J. Lal et al. / European Journal of Medicinal Chemistry 64 (2013) 579e588
587
CH3), 3.83 (s, 6H, 30,300, OCH3), 5.39 (s, 2H, 40,400, OH), 6.48 (d,
J ¼ 15.72 Hz, 2H, H-50,500), 6.55 (d, J ¼ 8.68 Hz, 1H, CH), 6.99 (d,
J ¼ 7.76 Hz, 2H, H-20,200), 7.05 (s, 1H, NH), 7.21 (m, 5H, C6H5), 7.46 (d,
J ¼ 8.52 Hz, 2H, H-1,7), 10.72 (s, 1H, NH); 13C NMR (400 MHz,
dilution method [25]. MuellereHinton broth solution was prepared
in distilled water and autoclaved for 30 min at 121 ꢁC and 15 IP
pressure. 150
well.150 L of tested compound was filled with each well containing
the broth and serially diluted to obtain 80, 40 and 20 M concen-
trations. The test organism (50 L) was pipetted into each of the well
mL of sterile nutrient broth was pipette out into each
m
DMSO-d6):
d
56.12, 95.12, 107.23, 112.22, 120.84, 135.14, 141.01,
m
151.11, 169.41; MS (ESI): m/z 705.11 (M þ H)þ.
m
containing the mixture of the broth and tested compound and then
finally incubated at 37 ꢁC for 24 h to allow maximum growth of the
organisms.
4.3.9. 4-Sulfanilamide curcumin oxazole (7a)
Dark brown solid; IR (
n
cmꢀ1, KBr): 3466 (OeHstr), 3230 (CeHstr),
3.82 (s, 6H, 30,300,
2939 (CeHstr), 1512 (NeHben), 1463 (C]Cstr), 1429 (CeOstr), 974e
833 (CeHdef); 1H NMR (400 MHz, DMSO-d6):
d
5.3. Antifungal activity assay
OCH3), 5.91 (s, 2H, 40,400, OH), 6.56 (d, J ¼ 2.16 Hz, 2H, H-60,600), 6.81 (d,
J ¼ 8.08 Hz, 2H, H-20,200), 7.05 (d, J ¼ 9.04 Hz, 2H, H-50,500), 7.08 (d,
J ¼ 9.76 Hz, 2H, H-1,7), 7.16 (s, 2H, Ar), 7.57 (m, 5H, C6H5); 13C NMR
The newly synthesized curcumin derivatives of sulfonamides
were tested for their in vitro antifungal activity against pathogenic
fungi viz. A. niger, A. flavus, T. viride and C. lunata by disc diffusion
(400 MHz, DMSO-d6): d 56.02, 100.08, 112.12, 116.23,122.08, 133.32,
139.20, 144.22, 150.06, 158.28; MS (ESI): m/z 549.18 (M þ H)þ.
method, at concentrations of 80, 40 and 20 mM. A spore suspension
was prepared in sterile distilled water from 4-day old culture of the
tested fungi growing on Potato Dextrose Agar (PDA) media. The final
spore concentration was adjusted to 1.5 ꢂ 105 CFU/mL and the spore
suspension was used for the antifungal activity assay. Inocula
(0.2 mL) were applied on the surface of Potato Dextrose Agar plate
and spread by using sterile spreader. The sterile filter paper discs
(5 mm diameter, Whatman filter paper No. 1) were soaked with
tested compound these soaked discs were placed in each plate. These
plates were then incubated for 48 h at 28 ꢁC. Zone of inhibition in mm
was determined after 48 h fluconazole was used as positive control
drug in order to check the sensitivity status of tested fungal strains
and acetone was used as negative control (Table 3).
4.3.10. 4-Sulfamethoxazole curcumin oxazole (7b)
Brown solid; IR (
1474 (NeHben), 1340 (C]Cstr), 1286 (CeOstr), 1106 (CeSstr), 962e
n
cmꢀ1, KBr): 3003 (OeHstr), 2942 (CeHstr),
871 (CeHdef); 1H NMR (400 MHz, DMSO-d6):
d
3.84 (s, 6H, 30,300,
OCH3), 5.83 (s, 2H, 40,400, OH), 6.49 (d, J ¼ 15.68 Hz, 2H, H-50,500), 6.81
(d, J ¼ 8.12 Hz, 1H, CH), 6.99 (d, J ¼ 9.28 Hz, 2H, H-20,200), 7.06 (s, 1H,
NH), 7.31 (s, 2H, Ar), 7.50 (m, 5H, C6H5), 7.56 (d, J ¼ 9.64 Hz, 2H, H-
1,7), 8.04 (1H, NH), 9.71 (s, H, NH),; 13C NMR (400 MHz, DMSO-d6):
d
56.11, 100.02, 112.08, 116.11, 120.22, 139.12, 144.01, 150.22, 158.06,
169.02; MS (ESI): m/z 630.17 (M þ H)þ.
5. Pharmacological activity assays
5.4. Minimum inhibitory concentration assay
5.1. Antibacterial activity assay
Minimum inhibitory concentration (MIC) of the synthesized
derivatives of curcumin against above mentioned fungal cultures
was evaluated. In this method the medium used was potato
dextrose broth, and test compounds were dissolved in acetone and
All the synthesized curcumin derivatives were evaluated for the
in vitro antimicrobial susceptibility test by determining zone of in-
hibition adopting KirbyeBaur’s method [24]. Stock solution (160 mM)
of curcumin and synthesized derivatives of sulfonamides were pre-
pared in acetone (which has no activityagainst test microorganisms).
Ciprofloxacin was used as a positive control; stock solution was
prepared in autoclaved double distilled water. The stock solutionwas
doubly diluted to make various test concentrations (80, 40 and
adjusted to get desired concentrations of 80, 40 and 20 mM. The test
tubes containing tested compounds and spore suspension were
shaken several times to ensure proper and uniform distribution of
the test compound and incubated at 28 ꢁC for 48 h.
20
5 mm diameter were sterilized by autoclaving for 20 min at 121 ꢁC
and 15 IP pressure. 4 L of above test sample solutions in different
mM) in sterile 2 mL tubes. Whatman filter paper No.1 paper disc of
5.5. Cytotoxic activity assay
m
Human cancer cell lines, HeLa, Hep-G2, QG-56 and HCT-116
were used for in vitro cytotoxic activity of the synthesized sulfon-
amides containing curcumin derivatives using standard MTT assay
[26]. At first, the concentration of curcumin derivatives was diluted
with RPMI 1640 culture media into 2.5, 5, 10, 20, 50, 100 and
concentrations were soaked on these sterile filter paper discs and
dried at 50 ꢁC for 30 min. MuellereHinton agar was prepared in
distilled water and autoclaved at same temperature and pressure,
cooled and poured into sterilized petri plates (25 mL in each plate) to
solidify. Bacterial inoculum of each tested bacteria viz. S. aureus,
B. cereus, S. typhi, P. aeruginosa and E. coli were at concentrations of
1.5 ꢂ 108 CFU/mL and then diluted to 3 ꢂ 102 CFU/mL. From these
200
m
M, Cells (5 ꢂ 104) were seeded in 96-well cell culture plates in
culture medium and grown over a period of 24 h. Nutrient media
without cells were used as a blank and adriamycin was used as
positive control. After the incubation period, 20 mL of MTT solution
were added to each sample. Samples were incubated for 4 h at
37 ꢁC. The formazan crystals generated during the incubation
period were dissolved by adding 0.1 mL of MTT solubilization so-
lution in each well and gently mixing the solution. After the crystals
were fully dissolved, the optical density of the solutions at 570 nm
was measured using ELISA reader.
diluted bacterial suspension, 50 mL were spread in each Muellere
Hinton agar plate with the help of sterilized spreader. The paperdiscs
soaked with tested curcumin derivatives were placed at equal dis-
tances aseptically over the inoculated MuellereHinton agar plates
using sterile forceps. The plates were then incubated at 37 ꢁC for 24 h
to allow growth of the organisms. The test material having antimi-
crobial activity inhibited the growth of the microorganisms and a
clear, distinct zone was visualized surroundings the discs. The anti-
microbial activity of the test agents was determined by measuring
the diameter of zone of inhibition after 24 h incubation (Table 2).
5.6. Anti-inflammatory activity assay
Carageenan-induced activity of the sulfonamide containing cur-
cumin derivatives was carried out as described in literature [27]. A
total number of 20 mice were weighed and randomly divided in four
groups (five per cage) and were kept for fasting for 2 h before
experiment. One of the groups acted as negative control (normal
5.2. Minimum inhibitory concentration assay
The minimum inhibitory concentration (MIC) of the tested cur-
cumin derivatives were determined using broth micro serial
Lal, Jaggi
