Synthesis and biological activity of some novel trifluoromethyl-substituted 1,2,4-triazole and bis(1,2,4-Triazole) mannich bases containing piperazine rings

Article abstract of DOI:10.1021/jf100300a

A series of trifluoromethyl-substituted 1,2,4-triazole Mannich base 6 and bis(1,2,4-triazole) Mannich base 7 containing pyrimidinylpiperazine rings via the Mannich reaction were synthesized and characterized by infrared (IR), ¹H nuclear magnetic resonance (NMR), and elemental analysis. The fungicidal tests indicated that most of compounds 6 and 7 possessed excellent fungicidal activity. Among 19 novel compounds, some showed superiority over commercial fungicides Dimethomorph, Thiophanate-methyl, Iprodione, and Zhongshengmycin. Some compounds also exhibited favorable herbicidal activity in the preliminary studies. On the basis of the comparative molecular field analysis (CoMFA), five novel compounds were subsequently synthesized, their activities were estimated fairly accurately, and compounds 6-A1 and 7-A2 displayed good fungicidal activity against Pseudoperonospora cubensis (96.9 and 84.9%) as 6h and 7c, respectively.

Full text of DOI:10.1021/jf100300a

J. Agric. Food Chem. 2010, 58, 5515–5522 5515
DOI:10.1021/jf100300a
Synthesis and Biological Activity of Some
Novel Trifluoromethyl-Substituted 1,2,4-Triazole and
Bis(1,2,4-Triazole) Mannich Bases Containing
Piperazine Rings
‡
B
AO-LEI
W
ANG
,
†,‡ YAN-XIA
S
HI
,
†,§ Y
I
M
A
,‡ XING-HAI
,*,§ AND
L
IU,‡ YONG-HONG
,‡
LI,
HAI-BIN
S
ONG,‡ BAO-J
U
L
I
Z
HENG-MING
LI*
‡State Key Laboratory of Elemento-organic Chemistry, Tianjin Key Laboratory of Pesticide Science,
Elemento-Organic Chemistry Institute, Nankai University, Tianjin 300071, China, and
§Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
† These authors contributed equally to this work.
A series of trifluoromethyl-substituted 1,2,4-triazole Mannich base 6 and bis(1,2,4-triazole) Mannich
base 7 containing pyrimidinylpiperazine rings via the Mannich reaction were synthesized and
characterized by infrared (IR), 1H nuclear magnetic resonance (NMR), and elemental analysis.
The fungicidal tests indicated that most of compounds 6 and 7 possessed excellent fungicidal
activity. Among 19 novel compounds, some showed superiority over commercial fungicides
Dimethomorph, Thiophanate-methyl, Iprodione, and Zhongshengmycin. Some compounds also
exhibited favorable herbicidal activity in the preliminary studies. On the basis of the comparative
molecular field analysis (CoMFA), five novel compounds were subsequently synthesized, their
activities were estimated fairly accurately, and compounds 6-A1 and 7-A2 displayed good fungicidal
activity against Pseudoperonospora cubensis (96.9 and 84.9%) as 6h and 7c, respectively.
KEYWORDS: 1,2,4-Triazole; pyrimidinylpiperazine; Mannich base; fungicidal activity; herbicidal activity
INTRODUCTION
used successfully in the control of several woody species during
the mid-1950s (12). Also, in the early 1990s, 3-trifluoromethyl-
4-aryl-1,2,4-triazole-5(4H)-thiones were reported to possess good
herbicidal activity (13). Furthermore, bioactive compounds pos-
sess a trifluoromethyl moiety that might be expected to enhance
their biological activity (14-17), which is often considered an
important point by researchers for designing various biological
molecules. All of these results encouraged us to synthesize some
other novel compounds containing such structural moiety.
In our previous work, we reported some interesting Mannich
bases derived from 1,2,4-triazole Schiff base (18) and benzotria-
zoles containing a pyrimidinylpiperzine ring, which are associated
with various useful biological activities (19). As a continuation of
our work, a series of novel Mannich base and bis-Mannich base
with trifluoromethyl-1,2,4-triazole and pyrimidinylpiperazine
moiety were synthesized and their fungicidal and herbicidal
activities were investigated in this paper.
The application of agrochemicals to protect vegetable and
cereal crops is an established part of conventional agriculture.
This has provided healthy crops and increased yields as well as
economic benefits for over many years. The main purpose of the
research for agrochemicals is to develop novel active compounds
with lower application doses and high selectivity and that are
environmentally friendly (1, 2). Compounds containing a 1,2,4-
triazole ring are often associated with various useful pesticidal
activity (3-6). In the early 1970s, the azole fungicides were
synthesized and used for the protection of various crops. Since
the discovery of these fungicides, a variety of other 1,2,4-triazole
compounds have been discovered (7-9). The introduction of
these 1,2,4-triazole sterol biosynthesis inhibitors represented
significant progress in the chemical control of fungal diseases.
This class of pesticides includes several excellent systemic fungi-
cides with long, protective, and curative activity against a broad
spectrum of foliar, root, and seedling diseases caused by many
ascomycetes, basidiomycetes, and imperfect fungi (10,11). On the
other hand, some structures with a 1,2,4-triazole ring also
exhibited outstanding herbicidal activity. For example, 3-amino-
1,2,4-triazole was introduced as a selective herbicide and has been
MATERIALS AND METHODS
Instruments and Materials. The melting points were determined on a
X-4 binocular microscope melting point apparatus (Beijing Tech Instru-
ment Co., Beijing, China) and were uncorrected. Infrared spectra were
recorded on a Nicolet MAGNA-560 spectrophotometer as KBr tablets.
1H nuclear magnetic resonance (NMR) spectra were measured on a
Bruker AC-P500 instrument (400 MHz) using tetramethylsilane (TMS)
as the internal standard and dimethylsulfoxide (DMSO)-d6 or CDCl3 as
the solvent. Elemental analyses were performed on a Vario EL elemental
*To whom correspondence should be addressed. Telephone: þ86-
10-62197975. E-mail: libj@mail.caas.net.cn (B.-J.L.); Telephone:
þ86-22-23503732. E-mail: nkzml@vip.163.com (Z.-M.L.).
© 2010 American Chemical Society
Published on Web 04/12/2010
pubs.acs.org/JAFC

5516 J. Agric. Food Chem., Vol. 58, No. 9, 2010
Wang et al.
Scheme 1
(s, 1H, Ph-H), 7.62 (d, J=8.0 Hz, 1H, Ph-H), 7.35 (d, J=8.0 Hz, 1H,
Scheme 2
Ph-H), 2.31 (s, 6H, CH3).
1
5e (R3=4-Cl): white crystals, yield 72%, mp 208-209 °C. H NMR
(DMSO-d6, 400 MHz) δ: 14.92 (s, 1H, SH), 10.05 (s, 1H, CH), 7.93 (d, J=
8.4 Hz, 2H, Ph-H), 7.67 (d, J=8.4 Hz, 2H, Ph-H).
5f (R3=2-NO2): yellow crystals, yield 89%, mp 201-202 °C. 1H NMR
(DMSO-d6, 400 MHz) δ: 14.96 (s, 1H, SH), 10.88 (s, 1H, CH), 7.87-8.23
(m, 4H, Ph-H).
General Synthetic Procedures for 1-[4-(4,6-Disubstituted-pyrimidin-
2-yl)piperazin-1-yl)methyl]-4-(substituted)benzylideneamino-3-trifl-
uoromethyl-1H-1,2,4-triazole-5(4H)-thione 6. Schiff base 5 (1 mmmol)
and 40% formalin (1.2 mmol) were dissolved in ethanol (15 mL), and the
mixture was stirred at room temperature for 10 min. A solution of
pyrimidylpiperazine 2 (1 mmol) in ethanol (2 mL) was slowly added
dropwise. Then, the reaction mixture was stirred for 2-3 h and placed in a
refrigerator overnight. The resulting precipitate was filtered and recrys-
tallized from ethanol to give Mannich base 6.
General Synthetic Procedures for 1,10-[Piperazin-1,4-diylbis-
(methylene)]bis[4-(substituted)benzylideneamino-3-trifluoromethyl-
1H-1,2,4-triazole-5(4H)-thione] 7. Schiff base 5 (1.8 mmmol) and 40%
formalin (2 mmol) were dissolved in ethanol (30 mL), and the mixture was
stirred at room temperature for 10 min. A solution of piperazine (0.9 mmol)
in ethanol (2 mL) was slowly added dropwise. Then, the reaction mixture
was stirred for 2-3 h and placed in a refrigerator overnight. The result-
ing precipitate was filtered and recrystallized from ethanol to give bis-
Mannich base 7.
Fungicidal Activity Tests. Fungicidal activity of compounds 6 and 7
against Corynespora cassiicola, Pseudomonas syringae pv. lachrymans,
Ascochyta citrallina Smith, Pseudoperonospora cubensis, and Sclerotinia
sclerotiorum were evaluated according to ref 24, and a potted plant test
method was adopted. Four commericial fungicides, Dimethomorph,
Thiophanate-methyl, Iprodione, and Zhongshengmycin, were evaluated
as controls at the same condition. 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. After 2 h, inoculations of
C. cassiicola, A. citrullina Smith, and P. cubensis were carried out by
spraying a conidial suspension, inoculation of P. syringae pv. lachrymans
was carried out by spraying a suspension, and inoculation of S. sclerotiorum
was carried out by spraying a mycelial 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 (24). The relative
analyzer. Crystallographic data of the compound were collected on a
Rigaku MM-07 Saturn 724 charge coupled device (CCD) diffractometer.
All of the solvents and materials were analytical-grade.
Synthetic Procedures. 4-Amino-5-trifluoromethyl-4H-1,2,4-triazole-
3-thiol 4 was prepared according to the literature (20).
General Synthetic Procedures for 4-(4,6-Disubstituted-pyrimidin-
2-yl)piperazine 2. 2-Chloro-4,6-disubstituted-pyrimidines 1 were pre-
pared by the reaction of the diazonium salts of 4,6-disubstituted-pyrimidin-
2-amines with concentrated HCl and ZnCl2 (21). Compound 2 was
prepared according to ref 22, and the method was improved. To a stirred
solution of piperazine (45 mmol) and K2CO3 (16.5 mmol) in water (20 mL)
was added chloropyrimidine 1 (18 mmol) in small portions at 50-65 °C.
The mixture was stirred for 1 h at 60-65 °C and cooled to 35 °C. The
yellow solid, 1,4-bis(4,6-disubstituted-pyrimidin-2-yl)piperazine 3, was
filtered off, and the filtrate was then extracted 3 times with chloroform,
dried over Na2SO4, and evaporated in vacuum to give compound 2, which
was used for the following reactions without further purification.
2a (R1=R2=H): yellow oil, yield 88%. 2b (R1=H; R2=Me): yellow
solid, yield 81%, mp 45-48 °C. 2c (R1=R2=Me): yellow solid, yield 79%,
mp 82-84 °C.
3a (R1=R2=H): yellow solid, yield 4%, mp 273-274 °C [literature mp
275-278 °C (23)]. 3b (R1=H, R2=Me): yellow crystal, yield 14%, mp
180-182 °C. 3c (R1=R2=Me): yellow crystal, yield 19%, mp 216-218 °C.
General Synthetic Procedures for 4-(Substituted)benzylideneamino-
5-trifluoromethyl-4H-1,2,4-triazole-3-thiol 5. Compound 4 (10 mmol)
and aromatic aldehyde (10.5 mmol) were mixed in acetic acid (15 mL).
After the reaction mixture was stirred and refluxed for 15 min, it was
cooled to room temperature. The resulting crystals were filtered and
washed with ethanol to give Schiff base 5.
5a (R3=H): white crystal, yield 74%, mp 198-199 °C [literature mp
200-201 °C (18)]. 1H NMR (DMSO-d6, 400 MHz) δ: 14.90 (s, 1H, SH),
9.97 (s, 1H, CH), 7.58-7.92 (m, 5H, Ph-H).
5b (R3=2-F): white crystals, yield 82%, mp 192-193 °C. 1H NMR
(DMSO-d6, 400 MHz) δ: 14.93 (s, 1H, SH), 10.44 (s, 1H, CH), 7.41-8.03
(m, 4H, Ph-H).
5c (R3=4-MeO): white crystals, yield 71%, mp 195-196 °C. 1H NMR
(DMSO-d6, 400 MHz) δ: 14.84 (s, 1H, SH), 9.73 (s, 1H, CH), 7.86 (d, J=
8.8 Hz, 2H, Ph-H), 7.13 (d, J=8.8 Hz, 2H, Ph-H), 3.86 (s, 3H, OCH3).
5d (R3=3,4-Me2): white crystals, yield 73%, mp 205-206 °C. 1H NMR
(DMSO-d6, 400 MHz) δ: 14.86 (s, 1H, SH), 9.79 (s, 1H, CH), 7.66

Article
J. Agric. Food Chem., Vol. 58, No. 9, 2010 5517
elemental analysis (calculated %)
Table 1. Analytical Data for Compounds 6 and 7
compound
R1
R2
R3
yield (%)
mp (°C)
appearance
formula
C
H
N
6a
6b
H
H
H
2-F
48
65
64
44
42
72
58
48
58
54
46
52
47
63
48
60
63
75
80
52
72
63
88
72
156-158
150-152
154-156
138-140
113-115
163-164
158-159
160-162
88-89
154-156
153-154
160-161
162-164
171-173
143-145
198-199a
201-202a
200-201a
204-206a
166-168
191-192
153-155
214-215a
196-197a
white crystal
white crystal
white crystal
white crystal
white crystal
white crystal
white crystal
white crystal
yellow crystal
white crystal
white crystal
white crystal
white crystal
white crystal
yellow crystal
white crystal
white crystal
white crystal
white crystal
white crystal
white crystal
yellow crystal
white crystal
yellow crystal
C19H19F3N8S
C19H18F4N8S
50.68 (50.89)
48.64 (48.92)
49.96 (50.20)
51.67 (51.94)
49.96 (49.99)
50.97 (51.21)
53.87 (53.87)
48.05 (48.34)
47.08 (47.33)
52.91 (52.93)
50.26 (51.00)
52.12 (52.16)
54.33 (54.75)
48.95 (49.36)
48.43 (48.36)
47.64 (47.70)
45.14 (45.22)
47.13 (47.05)
50.42 (50.69)
52.90 (52.93)
46.92 (47.26)
45.98 (46.25)
42.91 (43.16)
41.92 (41.94)
4.53 (4.27)
4.21 (3.89)
4.65 (4.42)
4.94 (4.58)
4.49 (4.20)
4.98 (4.71)
5.28 (5.14)
4.29 (4.06)
3.81 (3.97)
4.65 (4.86)
4.31 (4.48)
5.02 (4.97)
5.46 (5.39)
4.72 (4.34)
4.32 (4.25)
4.01 (3.70)
3.50 (3.21)
4.08 (3.95)
4.38 (4.54)
4.78 (4.86)
3.84 (3.76)
3.74 (3.68)
3.30 (3.06)
3.20 (2.98)
25.10 (24.99)
23.88 (24.02)
23.27 (23.42)
24.38 (24.23)
23.56 (23.32)
23.02 (22.75)
23.24 (22.84)
22.69 (22.55)
24.53 (24.84)
23.54 (23.51)
22.71 (22.66)
22.23 (22.12)
21.81 (22.21)
21.61 (21.93)
23.87 (24.17)
20.96 (21.40)
20.16 (20.28)
19.45 (19.60)
19.38 (19.71)
23.67 (23.51)
22.88 (23.20)
25.26 (25.55)
18.80 (19.36)
22.23 (22.57)
H
H
6c
6d
H
H
4-MeO
H
2-F
C20H21F3N8OS
C20H21F3N8S
C20H20F4N8S
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
6e
H
6f
6g
H
4-MeO
3,4-Me2
4-Cl
C21H23F3N8OS
C22H25F3N8S
H
6h
6i
H
C20H20ClF3N8S
C20H20F3N9O2S
C21H23F3N8S
H
2-NO2
H
2-F
6j
Me
Me
Me
Me
Me
Me
6k
C21H22F4N8S
C22H25F3N8OS
C23H27F3N8S
6l
4-MeO
3,4-Me2
4-Cl
6m
6n
C21H22ClF3N8S
C21H22F3N9O2S
C26H24F6N10S2
C26H22F8N10S2
C28H28F6N10O2S2
C30H32F6N10S2
C21H23F3N8S
6o
2-NO2
H
2-F
7a
7b
7c
7d
4-MeO
3,4-Me2
3,4-Me2
4-Cl
6-A1
6-A2
6-A3
7-A1
7-A2
H
H
H
H
H
H
C19H18ClF3N8S
C19H18F3N9O2S
C26H22Cl2F6N10S2
C26H22F6N12O4S2
2-NO2
4-Cl
2-NO2
a Decomposed.
3
3
˚
control efficacy of compounds compared to the blank assay was calculated
via the following equation:
V=2578.2(9) A , Z=4, Dc=1.367 mg/m , μ=0.186 mm-1, and F(000)=
1100. The final refinement converged at R = 0.0603 and wR = 0.1494
for 3552 observed reflections with I > 2σ(I), where W= 1/[σ2(Fo2) þ
(0.0980P)2 þ 0.0000P] with P=(Fo þ 2Fc )/3, S=1.073, (Δ/σ)max
<
2
2
relative control efficacy ð%Þ ¼ ½ðCK - PTÞ=CKꢀ ꢁ 100%
3
3
˚
˚
0.0001, (ΔF)max=0.434 e/A , and (ΔF)min=-0.265 e/A .
where CK is the average disease index during the blank assay and PT is the
average disease index after treatment during testing.
Three-Dimensional Quantitative Structure-Activity Relationship
(3D QSAR) Analysis. Molecular modeling was performed using SYBYL
6.91 software, and the comparative molecular field analysis (CoMFA)
method has been performed according to our previous papers (25,26). The
fungicidal activities of 19 compounds (6a-6o and 7a-7d, for training sets
compounds) against P. cubensis (% I ) at 500 μg/mL used to derive the
CoMFA analysis model were listed in Table 7. The activity was
expressed in terms of D by the formula D=log{[I/(100 - I)] ꢁ MW},
where I is the percent control efficacy and MW is the molecular weight of
the tested compounds. The compound 6i, owing to the determination of
the crystal structure, was used as a template to build the other molecular
structures. Each structure was fully geometry-optimized using a con-
jugate gradient procedure based on the TRIPOS force field and
Herbicidal Activity Tests. Inhibition of the Root Growth of Rape
(Brassica campestris). The evaluated compounds were dissolved in water
and emulsified if necessary. Rape seeds were soaked in distilled water for
4 h before being placed on a filter paper in a 6 cm Petri plate, to which 2 mL
of inhibitor solution had been added in advance. Usually, 15 seeds were
used on each plate. The plate was placed in a dark room and allowed to
germinate for 65 h at 28 ( 1 °C. The lengths of 10 rape roots selected from
each plate were measured, and the means were calculated. The check test
was carried out in distilled water only. The percentage of the inhibition was
calculated.
Inhibition of the Seedling Growth of Barnyardgrass (Echinochloa
crusgalli). The evaluated compounds were dissolved in water and emulsi-
fied if necessary. A total of 10 barnyardgrass seedswereplaced into a 50 mL
cup covered with a layer of glass beads and a piece of filter paper at the
bottom, to which 5 mL of inhibitor solution had been added in advance.
The cup was placed in a bright room and allowed to germinate for 65 h at
28 (1°C. The heights of seedlings of above-ground plant parts from each cup
were measured, and the means were calculated. The check test was carried
out in distilled water only. The percentage of the inhibition was calculated.
Crystal Structure Determination. Compound 6i was dissolved in hot
alcohol, and the resulting solution was allowed to stand in air at room
temperature to give a single crystal of compound 6i. A yellow crystal
of compound 6i suitable for X-ray diffraction with dimensions of 0.16 ꢁ
0.14 ꢁ 0.10 mm was mounted on a Rigaku MM-07 Saturn 724 CCD
€
Gasteiger and Huckel charges. Because these compounds share a
common skeleton, 19 atoms marked with an asterisk were used for
root-mean-square (rms) fitting onto the corresponding atoms of the
template structure. CoMFA steric and electrostatic interaction fields
were calculated at each lattice intersection on a regularly spaced grid of
˚
2.0 A. The grid pattern was generated automatically by the SYBYL/
CoMFA routine, and an sp3 carbon atom with a van der Waals radius of
˚
1.52 A and a þ1.0 charge was used as the probe to calculate the steric
(Lennard-Jones 6-12 potential) field energies and electrostatic
(Coulombic potential) fields with a distance-dependent dielectric at each
lattice point. Values of the steric and electrostatic fields were truncated at
30.0 kcal/mol. The CoMFA steric and electrostatic fields generated were
scaled by the CoMFA-STD method in SYBYL. The electrostatic fields
were ignored at the lattice points with maximal steric interactions. A
partial least-squares approach was used to derive the 3D QSAR, in
which the CoMFA descriptors were used as independent variables and
ED values were used as dependent variables. The cross-validation
with the leave-one-out option and the SAMPLS program (27) rather
than column filtering was carried out to obtain the optimal number
of components to be used in the final analysis. After the optimal
number of components was determined, a non-cross-validated analysis
was performed without column filtering. The modeling capability
˚
diffractometer with Mo KR radiation (λ=0.710 73 A) for data collection.
A total of 17003 reflections werecollected inthe range of 2.01 < θ < 25.02
using phi and scan modes at 113(2) K, of which 4363 were indepen-
dent with Rint=0.0614. All calculations were refined anisotropically
(SHELXS-97). All hydrogen atoms were located from a difference Fourier
map, placed at calculated positions, and included in the refinements in the
riding mode with isotropic thermal parameters. The compound crystallizes
in space group Pca21 of the orthorhombic system with cell parameters:
˚
˚
˚
a=20.312(4) A, b=14.395(3) A, c=8.8173(18) A, R=90°, β=90°, γ=90°,

5518 J. Agric. Food Chem., Vol. 58, No. 9, 2010
Wang et al.
Table 2. 1H NMR Spectral Data of Compounds 6 and 7
compound
δ (CDCl3, 400 MHz)
6a
6b
6c
10.34 (s, 1H, CH), 6.46-8.29 (m, 8H, Ar-H), 5.28 (s, 2H, CH2), 3.87 (t, J = 4.8 Hz, 4H, piperazine-H), 2.90 (t, J = 4.8 Hz, 4H, piperazine-H)
10.70 (s, 1H, CH), 6.45-8.29 (m, 7H, Ar-H), 5.28 (s, 2H, CH2), 3.86 (t, J = 4.8 Hz, 4H, piperazine-H), 2.90 (t, J = 4.8 Hz, 4H, piperazine-H)
10.07 (s, 1H, CH), 8.28 (d, J = 4.8 Hz, 2H, pyrimidine-H), 7.82 (d, J = 8.8 Hz, 2H, Ph-H), 6.98 (d, J = 8.8 Hz, 2H, Ph-H), 6.47 (t, J = 4.8 Hz, 1H, pyrimidine-H), 5.28
(s, 2H, CH2), 3.88 (s, 3H, OCH3), 3.86 (t, J = 4.8 Hz, 4H, piperazine-H), 2.90 (t, J = 4.8 Hz, 4H, piperazine-H)
10.35 (s, 1H, CH), 8.14 (d, J = 4.4 Hz, 1H, pyrimidine-H), 7.47-7.88 (m, 5H, Ph-H), 6.35 (d, J = 4.4 Hz, 1H, pyrimidine-H), 5.29 (s, 2H, CH2), 3.87 (t, J = 4.0 Hz, 4H,
piperazine-H), 2.89 (t, J = 4.0 Hz, 4H, piperazine-H), 2.31 (s, 3H, CH3)
6d
6e
6f
10.70 (s, 1H, CH), 8.13 (d, J = 4.4 Hz, 1H, pyrimidine-H), 7.15-8.09 (m, 4H, Ph-H), 6.35 (d, J = 4.4 Hz, 1H, pyrimidine-H), 5.28 (s, 2H, CH2), 3.88 (bs, 4H, piperazine-
H), 2.89 (bs, 4H, piperazine-H), 2.31 (s, 3H, CH3)
10.08 (s, 1H, CH), 8.13 (d, J = 4.8 Hz, 1H, pyrimidine-H), 7.82 (d, J = 8.0 Hz, 2H, Ph-H), 6.99 (d, J = 8.0 Hz, 2H, Ph-H), 6.35 (d, J = 4.8 Hz, 1H, pyrimidine-H), 5.28
(s, 2H, CH2), 3.88 (bs, 7H, CH3O þ piperazine-H), 2.89 (bs, 4H, piperazine-H), 2.31 (s, 3H, CH3)
6g
10.11 (s, 1H, CH), 8.13 (d, J = 4.8 Hz, 1H, pyrimidine-H), 7.63 (s, 1H, Ph-H), 7.60 (d, J = 8.0 Hz, 1H, Ph-H), 7.24 (d, J = 8.0 Hz, 1H, Ph-H), 6.35 (d, J = 4.8 Hz,
1H, pyrimidine-H), 5.28 (s, 2H, CH2), 3.87 (bs, 4H, piperazine-H), 2.89 (bs, 4H, piperazine-H), 2.33 (s, 3H, Ph-CH3), 2.32 (s, 3H, Ph-CH3), 2.31 (s, 3H,
pyrimidine-CH3)
6h
6i
10.44 (s, 1H, CH), 8.13 (d, J = 4.8 Hz, 1H, pyrimidine-H), 7.80 (d, J = 8.0 Hz, 2H, Ph-H), 7.47 (d, J = 8.0 Hz, 2H, Ph-H), 6.35 (d, J = 4.8 Hz, 1H, pyrimidine-H), 5.28
(s, 2H, CH2), 3.87 (bs, 4H, piperazine-H), 2.89 (bs, 4H, piperazine-H), 2.31 (s, 3H, CH3)
11.21 (s, 1H, CH), 7.70-8.18 (m, 5H, Ph-H þ pyrimidine-H), 6.36 (d, J = 4.8 Hz, 1H, pyrimidine-H), 5.28 (s, 2H, CH2), 3.88 (bs, 4H, piperazine-H), 2.90 (bs, 4H,
piperazine-H), 2.31 (s, 3H, CH3)
6j
10.35 (s, 1H, CH), 7.47-7.88 (m, 5H, Ph-H), 6.25 (s, 1H, pyrimidine-H), 5.29 (s, 2H, CH2), 3.89 (t, J = 4.4 Hz, 4H, piperazine-H), 2.89 (t, J = 4.4 Hz, 4H, piperazine-H),
2.26 (s, 6H, CH3)
6k
6l
10.70 (s, 1H, CH), 7.15-8.09 (m, 4H, Ph-H), 6.25 (s, 1H, pyrimidine-H), 5.29 (s, 2H, CH2), 3.88 (t, J = 4.0 Hz, 4H, piperazine-H), 2.89 (t, J = 4.0 Hz, 4H, piperazine-H),
2.26 (s, 6H, CH3)
10.08 (s, 1H, CH), 7.83 (d, J = 8.0 Hz, 2H, Ph-H), 6.99 (d, J = 8.0 Hz, 2H, Ph-H), 6.25 (s, 1H, pyrimidine-H), 5.29 (s, 2H, CH2), 3.89 (bs, 7H, CH3O þ piperazine-H),
2.88 (bs, 4H, piperazine-H), 2.26 (s, 6H, CH3)
6m
6n
10.11 (s, 1H, CH), 7.63 (s, 1H, Ph-H), 7.60 (d, J = 8.0 Hz, 1H, Ph-H), 7.25 (d, J = 8.0 Hz, 1H, Ph-H), 6.25 (s, 1H, pyrimidine-H), 5.28 (s, 2H, CH2), 3.88 (t, J = 4.4 Hz, 4H,
piperazine-H), 2.88 (t, J = 4.4 Hz, 4H, piperazine-H), 2.34 (s, 3H, Ph-CH3), 2.33 (s, 3H, Ph-CH3), 2.26 (s, 6H, pyrimidine-CH3)
10.44 (s, 1H, CH), 7.80 (d, J = 8.0 Hz, 2H, Ph-H), 7.47 (d, J = 8.0 Hz, 2H, Ph-H), 6.25 (s, 1H, pyrimidine-H), 5.28 (s, 2H, CH2), 3.88 (bs, 4H, piperazine-H), 2.88 (bs, 4H,
piperazine-H), 2.26 (s, 6H, CH3)
6o
7a
7b
7c
7d
11.21 (s, 1H, CH), 7.70-8.18 (m, 4H, Ph-H), 6.25 (s, 1H, pyrimidine-H), 5.29 (s, 2H, CH2), 3.89 (bs, 4H, piperazine-H), 2.89 (bs, 4H, piperazine-H), 2.26 (s, 6H, CH3)
10.35 (s, 2H, CH), 7.48-7.59 (m, 10H, Ph-H), 5.19 (s, 4H, CH2), 2.88 (s, 8H, piperazine-H)
10.71 (s, 2H, CH), 7.15-8.10 (m, 8H, Ph-H), 5.18 (s, 4H, CH2), 2.88 (s, 8H, piperazine-H)
10.08 (s, 2H, CH), 7.83 (d, J = 8.8 Hz, 4H, Ph-H), 6.99 (d, J = 8.8 Hz, 4H, Ph-H), 5.18 (s, 4H, CH2), 3.89 (s, 6H, OCH3), 2.87 (s, 8H, piperazine-H)
10.12 (s, 2H, CH), 7.24-7.64 (m, 6H, Ph-H), 5.18 (s, 4H, CH2), 2.88 (s, 8H, piperazine-H), 2.34 (s, 6H, CH3), 2.33 (s, 6H, CH3)
6-A1 10.11 (s, 1H, CH), 8.28 (d, J = 4.8 Hz, 2H, pyrimidine-H), 7.63 (s, 1H, Ph-H), 7.60 (d, J = 8.0 Hz, 1H, Ph-H), 7.24 (d, J = 8.0 Hz, 1H, Ph-H), 6.47 (t, J = 4.8 Hz, 1H,
pyrimidine-H), 5.28 (s, 2H, CH2), 3.87 (t, J = 4.8 Hz, 4H, piperazine-H), 2.90 (t, J = 4.8 Hz, 4H, piperazine-H), 2.33 (s, 3H, CH3), 2.32 (s, 3H, CH3)
6-A2 10.44 (s, 1H, CH), 8.28 (d, J = 4.8 Hz, 2H, pyrimidine-H), 7.80 (d, J = 8.8 Hz, 2H, Ph-H), 7.46 (d, J = 8.8 Hz, 2H, Ph-H), 6.47 (t, J = 4.8 Hz, 1H, pyrimidine-H), 5.28
(s, 2H, CH2), 3.87 (t, J = 4.4 Hz, 4H, piperazine-H), 2.90 (t, J = 4.4 Hz, 4H, piperazine-H)
6-A3 11.22 (s, 1H, CH), 8.29 (d, J = 4.8 Hz, 2H, pyrimidine-H), 7.70-8.19 (m, 4H, Ph-H), 6.47 (t, J = 4.8 Hz, 1H, pyrimidine-H), 5.28 (s, 2H, CH2), 3.87 (t, J = 4.4 Hz, 4H,
piperazine-H), 2.91 (t, J = 4.4 Hz, 4H, piperazine-H)
7-A1 10.45 (s, 2H, CH), 7.81 (d, J = 8.4 Hz, 4H, Ph-H), 7.47 (d, J = 8.4 Hz, 4H, Ph-H), 5.17 (s, 4H, CH2), 2.87 (s, 8H, piperazine-H)
7-A2 11.21 (s, 2H, CH), 7.71-8.19 (m, 8H, Ph-H), 5.18 (s, 4H, CH2), 2.89 (s, 8H, piperazine-H)
(goodness of fit) was judged by the correlation coefficient squared, r2,
and the prediction capability (goodness of prediction) was indicated by
the cross-validated r2 (q2).
Table 3. IR Spectral Data of Compounds 6 and 7
ν (cm-1) (KBr)
compound
6a
2860, 2825 (C-H), 1587 (CdN), 1550, 1506, 1483, 1465 (Ar),
1310, 1205 (C-F), 1161 (CdS)
RESULTS AND DISCUSSION
6d
6j
2980, 2830 (C-H), 1578 (CdN), 1566, 1489, 1467, 1449 (Ar),
1316, 1194 (C-F), 1161 (CdS)
2941, 2853 (C-H), 1577 (CdN), 1504, 1467, 1449 (Ar), 1311,
1194 (C-F), 1156 (CdS)
2935, 2822 (C-H), 1599 (CdN), 1583, 1463, 1452 (Ar), 1317,
1193 (C-F), 1156 (CdS)
Synthesis. The synthesis procedures for compounds 6 were
shown in Scheme 1, and the synthesis procedures for compounds
7 were shown in Scheme 2. According to the method described in
ref22 for the synthesis of required 4-(4,6-disubstituted-pyrimidin-
2-yl)piperazine 2, a small amount of 1,4-bis(4,6-disubstituted-
pyrimidin-2-yl) piperazine 3 was also obtained, which was not
mentioned in the literature. Compound 3 was further confirmed
by 1H NMR spectra. In reference to an acetic acid solvent method
reported by Wu et al. for the condensation of amine and
aldehyde (28), Schiff base 5, namely, 4-amino-5-trifluoromethyl-
4H-1,2,4-trizole-3-thiol, was prepared successfully. The Mannich
reaction of compound 5 with formaldehyde and pyrimidyl-
piperazine 2 in ethanol at room temperature led to novel tri-
fluoromethyl-substituted 1,2,4-triazole Mannich base 6. In a 2:1
molar ratio of Schiff base 5 and piperazine, novel trifluoromethyl-
substituted bis(1,2,4-triazole) Mannich base 7 was prepared
conveniently in 60-88% yield using the same procedures for
compound 6. Compounds 6 and 7 were identified by 1H NMR
and infrared (IR) spectra (Tables 2 and 3). The measured
7a
elemental analyses were also consistent with the corresponding
calculated ones (Table 1).
Crystal Structure. The structure of compound 6i was further
confirmed by single-crystal X-ray diffraction analysis (Figure 1).
From the molecular structure, it can be seen that both groups on
the N atoms of the piperazine ring (triazole-CH2 and pyrimidine)
are in the e-bond positions of chair conformation in the six-
membered ring. The dihedral angle between the 2-nitrobenzene
ring and the triazole ring is 32°, which indicates that the two rings
are not coplanar in the molecular structure. The X-ray analysis
also reveals that, in this typical compound 6i, the substi-
tuted benzene ring and the triazole ring are on the opposite
sides of the CdN double bond (Figure 1). The torsion angle of

Article
J. Agric. Food Chem., Vol. 58, No. 9, 2010 5519
Table 5. Herbicidal Activity of Compounds (Percent Inhibition)
rape (B. campestris) root test barnyardgrass (E. crusgalli) cup test
compound
100 μg/mL
10 μg/mL
100 μg/mL
10 μg/mL
6a
6b
66.8
74.5
78.8
12.5
24.4
30.3
11.2
68.6
0
49.2
70.8
53.5
0
0
0
15.3
18.6
15.3
8.0
9.9
0
6c
6d
8.7
0
6e
0
6f
6g
0
34.3
27.8
96.7
33.9
9.8
15.3
0
0
6h
6i
0
10.2
11.5
0
0
6j
71.1
72.4
67.7
20.5
88.5
11.2
35.1
4.3
36.9
67.1
30.6
6.0
9.5
0
6k
33.2
29.5
8.7
17.1
0
6l
6m
0
6n
6o
36.6
10.2
25.1
26.6
25.5
28
12.3
0
Figure 1. Molecular structure of compound 6i.
7a
7b
0
16.2
10.7
5.5
1.5
8.2
0
7c
7d
29.5
27.4
80.4
0
Table 4. Fungicidal Activity of Compounds (Percent Relative Control
Efficacy)
0
76.0
Chlorsulfuron
29.9
C.
cassiicola
P. syringae pv A. citrullina
Smith
P.
cubensis sclerotiorum
S.
compound
lachrymans
C. cassiicola. When R1 = R2 = Me and a bulky group at the
4 position of the benzene ring in Mannich base 6 (e.g., compound
6n, R3=Cl), there is an apparent increase of fungicidal activity,
while the introduction of the electron-withdrawing group in the
benzene ring of bis-Mannich base 7 is favorable to the improvement
of activity. (b) Compounds 6c, 6e, and 6n possessed efficacy rates
68.1, 62.3, and 61.5% against P. syringae pv. lachrymans, respec-
tively. All of the three compounds were more effective than
Dimethomorph, Thiophanate-methyl, and Iprodione. Among
them, compound 6c had almost the same activity as that of
Zhongshengmycin against P. syringae pv. lachrymans. For this
fungi, when R1=H and R2=Me (Mannich base 6), compounds
with an electron-donating group in the benzene ring will affect
lower fungicidal activities than those of others. However, the small
and electron-donating group in the benzene ring of bis-Mannich
base 7 is favorable. (c) Compounds 6l, 6o, 7b, and 7c held
86.5-89.7% efficacy rates against A. citrallina Smith, while all of
them were less effective than all of the contrasts. It was found
that compounds with two methyl groupds in the pyrimidine ring
(R1 = R2 = Me) have a higher level of fungicidal activity than
others (R1=R2=H and R1=H and R2=Me), which indicates
that the introduction of hydrophobic groups at 4 and
6 positions of the pyrimidine ring could be an activation process
for this group of compounds. (d) All of the compounds exhibited
good control efficacy against P. cubensis (exceeding 64% efficacy
rate), and most of them had higher activity than Thiophanate-
methyl, Iprodione, and Zhongshengmycin. It was worthy to note
that compounds 6h, 6l, 6n, and 7c, whose efficacy rates were 91.2,
87.3, 91.6, and 84.5%, respectively, were found to be more effective
compared to the most active fungicide Dimethomorph (83.8%)
against P. cubensis. To further explore the comprehensive struc-
ture-activity relationship on the basis of these data, CoMFA was
subsequently performed. (e) For S. sclerotiorum, compounds 6c, 6h,
and 6k exhibited favorable fungicidal activity and held 75.3, 70.3,
and 72.0% efficacy rates, respectively. However, all of them were
less effective than all of the contrasts.
6a
6b
6c
6d
6e
6f
56.2
40.3
73.4
49.2
-5.3
52.8
38.7
71.1
73.8
22.1
78.7
83.7
85.9
94.4
87.8
70.9
84.7
44.1
67.4
95.3
78.8
55.8
45.4
68.1
20.4
62.3
30.6
22.0
21.1
44.5
55.0
56.1
50.3
59.5
61.5
46.5
56.4
6.8
37.1
61.3
73.4
62.4
57.8
67.7
70.0
61.3
44.3
61.5
83.5
87.6
82.9
62.8
88.4
25.6
86.5
89.7
26.0
98.4
100
66.3
64.9
79.5
78.6
75.5
72.5
69.5
91.2
76.8
74.1
79.2
87.3
67.8
91.6
77.2
78.9
79.9
84.5
78.7
83.8
68.7
41.7
47.3
75.3
36.8
52.2
50.9
51.9
70.3
32.8
48.0
72.0
62.6
48.9
38.0
39.3
67.6
25.9
62.0
32.7
98.4
83.4
6g
6h
6i
6j
6k
6l
6m
6n
6o
7a
7b
7c
7d
55.6
36.7
14.0
54.2
Dimethomorph
Thiophanate-
methyl
Iprodione
Zhongshengmycin
53.5
90.2
38.5
69.7
98.4
100
55.9
62.6
97.8
84.4
C(6)-C(7)-N(2)-N(3) is 176.52°, which indicates that the CdN
double bond is in the E configuration.
Fungicidal Activity. The in vivo fungicidal results of the Man-
nich base 6a-6o and bis-Mannich base 7a-7d against C. cassiicola,
P. syringae pv. lachrymans, A. citrallina Smith, P. cubensis, and
S. sclerotiorum were listed in Table 4. Most of the compounds
showed promising results in inhibiting the mycelial growth of all
of the test fungi at a concentration of 500 μg/mL. Meanwhile, all
of these tested compounds were found safe for the cucumber
plants. The comparison of the fungicidal activity of compounds
6 and 7 for five test fungi to those of commercial fungicides leads to
the following conclusions: (a) Compounds 6l, 6m, 6n, 6o, and
7b exhibited a significant inhibition effect against C. cassiicola, and
the fungicidal activities (control efficacy of 83.7-94.4%) were
higher than those of Thiophanate-methyl and Iprodione. Espe-
cially, compound 6n showed a control efficacy of 94.4%, which
was similar to that of the most active fungicide Dimethomorph
(95.3%). It can be seen that variances among R1, R2, and R3
can greatly effect the fungicidal activity of compounds against
Herbicidal Activity. As shown in Table 5, most compounds
6 and 7 displayed herbicidal activity based on the rape
(B. campestris) root and barnyardgrass (E. crusgalli) cup tests
at a concentration of 100 μg/mL. Compounds 6a-6c, 6h, 6j-6l,
and 6n showed inhibition rates of 66.8-88.5% to the root growth
of dicotyledonous rape at 100 μg/mL. Especially, compounds 6b

5520 J. Agric. Food Chem., Vol. 58, No. 9, 2010
Wang et al.
Figure 2. Superimposition of compounds 6 and 7 for 3D QSAR studies.
Table 6. Summary of CoMFA Analysis
contribution (%)
Table 7. Experimental and Predicted Fungicidal Activity (P. cubensis) of 3D
QSAR
compound
MW
I
EDa
PDb
residual
q2
r2
S
F
compound steric electrostatic
6h 74.6 25.4
6a
6b
448
466
478
462
480
492
490
496
507
476
494
506
504
510
521
654
690
714
710
476
482
493
722
744
66.3
64.9
79.5
78.6
75.5
72.5
69.5
91.2
76.8
74.1
79.2
87.3
67.8
91.6
77.2
78.9
79.9
84.5
78.7
96.9
74.2
76.2
81.0
84.9
2.945
2.935
3.268
3.230
3.170
3.113
3.048
3.711
3.225
3.134
3.274
3.541
3.026
3.745
3.247
3.388
3.438
3.590
3.419
4.173
3.142
3.198
3.488
3.622
3.002
3.003
3.162
3.249
3.139
3.272
3.072
3.701
3.265
3.250
3.250
3.605
3.028
3.599
3.259
3.296
3.394
3.606
3.458
4.002
3.341
2.903
3.568
3.677
-0.057
-0.068
0.106
CoMFA 0.526 0.913 0.085 24.448
6c
6d
-0.019
0.031
and 6k held by inhibition rates 70.8 and 67.1% at the concentra-
tion of 10 μg/mL, respectively, which were similar to that of
contrast Chlorsulfuron. Against the seedling growth of mono-
cotyledonous barnyardgrass, all of the compounds showed little
inhibitory activitity at 10 μg/mL, but compounds 6h exhibited
96.7% inhibition at 100 μg/mL. On the whole, these compounds
might exhibit less of an effect in comparison to the commercial
sulfonylureas against rape root at 1 μg/mL [e.g., Chlorsulfuron,
64.2% inhibition; Metsulfuron-methyl, 81.0% inhibition (29)];
however, some showed favorable herbicidal activity in these
preliminary studies and might be further optimized to enhance
their activity.
CoMFA Analysis. The CoMFA method is widely used in drug
design, because it allows for rapid generation of predictive
information of QSAR from the biological activity of newly
designed molecules (30). Starting from the structural alignment
of Figure 2, comprehensive CoMFA analyses were performed.
The results of these computations were summarized in Table 6. A
comparison of experimental and predicted activity by CoMFA
for all of the compounds in this study was presented in Table 7.
The cross-validated results were assessed by their q2 value (see the
Materials and Methods), where a value above 0.3 indicates that
the probability of chance correlation is less than 5% and a value
over 0.5 is highly significant. As listed in Table 6, a predictive
CoMFA model was established with the cross-validated co-
efficient q2=0.526 and the conventional correlation coefficient
r2 = 0.913. The contributions of steric and electrostatic fields
are 74.6 and 25.4% as shown in panels a and b of Figure 3,
respectively.
6e
6f
6g
-0.159
-0.024
0.010
6h
6i
-0.040
-0.116
0.024
6j
6k
6l
-0.064
-0.002
0.146
6m
6n
6o
-0.012
0.092
0.044
7a
7b
7c
-0.016
-0.039
0.171
7d
6-A1c
6-A2c
6-A3c
7-A1c
7-A2c
-0.199
0.295
-0.080
-0.055
a ED = experimental D value. b PD = predicted D value. c Test set compounds.
3 position will decrease the fungicidal activity. For example, some
compounds bearing substituents at the 2 position of the benzene
ring, such as 6b, 6e, 6i, 6j, and 6o, displayed lower fungicidal
activity. The electrostatic contour plot is shown in Figure 3b. The
blue contour defines a region where an increase in the positive
charge will result in an increase in the activity, whereas the red
contour defines a region of space where increasing electron
density is favorable. As shown in Figure 3b, the target compounds
bearing an electron-donating group at the 4 or 6 position of the
pyrimidine ring or an electron-withdrawing group at the 3 or 4
position of the benzene ring, such as 6h, 6n, and 7c, displayed higher
activity. According to the 3D QSAR model obtained, five novel
Mannich bases (6-A1-6-A3, 7-A1, and 7-A2) as test set
compounds were subsequently synthesized and bioassayed.
As shown in Table 7, their activities were estimated fairly
accurately from the analysis, indicating that the alignment of
the molecules was valid. For example, compounds 6-A1 and
7-A2 were found to display good fungicidal activity against
In Figure 3, the isocontour diagrams of the steric and electro-
static field contributions (“standard deviation ꢁ coefficient”)
obtained from the CoMFA analysis are illustrated. The steric
field contour map is plotted in Figure 3a. The green displays 2
positions, where a bulky group would be favorable for higher
fungicidal activity. In contrast, yellow indicates positions where a
decrease in the bulk of the target molecules is favored. As shown
in Figure 3a, the CoMFA steric contour plots obviously indicated
that a yellow region is located around the 2 and 3 position of the
benzene ring. This means that the bulky substituents at the 2 and

Article
J. Agric. Food Chem., Vol. 58, No. 9, 2010 5521
Figure 3. (a) Steric map from the CoMFA model. (b) Electrostatic map from the CoMFA model.
P. cubensis (96.9 and 84.9%) as compounds 6h and 7c, res-
pectively. These results provide useful information for further
optimization of the compounds.
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trifluoromethyl-substituted 1,2,4-triazole Mannich base 6 and
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Received for review January 24, 2010. Revised manuscript received
March 25, 2010. Accepted March 30, 2010. This work was supported by
the National Basic Research Key Program of China (2003CB114406),
the Tianjin Natural Science Foundation (08JCYBJC00800 and
07JCYBJC00200), and the Specialized Research Fund for the
Doctoral Program of Higher Education (20070055044).