48Review of literature

Azapeptides are formed by the replacement of the Cα of one or more amino acid

residues with a nitrogen atom are promising peptidomimetic compounds. Azaamino acids

impart a unique conformational property to peptide structure because of the loss of chirality

and reduction of the flexibility of the parent linear peptide (Proulx et al., 2011).

H2N

R1

O

NH

R2

COOH

H2N

R1

O

NH

N

R2

COOH

Peptide

Azapeptide

The peculiar conformational properties make azaamino acids an attractive tool for

drug design based on specific secondary structure in peptides and proteins. One of the

advantages of azapeptides is their unproblematic synthesis allowing retention of the side

chain in the proteinogenic amino acid. The presence of an azaamino acid residue may

increase the biological activity and/or improve the pharmacokinetic properties of the parent

peptide (Zega, 2004).

De novo cyclic pseudopeptides composed of α-amino and azaamino acids were

designed with the aim to obtain potential new antimicrobial agents. Compound (58) exhibited

broad spectrum of antibacterial activities (Laurencin M et al., 2012).

Trp-azaLeu-Lys-azaLys-Trp-azaLeu58

48Peptidomimetic analogs of the peptide RRASVA (Arg-Arg-Alanine-Serine-Val-Ala),

RRASVazaβ3A , RRASazaβ3VA , RRazaβ3ASVA , Razaβ3RASVA , azaβ3RRASVA known

as the “minimal substrate” of the catalytic subunit of the cAMP-dependent protein kinase

(PKA), were synthesized by consecutive replacement of natural amino acids by their aza-β3

analogs. It was found that the interaction of these peptidomimetics with the enzyme active

center was sensitive to the location of the backbone modification (Ksenija Kisseljova et al.,

2010).

H2N

R1

O

OH N

O

OHH2N

R1

amino acid aza-beta 3 amino acid

A series of azapeptides as potential inhibitors of cysteine proteases were synthesized..

The majority of synthesized azapeptides shows high inhibitory potency toward the

investigated cysteine proteases, papain, cathepsin B, and cathepsin K. Z-Arg-Leu-Val-

azaGly-Ile-Val-OMe was proved to be a highly potent and selective inhibitor of cathepsin B

(Wieczerzak et al., 2002).

Azapeptide-based inhibitor(59) designed against the Hepatitis C virus (HCV) NS3

serine protease exhibited predominantly non-acylating, competitive inhibition (Zhang et al.,

2002)

NH

N

O

Ac-Asp-Thr-Glu-Asp-Val-Val-Pro

59

48Certain aza analogue (60) of functionalized amino acids exhibited significant

anticonvulsant activity in the maximal electroshock seizure test, but most are less potent than

their amino acid counterparts (Andrukar et al., 2001)

O

NH

N

O

HN

SN

60

Insertion of azaglycine instead of glycine in the cell adhesion motif Arg-Gly-Asp

(RGD) (61) demonstrated that both activity and selectivity can be influenced through the

substitution pattern of the azabuilding block (Gibson et al., 1999)

H2N

NH

HN

O

NN

R2 O

HN

COOH

O

NH2

R1

spacer

61

Aza analogues of known potent growth hormone secretagogues (3) with possible

applications including treatment of burns, Turners syndrome, sleep enhancement and

reduction of some age–related effects, were synthesized and their biological potencies were

measured. Compound (62) showed good results (Hansen et al., 1999)

48

NNH

O

H2N

O

NN S

O

O62

3-amino quinazolin-4-one nucleus has attracted the attention of medicinal chemists

with a wide range of pharmacalogical activities. It is also considered as topographically

constrained as one of the aromatic α-aza-amino acids (Ines Torriniet al., 1999).

N

N

O

NH2

Aza amino acid nature

3-amino-quinazolin-4-one

Construction of small molecule mimics of biological structures is a key contribution

organic chemistry can make to the discovery of new pharmaceuticals with wide range of

biological activities. An update on pharmacologically active compounds with

3-aminoquinazolinone template is discussed.

Antimicrobial activity

Abdul Jabar et al reported the synthesis of 2-(2-methyl-4-oxoquinazolin-3(4H)-

ylamino)-N'-(benzylidene) acetohydrazides. These synthesized compounds showed moderate

to good antibacterial activity. Compounds (63a) and (63d) were found active aginst E. Coli ,

S. Aureus and P.mirabilis (Abdul Jabar et al., 2012).

48

N

N

O

HN

O

NH

N

R

a. R=OHb. R= NO2c. R=Brd. R=Cl63

Some 3- amino quinazolinone derivatives were reported for their synthesis and

antimicrobial activity and among them, compound (64) showed good activity against C.

albicans and A. niger (F. Hassanzadeh et al., 2012)

N

N

HN

O

Cl

Cl

BrO

64

Ganguly and coworkers reported the synthesis of 3-[(E)-(furan-3-ylmethylidene)amino]-2-

phenylquinazolin-4(3H)-one (65), 3-(methylidene amino) -2-phenylquinazolin-4(3H)-one

(66) and 2-phenyl-3-{(E)-[(2E)-3-phenylprop-2-en-1-ylidene]amino}quinazolin-4(3H)-one

(67) . These compounds showed potent antibacterial activity against S. aureus and B. subtilis

and E. coli (Subarna Ganguly et al., 2012).

N

N

O

N

N

O

N

O

NCH2

65 66

48

N

N

O

N

67

A series of 2-Benzyl-3-{4-[N-(3-substituted -1,5-dihydropyrazole-4-

yliene)hydrazino]phenyl}-3H-quinazoline-4-one derivatives were synthesized and screened

for their antibacterial and antifungal activity against pathogenic bacteria and pathogenic

fungus. Antimicrobial results indicated that compound (68) showed significant activity

(Hurmathunnisa sulthan etal., 2012)

N

N

O

HN

NH

N

N

N

CH3

R

CH3

C6H5

68

Siddappa et al reported the synthesis and antimicrobial activity of 3-[(2-hydroxy-6-

methoxyquinolin-3-ylmethylene) amino]-2-methyl-3H-quinazoline-4-one (69) against

selected fungi and bacteria (Siddappa et al., 2012)

N

N

O

N

CH3

N

H

OH

OCH3

69

N-(4-Oxo-2-substituted phenylquinazolin-3(4H)-yl)-2-[(5-aryl-1,3,4-

oxadiazol-2-yl)sulfanyl] acetamides were synthesized and antibacterial and

48antioxidant activities were performed by agar diffusion and DPPH method.

Compounds (70a) and (70b) showed good antibacterial and moderate antioxidant activities

(Rajasekaran S and Gopalkrishna Rao., 2012).

N

N

O

NH

OS Het

NN

ON

NN

O

Het

a.

b.70

Osman et al reported the synthesis of 4-(4–oxo–3,3–(dibenzamido)–3,4–

dihydroquinazolin –2–yl)phenyl– 4– methyl benzenesulfonate(71) and 4-(3,3–diacetamido–

4–oxo–3,4–dihydro quinazolin–2–yl) phenyl-4- methylbenzenesulfonate. The prepared

compounds exhibited good antimicrobial activities (Osman et al.,2012).

N

N

O

Ar

CN

O

Ph

CO Ph

N

N

O

ArO

CN

C

cH3

CH3

O

O SO

O

Ar =

71 72

Ethyl-2-((2-methyl-4-oxoquinazolin-3(4H)-yl)diazenyl)-3-oxobutanoate was synthesized and

reported for in vitro antimicrobial activity against a number of microorganisms

(Staphylococcus aurous, E.coli, Proteus vulgaris, Pseudomonas, and Klebsiella) and two

fungal Aspergillus niger and Candida albicans (Ahmed A. H. Al-Amiery et al., 2010).

48

N

N

O

N

N

O

O

O

73

Deepthi Kohli and coworkers synthesized N-(4-oxo-2-phenylquinazolinyl)-2-

phenoxy acetamide derivatives and evaluated for their antibacterial activity by cup plate

method by measuring inhibition zone. Compounds (74a) and (74b) showed more potent

antibacterial activity than the standard drug ampicillin(Deepthi Kohli et al.,2009)

N

N

HN

O

O

O

R

a. R = Clb. R = NO2

74

Synthesis and antimicrobial activities of some novel substituted 2-(4,5-dihydro

imidazolyl)-N-(4-oxo-phenylquinazolin-3(4H)-yl)-acetamides were reported. Compound(74)

exhibited potent activity ( Raghavendra M et al.,2007)

N

N

OHN

O

NH

N

75

Analgesic and antiinflammatory activities

48 Mariappan et al synthesized and reported anti-inflammatory activity of 2-phenyl-

3-(propylideneamino) quinazolin-4(3H)-one (Mariappan et al., 2011)

N

N

O

N

76

A series of novel 2-benzylamino-3-substituted quinazolin-4(3H)-ones

have been synthesized by treating 3- amino-2-benzylamino quinazolin-4(3H)-one,

with different aldehydes and ketones. The title compounds were investigated for

analgesic and anti-inflammatory activities. Compounds (77a), (77b) and (77c) exhibited

significant analgesic activity (Alagaraswamy et al., 2003)

N

N

O

NHCH2C6H5

N Ra. R =

b. R =

c. R =77

Alagarsamy et al synthesized some novel 2-phenyl-3-substituted

quinazolin-4(3H) ones derivatives and evaluated them for analgesic and anti-

inflammatory activity compared with Diclofenac sodium as standard drug. 1-(4-oxo-2-

phenylquinazolin-3(4H)-yl)-3-phenylthiourea (78) exhibited potent activities (Alagaraswamy

et al ., 2002)

48

N

N

OHN

SNH

78

Antioxidant activity

A series of 3-substituted Schiff bases of quinazoline-2,4-dione have been

synthesized from the reactions of quinazoline-2,4-dione with substituted aromatic

aldehydes. DPPH inhibition potential and FRAP (Ferric reducing antioxidant

power) assay were carried out using in vitro models. Compounds 79(a-e) showed a dose

dependent antioxidant activities (Zaranappa et al 2012).

NH

N

O

O

N R

a. R = 2-OHb. R = 4-OHc. R = 4-Cld. R = 4-Fe. R = 4-OCH379

A series of novel glutamine linked 2,3-disubstituted quinazolinone conjugates

was synthesized from methyl anthranilate and different substituted acids and acid chlorides..

When tested for their antioxidant activity, showed potent radical scavenging activity against

2,2-diphenyl-1-picrylhydrazyl, hydroxyl, nitric oxide, and superoxide radical scavenging

48assays. These results suggest that these quinazolinone analogs could be considered as useful

templates for future development to obtain more potent antioxidant agents (Prashanth et al.,

2012).

A series of new compounds were prepared by condensation reaction of

3-amino-2-methyl- 4(3H)quinazolinone (AMQ) with different substituted aromatic

aldehydes in methanol. They were also evaluated for their antioxidant activities and the

results suggest that few of the synthesized compounds,(80a), (80b), (80c) and (80d) showed

better scavenging activity (Hosakere. D. Revanasiddappa et al., 2010)

N

N

CH3

O

C

HR

a. R = 4-OHb. R = 4-OH,3-OCH3c. R = 4-NO280

Al Omar reported the synthesis of quinazolinone derivative, 1-(6-iodo-4-oxo-2-

propylquinazolin-3(4H)-yl) urea (81) and screened for their antioxidant activity (Al Omar et

al.,2006)

N

N

O

HN

O

NH2I

81

Anticancer activity

3-(2-chloro benzylideneamine)-2-(furan-2-yl) quinazoline-4(3H)-one was found

to be the most active candidate of the series at five dose level screening against Ovarian

OVCAR-4 and Non-small cell lung cancer NCI-H522. Rational approach and QSAR

48techniques enabled the understanding of the pharmacophoric requirement (Malleshappa N.

Noolvi et al., 2011)

N

N

O

HN

S

I

Cl

82

The synthesis of some new 2-thieno-4(3H)-quinazolinone derivatives and their

biological evaluation as antitumor agents using the National Cancer Institute (NCI) disease

oriented antitumor screen protocol were investigated. Compound 2-(2-thieno)-6-iodo-3-

phenylamino-3,4-dihydro-quina-zolin-4-one (83) was proved to be the most active members

in this study (Abdulrahman M. Al-Obaid et al., 2009)

N

N

O

NHI Ph

S

83

Other activities

A series of novel 2-[4-substituted-piperazinyl-methyl]-3-[N-isonicotinamide-yl]-

quinazoline- 4-ones were designed, synthesized, characterized and evaluated for in

vitro antitubercular, activity. Compounds (84a) and (84b) exhibited excellent antitubercular

activity against Mycobacterium tuberculosis H37Rv (Myangar et al., 2012)

48

N

N

O

CH2

HN C N

O

N N

R a. R = 2-Cl,3-Clb. R = 4-OCH3

84

A series of 3-(benzylideneamino)-2-phenyl quinazoline-4(3H) ones was synthesized

and were investigated for their aniviral and anticancer activities. Compound (85) was found

to inhibit viral replication of para influenza-3virus, reovirus-1, Sindbis virus, Coxsackie virus

B4, Punta Toro virus in Vitro cell cultures (S.K.Krishnan et al., 2011)

N

N

O

N

OCH3

OCH3

85

Saravanan et al reported the synthesis and antiviral activity of a series of 2-phenyl

3-substituted quinazolin-4(3H)ones. Compound (86) displayed potent antiviral activity

(Saravanan et al.,2010)

N

N

O

HN NH

S

O

O

NH

O

86

Schiff bases of 3-amino-6,8-dibromo-2-phenyl-quinazolin-4-(3H)-ones with various

substituted aldehydes were synthesized and evaluated for their anticonvulsant activity on

48albino mice by maximal electroshock method using phenytoin as a standard. The compound

(9l) bearing a cinnamyl function displays a very high activity (Paneerselvam et al., 2010).

N

N

O

Br

C6H5

NBr CH

87

Ponnilavarasan ilangovan et al synthesized a series of 3-N'(benzylidene

semicarbazone)-2-phenyl 3H-quinazolin-4-one and evaluated for anticonvulsant and

neurotoxicity. Compound (Ponnilavarasan ilangovan et al., 2010)

N

NNH C

O O

NHC

88

Kashawa and coworkers synthesized several new 1-(4-substituted-phenyl)-3-(4-

oxo-2-phenyl/ethyl-4H-quinazolin-3-yl)-urea and screened for CNS depressant

activity by maximal electroshock induced seizures (MES) and subcutaneous

pentylenetetrazole scPTZ) induced seizure models in mice and they found that

compounds 89(a-d) were found to be active in both methods (Kashawa et al., 2009).

48

N

N

O

NH NH

O

R

a. R=Hb. R=Clc. R=CH3d. R=OCH389

Azapeptides, formed by replacing the C(α) of amino acid residues by nitrogen, are

promising peptidomimetics. In biologically active peptide analogs, the aza-substitution has

led to enhanced activity and selectivity as well as improved properties. Azaamino acids

impart an unique conformational property to peptide structures because of the loss of chirality

and reduction of the flexibility. 3-amino-2-phenyl-quinazolin-4(3H)-one is a very interesting

moeity with potential quinazolinone nucleus as a well-established peptiomimetic scaffold and

also it can be considered as a constrained azaamino acid. This rational approach enabled the

understanding of 3-amino-2-phenyl quinazolin-4-one nucleus as a pharmacophoric

requirement. Thus a series of novel quinazolinyl azapeptide derivatives were synthesized and

evaluated for biological activities.

48Experimental Methodology

Chemistry

Materials: As described in section_ of chapter_

Methods:

Synthesis of 2-phenyl-4H-benzo[1,3]oxazin-4-one(I): The procedure followed was

discussed in section_ of chapter_.

Synthesis of 2-phenyl-3-amino-quinazilon-4-one (II): A mixture of 2-phenyl-4H-benzo

[1,3]oxazin-4-one (I) (0.01mol) was taken in round bottom flask and treated with 99%

hydrazine hydrate in ethanol and refluxed for 3hrs at 60-900C. The resulting solution was

poured into the crushed ice. A white precipitated was obtained which was recrystallized with

ethanol and dried (Sridhar et al., 2009).Yield:75%, M.P:2210C (Lit.220-222 0C), UV(λmax)

nm:309,243 ; IR (KBr)cm-1: 3350(NH2), 3050(Ar C-H), 1695(ring C=O), 1340(C-N),

1020(N-N), 810(Ar C-H)

General Method of Synthesis of N-Benzylidene glycines, III (g-o): Synthesis of 2-

benzylidene amino acetic acid (IIIg): In a 50 ml round bottom flask attached with a reflux

condenser benzaldehyde /substituted benzaldehyde (0.01mol), glycine (0.01 mol) and sodium

hydroxide (0.1 mol) were mixed. 20 ml of ethanol was added to the mixture and the final

mixture was refluxed for 3 hours. The mixture was cooled, neutralized with dilute HCl

solution and filtered. Product obtained were washed with cold ethanol and dried to give IIIa

(Azzouz et al., 2010); Yield:65%, M.P:1940C (Lit.1950C),UV(λmax) nm:282, 247; IR(KBr)cm-

1: 3250(COOH),3110(Ar C-H),2985 (aliphatic C-H) 1645(C=O),1520 (C=N),710(Ar C-H).

Similar procedure was followed for the synthesis of IIIh-IIIo using different substituted

benzaldehydes.

482-(2-hydroxybenzylideneamino)acetic acid(IIIh): prepared by following the general

procedure above using 0.01mol 2-hydroxy benzaldehyde ,0.01 mol glycine and 0.1 mol

sodium hydroxide, Yield:66.5%, M.P:134-1360C; UV(λmax) nm:292,271; IR(KBr)cm-1:

3300(OH), 3110 (Ar C-H), 3050(COOH), 2887 (CH2) ,1620(C=O), 1590 (C=N), 780(Ar C-

H).

2-(4-hydroxybenzylideneamino)acetic acid(IIIi): prepared by following the general

procedure above using 0.01mol 4-hydroxy benzaldehyde ,0.01 mol glycine and 0.1 mol

sodium hydroxide,Yield:56.2%, M.P:121-1220C, UV(λmax) nm:290,276; IR(KBr)cm-

1:3211(OH),3015 (Ar C- H), 3009(COOH), 2879 (CH2) , 1610(C=O),1540 (C=N), 880(Ar C-

H).

2-(4-chlorobenzylideneamino)acetic acid(IIIj): prepared by following the general

procedure above using 0.01mol 4-chloro benzaldehyde ,0.01 mol glycine and 0.1 mol sodium

hydroxide,Yield:68.8%, M.P:1860C; UV(λmax) nm:322,201; IR(KBr)cm-1: 3204(OH),

3016(Ar C-H), 2952(COOH), 2789 (CH2) , 1600(C=O), 1590 (C=N), 813(C-Cl), 782(Ar C-

H).

2-(4-fluorobenzylideneamino)acetic acid(IIIk): prepared by following the general

procedure above using 0.01mol 4-fluoro benzaldehyde ,0.01 mol glycine and 0.1 mol sodium

hydroxide,Yield:65.3%,M.P:166-1680CUV(λmax) nm:312,219;IR(KBr)cm-1: 3224(OH), 3118

(Ar C-H),3062(COOH),2986 (CH2) ,1609(C=O),1537 (C=N),1113(C-F)882(Ar C-H).

2-(4-methoxybenzylideneamino)acetic acid(IIIl): prepared by following the general

procedure above using 0.01mol 4-methoxybenzaldehyde ,0.01 mol glycine and 0.1 mol

sodium hydroxide,Yield:75%, M.P:1560C; UV(λmax) nm:302,261; IR(KBr)cm-1: 3412(OH),

3163(Ar C-H), 3005(COOH), 2920(CH3), 2899 (CH2) , 1628(C=O), 1410 (C=N), 713(Ar C-

H).

482-(4-hydroxy-3-methoxybenzylideneamino)acetic acid(IIIm): prepared by following the

general procedure above using 0.01mol 4-hydroxy-3-methoxybenzaldehyde ,0.01 mol

glycine and 0.1 mol sodium hydroxide,Yield:79.5%,M.P:176-1740C UV(λmax)

nm:342,291;IR(KBr) cm-1:3249(OH),3116(ArC-

H),3052(COOH),2989(CH3),2756(CH2),1600(C=O),1490 (C=N), 762(Ar C-H).

2-(4-methylbenzylideneamino)acetic acid(IIIn): prepared by following the general

procedure above using 0.01mol 4-methylbenzaldehyde ,0.01 mol glycine and 0.1 mol sodium

hydroxide,Yield:59.8%,M.P:143-1410CUV(λmax) nm:299,285;IR(KBr)cm-1:3390(OH),31688

(ArC-H), 3112(COOH),2922(CH3),2656(CH2),1637(C=O),1488 (C=N),862(Ar C-H).

2-[(4-dimethylamino)benzylideneamino)]acetic acid(IIIo): prepared by following the

general procedure above using 0.01mol 4-dimethylaminobenzaldehyde ,0.01 mol glycine and

0.1 mol sodiumhydroxide,Yield:89.5%,M.P:2110C,UV(λmax)nm:442;IR(KBr)cm-1:3249(OH),

3116 (ArC- H),3052(COOH),2962(CH3),2756(CH2),1600(C=O),1490 (C=N), 762(Ar C-H).

General method for synthesis of quinazolinyl azapeptide derivatives (B1-B15): Synthesis

of 2-acetamido-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)acetamide (B1): Solution

of N-acetyl glycine, IIIa (1.5 mmol) in 30 ml of DMF was treated sequentially with HOBT

(16.30 mmol), DCC (1.63 mmol), and 1.95 mmol of 2-phenyl-3-amino-quinazilon-4-one (II).

The suspension was stirred for 15 min at 4-50C, TEA (2.7 mmol) was then added, and the

mixture was again stirred for 24 hours at room temperature. The reaction was monitored by

TLC and then quenched by pouring the mixture into 30 ml of ethyl acetate. It was extracted

with 3 portions of 1 N aqueous HCl, 2 portions of water, 1 portion of brine and 1 portion of

saturated aqueous NaHCO3. The solution was then dried over Na2SO4, filtered and

concentrated (Augeri et al., 1998). The residue was purified by ethyl acetate:hexane (7:3)

mixture to provide product that solidified upon standing. After complete drying, melting

48points were determined and TLC was run in moblie phase [ethyl acetate: chloroform:

methanol (1:2:1)]

Scheme-1: Scheme for synthesis of quinazolinyl azapeptide derivatives (B1-B15)

2-acetamido-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)acetamide(B1):white

amorphous solid; Rf :0.88(ethyl acetate: chloroform: methanol; 1:2:1); UV (λmax)

(nm):295,239; IR(KBr)cm-1 3322.02 (NH), 3077.9(Ar-H), 2927.48(CH3), 2847.92 (CH2),

481771.08(ringC=O), 1657.6(C=O,amide), 1560.75(C=O,acetyl), 1239(C-N); 1H-NMR

(300MHz,CDCl3) δ=2.1(s, 3H, CH3), 4.25( d,J=3.8Hz 2H,CH2 ),6.9-8.2(m,11H,(9Ar-H,2NH);

MS(EI+): m/z(%)338.41(19) [M+H]+,221(100)[m/z,phenylquinazolinone], 107(60) [m/z,CN-

C6H5),237.48(43)[m/z,3-amino quinazolinone], 78.91(41)(m/z,benzene), 294(16)[M-CO

CH3]. Anal.Calc:C, 64.28; H, 4.79;N,16.66.Found: C, 63. 92 ;H, 4.66 ; N, 16.48.

2-acetamido-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)-3-sulfanyl propanamide

(B2): 1.5 mmol of acetyl cysteine(IIIb) and 1.95mmol of II were used ; white amorphous

solid; Rf :0.63 (ethyl acetate: chloroform,:methanol; 1:1:2) UV (λmax) (nm) :295,232; IR

(KBr) cm-1: 3401.5(NH), 3188.76(Ar-H),3049.03(aliphatic C-H), 2611.12(S-H),1684.73(ring

C=O), 1643.5(C=O,amide), 1583.28 (C=O, acetyl),1232.33(C-N),1079.49(N-

N)899,702.46(ArC-H) 757.88(C-S); 1H-NMR (300MHz, CDCl3, δ) 1.6(t, J=12Hz,1H, SH),

1.9( s,3H,CH3 ), 4.5(t,J=12Hz,3H,CH,CH2) ,7.1-8.4(m,11H, (9Ar-H,2NH),

MS(EI+):m/z(%)382(36)[M+H]+, 222.83 (100) [m/z of 2-phenyl quinazolinone], 78.81 (64)

(m/z of benzene), 238.86 (43)(m/z of 3-amino quinazolinone], 296.77 (32)[M-COCH3],

106.63(26) (m/z of CN-C6H5). Anal.Calc.C, 59.67; H, 4.74; N, 14.65.Found: C, 59.58; H,

4.58; N, 14.59.

2-acetamido-4-(methylsulfanyl)-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-

yl)butanamide (B3): 1.5 mmol of acetyl methionine (IIIc) and 1.95mmol of II were used ;

creamy white crystalline solid; Rf :0.76(ethyl acetate: chloroform: methanol; 1:2:1); UV

(λmax) (nm) :299,226; IR(KBr) cm-1:3456.7(NH), 3281.6(Ar-H),2993.55(aliphatic C-H),

1673.8(ring C=O), 1613.45 (C=O ,amide), 1532.8 (C=O,acetyl), 1222.3(C-N), 1194.9(N-N),

792.6(ArC-H), 788.2(C-S); 1H-NMR (300MHz,CDCl3) δ=2.5(s, 6H, CH3), 3.2(q,J=13.4Hz,

2H,CH2), 3.4(t,J=5.6Hz,2H,CH2), 4.25(q, J=3.8Hz ,1H,CH),6.9-8.2(m,11H,(9Ar-H,2NH).

48N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)-2-(phenylformamido)acetamide(B4): 1.5

mmol of benzoyl glycine (IIId) and 1.95mmol of II were used; creamy amorphous

solid;Rf :0.9; UV (λmax) (nm) :294.5,224 ; IR(KBr) cm-1: :3556.7(NH), 3216(Ar-

H),2935.5(aliphatic C-H), 1713.8(ring C=O), 1634.5 (C=O,amide), 1632.8 (C=O, benzoyl),

1332.8(C-N),1019.4(N-N), 892(ArC-H); 1H-NMR (300MHz,CDCl3) δ=

4.1( d,J=3.8Hz,2H,CH2 ), 6.9-8.2(m,16H,(14Ar-H, 2NH) Anal .Calc. C,69.34; H,4.55;

N,14.06.Found: C,68.99; H,4.50 ; N,13.99.

N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)-2-(phenylformamido)propanemide(B5):

1.5 mmol of benzoyl alanine (IIIe) and 1.95mmol of II were used; creamy amorphous solid;

Rf : 0.66; UV (λmax) (nm) :289,247 ; IR(KBr) cm-1: 3446.56(NH), 3016(Ar-H),

2959.4(aliphatic C-H), 1738(ringC=O), 1645 (C=Oamide), 1602 (C=O benzoyl), 1328(C-N),

1214(N-N), 699(Ar C-H)

4-methyl-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)-2-(phenylformamido)

pentanamide (B6) : 1.5 mmol of benzoyl leucine (IIIf) and 1.95mmol of II were used.; white

amorphous solid; Rf :0.83; UV (λmax) (nm) :293.3; IR(KBr) cm-1: :3346.6(NH), 3116(Ar-

H),2994.4 (broad peak, aliphatic C-H), 1701(ring C=O), 1622 (C=O,amide), 1620

(C=O,benzoyl), 1283(C-N),1114(N-N),899(ArC-H) 1H-NMR (300MHz, CDCl3, δ) 1.6(t,

J=12Hz,1H, SH), 1.1( d, J= 2.5Hz,6H,CH3 ), 2.2(m, 1H,CH), 4.5(t,J=12Hz,3H,CH,CH2) ,7.1-

8.4(m,15H, (13Ar-H,2NH)

2-(benzylideneamino)-N-(4-oxo-2-phenylquinazolin-3(4H)-yl)acetamide(B7): 1.5 mmol of

2-benzylidene amino acetic acid (IIIg) and 1.95mmol of II were used; white amorphous

solid; Rf : 0.81; UV (λmax) (nm) :302,201 ; IR(KBr) cm-1: 3546(NH), 3016(Ar-H),2904

(aliphatic C-H), 1729(ring C=O), 1652 (C=O,amide),1585(CH=N), 1263(C-N),1217(N-

N),809 (ArC-H) 1H-NMR (300MHz,CDCl3) δ= 4.5(s,2H,CH2 ), 7.0-7.8(m,15H,(14Ar-H,

481NH), 7.9(s,1H, N=CH); Anal.Calc: C,72.24; H,4.74;N,14.65.Found:C,71.96;H, 4.71;

N,14.46.

2-[(2-hydroxybenzylidene)amino]-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-

yl)acetamide (B8): 1.5 mmol of 2-(2-hydroxybenzylideneamino)acetic acid (IIIh) and

1.95mmol of II were used creamy white crystals; Rf :0.76; UV (λmax) (nm) :300,221 ; IR(KBr)

cm-1: :3556 (NH),3190(O-H),3006.33(Ar-H),2974(aliphatic C-H), 1709(ring C=O), 1632.8

(C=O, amide), 1545 (CH=N),1463(C-N),1017(N-N),763(ArC-H) Anal. Calc. C, 69.34; H,

4.55; N, 14.06 Found: C, 68.99; H, 4.49; N, 13.94

2-[(4-hydroxybenzylidene)amino)]-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)

acetamide (B9): 1.5 mmol of 2-(4-hydroxybenzylideneamino)acetic acid (IIIi) and 1.95mmol

of II were used; buff crystals; Rf :0.9; UV (λmax) (nm) :304,224 ; IR(KBr) cm-1: :3350.64

(NH), 3330.69(O-H),3195.2(Ar-H),2979.6,2932.8 (aliphatic C-H), 1729.48(ring C=O),

1657.6 (C=O,amide), 1547.39(CH=N), 1426.57(C-N),1031.58(N-N), 633 (Ar C-H); 1H-NMR

(300 MHz,CDCl3) δ=4.4 (s,2H,CH2), 5.1(s,1H,OH),6.5-7.8(m,13H,(11Ar-H,2NH),8.3

(s,1H,N= CH); MS(EI+):m/z(%)399.71(10)[M+H]+, 224.1(40) [m/z of phenyl

quinazolinone], 77.08 (25)(m/z of benzene), 237.08 (16)(m/z of 3-amino quinazolinone),

382(8), 294.9 (4)[M-benzylidene], 106.63(26)[ m/z, CN-C6H5).

Anal.Calc.forC,69.34 ;H,4.55;N,14.06. Found: C,69.51;H, 4.51; N,13.96

2-[(4-chlorolbenzylidene)amino)]-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)

acetamide (B10): 1.5 mmol of 2-(4-chlorobenzylideneamino)acetic acid (IIIj) and 1.95mmol

of II were used; shiny white crystals; Rf :0.83; UV (λmax) (nm) :303,220 ; IR(KBr) cm-

1:3401.5(NH),3188.76(Ar-H), 3049.03(aliphaticC-H), 1684.73 (ring C=O),

1643.5(C=O,amide), 1583(CH=N), 1232.33(C-N), 1079(N-N), 757(ArC-H),7 02.48(C-Cl);

Anal. Calc: C, 66.27; H, 4.11; N, 13.44. Found: C, 65.68; H, 4.08; N, 13.01

482-[(4-fluorobenzylidene)amino]-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)

acetamide (B11): 1.5 mmol of 2-(4-fluorobenzylideneamino)acetic acid (IIIk) and 1.95mmol

of II were used; light yellow crystals; Rf :0.75; UV (λmax) (nm) :343,290 ; IR(KBr) cm-1:

3415.77(NH) , 3176.9(Ar-H), 2903(aliphatic-H), 1673 (ring C=O), 1635 (C=O

amide),1539(CH=N),1293(C-N),1179 (C-F), 757(Ar C-H). MS(EI+):m/z (%) 401.03 (25)

[M+],223 (100) [m/z,phenyl quinazolinone], 104.98 (68)[ m/z of CN-C6H5), 77.08 (50)(m/z

benzene), 296.7 (4)[M+2H-benzylidene].

2-[(4-methoxybenzylidene)amino]-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)

acetamide (B12): 1.5 mmol of 2-(4-methoxybenzylideneamino)acetic acid (IIIl) and

1.95mmol of II were used; light brown crystals; Rf :0.75; UV (λmax) (nm) :333,290 ; IR(KBr)

cm-1: 3415.77 (NH),3176.9(Ar-H),2997,2903(aliphatic C-H), 1703 (ring C=O), 1658

(C=O,amide), 1579 (CH=N), 1293(C-N),778(ArC-H); 1H-NMR(300MHz,CDCl3 δ)

3.3(s,3H,OCH3),4.4(s,2H, CH2), 5.4(s,1H,OH),6.5-7.8(m,14H,(13Ar-

H,1NH),8.8(s,1H,N=CH); Anal. Calc. C, 69.88; H, 4.89 N, 13.58.Found: C, 69.33; H, 4.86;

N, 13.09

2-[(4-hydroxy-3-methoxybenzylidene)amino]-N-(4-oxo-2-phenyl-3,4-dihydro

quinazolin-3-yl) acetamide(B13): 1.5 mmol of 2-(4-hydroxy-3-

methoxybenzylideneamino)acetic acid (IIIm) and 1.95mmol of II were used; white crystals;

Rf :0.81; UV (λmax) (nm) :290,212 ; IR(KBr) cm-1: 3529(NH), 3301(O-H),3077(Ar-H),

2980&2935(aliphatic C-H), 1741 (ring C=O), 1666(C=O, amide), 1543(CH=N), 1212(C-O),

694(Ar C-H). 1H-NMR (300MHz,CDCl3,δ) 3.3(s,3H,CH3) 4.4 (s,2H,CH2), 5.3(s,1H,OH) 6.5-

7.8(m,13H,(12Ar-H,1NH), 8.1 (s,1H,N=CH);MS(EI+):m/z(%) 429.79 (42)[M+], 223.53

(100) [m/z of phenyl quinazolinone], 106. 63(64)[m/z,CN-C6H5), 78.67 (28) (m/z of

48benzene), 294.9 (16) Anal. Calc. for: C, 67.28; H, 4.71; N, 13.08. Found: C, 66.66; H, 4.62;

N, 12.96

2-[(4-methyl benzylidene)amino]-N-(4-oxo-2-phenyl-3,4-dihydroquinazolin-3-yl)

acetamide (B14): 1.5 mmol of 2-(4-methylbenzylideneamino)acetic acid (IIIn) and 1.95mmol

of II; cream crystals; yield:53%; M.P:1300C;Rf :0.59; UV (λmax) (nm) :298,229 ;IR(KBr) cm-1:

:3444 (NH),3211(Ar-H), 2991,2835(aliphatic C-H), 1709 (ring C=O), 1644 (C=O amide ),

1603 (CH= N),1211(N-N),794(Ar C-H).

2-[4-(dimethylaminobenzylidene)amino]-N-(4-oxo-2-phenyl-3,4-dihydro quinazolin-3-

yl) acetamide(B15): 1.5 mmol of 2-[(4-dimethylamino)benzylideneamino)]acetic acid (IIIo)

and 1.95mmol of II were used; white crystals; yield:75%; M.P:1830C; Rf =0.81; UV (λmax)

(nm) 290,212 ; IR(KBr) cm-1: 3326.68(NH),3059.68(Ar-H),2929.6,2853.25 (aliphatic C-H),

1745.5 (ring C=O), 1625.25(C=O amide),1441.67(CH=N),1310(C-N),1088.93 (N-

N),639(ArC-H); 1H-NMR(300MHz,CDCl3,δ) 2.6(s,6H,CH3), 4.4(s,2H,CH2), 6.8-7.8(m,

14H, (13Ar-H,1NH), 8.2(s,1H,N=CH); MS (EI+): m/z (%) 426.76 (24)[M+], 224(100) (m/z

of phenyl quinazolinone), 78.67 (28)[m/z of benzene), Anal.Calc: C,64.27; H,4.79 ;

N,16.66 .Found: C,69.4 ; H, 5.11; N,16.30

48

Pharmacological studies

Methods:

Pharmacological studies:

Anti-inflammatory, analgesic, antimicrobial and preliminary screening of cytotoxic

activities were carried out according to the procedures mentioned in section_ of chapter_

In vitro antioxidant studies:

Reduction of DPPH stable free radical, nitric oxide scavenging activity, inhibition of

iron induced lipid peroxidation, superoxide anion scavenging activity, hydroxyl radical

scavenging activity, reducing power assay and CUPRAC (Cupric Reducing Antioxidant

Capacity) were carried out according to the procedures mentioned in section _ of chapter_

Results and discussion

Chemistry

The title compounds were synthesized according to the scheme described ( Scheme

2). 2-Phenyl-4-(3H)-benzoxazin-4-one, I was obtained by the reaction of anthranilic acid

with benzoyl chloride in pyridine. The formation of I was also discussed in previous chapter.

48The subsequent reaction between I and hydrazine hydrate in ethanol afforded 2-phenyl-3-

amino-quinazilon-4-one II in good yield. The appearance of bands due to N-H stretch for

compound II in the IR spectrum, which are not seen in starting material, I and other similar

bands as observed in I, indicate the formation of compound II by the replacement of

hydrazide moiety in place of ring oxygen of compound I with removal of water molecule.

III(a-f)N-benzylidene amino acids III (g-o) were prepared by reacting different

substituted benzaldehydes with glycine which involved nucleophilic substitution followed by

dehydration. IR spectrum of these compounds displayed bands at 3400-3250 cm-1 (COOH),

3300-3110 cm-1 (Ar C-H), 3000-2800 cm-1 (aliphatic C-H), 1660-1645 cm-1 (C=O), 1520-

1450 cm-1 (C=N), 900-710 cm-1 (Ar C-H). Other characteristic bands due to C-Cl, C-F also

were observed with compounds IIIj and IIIk.

The synthesis of title compounds (B1-B15) involved azapeptide bond formation

between the amino group of II with carboxylic group of appropriate N-protected amino acids,

III (a-o) using DCC as coupling reagent in presence of HOBT. The probable mechanism

involved in the formation of title compounds is described in figure _. A series of fifteen

compounds were obtained in good yields. The physical data of the synthesized compounds is

given in table _. The formation of these compounds was supported by the IR spectral data

showing isomethine, CH=N stretching band at 1595-1655 cm-1 and amide band ,NH-C=O at

1620-1670 cm-1 and N-N band at 1000-1060 cm-1 in the IR spectra. Other characteristic

bands in the region 720-790 cm-1correlated to C-S moiety,2550-2660 cm-1 of S-H, C-Cl

around 750-800 cm-1,O-H around 3200-3400 cm-1,aliphatic C-H around 2800-2900 cm-1.

1H-NMR of the compounds in general showed characteristic singlet in the region of δ

8.0-8.8 (NH), multiplets at 6.5-8.0 (Ar-H), singlet at 8.0-8.3(N=CH), doublet at 4.0-5.0(CH2),

singlet at 1.5-3.0(aliphatic CH) singlet at 4.5-5.0 (Ar-OH) and triplet at 2.0-3.0(SH). The

48molecular ion peaks were found at their characteristic m/z at M+, common fragment ions at

234-236(m/z of 3-amino 2-phenyl quinazolinone), 222(m/z of 2-phenyl quinazolinone), 146

(m/z of quinazolinone), 118(m/z of quinazolinone-CO) and 78(m/z of benzene). Elemental

analysis reports added further contribution for the assigned chemical structures and were

within ± 0.4% of the theoretical values. The detailed spectral data obtained for title

compounds is given in section_

Fig _: Plausible mechanism in formation of 2-phenyl-3-amino-quinazilon-4-one (II)

48

Fig _: Plausible mechanism in formation of quinazolinyl azapeptide derivatives (B1-B15)

48

Fig_: General mass fragmentation pattern of the title compounds

48

Fig_: IR spectrum of compound B1

48

Fig_: IR spectrum of compound B2

Fig_: IR spectrum of compound B9

48

Fig_: IR spectrum of compound B10

Fig_: IR spectrum of compound B13

48

Fig_: IR spectrum of compound B15

Fig_: 1H-nmr and mass spectra of compound B1

48

Fig_: 1H-nmr and mass spectra of compound B2

48

Fig_: 1H-nmr and mass spectra of compound B9

48

Fig_: 1H-nmr and mass spectra of compound B13

48

Fig_: 1H-nmr and mass spectra of compound B15

48

Pharmacological activities

Acute toxicity studies:

48Acute toxicity studies showed that the compounds were safe even up to 2000 mg/kg p.o dose

and no mortality or gross behavioral changes were observed in the animals used.

Anti-inflammatory activity:

Anti-inflammatory activity of the synthesized compounds (B1-B15) was evaluated by

carrageenan induced rat paw edema method. The test compounds and standard drug,

diclofenac were evaluated at 100 mg/kg p.o and their effects were measured at 30, 60, 120

and 180 min. All compounds exhibited significant decrease in edema volume at 180 min and

given in table _. Compound B1 bearing N-acetyl glycine moiety exhibited moderate inhibition

of edema of 40.12%.

Compound B2 bearing N-acetyl cysteine in place of N-acetyl glycine was found to be

most active among the series with 61.5% inhibition and is comparable to that of standard at

equidose (66.75%). It is in accordance with the earlier reports on N-acetyl cysteine as a

suppressor of prostaglandin production in monocytes and enhances the action of anti-

inflammatory drugs (Erica Hofferet al., 2002). Compound B3 with acetyl methionine showed

less reduction in paw edema (30.5%) which may be due to the absence of free thiol moiety.

Introduction of benzoyl group in place of acetyl group as in compounds B4, B5 and B6

did not result in good activity. Incorporation of unsaturated aromatic moiety such as

benzylidene ring in place of benzoyl group gave compound B7 which exhibited an activity of

52.73%. Compounds B8 and B9 with phenolic substitutions on the benzylidene ring either at

position 4 or 2 resulted in similar activity to that of compound B7. Methoxy substitution ortho

to the phenolic group, as in compound B13 resulted in increased activity (57.5%). Among the

halogen substituted compounds, compound B10 with 4-chloro exhibited higher activity than

compound B11 with 4-fluoro substitution. Other substitutions such as methyl and diethyl

amino groups did not result in improvement in activity.

48Analgesic activity

Acetic acid induced writhing method:

The compounds were tested for peripheral analgesic activity by acetic acid induced

writhing method at 100 mg/ kg i.p. dose (table 1). Compound B2 with NAC moiety was found

to possess significantly potent analgesic activity by reducing number of writhes by 67.1%,

which is comparable to standard drug, aspirin (63.15%). NAC was reported to possess

analgesic activity, thus the presence of this moiety might have influenced the activity

(L.Borgström et al.,1986).

Compound B9 with phenolic group in addition to quinazolinyl frame work exhibited

64.86% activity. Compounds B4 and B10 with benzoyl moiety and 4-Chloro substituted

benzylidene ring also analgesic good activities of 60.08% and 59.42%.

Analgesic activity by Eddy’s hot plate method:

The compounds tested above were also tested for central analgesic activity by Eddy’s

hot plate method (Table _). Compounds B8 and B9 possessing phenolic substituted

benzylidene functionality at position-4 on benzylidene ring exhibited good increase in

reaction time. The activity observed is in compliance with the earlier reports on other

quinazolinyl derivatives bearing phenolic substitution on phenyl ring (MA.Soobrattee et al.,

2005). Compounds B2, B7 and B14 were also found to possess analgesic activities.

Antimicrobial activity:

All the title compounds were screened for antimicrobial activity against gram positive

bacteria such as B.subtilis (MTCC441), S.aureus (MTCC96), P.Aeuroginosa (MTCC1688),

gram negative bacteria such as E.Coli (ESS2231) and fungi such as A.niger (MTCC292)

and C.albicans (MTCC227). Among the series, phenolic group containing compounds B8 and

48B9 were found to be active against both gram+ve and gram-ve bacteria compared to the

standard drug, amoxicillin (Table _). The result is analogous to the previous reports on the

antibacterial, antifungal, and antiviral activities of natural and synthetic phenolic compounds.

Compound B13 possessing both the phenolic and methoxy substitutions exhibited good

activity against E.Coli.

Compound B2 with N-acetyl cysteine moiety also exhibited potent activity against

gram+ve bacteria. As per earlier reports N-acetyl cysteine (NAC) was used as an auxiliary

medication in certain pathological conditions along with the antibiotic therapy (R.N.Dilger et

al., 2007). It is also used as a mucolytic agent in combination with clinically relevant

antibiotics for treatment of lower respiratory tract infection and can enhance the efficacy of β-

lactams against several bacterial strains (Chan Prenner et al., 2006).

Compounds B10 and B11 possessing chloro and fluoro substituents on position-4 of

benzylidene ring also exhibited good activity against the bacterial strains. Compounds

possessing phenolic substituions such as B7, B9 and B13 showed significant antifungal activity

comparable to standard drug, fluconazole. Among the compounds with electron donating

substituents, B11 with 4-fluoro, B10 with 4-chloro and B12 bearing methoxy substituents also

exhibited good antifungal activity.

Brine shrimp lethality test:

Compounds that exhibited good activities in the above models were tested by brine

shrimp lethality test for cytotoxic activities. The results were given as ED50 values and are

compared with standard drug, podophyllotoxin (3.77µg/ml) (Table _). Among the

compounds tested the lower ED50 of 4.02µg/ml was observed with compound B2 possessing

active NAC moiety which is comparable to the standard. A research study had shown that

48NAC has potential both as chemo protective agent and in treatment of lung, skin, head and

neck, mammary and liver cancer (Radomska-Leśniewska et al., 2010).

The next comparable activity was observed with compounds B13 and B9 with ED50 of

4.38 µg/ml & 4.53 µg/ml is in relevance to the earlier reports on antioxidant ability as well as

anticancer property of various natural and synthetic phenolic compounds (Rico Munoz et al.,

1987).

The ED50 obtained with 4-methoxy derivative B12 was 6.68 µg/ml, 4-hydroxy

derivative B9 exhibited 4.53 µg/ml and an increased activity with decreased ED50 of 4.38

µg/ml was observed with compound B13 with these two functionalities in the same molecule.

In vitro antioxidant studies

The synthesized compounds (B1-B15) were evaluated for free radical scavenging

activities at different concentrations (25, 50,100,125 µM) of test compounds and standard.

IC50 was calculated for each compound as well as standard graphically and summarized in

table _.

Reduction of DPPH:

Among the tested compounds the lowest IC50 of 39.49 µM was exhibited by

compound B2 which is comparable to standard drug, BHT (42.28 µM). The potentiality of

this compound can be attributed to the presence of acetyl cysteine moiety which may be

synergizing the activity due to its capability of donating the hydrogen of SH group (Suetsuna

et al., 2000). Compounds B10 and B9 possessing chloro and hydroxyl at position-4 on

benzylidene ring exhibited potent activity with IC50 40.73 and 41.16 µM respectively. The

antioxidant capacity of phenolic group containing quinazolinyl derivatives were also reported

(Zaranappa et al., 2012).

48Nitric oxide scavenging activity:

Of these, two of them exhibited good activity with lower IC50 compared to that of

standard drug, tocopherol (45.21). Compound B9 possessing phenolic group exhibited IC50 of

37.80 µM, followed by compound B2 with NAC exhibiting IC50 of 41.01 µM. NAC has been

reported to reduce nitrite production, this effect may be dependent on a direct reaction of

NAC thiol group and nitric oxide producing a nitrothiol compound (Dorota et al.,2012).

Compounds B13, B7 and B15 also exhibited significant activities comparable to the standard.

Hydroxyl radical scavenging activity:

Compound B9 with phenolic substitution and compound B152 with NAC exhibited

good activity with IC50 of 46.06 µM and 48.37 respectively which is comparable to standard

(Ascorbic acid) with IC50 49.89 µM. Sulfhydryl group (–SH) in the molecule make possible

to directly scavenge reactive oxygen species (ROS) such as hydroxyl radical (AM. Sadowska

et al.,2006). Compounds B13, B15 and B11 also exhibited good activities.

Inhibition of lipid peroxidation:

Interestingly, similar results were obtained as in the case of above methods.

Compounds B9 and B2 were proved to possess good activity by exhibiting lower IC 50 of

51.96 and 54.10 µM comparable to standard drug, ascorbic acid (58.81 µM). Compound B15

with dimethyl amino group on benzylidene ring was observed to be equipotent to that

standard. The activity of compound B2 with NAC is supported by a report on the effect of

oral administration of N-acetyl cysteine as a lipid peroxidation inhibitor in a biomarker study

in smokers(Van Schooten et al., 2002).The ability to scavenge hydroxyl radicals probably

plays the most important role in the inhibition of brain lipid peroxidation(Horáková et

al.,2000).

48Superoxide scavenging activity:

The lowest IC50 of 48.76 µM was observed with compound B2 possessing NAC

moiety. In some earlier studies the oxidation rate constant for sulfhydryl group was proved as

significantly larger than the other proton donating groups, thus making it potent radical

scavenger. The phenolic group containing compound B9 also showed IC50 of 49.4 µM which

is comparable to the standard (50.8 µM). Compound B15 with dimethyl amino group also

exhibited significant activity comparable to the standard. Overproduction of superoxide

anion radical contributes to redox imbalance and associated with harmful physiological

consequences (N.Suzuki etal.,1992).

Reducing power assay:

The strongest reducing capacity at 100µM was observed with compound B15 showing

highest absorbance of 0.73 compared to the standard (0.726). This result suggests the

importance of reducing ability of electron donating substituent, dimethyl amino on the

benzylidene ring of the compound. An absorbance of 0.72 was observed with compound B2

which is in compliance with the earlier reports that sulfhydryl group normalized disturbed

redox status of the cells and thus influence redox – sensitive cell signaling and transcription

pathways (I.Gülçin, 2004). The compounds B9 and B13 also showed good activity. The result

may be attributed to the reducing capabilities of these compounds.

CUPRAC (Cupric Reducing Antioxidant Capacity) assay:

The highest activity was exhibited by compound B2 possessing NAC moiety with an

absorbance of 0.6421 and TEAC (Trolox Equivalent Antioxidant Capacity) of 3.845 while

ascorbic acid showed TEAC of 2.635.This result can be supported by the previous reports on

the potent activity of thiol containing compounds by CUPRAC assay (S.D.Cekiç et al.,2009).

48The compounds with dimethyl amino B15, methoxy and hydroxyl substituted derivatives, B9

and1 B13 also exhibited good TEAC values.

An overall outcome reveals that N-acetyl cysteine moiety and electron donating

functionalities on benzylidene ring influenced the biological activities of these compounds in

addition to the quinazolinyl framework.

Molecular descriptors and drug likeliness:

In silico evaluation was conducted for compounds that exhibited good activities in

almost all of the biological methods tested above. Table _ lists the predicted values of

selected parameters for compounds as well as representative standard anti-inflammatory,

analgesic drugs such as diclofenac and aspirin, antimicrobial drugs such as amoxicillin and

fluconazole.

The results obtained revealed that the compounds tested obeyed Lipinski rule of five

with not more than 5 hydrogen bond donors (OH and NH groups), not more than 10

hydrogen bond acceptors (notably N and O), not more than 15 rotatable bonds (rotb),

molecular weight (M.W) under 500 g/mol and a partition coefficient log P (mi LogP) less

than 5. The compounds tested also passed the other Lipinski like filters such as

bioavailability, Ghose filter, lead likeness, Muegge filter and Veber filters thus provide

estimation on solubility and permeability of orally active compounds.

However, descriptors such as Log P, solvent accessible surface area, polarizability

and refractivity contributed towards the activity of the molecules. Absorption is defined as

the process involved in getting a drug from its dosage form into the body and the ability to

predict the percent oral absorption is primary goal in the design, optimization, and selection

of potential candidates in the development of oral drugs.  HIA% of the compounds tested was

48desirable and for compound B2 was found to be 84.7% which is nearer to the diclofenac

(86.1%). Predicted HIA% of compounds 9, 13 and 15 were also found to be good.

TPSA is another key property that has been linked to bioavailability and it was found

that passively absorbed molecules with a TPSA more than 140 are thought to have low oral

availability. TPSA obtained for the tested compounds were below 140 indicating their good

oral bioavailability. Thus these results predicted the good drug likeliness, solubility,

permeability and oral bioavailability of the tested compounds.

Bioactivity score prediction:

The bioactivity scores of the above mentioned compounds were calculated for their

GPCR ligand, kinase inhibitor, protease inhibitor and enzyme inhibitor activities (Table _).

For average organic molecules the probability is that if the bioactivity score is more than 0

then it is active, if -0.5 to 0 then moderately active . On comparing the scores, compound B2

showed scores of more than 0 in all the four bioactivities supported its biological activity

profile. The kinase inhibitor score of compound B2 was predicted as 0.1 which was

comparable to the score obtained with diclofenac (0.2). Good kinase inhibitor and enzyme

inhibition scores of compound B2 also supported the result of its potent as anti-inflammatory

and analgesic activity (Amita Verma et al., 2012). The protease inhibitor score of 0.4 for the

compound B2 indicates its antibacterial efficacy.

PASS prediction:

The PASS prediction of compound B2 revealed interesting results with Pa: Pi (active:

inactive) ratio of 0.897:0.004 for inflammatory bowel disease treatment, 0.798:0.003 for

rheumatoid arthritis treatment and 0.75:0.001 as oxygen radical scavenger. Pa and Pi are the

estimates of probability for the compound to be active and inactive respectively for each type

48of activity from the biological activity spectrum. If Pa > 0.7 the compound is very likely to

reveal this activity in experiments, usually potent anti-inflammatory drugs with antibiotic

activity are preferred in treatment of inflammatory bowel disease and rheumatoid arthritis

treatment (D.C. Baumgart et al.,2007). This result emphasizes the potentiality of this

molecule to possess good anti-inflammatory, analgesic and antioxidant activities.

Table _: Physical data of quinazolinyl azadipeptide derivatives (B1-B6)

Compd code

R X Molecular formula

M.P(0C) Yield (%)

B1 H CO-CH3 C18H16N4O3 148-150 55

B2 CH2SH CO-CH3 C19H18N4O3S 165 70

B3 CH2-CH2-SCH3 CO-CH3 C21H22N4O3S 120-121 60

B4 H CO-C6H5 C23H18N4O3 118-119 69

B5 CH3 CO-C6H5 C24H20N4O3 148 78

B6 CH2-CH-(CH3)2 CO-C6H5 C27H26N4O3 153-155 41

Table _: Physical data of quinazolinyl azadipeptide derivatives (B7-B15)

Compd code

R Molecular formula

M.P(0C) Yield (%)

B7 H C23H18N4O2 134-136 68

48B8 2-OH C23H18N4O3 143-144 79

B9 4-OH C23H18N4O3 120-122 75

B10 4-Cl C23H17ClN4O2 160-161 69

B11 4-F C23H17FN4O2 128 78

B12 4-OCH3 C24H20N4O3 125-126 77

B13 4-OH,3-OCH3 C24H20N4O4 115-117 81

B14 4-CH3 C24H20N4O2 138-140 76

B15 4-N(CH3)2 C25H23N5O2 155-157 85

Table _: Anti-inflammatory and analgesic activities of quinazolinyl azapeptide derivatives (B1-B15)

Carrageenan induced rat paw edema method

Acetic acid induced writhing method b

Eddy’s hot plate method c

Compd code

Edema volume after 3hr

(ml±SEM) a

% inhibition of

inflammation

Number of writhings Mean±SEM b

%Analgesic activity

Maximum increase in

reaction time (sec) c

%Analgesic activity

B1 0.86±0.22 40.12* NT NT NT NT

B2 0.55±0.002 61.53*** 7.5±0.002 67.10*** 12.7±0.22 46.1*

B3 1.00±0.25 30.49* NT NT NT NT

B4 0.77±0.15 46.22* 9.1±0.030 60.08** 7.1±0.12 36.6*

B5 0.94±0.12 34.72* NT NT NT NT

B6 0.82±0.3 42.82* NT NT NT NT

B7 0.68±0.01 52.73** 11.2±0.020 50.87** 10±0.02 42.5**

B8 0.71±0.17 50.72* 12.8±0.120 43.85* 10.6±0.22 50.0*

B9 0.67±0.02 53.49* 8.01±0.010 64.86** 12.6±0.03 50.6***

B10 0.75±0.05 51.14** 9.2±0.014 59.42** 7.1±0.42 34.5

B11 0.79±0.14 45.25* 10.9±0.32 52.19* 7.1±0.21 37.2*

B12 0.80±0.022 44.95** 13.1±0.110 42.98* 5.7±0.12 30.5*

48B13 0.61±0.006 57.58*** 8.5±0.002 62.71*** 7.5±0.152 37.0*

B14 0.72±0.10 46.24* 12.0±0.200 47.36* 10±0.0111 42.7**

B15 0.69±0.1 52.18* 10.4±0.082 54.25* 8.8±0.022 41.3*

Control 1.43±0.14 - 22.8±0.100 - - -

standard 0.47±0.001(Diclofenac

sodium)

66.75*** 8.4±0.022(Aspirin)

63.15*** - -

a At 100mg/kg (p.o) edema volume was measured 3 hr after carrageenan injection and each value represents as the mean ± sem (n=5), activity presented as %inhibition of inflammation. b Number of writhings in 15 minutes beginning 5 minutes after acetic acid injection and each value represents as the mean ± SEM (n=5) . c At basal time and after 2 hr of drug administration maximum reaction time for paw licking is noted and each value represents as the mean ± SEM (n=5).Significance levels *p<0.5, **p<0.01 and ***p<0.001 by dunnets t-test, NT-not testedTable 2: Antimicrobial and cytotoxic activities of quinazolinyl azapeptide derivatives (B1-B15)

Antimicrobial activity by cup plate method Brine shrimp

lethality testCompd code Diameter of zone of inhibition(mm) a ED 50 b

B.Subtilis S.Aureus P.aeuroginsa E .Coli C.albicans A.nigerB1 12 11 13 14 9 10 NT

B2 21 19 20 17 19 18 4.02

B3 13 15 17 21 13 14 NT

B4 12 13 8 19 13 12 NT

B5 11 14 10 15 19 18 NT

B6 13 11 9 16 12 13 NT

B7 19 18 19 20 20 19 NT

B8 19 21 20 22 16 17 NT

B9 21 24 20 23 21 21 4.53

B10 20 19 17 16 19 20 NT

B11 18 16 17 18 13 21 4.38

B12 14 11 16 14 17 18 25.01

B13 20 22 20 24 20 19 14.37

B14 15 12 15 11 12 14 6.68

48B15 14 13 15 16 12 11 NT

Amoxicillin 21 22 21 22 - - -Fluconazole - - - - 23 24 -

Podophyllotoxin - - - - - - 3.77

a Test compounds (0.02 ml of 1mg/ml),Amoxicillin and Fluconazole(200 and 500 µg/ml respectively) and DMSO as a control.Activity is measured as zone of inhibition in mm. b ED50 was calculated based on the % of larvae survived at different concentrations of test and standard drugs.

Table _: Invitro antioxidant activities of quinazolinyl azapeptide derivatives (B1-B15)

Compd code

Reduction of DPPH

IC50(µM) a

Nitric oxide scavenging

IC50(µM) a

Hydroxyl radical scavenging IC50(µM) a

Inhibition of lipid peroxidationIC50(µM) a

Superoxide scavenging

IC50(µM) a

Reducing power(Absorbance±SEM)

CUPRAC assay(Absorbance±SEM)

TEAC b

B1 71.91 113.42 101.11 127.36 82.73 0.535±0.05 0.212±0.0085 1.27

B2 39.49 41.01 48.39 54.1 48.76 0.824±0.59 0.6421±0.0478 3.845

B3 47.16 54.02 74.44 72.53 108.54 0.699±0.01 0.4895±0.0085 2.931

B4 79.03 104.17 92.71 108.36 117.42 0.62±0.34 0.2041±0.004 1.222

B5 64.82 87.87 83.66 96.78 118.39 0.692±0.74 0.1567±0.0044 0.938

B6 55.83 89.39 88.66 97.2 126.63 0.556±0.002 0.2146±0.0045 1.285

B7 42.44 48.05 61.02 60.37 100 0.478±0.003 0.2003±0.015 1.199

B8 59.14 55.29 66.6 60.18 69 0.676±0.45 0.3099±0.0027 1.855

B9 41.16 37.8 46.06 51.96 49 0.792±0.01 0.3948±0.0031 2.364

B10 40.73 54.64 75 80.52 104.2 0.79±0.02 0.6007±0.0064 3.59

B11 42.29 60.9 57.23 77.58 84.09 0.72±0.03 0.4861±0.006 2.91

B12 45.01 83.99 66.28 113.05 75.15 0.631±0.027 0.2512±0.0056 1.504

B13 43.03 47.53 50.95 74.69 65.57 0.829±0.05 0.6125±0.0076 3.667

B14 51.7 82.61 82.24 92.82 81.62 0.438±0.08 0.2125±0.0086 1.272

B15 49.75 50.41 57.13 58.81 52.21 78.2±0.2 0.6136±0.0185 3.674

std 42.28 45.21 49.89 57.81 50.8 0.878±0.05 0.4401±0.0007 2.635

48(BHT)

(tocopherol) (Ascorbic acid)

(Ascorbic acid)

(BHT)

(tocopherol)

(Ascorbic acid)

(Trolox)

a IC 50 values were calculated graphically by using the data obtained in each test for % scavenging at different concentrations of (25, 50, 75,100 and 125µM). b Trolox Equivalent Antioxidant Capacity (TEAC); TEAC test= ε test /ε TR

Table _: Prediction of molecular properties descriptors and druglikeliness parameters

Parameters Comp

d B2

Comp

d B9

Comp

d B13

Comp

d B15

Diclofena

c

Indomethaci

n

Aspiri

n

Amoxicilli

n

Fluconazol

e

Mol. Wt a 383.3 398.4 428.5 426.1 296.1 255.2 365.4 180.1 306.2

miLog P b 2.70 3.23 3.05 3.81 4.56 3.9 1.43 -1.35 0.56

TPSA c 93.09 96.58 96.58 105.82 99.32 88.53 63.6 132.9 81.66

SASA d 499.75 525.07 571.39 599.97 361.15 476.23 247.24 472.98 399.2

Polarizabilty 39.26 42.21 44.74 46.42 29.02 37.34 17.51 35.52 26.92

Refractivity 105.95 115.94 122.4 128.3 75.46 94.8 44.47 89.5 97.19

NON e 7 7 8 7 3 5 4 8 7

NOHNH f 2 2 2 1 2 1 1 5 1

Ntrob g 5 5 6 6 4 4 3 4 4

Violations 0 0 0 0 0 0 0 0 0

HIA(%) h 84.7 80.4 78.5 76 86.1 91.6 89 24.2 83.6

BBB i -0.8 0.06 -0.22 0.13 -1 -1.1 -1.12 -0.77 -0.8

LROF j Yes Yes Yes Yes Yes Yes Yes Yes Yes

Bioavailabilit

y

Yes Yes Yes Yes Yes Yes Yes Yes Yes

Ghose filter Yes Yes Yes Yes Yes Yes Yes No Yes

Lead likeness Yes Yes Yes Yes Yes Yes Yes Yes Yes

Muegge filter Yes Yes Yes Yes Yes Yes No No Yes

Veber filter Yes Yes Yes Yes Yes Yes Yes Yes Yes

GPCR-L score k 0.04 -0.01 -0.03 -0.13 0.14 0.24 0.05 0.07 0.04

K I score l 0.10 -0.21 -0.03 -0.16 0.20 -0.11 0.3 -0.65 -0.09

P I score m 0.40 -0.11 -0.70 -0.64 -0.10 -0.11 -0.21 0.80 -0.09

E I score n 0.17 -0.07 -0.19 -0.26 0.25 0.27 0.22 0.30 -0.03

a Mol.wt-Molecular wt in g/mol, b Log P-Partition coefficient, c TPSA-Topological polar surface area(A2), d

SASA-solvent accessible surface area(A2) ,e NON=No of hydrogen bond acceptors,f NOHNH_no. of hydrogen

48bond donors, g Ntrob,No.of rotatable bonds, h HIA- %human intestinal absorption, I BBB- Log Blood brain

barrier , j LROF-Lipinski’s rule of five,k GPCR-L-G-protein coupled receptor ligand ,l KI-kinase inhibitor, m PI-

protease inhibitor, n EI-Enzyme inhibitor