Synthesis, antimicrobial and anti-inflammatory activities of some 1,2,4-triazolo[3,4-b][1,3,4]thiadiazoles and 1,2,4-triazolo[3,4-b][1,3,4]thiadiazines bearing trichlorophenyl moiety

Article abstract of DOI:10.1016/j.ejmech.2007.06.026

The reaction of 2,3,5-trichlorobenzoic acid hydrazide with carbon disulfide and potassium hydroxide followed by treatment with hydrazine hydrate afforded 3-(2,3,5-trichlorophenyl)-4-amino-1,2,4-triazole-5-thione (6). Alternatively, this triazole was also synthesized by fusing 2,3,5-trichlorobenzoic acid with thiocarbohydrazide. Condensation of (6) with various aromatic carboxylic acids in the presence of phosphorous oxychloride or with phenacyl bromides afforded two series of fused heterocycles namely 6-(substituted aryl)-3-(2,3,5-trichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadizoles (7) and 6-(substituted aryl)-3-(2,3,5-trichlorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiaz ines (8), respectively. The structures of these newly synthesized compounds are characterised by elemental analysis, IR, ¹H NMR and mass spectroscopic studies. All the synthesized compounds were screened for their antimicrobial and anti-inflammatory activities. Some of the compounds exhibited promising antimicrobial and anti-inflammatory activities.

Full text of DOI:10.1016/j.ejmech.2007.06.026

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 808e815
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antimicrobial and anti-inflammatory activities of some
1,2,4-triazolo[3,4-b][1,3,4]thiadiazoles and 1,2,4-triazolo
[3,4-b][1,3,4]thiadiazines bearing trichlorophenyl moiety
a
b
Prakash Karegoudar b, D. Jagdeesh Prasad a, , Mithun Ashok , Manjathuru Mahalinga ,
*
Boja Poojary a, Bantwal Shivarama Holla a
a Department of Chemistry, Mangalore University, Mangalagangothri-574199, Mangalore, Karnataka, India
b Sequent Scientific Limited, 120 A & B, Industrial Area, Bikampady, New Mangalore, Karnataka, India
Received 1 April 2007; received in revised form 10 June 2007; accepted 12 June 2007
Available online 17 July 2007
Abstract
The reaction of 2,3,5-trichlorobenzoic acid hydrazide with carbon disulfide and potassium hydroxide followed by treatment with hydrazine
hydrate afforded 3-(2,3,5-trichlorophenyl)-4-amino-1,2,4-triazole-5-thione (6). Alternatively, this triazole was also synthesized by fusing 2,3,5-
trichlorobenzoic acid with thiocarbohydrazide. Condensation of (6) with various aromatic carboxylic acids in the presence of phosphorous
oxychloride or with phenacyl bromides afforded two series of fused heterocycles namely 6-(substituted aryl)-3-(2,3,5-trichlorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadizoles (7) and 6-(substituted aryl)-3-(2,3,5-trichlorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (8), respec-
tively. The structures of these newly synthesized compounds are characterised by elemental analysis, IR, 1H NMR and mass spectroscopic stud-
ies. All the synthesized compounds were screened for their antimicrobial and anti-inflammatory activities. Some of the compounds exhibited
promising antimicrobial and anti-inflammatory activities.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Trichlorophenyl moiety; Triazolothiadiazoles; Triazolothiadiazines; Anti-inflammatory; Antimicrobial screening
1. Introduction
represent an important group of sulphur compounds that are
promising for use in practical applications.
In the last few decades, the chemistry of 1,2,4-triazoles and
their fused heterocyclic derivatives has received considerable
attention owing to their synthetic and effective biological im-
portance. 1,2,4-Triazole moieties have been incorporated into
a wide variety of therapeutically interesting drug candidates
including anti-inflammatories, CNS stimulants, sedatives, anti-
anxiety compounds, antimicrobial agents [1e3] and antimy-
cotic ones such as fluconazole, intraconazole, voriconazole
[4,5]. There are marketed drugs containing the 1,2,4-triazole
group, e.g., Triazolam [6], Alprazolam [7], Etizolam [8], and
Furacylin [9]. Moreover, sulphur containing heterocycles
Among these heterocycles, the mercapto- and thione-
substituted 1,2,4-triazole ring systems (A and B, Fig. 1) have
been well studied and so far a variety of biological activities
have been reported for a large number of their derivatives,
such as antibacterial [10e15], antifungal [16,17], anti-
tubercular [18], antimycobacterial [19], anticancer [20,21],
diuretic [22,23], and hypoglycemic [24] properties. In addi-
tion to these important biological applications, mercapto-
1,2,4-triazoles are also of great utility in preparative organic
chemistry, for example, in the presence of various reagents,
undergo different types of reactions to yield other heterocy-
clic compounds, e.g., thiazolotriazoles, triazolothiadiazoles,
triazolothiazines, triazolothiazepines and triazolothiadiazines.
In this paper the most common and useful procedures for the
* Corresponding author.
E-mail address: jprasad_2003@yahoo.co.in (D.J. Prasad).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.06.026

P. Karegoudar et al. / European Journal of Medicinal Chemistry 43 (2008) 808e815
809
H
N
4-b][1,3,4]thiadiazoles and 1,2,4-triazolo[3,4-b][1,3,4]thiadia-
zines containing 2,3,5-trichlorophenyl moiety would result in
compounds with enhanced biological activities. Prompted by
these investigations and in continuation of our research work
on the synthesis of novel heterocyclic compounds exhibiting
biological activity, it was thought to be interesting to synthe-
size compounds containing the features namely, having
1,2,4-triazole moiety fused with the 1,3,4-thiadiazole/1,3,
4-thiadiazine rings.
N
N
N
R
R
S
SH
N
N
NH2
NH2
A
B
Fig. 1.
preparation of the 4-amino-5-substituted-4H-[1,2,4]triazole-
3-thiol and their utility for the synthesis of well known
heterocyclic ring systems namely triazolothiadiazoles and
triazolothiazines are compiled and evaluation of their anti-
microbial and anti-inflammatory activities are discussed.
A recent literature survey reveals that many of the halogen
containing heterocycles have attracted attention due to the
ability of halogen to act as polar hydrogen or hydroxy mimic.
Substitution of hydrogen by halogen has been a strategy in de-
signing molecules for biological studies [25]. Therefore it was
envisaged that chemical entities with both 1,2,4-triazolo[3,
2. Chemistry
Reaction sequence employed for the synthesis of title
compounds is shown in Scheme 1. The starting material 2,3,5-
trichlorobenzaldehyde (1) was converted into 2,3,5-trichloro-
benzoic acid (2) by oxidation with KMnO4 and then it is
esterified with methanol in the presence of acid to get
methyl-2,3,5-trichlorobenzoate (3). 2,3,5-Trichlorophenyl car-
bohydrazide (4) was obtained by refluxing (3) with hydrazine
hydrate in methanol. The compound (4) on reaction with car-
bon disulphide in methanolic potassium hydroxide yielded
Cl
Cl
Cl
CHO
MeOH,H+
KMnO4
CO2H
CO2CH3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3
1
2
MeOH/ NH2.NH2.H2O
Cl
Cl
Cl
Cl
KS
NH NH
CS2, KOH, MeOH
S
O
CONHNH2
Cl
5
4
NH2.NH2.H2O, Refluxe
Cl
N
N
SH
N
6
NH2
Cl
Cl
ArCOCH2Br / NaHCO3
ArCO2H / POCl3
Cl
Cl
N
N
N
N
S
N
N
S
N
N
Cl
Cl
Cl
7 (a-j)
Cl
Ar
Ar
8(a-g)
Scheme 1. Synthesis of substituted Triazolothiadiazoles and Triazolothiadiazines.

810
P. Karegoudar et al. / European Journal of Medicinal Chemistry 43 (2008) 808e815
corresponding dithiocarbazinate (5) in good yield and was di-
rectly used for the next step without further purification. The
triazole (6) was synthesized by refluxing (5) with hydrazine
hydrate. Condensation of (6) with various aromatic carboxylic
acids in phosphorous oxychloride under reflux condition
yielded, 6-(substituted aryl)-3-(2,3,5-trichlorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazoles (7aej) and with various phe-
nacyl bromides in ethanol under reflux condition gave 6-
(substituted aryl)-3-(2,3,5-trichlorophenyl)-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazines (8aeg) in good yield. Assignment
of new compounds was based on their elemental analysis
and spectroscopic data. Characterization data of all new com-
pounds are summarized in Table 1.
were added into each labelled well. A control was also pre-
pared for the plates in the same way using solvent DMSO.
The Petri dishes were prepared in triplicate and maintained
at 37 ꢀC for 3e4 days. Antibacterial activity was determined
by measuring the diameter of inhibition zone. Activity of
each compound was compared with Ciprofloxacin as standard
[28,29]. Zone of inhibition was determined for (7aej) and
(8aeg) and the results are summarized in Table 2.
3.2. Antifungal
Newly prepared compounds were screened for their anti-
fungal activity against Aspergillus flavus [NCIM No.524],
Aspergillus fumigatus [NCIM No. 902], Penicillium marneffei
[recultured] and Trichophyton mentagrophytes [recultured] in
DMSO by serial plate dilution method [30,31]. Agar media
were prepared by dissolving peptone (1 g), D-glucose (4 g)
and agar (2 g) in distilled water (100 mL) and adjusting the
pH to 5.7. Normal saline was used to make a suspension of
spore of fungal strains for lawning. A loopful of particular fun-
gal strain was transferred to 3 mL saline to get a suspension of
corresponding species. Agar media of 20 mL were poured into
each Petri dish. Excess of suspension was decanted and plates
were dried by placing in an incubator at 37 ꢀC for 1 h. Using
an agar punch each labelled well were made on these seeded
agar plates and minimum inhibitory concentrations of the
test compounds in dimethylsulfoxide (DMSO) were added
into each labelled well. A control was also prepared for the
plates in the same way using solvent DMSO. The Petri dish
were prepared in triplicate and maintained at 37 ꢀC for 3e4
days. Antifungal activity was determined by measuring the di-
ameter of the inhibition zone. Activity of each compound was
compared with Ciclopiroxolamine as standard. Zones of inhi-
bition were determined for (7aej) and (8aeg) and the results
are summarized in Table 3.
3. Biological activities
3.1. Antibacterial
The newly synthesized compounds were screened for their
antibacterial activity against Escherichia coli (ATTC-25922),
Staphylococcus aureus (ATTC-25923), Pseudomonas aerugi-
nosa (ATCC-27853) and Klebsiella pneumoniae (recultured)
bacterial strains by serial plate dilution method [26,27]. Serial
dilutions of the drug in MullereHinton broth were taken in
tubes and their pH was adjusted to 5.0 using phosphate buffer.
Standardized suspension of the test bacterium was inoculated
and incubated for 16e18 h at 37 ꢀC. The minimum inhibitory
concentration (MIC) was noted by observing the lowest con-
centration of the drug at which there was no visible growth.
A number of antimicrobial discs are placed on the agar for
the sole purpose of producing zones of inhibition in the bacte-
rial lawn. Agar media were poured into each Petri dish. Excess
of suspension was decanted and placing in incubator at 37 ꢀC
for 1 h dried the plates. Using an agar punch, wells were made
on these seeded agar plates and minimum inhibitory concen-
trations of the test compounds in dimethylsulfoxide (DMSO)
Table 1
Characterization data of substituted-[1,2,4]triazolo[3,4-b][1,3,4]thiadizoles (7aej) and substituted-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines (8aeg)
Compd.
Ar
Molecular formula (MW)
MP (ꢀC)
Yield (%)
% Analysis of C, H, N found (calculated)
C
H
N
7a
7b
7c
7d
7e
7f
4-Methylphenyl
4-Methoxyphenyl
3,5-dichlorophenyl
3,5-Dimethylphenyl
Phenoxymethyl
5-Quinolyl
C
16H9Cl3N4S (394)
C16H9Cl3N4OS (410)
15H5Cl5N4S (448)
C17H11Cl3N4S (408)
16H9Cl3N4OS (410)
C18H8Cl3N5S (431)
15H7Cl3N4S (380)
182e184
180e182
232e233
243e245
220e222
236e238
255e257
267e269
280e282
256e258
220e222
220e222
242e244
248e250
256e258
252e254
240e242
80
85
84
86
82
85
86
81
85
84
83
85
86
84
86
85
82
48.70 (48.73)
46.71 (46.82)
40.13 (40.17)
49.96 (50.00)
46.12 (46.82)
50.00 (50.11)
47.26 (47.36)
44.00 (44.09)
50.09 (50.11)
36.11 (36.52)
56.10 (56.17)
45.00 (45.03)
44.75 (44.85)
46.25 (46.36)
48.73 (48.73)
48.11 (48.00)
49.12 (50.00)
2.24 (2.28)
2.14 (2.19)
1.09 (1.11)
2.62 (2.69)
2.10 (2.19)
1.80 (1.85)
1.82 (1.84)
2.28 (2.26)
1.82 (1.85)
1.04 (1.08)
2.71 (2.76)
2.01 (2.20)
1.80 (1.86)
2.4 (2.5)
14.20 (14.21)
13.15 (13.65)
12.47 (12.50)
13.68 (13.72)
13.50 (13.65)
16.21 (16.29)
14.62 (14.73)
14.85 (14.96)
16.23 (16.24)
15.08 (15.21)
11.08 (11.91)
15.40 (15.45)
13.02 (13.08)
12.64 (12.72)
14.28 (14.21)
14.28 (14.26)
13.60 (13.72)
C
C
7g
7h
7i
Phenyl
Pyridyl
C
C14H6Cl3N5S (381)
C18H8Cl3N5S (431)
C14H5BrCl3N5S (460)
5-Isoquinolyl
2-Bromopyridyl
Biphenyl
7j
8a
8b
8c
8d
8e
8f
C22H13Cl3N4S (470)
4-Hydroxy-3-benzamide-1-yl
4-Chlorophenyl
4-Methylthiophenyl
Phenyl
C17H10Cl3N5O2S (453)
C16H8Cl4N4S (428)
C17H11Cl3N4S2 (440)
C
C
16H9Cl3N4S (394)
17H11Cl3N4S (424)
2.20 (2.28)
2.59 (2.51)
2.50 (2.61)
4-Methoxyphenyl
4-Methylphenyl
8g
C17H11Cl3N4S (408)

P. Karegoudar et al. / European Journal of Medicinal Chemistry 43 (2008) 808e815
811
Table 2
Antibacterial activity of compounds (7aej) and (8aeg)
Compd.
MIC in mg mLꢁ1 and zone of inhibition (mm)
E. coli
12 (11e15)
K. pneumoniae
P. aeruginosa
S. aureus
7a
7b
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15))
6.25 (16e20)
6.25 (16e20)
25 (<10)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
7c
7d
7e
6.25 (16e20)
6.25 (16e20)
12 (11e15))
6.25 (16e20)
25 (<10)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
7f
7g
7h
7i
7j
8a
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
25 (<10)
8b
8c
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
8d
8e
8f
8g
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
1.56 (22e30)
Ciprofloxacin
6.25 (16e20)
6.25 (16e20)
Note: the MIC values were evaluated at concentration range, 1.56e25 mg mLꢁ1
.
3.3. Anti-inflammatory activity
measured before and after 3 h of administration and the results
were compared. Fifty-four healthy albino rats of body weight
180e250 g were selected and made into nine groups of six an-
imals each. All the animals were kept on fasting for 18 h. One
group of animals was kept as control, which received 2% w/v
acacia mucilage, which was used to suspend the sample. An-
other group received the standard drug Indomethacin
1.5 mg kgꢁ1 body weight intraperitonially. Remaining seven
groups of animals received seven different test compounds
(1.5 mg kgꢁ1 body weight) intraperitonially. After 30 min,
0.1 mL of w/v carrageenan was injected subcutaneously into
the right hind paw of the rats. A mark was made at the right
hind paw, which was dipped in the plethismograph up to the
The 6-substituted-3-(2,3,5-trichlorophenyl)-[1,2,4]triazolo
[3,4-b][1,3,4]thiadizoles and 6-substituted-3-(2,3,5-trichloro-
phenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were also
screened for their anti-inflammatory activity. The screening
was conducted in acute inflammatory model. Carrageenan in-
duced rat paw oedema method [32] was used. Carrageenan (an
irritant) at a concentration of 1 mg mLꢁ1 was injected subcu-
taneously into the hind paw of the rat to produce oedema. Dif-
ferent groups of animals were administered with standard drug
Indomethacin, the test samples and the vehicle used for the
preparation of samples. The increase in the paw volume was
Table 3
Antifungal activity data of compounds (7aej) and (8aeg)
Compd.
MIC in mg mLꢁ1 and zone of inhibition (mm)
P. marneffei
T. mentagrophytes
A. flavus
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
25 (<10)
A. Fumigatus
7a
7b
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
25 (<10)
6.25 (16e20)
6.5 (16e20)
6.25 (16e20)
12 (11e15)
7c
7d
7e
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
7f
7g
7h
7i
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
7j
8a
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
12 (11e15)
8b
8c
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
12 (11e15)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
8d
8e
8f
8g
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
6.25 (16e20)
3.125 (27e33)
6.25 (16e20)
6.25 (16e20)
3.125 (25e30)
Ciclopiroxolamine
Note: the MIC values were evaluated at concentration range, 1.56e25 mg mLꢁ1
.

812
P. Karegoudar et al. / European Journal of Medicinal Chemistry 43 (2008) 808e815
mark and the volume was measured immediately and after 3 h.
The change in paw volume was compared with that in the vehi-
cle treated control animals. The percentage inhibition of oe-
dema was calculated using the formula,
biological activity relationship of title compounds showed
that presence 2,3,5-trichloro groups and biologically active
groups like eOCH3, 2,3-dichloro, 4-hydoxy-3-amido, 4-
chloro, SCH3 groups attached to phenyl ring, pyridyl, and bro-
mopyridyl groups attached to the thiadiazole and thiadiazine
ring of the title compounds are responsible for good antimicrobial
activity. In case of anti-inflammatory activity, compound
8c (6-(4-chlorophenyl)-3-(2,3,5-trichlorophenyl)-7H-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazine) showed good activity among
the tested compounds. Compound 8c structurally resembles
a known anti-inflammatory agent sodium dichlofenic [33]
and these tested compounds showed moderate anti-inflamma-
tory activity.
% Oedema inhibition ¼ 100 ꢁ ðVtest=VcontrolÞ ꢂ 100:
The percentage of inhibition was compared with that of the
standard drug Indomethacin. Interestingly five compounds 7a,
7b, 7i and 8c carrying 4-methylphenyl, 4-methoxyphenyl, iso-
quinolyl and 4-chlorophenyl substituents exhibited good anti-
inflammatory activity against Indomethacin (Table 4).
4. Results and discussion
6. Experimental protocols
The investigation of antibacterial and antifungal screening
data revealed that all the tested compounds 7aej and 8aeg
showed moderate to good inhibition at 1.56e25 mg mLꢁ1 in
DMSO. The compounds 7b, 7c, 7e, 7h, 7j, 8b, 8c, 8d, 8f
and 8g showed comparatively good activity against all the bac-
terial strains. The good activity is attributed to the presence of
pharmacologically active eOCH3, 2,3-dichloro, 4-hydoxy-3-
amide, 4-chloro, SCH3 groups attached to phenyl ring, pyridyl,
bromopyridyl and aryloxy groups of the thiadiazole and thia-
diazine ring. The compounds 7b, 7c, 7f, 7h, 7j, 8a, 8b, 8c, 8d
and 8f showed comparatively good activity against all the
fungal strains. The good activity is attributed to the presence
of pharmacologically active eCH3, OCH3, 2,3-dichloro, 4-
hydoxy-3-amide, 4-chloro, SCH3, phenyl groups attached to
phenyl ring, pyridyl, and bromopyridyl groups of the thiadia-
zole and thiadiazine ring. It has been observed that the thiadia-
zole derivatives are found to be more active than thiadiazines.
Melting points were determined in open capillaries and are
uncorrected [melting point apparatus: SERWELL Instruments
Inc., India]. Purity of the compounds was checked by thin
layer chromatography (TLC) on a silica coated aluminum
sheet (silica gel 60F254) using chloroform and methanol (9:1,
v/v). IR spectra were recorded on NICOLET AVATAR 330-
1
FTIR Spectrometer. H NMR spectra recorded on a Varian
300 MHz NMR spectrometer using TMS as an internal stan-
dard. Chemical shifts are reported in parts per million (d)
and signals are described as singlet (s), doublet (d), triplet
(t), quartet (q), broad (br) and multiplet (m). The FAB mass
spectra were recorded on a JEOL SX 102/DA-6000 spectro-
photometer/Data system using argon/xenon (6 kV, 10 mA)
FAB gas, at 70 eV. Elemental analysis was carried out using
Flash EA 1112 Series, CHNSO Analyzer (Thermo). Solvents
and reagents were purchased from the commercial venders
in the appropriate grade and were used without purification.
5. Conclusions
6.1. 2,3,5-Trichlorobenzoic acid (2)
The research study reports the successful synthesis and an-
timicrobial activity of new 1,2,4-triazolothiadiazoles and
1,2,4-triazolothiadiazines bearing 2,3,5-trichlorophenyl moi-
ety. The antimicrobial activity study revealed that all the com-
pounds tested showed moderate to good antibacterial and
antifungal activities against pathogenic strains. Structure and
To a solution of 2,3,5-trichlorobenzaldehyde (37 g, 0.176 mol)
in 30 mL water, a solution of potassium permanganate (30 g,
0.191 mol) in 60 mL water was added at 70e80 ꢀC over
a period of 2 h. The reaction mass was stirred at same
temperature for 2 h and filtered. The filtrate was acidified
using conc. hydrochloric acid at 0e5 ꢀC. The product ob-
tained was filtered, washed with water and dried. The crude
product was recrystallized from methanol. Yield 75%, mp
167e168 ꢀC [34].
Table 4
Compd.
Paw oedema
volume
Percentage
inhibition
Dose
(mg kgꢁ1 p.o)
Mean þ S.E. (mL) of paw oedema
2% Gumacacia (control) 0.62 ꢃ 0.029
10 mL kgꢁ1
6.2. Methyl-2,3,5-trichlorobenzoate (3)
Indomethacin
0.25 ꢃ 0.012
0.48 ꢃ 0.012
0.46 ꢃ 0.19
0.52 ꢃ 0.178
0.55 ꢃ 0.015
0.55 ꢃ 0.013
0.48 ꢃ 0.013
0.50 ꢃ 0.019
0.56 ꢃ 0.019
0.56 ꢃ 0.020
0.50 ꢃ 0.019
0.50 ꢃ 0.018
59.67
22.58
25.8
1.5
50
50
50
50
50
50
50
50
50
50
50
7a
7b
7f
To a solution of 2,3,5-trichlorobenzoic acid (2) (30 g) in
methanol (150 mL). Conc. sulphuric acid was added slowly
at 0e5 ꢀC over a period of 30 min and refluxed for 2 h. After
quenching into cold water, precipitated solid was filtered,
washed with water and dried. It has been judged to be pure
(95e97%) by TLC. Yield 85%, mp 72e74 ꢀC.
9.67
7g
7h
7i
16.12
11.29
22.58
19.35
30.64
9.67
7j
8c
8d
8f
IR (KBr, cmꢁ1): 3038 (aromatic CeH), 1612 (aromatic
1
C]C), 1736 (eCO2CH3), 878 (CeCl); H NMR (DMSO-
19.35
19.35
8g
d6): d 3.82 (s, 3H, eOCOCH3), 7.72 (d, 1H, J ¼ 2.4 Hz,

P. Karegoudar et al. / European Journal of Medicinal Chemistry 43 (2008) 808e815
813
2,3,5-trichlorophenyl), 8.22 (d, 1H, J ¼ 2.7 Hz, 2,3,5-
6.5.1. 6-(4-Methylphenyl)-3-(2,3,5-trichlorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (7a)
trichlorophenyl).
IR (KBr, cmꢁ1): 2873 (CeH of CH3), 3042 (aromatic CeH),
1623 (C]N), 887 (CeCl); 1H NMR (DMSO-d6): d 2.45 (s, 3H,
CH3), 7.34 (d, 2H, J ¼ 8.1 Hz, 4-methylphenyl), 7.65 (d, 2H,
J ¼ 8.1 Hz, 4-methylphenyl), 7.75 (d, 1H, J ¼ 2.4 Hz, 2,3,
5-trichlorophenyl), 7.85 (d, 1H, J ¼ 2.7 Hz, 2,3,5-trichloro-
phenyl); FAB MS (m/z, %): 394 (Mþ, 10), 395 (M þ 1, 10),
396 (M þ 2, 28), 398 (M þ 4, 28), 400 (M þ 6, 6).
6.3. 2,3,5-Trichlorophenylcarbohydrazide (4)
A mixture of methyl-2,3,5-trichlorobenzoate (3) (1 mmol)
and hydrazine hydrate (2 mmol) in 10 mL of methanol was
heated under reflux for 5e6 h and after cooling solid obtained
was collected by filtration. It was washed with methanol, dried
and recrystallized from methanol. Yield 90%, mp 196e
198 ꢀC.
6.5.2. 6-(4-Methoxyphenyl)-3-(2,3,5-trichlorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (7b)
IR (KBr, cmꢁ1): 3310, 3246 (eNH2), 3038 (aromatic Ce
IR (KBr, cmꢁ1): 3055 (aromatic CeH), 1616 (C]N), 890
(CeCl); 1H NMR (DMSO-d6): d 3.83 (s, 3H, OCH3), 7.20 (d,
2H, J ¼ 8.7 Hz, 4-methoxyphenyl), 7.82 (d, 2H, J ¼ 8.7 Hz,
4-methoxyphenyl), 7.81 (d, 1H, J ¼ 2.4 Hz, 2,3,5-trichloro-
phenyl), 8.10 (d, 2H, J ¼ 2.7 Hz, 2,3,5-trichlorophenyl); FAB
MS (m/z, %): 394 (Mþ, 10), 395 (M þ 1, 10), 396 (M þ 2,
28), 398 (M þ 4, 28), 400 (M þ 6, 6).
1
H), 1680 (pC]O), 1544 (pC]Co), 870 (CeCl); H NMR
(DMSO-d6): d 7.34 (d, 1H, J ¼ 2.7 Hz, 2,3,5-trichlorophenyl),
7.51 (d, 1H, J ¼ 2.7 Hz, 2,3,5-trichlorophenyl).
6.4. 4-Amino-5-(2,3,5-trichlorophenyl)-4H-[1,2,4]-
triazole-3-thiol (6)
2,3,5-Trichlorophenylcarbohydrazide (4) (20 g, 0.083 mol)
was treated with a solution of potassium hydroxide (6.4 g,
0.125 mol) dissolved in methanol (50 mL) at 0e5 ꢀC under
stirring. Then (9.6 g, 0.125 mol) carbon disulfide was added
slowly and the reaction mixture was stirred overnight at
room temperature. The solid product of potassium dithiocarba-
zinate (5) was filtered, washed with chilled methanol and
dried. It was directly used for next step without purification.
The above potassium dithiocarbazinate (5) was taken in water
(8 mL) and hydrazine hydrate (2 mmol) and refluxed for 4e
5 h. During progress of the reaction, the reaction mixture
turned to green with evolution of hydrogen sulphide and fi-
nally it became homogeneous. It was then diluted with little
cold water and acidified with conc. hydrochloric acid. The
white precipitate was filtered, washed with cold water and
recrystallized from aqueous methanol. Yield 85%, mp 200e
201 ꢀC (methanol).
6.5.3. 6-(3,5-Dichlorophenyl)-3-(2,3,5-trichlorophenyl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (7c)
IR (KBr, cmꢁ1): 3075 (aromatic CeH), 1616 (C]N), 894
(CeCl); FAB MS (m/z, %): 450 (Mþ, 5), 451 (M þ 1, 60), 452
(M þ 2, 10), 454 (M þ 4, 7), 456 (M þ 6, 10), 417 (10), 289
(15), 165 (5), 155 (20), 120 (5), 107 (20).
6.5.4. 6-(3,5-Methylphenyl)-3-(2,3,5-trichlorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (7d)
IR (KBr, cmꢁ1): 3019 (aromatic CeH), 1640 (C]N), 893
1
(CeCl); H NMR (DMSO-d6): d 2.41 (s, 6H, CH3), 7.26 (s,
1H, 2,6-dimethylphenyl), 7.48 (s, 1H, 2,6-dimethylphenyl),
7.49 (d, 1H, J ¼ 2.7 Hz, 2,3,5-trichlorophenyl), 7.7 (d, 1H,
J ¼ 2.4 Hz, 2,3,5-trichlorophenyl).
IR (cmꢁ1): 3313 (NH2), 2667 (SH), 1617 (C]N), 964
6.5.5. 6-(Phenoxymethyl)-3-(2,3,5-trichlorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (7e)
IR (KBr, cmꢁ1): 3066 (aromatic CeH), 1632 (C]N), 872
(CeCl); 1H NMR (DMSO-d6): d 5.21 (s, 2H, OCH2), 7.64 (d,
1H, 2,3,5-trichlorophenyl), 7.26e7.38 (m, 5H, 2,6-phenoxy),
7.86 (d, 1H, 2,3,5-trichlorophenyl).
1
(NeC]S); H NMR (DMSO-d6): d 7.78 (d, 1H, J ¼ 2.4 Hz,
2,3,5-trichlorophenyl), 8.07 (d, 1H, J ¼ 2.7 Hz, 2,3,5-trichlor-
ophenyl), 14.11 (s, 1H, SH); FAB MS (m/z, %): 295 (Mþ,
100), 296 (M þ 1, 50), 297 (M þ 2, 95), 299 (M þ 4, 40),
301 (M þ 6, 5), 289 (45), 263 (15), 20 (7), 166 (4), 107 (20).
6.5.6. 6-(5-Quinolyl)-3-(2,3,5-trichlorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (7f)
IR (KBr, cmꢁ1): 3044 (aromatic CeH), 1648 (C]N), 868
(CeCl); FAB MS (m/z, %): 432 (Mþ þ 1, 90), 431 (Mþ, 50),
289 (10), 154 (30), 136 (40), 120 (10).
6.5. General procedure for the preparation of 6-aryl-3-
(2,3,5-trichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (7aej)
A
mixture 4-amino-5-(2,3,5-trichlorophenyl)-4H-[1,2,4]
triazole-3-thiol (6) (1 mmol) and substituted benzoic acid
(1.1 mmol) in POCl3 (5 mL) was refluxed for 6e7 h. The re-
action mixture was slowly poured into crushed ice with stirring
and neutralized with solid sodium bicarbonate. Solid material
was filtered, washed with cold water, dried, and recrystallized
from chloroform.
6.5.7. 6-(Phenyl)-3-(2,3,5-trichlorophenyl)-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazole (7g)
IR (KBr, cmꢁ1): 3053 (aromatic CeH), 1655 (C]N), 842
(CeCl); FAB MS (m/z, %): 381 (Mþ þ 1, 100), 8031 (Mþ,
60), 165 (10), 136 (40), 107 (30).

814
P. Karegoudar et al. / European Journal of Medicinal Chemistry 43 (2008) 808e815
6.5.8. 6-Pyridin-4-yl-3-(2,3,5-trichlorophenyl)-[1,2,4]
6.6.3.
6-(4-Chlorophenyl)-3-(2,3,5-trichlorophenyl)-7H-
triazolo[3,4-b][1,3,4]thiadiazole (7h)
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (8c)
IR (KBr, cmꢁ1): 3056 (aromatic CeH), 864 (CeCl), 1648
(C]N); 1H NMR (DMSO-d6): d 7.59 (d, 1H, J ¼ 2.4 Hz,
2,3,5-trichlorophenyl), 7.62 (d, 1H, J ¼ 2.4 Hz, 2,3,5-trichloro-
phenyl), 7.72 (d, 1H, J ¼ 1.7 Hz, pyridyl), 8.29 (d, 1H,
J ¼ 1.7 Hz, pyridyl), 8.85 (d, 1H, J ¼ 1.7 Hz, pyridyl), 9.91
(d, 1H, J ¼ 1.7 Hz, pyridyl); FAB MS (m/z, %): 382 (Mþ, 96),
383 (M þ 1, 20), 384 (M þ 2, 100), 386 (M þ 4, 30), 388
(M þ 6, 5), 367 (20), 341 (15), 305 (10), 279 (50), 240 (70),
197 (60), 179 (60), 149 (50), 123 (20), 105 (40), 91 (70), 80 (20).
1H NMR (DMSO-d6): d 4.35 (s, 2H, CH2), 7.46 (d, 2H,
J ¼ 8.6 Hz, p-chlorophenyl), 7.6 (d, 1H, J ¼ 2.4 Hz, 2,3,5-tri-
chlorophenyl), 7.73 (d, 1H, J ¼ 2.4 Hz, 2,3,5-trichlorophenyl),
7.83 (d, 2H, J ¼ 8.6 Hz, p-chlorophenyl); FAB MS (m/z, %):
430 (Mþ, 120), 431 (M þ 1, 100), 432 (M þ 2, 25), 434
(M þ 4, 10), 436 (M þ 6, 5), 395 (15), 325 (3), 272 (10),
180 (5), 165 (10), 120 (10), 107 (20), 89 (20).
6.6.4. 6-(4-Methylthiophenyl)-3-(2,3,5-trichlorophenyl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (8d)
6.5.9. 6-(5-Isoquinolyl)-3-(2,3,5-trichlorophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole (7i)
IR (KBr, cmꢁ1): 3076 (aromatic CeH), 1624 (C]N), 884
(CeCl); FAB MS (m/z, %): 431 (Mþ, 100), 289 (25), 155 (56),
120 (10), 107 (20).
IR (KBr, cmꢁ1): 3039 (aromatic CeH), 861 (CeCl), 1653
1
(C]N); H NMR (DMSO-d6): d 2.52 (s, 3H, SCH3), 7.22 (d,
2H, 4-methylthiophenyl), 7.36 (d, 2H, 4-methylthiophenyl),
7.54 (d, 1H, 2,3,5-trichlorophenyl), 7.74 (d, 1H, 2,3,5-trichlor-
ophenyl); FAB MS (m/z, %): 440 (Mþ, 100), 431 (M þ 1,
100), 432 (M þ 2, 25), 434 (M þ 4, 10), 395 (10), 165 (20),
155 (10), 120 (10).
6.5.10. 6-(3-Bromopyridin-4-yl)-3-(2,3,5-trichlorophenyl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (7j)
IR (KBr, cmꢁ1): 3072 (aromatic CeH), 1593 (C]N), 892
6.6.5. 6-(Phenyl)-3-(2,3,5-trichlorophenyl)-7H-[1,2,4]-
triazolo[3,4-b][1,3,4]thiadiazine (8e)
1
(CeCl), 753 (CeBr); H NMR (DMSO-d6): d 7.71 (d, 1H,
J ¼ 2.4 Hz, 2,3,5-trichlorophenyl), 7.73 (d, 1H, J ¼ 2.4 Hz,
2,3,5-trichlorophenyl), 8.36 (d, 1H, J ¼ 2.4 Hz, 3-bromonico-
tyl), 8.89 (d, 1H, J ¼ 2.0 Hz, 3-bromopyridyl), 9.02 (d, 1H,
J ¼ 1.7 Hz, 3-bromopyridyl); FAB MS (m/z, %): 460 (Mþ,
50), 461 (M þ 1, 5), 462 (M þ 2, 75), 464 (M þ 4, 55), 466
(M þ 6, 15), 429 (50), 411 (100), 391 (40), 223 (25), 229
(25), 217 (60), 181 (25), 123 (5), 109 (20), 77 (5).
IR (KBr, cmꢁ1): 3062 (aromatic CeH), 1649 (C]N), 863
1
(CeCl); H NMR (DMSO-d6): d 4.48 (s, 2H, SCH2), 7.10e
7.28 (m, 5H, phenyl), 7.60 (d, 1H, 2,3,5-trichlorophenyl),
7.84 (d, 1H, 2,3,5-trichlorophenyl).
6.6.6. 6-(4-Methoxyphenyl)-3-(2,3,5-trichlorophenyl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (8f)
IR (KBr, cmꢁ1): 1689 (C]O), 1592 (C]N), 1230 (Ne
1
6.6. General procedure for the preparation of 6-aryl-3-
(2,3,5-trichlorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]-
thiadiazines (8aeg)
N]C), 688 (CeSeC); H NMR (DMSO-d6): d 3.83 (s, 3H,
OCH3), 4.43 (s, 2H, CH2), 7.08 (d, 2H, J ¼ 8.4 Hz, 4-methox-
yphenyl), 7.82 (d, 1H, J ¼ 2.7 Hz, 2,3,5-trichlorophenyl), 7.83
(d, 2H, J ¼ 8.4 Hz, 4-methoxyphenyl), 8.14 (d, 1H,
J ¼ 2.4 Hz, 2,3,5-trichlorophenyl); FAB MS (m/z, %): 424
(Mþ, 10), 425 (M þ 1, 80), 426 (M þ 2, 25), 428 (M þ 4,
20), 430 (M þ 6, 5), 391 (70), 373 (15), 220 (5), 178 (5),
167 (15), 120 (30), 107 (50), 89 (40).
A
mixture 4-amino-5-(2,3,5-trichlorophenyl)-4H-[1,2,4]
triazole-3-thiol 6 (1 mmol) and substituted phenacyl bromides
(1.2 mmol) in 10 mL of absolute ethanol was refluxed for 6e
7 h. The reaction mass was poured into crushed ice and neu-
tralized with sodium bicarbonate. Solid product obtained
was filtered, washed with water, dried and recrystallized
from absolute ethanol.
6.6.7.
6-(4-Methylphenyl)-3-(2,3,5-trichlorophenyl)-7H-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (8g)
IR (KBr, cmꢁ1): 2944 (CeH of CH3), 3014 (aromatic CeH),
1620 (C]N), 872 (CeCl); 1H NMR (DMSO-d6): d 2.42 (s, 3H,
CH3), 4.06 (s, 2H, CH2), 7.28 (d, 2H, J ¼ 8.0 Hz,
4-methylphenyl), 7.61 (d, 1H, J ¼ 2.4 Hz, 2,3,5-trichloro-
phenyl), 7.67 (d, 1H, J ¼ 2.7 Hz, 2,3,5-trichlorophenyl), 7.69
(d, 2H, J ¼ 8.2 Hz, 4-methylphenyl).
6.6.1. 6-(Biphenyl)-3-(2,3,5-trichlorophenyl)-7H-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazine (8a)
IR (KBr, cmꢁ1): 3033 (aromatic CeH), 866 (CeCl), 1636
1
(C]N); H NMR (DMSO-d6): 4.06 (s, 2H, eSeCH2), 7.60
and 7.66 (2d, 2H, 2,3,5-trichlorophenyl), 7.42e7.86 (m, 9H,
biphenyl); FAB MS (m/z, %): 470 (Mþ), 472 (M þ 2, 45),
474 (M þ 4, 25), 476 (M þ 6, 10).
Acknowledgments
6.6.2. 6-(3-Amido-4-hydroxyphenyl)-3-
(2,3,5-trichlorophenyl)-7H-[1,2,4]triazolo-
[3,4-b][1,3,4]thiadiazine (8b)
IR (KBr, cmꢁ1): 3356 (CONH2), 3041 (aromatic CeH),
866 (CeCl), 1636 (C]N); FAB MS (m/z, %): 454 (Mþ þ 1,
100), 453 (Mþ, 40).
The authors are thankful to the Chairman, Department of
Biochemistry, Gulbarga University for providing the antibac-
terial and antifungal activity data for the compounds reported
herein. The authors are also thankful to Dr. H.M.V. Swamy,
Gulbarga University, SAIF, CDRI Lucknow for NMR and
Mass spectroscopic data of the compounds reported herein.

P. Karegoudar et al. / European Journal of Medicinal Chemistry 43 (2008) 808e815
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