Synthesis and biological activities of 3,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivatives

Article abstract of DOI:10.4314/bcse.v31i3.12

Twelve novel triazolothiadiazole derivatives were synthesized from 4-amino-5-substituted-4H-1,2,4-triazole-3-thiols with various aromatic carboxylic acids by cyclization in the presence of phosphorous oxychloride. All the newly synthesized compounds were characterized by FTIR, ¹H NMR, mass spectroscopy and elemental analysis. The antimicrobial activities of the title compounds were examined by disc diffusion method against Escherichia coli, Staphylococcus aureus, Pyricularia oryzae and Rhizoctnia solani. The bioassay indicated all synthesized triazolothiadiazole derivatives possessed moderate to good antibacterial and antifungal activities against the tested organisms. Especially, compounds 2e and 2k exhibited excellent antibacterial and antifungal activities among these triazolothiadiazole derivatives.

Full text of DOI:10.4314/bcse.v31i3.12

Bull. Chem. Soc. Ethiop. 2017, 31(3), 481-489.
 2017 Chemical Society of Ethiopia and The Authors
DOI: http://dx.doi.org/10.4314/bcse.v31i3.12
ISSN 1011-3924
Printed in Ethiopia
SYNTHESIS AND BIOLOGICAL ACTIVITIES OF 3,6-DISUBSTITUTED-1,2,4-
TRIAZOLO-1,3,4-THIADIAZOLE DERIVATIVES
Lu Lin, Hua Liu, Dun-Jia Wang*, Yan-Jun Hu and Xian-Hong Wei
College of Chemistry and Chemical Engineering, Hubei Collaborative Innovation Center for
Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis and Reuse Technology,
Hubei Normal University, Huangshi 435002, China
(Received November 21, 2014; Revised November 19, 2017; Accepted November 27, 2017)
ABSTRACT. Twelve novel triazolothiadiazole derivatives were synthesized from 4-amino-5-substituted-4H-
1,2,4-triazole-3-thiols with various aromatic carboxylic acids by cyclization in the presence of phosphorous
oxychloride. All the newly synthesized compounds were characterized by FTIR, 1H NMR, mass spectroscopy and
elemental analysis. The antimicrobial activities of the title compounds were examined by disc diffusion method
against Escherichia coli, Staphylococcus aureus, Pyricularia oryzae and Rhizoctnia solani. The bioassay indicated
all synthesized triazolothiadiazole derivatives possessed moderate to good antibacterial and antifungal activities
against the tested organisms. Especially, compounds 2e and 2k exhibited excellent antibacterial and antifungal
activities among these triazolothiadiazole derivatives.
KEY WORDS: Triazolothiadiazole, Triazole, Thiadiazole, Antimicrobial activity
INTRODUCTION
Since Kanaoka reported the triazole[3,4-b]-1,3,4-thiadiazole derivatives by condensing the
triazole and thiadiazole molecules in 1956 [1], the triazolothiadiazole derivatives have received
considerable interest due to their diverse biological activities such as fungicidal [2], bactericidal
[3], insecticidal [4], herbicidal [5], anti-inflammatory [6], anticonvulsant [7], anticancer [8] and
analgesic [9] activities. Thus, many chemists reported synthesis and antimicrobial activity of
some 1,2,4-triazolothiadiazole derivatives in recent years [1015]. Especially, Karegoudar
synthesized some new 1,2,4-triazolothiadiazoles bearing 2,3,5-trichlorophenyl moiety and
studied their antimicrobial activity [16], which revealed that these compounds showed good
antibacterial and antifungal activities.
Therefore, with the purpose of broadening the class of compounds exhibiting good
antimicrobial activity, we introduce the fluorophenyl, chlorophenyl moieties into the triazole
ring and the phenyl, nitrophenyl, tert-butylphenyl moieties into the thiadiazole ring to
investigate their antimicrobial activities. Herein we report the synthesis and biological activities
of some new 3,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivatives. Structures and
properties of the obtained compounds were demonstrated by means of elemental analysis, FT-
IR, 1H NMR, ESI-MS spectroscopy.
EXPERIMENTAL
General
Melting points were determined using X-4 digital melting-point apparatus and are uncorrected.
Elemental analysis (C, H, N) was performed with a Perkin Elmer 2400 elemental analyzer.
Infrared spectra were recorded on a Nicolet FTIR 5700 spectrophotometer with KBr pellets.
__________
*Corresponding author. E-mail: dunjiawang@163.com
This work is licensed under the Creative Commons Attribution 4.0 International License

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Lu Lin et al.
Electrospray ionization mass spectra (ESI–MS) were performed with a Finnigan LCQ
1
Advantage Max spectrometer. H NMR spectra were measured on an Avance IIITM 300 MHz
NB Digital NMR spectrometer in CDCl3 or DMSO–d6 solution with TMS as internal standard.
Substituted benzoyl hydrazines were synthesized by our group according to the method reported
in the literature [17]. Hydrazine hydrate, phosphorus oxychloride and substituted benzoic acid
were purchased from Shanghai Chemical Reagent Company Ltd. (Shanghai, China). Other
reagents were of analytical grade purity and used without further purification.
General synthetic procedure for 4-amino-5-substituted-4H-1,2,4-triazole-3-thiols (1a1d)
Substituted benzoyl hydrazines (0.02 mol) and KOH (0.06 mol) were dissolved in ethanol (25
mL). To this, CS2 (0.2 mol) was added dropwise with stirring at room temperature and
maintained for 12 h. The yellow potassium dithiocarbazinate salts were formed, filtered and
washed with absolute ethanol three times. The intermediate potassium dithiocarbazinates were
dissolved in hot water (25 mL) and hydrazine hydrate (0.2 mol) was added. This mixture was
heated and refluxed for 3 h, then poured into ice-water and acidified with concentrated
hydrochloric acid. The precipitates were filtered, washed with water and recrystallized from
ethanol to obtain 4-amino-5-substituted-4H-1,2,4- triazole-3-thiols (1a1d).
4-Amino-5-(2-chlorophenyl)-4H-1,2,4-triazole-3-thiol (1a). White crystals, yield 65%, m.p.
165167 oC; IR (KBr): ν 3326 (s), 3256 (s), 2622 (w), 1627 (m), 1561 (m), 1461 (s), 1302 (s),
1077 (m), 1026 (s), 952 (s), 757 (s), 726 (s) cm1; 1H NMR (300 MHz, CDCl3): δ 11.39 (s, 1H,
SH), 7.417.57 (m, 4H, ArH), 4.82 (s, 2H, NH2) ppm; ESI-MS m/z: 226.95 [M+]; anal. calcd.
for C8H7ClN4S: C, 42.39; H, 3.11; N, 24.72%. Found: C, 42.15; H, 3.08; N, 24.98%.
4-Amino-5-(2-fluorophenyl)-4H-1,2,4-triazole-3-thiol (1b). White crystals, yield 61%, m.p.
166168 oC; IR (KBr): ν 3314 (s), 3226 (m), 2629 (w), 1621 (s), 1457 (s), 1323 (s), 1238 (s),
1
1055 (s), 949 (s), 765 (s) cm1; H NMR (300 MHz, CDCl3): δ 10.75 (s, 1H, SH), 7.557.63
(m, 2H, ArH), 7.237.34 (m, 2H, ArH), 4.86 (s, 2H, NH2) ppm; ESI-MS m/z: 210.90 [M+];
anal. calcd. for C8H7FN4S: C, 45.70; H, 3.36; N, 26.65%. Found: C, 45.35; H, 3.32; N, 26.84%.
4-Amino-5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (1c). White crystals, yield 75%, m.p.
186188 oC; IR (KBr): ν 3320 (s), 3238 (s), 2631 (w), 1626 (m), 1575 (m), 1461 (s), 1236 (s),
1032 (s), 926 (s), 731 (s) cm1; 1H NMR (300 MHz, CDCl3): δ 11.93 (1H, SH), 7.92 (d, 2H, J
= 7.5 Hz, ArH), 7.48 (d, 2H, J = 7.5 Hz, ArH), 5.02 (s, 2H, NH2) ppm; ESI-MS: m/z 226.89
[M+]; anal. calcd. for C8H7ClN4S: C, 42.39; H, 3.11; N, 24.72%. Found: C, 42.16; H, 3.09; N,
24.96%.
4-Amino-5-(4-fluorophenyl)-4H-1,2,4-triazole-3-thiol (1d). Yellow crystals, yield 68%, m.p.
202204 oC; IR(KBr): ν 3319 (s), 3240 (s), 2625 (w), 1632 (m), 1615 (m), 1452 (s), 1325 (m),
1216 (m), 1161 (m), 1064 (s), 955 (m), 742 (m) cm1; 1H NMR (300 MHz, CDCl3): δ 11.72 (s,
1H, SH), 7.76 (d, 2H, J = 8.1 Hz, ArH), 7.15 (d, 2H, J = 8.1 Hz, ArH ), 4.93 (s, 2H, NH2)
ppm; ESI-MS: m/z 210.36 [M+]; anal. calcd. for C8H7FN4S: C, 45.71; H, 3.36; N 26.65%.
Found: C, 45.38; H, 3.31; N, 26.83%.
General synthetic procedure for 3,6-disubstituted-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles
(2a2l)
To a solution of 4-amino-5-substituted-4H-1,2,4-triazole-3-thiols (1a1d) (3.0 mmol) in
phosphorus oxychloride (25 mL), the substituted benzoic acid (4.5 mmol) was added dropwise.
The reaction mixture was stirred for 3 h under reflux. Then the mixture was poured into ice-
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water and adjusted to pH = 8 with NaOH solution. The precipitates were formed and filtered,
washed three times with ethanol. The products were recrystallized from absolute ethanol and
dried to obtain 3,6-disubstituted-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles (2a2l).
3-(2-Chlorophenyl)-6-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2a). White needle
crystals, yield 38%; m.p. 163–165 oC; IR (KBr): ν 3057(m), 1623 (m), 1520 (m), 1457 (s), 1380
1
(m), 1242 (m), 1062 (m), 964 (m), 762 (s), 688 (s) cm1; H NMR (300 MHz, CDCl3): δ
7.817.91 (m, 3H, ArH), 7.627.27 (m, 6H, ArH) ppm; ESI-MS: m/z 313.12 [M+]; anal.
calcd. for C15H9ClN4S: C, 57.60; H, 2.90; N, 17.91%. Found: C, 57.28; H, 2.87; N, 18.07%.
3-(2-Chlorophenyl)-6-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole(2b).
Yellow
crystals, yield 43%; m.p. 167–169 oC; IR (KBr): ν 3103 (m), 3030 (w), 1604 (m), 1529 (s), 1466
1
(s), 1348 (s), 1055 (m), 962 (m), 856 (m), 752 (m) cm1; H NMR(300 MHz, CDCl3): δ 8.38–
8.42 (m, 2H, ArH), 8.08–8.13 (m, 2H, ArH), 7.82 (d, 1H, J = 7.2 Hz, ArH), 7.51–7.64 (m,
3H, ArH) ppm; ESI-MS: m/z 358.18 [M+]; anal. calcd. for C15H8ClN5O2S: C, 50.36; H, 2.25;
N, 19.57%. Found: C, 50.74; H, 2.21; N, 19.79%.
6-(4-(tert-butyl)phenyl)-3-(2-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (2c). White
powder, yield 41%; m.p. 133–135 oC; IR (KBr): ν 3056 (m), 2961 (s), 1607 (s), 1467 (s), 1400
(m), 1235 (s), 1115 (m), 970 (m), 841 (m), 765 (m), 706 (m) cm1; 1H NMR(300 MHz, CDCl3):
δ 8.05 (d, 2H, J = 6.9 Hz, ArH), 7.80–7.85 (m, 2H, ArH), 7.46–7.62 (m, 4H, ArH), 1.36 (s,
9H, t-BuH) ppm; ESI-MS: m/z 369.19 [M+]; anal. calcd. for C19H17ClN4S: C, 61.86; H, 4.65;
N, 15.19%. Found: C, 61.54; H, 4.61; N, 15.35%.
3-(2-Fluorophenyl)-6-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2d). White needle
crystals, yield 49%, m.p. 190–191 oC; IR (KBr): ν 3056 (s), 1621 (m), 1467 (s), 1388 (m), 1230
(m), 1082 (m), 984 (m), 764(m) cm1; 1H NMR (300 MHz,CDCl3): δ 8.06–8.12 (t, 1H, J = 8.1
Hz, ArH), 7.93 (d, 2H, J = 7.6 Hz, ArH), 7.52– 7.61 (m, 4H, ArH), 7.34–7.39 (m, 2H,
ArH) ppm; ESI-MS: m/z 297.07 [M+1]+; anal. calcd. for C15H9FN4S: C, 60.80; H, 3.06; N,
18.91%. Found: C, 60.58; H, 3.02; N, 19.09%.
3-(2-Fluorophenyl)-6-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
(2e).
White
crystals, yield 59%; m.p. 254–257 oC; IR (KBr): ν 3058 (m), 1618 (m), 1530 (s), 1473 (s), 1390
(m), 1349 (s), 1106 (m), 969 (m), 857 (m), 755 (m) cm1; 1H NMR (300 MHz, CDCl3): δ 8.40–
8.44 (m, 2H, ArH), 8.08–8.16 (m, 3H, ArH), 7.56–7.63 (m, 1H, ArH), 7.27–7.41 (m, 2H,
ArH) ppm; ESI-MS: m/z 342.30 [M+1]+; anal. calcd. for C15H8FN5O2S: C, 52.78; H, 2.36; N,
20.52%. Found: C, 52.46; H, 2.31; N, 20.68%.
6-(4-(tert-butyl)phenyl)-3-(2-fluorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (2f). Light
brown crystals, yield 36%; m.p. 175–177 oC; IR (KBr): ν 3056 (m), 2964 (m), 1610 (m), 1466
1
(s), 1369 (m), 1226 (m), 1110 (m), 957 (m), 830 (m), 765 (m) cm1; H NMR (300 MHz,
CDCl3): δ 8.09 (t, 1H, J = 8.4 Hz, ArH), 7.84 (d, 2H, J = 8.4 Hz, ArH), 7.527.62 (m, 3H,
ArH), 7.30–7.38 (m, 2H, ArH), 1.37 (s, 9H, t-BuH) ppm; ESI-MS: m/z 353.18 [M+1]+;
anal. calcd. for C19H17FN4S: C, 64.75; H, 4.86; N, 15.90%. Found: C, 64.93; H, 4.81; N,
15.99%.
3-(4-Chlorophenyl)-6-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2g). White crystals, yield
44%; m.p. 212–214 oC; IR (KBr): ν 3050 (m), 1616 (m), 1524 (m), 1463 (s), 1387 (m), 1088
1
(m), 973 (m), 830 (m), 767(m), 686(m) cm1; H NMR (300 MHz, CDCl3): δ 8.38 (d, 2H, J =
8.7 Hz, ArH), 7.95–7.98 (m, 2H, ArH), 7.53–7.61 (m, 5H, ArH) ppm; ESI-MS: m/z
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312.92[M+]; anal. calcd. for C15H9ClN4S: C, 57.60; H, 2.90; N, 17.91%. Found: C, 57.39; H,
2.85; N, 18.10%.
3-(4-Chlorophenyl)-6-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole(2h).
White
crystals, yield 33%; m.p. 157–159 oC; IR (KBr): ν 3072 (m), 1615 (s), 1534 (s), 1414 (m), 1342
(s), 1315 (m), 1267 (m), 1183 (s), 1112 (m), 1069 (s), 1016 (m), 969 (s), 943 (s), 837 (s), 702 (s)
1
cm1; H NMR (300 MHz, CDCl3): δ 7.87 (d, 4H, J = 6.7 Hz, ArH), 7.53 (d, 4H, J = 6.7 Hz,
ArH) ppm; ESI-MS: m/z 358.18 [M+]; anal. calcd. for C15H8ClN5O2S: C, 50.36; H, 2.25; N,
19.57%. Found: C, 50.21; H, 2.20; N, 19.74%.
6-(4-(tert-butyl)phenyl)-3-(4-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (2i). White
crystals, yield 43%; m.p. 237–239 oC; IR (KBr): ν 3051 (m), 2962 (s), 1609 (m), 1463 (s), 1093
(m), 971 (m), 832 (m) cm1; 1H NMR (300 MHz, CDCl3): δ 8.36 (d, 2H, J = 8.5 Hz, ArH),
7.87 (d, 2H, J = 8.4 Hz, ArH), 7.51–7.60 (m, 4H, ArH), 1.38 (s, 9H, t-BuH) ppm; ESI-MS:
m/z 369.16[M+]; anal. calcd. for C19H17ClN4S: C, 61.86; H, 4.65; N, 15.19%. Found: C, 61.57;
H, 4.62; N, 15.38%.
3-(4-Fluorophenyl)-6-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2j). Light brown crystals,
yield 33%; m.p. 164–166 oC; IR (KBr): ν 3057 (m), 1604 (m), 1535 (m), 1469 (s), 1230 (s),
1155 (m), 970 (m), 834 (m), 762 (m) cm1; 1H NMR(300 MHz, CDCl3): δ 8.39–8.44 (m, 2H,
ArH), 7.93 (d, 2H, J = 7.6 Hz, ArH), 7.557.63 (m, 3H, ArH), 7.22–7.28 (m, 2H, ArH)
ppm; ESI-MS: m/z 297.07 [M+1]+; anal. calcd. for C15H9FN4S: C, 60.80; H, 3.06; N, 18.91%.
Found: C, 60.69; H, 3.01; N, 19.02%.
3-(4-Fluorophenyl)-6-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2k). Brick-red
powder, yield 39%; m.p. 270–272 oC; IR (KBr): ν 3068 (m), 1604 (m), 1525 (s), 1468 (s), 1345
(s), 1225 (m), 1156 (m), 1104 (m), 969 (m), 848 (s), 684 (m) cm1; 1H NMR (300 MHz, CDCl3):
δ 8.38–8.47 (m, 4H, ArH), 8.16 (d, 2H, J = 7.9 Hz, ArH), 7.25–7.38 (m, 2H, ArH) ppm;
ESI-MS: m/z 341.92[M+1]+; anal. calcd. for C15H8FN5O2S: C, 52.78; H, 2.36; N, 20.52%.
Found: C, 52.57; H, 2.33; N, 20.65%.
6-(4-(tert-butyl)phenyl)-3-(4-fluorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2l). Light
brown crystals, yield 32 %; m.p. 162–164 oC; IR (KBr): ν 3031 (m), 2962 (s), 1608 (m), 1540
(m), 1477 (s), 1235 (s), 1158 (m), 832 (m), 725 (m) cm1; 1H NMR (300 MHz, CDCl3): δ 8.40–
8.44 (m, 2H, ArH), 7.87 (d, 2H, J = 6.7 Hz, ArH), 7.58 (d, 2H, J = 6.7 Hz, ArH), 7.25 (t,
2H, J = 8.7 Hz, ArH), 1.38 (s, 9H, t-BuH) ppm; ESI-MS: m/z 353.18 [M+1]+; anal. calcd. for
C19H17FN4S: C, 64.75; H, 4.86; N, 15.90%. Found: C, 64.54; H, 4.81; N, 16.01%.
Biological activity tests
The newly synthesized compounds (2a2l) were tested for their in vitro antibacterial activity
against Escherichia coli and Staphylococcus aureus and antifungal activity against Pyricularia
oryzae and Rhizoctnia solani by the disc diffusion method [18, 19] at a concentration of 50 mg
L1. Muller-Hinton agar (Hi-Media) was employed as culture medium and DMSO was used as
solvent control for antimicrobial activity. Norfloxacin and Triadimefon were used as standard
for antibacterial and antifungal activities, respectively. The inhibition zones were measured in
mm at the end of an incubation period of 24 h at 37 oC for bacteria and 72 h at 24 oC for fungi.
Generally, the results were taken in duplicate. Results with difference higher than 5% were
neglected and repeated. In addition, the relative inhibition percentage of the tested compounds
with respect to standard drug was calculated according the formula RI = Dt / Ds  100%, where
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RI is the relative inhibition percentage, Dt is the diameter of inhibition zone for the tested
compounds, Ds is the diameter of inhibition zone for the standard drug.
RESULTS AND DISCUSSION
Chemistry
Synthetic pathway for target compounds, 3,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole
(2a2l), is shown in Scheme 1. The substituted benzoyl hydrazines were prepared by treatment
benzoic acid esters and hydrazine hydrate in ethanol and followed by reaction with carbon
disulfide in the presence of potassium hydroxide in ethanol to afford the corresponding
intermediate potassium dithiocarbazinate. This salt underwent cyclization with the excess
hydrazine hydrate to give 4-amino-5-substituted-phenyl-4H-1,2,4-triazole-3-thiols (1a1d). The
resultant triazoles (1a1d) were further converted to 3,6-disubstituted-1,2,4-triazolo-1,3,4-
thiadiazole (2a2l) by one-pot reaction with substituted aromatic carboxylic acids in the
presence of phosphorus oxychloride.
O
O
NH2
O
KOH
CS2
N
N2H4 H2O
+
R1
R1
H
O
N
N
N
NH
SK
N2H4 H2O
R1
N
R1
H
SH
S
NH2
1a 1d
R1 = 2-Cl, 2-F, 4-Cl, 4-F
1a 1b 1c 1d
R1 = 2-Cl; R2 = H, NO2, C(CH3)3
2a 2b 2c
R1 = 2-F; R2 = H, NO2, C(CH3)3
2d 2e 2f
R1 = 4-Cl; R2 = H, NO2, C(CH3)3
2g 2h 2i
R1 = 4-F; R2 = H, NO2, C(CH3)3
2j 2k 2l
N
N
COOH
R2
S
N
N
R1
POCl3
2a 2l
R2
Scheme 1. Synthetic pathway for the title compounds 2a2l.
1
The newly synthesized compounds were confirmed by elemental analysis, FTIR, H NMR,
and mass spectral data. In the IR spectra, the relatively strong peaks at 33263226 cm1 were
attributed to the NH2 stretching vibrations and the weak single peaks at 26222631 cm1 due to
the SH stretching vibrations for the triazole compounds 1a1d. However, in the title
compounds 2a2l, their IR spectra did not exhibited the characteristic absorptions of the NH2
and SH stretching vibrations of parent compounds 1a1d, which clearly indicated the fusing
between compounds 1a1d and substituted aromatic carboxylic acids [20].
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Lu Lin et al.
Figure 1. The 1H NMR spectrum of the compound 2l.
The 1H NMR spectra of the triazole compounds 1a1d showed two characteristic two
proton signals at 11.9310.75 and 5.024.82 ppm, which were attributed to the protons of SH
and NH2 group for the triazoles, respectively. But these two proton signals were disappeared in
1
the H NMR spectra of the title compounds 2a2l. The chemical shifts at 8.447.22 ppm were
assigned to aromatic protons of compounds 2a2l. Here, we chose the compound 2l as an
example to discuss their chemical shifts. The 1H NMR spectrum of the compound 2l was shown
in Figure 1. From Figure 1, the multiple peak at  8.448.40 ppm was assigned to Ha and Ha’
and the two doublet peaks (J = 6.7 Hz) at  7.87 and 7.58 ppm were due to Hc and Hc’ and Hd
and Hd’, respectively. The protons Hb and Hb’ exhibited a triplet peak (J = 8.7 Hz) at  7.25 ppm.
The nine-proton singlet peak at  1.38 ppm was attributed to the protons of the tert-butyl group.
These results also confirmed that the triazoles 1a1d had converted to target triazolothiadiazole
compounds 2a2l by reacting with substituted aromatic carboxylic acids in phosphorus
oxychloride.
The ESI mass spectra of the all synthesized compounds 1a1d and 2a2l were compared to
confirm elemental compositions. Their molecular ion peaks (M+) for these compounds were
observed in accordance with the Nitrogen Rule. For example, the electrospray ionization mass
spectrum (Figure 2) of the compound 2l displayed a peak at m/z 353.18 [M+1]+, which was due
to its molecular ion peak.
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Synthesis of 3,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivatives
487
1_130421110402 #146 RT: 2.46 AV: 1 SB: 19 0.69-0.99 NL: 5.47E6
T: + c NSI Full ms [ 50.00-1000.00]
353.18
100
90
80
70
60
50
40
30
20
411.94
434.65
10
0
282.31
335.12
100.34
100
135.74
200.00
200
555.73
300
400
500
m/z
600
Figure 2. Electrospray ionization mass spectrum of the compound 2l.
Antimicrobial activity
In the bioassay screening, the triazolothiadiazole compounds 2a2l were tested for their anti-
microbial activity against Escherichia coli, Staphylococcus aureus, Pyricularia oryzae and
Rhizoctnia solani. The inhibition zones and relative inhibition percentage of these compounds
against bacteria and fungi are listed in Table 1. The results showed that all tested compounds
exhibited moderate to good antibacterial and antifungal activities in DMSO. Comparatively,
compounds 2e (R1 = 2-fluorophenyl, R2 = 4-nitrophenyl), and 2k (R1 = 4-fluorophenyl, R2 = 4-
nitrophenyl) revealed much significant antimicrobial activities than the other triazolothiadiazole
derivatives, which could be due to the presence of fluorophenyl and nitrophenyl moieties in
triazolothiadiazole. However, compounds 2c (R1 = 2-chlorophenyl, R2 = 4-tert-butylphenyl), 2f
(R1 = 2-fluorophenyl, R2 = 4-tert-butylphenyl) and 2i (R1 = 4-chlorophenyl, R2 = 4-tert-
butylphenyl) revealed much lower activities against the tested microorganisms among all
synthesized triazolothiadiazole derivatives, which probably assigned to tert-butylphenyl moiety
in triazolothiadiazole. By the preliminary structure-activity relationship analysis, it was
concluded that the introduction of the electron-withdrawing group in triazolothiadiazole can
distinctly improve their antimicrobial activities and the introduction of the electron-donating
group leads to the decrease of their antimicrobial activities. Especially, the triazolothiadiazole
derivatives with fluorophenyl and nitrophenyl moieties at 3,6-position of the triazolothiadiazole
ring showed potent antimicrobial activities against the tested microorganisms. As a result, it
indicated that the electronic nature of the substituent groups at 3,6-positions in the
triazolothiadiazole ring played a significant role in antimicrobial activities.
Bull. Chem. Soc. Ethiop. 2017, 31(3)

488
Lu Lin et al.
Table 1. Antimicrobial data of 1,2,4-triazolo-1,3,4-thiadiazole derivatives.
Inhibition zone / mm
Inhibition zone / mm
(relative inhibition percentage)
(relative inhibition percentage)
Compounds
E. coli
S. aureus
13 (41.9%)
19 (61.3%)
10 (32.3%)
16 (51.6%)
26 (83.9%)
13 (41.9%)
12 (38.7%)
23 (74.2%)
10 (32.3%)
17 (54.8%)
28 (90.3%)
12 (38.7%)
31
P. oryzae
14 (46.7%)
20 (66.7%)
11 (36.7%)
17 (56.7%)
26 (86.7%)
12 (40.0%)
14 (46.7%)
21 (70.0%)
12 (40.0%)
18 (60.0%)
25 (83.3%)
13 (43.3%)

R. solani
12 (41.4%)
20 (69.0%)
10 (34.5%)
15 (51.7%)
24 (82.8%)
12 (41.4%)
13 (44.8%)
20 (69.0%)
11 (37.9%)
19 (65.5%)
27 (93.1%)
12 (41.4%)

2a
2b
2c
2d
2e
2f
2g
2h
12 (37.5%)
21 (65.6%)
10 (31.3%)
16 (50.0%)
25 (78.1%)
11 (34.4%)
14 (43.8%)
22 (68.8%)
11 (34.4%)
18 (56.3%)
30 (93.8%)
13 (40.6%)
32
2i
2j
2k
2l
Norfloxacin
Triadimefon
30
29


CONCLUSION
In summary, some new 3,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivatives were
1
synthesized. Their structures were confirmed by elemental analysis, IR, H NMR, and mass
spectroscopy. The biological activities of these compounds were evaluated against Escherichia
coli, Staphylococcus aureus, Pyricularia oryzae and Rhizoctnia solani by disc diffusion method.
The results showed that these triazolothiadiazole derivatives exhibited moderate to good
antibacterial and antifungal activities. Especially, compounds 2e and 2k displayed much higher
antibacterial and antifungal activities in all the synthesized triazolothiadiazole derivatives. It
demonstrated that the triazolothiadiazole derivatives with fluorophenyl and nitrophenyl moieties
at 3,6-position in the triazolothiadiazole ring showed potent antimicrobial activities. Therefore,
it is helpful for further structural modification of the triazolothiadiazole derivatives to improve
their antimicrobial activities.
ACKNOWLEDGEMENT
The authors would like to acknowledge the support from National Natural Science Foundation
of China (No. 21273065).
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