Synthesis, antiinflammatory and antimicrobial activities of some 2,4-dichloro-5-fluorophenyl substituted arylidenetriazolothiazolidinones

Article abstract of DOI:10.1007/s00706-007-0794-z

A series of 2,4-dichloro-5-fluorophenyl substituted arylidenetriazolothiazolidinones were obtained by one-pot reaction of 3-(2,4-dichloro-5-fluorophenyl)-4H-1,2,4-triazole-5-thiol with substituted benzaldehydes and monochloroacetic acid in the presence of acetic anhydride, acetic acid, and sodium acetate. The structures of the newly synthesized compounds were characterized and confirmed by IR, ¹H NMR, mass spectra, and elemental analysis. Compounds bearing the 4-methylthiophenyl, 3,4-methylenedioxyphenyl, and 2,3,5-trichlorophenyl moiety showed excellent antiinflammatory activity. The antimicrobial screening studies revealed that compounds with 4-anisyl, 4-methylthiophenyl, 3,4-methylenedioxyphenyl, and 2,3,5-trichlorophenyl at position 5 of the arylidenetriazolothiazolidinone moiety showed excellent activity against all tested strains at 6.25 μg cm^-3 concentrations.

Full text of DOI:10.1007/s00706-007-0794-z

Monatsh Chem 139, 691–696 (2008)
DOI 10.1007/s00706-007-0794-z
Printed in The Netherlands
Synthesis, antiinflammatory and antimicrobial activities of some
2,4-dichloro-5-fluorophenyl substituted arylidenetriazolothiazolidinones
Mari S. Karthikeyan, Bantwal S. Holla
Department of Chemistry, Mangalore University, Mangalagangothri, Karnataka, India
Received 6 August 2007; Accepted 31 August 2007; Published online 21 April 2008
# Springer-Verlag 2008
Abstract A series of 2,4-dichloro-5-fluorophenyl
substituted arylidenetriazolothiazolidinones were ob-
tained by one-pot reaction of 3-(2,4-dichloro-5-fluor-
ophenyl)-4H-1,2,4-triazole-5-thiol with substituted
benzaldehydes and monochloroacetic acid in the
presence of acetic anhydride, acetic acid, and sodium
acetate. The structures of the newly synthesized com-
din-4-one ring systems, which is a core structure in
various synthetic pharmaceuticals displaying a broad
spectrum of biological activities [1]. The thiazo-
lidin-4-one ring system also occurs in Nature as
actithiazic acid, ((ꢀ)2-(5-carboxypentyl)thiazolidin-
4-one), which is isolated from Streptomyces strains [2].
Thiazolidinone derivatives are known to exhibit
diverse bioactivities such as anticonvulsant [3], anti-
diarrheal [4], antimicrobial [5], antidiabetic [6], cy-
cloxygenase inhibitory [7], Ca2þ channel blocker [8],
cardioprotective [9], anticancer [10], anti HIV [11],
and tumor necrosis factor-ꢁ-antagonist activities [12].
1,2,4-Triazoles represent an overwhelming and rap-
id developing field in modern heterocyclic chemistry.
From literature, it is predictable that 1,2,4-triazoles
represent important pharmacophores, and have a wide
range of therapeutic properties. They play a vital role
as medicinal agents due to different biological activi-
ties and lot of work might be carried out on this moiety
for obtaining better therapeutic molecules. At present
several triazole bearing compounds like Flutrox,
Nefazodone, Trazodone, Triazoledione, etc., are used
in modern medicine. A degree of respectability has
been bestowed for 1,2,4-triazole derivatives due to
their antibacterial, antifungal [13], antitubercular [14],
and anticancer [15] properties. Certain 1,2,4-triazoles
also find applications in the preparation of photograph-
ic plates, polymers, and as analytical agents [16].
The importance of fluorine containing compounds
in general, and heterocyclic in particular, has initiated
active research on fluorine containing heterocycles.
1
pounds were characterized and confirmed by IR, H
NMR, mass spectra, and elemental analysis. Com-
pounds bearing the 4-methylthiophenyl, 3,4-methyle-
nedioxyphenyl, and 2,3,5-trichlorophenyl moiety
showed excellent antiinflammatory activity. The anti-
microbial screening studies revealed that compounds
with 4-anisyl, 4-methylthiophenyl, 3,4-methylene-
dioxyphenyl, and 2,3,5-trichlorophenyl at position
5 of the arylidenetriazolothiazolidinone moiety
showed excellent activity against all tested strains at
6.25ꢀg cmꢀ3 concentrations.
Keywords 3-(2,4-Dichloro-5-fluorophenyl)-4H-1,2,4-tria-
zole-5-thiol; One-pot reaction; Arylidenetriazolothiazolidi-
nones; Antiinflammatory; Antimicrobial activity.
Introduction
Thiazolidinone derivatives constitute an important
class of heterocyclic compounds. There has been
considerable interest in the chemistry of thiazoli-
Correspondence: Mari Sithambaram Karthikeyan, Department
of Chemistry, Mangalore University, Mangalagangothri
574199, Karnataka, India. E-mail: karthims02@rediffmail.com

692
M. S. Karthikeyan, B. S. Holla
The replacement of hydrogen or hydroxyl by a fluo-
rine atom can alter the pKa, dipole moments, and
even the chemical reactivity and stability of neigh-
boring functional groups [17].
Fluorine containing heterocycles, triazoles, and
thiazolidinones displayed varied pharmacological
properties. Since there has been no report on dichlo-
rofluorophenyl containing arylidenetriazolothiazolidi-
nones, it was contemplated to synthesize them and to
pursue their antibacterial and antifungal screenings.
The corresponding results are presented in this paper.
Ref. [19]. 2-(2,4-Dichloro-5-fluorobenzoyl)hydrazine
carbothioamide (4) was obtained by the treatment of
2,4-dichloro-5-fluorobenzoyl hydrazide (3) with po-
tassium thiocyanate in presence of concentrated
hydrochloric acid, which on further cyclization in
the presence of base yielded 3-(2,4-dichloro-5-fluoro-
phenyl)-4H-1,2,4-triazole-5-thiol (5) in good yield.
A series of 2,4-dichloro-5-fluorophenyl containing
arylidenetriazolothiazolidinones (6) were obtained
in good yields by an one-pot reaction of 3-(2,4-di-
chloro-5-fluorophenyl)-4H-1,2,4-triazole-5-thiol (5)
with substituted benzaldehydes, and monochloro-
acetic acid in the presence of acetic anhydride, acetic
acid, and sodium acetate.
Results and discussion
Chemistry
Pharmacological studies
Antiinflammatory studies
2,4-Dichloro-5-fluorobenzoic acid (2) was obtained
(Scheme 1) from 2,4-dichloro-5-fluoroacetophenone
(1) according to Ref. [18]. 2,4-Dichloro-5-fluoro-
benzoyl hydrazide (3) was prepared according to
Few selected compounds were screened for their an-
tiinflammatory activity by Carrageenan induced rat
Scheme 1

2,4-Dichloro-5-fluorophenyl substituted arylidenetriazolothiazolidinones
693
Table 1 Antiinflammatory activity data of arylidenetriazo-
lothiazolidinones
Staphylococcus aureus, Pseudomonas aeruginosa,
Streptococcus pyogenes, and Klebsiella pneumo-
niae (recultured) bacterial strains by the disc diffu-
sion method [21, 22]. The diameter of the zone of
inhibition and minimum inhibitory concentration
values are given in Table 2. The antibacterial
screening data revealed that 6a, 6b, 6e, 6g, and
6l were active against S. aureus and E. coli. Com-
pounds 6a, 6b, 6e, 6i, and 6l were active against
P. aeruginosa and S. pyogenes. Especially com-
pounds 6a, 6b, 6e, 6j, and 6l exhibited good
antibacterial activity against all tested bacterial
strains almost equivalent to that of the standard
drug Ciprofloxacin.
Compd.
no.
Dose
(mg=kg,
p.o.)
Increase
in paw
volume=cm3
Inhibition
of paw
oedema=%
6a
6b
6c
6d
6e
6f
6g
6h
6j
6l
control
standard
50
50
50
50
50
50
50
50
0.46 ꢂ 0.0027
0.34 ꢂ 0.0037
0.52 ꢂ 0.0016
0.58 ꢂ 0.0017
0.37 ꢂ 0.0031
0.69 ꢂ 0.0017
0.48 ꢂ 0.0020
0.65 ꢂ 0.0011
0.26 ꢂ 0.0020
0.16 ꢂ 0.0026
0.65 ꢂ 0.0096
0.24 ꢂ 0.0014
26.6
48.4
18.8
9.4
42.2
0
25
0
59.4
75
50
50
10 cm3=kg
–
62.9
ꢁ
2
ꢁ
Antifungal studies
Indomethacin is used as the standard
The newly prepared compounds were screened
for their antifungal activity against Aspergillus
niger, Candida albicans, Aspergillus fumigatus,
Penicillium marneffei, and Trichophyton mentagro-
phytes (recultured) by the agar diffusion method [23,
24]. The diameter of zone of inhibition and minimum
inhibitory concentration values are given in Table 3.
The antifungal screening data showed that compounds
6a, 6g, 6j, and 6l showed good activity against
A. fumigatus and P. marneffei at 6.25ꢀg cmꢀ3 concen-
trations. Compounds 6a, 6j, and 6l exhibited good
antifungal activity against all tested fungal strains
almost equivalent to that of the standard drug,
Amphotericin B.
paw oedema (Acute-inflammation model) as described
by Winter et al. [20]. The results were expressed as %
inhibition of oedema over the untreated control group.
The anti-inflammatory results revealed that com-
pounds 6b, 6j, and 6l showed excellent antiinflamma-
tory activity whereas compound 6e showed moderate
antiinflammatory activity compared to that of the
standard drug, Indomethacin. The results of antiin-
flammatory studies are given in Table 1.
Antibacterial studies
The newly prepared compounds were screened for
their antibacterial activity against Escherichia coli,
Table 2 Antibacterial activity (diameter zones=mm) of inhibition (MIC=ꢀg cmꢀ3) of arylidenetriazolothiazolidinones
Compd.
no.
Staphylococcus
aureus
Escherichia
coli
Pseudomonas
aeruginosa
Klebsiella
pneumoniae
Streptococcus
pyogenes
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
standard
21 (6.25)
19 (6.25)
14 (12.5)
9 (25)
23 (6.25)
12 (12.5)
18 (6.25)
–
12 (12.5)
23 (6.25)
10 (25)
21 (6.25)
23 (6.25)
25 (6.25)
29 (6.25)
24 (6.25)
15 (12.5)
29 (6.25)
–
27 (6.25)
15 (12.5)
19 (12.5)
28 (6.25)
8 (25)
29 (6.25)
26 (6.25)
8 (25)
19 (6.25)
17 (6.25)
–
10 (25)
20 (6.25)
12 (12.5)
–
24 (6.25)
19 (6.25)
–
15 (12.5)
18 (6.25)
–
19 (6.25)
10 (25)
20 (6.25)
21 (6.25)
–
–
32 (6.25)
25 (6.25)
15 (12.5)
20 (12.5)
28 (6.25)
30 (6.25)
–
–
15 (12.5)
18 (6.25)
15 (12.5)
18 (6.25)
21 (6.25)
26 (6.25)
29 (6.25)
28 (6.25)
32 (6.25)
23 (6.25)
25 (6.25)
ꢁ
Ciprofloxacin is used as the standard, – indicates bacteria is resistant to the compounds >100 ꢀg cmꢀ3
ꢁ
MIC (ꢀg cmꢀ3) ¼ minimum inhibitory concentration, i.e., the lowest concentration to completely inhibit bacterial growth

694
M. S. Karthikeyan, B. S. Holla
Table 3 Antifungal activity (diameter zones=mm) of inhibition (MIC=ꢀg cmꢀ3) of arylidenetriazolothiazolidinones
Compd.
no.
Candida
albicans
Aspergillus
niger
Aspergillus
fumigatus
Trichophyton
mentagrophytes
Penicillium
marneffei
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
standard
19 (6.25)
8 (25)
12 (12.5)
–
10 (25)
–
14 (12.5)
17 (6.25)
9 (25)
21 (6.25)
15 (12.5)
18 (6.25)
21 (6.25)
23 (6.25)
–
15 (12.5)
18 (6.25)
–
10 (25)
14 (12.5)
12 (12.5)
7 (25)
20 (6.25)
15 (12.5)
24 (6.25)
25 (6.25)
18 (6.25)
12 (12.5)
10 (25)
8 (25)
16 (6.25)
14 (12.5)
–
10 (25)
13 (12.5)
9 (25)
20 (6.25)
11 (12.5)
14 (12.5)
–
9 (25)
–
18 (6.25)
12 (12.5)
–
18 (6.25)
–
–
14 (12.5)
16 (6.25)
–
11 (12.5)
20 (6.25)
9 (25)
–
17 (6.25)
13 (12.5)
19 (6.25)
12 (12.5)
18 (6.25)
19 (6.25)
22 (6.25)
22 (6.25)
21 (6.25)
23 (6.25)
ꢁ
Amphotericin B is used as the standard, – indicates fungus is resistant to the compounds >100 ꢀg cmꢀ3
ꢁ
MIC (ꢀg cmꢀ3) ¼ minimum inhibitory concentration, i.e., the lowest concentration to completely inhibit fungal growth
mixture was cooled, and poured onto crushed ice. The result-
ing solid was filtered, dried, and recrystallized from ethanol.
Conclusion
Yield 90%; mp 203–205ꢃC; FABMS: m=z (%) ¼ 282 (Mþ 1,
The antiinflammatory study revealed that com-
100), 191 (85).
pounds with 4-methylthiophenyl, 3,4-methylene-
dioxyphenyl, and 2,3,5-trichlorophenyl residues at
position 5 of the arylidenetriazolothiazolidinone
moiety showed excellent antiinflammatory activity.
The antimicrobial screening studies revealed that
compounds with 4-anisyl, 4-methylthiophenyl, 3,4-
methylenedioxyphenyl, and 2,3,5-trichlorophenyl at
position 5 of the arylidenetriazolothiazolidinones
moiety showed excellent activity.
5-(2,4-Dichloro-5-fluorophenyl)-4H-1,2,4-triazole-3-thiol
(5, C8H4Cl2FN3S)
To a solution of 100 cm3 of 5% NaOH, 0.01mol of 2-(2,4-
dichloro-5-fluorobenzoyl)hydrazine carbothioamide (4) was
added and refluxed for 3 h. The reaction mixture was cooled,
poured onto crushed ice, and neutralized with conc. HCl. The
resulting solid was filtered, dried, and recrystallized from a
mixture of ethanol and dimethylformamide. Yield 82%; mp
256–258ꢃC; 1H NMR (300 MHz, DMSO-d6): ꢂ ¼ 7.86 (d, 1H-
dichlorofluorophenyl, JHꢀF ¼ 9.6Hz), 8.05 (d, 1H-dichloro-
ortho
fluorophenyl, JHꢀF ¼ 7.2Hz), 13.84 (s, 1H–NH=SH) 13.92
meta
Experimental
(s, 1H–NH=SH) ppm.
Melting points were determined by open capillary method. The
IR spectra (in KBr pellets) were recorded on a Shimadzu FT-IR
1
157 spectrophotometer. H NMR spectra were recorded (in
5-Arylidene-2-(2,4-dichloro-5-fluorophenyl)thiazolo[3,2-b]-
1,2,4-triazol-6(5H)-one (6)
CDCl3=DMSO-d6) on a Bruker 300=400 MHz, NMR spec-
trometer using TMS as an internal standard. The mass spectra
were recorded on a MASPEC=FAB mass spectrometer oper-
ating at 70 eV. The purity of the compounds was checked by
thin layer chromatography (TLC) on silica gel plates using a
mixture of petroleum ether and ethyl acetate. Iodine was used
as visualizing agent.
2,4-Dichloro-5-fluorobenzoic acid (2) was obtained from
2,4-dichloro-5-fluoroacetophenone (1) according to Ref. [18].
2,4-Dichloro-5-fluorobenzoyl hydrazide (3) was prepared
according to Ref. [19].
To 0.01 mol of 5-(2,4-dichloro-5-fluorophenyl)-4H-1,2,4-tria-
zole-3-thiol (5), 0.015 mol of monochloroacetic acid,
0.01 mol of substituted benzaldehyde, 5 cm3 of acetic anhy-
dride, 0.01 mol of anhydrous sodium acetate, and 10cm3 of
glacial acetic acid was added and refluxed for 3 h. The reac-
tion mixture was cooled, and poured onto crushed ice. The
resulting solid was filtered, dried, and recrystallized from a
mixture of ethanol and dimethylformamide.
2-(2,4-Dichloro-5-fluorophenyl)-5-(4-methoxybenzylidene)-
1,3-thiazolo[3,2-b]-1,2,4-triazol-6(5H)-one
(6a, C18H10Cl2FN3O2S)
Yield 79%; mp 226–228ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
2-(2,4-Dichloro-5-fluorobenzoyl)hydrazine carbothioamide
(4, C8H6Cl2FN3OS)
ꢃꢀ¼ 3090 (Ar–H), 2952 (C–H), 1706 (C¼O), 1109 (C–F),
1
836 (C–Cl) cmꢀ1; H NMR (400MHz, DMSO-d6): ꢂ ¼ 3.87
A mixture of 0.01mol 2,4-dichloro-5-fluorobenzoyl hydrazide
(3), 0.01 mol, and then 0.02 mol of potassium thiocyanate was
added to 10cm3 of water containing 2 cm3 of conc. HCl. The
mixture was warmed on a water bath for 2 h. The reaction
(s, OCH3), 7.19 (d, 2H-p-anisyl, J ¼ 8.8 Hz), 7.78 (d, 2H-p-
anisyl, J ¼ 8.8 Hz), 8.04–8.08 (m, 2H-dichlorofluorophenyl),
8.29 (s, ¼CH) ppm; FABMS: m=z (%) ¼ 422 (M þ 1, 25).

2,4-Dichloro-5-fluorophenyl substituted arylidenetriazolothiazolidinones
695
2-(2,4-Dichloro-5-fluorophenyl)-5-[4-(methylthio)-
benzylidene]-1,3-thiazolo[3,2-b]-1,2,4-triazol-6(5H)-one
(6b, C18H10Cl2FN3OS2)
2-(2,4-Dichloro-5-fluorophenyl)-5-(2,3,5-trichloro-
benzylidene)-1,3-thiazolo[3,2-b]-1,2,4-triazol-6(5H)-one
(6l, C17H5Cl5FN3OS)
Yield 63%; mp 235–237ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
Yield 88%; mp 228–230ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢃꢀ¼ 3092 (Ar–H), 1702 (C¼O), 1089 (C–F), 852, 736 (C–
ꢃꢀ¼ 3090 (Ar–H), 2922 (C–H), 1702 (C¼O), 1105 (C–F),
Cl) cmꢀ1
;
1H NMR (300 MHz, DMSO-d6): ꢂ ¼ 3.50 (s,
812, 731 (C–Cl) cmꢀ1 1H NMR (300MHz, DMSO-d6):
;
SCH3), 7.48 (d, 2H-p-methylthio, J ¼ 8.8 Hz), 7.73 (d, 2H-p-
methylthio, J ¼ 8.8Hz), 8.05–8.09 (m, 2H-dichlorofluorophe-
nyl), 8.28 (s, ¼CH) ppm.
ꢂ ¼ 8.04 (s, 1H-trichloro), 8.09 (d, 1H-dichlorofluorophenyl,
JHꢀF ¼ 7.8 Hz), 8.12–8.13 (m, 2H-trichloro and dichloro-
ortho
fluorophenyl), 8.25 (s, ¼CH) ppm.
Antiinflammatory assay
2-(2,4-Dichloro-5-fluorophenyl)-5-[4-(dimethylamino)-
benzylidene]-1,3-thiazolo[3,2-b]-1,2,4-triazol-6(5H)-one
(6c, C19H13Cl2FN4OS)
Wister albino rats of either sex weighing 180–250g were used
for the experiment. They were housed in clean polypropylene
cages and kept under room temperature (25ꢃC), relative
humidity (60–70%) in 112h light-dark cycle. The animals
were given standard laboratory diet and water ad libitum.
Food was withdrawn 12 h before and during experimental
hours. Experiments were approved by the Institutional ethics
committee.
Yield 63%; mp 271–273ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢃꢀ¼ 3093 (Ar–H), 2958 (C–H), 1702 (C¼O), 1089 (C–F),
1
854 (C–Cl) cmꢀ1; H NMR (300 MHz, DMSO-d6): ꢂ ¼ 3.09
(s, N(CH3)2), 6.90 (d, 2H-p-dimethylamino, J ¼ 9 Hz), 7.73 (d,
2H-p-dimethylamino, J ¼ 9 Hz), 8.02–8.09 (m, 2H-dichloro-
fluorophenyl), 8.18 (s, ¼CH) ppm.
The animals were divided into 12 groups each group con-
tained six animals. A mark was made on the hind paw (left)
just beyond the tibio-tarsal junction, so that every time the
paw was dipped in the mercury column up to fixed mark
constant paw volume was ensured. The initial paw volume
of each rat was noted by plethysmometrically (Ugo Basile,
Italy). First group received normal saline and the second group
received Indomethacin orally at a dose of 1.5 mg=kg. The 3rd
to 12th groups were administered the test compounds (at a
dose of 50 mg=kg suspended in 10cm3=kg of 2% gum acacia)
orally. After 30min of treatment of test compounds, 0.1 cm3 of
1% (w=v) carrageenan was injected in the subplantar region of
the left hind paw. The right paw served as a reference to non-
inflammed paw for comparison. The initial paw volume was
measured with in 30sec of the injection. The relative increase
in paw volume was measured in control, standard and test
compounds at 1, 2, and 3 h after the carrageenan injection.
The difference between the two readings was taken as the
volume of oedema and the percentage inhibition by the drugs
was calculated using the formula,
5-(4-Chlorobenzylidene)-2-(2,4-dichloro-5-fluorophenyl)-
[1,3]thiazolo[3,2-b]-1,2,4-triazol-6(5H)-one
(6e, C17H7Cl2FN3OS)
Yield 87%; mp 232–234ꢃC (EtOH:DMF ¼ 2:1); 1H NMR
(400 MHz, CDCl3): ꢂ ¼ 7.54–7.63 (m, 3H-p-chloro, dichloro-
fluorophenyl) 7.92 (d, 1H-dichlorofluorophenyl, JHꢀF
¼
ortho
9.4 Hz), 8.22 (s, ¼CH) ppm; FABMS: m=z (%) ¼ 426 (M þ
1, 28).
5-(2-Chloro-5-nitrobenzylidene)-2-(2,4-dichloro-5-fluoro-
phenyl)-1,3-thiazolo[3,2b]-1,2,4-triazol-6(5H)-one
(6g, C17H6Cl3FN4O3S)
Yield 78%; mp 216–218ꢃC (EtOH:DMF ¼ 2:1); 1H NMR
(400 MHz, CDCl3): ꢂ ¼ 7.62 (d, 1H-dichlorofluorophenyl,
JHꢀF ¼ 6.4 Hz), 7.77 (d, 1H-chloronitro, J ¼ 8.7 Hz), 7.92
meta
(d, 1H-dichlorofluorophenyl, JHꢀF ¼ 9.2Hz), 8.32 (d, 1H-
ortho
chloronitro, J ¼ 7.2 Hz), 8.52 (s, 1H-chloronitro), 8.56 (s,
¼CH) ppm; FABMS: m=z (%) ¼ 471 (Mþ 1, 50).
Percentage of oedema inhibition
¼ 100 ꢀ ðVtest=VcontrolÞꢄ100;
where, Vcontrol ¼ volume of paw oedema in control group;
Vtest ¼ volume of paw oedema in the test compounds treated
group.
5-(2,4-Dichlorobenzylidene)-2-(2,4-dichloro-5-fluoro-
phenyl)-1,3-thiazolo[3,2-b]-1,2,4-triazol-6(5H)-one
(6h, C17H6Cl4FN3OS)
Yield 65%; mp 222–224ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢃꢀ¼ 3090 (Ar–H), 1712 (C¼O), 1105 (C–F), 882, 726 (C–
1
Cl) cmꢀ1; H NMR (300 MHz, DMSO-d6): ꢂ ¼ 7.95 (d, 1H-
dichloro, J ¼ 2 Hz), 7.66–7.75 (m, 2H-dichloro), 8.05–8.09
(m, 2H-dichlorofluorophenyl), 8.28 (s, ¼CH) ppm.
Antibacterial assay
The newly prepared compounds were screened for their anti-
bacterial activity against five bacterial strains by disc diffu-
sion method. A standard inoculum (1–2ꢄ107 c.f.u. cmꢀ3 0.5
McFarland standards) was introduced on to the surface of
sterile agar plates, and a sterile glass spreader was used
for even distribution of the inoculum. The discs measuring
6.25mm in diameter were prepared from Whatman no.1 filter
paper and sterilized by dry heat at 140ꢃC for 1 h. The sterile
disc previously soaked in a known concentration of the test
compounds were placed in nutrient agar medium. Solvent and
growth controls were kept. The plates were inverted and incu-
bated for 24h at 37ꢃC. The inhibition zones were measured
5-(1,3-Benzodioxol-5-ylmethylene)-2-(2,4-dichloro-5-fluoro-
phenyl)-1,3-thiazolo[3,2-b]-1,2,4-triazol-6(5H)-one
(6j, C18H8Cl2FN3O3S)
Yield 82%; mp 238–240ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢃꢀ¼ 3098 (Ar–H), 2927 (C–H), 1700 (C¼O), 1098 (C–F),
825, 736 (C–Cl) cmꢀ1 1H NMR (300MHz, DMSO-d6):
;
ꢂ ¼ 6.19 (s, OCH2O), 7.19 (d, 1H-methylenedioxy, J ¼
8.1 Hz), 7.34–7.42 (m, 2H-methylenedioxy), 8.04–8.1 (m,
2H-dichlorofluorophenyl), 8.25 (s, ¼CH) ppm; FABMS:
m=z (%) ¼ 436 (M þ 1, 27).

696
M. S. Karthikeyan, B. S. Holla
and compared with the controls. Minimum inhibitory concen-
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nutrient broth, which contained logarithmic serially two fold
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cells. The cultures were incubated for 24h at 37ꢃC and the
growth was monitored visually and spectrophotometrically.
The lowest concentration (highest dilution) required to ar-
rest the growth of bacteria was regarded as minimum in-
hibitory concentrations (MIC). Ciprofloxacin was used as a
standard drug.
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Antifungal assay
The newly prepared compounds were screened for their anti-
fungal activity against five fungal strains by the agar diffusion
method. Sabouraud agar media was prepared by dissolving 1 g
peptone, 4 g D-glucose, and 2 g agar in 100 cm3 distilled water,
and adjusting pH to 5.7 using buffer. Normal saline was used
to make a suspension of spore of fungal strain for lawning. A
loopful of particular fungal strain was transferred to 3 cm3
saline to get a suspension of corresponding species. 20cm3
of agar media was poured in to each Petri dish. Excess of
suspension was decanted and the plates were dried by plac-
ing in a incubator at 37ꢃC for 1 h. Using an agar punch, wells
were made and each wells are labeled. A control was also
prepared in triplicate and maintained at 37ꢃC for 3–4d. The
inhibition zones in diameter were measured and compared
with the controls. The Nutrient Broth, which contained loga-
rithmic serially two fold diluted amount of test compound and
controls were inoculated with approximately 1.6ꢄ104–
6ꢄ104 c.f.u. cmꢀ3 was used. The cultures were incubated
for 48 h at 35ꢃC and the growth was monitored. The lowest
concentration (highest dilution) required to arrest the growth
of fungus was regarded as minimum inhibitory concentrations
(MIC). Amphotericin B was used as the standard drug.
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Acknowledgements
18. a) Karthikeyan MS, Prasad DJ, Poojary B, Bhat KS,
Holla BS, Kumari NS (2006) Bioorg Med Chem
14:7482; b) Karthikeyan MS, Holla BS, Kalluraya B,
Kumari NS (2007) Monatsh Chem 138:1309
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Song G (2003) J Fluorine Chem 123:163; b) Shi W,
Quian X, Zhang R, Song G (2001) J Agric Food Chem
49:124
The authors are grateful to K.V. Ramanathan, The Head, NMR
Research Center, IISc-Bangalore, and CDRI-Lucknow for
providing 1H NMR, and mass spectral data. M.S.K. gratefully
acknowledges the financial support (Senior Research
Fellowship) rendered by CSIR, New Delhi.
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