Analgesic and antimicrobial studies of some 2,4-dichloro-5-fluorophenyl containing arylidenetriazolothiadiazines

Article abstract of DOI:10.1007/s00706-007-0797-9

2,4-Dichloro-5-fluorophenyl containing 7-arylidenetriazolothiadiazines were obtained by the reaction of 4-amino-3-(2,4-dichloro-5-fluorophenyl)-5-mercapto- 1,2,4-triazole with 2,3-dibromo-1,3-diarylpropan-1-ones, and also by the reaction of 4-amino-3-(2,4

Full text of DOI:10.1007/s00706-007-0797-9

Monatsh Chem 139, 707–716 (2008)
DOI 10.1007/s00706-007-0797-9
Printed in The Netherlands
Analgesic and antimicrobial studies of some 2,4-dichloro-5-fluorophenyl
containing arylidenetriazolothiadiazines
Mari S. Karthikeyan1, Bantwal S. Holla1, Shalini Shenoy2
1
Department of Chemistry, Mangalore University, Mangalagangothri, Karnataka, India
2
Department of Microbiology, K.M.C., Mangalore, Karnataka, India
Received 3 August 2007; Accepted 3 September 2007; published online 12 May 2008
# Springer-Verlag 2008
Abstract 2,4-Dichloro-5-fluorophenyl containing 7-
arylidenetriazolothiadiazines were obtained by the
reaction of 4-amino-3-(2,4-dichloro-5-fluorophenyl)-
5-mercapto-1,2,4-triazole with 2,3-dibromo-1,3-
diarylpropan-1-ones, and also by the reaction of
4-amino-3-(2,4-dichloro-5-fluorophenyl)-5-mercapto-
1,2,4-triazolewithꢀ-bromopropenones in the presence
of a base. The structure of the 7-arylidenetriazolothia-
diazines was confirmed by an alternative synthesis. A
plausible mechanism for the formation of 7-arylidene-
triazolothiadiazines is proposed. All newly synthe-
sized compounds were screened for their analgesic
and antimicrobial activities. Compounds bearing 4-
chlorophenyl or 3,4-methylenedioxyphenyl moieties
at position 7 of the arylidenetriazolothiadiazines
showed excellent analgesic activity. Arylidenetria-
zolothiadiazines carrying a phenyl, 4-chlorophenyl,
4-methylphenyl, 3,4-dimethoxyphenyl, and 2,4-di-
chlorophenyl moieties at position 7 showed excellent
antibacterial and antifungal activities.
Introduction
In recent days, active research has been initiated
on halogen containing heterocycles, particularly on
fluorine containing heterocycles. 2,4-Dichloro-5-
fluoroacetophenone is used in the synthesis of
drugs like Ciprofloxacin and their analogues [1].
Moreover, incorporation of a fluorine atom can alter
the course of reaction as well as biological activities
[2–4]. Furthermore, the introduction of a fluorine
atom or a CF3 group into an organic molecule large-
ly enhances the pharmacological properties as com-
pared with the non-fluorinated analogues.
Incorporation of fluorine may also lead to in-
creased lipid solubility thereby enhancing the rates
of absorption and transport of drugs in vivo. The
replacement of hydrogen or a hydroxyl group by
fluorine is a strategy widely used in drug develop-
ment to alter biological function. Despite the fact
that fluorine is bigger than hydrogen, several studies
have demonstrated that fluorine is a reasonable hy-
drogen mimic and exerts only a minor steric demand
at receptor sites [5].
Keywords 2,4-Dichloro-5-fluorophenyltriazole; Dibromo-
propan-1-ones; ꢀ-Bromopropenones; Analgesic; Antimicro-
bial activities.
1,2,4-Triazole derivatives are known to exhibit an-
tibacterial, antifungal [6], antitubercular [7], antican-
cer [8], anticonvulsant [9], anti-inflammatory, and
analgesic properties [10]. Among the pharmacologi-
cal profiles of 1,2,4-triazoles, their antimicrobial, an-
ticonvulsant, and antidepressant properties seem to
be best documented. The arrangement of three basic
Correspondence: Mari Sithambaram Karthikeyan, Depart-
ment of Chemistry, Mangalore University, Mangalagangothri
574199, Karnataka, India. E-mail: karthims02@rediffmail.
com

708
M. S. Karthikeyan et al.
nitrogen atoms in the triazole ring induces antiviral
activity in the compounds containing a triazole ring
[11]. The 1,2,4-triazole nucleus has been incorporat-
ed into a wide variety of therapeutically interesting
drug candidates including H1=H2 histamine receptor
blockers, cholinesterase active agents, CNS stimu-
lants, antianxiety, and sedatives agents [12]. Some
of the modern day drugs with triazole nucleus are
Ribavirin (antiviral agent), Alprazolam (anxiolytic
agent), Flucanozole, Itraconazole (antifungal agent)
[13], and Rizatriptan (antimigrane agent).
The ambident nucleophilic centers present in 3-
substituted-4-amino-5-mercapto-1,2,4-triazoles ren-
der them as useful synthons for the synthesis of
various N-bridged heterocycles. Moreover, synthesis
of triazole fused to other heterocycles has attracted
attention widely due to their diverse applications.
Recently, a number of N-bridged heterocycles are
also derived from substituted chalcones and have
been reported to possess some interesting biological
properties [14–16].
Prompted by these observations, it was contem-
plated to synthesize some dichlorofluorophenyl con-
taining arylidenetriazolothiadiazines with a view
to explore their potency as better chemotherapeutic
agents. All newly synthesized compounds were
screened for the analgesic, antibacterial, and antifun-
gal activities.
Results and discussion
Chemistry
Substituted acetophenones 1 and substituted benz-
aldehydes 2 were allowed to react in the presence
of aqueous potassium hydroxide to yield 1,3-
diaryl-2-propen-1-ones 3. Bromination of these
propenones 3 employing bromine in chloroform
yielded 2,3-dibromo-1,3-diarylpropan-1-ones 4.
The dibromopropanones 4 on dehydrobromination
using triethylamine in dry benzene yielded 1,3-
diaryl-2-bromo-2-propen-1-ones 5 [17, 18]. The
reaction sequences are outlined in Scheme 1.
4-Amino-3-(2,4-dichloro-5-fluorophenyl)-5-mercapto-
1,2,4-triazole 7 was obtained from 2,4-dichloro-5-
fluorobenzoic acid according to Refs. [19, 20].
Scheme 1
triazolothiadiazines 8 nor triazolothiadiazepines 9,
but unexpectedly chalcones 3 were obtained as the
major products, which was confirmed by spectroscopic
and analytical data. The possibility could be that 2,3-
dibromo-1,3-diarylpropan-1-ones 4 might undergo
debromination under mild basic condition and yielded
chalcones 3 as the major products (Scheme 2).
The same reaction was carried out in two steps.
Initially triethylamine was added to 2,3-dibromo-
1,3-diarylpropan-1-ones 4 in absolute ethanol and
refluxed for 1 h. To this reaction mixture 4-amino-
3-(2,4-dichloro-5-fluorophenyl)-5-mercapto-1,2,4-
triazole 7 was added and further refluxed for 6 h,
In a one-pot reaction between 2,3-dibromo-1,3-dia-
rylpropan-1-ones 4 and 4-amino-3-(2,4-dichloro-5-
fluorophenyl)-5-mercapto-1,2,4-triazole 7 in presence
of triethylamine in absolute ethanol neither arylidene-

2,4-Dichloro-5-fluorophenyl containing arylidenetriazolothiadiazines
709
Scheme 2
which yielded arylidenetriazolothiadiazines 8 rather
than triazolothiadiazepines 9 (Scheme 3).
of 6-substituted 3-(2,4-dichloro-5-fluorophenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines 11 with substi-
tuted benzaldehyde in presence of piperidine yielded
arylidenetriazolothiadiazines 8 (Scheme 4).
When ꢀ-bromopropenones 5 were treated with
4-amino-3-(2,4-dichloro-5-fluorophenyl)-5-mercapto-
1,2,4-triazole 7 in presence of potassium hydro-
xide in ethanol, arylidenetriazolothiadiazines 8
rather than triazolothiadiazepines 9 were formed
(Scheme 3).
Arylidenetriazolothiadiazines 8 were also synthe-
sized by an alternate route to confirm their formation.
6-Substituted-3-(2,4-dichloro-5-fluorophenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines 11 were
obtained by the initial reaction of 4-amino-3-(2,4-
dichloro-5-fluorophenyl)-5-mercapto-1,2,4-triazole
(7) with phenacyl bromides [21] 10 in presence of
sodium acetate in absolute ethanol. Condensation
Compounds 8 obtained by the above method
were found to be identical with the compounds
prepared by the direct condensation of triazole 7
with 2,3-dibromo-1,3-diaryl propan-1-ones 4 and
triazole 7 with ꢀ-bromopropenones 5. By the mixed
melting point and superimposable IR spectra the
compounds formed were found to be arylidene-
triazolothiadiazines 8 and not triazolothiadiaze-
pines 9. Although stereochemistry of 8 was not
investigated, analytical data suggest that only one
diastereomer was formed which is due to previous
work [22] very likely to be the (Z)-isomer.

710
M. S. Karthikeyan et al.
Scheme 3
Mechanism
erated in situ by refluxing 2,3-dibromo-1,3-diarylpro-
pan-1-ones 4 with triethylamine in absolute ethanol.
When triazole 7 was added to the reaction mixture
Schiff base was formed, further nucleophilic attack at
A plausible mechanism for the formation of arylid-
enetriazolothiadiazines 8 was proposed on the basis of
earlier observations. ꢀ-Bromopropenones were gen-

2,4-Dichloro-5-fluorophenyl containing arylidenetriazolothiadiazines
711
Scheme 4
C2 resulted in cyclization and subsequent dehydrobro-
mination yielding arylidenetriazolothiadiazines 8 rath-
er than triazolothiadiazepines 9 (Scheme 5).
Formation of arylidenetriazolothiadiazines 8 was
confirmed on the basis of IR, 1H NMR, mass spectral
data, and elemental analysis. All the newly synthe-
sized compounds were screened for their biological
activities.
Pharmacological studies
Analgesic studies
Selected compounds were screened for analgesic
activity by the hot plate test according to Eddy
and Leimbach [23]. The analgesic screening
results are given in Table 1. The analgesic screen-
ing results revealed that compounds 8c, 8e, and

712
M. S. Karthikeyan et al.
Scheme 5
Antibacterial studies
8h showed excellent analgesic activity whereas
compounds 8b, 8d, 8f, 8g, and 8k showed mod-
erate to good analgesic activity compared with
Pethidine.
The newly prepared compounds were screened for
their antibacterial activity against Staphylococcus
aureus (ATCC-25923), Escherichia coli (ATCC-

2,4-Dichloro-5-fluorophenyl containing arylidenetriazolothiadiazines
713
Table 1 Analgesic activity data of arylidenetriazolothiadia-
zines 8a–8l
Antifungal studies
The newly prepared compounds were screened for
their antifungal activity against Aspergillus niger,
Aspergillus flavus, Candida albicans, and Tricho-
phyton mentagrophytes (recultured) in DMSO by
the agar diffusion method [26, 27]. The diameter
of zone of inhibition and minimum inhibitory con-
centration values are given in Table 3. The anti-
fungal screening data showed that compounds 8e
showed good activity against Trichophyton men-
tagrophytes at 6.25 ꢁg cmꢀ3 concentration Com-
pounds 8a, 8c, 8f, 8g, and 8j exhibited good
antifungal activity against all tested fungal strains
almost equivalent to that of the standard drug
Griseofulvin.
Compd.
no.
Dose=
mg kgꢀ1
Time of reaction to pain
stimulus at time=h
[s] ꢁ SEM
0
1
3
8a
8b
8c
8d
8e
8f
8g
8h
8i
8k
control
standard
50
50
50
50
50
50
50
50
50
50
10
5
8.6
7.5
8.5
9.2
9.4
8.5
8.9
8.2
8.8
9.1
8.25
8.5
8.86
9.14
11.71
14.41
10.21
14.9
11.1
10.4
14.1
10.21
11.4
8.2
10.76
13.3
10.7
13.5
10.7
11.5
13.31
11.04
12.4
8.8
ꢂ
16.4
14.3
Conclusion
ꢂ
Pethidine is used as the standard
The analgesic activity study revealed that com-
pounds with 4-chlorophenyl, and 3,4-methyl-
enedioxyphenyl moieties at position 7 of the
arylidenetriazolothiadiazines showed excellent an-
algesic activity. The investigation of antibacterial
screening data revealed that among the twelve
compounds screened six compounds showed good
bacterial inhibition almost equivalent to that of the
standard. Only five of the compounds displayed
good antifungal activity. Arylidenetriazolothiadia-
zines carrying phenyl, 4-chlorophenyl, 4-methylphe-
nyl, 3,4-dimethoxyphenyl, and 2,4-dichlorophenyl
moieties at position 7 of the arylidenetriazolothiadia-
25922), Pseudomonas aeruginosa (ATCC-27853),
and Klebsiella pneumoniae (recultured) bacterial
strains by the disc diffusion method [24, 25]. The
diameter of the zone of inhibition and minimum in-
hibitory concentration MIC i.e., the lowest concen-
tration to completely inhibit bacterial growth values
are given in Table 2. The antibacterial screening data
revealed that the compounds 8a, 8c, 8f, 8g, 8j, and 8l
exhibited good antibacterial activity against all test-
ed bacterial strains almost equivalent to that of the
standard drug Norfloxacin.
Table 2 Antibacterial activity data of arylidenetriazolothiadiazines 8a–8l
Compd.
no.
Staphylococcus
aureus
Escherichia
coli
Pseudomonas
aeruginosa
Klebsiella
pneumoniae
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
standard
21 (6.25)
14 (25)
18 (6.25)
12 (25)
14 (12.5)
22 (6.25)
20 (6.25)
12 (12.5)
17 (6.25)
22 (6.25)
11 (25)
25 (12.5)
17 (6.25)
27 (6.25)
17 (6.25)
19 (6.25)
28 (6.25)
24 (6.25)
18 (6.25)
12 (25)
26 (6.25)
19 (6.25)
27 (6.25)
28 (6.25)
20 (6.25)
12 (25)
23 (6.25)
11 (25)
15 (12.5)
20 (6.25)
18 (6.25)
13 (25)
16 (6.25)
24 (6.25)
10 (25)
25 (6.25)
15 (12.5)
21 (6.25)
19 (6.25)
12 (25)
23 (12.5)
25 (6.25)
9 (25)
19 (6.25)
22 (6.25)
17 (6.25)
23 (6.25)
25 (6.25)
20 (6.25)
22 (6.25)
20 (6.25)
24 (6.25)
ꢂ
Norfloxacin is used as the standard. Diameter=mm of zone of inhibition. MIC=ꢁg cmꢀ3 values are given in brackets.
ꢂ

714
M. S. Karthikeyan et al.
Table 3 Antifungal activity data of arylidenetriazolothiadiazines 8a–8l
Compd.
no.
Aspergillus
niger
Aspergillus
flavus
Candida
albicans
Trichophyton
mentagrophytes
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
standard
28 (6.25)
10 (25)
25 (6.25)
19 (6.25)
27 (6.25)
12 (12.5)
10 (25)
25 (12.5)
28 (12.5)
15 (12.5)
22 (6.25)
26 (6.25)
20 (6.25)
19 (6.25)
28 (6.25)
33 (6.25)
30 (6.25)
28 (6.25)
20 (12.5)
24 (6.25)
34 (6.25)
29 (6.25)
19 (12.5)
24 (6.25)
32 (6.25)
18 (12.5)
21 (12.5)
33 (6.25)
25 (6.25)
12 (12.5)
21 (6.25)
17 (6.25)
21 (6.25)
24 (6.25)
20 (6.25)
18 (6.25)
15 (12.5)
23 (6.25)
12 (25)
29 (6.25)
23 (6.25)
15 (12.5)
28 (6.25)
30 (6.25)
16 (12.5)
23 (6.25)
27 (6.25)
20 (12.5)
15 (12.5)
30 (6.25)
10 (25)
25 (6.25)
ꢂ
Griseofulvin is used as the standard. Diameter=mm of zone of inhibition. MIC=ꢁg cmꢀ3 values are given in brackets.
ꢂ
off, washed with water, and recrystallized from a mixture of
ethanol and dimethylformamide to yield the title compound 8.
zines emerged as active compounds in both antibac-
terial and antifungal screening.
Substituted phenacyl bromides 10 were prepared according
to the reported procedure [21].
Experimental
General procedure for preparation of 6-(substituted phenyl)-
3-(2,4-dichloro-5-fluorophenyl)-7H-1,2,4-triazolo[3,4-b]-
1,3,4-thiadiazines 11
A mixture of 0.01 mol triazole 7, 0.01 mol substituted phena-
cyl bromides 10, and 0.01 mol anhydrous sodium acetate in
30cm3 absolute ethanol was refluxed on a water bath for 5 h.
The reaction mixture was cooled, the precipitated solid was
filtered off, and recrystallized from ethanol.
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
CDCl3=DMSO-d6) on a Bruker 400=300 MHz NMR spectrom-
eter using TMS as an internal standard. The mass spectra were
recorded on a MASPEC=FAB mass spectrometer operating at
70eV. 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 visualiz-
ing agent. Compounds 3, 4, and 5 were prepared according to
Refs. [17, 18] and 4-amino-3-(2,4-dichloro-5-fluorophenyl)-5-
mercapto-1,2,4-triazole (7) was prepared according to the
reported method [19, 20].
General procedure for preparation of 6-(substituted phenyl)-
7-(arylidene)-3-(2,4-dichloro-5-fluorophenyl)-1,2,4-triazolo-
[3,4-b]-1,3,4-thiadiazines 8 from 6-(substituted phenyl)-3-
(2,4-dichloro-5-fluorophenyl)-7H-1,2,4-triazolo[3,4-b]-
1,3,4-thiadiazines 11
A mixture of 0.01 mol 6-(substituted phenyl)-3-(2,4-dichloro-
5-fluorophenyl)-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine 11,
0.01mol substituted benzaldehyde 2, and 0.01 mol piperidine
in 25 cm3 absolute ethanol was refluxed for 5 h. The reaction
mixture was cooled. The precipitated solid was filtered off,
washed with water, and recrystallized from a mixture of abso-
lute ethanol and dimethylformamide to yield the title com-
pound 8. This product was identical with the sample prepared
by the direct condensation of triazole 7 with 1,3-diaryl-2,3-
dibromopropan-1-ones 4.
Procedure for the preparation of 6-(substituted phenyl)-7-
(arylidene)-3-(2,4-dichloro-5-fluorophenyl)-1,2,4-triazolo-
[3,4-b]-1,3,4-thiadiazines 8
A mixture of 0.01 mol of 1,3-diaryl-2,3-dibromo-propan-1-
ones 4 and 0.02 mol triethylamine in 35 cm3 absolute ethanol
was refluxed for 1 h. To the resulting reaction mixture
0.01mol triazole 7 was added and refluxed for 6 h. The result-
ing solid was filtered off, washed with water, and recrystal-
lized from a mixture of ethanol and dimethylformamide.
General procedure of alternative route for preparation
of 6-(substituted phenyl)-7-(arylidene)-3-(2,4-dichloro-5-
fluorophenyl)-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazines 8
A mixture of 0.01 mol 4-amino-3-(2,4-dichloro-5-fluorophe-
nyl)-5-mercapto-1,2,4-triazole (7), 0.01 mol 1,3-diaryl-2-bro-
mo-2-propen-1-ones 5, and 0.015 mol alcoholic KOH was
refluxed in ethanol for 6 h. The precipitated solid was filtered
7-Benzylidene-3,6-bis(2,4-dichloro-5-fluorophenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (8a, C23H10Cl4F2N4S)
Yield 87%; mp 200–02ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢂꢀ¼ 3090 (Ar–H), 2935 (C–H), 1567 (C¼N), 1096 (C–F),
1
734 (C–Cl) cmꢀ1; H NMR (400MHz, DMSO-d6): ꢃ ¼ 7.11
(s, 1H- exocyclic vinylic), 7.53 (m, 5H-phenyl), 7.8, 7.89
(d, 2H-dichlorofluorophenyl, JH–F ¼ 9.3 Hz), 8.04, 8.08 (d,
ortho

2,4-Dichloro-5-fluorophenyl containing arylidenetriazolothiadiazines
715
2H-dichlorofluorophenyl, JH–F ¼ 6.6 Hz) ppm; FABMS:
7.18 (s, 1H-exocyclic vinylic), 7.29 (d, 2H-p-tolyl, J ¼
8 Hz), 7.40 (d, 2H-p-tolyl, J ¼ 8 Hz), 7.48 (m, 3H-p-chloro-
phenyl, dichlorofluorophenyl), 7.65 (m, 3H-p-chlorophenyl,
dichlorofluorophenyl) ppm.
meta
m=z ¼ 552 (Mþ).
3,6-Bis(2,4-dichloro-5-fluorophenyl)-7-(4-methoxy-
benzylidene)-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine
(8b, C24H12Cl4F2N4OS)
6-(4-Chlorophenyl)-3-(2,4-dichloro-5-fluorophenyl)-7-(4-
N,N-dimethylamino benzylidene)-7H-1,2,4-triazolo[3,4-b]-
1,3,4-thiadiazine (8i, C25H17Cl3FN5S)
Yield 68%; mp 183–85ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢂꢀ¼ 3085 (Ar–H), 1583 (C¼N), 1087 (C–F), 825, 727 (C–
Cl) cmꢀ1
OCH3), 7.00 (s, 1H- exocyclic vinylic proton), 7.09 (m, 2H-
;
1H NMR (400 MHz, DMSO-d6): ꢃ ¼ 3.82 (s,
Yield 75%; mp 242–44ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢂꢀ¼ 3087 (Ar–H), 1567 (C¼N), 1093 (C–F) 816, 736
1
p-anisyl), 7.59 (m, 2H-p-anisyl), 7.78, 7.88 (d, 1H, dichloro-
fluorophenyl, JH–F ¼ 9.2 Hz), 8.06 (m, 2H-dichlorofluoro-
(C–Cl) cmꢀ1; H NMR (300MHz, DMSO-d6): ꢃ ¼ 3.03 (s,
N(CH3)), 6.82, 7.52 (d, 2H-N,N-dimethylamino, J ¼ 8.7 Hz),
ortho
phenyl) ppm; FABMS: m=z ¼ 582 (Mþ, 11), 583 (Mþ þ 1,
7.16 (s, 1H-exocyclic vinylic), 7.64 and 7.91 (d, 2H-p-chloro-
phenyl), 7.69 (d, 1H-dichlorofluorophenyl, JH–F ¼ 9.3 Hz),
42), 460 (18).
ortho
7.93 (d, 2H-dichlorofluorophenyl, JH–F ¼ 6.8 Hz) ppm;
meta
FABMS: m=z ¼ 544 (Mþ).
7-(4-Chlorobenzylidene)-3,6-bis(2,4-dichloro-5-fluoro-
phenyl)-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine
(8c, C23H9Cl5F2N4S)
Analgesic assay
Yield 75%; mp 199–201ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
Male Albino mice of either sex with weight between 20 and
25g were used for analgesic study. The animals were divided
into 12 experimental groups each consisted of 6 animals. Gum
acacia (2%) was administered to group 1. Group 2 received
Pethidine at a dose 5 mg=kg by intraperitoneal injection. Other
groups were given the test compounds (8a, 8b, 8c, 8d, 8e, 8f,
8g, 8h, 8i, and 8k) at a dose of 50mg=kg orally. The animals
were housed and fed in laboratory kept at constant tempera-
ture of 22ꢃC under standard conditions (12:12h light-dark
cycle, standard pellet diet, tap water). In this test, the reaction
of mice to painful stimulus was measured. Mice were placed
on the metal plate heated to 55 ꢁ 0.4ꢃC and covered with a
glass cylinder (25 cm high, 15cm in diameter). The time(s)
elapsing to the first pain response (licking or jumping) was
determined and then recorded as response latency, prior to 60,
180 min following the po administration of the investigated
compounds. Institutional ethics committee approved all the
experiments.
ꢂꢀ¼ 3098 (Ar–H), 1566 (C¼N), 1106 (C–F), 735 (C–Cl)
1
cmꢀ1; H NMR (400MHz, DMSO-d6): ꢃ ¼ 7.11 (s, 1H-exo-
cyclic vinylic), 7.61 (m, 4H-p-chlorophenyl), 7.78 and 7.88
(d, 1H-dichlorofluorophenyl, JH–F ¼ 9 Hz), 8.05 and 8.08
ortho
(d, 1H, dichlorofluorophenyl proton, JH–F ¼ 6.6 Hz) ppm;
meta
FABMS: m=z ¼ 586 (Mþ, 10), 587 (Mþ þ 1, 38), 589
(Mþ þ 2, 60), 594 (Mþ þ 4, 41).
7-(2,4-Dichlorobenzylidene)-3,6-bis(2,4-dichloro-5-fluoro-
phenyl)-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine
(8d, C23H8Cl6F2N4S)
Yield 81%; mp 248–50ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢂꢀ¼ 3090 (Ar–H), 1589 (C¼N), 1105 (C-F), 736 (C-Cl)
1
cmꢀ1; H NMR (400MHz, DMSO-d6): ꢃ ¼ 6.99 (s, 1H-exo-
cyclic vinylic), 7.63 (m, 2H-dichlorophenyl), 7.83 (m, 2H,
dichlorophenyl and dichlorofluorophenyl), 7.89 (d, 1H- di-
chlorofluorophenyl, JH–F ¼ 9.3 Hz), 8.08 (m, 2H-dichloro-
ortho
fluorophenyl) ppm; FABMS: m=z ¼ 620 (Mþ, 10), 621
(Mþ þ 1, 55), 589 (Mþ þ 2, 30), 594 (Mþ þ 4, 25).
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.=cm3 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
and compared with the controls. Minimum inhibitory concen-
tration (MIC) was determined by broth dilution technique. The
nutrient broth, which contained logarithmic serially two fold
diluted amount of test compound and controls were inoculated
with approximately 5ꢄ105 c.f.u. of actively dividing bacteria
cells. The cultures were incubated for 24h at 37ꢃC and the
growth was monitored visually and spectrophotometrically.
The lowest concentration (highest dilution) required to arrest
3,6-Bis(2,4-dichloro-5-fluorophenyl)-7-(3,4-dimethoybenzyl-
idene)-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine
(8f, C25H14Cl4F2N4O2S)
Yield 70%; mp 196–98ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢂꢀ¼ 3091 (Ar–H), 1577 (C¼N), 1097 (C–F) 812, 732 (C–
Cl) cmꢀ1
;
1H NMR (300 MHz, DMSO-d6): ꢃ ¼ 3.78 (s,
OCH3), 3.83 (s, OCH3), 7.01 (s, 1H-exocyclic vinylic), 7.14
(m, 2H-3,4-dimethoxyphenyl), 7.26 (d, 1H-3,4-dimethoxy-
phenyl, J ¼ 8.4 Hz), 7.80, 7.90 (d, 1H-dichlorofluorophenyl,
JH–F ¼ 9 Hz), 8.06 (m, 2H-dichlorofluorophenyl) ppm;
ortho
FABMS: m=z ¼ 612 (Mþ, 15), 613 (Mþ þ 1, 45).
6-(4-Chlorophenyl)-3-(2,4-dichloro-5-fluorophenyl)-7-(4-
methylbenzylidene)-7H-1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazine (8g, C24H14Cl3FN4S)
Yield 78%; mp 232–34ꢃC (EtOH:DMF ¼ 2:1); IR (KBr):
ꢂꢀ¼ 3088 (Ar–H), 1585 (C¼N), 1097 (C–F), 743 (C–Cl)
cmꢀ1
;
1H NMR (300 MHz, DMSO-d6): ꢃ ¼ 2.42 (s, CH3),

716
M. S. Karthikeyan et al.
the growth ofbacteriawas regarded asminimum inhibitorycon-
centrations (MIC). Norfloxacin was used as the standard drug.
6. Zitouni GT, Kaplancikli ZA, Yildiz MT, Chevallet P,
Kaya D (2005) Eur J Med Chem 40:607
´
7. Walczak K, Gondela A, Suwinski J (2004) Eur J Med
Chem 39:849
Antifungal assay
The newly prepared compounds were screened for their anti-
fungal activity against five fungal strains by the agar diffusion
method. Sabouraud’s 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 lawn-
ing. 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 into each Petri dish.
Excess of suspension was decanted and the plates were dried
by placing them in an incubator at 37ꢃC for 1 h. Using an agar
punch, wells were made and each well was labeled. A control
was also prepared in triplicate and maintained at 37ꢃC for 3–
4 d. The inhibition zones in diameter were measured and com-
pared with the controls. The nutrient broth, which contained
logarithmic serially two fold diluted amount of test compound
and controls were inoculated with approximately 1.6ꢄ104–
6ꢄ104 c.f.u. cmꢀ3. The cultures were incubated for 48h 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).
Griseofulvin was used as the standard drug.
8. a) Holla BS, Poojary KN, Rao BS, Shivananda MK
(2002) Eur J Med Chem 37:511; b) Holla BS, Veerendra
B, Shivananda MK, Poojary B (2003) Eur J Med Chem
38:759
9. Amir M, Shikha K (2004) Eur J Med Chem 39:535
10. Almasirad A, Tabatabai SA, Faizi M, Kebriaeezadeh A,
Mehrabi N, Dalvandi A, Shafiee A (2004) Bioorg Med
Chem Letts 14:6057
11. Masuda K, Toga T, Hayashi N (1975) J Labelled
Compound 11:301; (1976) Chem Abstr 84:121730f
12. Schreier E (1976) Helv Chim Acta 59:585
13. Windholz M (1976) The Merck Index 9th edn. Merck &
Co. Inc., Rahway, New Jersey
14. Bauer DJ, Salway JWT, Batcheler JF, Fisdale M,
Coldwell IC, Young DAB (1981) Nature 292:369
15. Soulie P, Chiche P, Carlothic J, Baillet J (1954) Presse
Med 62:847 (1954) Chem Abstr 48:7755h
16. Willak DT, Eilam WP, Sankarappa SK, Gavestri RC,
Newman HAI (1975) J Med Chem 18:934
17. Udupa KV (1991) PhD Thesis Mangalore University
18. Rao BS (2000) PhD Thesis Mangalore University
19. Karthikeyan MS, Prasad DJ, Poojary B, Bhat KS, Holla
BS, Kumari NS (2006) Bioorg Med Chem 14:7482
20. Karthikeyan MS, Holla BS, Kalluraya B, Kumari NS
(2007) Monatsh Chem 138:1309
Acknowledgements
21. Vogel’s Text Book of practical Organic Chemistry (1996)
5th edn. Longman, England, p 1125
22 Shiradkar MR, Padhalingappa MB, Bhetalabhotala S,
Akula KC, Tupe DA, Pinninti RR, Thummanagoti S
(2007) Bioorg Med Chem 15:6397
The authors are grateful to The Head, Prof. K.V. Ramanathan,
NMR Research Center, IISc-Bangalore, and CDRI, Lucknow
for providing 1H NMR, and mass spectral data. M.S.K. grate-
fully acknowledges the financial support (Senior Research
Fellowship) rendered by CSIR, New Delhi.
23. Eddy NB, Leimbach D (1953) J Pharmacol Exp Ther
7:385
24. Cruickshank R, Duguid JP, Marmion BP, Swain RHA
(1975) Medicinal Microbiology, 12th edn, Vol. II.
Churchill Livingstone, London, p 196
25. Collins AH (1976) Microbiological Methods, 2nd edn.
Butterworth, London
References
1. Chu DTW, Fernandes PB, Claiborne AK, Pihuleac E,
Nordeen CW, Maleczka RE Jr, Pernet AG (1985) J Med
Chem 28:1558
2. Filler R (1974) Chem Technol 4:752
3. Plevey RG, Tatlow JC (1970) Sci Org Oxy 58:481
4. Joshi KC, Dandia A, Khanna S (1990) Ind J Chem
29B:1125
5. Kevin Park B, Kitteringham NR, O’Neill PM (2001) Ann
Rev Pharmacol Toxicol 41:443
26. Khan ZK (1997) In vitro and in vivo screening techniques
for bioactivity screening and evaluation. Proc Int work-
shop UNIDO-CDRI, p 210
27. Varma RS (ed) (1998) Antifungal Agents: Past, Present
and Future prospects. National Academy of Chemistry
and Biology. Lucknow, India