Microwave assisted synthesis, antifungal activity and DFT theoretical study of some novel,1,2,4-triazole derivatives containing the 1,2,3-thiadiazole moiety

Article abstract of DOI:10.3390/molecules181012725

In order to investigate the biological activity of 1,2,4-triazole compounds, seventeen novel 1,2,4-triazole derivatives containing 1,2,3-thiadiazole moieties were synthesized by multi-step reactions under microwave assisted conditions. The structures were characterized by ¹H-NMR, ¹³C-NMR, MS and elemental analyses. The target compounds were evaluated for their in vivo fungicidal activities against Corynespora cassiicola, Pseudomonas syringae pv. Lachrymans, and Pseudoperonospora cubensis, and the results indicated that some of the title compounds displayed good fungicidal activities. Theoretical calculations on the title compounds were carried out at the B3LYP/6-31G (d,p). level. The full geometry optimization was carried out using the 6-31G(d,p) basis set, and the frontier orbital energy, atomic net charges were discussed, and the structure-activity relationships were also studied.

Full text of DOI:10.3390/molecules181012725

Molecules 2013, 18, 12725-12739; doi:10.3390/molecules181012725
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Microwave Assisted Synthesis, Antifungal Activity and DFT
Theoretical Study of Some Novel 1,2,4-Triazole Derivatives
Containing the 1,2,3-Thiadiazole Moiety
Na-Bo Sun 1, Jian-Qun Fu 1, Jian-Quan Weng 2, Jian-Zhong Jin 1, Cheng-Xia Tan 2 and
Xing-Hai Liu 2,*
1
College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015,
Zhejiang, China; E-Mails: nabosun@gmail.com (N.-B.S.); fujianqunshuren@126.com (J.-Q.F.);
jinjianzhongshuren@gmail.com (J.-Z.J.)
2
College of Chemical Engineering and Materials Science, Zhejiang University of Technology,
Hangzhou 310014, Zhejiang, China; E-Mails: jqweng@zjut.edu.cn (J.-Q.W.);
tangchengxia@zjut.edu.cn (C.-X.T.)
* Author to whom correspondence should be addressed; E-Mail: xhliu@zjut.edu.cn;
Tel./Fax: +86-571-8832-0832.
Received: 3 September 2013; in revised form: 11 October 2013 / Accepted: 11 October 2013 /
Published: 15 October 2013
Abstract: In order to investigate the biological activity of 1,2,4-triazole compounds,
seventeen novel 1,2,4-triazole derivatives containing 1,2,3-thiadiazole moieties were
synthesized by multi-step reactions under microwave assisted conditions. The structures
1
were characterized by H-NMR, 13C-NMR, MS and elemental analyses. The target
compounds were evaluated for their in vivo fungicidal activities against Corynespora
cassiicola, Pseudomonas syringae pv. Lachrymans, and Pseudoperonospora cubensis, and
the results indicated that some of the title compounds displayed good fungicidal activities.
Theoretical calculations on the title compounds were carried out at the B3LYP/6-31G (d,p).
level. The full geometry optimization was carried out using the 6-31G(d,p) basis set, and the
frontier orbital energy, atomic net charges were discussed, and the structure-activity
relationships were also studied.
Keywords: 1,2,4-triazole; 1,2,3-thiadiazole; synthesis; fungicidal activity; thioether;
theoretical calculations

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1. Introduction
In recent years, heterocyclic compounds had been receiving considerable attention due to their
pharmacological and pesticidal importance [1–9]. Nitrogen-containing heterocycles exhibit excellent
biological activities, especially 1,2,4-triazoles and 1,2,3-thiadiazoles. 1,2,4-Triazole rings are typically
planar 6π-electron aromatic systems, featuring an extensive chemistry [10,11]. 1,2,4-Triazole and its
derivatives represent one of the most biologically active classes of compounds, displaying a diversity
bioactivities in the medicinal and agrochemical field, including anti-inflammatory [12,13],
antifungal [14,15], herbicidal [16], antimicrobial [16,17], antiparasitic [18], cytostatic [19], and
brassinosteroid biosynthesis inhibitory activities [20]. Some such compounds had been developed as
commercial fungicides or herbicides (Figure 1), such as triadimefon, triadimenol, flusilazole, flupoxam
and so on.
Figure 1. Commercial drugs and pesticides containing 1,2,4-triazole groups.
1,2,3-Thiadiazoles have also been claimed to have beneficial agricultural applications [21,22],
because they exhibit wide spectrum biological activities, such as antiviral [23], herbicidal [24,25],
antiamoebic [26] and insecticidal [27] activities. For example, after plant inducers such as tiadinil
(TDL) and acibenzolar-S-methyl (BTH) (Figure 2) were discovered, 1,2,3-thiadiazole pesticides have
become one of the focuses for developing agrochemicals in academia and industry.
Figure 2. Some commercial pesticides containing 1,2,3-thiadiazoles.
N
N
H
N
N
N
Cl
S
S
O
MeS
O
BTH
tiadinil
There are many reports about each of the two heterocycles, but the combination of 1,2,3-thiadiazole
ring with 1,2,4-triazole ring in one molecule is seldom reported either in chemistry or biological

Molecules 2013, 18
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activity studies. In view of these facts mentioned above, and also as a part of our work [28] on the synthesis
of bioactive lead compounds, the title compounds were designed by introducing the 1,2,3-thiadiazole
pharmacophore into a 1,2,4-triazole scaffold. Seventeen novel 1,2,4-triazole derivatives were
1
synthesized and characterized by H-NMR, 13C-NMR, MS and elemental analysis. The antifungal
activities of these compounds were tested in vivo.
2. Results and Discussion
2.1. Chemistry
The synthesis procedures for title compounds are shown in Scheme 1. The intermediates 1~6 were
synthesized according our previous method [25]. The intermediate 1 was easily synthesized from
diethyl carbonate and 85% hydrazine hydrate at room temperature. The method of synthesis of the Schiff
base 2 was conventional. The key intermediate ethyl 4-methyl-1,2,3-thiadiazole-5-carboxylate (3) was
synthesized using the Hurd-Mori method. Then intermediate 3 is reacted with 85% hydrazine hydrate
to give intermediate 4. The intermediate 5 can be obtained from the intermediate 4 and a substituted
isothiocyanic ester. The intermediate 5 was easily cyclized to give intermediate 6 under alkaline
conditions, such as NaOH. The intermediate 6 is reacted with substituted benzyl chlorides or alkyl
chlorides to afford compounds 7. The microwave irradiation assisted synthesis and conventional
method were also employed in these experiments. The NaOH/DMF/H2O system was applied under
microwave irradiation. The best reaction conditions were 90 °C for 15 min under microwave
irradiation. The yield is higher than that of the conventional method, and the reaction time is also
shorter (Table 1).
Scheme 1. The synthetic route of title compounds.
Reagents and conditions: (i) NH2NH2 H2O, EtOH, reflux; (ii) ethyl 3-oxobutanoate, EtOH, room temperature;
(iii) SOCl2, room temperature; (iv) NH2NH2 H2O, EtOH, reflux; (v) R1NCS, EtOH, reflux; (vi) a. NaOH,
reflux; b.HCl; (vii) R2CH2Cl, K2CO3, DMF, MW.

Molecules 2013, 18
Table 1. Comparison of yields of 7b with or without microwave irradiation.
12728
No.
7b
7b
7b
7b
Method
No-MW
MW
Time
24 h
Temperature/°C
Yield/%
r.t.
90
90
90
78
80
85
83
10 min
15 min
20 min
MW
MW
The signal of the CH2 protons of the thioether neighboring the triazole ring was observed at
δ 4.43~4.63 ppm, respectively. The chemical shifts around 3.0 ppm are from the methyl of the
1,2,3-thiadiazole ring. The ESI-MS spectra showed the m/z of the molecular ions, according with their
molecular formulas. The elemental analysis results were in accord with the calculated results.
2.2. Fungicidal Activities and Structure-Activity Relationship (SAR)
The in vivo fungicidal results of the title compounds against Corynespora cassiicola (CC),
Pseudomonas syringae pv. Lachrymans (PS) and Pseudoperonospora cubensis (PC) are listed in Table 2,
where iprodione, validamycin and topsin-M were used as controls. As shown in Table 2, lots of the
title compounds showed good control efficacy against Corynespora cassiicola at a concentration of
500 μg/mL. When the R1 is a phenyl group, only compound 7e exhibited moderate activity (46.9%)
against Corynespora cassiicola. Notably, compounds 7b, 7c, 7d and 7e displayed excellent activity
(>80%) against Corynespora cassiicola, especially compound 7b which exhibited higher activity than
the control. Also the activity of benzene substituted derivatives is higher than that of cyclopropyl
substituted ones. Compounds 7b, 7c, 7d, 7j, 7k, 7l, 7m exhibited fair inhibition effects against
Pseudomonas syringae pv. Lachrymans, and the fungicidal activities (control efficacy of 52%–79%)
were higher than those of iprodione, validamycin and topsin-M. All of them were less effective than
the control validamycin. Compound 7e (71.03%) and 7g (68.79%) showed about 70% control effect
against Pseudoperonospora cubensis, at the same level as iprodione, validamycin and topsin-M. In general,
the in vivo fungicidal activity of compounds 7 indicated that the substituent changes affect the activity.
In order to further research the structure–activity relationships of the title compounds, some
compounds (7o, 7p, 7q) containing cyclopropyl groups synthesized in our other paper [29] were used
for comparison. From the data in Table 2, for Corynespora cassiicola, the compounds containing
phenyl groups exhibited higher activity than those with cyclopropyl groups, but for
Pseudoperonospora cubensis, the results are the opposite, and the compounds containing cyclopropyl
groups displayed higher activity than that of phenyl group-containing compounds. For Pseudomonas
syringae pv. Lachrymans, the structure–activity relationships are not obvious. Further biological
evaluation of all compounds is in progress. The mode of action of title compounds will be explored by
molecular modeling.

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Table 2. The antifungal activity of title compounds in vivo.
No.
7a
7b
7c
7d
7e
7f
R1
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
R2
CC
PS
PC
2,4-Cl2Ph
Ph
72.97
93.19
82.77
82.30
46.90
75.93
84.96
70.17
75.22
75.15
75.75
59.92
−1.71
60.67
55.08
53.92
36.71
17.59
47.16
11.49
48.20
61.48
67.06
61.00
50.34
54.51
44.40
39.15
71.03
52.88
68.79
19.65
42.25
28.64
26.15
49.38
4-F Ph
3-Cl Ph
3-F Ph
2-F Ph
4-MePh
4-Et Ph
CH3CH2
2-Cl Ph
CN
7g
7h
7i
7j
7k
7l
4-Bu Ph
7m
Ph
Ph
70.64
63.09
18.28
7n
7o
7p
3,4-Cl2Ph
3-CN Ph
71.07
28.99
79.76
24.41
39.35
69.17
21.38
54.03
6.47
71.07
81.62
30.17
7q
iprodione
validamycin
topsin-M
53.52
78.20
78.75
38.49
−11.42
54.15
55.92
56.31
68.71
(a) CC: Corynespora cassiicola; PS: Pseudomonas syringae pv. Lachrymans; PC: Pseudoperonospora
cubensis; (b) The test concentration of antifungal activity is at 500 mg/mL.
2.3. Molecular Total Energies and Frontier Orbital Energy Analysis
Molecular total energy and frontier orbital energy levels are listed in Table 3. Energy gap between
HOMO and LUMO was calculated by B3LYP.
Table 3. Total energy, frontier orbital energy.
DFT
Etotal/Hartree b
EHOMO/Hartree
−1765.0575
−0.221
E
LUMO/Hartree
−0.069
ΔEa/Hartree
0.152
a ΔE= ELUMO−EHOMO; b 1 Hartree = 4.35974417 × 10−18 J = 27.2113845 eV.
According to the frontier molecular orbital theory, HOMO and LUMO are the most important
factors that affect the bioactivity. HOMO has the priority to provide electrons, while LUMO can
accept electrons first [30]. Thus study on the frontier orbital energy can provide useful information
about the biological mechanism. Taking the DFT result for example, the geometry of the frame of the
title compound is hardly influenced by the introduction of either a 1,2,3-thiadiazole ring, 1,2,4-triazole
ring, thioether group or phenyl ring (Figure 3). The HOMO of the title compound is mainly located on

Molecules 2013, 18
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the 1,2,3-thiadiazole ring, 1,2,4-triazole ring and thioether group, while, the LUMO of the title
compound is located on the 1,2,3-thiadiazole ring, 1,2,4-triazole ring, thioether group and phenyl ring.
The fact that the title compound has strong affinity suggests the importance of the frontier molecular
orbital in the π-π stacking or hydrophobic interactions. This also implies that the orbital interactions
between the title compound and the aromatic ring or some other side of residue chains of receptors is
dominated by π-π or hydrophobic interactions among the frontier molecular orbitals.
Figure 3. The HOMO and LUMO of compound 7b.
LUMO+1 of compound 7b
LUMO Plot
(First excited state)
LUMO of compound 7b
(Ground state)
HOMO Plot
HOMO of compound 7b
HOMO-1 of compound 7b

Molecules 2013, 18
12731
2.4. Mulliken Atomic Charges
Table 4 lists the calculated Mulliken atomic charges except for the H atoms. Taking DFT for
example again, three atoms C1, C2, C17, S1 and S2 are the most positively charged ones, which can
interact with the negative charged part of the receptor easily. The negative charges are mainly located
on atoms N1, N2, N3, N4 and N5, so they can interact easily with the positive part of the receptor.
Therefore, we supposed that this compound can combine the amino-acid residues on its surface by the
interaction of S2-N4-N5 and S1-C1-N3, which may account in part for the bioactivity.
Table 4. Mulliken atomic charges except for H atoms (e).
Charge
Atom
DFT
C1
C2
0.150691
0.374005
0.208092
−0.011153
−0.003467
−0.000164
−0.000969
0.001182
−0.123948
0.085700
−0.009788
0.010835
0.013612
0.011859
−0.010715
−0.199074
0.267633
0.042000
−0.339205
−0.155032
−0.504897
−0.242781
−0.267556
0.295248
0.407893
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
N1
N2
N3
N4
N5
S1
S2

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3. Experimental
3.1. Materials and Reagents
All the reagents were of analytical grade or freshly prepared before use. The monitoring of the
progress of all reactions and homogeneity of the synthesized compounds were carried out by thin
chromatography (TLC), performed on silica gel plates obtained from Qingdao Ocean Chemicals
(Qing Dao, China). Melting points were determined using an X-4 apparatus and are uncorrected. 1H-NMR
13
and C-NMR spectra were measured on a Bruker AV 400 or 500 instrument using TMS as an internal
standard and CDCl3 as the solvent. Mass spectra were recorded on a Thermo Finnigan LCQ Advantage
LC/mass detector instrument. Elemental analyses were performed on a Vario EL elemental analyzer.
Microwave activation was carried out with a CEM DiscoverTM focused microwave (2,450 MHz, 300 W).
3.2. Therotical Calculations
On the basis of the above structure shown in Scheme 1, compound 7b was selected as the initial
structure, and the DFT-B3LYP/6-31G (d,p) [31] methods in the Gaussian 03 package [32] were
used to optimize the structure of the title compound. Vibration analysis showed that the optimized
structures were in accordance with the minimum points on the potential energy surfaces. All the
convergent precisions were the system default values, and all the calculations were carried out on a
personal computer.
3.3. Chemical Synthesis
3.3.1. Synthesis of Intermediates
3.3.1.1. Preparation of 4-methyl-1,2,3-thiadiazole-5-carbohydrazide (compounds 1~4)
The synthetic route is shown in Scheme 1. The intermediate 4-methyl-1,2,3-thiadiazole-5-
carboxylic acid hydrazide (2) was synthesized according to the literature [25]. Carbonic acid diethyl
ester (11.8 g, 0.1 mol) and hydrazine hydrate (5.6 mL, 0.095 mol, 85%) were added into a 250 mL
round-bottom flask equipped with a condenser. The reaction mixture was heated to 50 °C and stirred
for 20 min, and then cooled down to room temperature and further stirred for 30 h. Water, ethanol, and
excess carbonic acid diethyl ester were distilled off under reduced pressure. After drying, compound (2)
(9.88 g, 95% yield) was obtained as white crystals. To a stirred solution of compound 2 (6.36 g, 0.06 mol)
in ethanol (16.7 mL), a solution of ethyl acetoacetate (7.8 g, 0.06 mol) in ethanol (3.7 mL) was added
at room temperature. Stirring was continued for 6 h. Then, the solvent was removed in vacuo and the
crude 3-ethoxycarbonyl hydrazonoacetic acid ethyl ester was directly used in the next step without
further purification. 3-Ethoxycarbonyl hydrazonoacetic acid ethyl ester (12.8 g, 0.06 mol) was
dissolved in dry dichloromethane (25 mL), and thionyl chloride (20 mL) was added to the stirred
reaction mixture dropwise at 0 °C for 1 h. Next, the reaction mixture was allowed to stand for 20 h at
room temperature. The excess thionyl chloride and dichloromethane were distilled off, and the
remaining residue was subjected to fractional distillation under reduced pressure. A slightly yellowish
oil of 3 (7.95 g, 77% yield) was obtained. A mixture of 4-methyl-1,2,3-thiadiazole-5-carboxylic acid

Molecules 2013, 18
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ethyl ester (1.72 g, 10 mmol) and hydrazine hydrate (12 mmol) in 10 mL of methanol was stirred
vigorously for 0.5 h at room temperature, the resulting mixture was then filtered, and the solid was
washed with cold methanol. After drying, the solid was recrystallized from methanol to give
intermediate (4).
3.3.1.2. 2-(4-Methyl-1,2,3-thiadiazole-5-carbonyl)-N-phenylhydrazinecarbothioamide (5)
A mixture of 4-methyl-1,2,3-thiadiazole-5-carbohydrazide and isothiocyanatobenzene was refluxed
for 3 h in ethanol. After cooling down to room temperature, the product obtained was recrystallized
from methanol to give 5.
3.3.1.3. 5-(4-Methyl-1,2,3-thiadiazol-5-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol (6)
A mixture of compound 5 (10 mmol) in aqueous NaOH solution (5 mL, 2N) was refluxed for 4 h.
After cooling down to room temperature, HCl aqueous solution (4N) was added to afford a large
amount of precipitate. The solid was filtered, dried and recrystallized from methanol to give
intermediate 6.
3.3.1.4. General Procedure for the Preparation of Thioethers 7
A CEM-designed 10 mL pressure-rated vial was charged with DMF (15 mL), 6 (1.5 g, 5.1 mmol)
and K2CO3 (0.2 g, 5.6 mmol). The mixture was irradiated in a CEM Discover Focused Synthesizer
(150 w, 90 °C, 200 psi, 15 minutes). The mixture was cooled to room temperature by passing
compressed air through the microwave cavity for 2 minutes. It was poured into cold ice (40 mL) and
the precipitate formed was filtered. The crude solid was recrystallized from EtOH to give the title
compound 7a. All the other compounds are synthesized according to the same procedure.
5-(5-((2,4-Dichlorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7a). White
crystals, yield 74%, m.p. 102–103 °C; 1H-NMR (CDCl3, 400 MHz) δ: 3.07 (s, 3H, Het-Me), 4.61 (s, 2H,
SCH2), 7.14–7.22 (m, 4H, ArH), 7.35–7.40 (m, 2H, ArH), 7.56–7.60 (m, 1H, ArH), 7.64–7.66 (m, 1H,
ArH). MS (ESI), m/z: 435 (M+1)+. Elemental anal. (%), calculated: C, 49.77; H, 3.02; N, 16.12; found:
C, 49.98; H, 3.12; N,16.09.
5-(5-Benzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7b). White crystals, yield
75%, m.p. 120–121 °C; 1H-NMR (CDCl3, 400 MHz) δ: 3.07 (s, 3H, Het-Me), 4.63 (s, 2H, SCH2), 7.08
(d, J = 7.99 Hz, 2H, ArH), 7.28–7.32 (m, 3H, ArH), 7.36–7.38 (m, 2H, ArH), 7.54 (t, J = 8.00 Hz, 2H,
ArH), 7.64 (t, J = 7.38 Hz, 1H, ArH). MS (ESI), m/z: 366 (M+1)+. Elemental anal. (%), calculated: C,
59.15; H, 4.14; N, 19.16; found: C, 58.89; H, 4.43; N,19.32.
5-(5-((4-Fluorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7c). White crystals,
yield 82%, m.p. 88–89 °C; 1H-NMR (CDCl3, 400 MHz) δ: 3.06 (s, 3H, Het-Me), 4.51 (s, 2H, SCH2),
6.97 (d, J = 8.65 Hz, 2H, ArH), 7.13 (d, J = 7.30 Hz, 2H, ArH), 7.36–7.39 (m, 2H, ArH), 7.57
(t, J = 8.05 Hz, 2H, ArH), 7.65 (t, J = 7.47 Hz, 1H, ArH). MS (ESI), m/z: 384 (M+1)+. Elemental anal. (%),
calculated: C, 56.38; H, 3.68; N, 18.26; found: C, 56.12; H, 3.43; N,18.32.

Molecules 2013, 18
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5-(5-((3-Chlorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole
(7d).
White
1
crystals, yield 86%, m.p. 101–102 °C; H-NMR (CDCl3, 400 MHz) δ: 3.04 (s, 3H, Het-Me), 4.47 (s,
2H, SCH2), 7.10 (d, J = 7.60 Hz, 2H, ArH), 7.21–7.28 (m, 3H, ArH), 7.36 (s, 1H, ArH), 7.56 (t,
J = 7.25 Hz, 2H, ArH), 7.65 (t, J = 7.18 Hz, 1H, ArH). MS (ESI), m/z: 400 (M+1)+. Elemental anal.
(%), calculated: C, 54.06; H, 3.53; N, 17.51; found: C, 54.33; H, 3.63; N,17.67.
5-(5-((3-Fluorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7e). White crystals,
yield 88%, m.p. 98–99 °C; 1H-NMR (CDCl3, 400 MHz) δ: 3.06 (s, 3H, Het-Me), 4.52 (s, 2H, SCH2),
6.97 (t, J = 8.25 Hz, 1H, ArH), 7.09–7.24 (m, 4H, ArH), 7.56 (t, J = 7.86 Hz, 2H, ArH), 7.63–7.69 (m,
2H, ArH). MS (ESI), m/z: 384 (M+1)+. Elemental anal. (%), calculated: C, 56.38; H, 3.68; N, 18.26;
found: C, 56.75; H, 3.63; N,18.49.
5-(5-((2-Fluorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7f). White
1
crystals, yield 78%, m.p. 104–105 °C; H-NMR (CDCl3, 400 MHz) δ: 3.05 (s, 3H, Het-Me), 4.50
(s, 2H, SCH2), 7.05 (d, J = 7.70 Hz, 2H, ArH), 7.17–7.32 (m, 4H, ArH), 7.52 (t, J = 8.09 Hz, 2H, ArH),
7.61 (t, J = 6.97 Hz, 1H, ArH). MS (ESI), m/z: 384 (M+1)+. Elemental anal. (%), calculated: C, 56.38;
H, 3.68; N, 18.26; found: C, 56.13; H, 3.77; N,18.34.
4-Methyl-5-(5-((4-methylbenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1,2,3-thiadiazole (7g). White crystals,
yield 86%, m.p.115–116 °C; 1H-NMR (CDCl3, 400 MHz) δ: 2.33 (s, 3H, Ar-Me), 3.06 (s, 3H, Het-Me),
4.55 (s, 2H, SCH2), 7.02 (d, J = 7.31 Hz, 2H, ArH), 7.05–7.22 (m, 3H, ArH), 7.29 (d, J = 7.45 Hz, 1H,
ArH), 7.52 (t, J = 7.98 Hz, 2H, ArH), 7.62 (t, J = 7.47 Hz, 1H, ArH). MS (ESI), m/z: 380 (M+1)+.
Elemental anal. (%), calculated: C, 60.13; H, 4.52; N, 18.45; found: C, 59.89; H, 4.67; N,18.60.
4-Methyl-5-(5-((4-ethylbenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1,2,3-thiadiazole (7h). White
1
crystals, yield 89%, m.p. 135–136 °C; H-NMR (CDCl3, 400 MHz) δ: 1.28 (t, 3H, Me), 2.63 (q, 2H,
Ar-CH2), 3.06 (s, 3H, Het-Me), 4.55 (s, 2H, SCH2), 7.06–7.21 (m, 5H, ArH), 7.51–7.62 (m, 4H, ArH).
MS (ESI), m/z: 394 (M+1)+. Elemental anal. (%), calculated: C, 61.04; H, 4.87; N, 17.80; found: C,
60.99; H, 4.97; N,17.56.
4-Methyl-5-(4-phenyl-5-(propylthio)-4H-1,2,4-triazol-3-yl)-1,2,3-thiadiazole (7i). White crystals, yield
1
59%, m.p. 106–107°C; H-NMR (CDCl3, 400 MHz) δ: 1.03 (t, J = 7.37 Hz, 3H, Me), 1.79–1.86 (m,
2H, CH3CH2), 3.06 (s, 3H, Het-Me), 3.30 (t, J = 7.17 Hz, 3H, CH3CH2CH2), 4.55 (s, 2H, SCH2),
7.23–7.25 (m, 2H, ArH), 7.58–7.68 (m, 3H, ArH). MS (ESI), m/z: 318 (M+1)+. Elemental anal. (%),
calculated: C, 52.97; H, 4.76; N, 22.06; found: C, 52.76; H, 4.67; N,21.96.
5-(5-((3-Chlorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7j). White crystals,
yield 85%, m.p. 138–139 °C; 1H-NMR (CDCl3, 400 MHz) δ: 3.06 (s, 3H, Het-Me), 4.49 (s, 2H, SCH2),
7.10 (d, J = 7.16 Hz, 2H, ArH), 7.22–7.25 (m, 1H, ArH), 7.27–7.29 (m, 1H, ArH), 7.37 (s, 1H, ArH),
7.57 (t, J = 8.00 Hz, 2H, ArH), 7.66 (d, J = 7.58 Hz, 1H, ArH). MS (ESI), m/z: 400 (M+1)+. Elemental
anal. (%), calculated: C, 54.06; H, 3.53; N, 17.51; found: C, 53.89; H, 3.55; N,17.78.
2-((5-(4-Methyl-1,2,3-thiadiazol-5-yl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)acetonitrile (7k). White crystals,
yield 84%, m p. 118–119 °C; 1H-NMR (CDCl3, 500 MHz) δ: 3.08 (s, 3H, Het-Me), 4.16 (s, 2H, SCH2),

Molecules 2013, 18
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7.28 (d, J = 7.7 Hz, 1H, ArH), 7.34 (d, J = 7.7 Hz, 1H, ArH), 7.63–7.73 (m, 3H, ArH). 13C-NMR (CDCl3,
125 MHz) δ: 14.8, 17.8, 115.1, 127.8, 130.6, 131.7, 132.2, 148.8, 150.7, 160.4. MS (ESI), m/z: 315
(M+1)+. Elemental anal. (%), calculated: C, 49.66; H, 3.21; N, 26.73; found: C, 49.78; H, 3.11; N,26.55.
5-(5-((4-(tert-Butyl)benzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7l). White
1
crystals, yield 86%, m.p. 125–126 °C; H-NMR (CDCl3, 500 MHz) δ: 1.31 (s, 9H, Bu), 3.07 (s, 3H,
Het-Me), 4.52 (s, 2H, SCH2), 7.09 (d, J = 8.0 Hz, 2H, ArH), 7.29 (d, J = 8.5 Hz, 2H, ArH), 7.33 (d,
J = 8.5 Hz, 2H, ArH), 7.54 (t, J = 8.1 Hz, 2H, ArH), 7.63 (d, J = 7.5 Hz, 1H, ArH). 13C-NMR (CDCl3,
125 MHz) δ: 14.8, 31.3, 34.6, 36.9, 125.7, 127.9, 128.9, 130.5, 131.5, 132.1, 132.9, 133.7, 147.6,
151.1, 154.3, 159.9. MS (ESI), m/z: 423 (M+1)+. Elemental anal. (%), calculated: C, 62.68; H, 5.50; N,
16.61; found: C, 62.77; H, 5.55; N,16.45.
5-(5-(((2-Chlorothiazol-5-yl)methyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7m).
1
White crystals, yield 89%, m.p. 129–130 °C; H-NMR (CDCl3, 500 MHz) δ: 3.08 (s, 3H, Het-Me),
4.68 (s, 2H, SCH2), 7.10 (d, J = 7.8 Hz, 2H, ArH), 7.52 (s, 1H, thiazole-H), 7.61 (t, J = 8.0 Hz, 2H,
13
ArH), 7.70 (t, J = 7.5 Hz, 1H, ArH). C-NMR (CDCl3, 125 MHz) δ: 14.8, 28.3, 127.8, 130.8, 131.7,
131.9, 133.3, 136.4, 140.9, 148.2, 152.3, 152.9, 160.1. MS (ESI), m/z: 408 (M+1)+. Elemental anal.
(%), calculated: C, 44.27; H, 2.72; N, 20.65; found: C, 44.44; H, 2.81; N,20.77.
5-(5-((3,4-Dichlorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole
(7n).
1
White crystals, yield 81%, m.p. 109–110 °C; H-NMR (CDCl3, 500 MHz) δ: 3.06 (s, 3H, Het-Me),
4.49 (s, 2H, SCH2), 7.14 (d, J = 7.7 Hz, 2H, ArH), 7.30 (s, 1H, ArH), 7.44 (d, J = 8.3 Hz, 2H, ArH),
7.57 (t, J = 8.0 Hz, 2H, ArH), 7.66 (d, J = 7.5 Hz, 1H, ArH). MS (ESI), m/z: 423 (M+1)+. Elemental
anal. (%), calculated: C, 49.77; H, 3.02; N, 16.12; found: C, 49.58; H, 3.35; N,16.01.
3-(((4-Cyclopropyl-5-(4-methyl-1,2,3-thiadiazol-5-yl)-4H-1,2,4-triazol-3-yl)thio) methyl) benzonitrile (7o).
1
White solid, yield 82%, m.p. 133–134 °C; H-NMR (CDCl3, 400 MHz) δ: 0.79–0.83 (m, 2H,
cyclopropane-CH2), 1.13–1.18 (m, 2H, cyclopropane-CH2), 2.95 (s, 3H, CH3), 2.97–3.02 (m, 1H,
cyclopropane-CH), 4.53 (s, 2H, CH2), 7.37 (t, J = 7.22 Hz, 1H, Ph-H), 7.51 (d, J = 7.22 Hz, 1H, Ph-H),
7.71 (t, J = 7.84 Hz, 2H, Ph-H) ; MS (ESI), m/z: 355 (M+1)+; Elemental analysis for C16H14N6S2:
found C 54.21, H 3.70, N 23.99; calc. C, 54.22; H, 3.98; N, 23.71.
5-(4-Cyclopropyl-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7p). White
solid, yield 77%, m.p. 107–108 °C; 1H NMR (CDCl3, 400 MHz) δ: 0.78–0.94 (m, 2H, cyclopropane-CH2),
1.17–1.21 (m, 2H, cyclopropane-CH2), 2.24 (s, 1H, CH), 2.95 (s, 3H, CH3), 2.99-3.11 (m, 1H,
cyclopropane-CH), 4.09 (s, 2H, CH2).MS (ESI): m/z 278 (M+1)+; Elemental analysis for C11H11N5S2:
found C 47.88, H 4.23, N 25.14; calc. C, 47.63; H, 4.00; N, 25.25.
5-(4-Cyclopropyl-5-(octylthio)-4H-1,2,4-triazol-3-yl)-4-methyl-1,2,3-thiadiazole (7q). White solid,
1
yield 81%, m.p. 67–68 °C; H-NMR (CDCl3, 400 MHz) δ: 0.79–0.86 (m, 5H, cyclopropane-CH2 and
CH2), 1.14–1.24(m, 11H, cyclopropane-CH2 and CH2), 1.36–1.44 (m, 2H, CH2), 1.74–1.81 (m, 2H, CH2),
2.95(s, 3H, CH3), 2.99–3.05(m, 1H, cyclopropane-CH), 3.33 (t, 3H, CH3); MS (ESI) m/z: 352 (M+1)+;
Elemental analysis for C16H25N5S2: found C 54.86, H 7.24, N 20.20; calc. C, 54.67; H, 7.17; N, 19.92.

Molecules 2013, 18
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3.4. Fungicidal Activities
Fungicidal activity of compounds 7a–q against Pseudoperonospora cubensis(PC), Corynespora
cassiicola(CC), Pseudomonas syringae pv. Lachrymans(PS) were evaluated according to reference [11],
and a potted plant test method was adopted. Germination was conducted by soaking cucumber seeds in
water for 2 h at 50 °C and then keeping the seeds moist for 24 h at 28 °C in an incubator. When the
radicles were 0.5 cm, the seeds were grown in plastic pots containing a 1:1 (v/v) mixture of vermiculite
and peat. Cucumber plants used for inoculations were at the stage of two seed leaves. Tested
compounds and commercial fungicides were sprayed with a hand spray on the surface of the seed
leaves on a fine morning, at the standard concentration of 500 μg/mL, iprodione, validamycin and
topsin-M were used as a control. After 2 h, inoculations of Pseudoperonospora cubensis, Corynespora
cassiicola were carried out by spraying a conidial suspension, inoculation of Pseudomonas syringae pv.
Lachrymans was carried out by spraying fungal suspension. The experiment was repeated 4 times.
After inoculation, the plants were maintained at 18–30 °C [mean temperature of 24 °C and above 80%
relative humidity (RH)]. The fungicidal activity were evaluated when the nontreated cucumber plant
(blank) fully developed symptoms. The area of inoculated treated leaves covered by disease symptoms
was assessed and compared to that of nontreated ones to determine the average disease index. The relative
control efficacy of compounds compared to the blank assay was calculated via the following equation:
relative control efficacy (%) = (CK−PT)/CK × 100%
where CK is the average disease index during the blank assay and PT is the average disease index after
treatment during testing.
4. Conclusions
In summary, a series of 1,2,4-triazole derivatives containing 1,2,3-thiadiazole rings were
synthesized in good yields. The preliminary bioassays showed that some of the compounds had good
fungicidal activity. The present findings provided a powerful complement to the SARs of fungicides,
and warrant future investigation of the mechanism of action of these analogues.
Acknowledgments
The project was supported by Applied Chemistry Foundation of Zhejiang Shuren University
(YH2013G02, YH2012S09, YH2013X14) and Zhejiang Provincial Natural Science Foundation of
China (LQ13B020003), National Natural Science Foundation of China (No. 21002090), The Key
Innovation Team of Science and Technology in Zhejiang Province (2010R50018-06) and the National
Key Technologies R&D Program (2011BAE06B03-01). The fungicidal activity was determined by Li
Bao-Ju Group, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences,
Beijing, China.
Conflicts of Interest
The authors declare no conflict of interest

Molecules 2013, 18
12737
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Sample Availability: Samples of the compounds are available from the authors.
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distributed under the terms and conditions of the Creative Commons Attribution license
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