Synthesis and biological activity of novel dimethylpyrazole and piperazine-containing (bis)1,2,4-triazole derivatives

Article abstract of DOI:10.1080/10426507.2016.1223077

A series of novel dimethylpyrazole and piperazine-containing (bis)1,2,4-triazole derivatives have been conveniently synthesized via Mannich reaction in good yields. Their structures were identified by IR,¹H NMR,¹³C NMR, and elemental analysis. The preliminary bioassays showed that among 14 new compounds, the trifluoromethyl-containing compounds exhibited superior activity than the methyl-containing ones; some of the compounds displayed significant in vitro and in vivo fungicidal activity against several plant fungi and were comparable with the control Triadimefon; several compounds exhibited certain herbicidal activity against Brassica campestris; several compounds possessed favorable KARI inhibitory activity, especially 8D could be a promising KARI inhibitor for further study. The research results will provide useful information for the design and discovery of new agrochemicals with novel heterocyclic Mannich base structures containing piperazine moiety.

Full text of DOI:10.1080/10426507.2016.1223077

ACCEPTED MANUSCRIPT
Novel (Bis)1,2,4-Triazole Derivatives
Synthesis and Biological Activity of Novel Dimethylpyrazole and Piperazine -containing
(Bis)1,2,4-Triazole Derivatives
a,
a
b
a
a
a
Bao-Lei Wang *, Yan Zhang , Xing-Hai Liu , Li-Yuan Zhang , Yi-Zhou Zhan , Xiao Zhang , Li-Zhong
a
a
a,$
Wang , Yong-Hong Li , Zheng-Ming Li
a
State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of
Chemical Science and Enginnering(Tianjin), NanKai University, Tianjian 300071, China.
b
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China.
*
Address correspondence to Bao-Lei Wang, State-Key Laboratory of Elemento-Organic
Chemistry, Nankai University, Tianjin 300071, China, E-mails: wangbaolei@126.com
$
Address correspondence to Zheng-Ming Li, State-Key Laboratory of Elemento-Organic
Chemistry, Nankai University, Tianjin 300071, China, E-mails: nkzml@vip.163.com
A series of novel dimethylpyrazole and piperazine -containing (bis)1,2,4-triazole derivatives
have been conveniently synthesized via Mannich reaction in good yields. Their structures were
1
13
identified by IR, H NMR, C NMR and elemental analysis. The preliminary bioassays showed
that among 14 new compounds, the trifluoromethyl-containing compounds exhibited superior
activity than the methyl-containing ones; some of the compounds displayed significant in vitro
and in vivo fungicidal activity against several plant fungi and were comparable with the control
1
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Triadimefon; several compounds exhibited certain herbicidal activity against Brassica
campestris; several compounds possessed favorable KARI inhibitory activity, especially 8D could
be a promising KARI inhibitor for further study. The research results will provide useful
information for the design and discovery of new agrochemicals with novel heterocyclic
Mannich base structures containing piperazine moiety.
Keywords
1
,2,4-triazole, pyrazole, Mannich base, piperazine, biological activity
2
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INTRODUCTION
As one of the nitrogen-containing heterocycles, pyrazole occupies an important position in the
field of medicinal and pesticidal chemistry due to a variety of pharmacological and biological
activity of its derivatives -- on one hand, pyrazole and its derivatives possess significant
antibacterial, antiviral, anticoagulant, antimicrobial, anti-inflammatory and anticancer
[
1-3]
properties ; on the other hand, they also show versatile pesticidal activity and are used as
[
4-9]
insecticides, herbicides, fungicides and plant growth regulators.
For example, Fipronil, an
insecticide that is used to control lepidopteran and coleopteran insects, is a type of pyrazole
[
10]
derivative ; Pyraclostrobin, a pyrazole-containing strobilurin fungicide, possesses a broad
[
11]
fungicidal spectrum with preventive, curative, translaminar and locosystemic properties
.
Piperazine is another important nitrogen-containing heterocycle which has various drug-related
properties and have attracted extensive investigation for small molecule drug discovery
through decades. It has been found that N-substituted piperazine compounds have a wide
[
12]
[13]
[14]
range of biological activities, such as antimicrobial , anticancer , tuberculostatic
,
[
15,16]
[17]
antifungal
, herbicidal and insecticidal activities . Comparatively, there are much less
literatures about the the application of piperazine-containing compounds in agrochemical area
than in medicinal area.
In addition, there are lots of heterocyclic Mannich bases been reported, however most of the
[
18]
[19]
researches focused on their pharmacological activities, such as antimicrobial , anticancer
,
3
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[
20]
[21]
antitumor and antituberculous activities. Likewise, it is also necessary to carry out
extensive study on the various agrochemical activities of heterocyclic Mannich bases, which
might be a promising topic to produce more novel agrochemical candidates.
In our previous work, some interesting 1,2,4-triazole Mannich bases containing
[
22,23,16]
trifluoromethyl, piperazine and substituted phenyl groups were synthesized successfully
and found to exhibit favorable herbicidal activity against dicotyledon plant, and inhibitory
activity against ketol-acid reductoisomerase (KARI), one of the key enzyme involved in the
[24-26]
biosynthesis route of branched chain amino acids leucine, isoleucine and valine
.
Particularly, those kinds of compounds possessed significant fungicidal activities against
Phytophthora infestans (Mont.) de Bary, G. zeae Petch and other fungi, which provided an
important reference for us to make some further structural modifications. The preliminary SAR
studies illustrated the necessity for further optimization based on the general structure of this
type of compounds, especially for the phenyl group and the substituent on the piperazine part
[
22,23]
.
Encouraged by this, some 1,2,4-triazole Mannich bases with a
phenylpyrazolymethyleneamino group and a (substituted)piperazinomethyl group in the
triazole 4-position and 1-position respectively were recently synthesized in our lab; they were
[
6]
also found to possess favorable fungicidal activities against some plant fungi . In such type of
compounds, there are phenyl, trifluoromethyl/methyl and chlorine moieties in the pyrazole
ring, which mainly are electron-withdrawing groups. If more electron-donating groups could be
4
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introduced into the pyrazole ring of this type of structure, e. g. two methyl groups, the
corresponding intermediate probably would undergo the similar reaction as that of the
[6]
phenylprazole-intermediate in different reactivity, to generate some novel 1,2,4-triazole
Mannich bases with good biological activities. By taking into account all the facts mentioned
above, a series of novel methyl/trifluoromethyl-containing triazole Mannich bases with
dimethylpyrazole and substituted piperzaine moieties were synthesized. Meanwhile, in
piperazine ring there are two secondary amine structures (-NH-) that could both be used for
undertaking Mannich reaction to generate some new structures, with this in mind, novel
bis-1,2,4-triazole derivatives containing dimethylpyrazole and piperazine moieties were also
synthesized. The fungicidal, herbicidal and KARI inhibitory activities of these new
(bis)1,2,4-triazole derivatives were investigated in this paper.
RESULTS AND DISCUSSION
Synthesis and Spectra Characterization
The synthetic procedures for piperazine intermediates, triazole intermediates 7 and the title
compounds 8, 9 and 10 are shown in SCHEME 1, SCHEME 2 and SCHEME 3. The novel triazole
[
6]
thione intermediates (7A-B) were prepared using an acetic acid catalysis method via the
condensation of thione intermediate 6 and 5-chloro-1,3-dimethyl-1H-pyrazole-4-carbaldehyde
(5) in ethanol with ~ 80% yield. Foks et al. reported the similar Mannich reaction from
5
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heterocycle-thiol intermediate using methanol or dioxane as solvent under reflux condition (1
[
14]
h) , by contrast, our synthetic procedure for the title 1,2,4-triazole Mannich bases was to
carry out the reaction in ethanol at room temperature. Using this method, 8 and 9 can be
obtained in satisfying yields within 2-3 h, which results are similar with those of the Mannich
[
6]
reaction based on the phenylpyrazole-intermediats in our recent work . Under conditions of
excess formaldehyde and 2:1 mole ratio of 7 and piperazine, novel bis-1,2,4-triazole derivatives
1
0 can also be successfully synthesized at room temperature in high yield (82%-88%). It was
found that this type of Mannich reaction based on the pyrazolymethyleneamino-triazole thione
intermediates can undergo smoothly with high yield and under mild conditions in short time.
1
Intermediates 7 and the title compounds 8, 9 and 10 were identified by melting point, IR, H
1
3
NMR and C NMR spectra. The measured elemental analyses were also consistent with the
corresponding calculated values. In the IR spectra of the title compounds, the characteristic

1
stretching vibration absorption peaks of C = S and C = N appeared at 1155-1176 cm and

1
1
592-1612 cm respectively. The stretching vibrations ν (C-F) of the trifluoromethyl-containing

1
compounds were observed at 1186-1209 cm . For intermediates 7, absorption peaks of N-H

1
1
1
and C = N appeared at 3452 cm and 1638 cm , respectively. In H NMR, the --CH = N-- proton
appeared at δ 9.77-10.29 as a singlet in 8, 9 and 10. The chemical shift at δ 13.71-14.83 (singlet)
in 7 can be ascribed to the N-H active proton owing to the thione structure (--NH--C( = S)--). The
piperazine ring proton (CH ) in 8 and 9 appeared at δ 2.70-3.89 and δ 2.35-2.88 as two broad
2
6
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peaks or multiplets, respectively; however, due to the symmetrical structure of
1
3
bis-1,2,4-triazole derivatives 10, they were observed as a singlet at δ ~ 2.85. In the C NMR
spectra of 8, 9 and 10, the typical carbon resonance at δ 160.3-167.7 was indicative of a
thiocarbonyl group (C = S). The carbon signals of --CH = N-- group were observed at δ
1
1
43.2-150.0. The signals of C in the triazole ring and carbon adjacent to it were observed at δ ~
3
54.0 and δ ~ 14.9 (CH ) as a singlet respectively, while at δ ~138.0 (J ≈ 40.4 Hz) and δ ~116.0
3
(
CF , J ≈ 273.0 Hz) as quartet or multiplet, respectively in the trifluoromethyl-containing
3
compounds, which is due to the “F” splitting of the latter.
In addition, the carbon signal at δ 162.1-169.6 (C = S) for the compounds 5, 6 and 7 indicates
[
27]
these intermediates all existed in the thione tautomeric form . It was worthy of note that the
double bond in the imine part of these compounds (-CH = N-) should be all in E-configuration,
which can be supported by the single crystal and calculation work (optimal conformation) of
[
22]
such a similar parent structure reported in our previous work
.
Fungicidal activity
The in vitro fungicidal activity data of the title 1,2,4-triazole derivatives 8A-8D, 9A-9H and
bis-1,2,4-triazole derivatives 10A-10B for the inhibition of mycelial growth in six test fungi are
listed in Table S 1 (Supplemental Materials). The commercial fungicides Triadimefon,
Carbendazim and Chlorothalonil were used as controls. It was found that at the test
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concentration of 50 μg/mL, most of the compounds showed significant fungicidal activity
against Alternaria solani Sorauer, Gibberella sanbinetti, Physalospora piricola and Rhizoctonia
cerealis while some of these results were comparable with those of the control Triadimefon.
For examples, 9B, 9G, 9H, 10A and 10B possessed inhibition rates of 62% ~ 75% against
Physalospora piricola; 9G and 9H held inhibition rates of 94% ~ 96% against Rhizoctonia
cerealis. In addition, compound 10B whose activity was 75.0% and 65.5% against Cercospora
arachidicola and Gibberella sanbinetti, respectively, was more effective than Triadimefon.
According to the in vitro fungicidal activity data, several trifluoromethyl-containing
compounds were chosen to be tested for in vivo fungicidal activity against Puccinia sorghi
Schw.. From Table S 2 (Supplemental Materials), it was shown that at 200 μg/mL concentration,
compounds 8D, 9E, 9F, 9G and 9H held good activity against Puccinia sorghi Schw. with control
efficacy of 50% ~ 85%. At the test concentration of 100 μg/mL, 8D and 9F can still exhibit
control efficacy of 20% and 30%, respectively.
As a whole, these dimethylpyrazole and piperazine -containing (bis)1,2,4-triazole derivatives
displayed significant fungicidal activity against various plant fungi, while the CF group at
3
4
3
-position of 1,2,4-triazole ring (R = F) made an apparent influence on their fungicidal activity,
which could be well reflected particularly in the results of Rhizoctonia cerealis test (Table S 1).
By analyzing the SAR based on the fungicidal data against Rhizoctonia cerealis, it was found that
in most cases trifluoromethyl- containing compounds exhibit higher activity than methyl-
8
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containing ones, such as 8D vs. 8B, 9H vs. 9D, 10B vs. 10A. The activity of compounds 8A-8D,
4
when R is fixed as H or F atom, indicated the sequence of fungicidal activity is
2
,4-dichlorobenzyl > benzyl in the substituted benzyl moiety in piperazine ring; in the case of
4
compounds 9A-9H, when R is fixed as F atom, it showed the activity sequence of
4
,6-dimethylpyrimidyl ≈ 4-methylpyrimidyl > pyridyl ≈ phenyl in the piperazine ring.
Herbicidal activity
As shown in Table S 3 (Supplemental Materials), some of the title compounds exhibited
certain herbicidal activity in rape (Brassica campestris) root and barnyardgrass (Echinochloa
crusgalli) cup tests at 100 μg/mL concentration. For examples, 8C, 8D and 9G against Brassica
campestris possessed growth inhibition rates of 59.9%, 38.1% and 33.6%, respectively. Against
Echinochloa crusgalli, however, all the compounds showed poor herbicidal activity. In general,
trifluoromethyl-containing compounds displayed better herbicidal activity than
methyl-containing ones.
KARI inhibitory activity
From Table S 4 (Supplemental Materials), it was observed that most of the title compounds
exhibited apparent KARI inhibitory activity at the concentration of 200 μg/mL, such as 8D, 9A,
9
D, 9F and 10B (activity 34-88%). It was worthy to note that compound 8D whose in vitro
9
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inhibitory rate towards rice KARI is 87.5%, could be a promising KARI inhibitor for further
investigation in the future, although it is less active than the known potent inhibitor CPD
[
24]
.
EXPERIMENTAL
Melting points were determined using an X-4 binocular microscope apparatus and
uncorrected. Infrared spectra were recorded on a Bruker Tensor 27 spectrophotometer as KBr
1
13
tablets. H NMR and C NMR spectra were measured on a Bruker AV-400 instrument (400
MHz) using tetramethylsilane as an internal standard and DMSO-d or CDCl as solvent.
6
3
Elemental analyses were performed on a Vario EL CUBE elemental analyzer. KARI inhibitory
activity test was carried out on a VARIAN CARY 100 Bio Uv/Visibile spectrophotometer.
[
28,23]
4
4
-(Substituted)benzylpiperazine 1A and 1B were prepared according to the literature
.
-(4,6-Disubstituted pyrimidin-2-yl)piperazine 3A and 3B were synthesized referring to the
[
22]
literature method . 4-Phenylpiperazine (3C) and 4-(pyridine-2-yl)piperazine (3D) were
purchased from Aladin and Alfa Aesar reagent companies.
4
-Amino-3-methyl/trifluoromethyl-1H-1,2,4-triazole-5(4H)-thione were prepared according to
[29]
the literature . Anhydrous piperazine and other materials were purchased from Nanjing
Duodian Reagent Co. Ltd. All solvents were dried by standard methods in advance and distilled
1
13
before use. The Supplemental Materials contains sample H and C NMR and IR spectra of
products 7-10 (Figures S 1 -- S 54)
1
0
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Preparation of 5-Chloro-1,3-dimethyl-1H-pyrazole-4-carbaldehyde 5
2
,5-Dimethyl-2,4-dihydro-3H-pyrazol-3-one 4 was prepared by refluxing ethyl acetoacetate
[
5]
and methylhydrazine in ethanol . Intermediate pyrazole carbaldehyde 5 was prepared
[
30]
according to reference and the method was improved. To a violently stirred cold solution of
DMF (23 mL) was added dropwise phosphorus oxychloride (64 mL) at 0-10 C. The mixture was
stirred at this temperature for 30 min then compound 4 (0.1 mol) was added in portions. The
mixture was heated to 80-90 C and stirred for 4 h. After being cooled to room temperature,
the mixture was poured into ice-water (350 mL) and stirred for 10 min, then extracted with
ethyl acetate (3 × 25 mL). The organic layer was washed with saturated NaHCO solution (2 × 15
3
mL) and brine (15 mL) successively, and dried over anhydrous Na SO . The solvent was removed
2
4
under reduced pressure, the residue was recrystallized from ethyl acetate/petroleum ether
60-90°C) to give pyrazole carbaldehyde 5 as light yellow crystals, mp 77-78 C, yield 81.5% (Lit.
(
[
30]
mp 78-79 C).
General Synthetic Procedure for Novel Triazole Thione Intermediates 7
Compound 6A or 6B (10 mmol), pyrazole carbaldehyde 5 (10.5 mmol) and acetic acid (10
drops) were mixed in absolute ethanol (25 mL). After having been stirred and refluxed for 4-5 h,
the reaction mixture was cooled to room temperature. The resulting solid was filtered off and
recrystallized from ethanol to give thione intermediate 7.
1
1
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(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-3-methyl-1H-1,2,4-triazole-
5
(4H)-thione (7A)
1
Colorless crystals, yield 81.3%, mp 239-241 C; IR (νmax, cm ): 3452 (N-H), 1638(C = N),
1
1
159(C = S). H NMR (400 MHz, DMSO-d ): δ 13.71 (s, 1H, NH), 9.97 (s, 1H, CH = N), 3.81 (s, 3H,
6
1
3
Pyrazole-CH ), 2.39 (s, 3H, Triazole-CH ), 2.32 (s, 3H, Pyrazole-CH ). C NMR (101 MHz,
3
3
3
DMSO-d ): δ 162.1 (s, 1C, C = S), 155.9 (s, 1C, Triazole-C), 149.5 (s, 1C, Pyrazole-C), 149.2 (s, 1C,
6
N = CH), 131.5 (s, 1C, Pyrazole-C), 111.0 (s, 1C, Pyrazole-C), 37.3 (s, 1C, Pyrazole-CH ), 15.5 (s,
3
1
3
C, Triazole-CH ), 11.8 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for C H ClN S: C 39.93, H 4.10, N
3
3
9
11
6
1.04. Found: C 39.67, H 4.26, N 30.87.
(
E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-3-(trifluoromethyl)-1H-1,2,
-triazole-5(4H)-thione (7B)
Colorless crystals, yield 78.8%, mp 214-216 C; IR (νmax, cm ): 3452 (N-H), 1639(C = N). H
NMR (400 MHz, DMSO-d ): δ 14.84 (s, 1H, NH), 9.95 (s, 1H, CH = N), 3.82 (s, 3H, Pyrazole-CH ),
4

1
1
6
3
1
3
2
.36 (s, 3H, Pyrazole-CH ). C NMR (101 MHz, DMSO-d ): δ 163.7 (s, 1C, C = S), 156.9 (s, 1C,
3
6
Pyrazole-C), 156.8 (s, 1C, Pyrazole-C), 148.3 (s, 1C, N = CH), 138.6 (d, JC-F = 40.4 Hz, 1C,
Triazole-C ), 131.2 (s, 1C, Pyrazole-C), 114.1 (q, JC-F = 273.7 Hz, 1C, CF ), 36.2 (s, 1C,
3
3
Pyrazole-CH ), 14.0 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for C H ClF N S: C 33.29, H 2.48, N
3
3
9
8
3 6
2
5.88. Found: C 33.12, H 2.49, N 25.74.
General Synthetic Procedure for Novel 1,2,4-Triazole Derivatives 8
1
2
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A solution of 4-(substituted)benzylpiperazine 1 (1 mmol) in ethanol (2 mL) was added
dropwise to a mixture of thione intermediate 7 (1 mmol) and 37% formalin (1.5 mmol) in
ethanol (15 mL). Then the reaction mixture was stirred for 2-3 h at room temperature. The
resulting precipitate (in some cases, there is no solid precipitated out after reaction completed,
the mixture can be placed in a fridge or concentrated to accelerate precipitating) was filtered
off and recrystallized from ethanol to give novel 1,2,4-triazole derivative 8(A-D).
(E)-1-((4-Benzylpiperazin-1-yl)methyl)-4-((5-chloro-1,3-dimethyl-1H-pyrazol-4-yl)methylene
amino)-3-methyl-1H-1,2,4-triazole-5(4H)-thione (8A)

1
White crystals, yield 62.4%, mp 152-154 C; IR (νmax, cm ): 2945, 2825(C-H), 1610(C = N),
1
1
7
2
532, 1494, 1452(Ar), 1168(C = S). H NMR (400 MHz, DMSO-d ): δ 9.83 (s, 1H, CH = N),
6
.31-7.22 (m, 5H, Ph-H), 5.01 (s, 2H, NCH N), 3.81 (s, 3H, Pyrazole-CH ), 3.42 (s, 2H, PhCH ),
2
3
2
.70 (bs, 4H, Piperazine-H), 2.40 (s, 3H, Pyrazole-CH ), 2.35 (bs, 7H, Piperazine-H +
3
1
3
Triazole-CH ). C NMR (101 MHz, CDCl ): δ 162.5 (s, 1C, C = S), 153.9 (s, 1C, Triazole-C), 149.3
3
3
(s, 1C, Pyrazole-C), 147.5 (s, 1C, N = CH), 138.0 (1C, Ph-C), 131.3 (s, 1C, Pyrazole-C), 129.2 (s, 2C,
Ph-C), 128.2 (s, 2C, Ph-C), 127.0 (s, 1C, Ph-C), 110.7 (s, 1C, Pyrazole-C), 68.6 (s, 1C, CH ), 63.0 (s,
2
1
1
2
C, CH ), 53.0 (s, 2C, Piperazine-C), 50.5 (s, 2C, Piperazine-C), 36.2 (s, 1C, Pyrazole-CH ), 14.8 (s,
2
3
C, Triazole-CH ), 11.1 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for C H ClN S: C 54.95, H 5.93, N
3
3
21 27
8
4.41. Found: C 54.78, H 5.99, N 24.32.
1
3
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(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-1-((4-(2,4-dichlorobenzyl)pi
perazin-1-yl)methyl)-3-methyl-1H-1,2,4-triazole-5(4H)-thione (8B)

1
White solid, yield 65.5%, mp 157-159 C; IR (νmax, cm ): 2929, 2840(C-H), 1606(C = N), 1528,
1
1
2
474, 1453(Ar), 1166(C = S). H NMR (400 MHz, CDCl ): δ 10.29 (s, 1H, CH = N), 7.38-7.34 (m,
3
H, Ph-H), 7.19-7.18 (m, 1H, Ph-H), 5.12 (s, 2H, NCH N), 3.84 (s, 3H, Pyrazole-CH ), 3.55 (s, 2H,
2
3
ArCH ), 2.86 (bs, 4H, Piperazine-H), 2.53 (bs, 4H, Piperazine-H), 2.48(s, 3H, Pyrazole-CH ), 2.41
2
3
1
3
(
s, 3H, Triazole-CH ). C NMR (101 MHz, CDCl ): δ 162.5 (s, 1C, C = S), 154.0 (s, 1C, Triazole-C),
3
3
1
49.3 (s, 1C, Pyrazole-C), 147.6 (s, 1C, N = CH), 134.8, 134.6, 133.0 (3C, Ph-C), 131.4 (s, 1C,
Pyrazole-C), 131.3 (s, 1C, Ph-C), 129.1 (s, 1C, Ph-C), 126.8 (s, 1C, Ph-C), 110.7 (s, 1C, Pyrazole-C),
6
1
8.6 (s, 1C, CH ), 58.6 (s, 1C, CH ), 53.0 (s, 2C, Piperazine-C), 50.5 (s, 2C, Piperazine-C), 36.2 (s,
2
2
C, Pyrazole-CH ), 14.9 (s, 1C, Triazole-CH ), 11.1 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for
3
3
3
C H Cl N S: C 47.78, H 4.77, N 21.23. Found: C 47.87, H 4.57, N 21.14.
2
1
25
3 8
(E)-1-((4-Benzylpiperazin-1-yl)methyl)-4-((5-chloro-1,3-dimethyl-1H-pyrazol-4-yl)methylene
amino)-3-(trifluoromethyl)-1H-1,2,4-triazole-5(4H)-thione (8C)

1
White solid, yield 57.2%, mp 112-114 C; IR (νmax, cm ): 2935, 2813(C-H), 1606(C = N), 1533,
1
1
492, 1455(Ar), 1313, 1190(C-F), 1157(C = S). H NMR (400 MHz, DMSO-d ): δ 9.80 (s, 1H, CH =
6
N), 7.32-7.22 (m, 5H, Ph-H), 5.15 (s, 2H, NCH N), 3.81 (s, 3H, Pyrazole-CH ), 3.44 (s, 2H, PhCH ),
2
3
2
1
3
2
.75 (bs, 4H, Piperazine-H), 2.36 (bs, 7H, Piperazine-H + Pyrazole-CH ). C NMR (101 MHz,
3
1
4
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CDCl ): δ 165.0 (s, 1C, C = S), 155.4 (s, 1C, Pyrazole-C), 149.9 (s, 1C, N = CH), 138.5 (s, 1C,
3
Triazole-C ), 137.9 (s, 1C, Ph-C), 132.2 (s, 1C, Pyrazole-C), 129.2 (s, 2C, Ph-C), 128.3 (s, 2C, Ph-C),
3
1
6
1
4
27.1 (s, 1C, Ph-C), 118.8 (q, J = 273.7 Hz, 1C, CF ), 110.2 (s, 1C, Pyrazole-C), 69.9 (s, 1C, CH ),
C-F
3
2
3.1 (s, 1C, CH ), 52.9 (s, 2C, Piperazine-C), 50.4 (s, 2C, Piperazine-C), 36.2 (s, 1C, Pyrazole-CH ),
2
3
4.7 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for C H ClF N S: C 49.17, H 4.72, N 21.84. Found: C
3
21 24
3 8
9.34, H 4.90, N 22.01.
(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-1-((4-(2,4-dichlorobenzyl)pi
perazin-1-yl)methyl)-3-(trifluoromethyl)-1H-1,2,4-triazole-5(4H)-thione (8D)

1
White solid, yield 66.1%, mp 139-141 C; IR (νmax, cm ): 2936, 2814(C-H), 1604(C = N), 1531,
1
1
473, 1457(Ar), 1318, 1198(C-F), 1167(C = S). H NMR (400 MHz, DMSO-d ): δ 9.80 (s, 1H, CH =
6
N), 7.56 (s, 1H, Ph-H), 7.46-7.37 (m, 2H, Ph-H), 5.16 (s, 2H, NCH N), 3.81 (s, 3H, Pyrazole-CH ),
2
3
3
.51 (s, 2H, ArCH ), 2.76 (bs, 4H, Piperazine-H), 2.43 (bs, 4H, Piperazine-H), 2.36 (s, 3H,
2
1
3
Pyrazole-CH ). C NMR (101 MHz, CDCl ): δ 165.0 (s, 1C, C = S), 155.4 (s, 1C, Pyrazole-C), 149.9
3
3
(
s, 1C, N = CH), 138.8 (d, J = 40.4 Hz, 1C, Triazole-C ), 134.8 (s, 1C, Ph-C), 134.4 (s, 1C, Ph-C),
3
1
33.1 (s, 1C, Ph-C), 132.2 (s, 1C, Pyrazole-C), 131.4 (s, 1C, Ph-C), 129.2 (s, 1C, Ph-C), 126.9 (s, 1C,
Ph-C), 117.0 (q, J = 273.7 Hz, 1C, CF ), 110.2 (s, 1C, Pyrazole-C), 69.9 (s, 1C, CH ), 58.6 (s, 1C,
C-F
3
2
CH ), 53.0 (s, 2C, Piperazine-C), 50.4 (s, 2C, Piperazine-C), 36.2 (s, 1C, Pyrazole-CH ), 14.7 (s, 1C,
2
3
Pyrazole-CH ); Anal. Calcd. (%) for C H Cl F N S: C 43.35, H 3.81, N 19.26. Found: C 43.53, H
3
21 22 3 3
8
3
.62, N 19.09.
1
5
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General Synthetic Procedure for Novel 1,2,4-Triazole Derivatives 9
It was similar with that of 8, using 4-phenylpiperazine 3C, or 4-(pyridin-2-yl)piperazine 3D, or
4
1
-(4,6-disubstituted-pyrimidin-2-yl)piperazine 3(A-B) as amine material to give corresponding
,2,4-triazole derivative 9(A-H).
(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-3-methyl-1-((4-phenylpiper
azin-1-yl)methyl)-1H-1,2,4-triazole-5(4H)-thione (9A)

1
White solid, yield 71.6%, mp 190-192 C; IR (νmax, cm ): 2944, 2826(C-H), 1598(C = N), 1534,
1
1
502, 1453(Ar), 1162(C = S). H NMR (400 MHz, DMSO-d ): δ 9.83 (s, 1H, CH = N), 7.18 (bs, 2H,
6
Ph-H), 6.91 (bs, 2H, Ph-H), 6.76 (bs, 1H, Ph-H), 5.10 (s, 2H, NCH N), 3.81 (s, 3H, Pyrazole-CH ),
2
3
3
.12 (bs, 4H, Piperazine-H), 2.85 (s, 3H, Pyrazole-CH ), 2.40 (bs, 4H, Piperazine-H), 2.35 (s, 3H,
3
1
3
Triazole-CH ). C NMR (101 MHz, CDCl ): δ 162.6 (s, 1C, C = S), 154.1 (s, 1C, Triazole-C), 151.3
3
3
(s, 1C, Ph-C), 149.3 (s, 1C, Pyrazole-C), 147.7 (s, 1C, N = CH), 131.4 (s, 1C, Pyrazole-C), 129.1 (s,
2
C, Ph-C), 119.9 (s, 1C, Ph-C), 116.4 (s, 2C, Ph-C), 110.7 (s, 1C, Pyrazole-C), 68.6 (s, 1C, CH ), 50.5
2
(
s, 2C, Piperazine-C), 49.3 (s, 2C, Piperazine-C), 36.2 (s, 1C, Pyrazole-CH ), 14.9 (s, 1C,
3
Triazole-CH ), 11.1 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for C H ClN S: C 53.98, H 5.66, N
3
3
20 25
8
2
5.18. Found: C 53.68, H 5.62, N 24.92.
(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-3-methyl-1-((4-(pyridin-2-yl
)
piperazin-1-yl)methyl)-1H-1,2,4-triazole-5(4H)-thione (9B)
1
6
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
1
White solid, yield 76.2%, mp 160-162 C; IR (νmax, cm ): 2944, 2842(C-H), 1592(C = N), 1534,
1
1
478, 1453(Ar), 1161(C = S). H NMR (400 MHz, DMSO-d ): δ 9.81 (s, 1H, CH = N), 8.07 (d, J = 3.6
6
Hz, 1H, Pyridine-H), 7.49 (t, J = 7.2 Hz, 1H, Pyridine-H), 6.80 (d, J = 8.8 Hz, 1H, Pyridine-H),
.61-6.58 (m, 1H, Pyridine-H), 5.09 (s, 2H, NCH N), 3.80 (s, 3H, Pyrazole-CH ), 3.48 (bs, 4H,
6
2
3
Piperazine-H), 2.78 (bs, 4H, Piperazine-H), 2.38 (s, 3H, Pyrazole-CH ), 2.33 (s, 3H, Triazole-CH ).
3
3
1
3
C NMR (101 MHz, CDCl ): δ 162.6 (s, 1C, C = S), 159.3 (s, 1C, Pyridine-C) 154.1 (s, 1C,
3
Triazole-C), 149.3 (s, 1C, Pyrazole-C), 148.0 (s, 1C, Pyridine-C), 147.7 (s, 1C, N = CH), 137.4 (s, 1C,
Pyridine-C), 131.4 (s, 1C, Pyrazole-C), 113.2 (s, 1C, Pyridine-C), 110.7 (s, 1C, Pyrazole-C), 107.1
(
s, 1C, Pyridine-C), 68.7 (s, 1C, CH ), 50.4 (s, 2C, Piperazine-C), 45.2 (s, 2C, Piperazine-C), 36.2 (s,
2
1
C, Pyrazole-CH ), 14.8 (s, 1C, Triazole-CH ), 11.1 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for
3
3
3
C H ClN S: C 51.17, H 5.42, N 28.27. Found: C 50.98, H 5.39, N 27.99.
1
9
24
9
(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-3-methyl-1-((4-(4-methylpy
rimidin-2-yl)piperazin-1-yl)methyl)-1H-1,2,4-triazole-5(4H)-thione (9C)

1
White solid, yield 74.3%, mp 151-153 C; IR (νmax, cm ): 2948, 2846(C-H), 1612(C = N), 1573,
1
1
562, 1487, 1448(Ar), 1159(C = S). H NMR (400 MHz, DMSO-d ): δ 9.81 (s, 1H, CH = N), 8.18 (d,
6
J = 4.4 Hz, 1H, Pyrimidine-H), 6.48 (d, J = 4.8 Hz, 1H, Pyrimidine-H), 5.09 (s, 2H, NCH N), 3.81 (s,
2
3
H, Pyrazole-CH ), 3.74 (bs, 4H, Piperazine-H), 2.74 (bs, 4H, Piperazine-H), 2.39 (s, 3H,
3
1
3
Pyrazole-CH ), 2.32 (s, 3H, Triazole-CH ), 2.25 (s, 3H, Pyrimidine-CH ). C NMR (101 MHz,
3
3
3
CDCl ): δ 167.6 (s, 1C, C = S), 162.6 (s, 1C, Pyrimidine-C ), 161.5 (s, 1C, Pyrimidine-C ), 157.9 (s,
3
4
2
1
7
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1
1
5
C, Pyrimidine-C ), 154.1 (s, 1C, Triazole-C), 149.3 (s, 1C, Pyrazole-C), 147.7 (s, 1C, N = CH),
6
31.4 (s, 1C, Pyrazole-C), 110.7 (s, 1C, Pyrazole-C), 109.4 (s, 1C, Pyrimidine-C ), 68.9 (s, 1C, CH ),
5
2
0.6 (s, 2C, Piperazine-C), 43.5 (s, 2C, Piperazine-C), 36.2 (s, 1C, Pyrazole-CH ), 24.3 (s, 1C,
3
Pyrimidine-CH ), 14.8 (s, 1C, Triazole-CH ), 11.1 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for
3
3
3
C H ClN S: C 49.50, H 5.47, N 30.38. Found: C 49.44, H 5.42, N 30.09.
1
9
25
10
(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-1-((4-(4,6-dimethylpyrimidi
n-2-yl)piperazin-1-yl)methyl)-3-methyl-1H-1,2,4-triazole-5(4H)-thione (9D)

1
White solid, yield 80.4%, mp 189-191 C; IR (νmax, cm ): 2944, 2826(C-H), 1610(C = N), 1570,
1
1
1
543, 1477, 1450(Ar), 1170(C = S). H NMR (400 MHz, CDCl ): δ 10.26 (s, 1H, CH = N), 6.23 (s,
3
H, Pyrimidine-H), 5.17 (s, 2H, NCH N), 3.89-3.86 (m, 4H, Piperazine-H), 3.83 (s, 3H,
2
Pyrazole-CH ), 2.88-2.85 (m, 4H, Piperazine-H), 2.47 (s, 3H, Pyrazole-CH ), 2.38 (s, 3H,
3
3
1
3
Triazole-CH ), 2.25 (s, 6H, Pyrimidine-CH ). C NMR (101 MHz, CDCl ): δ 167.0 (s, 1C, C = S),
3
3
3
1
62.6 (s, 2C, Pyrimidine-C ), 161.6 (s, 1C, Pyrimidine-C ), 154.1 (s, 1C, Triazole-C), 149.3 (s, 1C,
4
,6
2
Pyrazole-C), 147.6 (s, 1C, N = CH), 131.4 (s, 1C, Pyrazole-C), 110.7 (s, 1C, Pyrazole-C), 108.8 (s,
2
1
C, Pyrimidine-C ), 68.9 (s, 1C, CH ), 50.7 (s, 2C, Piperazine-C), 43.5 (s, 2C, Piperazine-C), 36.2 (s,
5
2
C, Pyrazole-CH ), 24.1 (s, 2C, Pyrimidine-CH ), 14.8 (s, 1C, Triazole-CH ), 11.1 (s, 1C,
3
3
3
Pyrazole-CH ); Anal. Cacld. (%) for C H ClN S: C 50.57, H 5.73, N 29.49. Found: C 50.63, H
3
20 27
10
5
.65, N 29.25.
1
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(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-1-((4-phenylpiperazin-1-yl)
methyl)-3-(trifluoromethyl)-1H-1,2,4-triazole-5(4H)-thione (9E)

1
White crystals, yield 69.4%, mp 140-142 C; IR (νmax, cm ): 2952, 2828(C-H), 1612(C = N),
1
1
529, 1495, 1453(Ar), 1318, 1187 (C-F), 1164(C = S). H NMR (400 MHz, DMSO-d ): δ 9.82 (s, 1H,
6
CH = N), 7.19 (t, J = 7.1 Hz, 2H, Ph-H), 6.91 (d, J = 7.7 Hz, 2H, Ph-H), 6.76 (t, J = 7.0 Hz, 1H, Ph-H),
.23 (s, 2H, NCH N), 3.81 (s, 3H, Pyrazole-CH ), 3.13 (bs, 4H, Piperazine-H), 2.90 (bs, 4H,
5
2
3
1
3
Piperazine-H), 2.36 (s, 3H, Pyrazole-CH ). C NMR (101 MHz, CDCl ): δ 165.1 (s, 1C, C = S), 155.5
3
3
(s, 1C, Pyrazole-C), 151.3 (s, 1C, Ph-C), 150.0 (s, 1C, N = CH), 138.8 (d, J = 40.2, 1C, Triazole-C3),
1
=
32.2 (s, 1C, Pyrazole-C), 129.1 (s, 2C, Ph-C), 120.1 (s, 1C, Ph-C), 116.5 (s, 2C, Ph-C), 114.1 (q, JC-F
273.7 Hz, 1C, CF ), 110.2 (s, 1C, Pyrazole-C), 69.8 (s, 1C, CH ), 50.4 (s, 2C, Piperazine-C), 49.4
3
2
(
s, 2C, Piperazine-C), 36.2 (s, 1C, Pyrazole-CH ), 14.7 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for
3
3
C H ClF N S: C 48.14, H 4.44, N 22.46. Found: C 48.11, H 4.46, N 22.21.
2
0
22
3 8
(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-1-((4-(pyridin-2-yl)piperazi
n-1-yl)methyl)-3-(trifluoromethyl)-1H-1,2,4-triazole-5(4H)-thione (9F)

1
White crystals, yield 75.3%, mp 127-129 C; IR (νmax, cm ): 2953, 2829(C-H), 1612(C = N),
1
1
592, 1530, 1478(Ar), 1317, 1209(C-F), 1163(C = S). H NMR (400 MHz, DMSO-d ): δ 9.79 (s, 1H,
6
CH = N), 8.08-8.06 (m, 1H, Pyridine-H), 7.50 (t, J = 8.0 Hz, 1H, Pyridine-H), 6.81 (d, J = 8.8 Hz, 1H,
Pyridine-H), 6.60 (dd, J = 6.9, 5.0 Hz, 1H, Pyridine-H), 5.23 (s, 2H, NCH N), 3.81 (d, J = 8.4 Hz, 3H,
2
1
9
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Pyrazole-CH ), 3.51-3.48 (m, 4H, Piperazine-H), 2.85-2.82 (m, 4H, Piperazine-H), 2.35 (s, 3H,
3
1
3
Pyrazole-CH ). C NMR (101 MHz, CDCl ): δ 160.3 (s, 1C, C = S), 154.5 (s, 1C, Pyrazole-C), 150.7
3
3
(s, 1C, Pyridine-C), 145.2 (s, 1C, Pyridine-C), 143.2 (s, 1C, N = CH), 134.1 (d, J = 42.2 Hz, 1C,
Triazole-C ), 132.7 (s, 1C, Pyrazole-C), 127.4 (s, 1C, Pyridine-C), 111.1 (q, J = 272.7 Hz, 1C, CF ),
3
C-F
3
1
4
08.6 (s, 1C, Pyrazole-C), 105.4 (s, 1C, Pyridine-C), 102.4 (s, 1C, Pyridine-C), 65.2 (s, 1C, CH2),
5.5 (s, 2C, Piperazine-C), 40.4 (s, 2C, Piperazine-C), 31.5 (s, 1C, Pyrazole-CH ), 10.0 (s, 1C,
3
Pyrazole-CH ); Anal. Calcd. (%) for C H ClF N S: C 45.65, H 4.23, N 25.21. Found: C 45.66, H
3
19 21
3 9
4
.35, N 24.99.
(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-1-((4-(4-methylpyrimidin-2
-
yl)piperazin-1-yl)methyl)-3-(trifluoromethyl)-1H-1,2,4-triazole-5(4H)-thione (9G)

1
White solid, yield 77.3%, mp 114-116 C; IR (νmax, cm ): 2945, 2836(C-H), 1604(C = N), 1578,
1
1
531, 1487, 1450(Ar), 1318, 1206(C-F), 1158(C = S). H NMR (400 MHz, DMSO-d ): δ 9.79 (s, 1H,
6
CH = N), 8.16 (d, J = 4.2 Hz, 1H, Pyrimidine-H), 6.46 (d, J = 4.2 Hz, 1H, Pyrimidine-H), 5.22 (s, 2H,
NCH N), 3.80 (s, 3H, Pyrazole-CH ), 3.75 (bs, 4H, Piperazine-H), 2.78 (bs, 4H, Piperazine-H), 2.35
2
3
1
3
(
s, 3H, Pyrazole-CH ), 2.24 (s, 3H, Pyrimidine-CH ). C NMR (101 MHz, CDCl ): δ 167.7 (s, 1C, C =
3
3
3
S), 165.0 (s, 1C, Pyrimidine-C ), 161.5 (s, 1C, Pyrimidine-C ), 157.2 (s, 1C, Pyrimidine-C ), 155.4
4
2
6
(
s, 1C, Pyrazole-C), 149.9 (s, 1C, N = CH), 138.6 (d, J = 40.4 Hz, 1C, Triazole-C ), 132.2 (s, 1C,
3
Pyrazole-C), 114.1 (q, J = 273.7 Hz, 1C, CF ), 110.2 (s, 1C, Pyrazole-C), 109.5 (s, 1C,
C-F
3
Pyrimidine-C ), 70.1 (s, 1C, CH ), 50.5 (s, 2C, Piperazine-C), 43.5 (s, 2C, Piperazine-C), 36.2 (s, 1C,
5
2
2
0
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Pyrazole-CH ), 24.3 (s, 1C, Pyrimidine-CH ), 14.7 (s, 1C, Pyrazole-CH ); Anal. Calcd. (%) for
3
3
3
C H ClF N S: C 44.31, H 4.31, N 27.20. Found: C 44.48, H 4.45, N 27.06.
1
9
22
3 10
(E)-4-((5-Chloro-1,3-dimethyl-1H-pyrazol-4-yl)methyleneamino)-1-((4-(4,6-dimethylpyrimidi
n-2-yl)piperazin-1-yl)methyl)-3-(trifluoromethyl)-1H-1,2,4-triazole-5(4H)-thione (9H)

1
White solid, yield 84.4%, mp 140-142 C; IR (νmax, cm ): 2948, 2847(C-H), 1605(C = N), 1579,
1
1
530, 1488, 1449(Ar), 1318, 1195(C-F), 1155(C = S). H NMR (400 MHz, DMSO-d ): 9.77 (s, 1H,
6
CH = N), 6.35 (s, 1H, Pyrimidine-H), 5.21 (s, 2H, NCH N), 3.80 (s, 3H, Pyrazole-CH ), 3.75 (bs, 4H,
2
3
Piperazine-H), 2.77 (bs, 4H, Piperazine-H), 2.35 (s, 3H, Pyrazole-CH ), 2.19 (s, 6H,
3
1
3
Pyrimidine-CH ). C NMR (101 MHz, CDCl ): δ 167.1 (s, 1C, C = S), 165.0 (s, 2C, Pyrimidine-C ),
3
3
4,6
1
61.5 (s, 1C, Pyrimidine-C ), 155.4 (s, 1C, Pyrazole-C), 149.9 (s, 1C, N = CH), 139.5 (m, 1C,
2
Triazole-C ), 132.2 (s, 1C, Pyrazole-C), 116.9 (q, J = 272.7 Hz, 1C, CF ), 110.2 (s, 1C,
3
C-F
3
Pyrazole-C), 109.0 (s, 1C, Pyrimidine-C ), 70.1 (s, 1C, CH ), 50.5 (s, 2C, Piperazine-C), 43.4 (s, 2C,
5
2
Piperazine-C), 36.2 (s, 1C, Pyrazole-CH ), 24.1 (s, 2C, Pyrimidine-CH ), 14.7 (s, 1C, Pyrazole-CH );
3
3
3
Anal. Calcd. (%) for C H ClF N S: C 45.41, H 4.57, N 26.48. Found: C 45.52, H 4.60, N 26.22.
2
0
24
3 10
General Synthetic Procedure for Novel Bis-1,2,4-triazole Derivatives 10
A solution of anhydrous piperazine (0.9 mmol) in ethanol (2 mL) was added dropwise to a
mixture of thione intermediate 7 (1.8 mmol) and 37% formalin (2.5 mmol) in ethanol (30 mL).
After that, the reaction mixture was stirred at room temperature for 2-3 h. The resulting
2
1
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precipitate was filtered off and recrystallized from ethanol to give novel bis-1,2,4-triazole
derivative 10(A-B).
(E)-1,1'-(Piperazine-1,4-diylbis(methylene))bis(4-((E)-(5-chloro-1,3-dimethyl-1H-pyrazol-4-yl
)
methyleneamino)-3-methyl-1H-1,2,4-triazole-5(4H)-thione) (10A)
1
White solid, yield 87.6%, mp 226-228 C (Dec.); IR (νmax, cm ): 2937, 2826(C-H), 1610(C = N),
1
1
541, 1475(Ar), 1176(C = S). H NMR (400 MHz, CDCl ): δ 10.23 (s, 2H, CH = N), 5.05 (s, 4H,
3
NCH N), 3.82 (s, 6H, Pyrazole-CH ), 2.83 (s, 8H, Piperazine-H), 2.46 (s, 6H, Triazole-CH ), 2.37 (s,
2
3
3
1
3
6
1
H, Pyrazole-CH ). C NMR (101 MHz, CDCl ): δ 162.4 (s, 2C, C = S), 154.0 (s, 2C, Triazole-C),
3
3
49.3 (s, 2C, Pyrazole-C), 147.6 (s, 2C, N = CH), 131.4 (s, 2C, Pyrazole-C), 110.7 (s, 2C,
Pyrazole-C), 68.6 (s, 2C, CH ), 50.4 (s, 4C, Piperazine-C), 36.2 (s, 2C, Pyrazole-CH ), 14.8 (s, 2C,
2
3
Triazole-CH ), 11.1 (s, 2C, Pyrazole-CH ); Anal. Calcd. (%) for C H Cl N S : C 44.24, H 4.95, N
3
3
24 32
2 14 2
3
0.09. Found: C 44.31, H 5.04, N 30.02.
(E)-1,1'-(Piperazine-1,4-diylbis(methylene))bis(4-((E)-(5-chloro-1,3-dimethyl-1H-pyrazol-4-yl
)
methyleneamino)-3-(trifluoromethyl)-1H-1,2,4-triazole-5(4H)-thione) (10B)
1
White solid, yield 81.7%, mp 193-195 C (Dec.); IR (νmax, cm ): 2938, 2854(C-H), 1605(C = N),
1
1
528, 1489, 1459(Ar), 1317, 1186(C-F), 1161(C = S). H NMR (400 MHz, CDCl ): δ 10.18 (s, 2H,
3
CH = N), 5.16 (s, 4H, NCH N), 3.83 (s, 6H, Pyrazole-CH ), 2.86 (s, 8H, Piperazine-H), 2.45 (s, 6H,
2
3
1
3
Pyrazole-CH ). C NMR (101 MHz, CDCl ): δ 165.0 (s, 2C, C = S), 155.4 (s, 2C, Pyrazole-C), 149.9
3
3
2
2
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(
s, 2C, N = CH), 138.8 (q, J = 41.4 Hz, 2C, Triazole-C ), 132.2 (s, 2C, Pyrazole-C), 116.9 (q, J =
C-F
3
C-F
2
2
3
72.7 Hz, 2C, CF ), 110.2 (s, 2C, Pyrazole-C), 69.8 (s, 2C, CH ), 50.3 (s, 4C, Piperazine-C), 36.2 (s,
3
2
C, Pyrazole-CH ), 14.7 (s, 2C, Pyrazole-CH ); Anal. Calcd. (%) for C H Cl F N S : C 37.95, H
3
3
24 26 2 6 14 2
.45, N 25.82. Found: C 38.11, H 3.59, N 25.60.
Fungicidal Activity Tests
The in vitro fungicidal activity of the compounds against Fusarium omysporum, Cercospora
arachidicola, Physalospora piricola, Alternaria solani Sorauer and Gibberella sanbinetti were
[
31]
evaluated via the mycelium growth rate test according to the literature . The method for
testing the primary biological activity was performed in an isolated culture.
The in vivo fungicidal activity of the compounds against Puccinia sorghi Schw. were tested
[
32]
according to the reference , and the commercial fungicides Triadimefon and Azoxystrobin
were used as controls.
Herbicidal Activity Tests
The in vivo herbicidal activity of compounds were determined by the inhibition of the
root-growth of rape (Brassica campestris) and inhibition of the seedling-growth of
[
33]
barnyardgrass (Echinochloa crusgalli) tests according to the reported method
.
KARI Inhibitory Activity Test
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[
24]
The cloning of rice KARI has been described previously , and enzyme expression and
purification followed that protocol. KARI activity was measured with a continuous assay
[
24]
method, following the consumption of NADPH at 340 nm and 30 C . Assay solutions
contained 0.2 mmol/L NADPH, 1 mmol/L MgCl , 0.1 mmol/L substrate (2-acetolactate), and
2
inhibitor (synthesized compounds or CPD), in 0.1 mol/L Tris-HCl buffer (pH 8.0). Inhibitor was
preincubated with the enzyme, NADPH and MgCl in Tris-HCl buffer at 30 C for 10 min. The
2
reaction was then started by adding the substrate. The percentage of the inhibition was
calculated.
CONCLUSION
In summary, a series of novel dimethylpyrazole and piperazine -containing (bis)1,2,4-triazole
derivatives have been conveniently synthesized via Mannich reaction in good yields. The
preliminary bioassays showed that some of the compounds showed significant in vitro and in
vivo fungicidal activity against several tested plant fungi and were comparable with the control
Triadimefon. Several compounds also exhibited certain herbicidal activity against Brassica
campestris. Furthermore, several compounds possessed favorable KARI inhibitory activity in the
present study, especially 8D showed superiority than others and could be a promising KARI
inhibitor for further study. The research work in this manuscript will provide useful information
for the design and discovery of new agrochemicals with novel heterocyclic Mannich base
structures containing piperazine moiety.
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ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China (No. 21372133),
Zhejiang Province National Natural Science Foundation of China (No. LY16C140007) and China
Scholarship Council (CSC). We thank Dr. Cong-Wei Niu of Nankai University (China) for kind
biological testing assistance.
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SCHEME 1: Synthesis of the intermediates 1A-B and 3A-B.
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