Synthesis and antibacterial activity evaluation of 2,6-bis(6-substituted-1, 2,4-triazolo[3,4-b][1,3,4]thiadiazol-3-yl)pyridine derivatives

Article abstract of DOI:10.1007/s00044-013-0790-2

The reaction of 5,5′-(pyridine-2,6-diyl)bis(4-amino-3-mercapto-1,2,4- triazole) with various carboxylic acid in phosphorus oxychloride yielded the corresponding 2,6-bis(6-substituted-1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-3-yl) pyridine derivatives 6a-l. The structures of the newly synthesized compounds were confirmed by elemental analysis, ¹H NMR and ¹³C NMR spectral, mass spectral, and Infrared spectral studies. All the synthesized target compounds have been investigated for their in vitro antibacterial activity, and the preliminary results revealed that the compounds 6b and 6k exhibited very promising activity against Escherichia coli and Pseudomonas aeruginosa. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0790-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1941–1949
DOI 10.1007/s00044-013-0790-2
ORIGINAL RESEARCH
Synthesis and antibacterial activity evaluation of 2,6-bis(6-
substituted-1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-3-yl)pyridine
derivatives
•
Haihua Xiao Pingliang Li Dongcai Guo Jinhui Hu
•
•
•
•
Yuchao Chai Wei He
Received: 31 May 2013 / Accepted: 11 September 2013 / Published online: 26 September 2013
Ó Springer Science+Business Media New York 2013
Abstract The reaction of 5,50-(pyridine-2,6-diyl)bis(4-
amino-3-mercapto-1,2,4-triazole) with various carboxylic
acid in phosphorus oxychloride yielded the corresponding
2,6-bis(6-substituted-1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-
3-yl)pyridine derivatives 6a–l. The structures of the newly
synthesized compounds were confirmed by elemental
in literature that certain compounds bearing 1,2,4-triazole
nucleus possess significant antimicrobial activities (Sahoo
et al., 2010; El-Ashry et al., 2009; Feng et al., 2012).
Recently, triazole-fused thiadiazole compounds have been
frequently found to display a broad spectrum of biological
activities (Badr and Barwa, 2011; Mathew et al., 2007). A
triazolo-thiadiazole system may be viewed as a cyclic ana-
log of two important components—thiosemicarbazide and
biguanide (Fan et al., 2010; Gilani et al., 2010), which often
display diverse biological activities including anti-inflam-
matory (Karegoudar et al., 2008), antifungal (Sangshetti
et al., 2011), analgesic (Tozkoparan et al., 2009), anticancer
(Sunil et al., 2010), antimicrobial (Almajan et al., 2010a, b),
and anti-tumor (Ibrahim, 2009) activities. In continuation to
our efforts directed toward the synthesis of heterocyclic
compounds containing nitrogen, sulfur, and bicyclic systems
with anticipated antibacterial activities (Almajan et al.,
2010a, b; Dawood et al., 2005), in this paper we report the
synthesis and antimicrobial activities of 2,6-bis(6-substi-
tuted-1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-3-yl)pyridine deri-
vatives.
1
analysis, H NMR and 13C NMR spectral, mass spectral,
and Infrared spectral studies. All the synthesized target
compounds have been investigated for their in vitro anti-
bacterial activity, and the preliminary results revealed that
the compounds 6b and 6k exhibited very promising
activity against Escherichia coli and Pseudomonas
aeruginosa.
Keywords Pyridine-2,6-dicarboxylic acid derivatives ꢀ
1,2,4-Triazole ꢀ Synthesis ꢀ Antibacterial activity
Introduction
1,2,4-Triazole derivatives, an important group of heterocy-
clic compounds, have been the subject of extensive study in
the recent years (Eswaran et al., 2009). It has been reported
Results and discussion
Chemistry
H. Xiao ꢀ P. Li ꢀ D. Guo (&) ꢀ Y. Chai ꢀ W. He
College of Chemistry and Chemical Engineering, Hunan
University, Changsha 410082, People’s Republic of China
e-mail: dcguo2001@hnu.edu.cn
The target compounds were synthesized as outlined in
Scheme 1. The key intermediate compound 5,50-(pyridine-
2,6-diyl)bis(4-amino-3-mercapto-1,2,4-triazole) (5) was
prepared starting from pyridine-2,6-dicarboxylic acid (1) by
esterification with absolute methanol to give pyridine-2,6-
dicarboxylic acid dimethyl ester (2) (Yang et al., 2012), then
the compound 2 was treated with hydrazine hydrate in
absolute ethanol to yield the pyridine-2,6-dimethylhydrazine
P. Li
Guangzhou Mechanical Engineering Research Institute Co.,
Ltd., Guangzhou 510700, People’s Republic of China
J. Hu
Hunan Province People’s Hospital, Changsha 410005,
People’s Republic of China
123

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Med Chem Res (2014) 23:1941–1949
Cl
NO2
Cl
6a, R=
;
6b, R=
;
6c, R=
;
6d, R=
;
NO2
O
OCH3
N
6e, R=
;
6f, R=
;
6g, R=
;
6h, R=
6l, R=
;
I
N
CH3
6i, R=
;
6j, R= CH3
;
6k, R=
;
(i) CH3OH/CH3COCl/reflux
(ii) NH2NH2·H2O/reflux
(iii) CS2/KOH, EtOH, r.t.
(iv) NH2NH2·H2O/H2O/reflux
(v) RCOOH/ POCl3/reflux
Scheme 1 Synthetic pathway of the target compounds
(3) (Duke et al., 2012), which was allowed to react with
carbon disulfide in the presence of potassium hydroxide to
afford the corresponding potassium salt N,N0-(pyridine-2,6-
dicarbonyl)dithiocarbazate (4) (Said et al., 2011), this
potassium salt underwent ring closure with hydrazine
hydrate to yield the intermediate compound 5 (Tan et al.,
2007). The target compounds (6a–l) were prepared from the
reaction of the compound 5 with various carboxylic acid
under reflux in phosphorus oxychloride. All the synthesized
compounds are novel except the compounds 6a, 6f, 6i, and
6l which have been reported (Tan et al., 2007).
that the 5,50-(pyridine-2,6-diyl)bis(4-amino-3-mercapto-
1,2,4-triazole) 5 underwent ring closure to get the compound
6b. The 13CNMR spectralanalysesofcompound6b showeda
number of signals that are consistent with the number of
carbons in the molecule. The 13C NMR signals of triazolo-
thiadiazole-C-8,80, triazolo-thiadiazole-C-3,30, and triazolo-
thiadiazole-C-6,60 in compound 6b were observed at 166.11,
156.13, and 145.84 ppm, respectively. The other signals
present in 13C NMR spectra of compound 6b were recorded at
the expected chemical shifts.
The mass spectra of the target compounds showed that
all the fragmentation ions peaks were consistent with their
structures and could be clearly assigned. For the compound
6b, the molecular weight 547 was observed consistent with
the molecular formula C23H11Cl2N9S2. The values for the
remaining compounds have been presented under the
experimental part. The IR spectrum of the compound 6b
showed that the absorption band at 1,236 cm-1 was
assigned to the N–N=C group and the absorption band at
1,461 cm-1 was attributed to the C=C group, and a very
strong absorption band at 1,594 cm-1 was assigned to the
C=N group. The new absorption band appeared at
677 cm-1 which was attributed to the stretching frequency
of C–S–C group of the thiadiazole ring.
The structures of all the newly synthesized compounds
were characterized by elemental analysis, 1H NMR and 13
C
NMR spectral, mass spectral, and Infrared spectral studies.
The elemental analysis data along with other physical prop-
erties of these compounds are reported in Table 1. All the
newly synthesized compounds were analyzed satisfactorily
for their carbon, hydrogen, nitrogen, and sulfur content.
Since the structures of all the target compounds 6a–l are
very similar, only the compound 6b has been selected for
illustration. The 1H NMR spectrum of compound 6b showed
that the peaks of aromatic protons appeared at 7.27–7.85 ppm
and the peak of pyridine protons appeared at 8.27–8.41 ppm,
the peaks of SH and NH2 protons disappeared. This confirmed
123

Med Chem Res (2014) 23:1941–1949
1943
Table 1 Physico-chemical properties of the synthesized compounds
Compd.
R
Molecular
formula
Molecular
weight
(g/mol)
M.p. (°C)
Yield
(%)
Elemental analysis calc. (found)
C
H
N
S
5
–
C9H9N9S2
307.36
479
268–270
269–271
40
53
35.17 (35.08)
57.61 (57.36)
2.95 (2.96)
2.73 (2.75)
41.01 (41.06)
26.29 (26.24)
20.86 (20.80)
13.37 (13.56)
6a
C23H13N9S2
Cl
6b
6c
6d
6e
6f
C23H11Cl2N9S2
C23H11Cl2N9S2
C23H11N11O4S2
C23H11N11O4S2
C21H11N11S2
547
547
569
569
481
459
539
507
[300
[300
[300
[300
[300
[300
249–251
[300
45
48
55
56
42
40
42
61
50.37 (50.45)
50.37 (50.64)
48.50 (48.70)
48.50 (48.80)
52.38 (52.14)
49.67 (49.44)
55.65 (55.43)
59.16 (59.42)
2.02 (2.27)
2.02 2.23)
1.95 (1.91)
1.95 (1.90)
2.30 (2.35)
1.97 (2.00)
3.18 (3.24)
3.38 (3.44)
22.99 (22.76)
22.99 (22.76)
27.05 (26.91)
27.05 (27.06)
32.00 (32.05)
27.44 (27.40)
23.36 (23.40)
24.83 (24.75)
11.69 (11.58)
11.69 (11.63)
11.26 (11.21)
11.26 (11.21)
13.32 (13.93)
13.96 (13.23)
11.88 (11.58)
12.63 (12.49)
Cl
NO2
NO2
N
O
6g
6h
6i
C19H9N9O2S2
C25H17N9O2S2
C25H17N9S2
OCH3
CH3
6j
CH3
C13H9N9S2
355
731
279–281
40
50
43.93 (43.76)
37.77 (37.80)
2.55 (2.60)
1.52 (1.52)
35.47 (35.19)
17.24 (19.65)
18.04 (18.17)
8.77 (8.89)
I
6k
C23H11I2N9S2
[300
N
6l
C21H11N11S2
481
[300
48
52.38 (52.49)
2.30 (2.36)
32.00 (32.25)
13.32 (12.72)
Antibacterial activity
minimum inhibitory concentration (MIC) of the compounds
6a–l, the antibacterial antibiotic gentamicin and ampicillin
sodium (Table 3) were in accordance with the results
obtained in the primary screening. Moderate antibacterial
acticity was observed for the tested compounds 6a–l against
Staphylococcus aureus in comparison to reference genta-
micin and ampicillin, the diameter of the inhibition zone of
the most active compound 6k (MIC = 32 lg/mL) is
16 mm. The results indicated that the compounds 6b, 6h,
and 6k have a certain antibacterial activity against Bacillus
With gentamicin and ampicillin sodium as the reference
drug molecules, the newly synthesized compounds
6a–l were tested for in vitro antibacterial activity against two
Gram-negative and three Gram-positive bacterial strains.
The diameter of growth inhibition zone of the preliminary
antimicrobial testing of both the compounds 6a–l (250 lg/
tube) and the antibacterial antibiotic gentamicin and ampi-
cillin sodium (100 lg/tube) are shown in Table 2. The
123

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Med Chem Res (2014) 23:1941–1949
Table 2 Antibacterial activity of the compounds 6a–l as diameter of
growth inhibition zone
Table 3 Antibacterial activities of the compounds 6a–l as MIC
values (lg/mL)
Compd.
Diameter of growth inhibition zone (mm)
Compd.
Minimum inhibitory concentrations (MIC, lg/
mL)
Gram-positive
bacteriaa
Gram-negative
bacteriab
Gram-positive
bacteria
Gram-negative
bacteria
SA
BS
SP
EC
PA
SA
BS
SP
EC
PA
6a
12
15
14
13
13
12
14
14
13
12
16
12
22
10
14
13
11
11
11
12
14
10
10
14
10
22
21
12
16
16
15
14
13
12
14
13
12
14
13
21
18
12
17
15
15
14
14
15
16
14
14
18
14
25
17
10
15
14
12
12
13
15
12
10
12
15
13
21
17
6a
128
32
–
128
32
128
32
64
64
64
64
64
32
64
64
32
64
0.5
2
–
6b
6b
64
64
128
128
128
128
64
–
32
64
128
128
128
32
128
–
6c
6c
64
32
6d
6d
128
128
128
64
64
6e
6e
64
6f
6f
128
128
64
6g
6g
6h
6h
64
6i
6i
128
128
32
128
128
64
6j
6j
–
128
64
128
1
6k
6k
64
–
6l
6l
128
2
128
2
Gentamicin
Gentamicin
2
Ampicillin sodium 23
Ampicillin sodium 1
0.5
2
2
a
SA (S. aureus); BS (B. subtilis); SP (S. pneumoniae)
EC (E. coli); PA (P. aeruginosa)
b
(–) MIC values [128 lg/mL
antibacterial activity of the compound 6b was better than
that of the compound 6c. Otherwise, the antibacterial
activity of the derivatives substituted by the donative
electron group was better than that of the derivatives
substituted by the electrophilic group, the antibacterial
activity of the compound 6h was better than that of the
compound 6e. The preliminary results revealed that the
compounds 6b and 6k exhibited very promising activity
against E. coli and P. aeruginosa.
subtilis and the rest of the compounds have low antibacterial
activity. Most of the tested compounds have a good anti-
bacterial activity against Streptococcus pneumoniae, the
compounds 6b (MIC = 32 lg/mL) and 6c (MIC = 32 lg/
mL) were found to be as effective as reference drug ampi-
cillin. In addition, all the target compounds 6a–l displayed
potential antibacterial activity against Escherichia coli, the
most active compounds are 6b (MIC = 32 lg/mL) and 6k
(MIC = 32 lg/mL) with 17 and 18 mm diameters of the
inhibition zone, respectively. It can be observed that the
compounds 6b, 6g, and 6k exhibited a good antibacterial
activity against Pseudomonas aeruginosa.
Experimental
The relationship between the structures and biological
activity was discussed. The antimicrobial activity results of
the compounds 6a–l revealed that the antimicrobial activity
of aryl substituted derivatives was better than that of the
aliphatic substituted derivatives, and the phenyl substituted
derivatives were better than that of the heterocyclic
substituted derivatives. The results indicated that the anti-
bacterial activity of the derivatives with substituent in
phenyl was higher than that of the derivatives without
substituent in phenyl for the target compounds with the
same sketch structure. For example, the antibacterial
activity of the derivatives 6b–e, 6h, 6i, and 6k were better
than that of the compound 6a. The antibacterial activity of
the para-substituent derivatives were better than that of the
ortho- and meta-substituent derivatives. For example, the
Materials and methods
The five bacteria used were provided by the Hunan Uni-
versity of Chinese Medicine (HNUCM). Melting points
were determined with TECH XT-4 melting point apparatus
and were uncorrected. Infrared spectra were obtained in
solid state as KBr pallet on a PERKIN-ELMER Spectrum
1
One. H NMR spectra were registered on a Varian-400
NMR instrument (400 MHz) and 13C NMR spectra were
registered on a Bruker spectrometer at 400 MHz using
DMSO-d6 as solvent and the tetramethylsilane (TMS) as an
internal standard, chemical shifts are reported as d values
in units of ppm. The mass spectra were recorded on LC–
MS-Agilent 1,100 series. Elemental analyses were per-
formed on VARIO EL (III) elemental analyzer.
123

Med Chem Res (2014) 23:1941–1949
Chemistry
1945
portions of cold water, then recrystallized from ethanol–
water, and dried under vacuum. Yield 64 %. 1H NMR
(400 MHz, DMSO-d6) d/ppm: 6.382 (s, 4H, NH2); 8.124
(d, 2H, J = 6.8 Hz, pyridine protons); 8.169 (dd, 1H,
J1 = 9.6 Hz, J2 = 6.0 Hz, pyridine proton); 10.950 (s, 2H,
SH); 13C NMR (400 MHz, DMSO-d6) d/ppm: 165.65
Synthesis of pyridine-2,6-dicarboxylic acid dimethylester
(2)
A
mixture of pyridine-2,6-dicarboxylicacid (10.1 g,
0
(C3,3 -triazole ring); 146.55 (C2,6-pyridine); 145.14 (C5,5
0
-
60 mmol) and absolute methanol (80 mL) was heated to
30 °C with stirring in a 150-mL three-neck flask, then acetyl
chloride (3.5 mL, 90 mmol) was added portionwise. The
reaction mixture was refluxed for 20 h at 90 °C, cooled to
the room temperature (RT), and then filtered. The filter cake
was washed with absolute ethanol, and dried under vacuum
to get the pyridine-2,6-dicarboxylicaciddimethylester. Yield
triazole ring); 139.67 (C4-pyridine); 124.50 (C3,5-pyridine).
General synthesis procedure of 2,6-bis(6-substituted-1,2,4-
triazolo[3,4-b][1,3,4]thiadiazol-3-yl)pyridine (6a–l)
A mixture of 5,50-(pyridine-2,6-diyl)bis(4-amino-3-mer-
capto-1,2,4-triazole) 5 (0.307 g, 1 mmol) and the substi-
tuted carboxylic acid (4 mmol) in 10 mL POCl3 was
refluxed for 6 h at 110 °C in a 50-mL single-neck flask.
The reaction mixture was poured into 50 mL cold water
with vigorous stirring, alkalized with sodium hydroxide
(2 mol/L) to pH 10, and then filtered. The filter cake was
washed thoroughly with cold water, and then recrystallized
from dimethylformamide (DMF) to yield the target com-
pounds 6a–l.
1
86 %. H NMR (400 MHz, DMSO-d6) d/ppm: 2.10 (s, 6H,
2CH3), 8.18 (t, 1H, J = 7.8 Hz, pyridine proton); 8.41 (d,
2H, J = 7.8 Hz, pyridine protons).
Synthesis of pyridine-2,6-dimethylhydrazine (3)
A mixture of pyridine-2,6-dicarboxylicaciddimethylester
(9.75 g, 50 mmol) and 80 % hydrazine hydrate (20 mL) in
100 mL of ethanol was refluxed for 10 h at 80 °C with
stirring in a 150-mL three-neck flask. After cooled to RT,
the crude product was obtained. Which was filtered and
washed thoroughly with 10 mL ethanol and 10 mL diethyl
ether, then dried under vacuum to get a white needle
2,6-Bis(6-phenyl-1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-3-yl)
pyridine (6a) A light brown solid, Yield 53 %, m.p.
269–271 °C. 1H NMR (400 MHz, DMSO-d6) d/ppm: 7.361
(t, 4H, J = 7.8 Hz, aromatic protons); 7.506 (t, 2H, J = 7.4
Hz, aromatic protons); 7.938 (d, 4H, J = 7.2 Hz, aromatic
protons); 8.306 (dd, 1H, J1 = 8.8 Hz, J2 = 7.2 Hz, pyridine
proton); 8.436 (d, 2H, J = 7.6 Hz, pyridine protons); 13C
1
crystal. Yield 90 %. H NMR (400 MHz, DMSO-d6) d/
ppm: 2.12 (s, 4H, NH2), 7.84 (s, 2H, NH), 8.23 (t, 1H,
J = 8.0 Hz, pyridine proton); 8.31 (d, 2H, J = 8.0 Hz,
pyridine protons); MS (ESI) m/z (%): 194 (M-1, 100), 195
(M, 5).
0
NMR (400 MHz, DMSO-d6) d/ppm: 167.36 (C8,8 -triazolo-
0
thiadiazole ring); 155.85 (C3,3 -triazolo-thiadiazole ring);
Synthesis of potassium pyridine-2,6-dithiocarbazates (4)
0
150.15 (C2,6-pyridine); 145.91 (C6,6 -triazolo-thiadiazole
0
ring); 139.58 (C4-pyridine); 133.23 (C1,1 -aromatic ring);
Pyridine-2,6-dimethylhydrazine (0.975 g, 5 mmol), potas-
sium hydroxide (1.12 g, 20 mmol), and absolute ethanol
(30 mL) were added to a 150-mL single-neck flask and
then carbon disulfide (1.52 g, 20 mmol) was added drop-
wise. The reaction mixture was stirred for 14 h at RT, and
then diluted with 30 mL of dry ether. The solid product
was collected by filtration and washed with 2 9 10 mL
ether, then dried at 65 °C under vacuum. The potassium
salt was used for the next reaction directly without
purification.
0
129.82 (C2,2 ,6,6 -aromatic ring); 129.34 (C4,4 -aromatic
0
0
0
0
ring); 127.71 (C3,3 ,5,5 -aromatic ring); 123.62 (C3,5-pyri-
dine); IR (KBr) v/cm-1: 3081 (Aromatic C–H), 1591 (C=N),
1572 (Aromatic C–H), 1519 (Aromatic C–H), 1457 (C=C),
1365 (Aromatic C–H), 1237 (N–N=C), 686 (C–S–C); MS
(EI) m/z (%): 481 (M?2, 16), 480 (M?1, 31), 479 (M, 100),
478 (M-1, 15), 406 (5), 304 (10), 187 (4), 173 (5), 161 (18),
129 (9), 121 (46), 103 (85), 77 (16), 76 (26), 51 (7).
2,6-Bis(6-(4-chlorophenyl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)pyridine (6b) A light brown solid, Yield 45 %,
m.p. [300 °C. 1H NMR (400 MHz, DMSO-d6) d/ppm:
7.279 (d, 4H, J = 8.0 Hz, aromatic protons); 7.842 (d, 4H,
J = 8.4 Hz, aromatic protons); 8.279 (t, 1H, J = 8.0 Hz,
pyridine proton); 8.406 (d, 2H, J = 8.0 Hz, pyridine
protons); 13C NMR (400 MHz, DMSO-d6) d/ppm:
Synthesis of 5,50-(pyridine-2,6-diyl)bis(4-amino-3-
mercapto-1,2,4-triazole) (5)
A mixture of potassium salt (4) (1.30 g, 5 mmol) and 85 %
hydrazine hydrate (2 mL, 35 mmol) was refluxed for 6 h at
110 °C, then diluted with 30 mL of cold water and acidi-
fied with hydrochloric acid (6 mol/L) to pH 6. The white
precipitate was filtered and washed with 2 9 15 mL
0
166.11 (C8,8 -triazolo-thiadiazole ring); 156.13 (C3,3 -triaz-
0
olo-thiadiazole ring); 145.96 (C2,6-pyridine); 145.84
123

1946
Med Chem Res (2014) 23:1941–1949
0
(C6,6 -triazolo-thiadiazole ring); 139.52 (C4-pyridine);
2,6-Bis(6-(3-nitrophenyl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
0
137.84 (C4,4 -aromatic ring); 129.56 (C3,3 ,5,5 -aromatic
0
0
diazol-3-yl)pyridine (6e) A light yellow solid, Yield
1
0
0
0
56 %, m.p. [300 °C. H NMR (400 MHz, DMSO-d6) d/
ring); 129.29 (C2,2 ,6,6 -aromatic ring); 128.10 (C1,1 -aro-
matic ring); 123.27 (C3,5-pyridine); IR (KBr) v/cm-1: 3087
(Aromatic C–H), 1594 (C=N), 1572 (Aromatic C–H), 1523
(Aromatic C–H), 1461 (C=C), 1364 (Aromatic C–H), 1236
(N–N=C), 677 (C–S–C); MS (EI) m/z (%): 551 (M?4, 9),
550 (M?3, 11), 549 (M?2, 35), 548 (M?1, 17), 547 (M, 46),
512 (11), 338 (4), 195 (11), 155 (38), 139 (39), 137 (100), 102
(39), 77 (6), 76 (12), 75 (16), 51 (7).
ppm: 7.550 (t, 2H, J = 8.0 Hz, aromatic protons); 8.074
(d, 2H, J = 8.0 Hz, aromatic protons); 8.224 (d, 2H, J =
8.0 Hz, aromatic protons); 8.293 (dd, 1H, J1 = 8.4 Hz,
J2 = 7.2 Hz, pyridine proton); 8.417 (d, 2H, J = 8.4 Hz,
pyridine protons); 8.443 (s, 2H, aromatic protons); 13C
0
NMR (400 MHz, DMSO-d6) d/ppm: 165.15 (C8,8 -triazolo-
0
thiadiazole ring); 156.16 (C3,3 -triazolo-thiadiazole ring);
0
147.46 (C3,3 -aromatic ring); 146.08 (C2,6-pyridine);
0
145.67 (C6,6 -triazolo-thiadiazole ring); 139.62 (C4-pyri-
2,6-Bis(6-(3-chlorophenyl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)pyridine (6c) A gray white solid, Yield 48 %,
m.p. [300 °C. 1H NMR (400 MHz, DMSO-d6) d/ppm:
7.328 (t, 2H, J = 7.8 Hz, aromatic protons); 7.461 (d, 2H,
J = 8.0 Hz, aromatic protons); 7.830 (d, 2H, J = 7.6 Hz,
aromatic protons); 7.872 (s, 2H, aromatic protons); 8.280 (q,
1H, J = 5.2 Hz, pyridine proton); 8.397 (d, 2H, J = 8.0 Hz,
pyridine protons); 13C NMR (400 MHz, DMSO-d6) d/ppm:
0
dine); 133.90 (C6,6 -aromatic ring); 131.52 (C1,1 -aromatic
0
0
ring); 130.68 (C5,5 -aromatic ring); 126.91 (C4,4 -aromatic
0
0
ring); 123.16 (C2,2 -aromatic ring); 121.16 (C3,5-pyridine);
IR (KBr) v/cm-1: 3083 (Aromatic C–H), 1592 (C=N),
1574 (Aromatic C–H), 1533 (Aromatic C–H), 1457 (C=C),
1345 (Aromatic C–H), 1242 (N–N=C), 674 (C–S–C); MS
(EI) m/z (%): 571 (M?2, 15), 570 (M?1, 34), 569
(M, 100), 207 (6), 206 (15), 166 (34), 148 (20), 137 (11),
129 (12), 120 (30), 102 (45), 90 (8), 77 (7), 76 (16), 75
(18), 64 (9), 51 (10).
0
0
165.74 (C8,8 -triazolo-thiadiazole ring); 156.04 (C3,3 -triaz-
0
olo-thiadiazole ring); 146.04 (C2,6-pyridine); 145.75 (C6,6
-
triazolo-thiadiazole ring); 139.67 (C4-pyridine); 134.40
0
(C3,3 -aromatic ring); 132.75 (C1,1 -aromatic ring); 131.50
0
2,6-Bis(6-(pyridin-3-yl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
0
0
(C5,5 -aromatic ring); 131.14 (C6,6 -aromatic ring); 126.77
diazol-3-yl)pyridine (6f) A dark brown solid, Yield
1
42 %, m.p. [300 °C. H NMR (400 MHz, DMSO-d6) d/
ppm: 7.336 (dd, 2H, J1 = 8.0 Hz, J2 = 5.2 Hz, pyridine
protons), 7.943 (s, 2H, pyridine protons), 8.281 (d, 2H,
J = 7.6 Hz, pyridine protons), 8.323 (t, 1H, J = 8.0 Hz,
pyridine proton), 8.472 (d, 2H, J = 7.6 Hz, pyridine pro-
tons), 8.613 (d, 2H, J = 4.4 Hz, pyridine protons); 13C
0
0
(C4,4 -aromatic ring); 126.59 (C2,2 -aromatic ring); 123.29
(C3,5-pyridine); IR (KBr) v/cm-1: 3063 (Aromatic C–H),
1592 (C=N), 1572 (Aromatic C–H), 1517 (Aromatic C–H),
1454 (C=C), 1366 (Aromatic C–H), 1234 (N–N=C), 683 (C–
S–C); MS (EI) m/z (%): 551 (M?4, 14), 550 (M?3, 16), 549
(M?2, 59), 548 (M?1, 26), 547 (M, 75), 512 (11), 338 (13),
195 (21), 155 (70), 139 (34), 137 (100), 102 (40), 77 (15), 76
(16), 75 (22), 51 (11).
0
NMR (400 MHz, DMSO-d6) d/ppm: 164.88 (C8,8 -triazolo-
0
thiadiazole ring); 156.07 (C3,3 -triazolo-thiadiazole ring);
0
152.72 (C6,6 -triazolo-thiadiazole ring); 149.10 (C2,2 of
0
2,6-Bis(6-(4-nitrophenyl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
0
6-substituted pyridine); 148.09 (C6,6 of 6-substituted pyr-
diazol-3-yl)pyridine (6d) A aurantiaceous solid, Yield
1
55 %, m.p. [300 °C. H NMR (400 MHz, DMSO-d6) d/
0
idine); 146.00 (C2,6-pyridine); 145.83 (C4,4 of 6-substi-
0
tuted pyridine); 139.69 (C4-pyridine); 135.23 (C3,3 of
ppm: 7.910 (d, 4H, J = 8.0 Hz, aromatic protons); 8.085
(d, 4H, J = 8.4 Hz, aromatic protons); 8.346 (t, 1H, J =
8.8 Hz, pyridine proton); 8.463 (d, 2H, J = 7.2 Hz, pyri-
dine protons); 13C NMR (400 MHz, DMSO-d6) d/ppm:
0
6-substituted pyridine); 125.87 (C5,5 of 6-substituted pyr-
idine); 124.51 (C3,5-pyridine); IR (KBr) v/cm-1: 3083
(Aromatic C–H), 1590 (C=N), 1573 (Aromatic C–H), 1519
(Aromatic C–H), 1457 (C=C), 1360 (Aromatic C–H), 1260
(N–N=C), 670 (C–S–C); MS (EI) m/z (%): 483 (M?2, 6),
482 (M?1, 13), 481 (M, 56), 305 (15), 207 (26), 173 (7),
162 (4), 129 (14), 122 (41), 104 (100), 103 (22), 77 (60), 64
(6), 51 (17).
0
166.66 (C8,8 -triazolo-thiadiazole ring); 154.68 (C3,3
0
-
0
triazolo-thiadiazole ring); 149.42 (C6,6 -triazolo-thiadiazole
0
ring); 146.38 (C2,6-pyridine); 141.17 (C4,4 -aromatic ring);
0
139.76 (C4-pyridine); 138.27 (C1,1 -aromatic ring); 128.93
0
0
0
0
(C2,2 ,6,6 -aromatic ring); 124.27 (C3,3 ,5,5 -aromatic ring);
119.95 (C3,5-pyridine); IR (KBr) v/cm-1: 3060 (Aromatic
C–H), 1605 (C=N), 1576 (Aromatic C–H), 1528 (Aromatic
C–H), 1459 (C=C), 1348 (Aromatic C–H), 1263 (N–N=C),
688 (C–S–C); MS (EI) m/z (%): 571 (M?2, 8), 570 (M?1,
14), 569 (M, 43), 207 (14), 206 (8), 166 (15), 148 (50), 137
(19), 129 (16), 118 (36), 102 (100), 90 (28), 77 (17), 76
(31), 75 (46), 64 (33), 51 (28).
2,6-Bis(6-(furan-2-yl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)pyridine (6g) A brown solid, Yield 40 %,
m.p. [300 °C. 1H NMR (400 MHz, DMSO-d6) d/ppm:
6.674 (dd, 2H, J1 = 3.6 Hz, J2 = 2 Hz, furan protons);
7.428 (d, 2H, J = 2 Hz, furan protons), 8.068 (d, 2H, J2 =
2 Hz, furan protons); 8.338 (q, 1H, J = 7.4 Hz, pyridine
123

Med Chem Res (2014) 23:1941–1949
1947
proton); 8.397 (d, 2H, J = 7.2 Hz, pyridine protons); 13C
2,6-Bis(6-methyl-1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-3-yl)
pyridine (6j) A light brown solid, Yield 40 %, m.p.
279–281 °C. 1H NMR (400 MHz, DMSO-d6) d/ppm: 2.819
(s, 6H, CH3); 8.261 (q, 1H, J = 7.4 Hz, pyridine proton);
8.331 (d, 2H, J = 6.8 Hz, pyridine protons); 13C NMR
0
NMR (400 MHz, DMSO-d6) d/ppm: 166.44 (C8,8 -triazolo-
0
thiadiazole ring); 161.64 (C6,6 -triazolo-thiadiazole ring);
0
153.22 (C3,3 -triazolo-thiadiazole ring); 148.14 (C2,6-pyri-
0
0
dine); 145.91 (C5,5 -furan); 145.84 (C2,2 -furan); 143.41
0
0
(400 MHz, DMSO-d6) d/ppm: 167.24 (C8,8 -triazolo-thia-
(C4-pyridine); 123.70 (C3,5-pyridine); 115.56 (C3,3 -furan);
113.77 (C4,4 -furan); IR (KBr) v/cm-1: 3114 (Aromatic C–
0
diazole ring); 156.76 (C3,3 -triazolo-thiadiazole ring);
0
0
145.95 (C2,6-pyridine); 145.37 (C6,6 -triazolo-thiadiazole
H), 1592 (C=N), 1572 (Aromatic C–H), 1505 (Aromatic
C–H),1453 (C=C), 1360 (Aromatic C–H), 1269 (N–N=C),
682 (C–S–C); MS (EI) m/z (%): 461 (M?2, 14), 460
(M?1, 28), 459 (M, 100), 405 (6), 294 (15), 173 (4), 151
(17), 129 (12), 111 (37), 93 (36), 76 (4), 64 (15).
ring); 139.35 (C4-pyridine); 123.48 (C3,5-pyridine); 18.54
(-CH3); IR (KBr) v/cm-1: 3040 (Aromatic C–H), 1596
(C=N), 1575 (Aromatic C–H), 1537 (Aromatic C–H), 1460
(C=C), 1389 (Aromatic C–H), 1242 (N–N=C), 682 (C–S–
C); MS (EI) m/z (%): 357 (M?2, 11), 356 (M?1, 20), 355
(M, 98), 244 (14), 243 (100), 242 (18), 187 (9), 173 (18), 147
(4), 130 (12), 129 (25), 103 (32), 99 (2), 76 (11), 59 (32).
2,6-Bis(6-(4-methoxyphenyl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)pyridine (6h) A dark brown solid, Yield 42 %,
1
m.p. 249–251 °C. H NMR (400 MHz, DMSO-d6) d/ppm:
2,6-Bis(6-(3-iodophenyl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)pyridine (6k) A yellow solid, Yield 50 %, m.
p.[300 °C. 1H NMR (400 MHz, DMSO-d6) d/ppm: 7.108
(t, 2H, J = 7.8 Hz, aromatic protons); 7.751 (d, 2H, J =
8.4 Hz, aromatic protons); 7.888 (d, 2H, J = 7.6 Hz, aro-
matic protons); 8.153 (s, 2H, aromatic protons); 8.302
(q, 1H, J = 5.2 Hz, pyridine proton); 8.408 (d, 2H, J = 8.0
Hz, pyridine protons); 13C NMR (400 MHz, DMSO-d6)
3.773 (s, 6H, OCH3); 6.839 (d, 4H, J = 8.4 Hz, aromatic
protons); 7.836 (d, 4H, J = 8.4 Hz, aromatic protons); 8.282
(t, 1H, J = 8.0 Hz, pyridine proton), 8.406 (d, 2H, J =
7.2 Hz, pyridine protons); 13C NMR (400 MHz, DMSO-d6)
0
d/ppm: 166.92 (C8,8 -triazolo-thiadiazole ring); 163.14
0
0
(C4,4 -aromatic ring); 155.77 (C3,3 -triazolo-thiadiazole
ring); 145.95 (C2,6-pyridine); 139.44 (C4-pyridine); 129.52
0
(C6,6 -triazolo-thiadiazole ring); 128.89 (C2,2 ,6,6 -aromatic
0
0
0
d/ppm: 165.63 (C8,8 -triazolo-thiadiazole ring); 156.00
0
ring); 123.39 (C1,1 -aromatic ring); 121.66 (C3,5-pyridine);
0
(C3,3 -triazolo-thiadiazole ring); 146.05 (C2,6-pyridine);
0
0
0
145.75 (C6,6 -triazolo-thiadiazole ring); 141.47 (C2,2 -aro-
0
115.08 (C3,3 ,5,5 -aromatic ring); 56.00 (–OCH3); IR (KBr)
v/cm-1: 3083 (Aromatic C–H), 1606 (C=N), 1574 (Aromatic
C–H), 1527 (Aromatic C–H), 1457 (C=C), 1364 (Aromatic
C–H), 1263 (N–N=C), 683 (C–S–C); MS (EI) m/z (%): 541
(M?2, 15), 540 (M?1, 38), 539 (M, 100), 538 (M-1, 34),
436 (5), 334 (12), 191 (11), 165 (7), 151 (56), 133 (49), 103
(24), 90 (18), 76 (7).
0
matic ring); 139.64 (C4-pyridine); 135.16 (C1,1 -aromatic
0
ring); 131.36 (C4,4 -aromatic ring); 131.12 (C5,5 -aromatic
0
0
ring); 127.43 (C6,6 -aromatic ring); 123.21 (C3,5-pyridine);
95.87 (C3,3 -aromatic ring); IR (KBr) v/cm-1: 3040 (Aro-
0
matic C–H), 1596 (C=N), 1573 (Aromatic C–H), 1513
(Aromatic C–H), 1452 (C=C), 1364 (Aromatic C–H), 1239
(N–N=C), 681 (C–S–C); MS (EI) m/z (%): 754 (M ? 23,
42), 755 (10), 1484 (2M?23, 100), 1485 (59), 1486 (41),
1487 (15).
2,6-Bis(6-(4-methylphenyl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)pyridine (6i) A yellow solid, Yield 61 %, m.p.
[300 °C. 1H NMR (400 MHz, DMSO-d6) d/ppm: 2.256 (s,
6H, CH3); 7.052 (d, 4H, J = 8.0 Hz, aromatic protons);
7.714 (d, 4H, J = 8.0 Hz, aromatic protons); 8.220 (t, 1H,
J = 7.6 Hz, pyridine proton); 8.344 (d, 2H, J = 8.0 Hz,
pyridine protons); 13C NMR (400 MHz, DMSO-d6) d/ppm:
2,6-Bis(6-(pyridin-4-yl)-1,2,4-triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)pyridine (6l) A dark brown solid, Yield
1
48 %, m.p. [300 °C. H NMR (400 MHz, DMSO-d6) d/
ppm: 7.841 (d, 4H, J = 6.0 Hz, pyridine protons), 8.327 (t,
1H, J = 8.0 Hz, pyridine protons), 8.462 (d, 2H, J =
8.0 Hz, pyridine protons), 8.518 (d, 4H, J = 5.6 Hz, pyr-
idine protons); 13C NMR (400 MHz, DMSO-d6) d/ppm:
0
0
167.22 (C8,8 -triazolo-thiadiazole ring); 155.72 (C3,3 -triaz-
0
olo-thiadiazole ring); 149.43 (C2,6-pyridine); 145.84 (C6,6
-
0
triazolo-thiadiazole ring); 143.44 (C4,4 -aromatic ring);
0
139.39 (C4-pyridine); 130.11 (C3,3 ,5,5 -aromatic ring);
0
0
165.46 (C8,8 -triazolo-thiadiazole ring); 157.13 (C3,3 -
0
0
0
0
0 0
triazolo-thiadiazole ring); 151.10 (C2,2 ,6,6 of 6-substituted
127.54 (C2,2 ,6,6 -aromatic ring); 126.53 (C1,1 -aromatic
ring); 123.37 (C3,5-pyridine); 21.46 (–CH3); IR (KBr)
v/cm-1: 3050 (Aromatic C–H), 1592 (C=N), 1575 (Aromatic
C–H), 1523 (Aromatic C–H), 1456 (C=C), 1383 (Aromatic
C–H), 1237 (N–N=C), 685 (C–S–C); MS (EI) m/z (%): 509
(M?2, 17), 508 (M?1, 33), 507 (M, 100), 434 (4), 318 (10),
175 (11), 151 (6), 135 (47), 117 (34), 116 (25), 103 (18), 90
(16), 77 (6).
0
pyridine); 148.24 (C2,6-pyridine); 145.80 (C6,6 -triazolo-
0
thiadiazole ring); 140.65 (C4-pyridine); 136.41 (C4,4 of
6-substituted pyridine); 123.52 (C3,5-pyridine); 121.18
(C3,3 ,5,5 of 6-substituted pyridine); IR (KBr) v/cm-1: 3030
(Aromatic C–H), 1596 (C=N), 1573 (Aromatic C–H), 1512
(Aromatic C–H), 1457 (C=C), 1369 (Aromatic C–H), 1243
(N–N=C), 685 (C–S–C); MS (EI) m/z (%): 483 (M?2, 10),
0
0
123

1948
Med Chem Res (2014) 23:1941–1949
Almajan GL, Barbuceanu SF, Bancescu G, Saramet I, Saramet G,
Draghici C (2010b) Synthesis and antimicrobial evaluation of
some fused heterocyclic[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
derivatives. Eur J Med Chem 45:6139–6146
482 (M?1, 23), 481 (M, 78), 305 (6), 207 (23), 173 (9),
162 (8), 129 (18), 122 (47), 104 (100), 103 (29), 77 (64), 64
(26), 51 (20).
Badr SMI, Barwa RM (2011) Synthesis of some new [1,2,4]triazol-
o[3,4-b][1,3,4]thiadiazines and [1,2,4]triazolo[3,4-b][1,3,4]thi-
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Antibacterial activity
The newly synthesized compounds were assayed in vitro for
antibacterial activity against the Gram-positive bacteria (S.
aureus; B. subtilis; S. pneumoniae) and Gram-negative
bacteria (E. coli; P. aeruginosa) by using the Oxford cup
method (cylinder-plate method) (Plein and Plein, 1957;
Mohammadzadeh et al., 2012). Gentamicin and ampicillin
sodium were used as the control drugs to compare the anti-
bacterial activity. The target compounds were dissolved in
dimethylsulfoxide (DMSO) of specific concentration
(250 lg/tube), the antibacterial antibiotics Gentamicin and
Ampicillin Sodium (100 lg/tube) were carefully placed on
the agarose gel culture plates that have been previously
inoculated separately with the microorganisms, and incu-
bated at 36 °C for 24 h. The diameter of the growth inhibi-
tion zone was measured. In order to ensure that the solvent
had no effect on bacterial growth, a control test was carried
out with only DMSO on the agarose gel culture plates, the
test results showed that the solvent had no antibacterial
activity against any of the test microorganisms.
The MIC was determined by using the serial dilutions
method. The synthesized compounds 6a–l, gentamicin, and
ampicillin sodium were dissolved in DMSO at concentra-
tion of 128 lg/mL. Nine different dilutions of the test
compounds between 128 and 0.5 lg/mL were prepared in
plates by serial dilutions from top to bottom. Then the
microorganisms suspensions at 106 CFU/mL (colony
forming unit/mL) concentrations were inoculated to the
corresponding wells. The MIC was defined as the lowest
concentration of the test compounds that inhibited visible
growth of microorganisms on the plate.
Acknowledgments The authors are grateful for the financial sup-
port of the National Natural Science Foundation of China (No.
J1103312), and the Natural Science Foundation of Hunan Province
(No. 11JJ5005), and the Innovative Research Team in University.
(No. IRT1238) as well as the Science and Technology Project of
Hunan provincial Science and Technology Department (No.
2012GK3156). We also thank the Hunan University of Chinese
Medicine (HNUCM) for providing the antibacterial screening, and
Dr. William Hickey, the U.S. professor of HRM, for the English
editing on this paper.
Mohammadzadeh T, Sadjjadi SM, Habibi P, Sarkari B (2012)
Comparison of agar dilution, broth dilution, cylinder plate and
disk diffusion methods for evaluation of anti-leishmanial drugs
on leishmania promastigotes. Iran J Parasitol 7:43–47
Plein EM, Plein JB (1957)
A cylinder-plate method for the
quantitative determination of diffusion of antibacterial drugs
from ointments. J Pharm Sci 46:716–720
Sahoo PK, Sharma R, Pattanayak P (2010) Synthesis and evaluation
of 4-amino-5-phenyl-4H-[1,2,4]-triazole-3-thiol derivatives as
antimicrobial agents. Med Chem Res 19:127–135
Said ASG, Mohammed AA, Abd EI-Galil EA et al (2011) Synthesis
and antimicrobial activity of some heterocyclic 2,6-bis(substi-
tuted)-1,3,4-thiadiazolo-, oxadiazolo-, and oxathiazolidino-pyri-
dine derivatives from 2,6-pyridine dicarboxylic acid dihydrazide.
J Heterocyclic Chem 48:1103–1110
Sangshetti JN, Lokwani DK, Sarkate AP, Shinde DB (2011)
Synthesis, antifungal activity, and docking study of some new
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