Synthesis and evaluation of fused pyrimidine derivatives as anti-inflammatory, antiproliferative and antimicrobial agents

Article abstract of DOI:10.1007/s00044-011-9826-7

In this study, condensation of 2-amino-5-chlorobenzoic acid with benzoyl chloride afforded 2-benzamido-5- chloro benzoic acid which on cyclization with different primary amines yielded the corresponding 6-chloro-2-phenyl- 3-substituted quinazolin-4(3H)-ones (V1-V8). The reaction was monitored by thin layer chromatographic analysis. The structure of the synthesized compounds was confirmed by spectral studies and elemental analysis. All these compounds were screened for their anti-inflammatory, antiproliferative and antimicrobial activities. Anti-inflammatory activity was done by rat paw oedema method. Compounds V6 and V5 showed a good percentage inhibition of inflammation at the 2nd and the 3rd h, respectively. Antiproliferative activity was carried out byMTT assay againstChang liver,Hep2 andHeLa cell lines. Compounds V1, V5 and V8 showed antiproliferative activity at low concentrations. The minimum inhibitory concentrations (MIC) of the synthesized compounds against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus flavus was studied by microdilution assay. Compound V4 was found to be a potent antimicrobial agent against the organisms tested. Further, itwas selected for time kill study. These data revealed that the compounds containing chloro- and/or fluoro-substituted phenyl ring or N,N-dimethyl ethyl substitutions in the quinazolin-4(3H)-ones led to increase of their biological activities. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9826-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2998–3005
DOI 10.1007/s00044-011-9826-7
ORIGINAL RESEARCH
Synthesis and evaluation of fused pyrimidine derivatives as
anti-inflammatory, antiproliferative and antimicrobial agents
•
Seekarajapuram Dinakaran Vachala
•
Keloth Kothari Srinivasan
Peralam Yegneswaran Prakash
Received: 1 June 2011 / Accepted: 19 October 2011 / Published online: 6 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, condensation of 2-amino-5-chloro-
benzoic acid with benzoyl chloride afforded 2-benzamido-5-
chloro benzoic acid which on cyclization with different
primary aminesyielded the corresponding 6-chloro-2-phenyl-
3-substituted quinazolin-4(3H)-ones (V1–V8). The reaction
was monitored by thin layer chromatographic analysis. The
structure of the synthesized compounds was confirmed by
spectral studies and elemental analysis. All these compounds
were screened for their anti-inflammatory, antiproliferative
and antimicrobial activities. Anti-inflammatory activity was
done by rat paw oedema method. Compounds V6 and V5
showed a good percentage inhibition of inflammation at the
2nd and the 3rd h, respectively. Antiproliferative activity was
carriedoutbyMTT assayagainstChangliver, Hep2andHeLa
cell lines. Compounds V1, V5 and V8 showed antiprolifera-
tive activity at low concentrations. The minimum inhibitory
concentrations (MIC) of the synthesized compounds against
Pseudomonas aeruginosa, Staphylococcus aureus, Esche-
richia coli, Candida albicans and Aspergillus flavus was
studied by microdilution assay. Compound V4 was found to
be a potent antimicrobial agent against the organisms tested.
Further, itwas selected for time killstudy. These datarevealed
that the compounds containing chloro- and/or fluoro-substi-
tuted phenyl ring or N,N-dimethyl ethyl substitutions in the
quinazolin-4(3H)-ones led to increase of their biological
activities.
Keywords 4(3H)-Quinazolinone ꢀ
Minimum inhibitory concentration ꢀ Time kill study ꢀ
Anti-inflammatory ꢀ Antiproliferative ꢀ MTT assay
Introduction
The chemistry of heterocyclic compounds has been a
thought-provoking field of study for a long time. The
synthesis of novel 4(3H)-quinazolinone derivatives and
investigation of their chemical and biological behaviours
have gained more importance in recent decades in view of
their biological and medicinal values.
Quinazolinone nucleus is a versatile lead molecule and
has been the focus of great interest recently because its
derivatives have been found to possess wide spectrum of
pharmacological activities. Quinazolin-4(3H)-one deriva-
tives are useful heterocycles, possessing potent pharma-
cological activities such as anticonvulsant (Ponnilavarasan
et al., 2010), anti-inflammatory (Mosaad et al., 2010),
antihelminthic (Rajiv et al., 2008), antitumor (Cao et al.,
2005), antifungal (Chatrasal et al., 2010), antibacterial
(Vachala et al., 2008), MAO inhibitory activities (Sri-
vastava et al., 1980) and antiproliferative activities, as well
as inhibitory effects for thymidylate synthase and poly-
(ADP-ribose) polymerase (PARP) activities (Griffin et al.,
1998). Furthermore, the 2-substituted quinazolinones in
particular have been utilized as peptidomimetic scaffolds
with specificity for cholecystokinin, angiotensin and cer-
tain cell adhesion receptors (Banihashemi et al., 1998).
Several 2,3-disubstituted benzopyrimidine derivatives were
synthesized and tested for different biological activities.
The reports showed that the aryl substitution at 3rd position
enhances the biological activity.
S. D. Vachala (&) ꢀ K. K. Srinivasan
Department of Pharmaceutical Chemistry, College
of Pharmaceutical Sciences, MCOPS, Manipal University,
Madav Nagar, Manipal, Karnataka 576 104, India
e-mail: sdvachu@yahoo.com
P. Y. Prakash
Microbiology Department, Kausthuriba Medical College,
Manipal University, Manipal, Karnataka 576 104, India
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Med Chem Res (2012) 21:2998–3005
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Several methods have been reported for the synthesis of
4(3H)-quinazolinone derivatives (McNamara et al., 1990).
However, these methods suffer from drawbacks, such as longer
reaction time, complicated workup, and use of expensive and
hazardous chemicals with low yield (Varma etal., 1997). Usage
ofmicrowave irradiation (MWI) is well known for the synthesis
of a variety of compounds wherein chemical reactions are
accelerated because of selective absorption of microwave by
polar molecules. The coupling of MWI with solid-supported
reagents under solvent-free conditions provides unique chem-
ical processes with special attribute, such as enhanced reaction
rate, higher yield and greater selectivity. However, this tech-
nique requires an appreciable amount of solvent for adsorption
ofreactants and elution of products. In view of ongoing research
on neat synthesis, when the solvent-free reactions are coupled
with MWI, the same proved to be advantageous considering
environmental reasons as well as due to their uniform heating
effect and shorter reaction times.
was added dropwise with continuous shaking. After adding
benzoyl chloride, the reaction mixture was stirred for 1 h.
The crude product that separated out was filtered, washed
with cold water and recrystallized using ethanol (Mahadik
and Bhosale, 2004). The reaction was monitored by TLC
analysis using CHCl3:EtOH (0.5:2.5).
General procedure for the synthesis of quinazolin-4(3H)-
one derivatives (V1–V8)
Equimolar amount of 2-benzamido-5-chloro benzoic acid
and different amines were condensed in the presence of
DCC under MWI for 8–14 min (Kidwai et al., 2004). Upon
completion of reaction as monitored by TLC, the reaction
mixture was cooled, ice cold water was added, and the
solid separated was filtered off. Then, the product was
washed with cold ethanol and recrystallized from ethanol.
A combination of two or more biologically active
moiety could increase or decrease the activity. Keeping this
aspect in mind, in view of the involvement of biological
activities and the eco-friendly synthesis of 4(3H)-quinaz-
olinone, the purpose of the present study is to construct
some novel 6-chloro-2-phenyl-3-substitutued-4(3H)-Qui-
nazolinone as potential anti-inflammatory, antiproliferative
and antimicrobial agents.
Spectral data of the representative compounds
6-Chloro-3-(3-chloro-4-fluorophenyl)-2-phenylquinazolin-
4(3H)-one (V1): IR (KBr) (cm-1): 3039 (aromatic CH),
1
1683 (C=O), 1581 (C=N), 1099 (C–Cl), 1209 (C–F); H-
NMR (DMSO-d6): 7.52–8.1 (m, 12H, aromatic-H); MS:
384(M?1), 345, 322, 225, 176; Anal. Calc. for
C20H11Cl2FN2O: C, 62.36; H, 2.88; N, 7.27. Found: C,
62.52; H, 2.76; N, 7.41.
6-Chloro-3-(4-fluorophenyl)-2-phenylquinazolin-4(3H)-
one (V4): IR (KBr) (cm-1): 2929 (aromatic CH), 1631
Materials and methods
Experimental
1
(C=O), 1575 (C=N), 1099 (C–Cl), 1188 (C–F); H-NMR
(DMSO-d6): 7.37–7.51 (m, 12H, aromatic-H); MS:
350(M?1), 317, 273, 176, 103. Anal. Calc. for
C20H12ClFN2O : C, 68.48; H, 3.45; N, 7.99. Found: C,
68.52; H, 3.63; N, 7.71.
Chemistry
The melting points of the synthesized compounds were deter-
mined in open capillary tubes and were uncorrected. The IR
spectra of the compounds were recorded in the range of
500–4,000 cm-1 on Shimadzu FT-IR 8310 using KBr pellets.
6-Chloro-3-(3-chlorophenyl)-2-phenylquinazolin-4(3H)-
one (V5): IR (KBr) (cm-1): 2924 (aromatic CH), 1631
1
(C=O), 1570 (C=N), 1097 (C–Cl); H-NMR (DMSO-d6):
7.53–7.8 (m, 12H, aromatic-H); MS: 366(M?1), 350, 336,
255, 201, 127, 104. Anal. Calc. for C20H12Cl2N2O: C, 65.41;
H, 3.29; N, 7.63. Found: C, 65.58; H, 3.51; N, 7.82.
6-Chloro-3-(2-(dimethyl amino)ethyl)-2-phenylquinaz-
olin-4(3H)-one (V6): IR (KBr) (cm-1): 2945 (aromatic
CH), 1630 (C=O), 1556 (C=N); 1H-NMR (DMSO-d6):
7.47–7.59 (m, 8H, aromatic-H), 2.48 (t, 2H, –CH2–), 3.21
(t, 2H, –CH2–); MS: 327(M?1), 317, 152, 111, 98. Anal.
Calc. for C18H18ClN3O: C, 65.95; H, 5.53; N, 12.82.
Found: C, 65.72; H, 5.28; N, 12.76.
1
The H-NMR spectra were recorded on Joel, model GSV-
400 MHz spectrometer using CDCl3/DMSO-d6 as solvent. The
chemical shifts were reported as parts per million downfield
from tetramethylsilane (Me4Si). Mass spectra were recorded on
the Shimadzu GC-MS QP5050. The reactions were monitored
by thin layer chromatographic analysis on precoated SiO2 gel
(HF254, 200 mesh)-coated glass plates. The spots were visual-
ized by UV light. Micro analyses for C, H and N were per-
formed in Heraeus CHN Rapid Analyser. Satisfactory C, H and
N analyses were obtained for all the compounds.
6-Chloro-3-(4-chlorophenyl)-2-phenylquinazolin-4(3H)-
one (V8): IR (KBr) (cm-1): 2958 (aromatic CH), 1645
1
Synthesis of 2-benzamido-5-chloro benzoic acid
(C=O), 1595 (C=N), 1085 (C–Cl); H-NMR (DMSO-d6):
7.83–8.1 (m, 12H, aromatic-H); MS: 366(M?1), 317, 211,
121, 104, 98. Anal. Calc. for C20H12Cl2N2O: C, 65.41; H,
3.29; N, 7.63. Found: C, 65.28; H, 3.53; N, 7.46.
To a cooled solution of 5-chloroanthranilic acid (0.5 g) in
5 ml of 10% sodium hydroxide, 1 ml of benzoyl chloride
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Med Chem Res (2012) 21:2998–3005
Biological activities
Anti-inflammatory activity
Animals
prepared. A 96-well flat bottom tissue culture plate was
seeded with 2 9 103 cells in 0.1 ml of MEM (Eagle’s min-
imum essential medium) medium supplemented with 10%
FBS (foetal bovine serum) and allowed to attach for 24 h.
After 24 h of incubation, the cells were treated with test
compounds. Dimethylsulfoxide (0.2% DMSO) was used as a
solvent to prepare the test solutions. The cells in the control
wells were treated only with the medium containing 0.2%
DMSO. Each treatment was performed in triplicate. After
24 h of incubation, drug containing media was removed and
washed with 100 ll of phosphate buffered saline. To each
well of the plate, 100 ll of MTT reagent was added and
incubated for 4 h at 37°C. After the 4th h of incubation, the
plate was inverted on tissue paper to remove the MTT
reagent. To solubilize the formazan crystals, 100 ll of
DMSO was added to each well. The absorbance was mea-
sured at 540 nm. The IC50 values were calculated.
Albino rats of Wistar strain of either sex, weighing
150–180 g, were selected by random sampling technique
and used in the study of anti-inflammatory activity. The
animals had free access to standard commercial diet and
water ad libitum, and rats were kept in rooms maintained at
22 1°C with a 12-h light/dark cycle.
Carrageenan-induced paw oedema
The anti-inflammatory activity of the test compounds was
carried out by carrageenan-induced paw oedema method
(Winter et al., 1962). Carboxy methyl cellulose (0.5% w/v
CMC) was selected as vehicle to suspend the standard drug
and the test compounds. The animals were weighed, marked
for identification and divided into 10 groups each group
containing six animals. Oedema was induced in the left hind
paw of all rats by subcutaneous injection of 0.1 ml of 1%
(w/v) carrageenan in normal saline (0.9%) into their foot-
pads. The 1st group was kept as control and was given the
respective volume of the solvent. The 2nd to 9th groups were
given an aqueous suspension of the synthesized compounds
in a dose of 100 mg/kg body mass. The 10th group was
administered ibuprofen in a dose of 100 mg/kg (standard).
All the test compounds and the standard drug were admin-
istered orally, 1 h before the carrageenean injection. The
paw volume of each rat was measured using a digital ple-
thysmometer (UGO Basil, Italy), just before the carragee-
nean injection (0 h) and then hourly for 5 h post
administration of the carrageenan injection. The percentage
inhibition of paw volume for each test group was calculated
using the following equation:
Antimicrobial activity
Microbial strains
Transfer culture of bacterial strains: Pseudomonas aeru-
ginosa (ATCC 27853), Staphylococcus aureus (ATCC
25923), Escherichia coli (ATCC 25922); and fungal
strains Candida albicans (ATCC 24433) and Aspergillus
flavus (ATCC 15517) from the stock collection were car-
ried out on to Muller Hinton Agar (MHA) and Sabourauds
Dextrose Agar (SDA) plates, respectively. The cultures
were passaged aseptically thrice to ensure purity.
Microdilution assay
The broth micro dilution test was performed using sterile,
disposable, multiwell microdilution plates (96 U-shaped
wells) in 12 9 8 format (Venugopal et al., 2007). Column
1 was the media control, containing 200 ll of the medium
to determine the sterility of the medium. Column 11 was
the test compound growth control with 100 ll of sterile
medium and 100 ll of the test compound. It determines the
purity of the compound tested. Column 12 was the growth
control which contains 100 ll of medium and 100 ll of
diluted inoculum suspension.
Percentage of inhibition ð%Þ ¼ 100ð1 ꢁ ða ꢁ x=b ꢁ yÞÞ
where a = mean paw volume of treated animals after
carrageenan injection, x = mean paw volume of treated
animals before carrageenan injection, b = mean paw vol-
ume of control animals after carrageenan injection, and
y = mean paw volume of control animals before carra-
geenan injection
Initially, 100 ll of medium (MHB for bacteria and
RPMI for fungi) were added to all the wells of the plate
except the media control, where 200 ll was added. Next,
100 ll of each of the test compounds were added to their
respective wells in column 2. Column 2 contains 100 ll of
the highest concentration of the drugs and 100 ll of the
medium. By performing a serial dilution from column 2 to
column 10, varying concentrations of the test compounds
ranging from 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 and
In vitro antiproliferation assay
In vitro antiproliferative activity was performed by MTT
assay method (Mosmann, 1983). Exponentially growing
cancerous cells were harvested from 75 cm2 tissue culture
flask and a stock cell suspension (1 9 105cell/ml) was
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Med Chem Res (2012) 21:2998–3005
3001
Cl
COOH
NH2
0.39 lg/ml were achieved. Column 10 had 100 ll of the
lowest concentration of the drugs and 100 ll of the med-
ium. Finally, 100 ll of the diluted inoculum suspension
was added to wells from column 10 to column 2. The micro
dilution plates were incubated at 37°C and observed for
interpretation of bacterial and fungal MIC values after 24
and 48 h respectively. The last well in the dilution series
that does not demonstrate growth indicated by turbidity
and/or a pellet of microorganisms in the bottom of the
vessel corresponds with the minimum inhibitory concen-
tration (MIC) of the antimicrobial agent.
2-amino-5-chlorobenzoic acid
C6H5COCl
NaOH, stirring for 1h
COOH
Cl
O
N
H
Time kill study
2-(benzamido)-5-chlorobenzoic acid
The compounds which showed good antimicrobial activity at
low concentration were considered for time kill study. Test
tubes with 5 ml Mueller–Hinton broth (Difco) for S. aureus,
P. aeruginosa and E. coli (Difco) and RPMI broth (Difco)
C. albicans, A. flavus were used. Test drug (V4) concentra-
tions were chosen to comprise dilutions at the MIC range
1 mg/ml. A sensitivity threshold of 5 9 106 CFU/ml was
used to determine the 99.9% killing. The inoculum for
S. aureus, P. aeruginosa and E. coli and C. albicans was
prepared by diluting a 16 h broth culture to obtain the correct
inoculum 5 9 106 CFU/ml. In case of A. flavus, a spore
suspension was adjusted to 5 9 106 CFU/ml by counting
using a cell counter. After vortexing briefly, 1 ml of each
diluted inoculum was delivered by pipette and plated for via-
bility counts (0 h). Only tubes with an initial inoculum within
the range 5 9 106 CFU/ml were acceptable for the study.
The viability counts of test drugs (V4) suspensions were
performed at 2, 4, 6, 12, and 24 h by plating 0.1 ml ali-
quots of inoculum of each test microorganisms onto Try-
pticase soy agar (TSA) for bacteria and Sabourauds
dextrose agar (SDA) for fungi. The plates were incubated
for up to 48 h, and colony counts were noted for the
recovery, yielding 30–300 colonies. The lower limit of
sensitivity of colony counts was 250 CFU/ml.
RNH2
DCC, MWI, 8-14 minutes
O
Cl
R
N
N
6-chloro-2-phenyl-3-substituted quinazolin-4(3H)-one
(V1-V8)
Fig. 1 Synthetic route to 6-chloro-2-phenyl-3-substituted quinazolin-
4(3H)-ones
chlorobenzoic acid with benzoyl chloride gave 2-benz-
amido-5-chloro benzoic acid which on cyclization with
different primary amines by MWI yielded the corre-
sponding 6-chloro-2-phenyl-3-substituted quinazolin-4(3H)-
ones (V1–V8). The physicochemical properties of the
synthesized compounds are given Table 1.
The postulated structures of the synthesized compounds
were in accordance with IR, 1H-NMR, mass, and elemental
analyses. In the IR spectra of compounds V1–V8, the
carbonyl absorption bands were observed in the region of
1,630–1,690 cm-1. The C–H stretching band was seen
around 2,929–3,052 cm-1, whereas the –C=N stretching
band was observed around 1,556–1,580 cm-1. In the
1H-NMR spectra of these compounds, the methylene pro-
tons appeared as singlet at region of 1.3–1.8, while the
aromatic protons were seen as multiplet in the range of
7.35–8.10. Mass spectra showed the corresponding
molecular ion peaks for all the compounds. Satisfactory C,
H and N analyses were obtained for all the compounds.
All the synthesized compounds (V1–V8) were screened
for the anti-inflammatory activity by carrageenan-induced
rat paw oedema method. Carrageenan is a pro-inflammatory
agent, induces the inflammation by releasing inflammatory
mediators such as histamine, serotonin and cytokines leads
Time-kill assay results were analysed by determining the
-1
numbers of strains which yielded log CFU/ml in com-
10
parison with counts at 0 h, for test drugs at different time
intervals (Pankuch et al., 1994). Test drugs were considered
microbicidal at the MIC concentration that reduced the ori-
-1
ginal inoculum by C3 log CFU/ml (99.9%) and microbi-
-1
10
static, if inoculum was reduced by\3 log CFU/ml.
10
Results and discussion
Chemistry
The synthesis of hitherto unreported title compounds were
prepared as outlined in Fig. 1. Condensation of 2-amino-5-
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Med Chem Res (2012) 21:2998–3005
Table 1 Physicochemical properties of the synthesized compounds
Compound
code
R
Mol. formula
Reaction
time (min)
Yield (%)
Melting
point (8C)
Retention
factor (Rf)
V1
V2
V3
V4
V5
V6
V7
V8
3-Chloro-4-fluorophenyl
4-Fluorobenzyl
C20H11Cl2FN2O
13
10
11
12
14
11
9
77
82
83
85
77
89
84
81
224
198
208
184
148
202
190
220
0.60
0.81
0.81
0.56
0.70
0.80
0.67
0.82
C
C
C
C
C
C
C
21H14ClFN2O
21H14Cl2N2O
20H12ClFN2O
20H12Cl2N2O
18H18ClN3O
21H15ClN2O2
20H12Cl2N2O
4-Chlorobenzyl
4-Fluorophenyl
3-Chlorophenyl
N,N-Dimethyl ethyl
4-Methoxyphenyl
4-Chlorophenyl
8
cascade inflammation reactions in short period time and
later (up to 6 h) further augmentation of inflammation
reaction is mediated by prostaglandins (PGs) derived from
arachidonic acid by the action of prostaglandin H synthase
[also referred as cyclooxygenase (COX)]. The present study
illustrated that all the compounds are having anti-inflam-
matory activity. The inhibition of inflammation was given
in Table 2. The percentage inhibition of paw oedema and
the percentage potency of the compounds were represented
in Figs. 2 and 3. At 1st h, compound V3 showed very good
anti-inflammatory activity. The percentage inhibition of
inflammation was found to be 72.9. Other compounds did
not show any inhibitory activity at 1st h. At the 2nd h, all
the compounds were showing good anti-inflammatory
activity. Compounds V6 and V5 showed 88.42 and 86.8%
inhibitions of paw oedema, respectively. The standard drug
showed 65.3% anti-inflammatory activity. At the 3rd h and
the 4th h, compound V5 showed maximum inhibition of
inflammation, and it was found to be 81.3 and 79.1%,
respectively. At the 5th h, compound V3 showed good
inhibition of inflammation when compared with all the
other compounds. Compound V2 was found to be active
only at the 2nd h. After the 2nd h, it was not showing any
anti-inflammatory activity. These data revealed that the
presence of 4-chlorophenyl, 4-fluorobenzyl and N,N-dime-
thyl ethyl substitution in the structure played a major role in
their anti-inflammatory activity.
In vitro antiproliferative activity was done for all the
compounds by MTT assay using different cell lines. The
IC50 values were summarized in Table 3. Compounds V1,
V5 and V8 showed antiproliferative activity at lower
concentrations (\6.25 lg/ml against Chang liver cells and
Hep2 cells and\25 lg/ml against HeLa cells). Compounds
V2, V4 and V7 showed the activity at moderate concen-
trations (IC50:10.87–50 lg/ml). The IC50 of the standard
drug was found to be 1.72, 18.27 and 1.25 lg/ml against
Chang liver cells, Hep2 cells and HeLa cells, respectively.
Compound V3 had IC50 value ꢂ50 lg/ml against Chang
liver cells and Hep2 cells and [200 for HeLa cells. These
results showed that the compounds containing fluoro- or
chloro-substituted phenyl ring at the 3rd position might be
responsible for their antiproliferative potency.
Minimum inhibitory concentration of the test com-
pounds were determined by micro dilution assay using 96
well plates and the results were given in Table 4. Com-
pound V4 showed very good antimicrobial activity, and it
Table 2 Anti-inflammatory activity of the synthesized compounds V1–V8
Compound
code
Mean paw volume SEM
0 h
1 h
2 h
3 h
4 h
5 h
Ctrl
V1
1.1150 0.0437
1.1175 0.0454
0.7875 0.2477
1.1125 0.0823
1.1050 0.0378
1.0225 0.0263
1.0850 0.0719
1.0225 0.0287
1.0450 0.0393
1.0925 0.0437
1.2075 0.0405
1.1875 0.0301
1.1525 0.1026
1.1375 0.0798
1.2200 0.0460
1.1325 0.0269
1.1825 0.0698
1.1200 0.0363
1.1350 0.0357
1.0350 0.0456
1.4175 0.1931
1.3525 0.0366
1.2850 0.0595
1.2650 0.0977
1.2675 0.0800
1.0625 0.0269
1.1200 0.0631
1.1875 0.0466
1.1100 0.0274
0.9875 0.0411
1.2250 0.0484
1.1700 0.0587
1.1350 0.0134
1.1950 0.0803
1.2100 0.0816
1.0600 0.0248
1.1875 0.0409
1.2100 0.0510
1.2300 0.0722
0.9600 0.0474
1.2825 0.0862
1.3075 0.0614
1.2400 0.0422
1.2325 0.0807
1.2750 0.0750
1.0575 0.0144
1.1650 0.0539
1.1400 0.0363
1.1425 0.0832
0.9500 0.0397
1.2400 0.0754
1.2500 0.0374
1.2875 0.0539
1.1825 0.0859
1.2100 0.0861
1.1250 0.0284
1.2150 0.0441
1.1750 0.0328
1.1825 0.0527
0.9600 0.0543
V2
V3
V4
V5
V6
V7
V8
Ibuprofen
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Med Chem Res (2012) 21:2998–3005
3003
Fig. 2 Percentage inhibition of
paw oedema at 100 mg/kg body
weight
90
80
70
60
50
40
30
20
10
0
V1
V2
V3
V4
V5
V6
V7
V8
Standard
1
2
3
4
5
Hour
Fig. 3 Percentage potency of
the test compounds
Hour
Table 3 In vitro anti-cancer activity of the test compounds by MTT
assay
Table 4 Minimum inhibitory concentration of the test compounds
V1–V8
Compound
code
50% inhibitory concentration (lg/ml)
Chang liver cells
HeLa cells
Hep2 cells
Compound P.
code
S.
aureus
E. coli C.
albicans
A.
flavus
aeroginosa
V1
\6.25
17.95
[[50
16.58
\6.25
33.39
[25
\25
41.48
[200
\25
\6.25
10.87
[[50
10.77
\6.25
20.49
12.5
V2
V1
V2
V3
V4
V5
V6
V7
V8
[100
3.12
[100
[100
0.78 1.56
[100
12.5
12.5
1.56
[100
25
V3
V4
3.12
0.78
0.78
0.39
3.12
1.56
3.12
3.12
1.56
0.39
25
V5
\25
6.25
V6
[200
\50
25
6.25 [100
[100
100
V7
6.25
3.12
3.12
1.56
50
V8
6.25
\25
\25
12.5
[100
[100
Cisplatin
1.72
18.27
\1.25
12.5
12.5 [100
123

3004
Med Chem Res (2012) 21:2998–3005
Table 5 Time kill study of the test compound V4
-1
No (%) of strains with the indicated log CFU/ml at time intervals
10
Test strains
2 h
4 h
6 h
12 h
24 h
P. aeroginosa (ATCC 27853)
S. aureus (ATCC 25023)
E. coli (ATCC 25922)
6 (66.7)
3 (33.3)
3 (33.3)
4 (44.4)
1 (11.1)
6 (66.7)
6 (66.7)
6 (66.7)
6 (66.7)
2 (22.2)
9 (100)
9 (100)
9 (100)
9 (100)
8 (88.9)
9 (100)
9 (100)
9 (100)
9 (100)
6 (66.7)
9 (100)
9 (100)
9 (100)
9 (100)
9 (100)
C. albicans (ATCC 24433)
A. Flavus (ATCC 15517)
microbiology, KMC, and the management of Manipal University,
Manipal for providing all the facilities to carry out this study.
inhibited the growth of all the test organisms at very low
concentration. The MICs of compound V4 against
P. aerugenosa, S. aureus, E. coli, C. albicans and A. flavus
was found to be 0.78, 0.39, 0.39, 1.56, and 6.5 lg/ml,
respectively. Compound, V2, V3 and V7 were having
almost the same MIC values. Compound V1 was not
showing microbial growth inhibition at 100 lg/ml con-
centration. Its MIC value was found to be [100 lg/ml in
all the test strains used. The most potent compound V4 was
further studied for the time kill study. The result was
produced in Table 5. The growth of P. aeroginosa,
S. aureus, E. coli and C. albicans was completely inhibited
at the 6th h. At the 24th h, it prevented the growth of
A. flavus. Compound V4 was found to be bactericidal
against the strains tested. It also worked as a fungicidal
against C. albicans. Initially compound V4 was not
effectively reducing the growth of A. flavus. However, after
24 h of incubation, it showed complete inhibition of the
fungal growth. These reports revealed that the presence of
4-fluorophenyl substitution at the 3rd position in the qui-
nazolin-4(3H)-one (benzopyrimidinone) nucleus showed
microbicidal activity at low concentrations.
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