Synthesis of some new glutamine linked 2,3-disubstituted quinazolinone derivatives as potent antimicrobial and antioxidant agents

Article abstract of DOI:10.1007/s00044-012-0269-6

A series of novel glutamine linked 2,3-disubstituted quinazolinone conjugates was synthesized from methyl anthranilate and different substituted acids and acid chlorides. The compounds 5a-l were prepared in good yields. All compounds were screened for their antibacterial activity against Gram-positive and Gram-negative bacteria and for antifungal activity against Candida albicans and Aspergillus flavus using paper disk diffusion technique. The minimum inhibitory concentrations of the compounds were also determined by agar streak dilution method. The compound 5b was found to exhibit the most potent in vitro anti-microbial activity. When tested for their antioxidant activity, compounds 5i and 5l showed potent radical scavenging activity, while compound 5g had moderate effect against 2,2-diphenyl-1-picrylhydrazyl, hydroxyl, nitric oxide, and superoxide radical scavenging assays. These results suggest that, the three quinazolinone analogs (5g, 5i, and 5l) could be considered as useful templates for future development to obtain more potent antioxidant agents.

Full text of DOI:10.1007/s00044-012-0269-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2665–2676
DOI 10.1007/s00044-012-0269-6
ORIGINAL RESEARCH
Synthesis of some new glutamine linked 2,3-disubstituted
quinazolinone derivatives as potent antimicrobial and antioxidant
agents
•
M. K. Prashanth H. D. Revanasiddappa
Received: 9 April 2012 / Accepted: 29 September 2012 / Published online: 9 October 2012
ꢀ Springer Science+Business Media New York 2012
Abstract A series of novel glutamine linked 2,3-disub-
stituted quinazolinone conjugates was synthesized from
methyl anthranilate and different substituted acids and acid
chlorides. The compounds 5a–l were prepared in good
yields. All compounds were screened for their antibacterial
activity against Gram-positive and Gram-negative bacteria
and for antifungal activity against Candida albicans and
Aspergillus flavus using paper disk diffusion technique.
The minimum inhibitory concentrations of the compounds
were also determined by agar streak dilution method. The
compound 5b was found to exhibit the most potent in vitro
anti-microbial activity. When tested for their antioxidant
activity, compounds 5i and 5l showed potent radical
scavenging activity, while compound 5g had moderate
effect against 2,2-diphenyl-1-picrylhydrazyl, hydroxyl,
nitric oxide, and superoxide radical scavenging assays.
These results suggest that, the three quinazolinone analogs
(5g, 5i, and 5l) could be considered as useful templates for
future development to obtain more potent antioxidant
agents.
and its derivatives are versatile lead molecules for designing
potential bioactive agents (Mhaske and Argade, 2006). Qui-
nazolinone derivatives are found to possess a number of
therapeutic activities including antimicrobial, antioxidant,
anti-inflammatory, and anti-cancer activities (Maggio et al.,
2001; Grover and Kini, 2006; Mani Chandrika et al., 2008).
4(3H)-quinazolinone is one of the most frequently encoun-
tered heterocycles in medicinal chemistry with wide appli-
cations. Among the various classes of quinazolinones,
2,3-disubstituted-4(3H)-quinazolinone has been reported to
be associated with anti-microbial properties. Examples of
such disubstituted derivatives include substituted phenyl ring
moieties, and bridged phenyl rings and aliphatic systems
(Mosaad et al., 2010). The 2,3-disubstituted quinazolinones
have been predicted to possess antiviral and antihypertensive
activities (Pandey et al., 2004) and exhibited good analgesic
and anti-inflammatory activities (Alagarsamy et al., 2007).
The presence of fluorine atom at appropriate position in the
molecule alters the properties of molecule by promoting
activity due to high lipid solubility and enhancement of
transport mechanism (Furuyal and Ritter, 2011).
Keywords Glutamine ꢀ Quinazolinone ꢀ Antimicrobial ꢀ
Free radicals and oxygen derivatives are constantly
generated in vitro by specific metabolic processes
(Suthakaran et al., 2008). These radicals can easily react
with most biological molecules including proteins, lipids,
lipoproteins, and DNA. These can be responsible for a
wide range of human conditions such as arthritis, hemor-
rhagic shock, coronary artery diseases, cataract, cancer,
AIDS as well as age-related degenerative brain diseases
(Parr and Bolwell, 2000). Thus, there is a constant need for
searching new and effective therapeutic agents.
Antioxidant
Introduction
Quinazolinone is a building block for naturally occurring
alkaloids isolated from a number of families of plant kingdom
General investigation revealed that the components with
antimicrobial activity have gained increasing importance
due to growing worldwide concern about the increase in the
rate of infection by pathogenic microbes (Davies, 1994).
M. K. Prashanth ꢀ H. D. Revanasiddappa (&)
Department of Studies in Chemistry, University of Mysore
Manasagangothri, Mysore 570006, Karnataka, India
e-mail: hdrevanasiddappa@yahoo.com
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Antibacterial is a general term for drug, chemical, or other
substance that either kills or slows the growth of microbe.
Thus, the development of new and different antimicrobial
drugs is a very important objective and much of the research
program efforts are directed toward the design of new
antibacterial agents.
combined organic portions were dried over anhydrous
Na2SO4. The crude product was obtained after evaporation
of the solvent, and recrystallization from absolute ethanol
gave the desired compounds 2a–f.
To the suspension of different substituted aromatic acids
(0.01 mol) in CH2Cl2 an excess of thionyl chloride was
added and then the resulting mixture was stirred at room
temperature for 2 h. The solvent was evaporated under
reduced pressure giving the acid chlorides. This acid
chloride was added to a solution containing methyl
anthranilate in CH2Cl2 and stirring was continued at room
temperature for 20 h, and the reaction mixture was diluted
with CH2Cl2 and washed with water (50 ml) and brine
solution (100 ml). The combined organic portions were
dried over Na2SO4. Evaporation of the solvent gave the
crude product, and recrystallization from absolute ethanol
gave desired compounds 2g–l.
Currently, antioxidants that exhibit DPPH radical scav-
enging activity are receiving increasing attention. Antiox-
idants act as a major defense against radical mediated
toxicity by protecting the damages caused by free radicals
and they have been reported to have interesting anticancer,
anti-aging, and anti-inflammatory activities (Finkel and
Holbrook, 2000). Literature is scanty on the study of
antioxidant activity of substituted quinazolinone deriva-
tives. This prompted the authors to synthesize a new series
of biologically active quinazolinones and evaluate their
antimicrobial activity by disk diffusion method and anti-
oxidant activity by DPPH free radical scavenging, hydro-
xyl radical scavenging, superoxide radical scavenging, and
nitric oxide (NO) radical scavenging assays. Thus, the
present work is aimed at designing new and more active
and less toxic synthetic antimicrobial and antioxidant
agents.
Methyl 2-(3,5-difluorobenzamido)benzoate (2a)
Yield: 72 %. IR (KBr, cm-1): 3190 (NH), 1687, 1630
(2C=O). 1H NMR (CDCl3, d ppm): 8.74 (s, 1H, NH),
8.21–7.20 (m, 7H, Ar–H), 3.80 (s, 3H, OCH3).
Methyl 2-(2,3,4-trifluorobenzamido)benzoate (2b)
Experimental
Yield: 70 %. IR (KBr, cm-1): 3248 (NH), 1695, 1637
(2C=O). 1H NMR (CDCl3, d ppm): 8.41 (s, 1H, NH),
8.19–7.12 (m, 6H, Ar–H), 3.87 (s, 3H, OCH3).
All chemicals and solvents were of AR grade. Solvents
were used as received without further purification. Ele-
mental analysis (C, H, N) was performed using a Carlo-
Erba 1160 elemental analyzer. IR spectra were recorded
on a JASCO FTIR-8400 spectrophotometer using KBr
Methyl 2-(2,5-difluorobenzamido)benzoate (2c)
1
method. The H-NMR spectra were recorded on a Varian
Yield: 78 %. IR (KBr, cm-1): 3196 (NH), 1681, 1640
(2C=O). 1H NMR (CDCl3, d ppm): 8.65 (s, 1H, NH),
8.23–7.22 (m, 7H, Ar–H), 3.79 (s, 3H, OCH3).
AC 400 spectrometer using TMS as the internal standard
and CDCl3 as a solvent. Mass spectra were obtained on a
Varian 1200L model mass spectrometer (solvent: CH3OH).
Melting points were determined with a Buchi 530 melting
point apparatus in open capillaries and are uncorrected.
Compound purity was checked by thin layer chromatog-
raphy (TLC) on precoated silica gel plates (Merck,
Kieselgel 60 F254, layer thickness 0.25 mm).
Methyl 2-(4-chlorobenzamido)benzoate (2d)
Yield: 72 %. IR (KBr, cm-1): 3219 (NH), 1690, 1624
(2C=O). 1H NMR (CDCl3, d ppm): 8.33 (s, 1H, NH),
8.21–7.15 (m, 8H, Ar–H), 3.80 (s, 3H, OCH3).
Synthesis
Methyl 2-(2-phenylacetamido)benzoate (2e)
General procedure for compounds (2a–f)/(2g–l)
Yield: 77 %. IR (KBr, cm-1): 3211 (NH), 1695, 1639
(2C=O). 1H NMR (CDCl3, d ppm): 8.60(s, 1H, NH),
8.22–7.26 (m, 8H, Ar–H), 3.80 (s, 3H, OCH3).
Compounds 2a–f were prepared by the reaction of methyl
anthranilate (0.01 mol) with corresponding substituted
benzoyl chlorides (0.01 mol) in CH2Cl2, and the reaction
mixture was stirred at room temperature for 20 h and the
progress of the reaction was monitored by TLC. The
reaction mixture was then diluted with CH2Cl2 and washed
with water (50 ml) and brine solution (100 ml), and the
Methyl 2-(2-chlorobenzyloxycarbonylamino)benzoate (2f)
Yield: 80 %. IR (KBr, cm-1): 3150 (NH), 1673, 1621
(2C=O). 1H NMR (CDCl3, d ppm): 9.27 (s, 1H, NH),
8.21–7.04 (m, 8H, Ar–H), 3.80 (s, 3H, OCH3).
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Methyl 2-(4-methoxybenzamido)benzoate (2g)
2-(2,3,4-Trifluorobenzamido)benzoic acid (3b)
Yield: 74 %. IR (KBr, cm-1): 3271 (NH), 1654, 1620
(2C=O). 1H NMR (CDCl3, d ppm): 8.64 (s, 1H, NH),
8.23–7.01 (m, 8H, Ar–H), 3.80, 3.89 (s, 6H, 2OCH3).
Yield: 92 %. IR (KBr, cm-1): 3248 (NH), 2700 (COOH),
1647 (C=O). 1H NMR (CDCl3, d ppm): 12.33 (s, 1H,
COOH), 8.63 (s, 1H, NH), 8.22–7.14 (m, 6H, Ar–H).
Methyl 2-(4-nitrobenzamido)benzoate (2h)
2-(2,5-Difluorobenzamido)benzoic acid (3c)
Yield: 70 %. IR (KBr, cm-1): 3280 (NH), 1692, 1644
(2C=O). 1H NMR (CDCl3, d ppm): 8.92 (s, 1H, NH),
8.27–7.12 (m, 8H, Ar–H), 3.80 (s, 3H, OCH3).
Yield: 92 %. IR (KBr, cm-1): 3199 (NH), 2699 (COOH),
1675 (C=O). 1H NMR (CDCl3, d ppm): 11.96 (s, 1H,
COOH), 8.69 (s, 1H, NH), 8.23–7.15 (m, 7H, Ar–H).
Methyl 2-(4-aminobenzamido)benzoate (2i)
2-(4-Chlorobenzamido)benzoic acid (3d)
Yield: 76 %. IR (KBr, cm-1): 3255 (NH), 1703, 1633
(2C=O). 1H NMR (CDCl3, d ppm): 9.03 (s, 1H, NH),
8.21–7.19 (m, 8H, Ar–H), 5.72 (s, 2H, NH2), 3.80 (s, 3H,
OCH3).
Yield: 96 %. IR (KBr, cm-1): 3221 (NH), 2784 (COOH),
1678 (C=O). 1H NMR (CDCl3, d ppm): 12.11 (s, 1H,
COOH), 8.47 (s, 1H, NH), 8.21–7.21 (m, 8H, Ar–H).
Methyl 2-(2-methylbenzamido)benzoate (2j)
2-(2-Phenylacetamido)benzoic acid (3e)
Yield: 81 %. IR (KBr, cm-1): 3214 (NH), 1652, 1621
(2C=O). 1H NMR (CDCl3, d ppm): 8.52 (s, 1H, NH),
8.24–7.33 (m, 8H, Ar–H), 3.80 (s, 3H, OCH3), 2.41 (s, 3H,
CH3).
Yield: 92 %. IR (KBr, cm-1): 3219 (NH), 2690 (COOH),
1680 (C=O). 1H NMR (CDCl3, d ppm): 12.07 (s, 1H,
COOH), 8.69 (s, 1H, NH), 8.21–7.26 (m, 8H, Ar–H), 3.80
(s, 3H, OCH3).
Methyl 2-benzamidobenzoate (2k)
2-((2-Chlorobenzyloxy)carbonylamino)benzoic acid (3f)
Yield: 70 %. IR (KBr, cm-1): 3207 (NH), 1697, 1630
(2C=O). 1H NMR (CDCl3, d ppm): 8.36 (s, 1H, NH),
8.22–7.24 (m, 8H, Ar–H), 3.80 (s, 3H, OCH3).
Yield: 95 %. IR (KBr, cm-1): 3150 (NH), 2751 (COOH),
1672 (C=O). 1H NMR (CDCl3, d ppm): 12.64 (s, 1H,
COOH), 9.61 (s, 1H, NH), 8.23–7.14 (m, 8H, Ar–H).
Methyl 2-(4-hydroxybenzamido)benzoate (2l)
2-(4-Methoxybenzamido)benzoic acid (3g)
Yield: 78 %. IR (KBr, cm-1): 3258 (NH), 1701, 1649
(2C=O). 1H NMR (CDCl3, d ppm): 10.04 (s, 1H, OH), 9.27
(s, 1H, NH), 8.24–7.12 (m, 8H, Ar–H), 3.80 (s, 3H, OCH3).
Yield: 98 %. IR (KBr, cm-1): 3290 (NH), 2739 (COOH),
1670 (C=O). 1H NMR (CDCl3, d ppm): 12.06 (s, 1H,
COOH), 9.11 (s, 1H, NH), 8.23–7.04 (m, 8H, Ar–H), 3.59
(s, 3H, OCH3).
General procedure for compounds (3a–l)
Compounds 2a–l (1.0 mmol) were dissolved separately in
1:1 mixture of methanol and CH2Cl2, then hydrolyzed by
aqueous NaOH (2 M, 5 ml), and then each mixture was
refluxed for 1–2 h. Afterward, the reaction mixture was
poured into water and acidification to pH 2 with 2 M HCl
gave a precipitate, which was collected by filtration, giving
3a–l as an amorphous powder.
2-(4-Nitrobenzamido)benzoic acid (3h)
Yield: 94 %. IR (KBr, cm-1): 3302 (NH), 2697 (COOH),
1700 (C=O). 1H NMR (CDCl3, d ppm): 12.71 (s, 1H,
COOH), 8.97 (s, 1H, NH), 8.24–7.12 (m, 8H, Ar–H).
2-(4-Aminobenzamido)benzoic acid (3i)
2-(3,5-Difluorobenzamido)benzoic acid (3a)
Yield: 96 %. IR (KBr, cm-1): 3255 (NH), 2700 (COOH),
1701 (C=O). 1H NMR (CDCl3, d ppm): 12.94 (s, 1H,
COOH), 9.33 (s, 1H, NH), 8.21–7.17 (m, 8H, Ar–H), 5.27
(s, 2H, NH2).
Yield: 98 %. IR (KBr, cm-1): 3201 (NH), 2735 (COOH),
1685 (C=O). 1H NMR (CDCl3, d ppm): 12.01 (s, 1H,
COOH), 8.94 (s, 1H, NH), 8.22–7.19 (m, 7H, Ar–H).
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2-(2-Methylbenzamido)benzoic acid (3j)
2-(2-Chlorobenzyloxy)-4H-benzo[d][1,3]oxazin-4-one (4f)
Yield: 91 %. IR (KBr, cm-1): 3216 (NH), 2730 (COOH),
1695 (C=O). 1H NMR (CDCl3, d ppm): 12.64 (s, 1H,
COOH), 8.90 (s, 1H, NH), 8.23–7.21 (m, 8H, Ar–H), 2.46
(s, 3H, CH3).
Yield: 74 %. IR (KBr, cm-1): 1665 (C=O), 1583 (C=N).
1H NMR (CDCl3, d ppm): 8.27–7.14 (m, 8H, Ar–H), 2.47
(s, 2H, CH2).
2-(4-Methoxyphenyl)-4H-benzo[d][1,3]oxazin-4-one (4g)
2-Benzamidobenzoic acid (3k)
Yield: 77 %. IR (KBr, cm-1): 1695 (C=O), 1587 (C=N).
1H NMR (CDCl3, d ppm): 8.23–7.11 (m, 8H, Ar–H), 3.66
(s, 3H, OCH3).
Yield: 90 %. IR (KBr, cm-1): 3248 (NH), 2714 (COOH),
1695 (C=O). 1H NMR (CDCl3, d ppm): 12.37 (s, 1H,
COOH), 8.61 (s, 1H, NH), 8.22–7.17 (m, 8H, Ar–H).
2-(4-Nitrophenyl)-4H-benzo[d][1,3]oxazin-4-one (4h)
2-(4-Hydroxybenzamido)benzoic acid (3l)
Yield: 73 %. IR (KBr, cm-1): 1700 (C=O), 1589 (C=N).
Yield: 97 %. IR (KBr, cm-1): 3279 (NH), 2690 (COOH),
1684 (C=O). 1H NMR (CDCl3, d ppm): 13.16 (s, 1H,
COOH), 10.01 (s, 1H, OH), 9.47 (s, 1H, NH), 8.23–7.11
(m, 8H, Ar–H).
1H NMR (CDCl3, d ppm): 8.21–7.20 (m, 8H, Ar–H).
2-(4-Aminophenyl)-4H-benzo[d][1,3]oxazin-4-one (4i)
General procedure for compounds (4a–l)
Yield: 80 %. IR (KBr, cm-1): 3390 (NH2), 1695 (C=O),
1582 (C=N). 1H NMR (CDCl3, d ppm): 8.22–7.15 (m, 8H,
Ar–H), 5.49 (s, 2H, NH2).
Compounds 3a–l (0.1 mmol) were refluxed in thionyl
chloride (0.1 mmol) for 2 h. The solvent was evaporated
under reduced pressure and recrystallization of the residue
from acetonitrile gave the products 4a–l.
2-o-tolyl-4H-benzo[d][1,3]oxazin-4-one (4j)
Yield: 81 %. IR (KBr, cm-1): 2732 (C–CH3), 1699 (C=O),
1601 (C=N). 1H NMR (CDCl3, d ppm): 8.23–7.21 (m, 8H,
Ar–H), 2.46 (s, 3H, CH3).
2-(3,5-Difluorophenyl)-4H-benzo[d][1,3]oxazin-4-one (4a)
Yield: 70 %. IR (KBr, cm-1): 1685 (C=O), 1597 (C=N).
1H NMR (CDCl3, d ppm): 8.23–7.19 (m, 7H, Ar–H).
2-Phenyl-4H-benzo[d][1,3]oxazin-4-one (4k)
2-(2,3,4-Trifluorophenyl)-4H-benzo[d][1,3]oxazin-4-one
(4b)
Yield: 74 %. IR (KBr, cm-1): 1703 (C=O), 1597 (C=N).
1H NMR (CDCl3, d ppm): 8.27–7.20 (m, 9H, Ar–H).
Yield: 79 %. IR (KBr, cm-1): 1695 (C=O), 1587 (C=N).
1H NMR (CDCl3, d ppm): 8.21–7.12 (m, 6H, Ar–H).
2-(4-Hydroxyphenyl)-4H-benzo[d][1,3]oxazin-4-one (4l)
2-(2,5-Difluorophenyl)-4H-benzo[d][1,3]oxazin-4-one (4c)
Yield: 79 %. IR (KBr, cm-1): 3184 (OH), 1695 (C=O),
1592 (C=N). 1H NMR (CDCl3, d ppm): 10.07 (s, 1H, OH),
8.23–7.11 (m, 8H, Ar–H).
Yield: 82 %. IR (KBr, cm-1): 1700 (C=O), 1580 (C=N).
1H NMR (CDCl3, d ppm): 8.26–7.10 (m, 7H, Ar–H).
2-(4-Chlorophenyl)-4H-benzo[d][1,3]oxazin-4-one (4d)
General procedure for compounds (5a–l)
Yield: 72 %. IR (KBr, cm-1): 1697 (C=O), 1600 (C=N).
Equimolar quantities (0.06 mmol) of 2-substituted-
3,1-benzoxazin-4-one and the primary amine (gluta-
mine) in glacial acetic acid (5 ml) were refluxed for 6 h.
The resulting reaction mixture was cooled to room
temperature and poured onto crushed ice. The separated
out solid was filtered, washed thoroughly with cold
distilled water, vacuum dried and recrystallized from
ethanol to obtain the products 5a–l.
1H NMR (CDCl3, d ppm): 8.23–7.20 (m, 8H, Ar–H).
2-Benzyl-4H-benzo[d][1,3]oxazin-4-one (4e)
Yield: 72 %. IR (KBr, cm-1): 1678 (C=O), 1593 (C=N).
1H NMR (CDCl3, d ppm): 8.24–7.16 (m, 8H, Ar–H), 2.36
(s, 2H, CH2).
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2669
2-Amino-5-(2-(3,5-difluorophenyl)-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic acid (5a)
Analysis Calcd. for C20H19N3O4, (%): C 65.74, H 5.24, N
11.50. Found (%): C 65.50, H 5.20, N 11.26.
IR (KBr, cm-1): 3341 (NH2), 2660 (COOH), 1673, 1591
(C=O), 1539 (C=N). 1H NMR (CDCl3, d ppm): 12.10 (s,
1H, COOH), 8.26–7.22 (m, 7H, Ar–H), 5.47 (s, 2H, NH2),
3.54–3.51 (t, 1H, CH), 2.53–2.50 (t, 2H, CH2), 2.29–2.26
(q, 2H, CH2). MS m/z: 387 (M?). Analysis Calcd. for
C19H15F2N3O4 (%): C 58.92, H 3.90, N 10.85. Found (%):
C 58.87, H 3.93, N 10.93.
2-Amino-5-(2-(2-chlorobenzyloxy)-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic acid (5f)
IR (KBr, cm-1): 3369 (NH2), 2671 (COOH), 1662, 1611
1
(C=O), 1564 (C=N). H NMR (CDCl3, d ppm): 12.16 (s,
1H, COOH), 8.29–7.19 (m, 8H, Ar–H), 5.40 (s, 2H, NH2),
4.36 (s, 2H, Ph–CH2), 3.53–3.50 (t, 1H, CH), 2.57–2.54 (t,
2H, CH2), 2.29–2.25 (q, 2H, CH2). MS, m/z: 415 (M?), 417
(M?2). Analysis Calcd. for C20H18ClN3O5 (%): C 57.77, H
4.36, N 10.11. Found (%): C 57.63, H 4.31, N 10.18.
2-Amino-5-oxo-5-(4-oxo-2-(2,3,4-trifluorophenyl)
quinazolin-3(4H)-yl)pentanoic acid (5b)
IR (KBr, cm-1): 3361 (NH2), 2670 (COOH), 1681, 1609
2-Amino-5-(2-(4-methoxyphenyl)-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic acid (5g)
1
(C=O), 1562 (C=N). H NMR (CDCl3, d ppm): 12.01 (s,
1H, COOH), 8.28–7.19 (m, 8H, Ar–H), 5.37 (s, 2H, NH2),
3.53–3.50 (t, 1H, CH), 2.51–2.48 (t, 2H, CH2), 2.24–2.21
(q, 2H, CH2). MS m/z: 405 (M?). Analysis Calcd. for
C19H14F3N3O4 (%): C 56.30, H 3.48, N 10.37. Found (%):
C 56.58, H 3.11, N 10.04.
IR (KBr, cm-1): 3370 (NH2), 2669 (COOH), 1682, 1602
1
(C=O), 1574 (C=N). H NMR (CDCl3, d ppm): 12.16 (s,
1H, COOH), 8.17–7.02 (m, 8H, Ar–H), 5.43 (s, 2H, NH2),
3.67 (s, 3H, OCH3), 3.64–3.61 (t, 1H, CH), 2.58–2.54 (t,
2H, CH2), 2.26–2.21 (q, 2H, CH2). MS m/z: 381 (M?).
Analysis Calcd. for C20H19N3O5 (%): C 62.99, H 5.02, N
11.02. Found (%): C 62.93, H 5.11, N 11.07.
2-Amino-5-(2-(2,5-difluorophenyl)-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic (5c)
2-Amino-5-(2-(4-nitrophenyl)-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic acid (5h)
IR (KBr, cm-1): 3370 (NH2), 2668 (COOH), 1694, 1628
1
(C=O), 1569 (C=N). H NMR (CDCl3, d ppm): 12.11 (s,
1H, COOH), 8.29–7.19 (m, 7H, Ar–H), 5.40 (s, 2H, NH2),
3.56–3.52 (t, 1H, CH), 2.51–2.48 (t, 2H, CH2), 2.29–2.26
(q, 2H, CH2). MS m/z: 387 (M?). Analysis Calcd. for
C19H15F2N3O4 (%): C 58.92, H 3.90, N 10.85. Found (%):
C 58.50, H 3.73, N 10.49.
IR (KBr, cm-1): 3367 (NH2), 2669 (COOH), 1693, 1632
1
(C=O), 1579 (C=N). H NMR (CDCl3, d ppm): 12.04 (s,
1H, COOH), 8.27–7.11 (m, 8H, Ar–H), 5.47 (s, 2H, NH2),
3.68–3.63 (t, 1H, CH), 2.47–2.44 (t, 2H, CH2), 2.24–2.20
(q, 2H, CH2). MS, m/z: 396 (M?). Analysis Calcd. for
C19H16N4O6 (%): C 57.58, H 4.07, N 14.14. Found (%): C
57.41, H 4.11, N 14.06.
2-Amino-5-(2-(4-chlorophenyl)-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic acid (5d)
2-Amino-5-(2-(4-aminophenyl)-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic acid (5i)
IR (KBr, cm-1): 3374 (NH2), 2669 (COOH), 1693, 1630
1
(C=O), 1587 (C=N). H NMR (CDCl3, d ppm): 12.01 (s,
IR (KBr, cm-1): 3477, 3362 (2 NH2), 2669 (COOH), 1698,
1626 (C=O), 1555 (C=N). 1H NMR (CDCl3, d ppm):
12.01(s, 1H, COOH), 8.28–7.19 (m, 8H, Ar–H), 5.39 (s,
2H, 2NH2), 3.64–3.60 (t, 1H, CH), 2.54–2.50 (t, 2H, CH2),
2.24–2.21 (q, 2H, CH2). MS, m/z: 366 (M?). Analysis
Calcd. for C19H18N4O4 (%): C 62.29, H 4.95, N 15.29.
Found (%): C 62.21, H 4.99, N 15.17.
1H, COOH), 8.23–7.20 (m, 8H, Ar–H), 5.35 (s, 2H, NH2),
3.52–3.49 (t, 1H, CH), 2.52–2.50 (t, 2H, CH2), 2.29–2.26
(q, 2H, CH2). MS m/z: 384 (M?), 386 (M?2). Analysis
Calcd. for C19H16ClN3O4 (%): C 59.15, H 4.18, N 10.89.
Found (%): C 59.11, H 4.09, N 10.94.
2-Amino-5-(2-benzyl-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic acid (5e)
2-Amino-5-oxo-5-(4-oxo-2-o-tolylquinazolin-
3(4H)-yl)pentanoic acid (5j)
IR (KBr, cm-1): 3354 (NH2), 2667 (COOH), 1675, 1627
1
(C=O), 1589 (C=N). H NMR (CDCl3, d ppm): 11.96 (s,
IR (KBr, cm-1): 3362 (NH2), 2669 (COOH), 1697, 1634
1H, COOH), 8.27–7.19 (m, 9H, Ar–H), 5.62 (s, 2H, NH2),
4.03 (s, 2H, Ph–CH2), 3.62–3.58 (t, 1H, CH), 2.47–2.44 (t,
2H, CH2), 2.19–2.15 (q, 2H, CH2). MS m/z: 365 (M?).
1
(C=O), 1582 (C=N). H NMR (CDCl3, d ppm): 11.61 (s,
1H, COOH), 8.23–7.19 (m, 8H, Ar–H), 5.42 (s, 2H, NH2),
123

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Med Chem Res (2013) 22:2665–2676
3.64–3.61 (t, 1H, CH), 2.54–2.50 (t, 2H, CH2), 2.47 (s, 3H,
CH3), 2.26–2.23 (q, 2H, CH2). MS m/z: 365 (M?). Analysis
Calcd. for C20H19N3O4 (%): C 65.74, H 5.24, N 11.50.
Found (%): C 65.46, H 5.27, N 11.59.
48 h at 28 ꢁC in the case of fungi. Streptomycin (10 lg/disk)
and fluconazole (10 lg/disk) were used as standards for
antibacterial and antifungal activity, respectively. Each
sample was assayed in triplicate and the zone of inhibition
was measured in millimeters.
2-Amino-5-oxo-5-(4-oxo-2-phenylquinazolin-3(4H)-
yl)pentanoic acid (5k)
Minimum inhibitory concentration (MIC)
IR (KBr, cm-1): 3367 (NH2), 2674 (COOH), 1699, 1617
The MIC of the synthesized compounds was determined by
dilution method (Jones et al., 1984). The compounds were
dissolved in DMSO; two-fold serial concentrations of the
compounds were employed to determine the MIC. In this
method, the test concentrations of chemically synthesized
compounds were made from 5 to 125 lg/ml. The MIC-
value was determined as the lowest concentration of the
compound that completely inhibited macroscopic growth
of microorganism.
1
(C=O), 1572 (C=N). H NMR (CDCl3, d ppm): 12.11 (s,
1H, COOH), 8.28–7.25 (m, 9H, Ar–H), 5.64 (s, 2H, NH2),
3.54–3.51 (t, 1H, CH), 2.50–2.47 (t, 2H, CH2), 2.14–2.12
(q, 2H, CH2). MS m/z: 351 (M?). Analysis Calcd. for
C19H17N3O4 (%): C 64.95, H 4.88, N 11.96. Found (%): C
64.75, H 4.72, N 11.99.
2-Amino-5-(2-(4-hydroxyphenyl)-4-oxoquinazolin-
3(4H)-yl)-5-oxopentanoic acid (5l)
IR (KBr, cm-1): 3361 (NH2), 3176 (OH), 2670 (COOH),
1691, 1635 (C=O), 1583 (C=N). 1H NMR (CDCl3, d ppm):
11.21 (s, 1H, COOH), 10.03 (s, 1H, OH), 8.28–7.06 (m,
8H, Ar–H), 5.48 (s, 2H, NH2), 3.58–3.55 (t, 1H, CH),
2.52–2.50 (t, 2H, CH2), 2.01–1.99 (q, 2H, CH2). MS, m/z:
367 (M?). Analysis Calcd. for C19H17N3O5 (%): C 62.12,
H 4.66, N 11.44. Found (%): C 62.01, H 4.53, N 11.49.
Antioxidant activity
DPPH free radical scavenging activity
The capacity to scavenge the stable free radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH) was monitored accord-
ing to the Blois method (Blois, 1958). The test samples
(10–100 ll) were mixed with 1 ml of DPPH solution
(0.1 mM) and filled up with methanol to a final volume of
4 ml. The absorbance of the resulting solution was measured
at 517 nm on a visible spectrophotometer (Model 166,
Systronics, India). The free radical scavenging rate of the
reaction solution was calculated as a percentage (%) of DPPH
discoloration using the equation
Antimicrobial activity
Microbial strains
The in vitro antimicrobial screening of the quinazolinone
compounds was individually performed against a panel of
bacteria and fungi including Staphylococcus aureus (NCIM
5021), Pseudomonas aeruginosa (NCIM 5029), E. coli
(NCIM 2574), Bacillus subtilis (NCIM 2999), Candida
albicans (NCIM 3102), and Aspergilus flavus (NCIM 524).
Microbial strains were cultured overnight at 37 ꢁC in
nutrient and potato dextrose agar medium. All the pure
microbial strains were obtained from National Chemical
Laboratory (NCL), Pune, India.
À
Á
I ð%Þ ¼ Ablank ꢁ Asample=Ablank ꢂ 100
where Ablank is the absorbance of the control reaction mixture
excluding the test compounds, and Asample is the absorbance
of the test compounds. Radical scavenging potential was
expressed as IC50 value, which represents the sample con-
centration at which 50 % of the DPPH radicals scavenged.
Tests were carried out in triplicate and the results were
expressed as mean values standard deviations.
Hydroxyl radical scavenging assay
Antimicrobial screening
This assay was performed by a standard method (Jayabha-
rathi et al., 2012), with a slight modification. Hydroxyl
radical scavenging of quinazolinone derivatives was carried
out by measuring the competition between 2-deoxyribose
and the synthesized compounds for hydroxyl radicals. The
assay is based on quantification of the degradation product of
2-deoxyribose by condensation with thiobarbituric acid. The
hydroxyl radicals (•OH) in aqueous media were generated
The antibacterial activity of compounds was determined by
agar disk diffusion method (Gillespie, 1994). Briefly, a
suspension of tested bacterial strains was spread on the
nutrient agar medium and potato dextrose agar for fungi. The
disks (6 mm in diameter) impregnated with test chemicals,
each 100 lg/ml in DMSO, were placed on the inoculated
agar. These plates were kept at 4 ꢁC for 2 h. Each plate was
then incubated at 37 ꢁC for 24 h in the case of bacteria and
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Med Chem Res (2013) 22:2665–2676
2671
through the Fenton system. The assay was performed by
mixing 0.36 ml of 2-deoxyribose (2.8 mM), 0.33 ml of
phosphate buffer (20 mM, pH-7.4), 1 ml of test solution
(10–100 ll), 0.1 ml of hydrogen peroxide (1 mM), 0.1 ml of
ascorbic acid (100 mM), 0.1 ml of EDTA (100 mM), and
0.01 ml of FeCl3 (100 mM) and this mixture was incubated
at 37 ꢁC for 1 h. Thereafter, 1 ml of cold 2.8 % trichloro-
acetic acid was added and reactivity was developed by
adding 1 ml of thiobarbituric acid (1 % w/v) followed by
heating at 100 ꢁC for 15 min, the absorbance of the cooled
mixture was measured at 532 nm. Butylated hydroxyanisole
(BHA) was used as a positive control. All samples and the
control were made in triplicate and the results were expres-
sed as mean values standard deviations.
Results and discussion
Chemistry
The strategy to synthesize the target quinazolinone deriva-
tives is depicted in Scheme 1. The first synthetic step
involves the amidation of methyl anthranilate with different
substituted acid chlorides (1a–f) in dichloromethane giving
rise to the respective diaryl amide intermediates. On the
other hand, different substituted aromatic acids (1g–l) were
converted to their corresponding acid chlorides by reaction
with an excess of thionyl chloride in THF and allowed to
react with methyl anthranilate to yield the respective diaryl
amide intermediates. The products (2a–l) were subjected to
hydrolysis using aqueous sodium hydroxide in methanol
and subsequent acidification, to afford the deprotected
carboxylic acid (Jeffrey et al., 2008) followed by cycliza-
tion of 3a–l using thionyl chloride to afford the desired
benzoxazinones 4a–l. Finally, benzoxazinones were cou-
pled with glutamine to give the respective 2,3-disubstituted
quinazolinone derivatives 5a–l in high yields. TLC was run
throughout the reaction to optimize the reaction for purity
and completion. The physicochemical data for the newly
synthesized compounds are presented. All the synthesized
compounds were characterized by their physical, analytical,
and spectral data. The chemical structures and physical data
of all the synthesized compounds are given in Table 1.
Compounds gave agreeing values in elemental analysis.
IR spectrum of compound 5e revealed the presence of
two carbonyl groups at 1,675 and 1,627 cm-1. It also
showed –NH2 and –COOH stretching bands at 3,354 and
2,667 cm-1, respectively, and these bands were observed
for other compounds at expected regions. A band in the
range of 2,660–2,674 cm-1 was obtained due to COOH
stretching. In general, the IR spectra of compounds 5a–
l exhibited C=N stretching absorption bands in the range of
1,539–1,589 cm-1. The IR spectra of all the compounds
showed characteristic band in the region 3,341–3,374 cm-1
indicating the presence of (–NH2) primary amine group.
The proton magnetic resonance spectra of synthesized
compounds were recorded in CDCl3. The chemical shift
and multiplicity patterns correlated well with the proposed
NO radical scavenging assay
NO radical scavenging capacity is based on the method of
Padmaja et al., (2011). The assay is based on generation of
NO from sodium nitroprusside (SNP) and it was measured
by the Griess reagent. SNP in aqueous solution at physio-
logical pH spontaneously generates NO which interacts
with oxygen to produce nitrite ions, which can be quantified
by the Griess reagent. The reaction mixture containing 1 ml
of SNP (10 mM), 1.5 ml of phosphate buffer (pH 7.4), and
test solution (10–100 ll) was incubated for 150 min at
25 ꢁC. Then, 1 ml of Griess reagent (1 % sulfanilamide in
3 % phosphoric acid and 0.2 % N-(1-naphthyl)ethylenedi-
amine dihydrochloride) was added to the reaction mixture
and allowed to stand for 3 min, the absorbance of this
solution was measured at 546 nm against the reagent blank.
All samples and controls were made in triplicate. IC50
values were determined and the results were expressed as
mean values standard deviations.
Superoxide radical scavenging assay
Superoxide radical scavenging activity was measured as
described by Kovala-Demertzi et al., (2004). The assay is
based on the reduction of nitroblue tetrazolium (NBT) by
superoxide ions generated by the xanthine/xanthine oxi-
dase (X–XO) system. The reaction mixture contained test
samples (10–100 ll), 0.2 mM xanthine, and 0.6 mM NBT
in 0.1 M phosphate buffer, pH 7.8. The reaction was started
by the addition of xanthine oxidase (0.07 U ml-1) to the
reaction mixture, an activity which allowed yielding the
absorbance change between 0.03 and 0.04 per minute, at
560 nm. The extent of NBT reduction was followed
spectrophotometrically, by measuring the increase of the
absorbance at 560 nm. All the experiments were replicated
three times. The IC50 of each compound was defined as the
concentration which inhibited 50 % of the NBT reduction
by superoxide ions produced by the X–XO system.
1
structures. H NMR spectra of all the synthesized quinaz-
olinone analogs showed NH2 proton as singlet at
5.35–5.64 ppm. The signal due to acidic OH in all the
analogs appeared as singlet at 11.21–12.16 ppm. In addi-
tion to acidic OH, –OCH3 protons present in the compound
5g resonated as a singlet at 3.67 ppm. Other aromatic
protons were observed at the expected regions.
The mass spectra of compounds are in agreement with
their structures. All the spectra exhibit parent peaks due to
molecular ions (M?) and the chlorine substituted com-
pounds give (M?2) peak. The proposed molecular formula
123

2672
Med Chem Res (2013) 22:2665–2676
Scheme 1 synthesis
of glutamine linked
2,3-disubstituted quinazolinone
derivatives (5a–l)
O
COOCH3
O
rt, 20 h
SOCl2, rt, 2h
R
C Cl
R
C OH
methyl
anthranilate
methyl anthranilate
rt, 20 h
NH
R
1g-l
1a-f
O
2a-l
O
COOH
SOCl2
aq NaOH
O
2a-l
-MeOH
reflux
NH
R
N
R
O
4a-l
3a-l
O
O
O
Glutamine
N
OH
NH2
N
R
5a-l
of each compound was confirmed by its molecular formula
weight with m/z values. The mass spectra of 5b and 5g
showed molecular ion peak M? at 405 and 381, respec-
tively, corresponding to their molecular formula.
structures of active compounds reveals that the halogen
substitution of phenyl ring increased antimicrobial activity.
The compound 5d possesses active inhibition against all
the tested strains and this may be attributed to the presence
of chlorine in the compound. It is interesting to note that
the electron withdrawing property of the phenyl ring is
important, which is corroborated by the eminent activity of
compounds with halogen group and decreased activity of
compounds with either methyl or methoxy group in the
phenyl ring. Hence, the replacement of methyl or methoxy
group in place of halogens shows decrease in antibacterial
activity. Other compounds exhibited moderate to good
antibacterial activity against all organisms. Similarly,
compound 5e exhibited poor antibacterial activity against
S. aureus, E. coli, and P. aeruginosa as compared to the
standard drug (streptomycin).
Antimicrobial activity
All the newly synthesized compounds were evaluated for
their in vitro antibacterial activity by disk diffusion method
and MIC against an assortment of two Gram-positive bac-
teria (B. subtilis and S. aureus), two Gram-negative bacteria
(P. aeruginosa and E. coli), and antifungal activity (against
C. albicans and A. flavus) was evaluated. Standard antibiotic,
streptomycin and antifungal drug fluconazole were used to
compare the antibacterial and antifungal activities shown by
compounds. Screening results are summarized in Table 2.
The newly synthesized compounds 5a–l exerted significant
inhibitory activity against the growth of tested bacterial
strains. The antibacterial data revealed that all tested com-
pounds of this investigation were found to exhibit moderate
to good activity against all the tested pathogenic bacteria as
compared to the standard drug streptomycin.
A notable observation being that all the compounds
containing COOH group showed better activity. This is due
to the increase in the polarity of these compounds (Suresha
et al., 2009), which would penetrate the molecules through
the lipid membrane and thus, they could inhibit the growth
of the microorganisms. It can be concluded that the anti-
microbial activity of such compounds may change by the
introduction or elimination of a specific group.
The most potent antibacterial activity was exhibited by
compound 5b (MIC 8.6 lg/ml) with halogen substituted
ring in the respective series compared to the compounds
bearing other electron donating or withdrawing groups.
The inclusion of nitro group to the phenyl ring (5h) pro-
duced moderate activity against S. aureus with a MIC of
9.6 lg/ml. A close analysis of the screening results and
The screening data of antifungal activity of these series of
compounds showed wide range of antifungal activity. Com-
pounds 5b and 5h exhibited good antifungal activity against
C. albicans and A. flavus compared to the standard drug
fluconazole. The remaining compounds were moderately
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Med Chem Res (2013) 22:2665–2676
2673
reactive oxygen species is becoming increasingly recog-
nized in the pathogenesis of many human diseases,
including cancer, aging, and atherosclerosis (Halliwell and
Whiteman, 2004). Antioxidants are playing a key role in
offering cure to various life-style related diseases (Wua
et al., 2011). So, their antioxidant capacity cannot be fully
described using a single method because antioxidant
capacity is influenced by many factors. Therefore, various
assays were performed to assess the radical scavenging
activity of the compounds in cell free system. Accordingly,
we have performed the following in vitro assays namely,
inhibition of DPPH radical, superoxide radical scavenging,
hydroxyl radical scavenging, and NO radical scavenging
methods to assess the antioxidant properties of synthesized
compounds, in comparison with the standards, such as
ascorbic acid and BHA.
Table 1 Chemical structure, yield, and physical characterization of
synthesized compounds
Compound
R
Yield (%)
85
m. p.(°C)
194–197
F
F
5a
5b
5c
78
80
196–198
216–218
F
F
F
F
F
5d
5e
76
73
190–193
214–216
Cl
A freshly prepared DPPH solution exhibited a deep
purple color with an absorption maximum at 517 nm. The
absorbance decreased when the antioxidant molecule
quenched DPPH free radical through donation of hydrogen
atom or electron to form a stable DPPH molecule, resulting
in a color change from purple to yellow. Hence, rapidity of
the absorbance decrease was linked the more potent anti-
oxidant activity of such compound.
5f
84
210–213
O
Cl
5g
5h
5i
82
76
80
82
212–215
198–200
211–213
214–216
OCH3
NO2
NH2
The scavenging effect of the synthesized compounds is
given in Table 3. All the compounds (5a–l) showed com-
parable or slightly less activity to the standards (ascorbic
acid and BHA). The antioxidant activity was expressed as
the 50 % inhibitory concentration (IC50) based on the
amount of compound required for a 50 % decrease in the
initial DPPH radical concentration. It was observed that all
the compounds notably reduced the concentration of DPPH
free radical. It was clearly demonstrated that free radical
scavenging increased with increasing concentration.
The difference in radical scavenging activity of the
synthesized quinazolinone compounds (5a–l) were due to
the difference in the stability of the oxygen centered radical
formed in these compounds. The better activity of
compound 5l (IC50 14.3 lg/ml) having hydroxyl group at
p-position in the aromatic ring is due to high electron-
releasing properties (positive mesomeric effect is higher
than negative inductive effect) and this activates the aro-
matic ring. The compound 5g (IC50 18.4 lg/ml) bearing an
electron donating methoxy group at para position showed
better DPPH radical scavenging activity compared to 5k
(IC50 29.7 lg/ml). The compound 5h (IC50 36.4 lg/ml)
exhibited less activity compared to compound 5g due to the
presence of electron withdrawing nitro group instead of a
methoxy group in the same position.
H3
C
5j
5k
5l
79
84
210–212
205–208
OH
active against these two microorganisms. In the whole series,
compound 5h with MIC 10 lg/ml showed the highest per-
centageinhibitionagainst both fungalstrains, whereasnoneof
the tested compounds restricted the fungal growth. The
compounds 5a and 5c displayed differences in activity due to
the presence of fluoro group at different positions. Instead of
methyl or methoxy groups, halogen substituted compounds
5a, 5b, and 5c seemed better in displaying pronounced
inhibitory power against C. albicans and A. flavus. Compound
5e showed very less potency in comparison with reference
fluconazole, while all other compounds exhibited MIC
between 10 and 100 lg/ml.
Antioxidant activity
Since the synthesized compounds exhibited good antimi-
crobial activity, it was considered worthwhile to study other
potential aspects of these compounds, such as antioxidant
and antiradical activity. The role of free radicals and
Observing the overall data for antioxidant activity, it is
clear that compounds 5g, 5i, and 5l were found to be the
most efficacious antioxidants among all the compounds,
obviously this confers great influence on radical scavenging
123

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Med Chem Res (2013) 22:2665–2676
Table 2 Antimicrobial activity of the synthesized compounds
Compounds
In vitro activity zone of inhibition in mm (MIC in lg/ml)
Gram positive
Gram negative
Fungi
S. aureus
B. subtilis
E. coli
P. aeruginosa
C. albicans
A. flavus
5a
11 (13)
13.2 (08)
14 (12.5)
13 (10)
10 (50)
12 (30)
14 (30)
14 (9.6)
10 (25)
11 (50)
11 (45)
13 (25)
14.1 (05)
–
16 (15)
13.5 (08)
12 (12.5)
12.7 (11.2)
07 (50)
14 (50)
10 (50)
16 (12.5)
13 (30)
12 (30)
11 (50)
09 (25)
13.4 (05)
–
12 (50)
12.7 (12)
11 (30)
11.5 (15)
11 ([100)
10 (50)
11 (20)
09 (25)
15 (50)
10 (50)
15 (50)
15 (50)
16.5 (05)
–
14 (25)
14.8 (10)
17 (35)
12 (15)
11 ([100)
13 (50)
13 (25)
11 (25)
16 (50)
11 (30)
12 (75)
13 (50)
14 (05)
–
11 (15)
13.6 (10)
11.4 (10)
13.6 (12)
14 (100)
13.7 (25)
10 (25)
14.1 (10)
12.5 (50)
12 (25)
14 (30)
09 (40)
–
10.4 (25)
14.4 (15)
10.9 (15)
12.1 (15)
10.2 (75)
11 (40)
14 (15)
13.6 (12.5)
11 (50)
15 (25)
10 (25)
15 (40)
–
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
Streptomycin
Flucanazole
16 (05)
19 (05)
activity due to the presence of electron donating OCH3,
NH2, and OH groups on the phenyl ring and their ability to
scavenge DPPH.
the compounds 5i and 5l were found to be 17.5 and 15.2
lg/ml, respectively. The NO generated from SNP at phys-
iological pH and its metabolic product peroxynitrite
(ONOO–) was formed after reacting with oxygen. The
nitrite formation was suppressed by compounds which
directly compete with oxygen in the reaction with NO. The
scavenging of NO was found to increase in dose-dependent
manner.
Hydroxyl radical scavenging capacity is directly related to
its antioxidant activity and hydroxyl radicals are the major
active oxygen species and they mediate various types of reac-
tions including enormous biological damage and lipid oxida-
tion (Boguslaw, 2011). Biologically, the hydroxyl radical is
widely believed to be generated when hydrogen peroxide reacts
with Fe(II) (Fenton reaction). This assay showed the abilities of
the compounds and standard to inhibit hydroxyl radical medi-
ated deoxyribose degradation in an Fe3?-EDTA-ascorbic acid
and H2O2 reaction mixture. The values of IC50 of compounds
for hydroxyl radical ranged from 17.8 to 55.1 lg/ml. The
compound 5l showed higher abilities of scavenging for
hydroxyl radical, possibly due to the key role of functional
group –OH, which can react with hydroxyl radical to form
stable macromolecular radicals by the typical H-abstraction
reaction (Ueda et al., 1996). The significant increase in the
scavenging activity, observed in compounds 5g and 5i com-
pared to that of compound 5k can be attributed to the presence
of methoxy and amine groups and for increased protection
against H2O2 rather than an increase in reactive oxygen species
concentrations. The scavenging of hydroxyl radical by the
compounds increased in dose-dependent manner.
Superoxide is biologically quite toxic and is deployed by
the immune system to kill invading microorganisms. It is
Table 3 Antioxidant activity of the synthesized compounds
Compounds IC50 (lg/ml)
DPPH
•OH
O2•
NO
5a
5b
5c
27.4 0.11 38.1 0.04 45.7 0.17 40.1 0.22
29.7 0.02 55.1 0.15 56.0 0.08 48.2 0.13
34.9 0.03 41.8 0.17 50.8 0.13 46.1 0.02
37.1 0.07 49.0 0.09 41.8 0.04 37.2 0.17
26.6 0.16 29.8 0.11 31.6 0.16 36.2 0.04
26.1 0.11 27.7 0.06 31.4 0.19 38.7 0.12
18.4 0.17 23.4 0.20 24.7 0.17 20.1 0.08
36.4 0.08 39.0 0.16 43.2 0.26 49.0 0.19
17.5 0.05 18.0 0.03 19.4 0.17 17.5 0.27
24.9 0.12 30.4 0.10 25.4 0.04 29.1 0.14
29.7 0.22 31.2 0.03 32.0 0.16 36.0 0.17
14.3 0.14 17.8 0.19 18.3 0.07 15.2 0.05
5d
5e
5f
5g
5h
5i
5j
In addition to reactive oxygen species, NO radical is
implicated in pathogenesis of several diseases and a potent
pleiotropic inhibitor of physiological processes. NO is a
very unstable species under aerobic condition. It reacts with
O2 to produce stable products nitrate and nitrite through
intermediates. Among the tested compounds, compounds 5i
and 5l showed the most potent activity. The IC50 values of
5k
5l
AAa
BHAb
a
12.6 0.43
–
–
–
–
15.3 0.76 13.4 0.29 14.6 0.11
Ascorbic acid
b
Butylated hydroxyanisole
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2675
Blois MS (1958) Antioxidant determinations by the use of a stable
free radical. Nature 26:1199–1200
Boguslaw L (2011) Hydroxyl radical and its scavengers in health and
disease. Oxid Med Cell Longev. doi:10.1155/2011/809696
Davies J (1994) Inactivation of antibiotics and the dissemination of
resistance genes. Science 264:375–382
Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the
biology of ageing. Nature 408:239–247
Furuyal T, Ritter T (2011) Catalysis for fluorination and trifluoro-
methylation. Nature 473:470–477
an oxygen centered radical with selective reactivity.
Superoxide anion is a precursor to active free radicals.
Superoxide is very harmful to cellular components (Liang
et al., 2010) and it has been observed to directly initiate
lipid peroxidation (Wickens, 2001). The superoxide radical
generated in vitro by X–XO can be measured by its ability
to reduce NBT. The synthesized compounds have strong
scavenging effect on superoxide radicals. The inhibitory
effects of the tested compounds on superoxide radical were
concentration related and the suppression ratio increased
with the increasing sample concentration. The compounds
clearly showed significant superoxide radical scavenging
activity compared with that of standard antioxidants like
BHA (IC50 10.19 lM). In the present study, the com-
pounds 5i and 5l showed the reduction of NBT and the IC50
values were found to be 19.4 and 18.3 lg/ml, respectively.
These values are higher than the value observed for other
synthesized compounds due to the presence of amine and
hydroxyl groups. Among others, the compounds 5a, 5b,
and 5c showed the least scavenging activity as shown by
the high value of IC50.
Gillespie SH (1994) Medical microbiology-illustrated Butterworth
Heinemann Ltd, Oxford
Grover G, Kini SG (2006) Synthesis and evaluation of new
quinazolone derivatives of nalidixic acid as potential antibacte-
rial and antifungal agents. Eur J Med Chem 41:256–262
Halliwell B, Whiteman M (2004) Measuring reactive species and
oxidative damage in vivo and in cell culture: how should you do
it and what do the results mean. J Pharmacol 142:231–255
Jayabharathi J, Thanikachalam V, Rajendraprasath N, Saravanan K,
Perumal MV (2012) Antioxidant potential and antimicrobial
screening of some novel imidazole derivatives: greenway
efficient one pot synthesis. Med Chem Res 21:1850–1860
Jeffrey JM, Jan B, Tomasz J (2008) Synthetic studies of cephalandole
alkaloids and the revised structure of cephalandole A. J Nat Prod
71:1447–1450
Jones RN, Barry AL, Gaven TL, Washington JA, Lennette EH, Balows
A, Shadomy WJ (1984) Manual of clinical microbiology, 4th edn.
American Society for Microbiology, Washington DC
Kovala-Demertzi D, Galani A, Demertzis MA, Skoulika S, Kotoglou
C (2004) Binuclear copper(II) complexes of tolfenamic: synthe-
sis, crystal structure, spectroscopy and superoxide dismutase
activity. J Inorg Biochem 98:358–364
Liang XL, Wang XL, Li Z, Hao QH, Wang SY (2010) Improved
in vitro assays of superoxide anion and 1,1-diphenyl-2-picrylhy-
drazyl (DPPH) radical-scavenging activity of isoflavones and
isoflavone metabolites. J Agric Food Chem 58:11548–11552
Maggio B, Daidone G, Raffa D, Plescia S, Mantione L, Cutuli VMC,
Mangano NG, Caruso A (2001) Synthesis and pharmacological
study of ethyl 1-methyl-5-(substituted 3,4-dihydro-4-oxoquinazo-
lin-3-yl)-1H-pyrazole-4-acetates. Eur J Med Chem 36:737–742
Mani Chandrika P, Yakaiah T, Raghu Ram Rao A, Narsaiah B,
Chakra Reddy N, Sridhar V, Venkateshwara Rao J (2008)
Synthesis of novel 4,6-disubstituted quinazoline derivatives,
their anti-inflammatory and anti-cancer activity (cytotoxic)
against U937 leukemia cell lines. Eur J Med Chem 43:846–852
Mhaske SB, ArgadeNP(2006)Thechemistryof recentlyisolatednaturally
occurring quinazolinone alkaloids. Tetrahedron 62:9787–9826
Mosaad SM, Mohsen MK, Emad MMK, Nageh A, Sherein IAM,
Marwa FA (2010) Novel 6,8-dibromo-4(3H)quinazolinone
derivatives of antibacterial and antifungal activities. Eur J Med
Chem 45:3311–3319
Compounds 5i and 5l showed the higher scavenging
effect than the other compounds. The compounds 5a and 5c
showed difference in activity due to the presence of fluoro
group at different positions. Compound 5h showed the
lowest radical scavenging activity due to the presence of
strong electron withdrawing nitro group situated at para
position, which would destabilize the ring.
Conclusion
In summary, we have synthesized a small library of novel
2,3-disubstituted quinazolinone analogs and assessed their
antimicrobial and antioxidant activities. Most of the new
compounds exhibited substantial antimicrobial and anti-
oxidant activity, while four analogs showed diverse anti-
microbial activity against selected kinds of bacteria and
fungi. In particular, three compounds (5g, 5i, and 5l)
showed higher potency of radical scavenging activity than
other analogs with IC50 values of 14.3–24.7 lg/ml.
Padmaja A, Rajasekhar C, Muralikrishna A, Padmavathi V (2011)
Synthesis and antioxidant activity of oxazolyl/thiazolylsulfonyl-
methyl pyrazoles and isoxazoles. Eur J Med Chem 46:5034–5038
Pandey VK, Tusi S, Tusi Z, Raghubir R, Dixit M, Joshi MN (2004)
Thiadiazolyl quinazolones as potential antiviral and antihyper-
tensive agents. Indian J Chem 43:180–183
Acknowledgments The authors are thankful to the University of
Mysore, Mysore for providing this project. The authors are grateful to
the MHRD and UGC, New Delhi for the funding through Institution
of Excellence Scheme. One of the authors M. K. Prashanth is thankful
to the University of Mysore, Mysore for the award of fellowship.
Parr A, Bolwell GP (2000) Phenols in the plant and in man. The
potential for possible nutritional enhancement of the diet by
modifying the phenols content or profile. J Sci Food Agric 80:
985–1012
References
Suresha GP, Prakasha KC, Shivakumara KN, Wethroe K, Gowda DC
(2009) Design and synthesis of heterocyclic conjugated peptides
as novel antimicrobial agents. Int J Pept Res Ther 15:25–30
Suthakaran R, Kavimani S, Venkapayya P, Suganthi K (2008)
Synthesis and antimicrobial activity of 3-(2-(4z)-4-substituted
Alagarsamy V, Vijayakumar S, Raja Solomon V (2007) Synthesis of
2-mercapto-3-substituted-5,6-dimethylthieno[2,3-d] pyrimidin-
4(3H)-ones as new analgesic, anti-inflammatory agents. Biomed
Pharmacother 61:285–291
123

2676
Med Chem Res (2013) 22:2665–2676
benzylidene-4,5-dihydro-5-oxo-2-phenylimidazol-1-yl)ethyl)-
6,8-un/dibromo subtituted-2-substituted quinazoline-(3h)-one.
Int J Pharm Bio Sci 1:22–29
Wickens AP (2001) Ageing and the free radical theory. Respir
Physiol 128:379–391
Wua CR, Lin WH, Hseu YC, Lien JC, Lin YT, Kuo TP, Ching H
(2011) Evaluation of the antioxidant activity of five endemic
Ligustrum species leaves from Taiwan flora in vitro. Food Chem
127:564–571
Ueda JI, Saito N, Shimazu Y, Ozawa T (1996) A comparison of
scavenging abilities of antioxidants against hydroxyl radicals.
Arch Biochem Biophys 333:377–384
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