-d6 with 40mg of the

sample dissolved in 0.5ml of solvent. Tetramethylsilane(TMS) was used as internal

standard for measuring the chemical shift values to within±0.001 ppm. A region from 0-

10 ppm was scanned for all the samples. Mass spectra of the synthesized compounds

were obtained using a Q-TOF waters Ultima instrument (No-Q-Tof GAA 082, Water

Corporation, Manchester, UK) fitted with an Electron spray ionization(ESI) source. The

data acquisition software used was Version 4.0. Element micro analytical data were

obtained by Elemental–Vario EL–III.


Page 216

Experimental

SECTION- I

General procedure for the synthesis of N-acylated analogues of 10-Methoxy-5H-

dibenz[b,f]azepine

To the well stirred solution of 10-methoxy-5H-dibenz[b,f]azepine (2 mM) and

triethylamine (2.2 mM) in 50 ml benzene, Phosgene or chloro acyl chloride (2.2 mM) in 25

ml benzene was added drop by drop for about 30 min(Scheme 4). Then the reaction

mixture is stirred at room temperature for about 8 hr. Progress of the reaction is monitored

by tlc using hexane: ethyl acetate mixture (9 : 1) as mobile phase. After the completion of

reaction the solvent was removed under pressure and the remaining residue was quenched

in ice cold water and extracted in to diethyl ether. The ether layer was washed twice with

5% NaHCO3 and once with distilled water. Finally the ether layer is dried over anhydrous

Na2SO4. The brownish yellow semi solid product was obtained by desolventation through

rotavapour.

(XXVI – XXVIII)

X= Cl, CH2-Cl, CH2-CH2-Cl

n= 0, 1, 2

Scheme.4. Schematic protocol for the synthesis of N-acylated analogues of 10-methoxy-5H-dibenz[b,f]azepine.

N

H

ClCO(CH2)nCl

Triethyl amine,C6H6, RT, 6 hr

N

OX

+ HCl

H3CO H3CO


Page 217

The synthesized compounds were purified by column chromatography. The

activated silica gel [60-120 mesh] was packed on to the glass column [450×40 mm] using

methanol as solvent. The obtained product was loaded and eluted using mixture of n-

hexane: ethyl acetate(90 : 10). The fractions were collected separately and the active

fraction was concentrated by using the rotary evaporator. The obtained compound was

monitored for single spot through thin layer chromatography(tlc). The plates were

developed using n-hexane: ethyl acetate( 90 : 10) as mobile phase. The spot was located

by exposing the tlc plates to iodine fumes.

All the N-acylated analogues of 10-methoxy-5H-dibenz[b,f]azepine are characterized and

the data are presented in the table 4.4-4.6. The IR, 1H NMR, and mass spectra of 10-

methoxy-5H-dibenz[b,f]azepine and 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-

yl)propan-1-one(XXVIII) are presented in figure 4.2-4.4.


Page 218

IUPAC Name 10-methoxy-5H-dibenz[b,f]azepine-5-carbonyl chloride

Molecular formula C16H12ClNO2

Nature Brown semi solid

Melting point -

Yield 82 %

Mass M+ 285.34

IR 3067.9 (Ar C–H), 1675.3 (C=O) cm-1

1H NMR 1H NMR (δ, CDCl3): δ 7.5-7.6 (m, 8H, Ar – H), 6.3 (s, 1H, Ar – H of seven membered ring),

3.5 (s, 3H, OCH3)

Elemental Analysis Anal.Calcd: C, 67.26; H, 4.23; N, 4.90 %

Found: C, 67.25; H, 4.24; N, 4.88 %

Table. 4.4. Physico-chemical and spectral properties of compound XXVI

N

OCl

H3CO


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IUPAC Name 2-chloro-1-(10-methoxy-5H-dibenz[b,f]azepin-5-yl)ethanone



Melting point -

Yield 85 %

Mass M+ 299.54

IR 3163.4 (Ar-H), 1681 (C=O) cm-1

1H NMR 1H NMR (δ, CDCl3): 7.3-7.6 (m, 8H, Ar-H), 6.0 (s, 1H, seven membered Ar-H),

4.2 (d, 2H, CH2-Cl), 3.5 (s, 3H, OCH3)

Elemental Analysis Anal.Calcd: C, 68.12; H, 4.71; N, 4.67 %

Found: C, 68.11; H, 4.72; N, 4.65 %

Table. 4.5. Physico-chemical and spectral properties of compound XXVII

N

O

H3CO

Cl


Page 220

IUPAC Name 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5yl)propan-1-one



Melting point -

Yield 89 %

Mass M+ 314.61

IR 3067.9 (Ar C–H), 1675.3 (C=O), 2971.0–3026.1 (CH2) cm-1

1H NMR 1H NMR (δ, CDCl3): 2.8 (d, 2H, CH2–C=O), 3.7 (d, 2H, CH2Cl), 7.5-7.6 (m, 8H, Ar – H),

6.9 (d, 1H, Ar – H of seven membered ring), 3.5(s, 3H, OCH3).

Elemental Analysis Anal. Calcd: C, 68.90; H, 5.14; N, 4.46 %.

Found: C, 68.88; H, 5.13; N, 4.45 %.

Table. 4.6. Physico-chemical and spectral properties of compound XXVIII

N

O

H3CO

Cl


Page 221

Figure 4.2. IR spectra for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one(XXVIII).

N

H3CO

O

Cl


Page 222

Figure 4.3. 1H NMR spectra for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one(XXVIII)

N

H3CO

O

Cl


Page 223

Figure 4.4.Mass spectra for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1- one(XXVIII) SECTION- II

General procedure for the synthesis of 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5-yl) propan-1-one conjugated with different amino acids.

Glycine (1.2 mM) in methanol (25 mL) was neutralized with triethylamine (1.2 mM)

and air is displaced by N2 gas. To this, K2CO3 (600 mg) was added in the prersence of N2

atmosphere. Later the solution of 3-chloro-1-(5H-dibenz[b,f]azepine-5-yl)propan-1-one (1

mM) in methanol (50 mL) was added drop by drop for 30 min. The reaction mixture was

refluxed for 6–8 hr. The progress of the reaction was monitored by tlc. Typically inert

atmosphere (N2 atmosphere) was maintained in order to couple the amino acids. The

reaction sequences are outlined in scheme 5. The reaction mixture was then desolventized

in rotavapour and the compound is extracted in to ethyl acetate. The ethyl acetate layer was

N

H3CO

O

Cl


Page 224

washed with water and dried over anhydrous Na2SO4. The brownish yellow semi solid

product(XXIX) was obtained by further desolventation in rotavapour.

Further, 3-chloro-1-(5H-dibenz[b,f]azepine-5-yl)propan-1-one conjugated with

alanine(XXX), threonine(XXXI), cysteine(XXXII), methionine(XXXIII),

proline(XXXIV), phenylalanine(XXXV), tyrosine(XXXVI), hydroxyproline(XXXVII),

tryptophan(XXXVIII) and histidine(XXXIX) by following the same procedure (Scheme

5). The products were separated and purified by column chromatography.

(XXIX – XXXIX)

Where,

Scheme 5. Reaction sequence to obtain the amino acid analogues of 3-chloro-1-(10- methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one.

Compound R Compound R

XXIX H XXXV

XXX -CH3 XXXVI

XXXI -CH(CH3)OH XXXVII

XXXII -CH2SH XXXVIII

XXXIII XXXIX

XXXIV

N

O

Cl

+

O

HO NH2

R

CH3OH, K2CO3

Nitrogen, reflux, 6-8 hr

N

O

O

HO

NH

R

+ HCl

H3CO

H3CO

N

N OH

NH

OH

SN

NH


Page 225

PURIFICATION OF newly synthesized COMPOUNDS

The products were purified by column

chromatography, where the activated silica gel [60-120 mesh] was packed on to the glass

column [450×40 mm] using methanol as solvent. The obtained product was loaded and

eluted using mixture of chloroform : methanol : acetic acid(85 : 12 : 3). The fractions

were collected and the active fraction was concentrated by using the rotary evaporator.

The plates were developed using chloroform : methanol : acetic acid(85 : 12 : 3) as

mobile phase. The spot was located by exposing the tlc plates to iodine fumes. The

obtained pure compounds were characterized by physico-chemical and spectral methods

and the data are presented in the table 4.7-4.17. The IR, 1HNMR, and mass spectra of 3-

chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one conjugated with

tyrosine(XXXIV) are presented in figure 4.5-4.7.


Page 226

Figure 4.5. IR spectra for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one conjugated with Tyrosine(XXXVI)

N

H3CO

O

NH

HO

O

HO


Page 227

Figure. 4.6. 1H NMR spectra for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one conjugated with Tyrosine(XXXVI)

N

H3CO

O

NH

HO

O

HO


Page 228

Figure 4.7. Mass spectra for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1- one conjugated with Tyrosine(XXXVI).

N

H3CO

O

NH

HO

O

HO


Page 229

IUPAC Name 2-(3-(10-methoxy-5H-dibenzo[b,f]azepin-5-yl)-3-

oxopropylamino)acetic acid [Glycine analogue]

Molecular formula C20H20N2O4


Melting point -

Yield 72%

Mass 352.14

IR 1669.7 (C=O), 3317.6 (N–H), 3428.5 (OH–carboxylic acid), 3052.6–2748.1(Ar – H), 3022.2 (CH2)cm-1

1H NMR 7.3–7.7 (m, 8H, Ar–H), 3.48 (s, 3H, seven memberd OCH3), 3.5 (d, 2H, CH2 of glycine),

3.0 (t, 2H, CH2–NH), 2.5 (t, 2H, CH2–C=O), 1.9 (s, 1H, NH), 12.8 (s, 1H, OH of COOH).

Elemental analysis Anal. Calcd:C, 68.17; H, 5.72; N, 7.95 %

Found: C, 68.15; H, 5.70; N, 7.94 %

Table. 4.7. Physico-chemical and spectral properties of compound XXIX

N

H3CO

O

NH

O OH


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oxopropylamino)propanoic acid [Alanine analogue]



Melting point -

Yield 78%

Mass 366.34

IR 1669.3 (C=O), 3315.5 (N–H), 3489.1 (OH–carboxylic acid), 3052.1–2748.0 (Ar – H), 3022.2 (CH2)cm -1

1H NMR 7.3– 7.7 (m, 8H, Ar–H), 3.48 (s, 3H, seven memberd OCH3), 3.0 (t, 2H, CH2C=O), 2.4 (t, 2H, CH2–NH),

1.2 (d, 3H, CH3 of alanine), 3.4 (q, 1H, CH of alanine), 2.0 (s, 1H, NH of alanine),

12.0 (s, 1H, OH of COOH of alanine).

Elemental analysis Anal. Calcd: C, 68.84; H, 6.05; N, 7.65 %.

Found: C, 68.80; H, 6.06; N, 7.63 %.

Table. 4.8. Physico-chemical and spectral properties of compound XXX

N

H3CO

O

HN

OHO


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IUPAC Name 3-hydroxy-2-(3-(10-methoxy-5H-dibenz[b,f]azepin-5-yl)-3-

oxopropylamino) butanoic acid [Threonine analogue ]



Melting point -

Yield 67%

Mass 396.44

IR 3308.8 (N-H), 2748.1-3066.4 (OH-carboxylic acid), 1676.8 (C=O), 1566.1and 1619.4 (CH2) cm-1

1H NMR 2.36 (d, 2, α C=O, 2H), 2.98 (d, 2, β C=O, 2H), 5.92 (s, 1, CH, 1H), 3.48 (s, 3H, seven memberd OCH3),

7.19 – 7.81 (m, 7, Ar – H, 7H), 11.5 (s, 1, COOH, 1H), 5.1 (s, 1, OH, 1H), 4.1 (t, 1, CH, 1H), 1.22 (d, 3, CH3, 3H),

3.65 (d, 1, CH, 1H).


Found: C, 67.60: H, 5.90; N, 6.88 %.

Table. 4.9. Physico-chemical and spectral properties of compound XXXI

N

H3CO

O

NH

HO O

OH


Page 232

IUPAC Name 3-mercapto-2-(3-(10-methoxy-5H-dibenzo[b,f]azepin-5-yl)-3-

oxopropylamino)propanoic acid [Cysteine analogue]

Molecular formula C21H22N2O4S


Melting point -

Yield 73%

Mass 398.33

IR 1669.6 (C=O), 3317.7 (N–H), 3457.4 (OH–carboxylic acid), 3052.6–2748.7 (Ar – H), 3022.2 (CH2)cm-1

1H NMR 7.1-7.8 (m, 8H, Ar–H), 3.48 (s, 3H, seven memberd OCH3), 1.6 (s, 1H, SH), 2.0 (s, 1H, NH),

2.6 (t, 2H, CH2C=O), 3.2 (m, 2H, CH2–NH), 3.6 (m, 2H, CH2 of cysteine), 3.8 (t, 1H, CH of cysteine),

12.5(s, 1H, OH of COOH of cysteine)


Found: C, 63.31; H, 5.55; N, 7.04 % .

Table. 4.10. Physico-chemical and spectral properties of compound XXXII

N

H3CO

O

NH

SH

O

OH


Page 233


oxopropylamino)-4-(methylthio)butanoic acid

[Methionine analogue]

Molecular formula C23H26N2O4S


Melting point -

Yield 70%

Mass 426.87


1H NMR 7.1– 7.8 (m, 8H, Ar–H), 3.48 (s, 3H, seven memberd OCH3), 2.0(s, 1H, NH), 1.9 (t, 2H, CH2 of methionine),

2.4 (s, 3H, CH3 of methionine), 2.5 (t, 2H, CH2C=O), 2.6 (t, 2H, CH2 of methionine),

3.0 (t, 2H, CH2–NH), 3.4 (t, 1H, CH of methionine), 12.2 (s, 1H, OH of COOH of methionine)

Elemental

analysis

Anal. Calcd:C, 64.77; H, 6.14; N, 6.57 %.

Found: 64.75; H, 6.15; N, 6.60 %.

Table. 4.11. Physico-chemical and spectral properties of compound XXXIII

N

H3CO

O

NH

S

O

OH


Page 234


oxopropyl)pyrrolidine-2-carboxylic acid [Proline analogue]



Melting point -

Yield 76%

Mass 352.32

IR 1669.2 (C=O), 3484.8 (OH–carboxylic acid), 3052.1–3748.2 (Ar – H), 3022.2 (CH2) cm-1

1H NMR 7.3– 7.7 (m, 8H, Ar–H), 3.48 (s, 3H, seven memberd OCH3), 3.4 (t, 1H, CH of pyrolidine group of

proline), 3.3 (t, 2H, CH2–NH), 2.4 (m, 2H, CH2–C=O), 2.6 (d, 1H, CHN of pyrolidin ring),

1.8 (t, 1H, CH2, C4 of pyrolidine ring), 1.6 (m, 3H, CH2, 2H of C3 and 1H of C4 of pyrolidine ring),

12.1 (s, 1H, OH of COOH of pyrolidine).


Found: C, 70.35; H, 6.15; N, 7.10 %.

Table. 4.12. Physico-chemical and spectral properties of compound XXXIV

N

H3CO

O

N

OHO


Page 235

IUPAC Name 2-(3-(10-methoxy-5H-dibenz[b,f]azepin-5-yl)-3-

oxopropylamino)-3-phenyl propanoic acid

[Phenylalanine analogue]



Melting point -

Yield 81%

Mass 442.57

IR 3318.0 (N-H), 2071.0 – 30628 (OH -carboxylic acid), 1675.9 (C=O), 1566.1 and 1620.3 (CH2) cm-1

1H NMR 2.36 (d, 2, α C=O, 2H), 2.98 (d, 2, β C=O, 2H), 5.92 (s, 1, CH, 1H), 3.48 (s, 3H, seven memberd

OCH3), 7.30 – 8.3 (m, 13, Ar – H, 13H), 11.5 (s, 1,COOH, 1H), 2.40 (s, 1, NH), 4.1 (t, 1, CH, 1H),

3.11 – 3.40 (t, 2, CH2, 2H).


Found: C, 73.25; H, 5.90; N, 6.30 %.

Table. 4.13. Physico-chemical and spectral properties of compound XXXV

N

H3CO

O

NH

O

OH


Page 236

IUPAC Name 3-(4-hydroxyphenyl)-2-(3-(10-methoxy-5H-

dibenz[b,f]azepin-5-yl)-3-oxopropylamino) propanoic acid

[Tyrosine analogue]



Melting point -

Yield 75%

Mass 458.33

IR 3321.4 (N-H), 2339.4 – 3062.8 (OH - carboxylic acid), 1671.8 (C=O), 1599.3 and 1618.5 (CH2) cm-1

1H NMR 2.36 (d, 2, α C=O, 2H), 2.98 (d, 2, β C=O, 2H), 5.92 (s, 1, CH, 1H), 3.48 (s, 3H, seven memberd OCH3),

6.78 – 8.33 (m, 12, Ar – H, 12H), 9.40 (s, 1, Ar – OH, 1H), 11.5 (s, 1, COOH, 1H), 2.40 (s, 1, NH, 1H),

4.1 (s, 1, CH, 1H) 3.11 – 3.40 (t, 2, CH2, 2H).


Found: C, 70.72; H, 5.71; N, 6.10 %.

Table. 4.14. Physico-chemical and spectral properties of compound XXXVI

N

H3CO

O

NH

HO

O

OH


Page 237

IUPAC Name

3-hydroxy-1-(3-((E)-10-methoxy-5H-dibenzo[b,f]azepin-5-yl)-3-

oxopropyl)pyrrolidine-2-carboxylicacid[Hydroxyproline analogue]



Melting point -

Yield 73%

Mass 408.43

IR 2311.4– 3418.8 (OH - carboxylic acid), 1670.6 (C=O), 1566.3 and 1619.1 (CH2) cm-1

1H NMR 2.36 (d, 2, α C=O, 2H), 2.98 (d, 2, β C=O, 2H), 5.92 (s, 1, CH, 1H), 3.48 (s, 3H, seven memberd OCH3)

7.19 – 7.81 (m, 7, Ar – H, 7H), 11.5 (s, 1, COOH, 1H), 1.72 – 1.95 (m, 2, CH2, 2H), 2.25 – 2.35 (q, 2, CH2, 2H),

3.80 (q, 1, CH, 1H), 3.31 (d, 1, CH, 1H)

Elemental analysis Anal. Calcd: For C, 73.28; H, 5.92; N, 6.33 %.

Found: C, 73.24; H, 5.89; N, 6.28 %.

Table. 4.15. Physico-chemical and spectral properties of compound XXXVII

N

H3CO

O

NHO

OHO


Page 238

IUPAC Name

3-(1H-indol-3-yl)-2-(3-(10-methoxy-5H-dibenzo[b,f]azepin-5-yl)-3-

oxopropylamino)propanoic acid [Tryptophane analogue]



Melting point -

Yield 69%

Mass 481.23

IR 1668.3 (C=O), 3317.2 (N–H), 3487.7 (OH–carboxylic acid), 3095.6–2747.3 (Ar – H), 3022.2 (CH2) cm-1

1H NMR 7.2–7.7 (m, 8H, Ar–H), 3.48, (s, 3H, seven memberd OCH3), 2.0 (s, 1H, NH, CH2NH),

2.4 (t, 2H, CH2C=O), 3.0 (t, 2H, CH2–NH), 3.2 (t, 2H, CH2 of tryptophan), 3.7 (t, 1H, CH of tryptophan),

10.2 (s, 1H, NH of indole ring), 7.3 (m, 1H, CH–NH of indole ring of tryptophan),

7.4–7.7 (m, 4H, Ar–H of tryptophan), 12.1 (s, 1H, OH of COOH of tryptophan);


Found: C, 72.32; H, 5.62; N, 8.70 %.

Table. 4.16. Physico-chemical and spectral properties of compound XXXVIII

N

H3CO

O

NH NH

O

HO


Page 239

IUPAC Name 3-(1H-imidazol-4-yl)-2-(3-(10-methoxy-5H-dibenzo[b,f]azepin-5-yl)-

3 oxopropylamino)propanoic acid [Histidine analogue]



Melting point -

Yield 66%

Mass 432.64


1H NMR 7.1–7.8 (m, 8H, Ar–H), 3.48 (s, 3H, seven memberd OCH3), 1.95 (s, 1H, NH, CH2NH),

2.5 (t, 2H, CH2C=O), 3.2 (d, 2H, CH2 of histidine), 3.3 (t, 2H, CH2–NH), 3.9 (t, 1H, CH–NH of histidine),

7.3 (m, 1H, CH of imedazole ring of histidine), 8.6 (m, 1H, Ar–CH, N=CHNH of histidine),

12.3 (s, 1H, OH of COOH of tryptophan), 13.5 (s, 1H,NH of imedazole ring).


Found: C, 66.62; H, 5.55; N, 12.94 %.

Table. 4.17. Physico-chemical and spectral properties of compound XXXIX

N

H3CO

O

NH NH

N

O

HO


Page 240

Section –iii

In this section of the thesis, a series of

aminophenols and substituted aminophenols are conjugated to 3-chloro-1-(10-methoxy-

5H-dibenz[b,f]azepine-5yl)propan-1-one in order to obtain a better antioxidant. The

evaluation of antioxidant activities and the structure-activity-relationships of the newly

synthesized compounds were also studied.

General procedure for the synthesis of 3-chloro-1-(5H-dibenz[b,f]azepine-5-yl)

propan-1-one conjugated with different aminophenols and substituted aminophenols.

p-Aminophenol (1.2 mM) in tetrahydrofuron (THF, 25 mL) was neutralized with

triethylamine (1.2 mM) and K2CO3 (600 mg) was added. Later the solution of 3-chloro-1-

(10-methoxy-5H-dibenz [b,f] azepine-5-yl)propan-1-one (1 mM) in THF (50 mL) was

added drop by drop for 30 min. The reaction mixture was refluxed for 6-8 hr. The

progress of the reaction mixture was monitored by tlc. The reaction mixture was then

desolventized in rotavapour and the compound is extracted into ethyl acetate. The ethyl

acetate layer was washed with water and dried over anhydrous Na2SO4. The drak brown

semi solid was obtained by further desolventation in rotavapour.

Similarly, o-aminophenol, m-aminophenol, 4-amino-2-methoxyphenol and 4-amino-

2-nitrophenol analogues of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-

1-one were obtained by following the same procedure (Scheme 6).


Page 241

Where, (XXXX – XXXXIV)

Compound R1 R2 R3 R4 R5

XXXX H H OH H H XXXXI OH H H H H XXXXII H OH H H H

XXXXIII H OH H NO2 H XXXXIV H OH H OCH3 H

Scheme 6. Reaction sequence to obtain the aminophenols and substituted aminophenol analogues of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one.

PURIFICATION OF SYNTHESIZED COMPOUNDS

The compounds were further purified by coloumn

chromatography using silica gel [60-120 mesh] as adsorbent and chloroform :

methanol(80 : 10) as elutant. The pure compounds were characterized by tlc, IR, 1H

NMR, Mass and elemental analysis. The data’s were presented in the table 4.18-4.22.

The IR, 1HNMR, and mass spectra of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-

yl)propan-1-one conjugated with p-aminophenol(XXXX) are presented in figure 4.8-

4.10.

N

OTHF, K2CO3

reflux, 6-8 hr

N

O

Cl

NHR1

R2R3

R4

R5

HCl+

NH2R1

R2R3

R4

R5+

H3CO

H3CO


Page 242

Figure.4.8. IR spectra for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one containing p-aminophenol(XXXX)

N

H3CO

O

NH

OH


Page 243

Figure4.9.1H NMR spectra for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one containing p-aminophenol(XXXX)

N

H3CO

O

NH

OH


Page 244

Figure. 4.10. Mass spectra for 3-chloro-1-(10-methoxy -5H-dibenz[b,f]azepine-5-yl)propan- 1-one containing p-aminophenol(XXXX)

N

H3CO

O

NH

OH


Page 245

IUPAC Name 3-(4-hydroxyphenylamino)-1-(10-methoxy-

5H-dibenzo[b,f]azepin-5-yl)propan-1-one

[Para-aminophenol analogue]



Melting point -

Yield 78%

Mass 386.54

IR 3053–2829.5 (Ar C–H), 1670.9 (C=O), 3234.5 (N-H), 3222.4–3500.8 (phenolic-OH),

2971.0–3026.1 (CH2) cm-1 1H NMR 7.19–7.18 (m, 8H, Ar-H), 3.48 (s, 3H, seven memberd OCH3), 2.68 (t, 2H, CH2, C=O), 3.55 (t, 2H,

CH2, CH2N-H), 5.82 (s, 1H, N-H), 6.71–6.73 (m, 4H, Ar-H of aminophenol, 9.43 (s, 1H, OH)

Elemental analysis Anal. Calcd: C, 74.59; H, 5.74; N, 7.25 %

Found: C, 74.55; H, 5.76; N, 7.24 %

Table.4.18. Physico-chemical and spectral properties of compound XXXX

N

H3CO

O

OH

NH


Page 246

IUPAC Name 3-(2-hydroxyphenylamino)-1-(10-methoxy-5H-

dibenz[b,f]azepin-5-yl)propan-1-one

[Ortho-Aminophenol analogue]



Melting point -

Yield 75%

Mass 386.44

IR 3052–2803.5 (Ar C–H), 1659.5 (C=O), 3360.4 (N-H), 3213.1–3450.6 (phenolic-OH),

2971.0–3026.1 (CH2) 1H NMR 7.19–7.18 (m, 8H, Ar-H), 3.48 (s, 3H, seven memberd OCH3), 2.68 (t, 2H, CH2, C=O),

3.55 (t, 2H, CH2, CH2N-H), 8.02 (s, 1H, N-H), 6.71–6.9 (m, 4H, Ar-H of aminophenol,

10.0 (s, 1H, OH) cm-1


Found: C, 74.56; H, 5.71; N, 7.24 %.

Table. 4.19. Physico-chemical and spectral properties of compound XXXXI

N

O

NH

H3CO

OH


Page 247

IUPAC Name 3-(3-hydroxyphenylamino)-1-(10-methoxy-5H-


[Meta-Aminophenol analogue]



Melting point -

Yield 73%

Mass 386.64

IR 3048.1–2834.3 (Ar C–H), 1662.4 (C=O), 3315.2 (N-H), 3210.3–350.5 (phenolic-OH),

2971.0–3026.1 (CH2) cm-1 1H NMR 7.19–7.18 (m, 8H, Ar-H), 3.48 (s, 3H, seven memberd OCH3), 2.68 (t, 2H, CH2, C=O),


9.0 (s, 1H, OH)


Found: C, 74.55; H, 5.73; N, 7.22 %.

Table. 4.20. Physico-chemical and spectral properties of compound XXXXII

N

O

NH

OH

H3CO


Page 248

IUPAC Name 3-(4-hydroxy-3-nitrophenylamino)-1-(10-methoxy-5H-


[Nitro-Aminophenol analogue ]



Melting point -

Yield 64%

Mass 431.45


2971.0–3026.1 (CH2) cm-1 1H NMR 7.19–7.18 (m, 8H, Ar-H), 3.48 (s, 3H, seven memberd OCH3), 2.68 (t, 2H, CH2, C=O),


10.0 (s, 1H, OH)


Found: C, 66.82; H, 4.92; N, 9.76 %.

Table. 4.21. Physico-chemical and spectral properties of compound XXXXIII

OH

O2N

NH

N

O


Page 249

IUPAC Name 3-(4-hydroxy-3-methoxyphenylamino)-

1-(10-methoxy-5H-dibenzo[b,f]azepin-5-yl)propan-1-one

[Methoxy-Aminophenol analogue]



Melting point -

Yield 71%

Mass 416.66


2971.0–3026.1 (CH2) cm-1 1H NMR 7.19–7.18 (m, 8H, Ar-H), 3.48 (s, 3H, seven memberd OCH3), 2.68 (t, 2H, CH2, C=O), 3.55 (t, 2H, CH2,

CH2N-H), 5.82 (s, 1H, N-H), 6.0–6.6 (m, 3H, Ar-H of aminophenol, 9.4 (s, 1H, OH), 3.8 (t, 3H, OCH3)


Found: C, 72.13; H, 5.84; N, 6.72 %.

Table. 4.22. Physico-chemical and spectral properties of compound XXXXIV

OH

H3CO

NH

N

O


Page 250

Chapter 4 Results and Discussion

Page 250

Results and Discussion

An enormous amount of work on the N-

substituted analogues of 10-methoxy-5H-dibenz[b,f]azepines has been reported in the

literature. No efforts were directed towards the study of the effect of antioxidant activities

on incorporation of the amino acids, aminophenols and substituted aminophenols as the

substrates attached to the N-acylated moiety. Thus, in order to couple different amino

acids, aminophenols and substituted aminophenls, chain extension of 10-methoxy-5H-

dibenz[b,f]azepines by N-acylation was carried out. Further compound XXVI, XXVII

and XXVIII were obtained from N-acylation of 10-methoxy-5H-dibenz[b,f]azepine by

applying triethylamine(TEA) base promoted reaction with COCl2, COCH2Cl and

COCH2CH2Cl2. The protocol of the reaction is simple, inexpensive, applied for good

yield. The products were purified through column chromatography using n-hexane and

ethyl acetate(90 : 10) as a mobile solvent and characterized by various analytical and

spectral methods. The relevant data’s were presented in the table 4.4-4.6.

IR spectra of compound XXVI, XXVII and XXVIII showed absorption peak in the

region 1700 cm-1 due to the carbonyl group and the absence of N-H band at 3300cm-1. This

data reveals that the N-acylation of 10-methoxy-5H-dibenz[b,f]azepines has taken place on

our simple experimental protocol. In the IR spectra of 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5yl)propan-1-one, C=O stretching band was observed at about 1687.1

cm-1 and CH2 stretching band was observed at 2835.7 cm-1, whereas CH2–Cl stretching

was signaled at 2958.4 cm-1. Absence of N–H band at 3360 cm-1 was found. An absorption

band at 3067–2700 cm-1 was observed due to aromatic stretching was observed.


Page 251

1H NMR of compound XXVII showed –CH2 protons peaks as triplet at 1.9 ppm.

Whereas, compound XXVIII showed two –CH2 resonated peaks at 2.8 (CH2–C=O), and

3.7 (CH2Cl) ppm as doublet, the OCH3 protons were found as triplet at 3.4 ppm.. The

entire aromatic proton peaks appeared as a broad multiplet in the region 7.5-7.6 ppm.

Mass spectra of all the N-acylated analogues (XXVI-XXVIII) showed the expected mass

corresponding to their molecular weight. A typical IR, 1H NMR, and mass spectra for 3-

chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one were showed in the

figure, 4.2, 4.3 and 4.4.

The newly synthesized three N-acylated analogues of 10-methoxy-

dibenz[b,f]azepine(XXVI-XXVIII) were evaluated for antioxidant activities by

following different assays namely

1. DPPH free radical scavenging activity

2. Inhibition of lipid peroxidation in β-carotene linoleate system

3. Reducing power assay

4. Inhibition of human low-density lipoprotein(LDL) oxidation

DPPH free radical scavenging activity

The DPPH assay is a simple method to measure the ability of antioxidants to trap

free radicals. The scavenging effects of the N-acylated analogues of 10-methoxy-

dibenz[b,f]azepine at different concentration(10, 50, 100, 200 and 500 µM) was studied

and showed in figure.4.11.


Page 252

Figure 4.11. Percentage scavenging effect on DPPH by N-acylated analogues of 10-methoxy-

5H-dibenz[b,f]azepine and standards. Each value represents means ± SD (n=3)

Two controls, traditional antioxidants like ascorbic acid and BHA, were also included.

All the N-acylated analogues(XXVI-XXVIII) even at higher concentration (500 µM)

showed less activity towards DPPH (11.02%, 13.56% and 15.48%) respectively, where

compound XXVIII showed better activity followed by compound XXVII and compound

XXVI. It can also be observed that the increase in the length of the alkyl chain increases

the radical scavenging capacity. This effect may probably due to the fact that as the alkyl

chain increases its electron-donating activity also increases. From the study N-acylated

analogues showed less activity over standards. The results were calculated on the

effective concentration IC50 of the test compound in relation to starting DPPH

concentration at which 50% of DPPH radical remained (table 4.23).


Page 253

Table 4.23. 50% Inhibition of DPPH radical by N-acylated analogues of 10-methoxy-5H- dibenz[b,f]azepine and standards. Where – corresponds to no significant 50% inhibition. Each value represents means ± SD (n=3)

Compound IC50 (µM)

XXVI -

XXVII -

XXVIII -

AA 13.2±0.10

BHA 14.1±0.21

The DPPH activity of all the N-acylated analogues and the standards increases as the

concentration increases. The experiments were carried out in triplicate and each values

represents the mean±SD (n=3). The increasing order of DPPH activity of N-acylated

analogues and the standards are as follows AA> BHA> compound XXVIII> compound

XXVII> compound XXVI

Inhibition of lipid peroxidation in β-carotene linoleate system

The antioxidant activity of N-acylated analogues of 10-methoxy-

dibenz[b,f]azepine(XXVI-XXVIII) and standards like ascorbic acid and BHA at

different concentration (10 and 25 µM) as measured by the bleaching of β-carotene, is

presented in figure 4.12.


Page 254

Figure 4.12. Bleaching of β-carotene with respect to time in the presence of N-acylated analogues of 10-methoxy-5H-dibenz[b,f]azepine(XXVI-XXVIII) and standrds at different concentrations (10 and 25 µM). Each value represents the mean ± SD (n=3) It can be seen that all the N-acylated analogues exhibit varying degree of antioxidant

activity. From the figure in the presence of all the N-acylated analogues bleaches the β-

carotene as the time left for oxidation, among the analogues compound XXVIII hinder

the extent of β-carotene bleaching in the better way followed by compound XXVII and

compound XXVI. On the other hand standards like ascorbic acid and BHA inhibit the

extent of β-carotene bleaching dominantly maintaining higher absorbance. But in case of


Page 255

blank the extent of bleaching of β-carotene was more and become colourless at the end of

180 min. On the basis of extent of bleaching of β-carotene the percentage(%) antioxidant

activity of N-acylated analogues of 10-methoxy-dibenz[b,f]azepine and standards were

also determined and showed in the figure 4.13.

Figure 4.13. % Antioxidant activity of N-acylated analogues of 10-methoxy-5H- dibenz[b,f]azepine(XXVI-XXVIII) and standards by β-carotene-linoleic acid model. Each value represents the mean ± SD (n=3)

From the figure all the N-acyl derivatives showed less antioxidant activity. Among the

analogues compound XXVIII showed 13.65% activity followed by compound XXVII

(8.12%) and compound XXVI (6.35%) at 25µM concentration. The experiments were

carried out in triplicate and each values represents the mean±SD (n=3). The increasing

order of % antioxidant activity of N-acylated analogues and the standards on β-carotene -

linoleic acid model are as follows AA> BHA> compound XXVIII> compound XXVII>

compound XXVI .

Reducing power assay

The antioxidant activity has been reported to be concomitant with the reducing

power11.The reducing power ability of N-acylated analogues of 10-methoxy-


Page 256

dibenz[b,f]azepine was determined by using potassium ferricyanide method. The

reducing power of N-acylated analogues at different concentration (25, 50, 100, and 200

µM) are showed in the figure 4.14.

Figure 4.14. Reducing power of N-acylated analogues of 10-methoxy-5H-dibenz[b,f]azepine and standards. Each value represents the mean ± SD (n=3) At higher concentration (500 µM), N-acylated analogues(XXVI, XXVII and XXVIII)

showed less absorbance of 0.1226, 0.0915 and 0.0625 respectively, indicating less

activity. Among the N-acyalted analogues compound XXVIII showed better reducing

power than the compound XXVII and compound XXVI.

The reducing power of N-acylated analogues were compared to the standards ascorbic

acid and BHA, where standards showed dominating reducing power over N-acylated

analogues of 10-methoxy-dibenz[b,f]azepine(XXVI-XXVIII). Here in this assay, all the

compounds and standards showed reducing power in concentration dependent manner.

The experiments were carried out in triplicate and the each values represents mean±SD

(n=3). The increasing order of reducing power of N-acylated analogues and the standards

are as follows AA> BHA> compound XXVIII> compound XXVII> compound XXVI.


Page 257

Inhibition of human low-density lipoprotein(LDL) oxidation

The antioxidant activity of N-acylated analogues of 10-methoxy-

dibenz[b,f]azepine(XXVI, XXVII and XXVIII) and the standards on human LDL

oxidation was determined.

The % inhibition of human LDL oxidation by N-acylated analogues and the standards at

different concentration (10 and 25 µM) at different time intervals (2, 4 and 6 hr) were

depicted in the figure 4.15.

Figure 4.15. Antioxidant activity(%) of N-acylated analogues of 10-methoxy-5H-dibenz[b,f]azepine and standards on human LDL oxidation in different concentrations (10 and 25 µM). Values represent means ± SD (n=3).


Page 258

From the figure even at higher concentration (25 µM) all the N-acylated analogues

(XXVI, XXVII and XXVIII) showed less activity over inhibition of LDL oxidation

(11.35%, 9.54% and 8.26%) at the end of 2 hr and showed only (17.36%, 14.62%

and12.12%) at the end of 6 hr. Whereas, standards (ascorbic acid and BHA) showed79.16

and 75.71% inhibition at the end of 2 hr and 92.19 and 90.89% inhibition at the end of 6

hr showed dominant activity over N-acylated analogues. Among the N-acylated

analogues compound XXVIII showed better activity compared to compound XXVII and

compound XXVI. Throughout the assay the % inhibition of LDL oxidation of all the

compounds and the standards increased in increase in the concentration. The experiments

were carried out in triplicate and the each values represents mean±SD (n=3). The

increasing order of inhibition of % human LDL oxidation of N-acylated analogues and

the standards are as follows AA> BHA> compound XXVIII> compound XXVII>

compound XXVI.

Thus, in order to explore the better antioxidant activities we have selected compound

XXVIII as a model compound for coupling of amino acids, aminophenols and substituted

aminophenols.

Further, a series of amino acid analogues of 10-methoxy-

5H-dibenz[b,f]azepine as target compounds were synthesized. Scheme 5 illustrates the

way to prepare the target compounds(XXIX-XXXIX). The structure of the compounds

was elucidated by IR, 1H NMR, mass spectral data and elemental analysis. The presence

of requisite peaks and absence of extraneous peaks confirms the synthesis.


Page 259

A series of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one

containing amino acid(XXIX-XXXIX) were evaluated by following the methodology

described earlier. Here also AA and BHA were used as standard antioxidants.


The scavenging effects of newly synthesized amino acid analogues of 3-chloro-1-

(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one and standards under the

investigation on DPPH with respect to their different concentrations is presented in figure

4.16.

Figure 4.16. Scavenging effect on DPPH by 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5- yl)propan-1-one containing amino acid and standards. Each value represents means ± SD (n=3) IC50 for DPPH activity of synthesized compounds were also done and showed in the

table 4.24.


Page 260

Table 4.24. 50% Inhibition of DPPH radical by the amino acid analogues of 3-chloro-1-(10- methoxy-5H-dibenz[b,f]azepine-5-yl) propan-1-one Where – corresponds to no significant 50% inhibition. Each value represents means ± SD (n=3)

Inhibition of lipid peroxidation in β-carotene linoleate system

Heat-induced oxidation of an aqueous emulsion system of β-carotene-linoleic acid

was employed as an antioxidant test reaction. The newly synthesized compounds were

screened for their antioxidant potential. Bleaching of β-carotene with respect to time for

newly synthesized compounds at two different concentrations (10 and 25 µM) is

represented in figure 4.17.

Compound IC50 (µM/mL)

XXVIII -

XXIX -

XXX -

XXXI 172.2±0.26

XXXII 15.36±0.12

XXXIII 38.22±0.30

XXXIV -

XXXV -

XXXVI 14.21±0.01

XXXVII 14.52±0.13

XXXVIII 14.46±0.16

XXXIX 14.62±0.32

BHA 14.10±0.21

AA 13.20±0.10


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Figure 4.17. Bleaching of β-carotene with respect to time in the presence of 3-chloro-1-(10- methoxy-5H-dibenz[b,f]azepine-5-yl) propan-1-one containing amino acid at different concentrations (10 µM and 25 µM). Each value represents the mean ± SD (n=3). The % antioxidant activity for 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-

yl)propan-1-one XXVIII containing amino acid was measured on the basis of extent of

bleaching of β-carotene is presented in figure 4.18.


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Figure 4.18. % Antioxidant activity of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5- yl)propan-1-one containing amino acid by β-carotene-linoleic acid model. Each value represents the mean ± SD (n=3). Reducing power assay

The reducing power of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-

yl)propan-1-one containing amino acid, which may sever as a significant reflection of the

antioxidant activity, was determined using a modified iron(Fe3+) to iron(Fe2+) reduction

assay( figure 4.19).

Figure 4.19.Reducing power of amino acid analogues of 3-chloro-1-(10-methoxy-5H- dibenz[b,f]azepine-5-yl) propan-1-one containing aminoacid. Each value represents the mean ± SD (n=3)


Page 263

Inhibition of human low-density lipoprotein (LDL) oxidation

The antioxidant capacities for the inhibition of LDL oxidation of newly synthesized

compounds were studied. The effect of compound on LDL oxidation in different

concentration (10 µM and 25 µM) is showed in the figure 4.20.

Figure 4.20. % Inhibition of LDL oxidation of amino acid analogues of 3-chloro-1-(10- methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one containing amino acid at different concentrations. Each value represents the mean ± SD (n=3)


Page 264

In all the four assays performed, analogues of tyrosine, tryptophan and

hydroxyproline, were effective as antioxidants and their efficacy is almost equal to the

standards, AA and BHA. This may be due to the combined effect of 3-chloro-1-(10-

methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one containing OCH3 group and presence

of phenolic –OH group in tyrosine, indole NH in tryptophan and alcoholic group in

hydroxyproline analogues. On the other hand histidine and cysteine analogues were also

effective in preventing oxidation. The threonine and methionine analogues showed

moderate activity, while all other analogues showed less activity. The increasing

antioxidant activities of synthesized compounds and the standards in all the assays

performed are showed in the following order.

AA>BHA>XXXVI>XXXVIII>XXXVII>XXXIX>XXXII>XXXIII>XXXI>XXX>XXI

X>XXXIV>XXXV>XXVIII

Comparison of antioxidant activities of free amino acids and their conjugated analogues

A comparative studies on the antioxidant

activities of free amino acids (standards) and their respective amino acid

conjugated(products) by various methods namely free radical scavenging activity by

DPPH free radical scavenging activity, inhibition of lipid peroxidation in β-carotene

linoleate system, inhibition of human low-density lipoprotein(LDL) oxidation and

reducing power assay. The antioxidant activities of standards and the respective products

at higher concentration with data including graphical representation are depicted below.


Page 265

DPPH radical scavenging activity

The DPPH activity of free amino acids and 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5-yl)propan-1-one conjugated with respective amino acids(product)

were depicted in the figure 4.21.

Figure 4.21. DPPH radical scavenging activity(%) of final products towards the standard amino acids at 500 µM. Values represent means ± SD (n=3). Inhibition of lipid peroxidation in β-carotene linoleate system

The % antioxidant activities of free amino acids and 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5-yl)propan-1-one conjugated with different amino acids(product) as

measured by bleaching of β-Carotene is presented in figure 4.22.


Page 266

Figure 4.22. Antioxidant activity(%) of final products towards the standard amino acids at 25 µM on β-Carotene linoeic acid model system. Values represent means ± SD (n=3). Reducing power assay

The reducing power of all the amino acids and 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5-yl)propan-1-one conjugated with amino acids(product) at 500 µM

concentration were depicted on figure 4.23.

Figure 4.23. Reducing power of final products towards the standard amino acids at 500 µM. Values represent means ± SD (n=3).


Page 267

Inhibition of human low-density lipoprotein(LDL) oxidation

The antioxidant capacities for the inhibition of human LDL oxidation of final

product towards the free amino acids at higher concentrations (25 µM) were studied. The

effect of compounds on LDL oxidation is showed in the figure 4.24.

Figure 4.24. Antioxidant activity(%) of final products towards the standard amino acids at 25 µM on human LDL oxidation at 25 µM. Values represent means ± SD (n=3). The free amino acids were showed less antioxidant properties than their conjugated

analogues in all the four assays performed. In other words, coupling of amino acids to

3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one increases the

antioxidant activities.

Section -II

Prompted by the new analogues of 10-methoxy-

5H-dibenz[b,f[azepine and to explore the antioxidant activity, continuation of our work on


Page 268

exploring newer compounds having different substrate were done. A series of six new

compounds were synthesized by coupling of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-

5-yl)propan-1-one with three different aminophenols(4-aminophenol, 2-aminophenol, 3-

aminophenol) and two different substituted aminophenols(4-amino-2-methoxyphenol and 4-

amino-2-nitrophenol) by base catalyzed condensation to get products(XXXX-XXXXIV) in

moderate to good yield. The reaction sequences are outlined in scheme 6. Structural

conformation was done using IR, 1HNMR, mass spectra and elemental analysis. The

presence of requisite peaks and the absence of extraneous peak confirm the synthesis. The

physico-chemical and spectral characterization of 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5-yl) propan-1-one containing different aminophenols and substituted

aminophenols were presented in the figure 4.18-4.22.

The synthesized compounds 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl)

propan-1-one conjugated with different aminophenols and substituted aminophenols

(XXXX-XXXXIV) were evaluated for their antioxidant capacity by various methods

described earlier. The antioxidant potential of standards like AA and BHA were also

investigated and a comparative study was carried out for the synthesized compounds.


Firstly, a series of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-yl) propan-1-

one conjugated with different aminophenols and substituted aminophenols(XXXX-

XXXXIV) were evaluated for antioxidant activity using a DPPH radical-generating

system (figure 4.25).


Page 269

Figure 4.25. Scavenging effect on DPPH by 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5- yl)propan-1-one containing aminophenols and substituted aminophenols and standards. Each value represents means ± SD (n=3) As parameter for radical scavenging activities, the IC50 values (i.e., the sample

concentration causing 50% inhibition of DPPH radical formation) was used (table 4.25).

Table 4.25. 50% Inhibition of DPPH radical by 3-chloro-1-(10-methoxy-5H- dibenz[b,f]azepine-5-yl)propan-1-one containing aminophenols and substituted aminophenols and standards. Where – corresponds to no significant 50% inhibition. Each value represents means ± SD (n=3)

Compound IC50 (µM/mL)

XXVIII -

XXXX 12.06±0.21

XXXXI 12.43±0.09

XXXXII 12.76±0.11

XXXXIII 11.33±0.36

XXXXIV 10.32±0.13

BHA 14.10±0.21

AA 13.20±0.10


Page 270

β-carotene linoleic acid assay

The extents of bleaching of β-carotene in the presence of synthesized

compounds(XXXX-XXXXIV) at two different concentrations were showed in the figure

4.26.

Figure 4.26. Bleaching of β-carotene with respect to time in the presence of 3-chloro-1-(10- methoxy-5H-dibenz[b,f]azepine-5-yl)propan-1-one containing aminophenols and substituted aminophenols and standards at different concentrations (10 µM and 25 µM). Each value represents the mean ± SD (n=3).


Page 271

The % antioxidant activity of synthesized compounds and the standards (ascorbic

acid and BHA) as measured by the bleaching of β-carotene at different concentration is

presented in the figure 4.27.

Figure 4.27. % Antioxidant activity of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5- yl)propan-1-one containing aminophenols and substituted aminophenols and standards by β-carotene-linoleic acid model. Each value represents the mean ± SD (n=3).

Reducing power assay

The synthesized compounds(XXXX-XXXXIV) were analyzed for their reducing

power at different concentration [10, 25, 50, 100, 200 and 500 µM] using the potassium

ferricyaninde reduction method(figure 4.28)


Page 272

Figure 4.28. Reducing power of amino acid analogues of 3-chloro-1-(10-methoxy-5H- dibenz[b,f]azepine-5-yl)propan-1-one containing aminophenols and substituted aminophenols and standards. Each value represents the mean ± SD (n=3)

Human LDL oxidation assay

The antioxidant activity of 3-chloro-1-(10-methoxy-5H-dibenz[b,f]azepine-5-

yl)propan-1-one (XXVIII) containing aminophenols and substituted aminophenols

(XXXX-XXXXIV) against human LDL oxidation with different concentrations (10 µM

and 25 µM) is shown in the figure 4.29.


Page 273

Figure 4.29. Antioxidant activity(%) of 3-chloro-1-(10- methoxy-5H-dibenz[b,f]azepine-5- yl)propan-1-one and its aminophenols and substituted aminophenols analogues on human LDL oxidation at different concentrations ( 10, and 25 µM/mL of LDL). Values represent means ± SD (n=3). From the antioxidant studies, all the aminophenols and substituted aminophenols exhibits

better antioxidant activity compared to the standards, AA and BHA. All the five

synthesized analogues exhibits even better antioxidant activity than the standards. Here,

the combined effects of OCH3 group present in the 3-chloro-1-(10-methoxy-5H-

dibenz[b,f]azepine-5-yl)propan-1-one and the presence of both phenolic OH and OCH3

increses the activity better than the standards. One surprising aspect of our result is that

the electron with drawing NO2 group is equally good in increasing antioxidant activity as

compared to –OH and –OCH3 groups which are electron releasing group. The increased

antioxidant activities of synthesized compounds and the standards are as follows.

XXXXIV>XXXXIII>XXXX>XXXXI>XXXXII>AA>BHA


Page 274

Comparison of antioxidant activities of free

aminophenol and substituted aminophenols and their

respective conjugated analogues

A comparative studies on the antioxidant

activities of amino phenols and substituted amino phenols (standards) and their conjugated

analogues by various methods namely free radical scavenging activity by using DPPH free

radical scavenging activity, inhibition of lipid peroxidation in β-carotene linoleate system,

inhibition of human low-density lipoprotein(LDL) oxidation and reducing power assay was

also done. The antioxidant activities of standards and the respective products at higher

concentration including graphical representation are depicted below.

DPPH radical scavenging activity

The comparison of DPPH free radical scavenging activity of the final product

towards aminophenols and substituted aminophenols (standards) were also studied and

showed in the figure 4.30.


Page 275

Figure 4.30. DPPH radical scavenging activity (%) of final products towards the standard aminophenols and substituted aminophenols at 500 µM. Values represent means ± SD (n=3). Inhibition of lipid peroxidation in β-carotene linoleate system

The extent of bleaching of final product towards the aminophenols and substituted

aminophenols (standards) from the hydroperoxide formed from linoelic acid was also

studied. Based on the extent of bleaching of β-carotene, the percentage antioxidant activity

of final product towards the aminophenols and substituted aminophenols (standards) at

higher concentration (25 µM) was calculated and depicted in the figure 4.31.


Page 276

Figure 4.31. Antioxidant activity(%) of final products towards the standard aminophenols and substituted aminophenols at 25 µM. Values represent means ± SD (n=3). Reducing power assay

The newly synthesized compounds (products) towards the aminophenols and

substituted aminophenols (standards) were analyzed for their reducing power at 500 µM

using the potassium ferricyaninde reduction method (figure 4.32)

Figure 4.32. Reducing power of final products towards the standard aminophenols and substituted aminophenols at 500 µM on human LDL oxidation at 25 µM. Values represent means ± SD (n=3).


Page 277

Inhibition of human low-density lipoprotein (LDL) oxidation

The antioxidant capacities for the inhibition of human LDL oxidation of final

product towards the aminophenols and substituted aminophenols (standards) at higher

concentrations (25 µM) were studied. The effect of compounds on LDL oxidation is

showed in the figure 4.33.

Figure 4.33. Antioxidant activity(%) of final products towards the standard aminophenols and substituted aminophenols at 25 µM on human LDL oxidation. Values represent means ± SD (n=3).

Chapter 4 References

Page 278

Reference

1. L. J. Kricka, and A. Ledwith, Chem. Rev., 1974, 74, 101.

2. A. Hempel, N. Camerman, A. Camerman, and D. Mastropaolo,. Acta Cryst., 2005, E61, o1313.

3. B. Nagaraja, H. S. Yathirajana and D. E. Lynch, Acta Cryst., 2005, E61, o1757.

4. Milanese, World Intellectual Property Organization Patent., 2005, No., 118550A1.

5. C. Parenky and R. Chaturvedi, U.S. Patent, 2007, No. 0032647 A1.

6. P. C. Fuenfschilling, W. Zaugg, U. Beutler, D. Kaufmann, O. Lohse, J. P. Mutz, U. Ouken, J. Louis Reber, and D. Shenton. Org. Pro. Res and Dev, 2005, 9, 272.

7. Vogel’s Text Book of Practical Organic Chemistry, Longman. UK 5th Edtn 1989.

8. W.L.F. Armarego, D.D Perrin, Purification of laboratory chemicals. Butterworth-Heinemann: Oxford,4th Edn,1996

9. Organicum, Practical Hand book of Organic chemistry. Ongley, P.A.Ed., Engl. Transl.Hazzard, B.J. Addison,Wesley, Massachusetts, 1973.

10. Reagents for Organic Synthesis, Fieser and L.F, Fieser, M.Ed,; Wiley: New Yark. 1967.

11. M. Tanaka, C.W. Huie, Y. Nagashima, ad T. Taguchi. Nippon Susian Gakkaishie., 1999, 54, 1409.

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Chapter - 4shodhganga.inflibnet.ac.in/bitstream/10603/45072/7/chapter 4.pdf · -Bill Cosby Synthesis, characterization and evaluation of antioxidant activities of 3-chloro-1-(10-methoxy-