V. R. Avupati et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1031–1035
1033
adjusted the pH to 8.5 using 1 N NaOH) under constant aeration for
38 h. After hatching, 10 nauplii were placed in each vial and added
various concentrations of drug solutions in a final volume of 5 mL,
maintained at 37 °C for 24 h under light of incandescent lamps and
surviving larvae were counted. Each experiment was conducted
along with control (vehicle treated) at various concentrations of
the test substances. The percentage lethality was determined by
comparing mean surviving larvae of test and control tubes. The
ED50 values were obtained using fenny probed analysis software.
The result for the test compound was compared with the positive
control podophyllotoxin. The results of cytotoxicity study are given
in Table 1.
The investigation of cytotoxicity screening data revealed that
the compounds 18, 22 and 17 demonstrated comparatively the
most potent cytotoxicity, with ED50 values of 3.96 0.21,
4.02 0.19 and 4.13 0.11 lg/mL, respectively (Table 1). It is inter-
esting to note that the compounds 23, 20 and 21 also showed
length of
enhancing the cytotoxicity as seen in case of compound 24 (ED50
18.24 0.11 g/mL).
a,b-unsaturated carbonyl system is also relevant for
:
l
With respect to the antimicrobial activity, the standard strains
were procured from the National Collection of Industrial Microor-
ganisms (NCIM), National Chemical Laboratory, Pune, India. The
antimicrobial activity of the synthesized compounds (1–28) was
determined by agar well diffusion method as recommended by
the National Committee for Clinical Laboratory Standards,
(NCCLS).26 The compounds were evaluated for antimicrobial activ-
ity against bacteria viz. Bacillus subtilis (NCIM 2063), Bacillus pumi-
lus (NCIM 2327), Staphylococcus aureus (NCIM 2079), Micrococcus
luteus (NCIM 2155), Pseudomonas aeruginosa (NCIM 2036), Klebsi-
ella pneumonia (NCIM 5082), Escherichia coli (NCIM 2065), Proteus
vulgaris (NCIM 2813), Candida albicans (NCIM 3102), Aspergillus ni-
ger (NCIM 548), Aspergillus oryzae (NCIM 643) and Penicillium
chrysogenum (NCIM 738). Serial solutions of compounds (1–28)
were diluted in dimethyl sulfoxide (1% DMSO) to give a final con-
appreciable cytotoxicity with ED50 values of 6.11 0.13,
6.92 0.11 and 8.34 0.22
lg/mL, respectively. The other com-
centration ranging from 16 to 512 lg/mL used for determining MIC
pounds such as 1, 2, 4–8, 11–13, 16, 19, 24, 26 and 27 showed mod-
erate level of activity at concentrations ranging from 12.78 0.11 to
value. The Minimum inhibitory concentration (MIC) was defined as
the lowest concentration of compound required for a complete
inhibition of the bacterial and fungal growth after incubation time.
For antibacterial activity nutrient agar was used seeded with
0.1 mL of the respective bacterial culture strains suspension pre-
pared in a sterile saline (0.85%) of 105 CFU/mL dilution. For anti-
fungal activity, different fungal spore suspensions in sterile
distilled water were adjusted to give a final concentration of
106 CFU/mL. An inoculum of 0.1 mL spore suspension of each fun-
gus was spread on Potato-Dextrose-Agar (PDA) plates. The wells of
6 mm diameter were filled with 0.1 mL of each compound having
different concentrations separately for each test of bacterial and
fungi strain. The DMSO (1%) alone was used as a control. The anti-
28.11 0.15
exhibited comparatively less activity with ED50 values ranging from
32.11 0.16 to 44.46 0.15 g/mL in comparison with the standard
drug (podophyllotoxin, ED50: 3.61 0.17 g/mL).
lg/mL. The compounds 3, 5, 9, 10, 14, 15, 25 and 28
l
l
A close look at the SAR (structure–activity relationship) of these
compounds clearly revealed the inherent cytotoxicity associated
with the basic skeleton consisting of diarylsulfonylurea22 and
a
,b-
unsaturated ketone moieties27 as seen in case of the unsubstituted
compound 1 with ED50 value of 25.01 0.15 g/mL, which in some
l
cases was enhanced by the influence of some substituents and de-
creased by some other substituents. For example, the compounds
18, 22 and 17 having halogen substituents either at meta or para
positions significantly enhanced the activity. A reduction in the
activity was observed when the substituted phenyl ring was re-
placed by a bulky anthracene moiety, as seen in the case of com-
biotic chloramphenicol (16 lg/mL) and ketoconazole (16 lg/mL)
are used as reference antibacterial and antifungal agents, respec-
tively for comparison. Inoculated plates in triplicate were then
incubated at 37 0.5 °C for antibacterial activity for 24 h and
48 h at 28 0.2 °C for antifungal activity. After incubation, the min-
imum inhibitory concentrations (MICs) were noted. The results of
antimicrobial activity studies are given in Table 2.
pound 28 with ED50 value 44.32 0.15
pyrrole ring in compound 25 in the place of substituted phenyl ring
of ,b-unsaturated carbonyl system enhanced the activity com-
lg/mL. The presence of a
a
pared to the one possessing anthracene moiety, but less than that
of the one having substituted phenyl ring. It is also interesting to
see the presence of pyridine ring in the place of substituted phenyl
ring contributing to an increase in activity compared to the one pos-
sessing pyrrole moiety, as seen in the case of compounds 26 and 27
The results of antimicrobial activity of the synthesized com-
pounds (1–28) against selected Gram-positive, Gram-negative bac-
teria and fungi are illustrated in Table 2. The compounds 14 and 23
were found to be more active than other compounds with MIC 16–
32
10, 11, 15, 25 and 28 showed less antimicrobial activity with
MIC 256–512 g/mL. From the results of antibacterial activity,
compounds 14, 16 and 17 were found to be more active against
all Gram-positive bacteria with MIC value 16 g/mL, but with less
lg/mL against all tested microorganisms. Compounds 1, 2, 9,
with ED50 values 14.09 0.16 and 16.83 0.15 lg/mL, respectively.
It is also reported that the cytotoxicity of compounds 2–8, substi-
tuted with electron releasing groups was found to be biologically
relevant (6 (3,4-diOCH3) > 2 (4-CH3) > 7 (2,4-diOCH3) > 8 (3,4,5-tri-
OCH3) > 4 (3-OCH3) > 5 (4-OCH3) > 3 (N(CH3)2)) with ED50 values
l
l
potency against M. luteus. Among all the tested compounds, com-
ranging from 12.86 0.14 to 38.12 0.12 lg/mL. It is important
pound 23 showed significant inhibition against all Gram-negative
that less activity was observed when the hydroxyl groups are
substituted at different positions on the phenyl ring as seen in the
case of compounds 9–11 with ED50 values 32.33 0.12,
bacteria with MIC 16
E. coli. A broad spectrum of antifungal activity of the compounds
14 and 23 was obtained against all the fungi with MIC 16 lg/mL,
lg/mL except a moderate potency against
39.26 0.17 and 24.30 0.17
loss of activity observed when the nitro groups are introduced on
the phenyl ring of ,b-unsaturated carbonyl system as seen in the
case of compounds 14 and 15 with ED50 values 40.12 0.11 and
l
g/mL, respectively. It is notable that
while other compounds displayed less antifungal activity.
From the obtained data, the following conclusions on antimi-
crobial activity can be made: the compounds substituted with hal-
ogens on the phenyl ring at ortho, meta and para positions
enhanced the antibacterial activity (F > Cl > Br) as seen in the case
of compounds 17–22. It is also interesting to observe an increase in
activity when the nitro group is present at the ortho position of the
phenyl ring when compared to the substitution at meta position as
seen in the case of compounds 14 and 15. The compound 16 having
nitro group at position 2 and hydroxyl group at position 5 of the
phenyl ring also contributed favorably to the antibacterial activity.
It is noteworthy that compounds 2–8 having electron donating
substituents (OCH3 > N(CH3)2 > CH3) on the phenyl ring at ortho,
a
44.46 0.15
mL), 13 (ED50: 17.80 0.11
l
g/mL. The compounds 12 (ED50: 19.20 0.12
l
l
g/
g/
lg/mL) and 16 (ED50: 18.29 0.13
mL) having the hydroxyl group substitution on the phenyl ring
along with the ethoxy group at position 3 (12), methoxy group at
3 (13) and nitro group at 2 (16) positions, respectively showed en-
hanced level of cytotoxicity when compared with that of the com-
pounds (9–11) possessing only hydroxyl group substitution. The
compound 23 (ED50: 6.11 0.13
lg/mL) having allyloxy group
exhibited good level of cytotoxicity. An increase in the carbon chain
Avupati, Vasudeva Rao
