Synthesis, structure and biological activity of novel 1,2,4-triazole Mannich bases containing a substituted benzylpiperazine moiety

Article abstract of DOI:10.1111/j.1747-0285.2011.01132.x

A series of novel Mannich bases with trifluoromethyl-1,2,4-triazole and substituted benzylpiperazine moieties were synthesized. Their structures were confirmed by IR, ¹H NMR and elemental analysis. The single crystal structure of compound 4r was also determined. The preliminary bioassays showed that most of the lead compounds had low herbicidal activity against Brassica campestris, Echinochloa crusgalli, and KARI enzyme. However, most of them exhibited significant fungicidal activity at the dosage of 50μg/mL toward five test fungi. Among the 18 novel compounds, several showed superiority over the commercial fungicide Triadimefon against Cercospora arachidicola and Fusarium oxysporum f. sp. cucumerinum during this study. Meanwhile, some compounds displayed plant growth regulatory activity at the dosage of 10μg/mL.

Full text of DOI:10.1111/j.1747-0285.2011.01132.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01132.x
Chem Biol Drug Des 2011; 78: 42–49
Research Article
Synthesis, Structure and Biological Activity of
Novel 1,2,4-Triazole Mannich Bases Containing
a Substituted Benzylpiperazine Moiety
In recent years, many 1,2,4-triazole derivatives such as Diniconazole,
Triadimefon, Triadimenol, Flusilazole, Fluconazole, Itraconazole, and
so on (Figure 1) have been found and developed as fungicides. These
compounds represent the most important category of fungicides to
date and have excellent protective, curative, and eradicant power
toward a wide spectrum of crop diseases (7–9). Meanwhile some
structures with a 1,2,4-triazole ring also exhibited outstanding herbi-
cidal activity (10,11). Because of their diverse properties, 1,2,4-triazole
pesticides may become one of the focuses in the research and devel-
opment of agrochemicals. Thus, the synthesis of broader spectrum
and highly bioactive 1,2,4-triazole compounds has become an active
research in agricultural chemistry. Further, piperazine derivatives were
also found to possess various biological activities (12–14). Many liter-
ature references have shown that Mannich bases possess potent bio-
logical activity such as antibacterial, antifungal, anti-inflammatory,
antimalarial, and pesticide properties (15–18).
Bao-Lei Wang, Xing-Hai Liu, Xiu-Lan Zhang,
Ji-Feng Zhang, Hai-Bin Song and
Zheng-Ming Li*
The Research Institute of Elemento-Organic Chemistry, State-Key
Laboratory of Elemento-Organic Chemistry, Nankai University,
300071 Tianjin, China
*Corresponding author: Zheng-Ming Li, nkzml@vip.163.com
A series of novel Mannich bases with trifluorom-
ethyl-1,2,4-triazole and substituted benzylpiper-
azine moieties were synthesized. Their structures
were confirmed by IR, 1H NMR and elemental anal-
ysis. The single crystal structure of compound 4r
was also determined. The preliminary bioassays
showed that most of the lead compounds had low
herbicidal activity against Brassica campestris,
Echinochloa crusgalli, and KARI enzyme. However,
most of them exhibited significant fungicidal
activity at the dosage of 50 lg ⁄ mL toward five test
fungi. Among the 18 novel compounds, several
showed superiority over the commercial fungicide
Triadimefon against Cercospora arachidicola and
Fusarium oxysporum f. sp. cucumerinum during
this study. Meanwhile, some compounds displayed
plant growth regulatory activity at the dosage of
10 lg ⁄ mL.
In our previous work, we reported some interesting Mannich bases
derived from 1,2,4-triazole Schiff bases (19), and benzotriazoles con-
taining benzylpiperzine ring, which are associated with various useful
herbicidal activity (12). In view of all these facts and as continuation
of our research on pesticidal important heterocycles, hereby a series
of novel Mannich bases with trifluoromethyl-1,2,4-triazole and substi-
tuted benzylpiperazine moieties were synthesized, and their herbi-
cidal, fungicidal, and PGR activity were investigated. It was worthy of
note that the structures we synthesized here are similar to those of
benzotriazoles Mannich bases we reported earlier, during our search
for novel inhibitors of KARI (one of the key herbicidal target enzymes
involved in the biosynthesis of chain amino acids) (12). So the in vitro
and in vivo herbicidal activity of lead compounds were both investi-
gated. The preliminary biological tests showed that some compounds
exhibit significant fungicidal activity and PGR activity.
Key words: 1,2,4-triazole, benzylpiperazine, fungicidal activity, her-
bicidal activity, Mannich base, plant growth regulatory activity
Received 10 October 2010, revised 21 February 2011 and accepted for
publication 11 April 2011
Sulfur- and nitrogen-linked heterocyclic compounds have received
considerable attention in recent times because of their medicinal
and pesticidal importance (1–4). Triazoles, like many other five-
membered heterocyclic compounds are used very often in pharma-
cological, medicinal, and agricultural applications. The compounds
containing a triazole ring, such as 1-(substituted phenyl)-3-(1-alkoxy
carboxyl alkoxy)-1,2,4-1H-triazoles have shown a versatility and
useful biological properties, and they have been developed as
fungicides, herbicides, or plant growth regulators (PGRS) (5). The
incorporation of different active functional groups into triazole
ring was proved to be a good way to produce novel active
pesticides (6).
Experimental
Instruments and materials
The melting points were determined on an X-4 binocular microscope
melting point apparatus (Beijing Tech Instrument Co., Beijing, China)
and are uncorrected. Infrared spectra were recorded on a Nicolet
MAGNA-560 spectrophotometer (Nicolet Instrument Corp., Madison,
1
WI, USA) as KBr tablets. H NMR spectra were measured on a Bru-
ker AC-P500 instrument (400 MHz) (Bruker, Fallanden, Switzerland)
using TMS as an internal standard and DMSO-d6 or CDCl3 as solvent.
Elemental analysis was performed on a Vario EL elemental analyzer
42

Synthesis, Structure and Biological Activity of Novel 1,2,4-Triazole Mannich Bases
F
O
OH
N
Cl
O
N
O
N
N
N
Si
N
N
N
N
N
Cl
Cl
Cl
HO
N
N
F
Diniconazole
Triadimefon
Triadimenol
Flusilazole
O
OH
O
N
N
N
N
N
N
N
O
N
N
N
N
O
F
F
N
N
N
F
F
Fluconazole
Itraconazole
Figure 1: Some representative structures of triazole fungicides.
(Elementar, Hanau, Germany). Crystallographic data of the compound
were collected on a Rigaku MM-07 Saturn 724 CCD diffractometer
(Rigaku International Corp., Tokyo, Japan). All of the solvents and
materials are of analytical grade.
reflux, and substituted benzyl chlorine (25 mmol) was added drop-
wise for over 5 min. The mixture was refluxed for 4–8 h with TLC
monitoring, then left to stand overnight at room temperature. The
solid precipitated was filtered and washed with ethanol, the filtrate
was evaporated in vacuum, and the residue was dissolved in 30 mL
of saturated K2CO3 aq., extracted with chloroform (8 mL · 5). The
chloroform solution was dried with anhydrous Na2SO4 and evapo-
rated in vacuum. The residue was then distilled under reduced pres-
sure to give compound 1 as a colorless liquid.
Synthesis
The lead compounds were synthesized according to the route
shown in Scheme 1.
1a (R1=R2=H): yield 58%, bp 131–134 ꢀC⁄10 mmHg [Lit. bp 154–
160 ꢀC⁄18 mmHg (22)]; 1b (R1=H, R2=Cl): yield 41%, bp 138–
141 ꢀC⁄10 mmHg [Lit. bp 92–96 ꢀC⁄0.1 mmHg (23)]; 1c (R1=R2=Cl): yield
36%, bp 147–150 ꢀC⁄6 mmHg [Lit. bp 106–108 ꢀC⁄0.05 mmHg (23)].
Synthesis of compound triazolthiol (2)
4-Amino-5-trifluoromethyl-4H-1,2,4-triazole-3-thiol (2) was prepared
according to the literature (20).
Preparation of 4-(substituted)benzylpiperazine
(1)
General synthetic procedures for 4-
(substituted)benzylideneamino-5-
The procedures are similar to that described previously (21). To a
solution of anhydrous piperazine (50 mmol) in 20 mL 96% of etha-
nol was added conc. HCl (25 mmol). The mixture was stirred under
trifluoromethyl-4H-1,2,4-triazole-3-thiol (3)
Compound 2 (10 mmol) and aromatic aldehyde (10.5 mmol) were
mixed in acetic acid (15 mL). After having been stirred and refluxed
HCl
EtOH, reflux
Cl
N
+
HN
NH
NH
R2
R1
R2
R1
1
N N
S
O
CHO
reflux
R3
HOAc, reflux
+
F3C
SH
N
NH2
H2NHN
NHNH2
F3C
OH
2
N
S
N
N N
N
1
N N
F3C
SH
R1
R2
37% CH2O, EtOH, r.t.
F3C
N
N CH
N CH
R3
R3
3
4
Scheme 1: Synthetic route of title compounds.
Chem Biol Drug Des 2011; 78: 42–49
43

Wang et al.
for 15 min, the reaction mixture was cooled to room temperature.
The resulting crystals were filtered and washed with ethanol to
give Schiff base 3.
The compound crystallizes in space group P21 ⁄ n of the monoclinic
system with cell parameters: a = 11.884(2) ꢀ, b = 8.6972(17) ꢀ,
c = 24.051(5) ꢀ, a = 90ꢀ, b = 96.33(3)ꢀ, c = 90ꢀ, V = 2470.7(9) ꢀ3,
Z = 4, Dc = 1.544 mg ⁄ m3, l = 0.406 per mm, and F(000) = 1176.
The final refinement converged at R = 0.0315, wR = 0.0814 for
3a (R3=H): white crystal, yield 74%, mp 198–199 ꢀC [Lit. mp 200–
201 ꢀC (19)]; 1H NMR (DMSO-d6, 400 MHz) d 14.90 (s, 1H, SH),
9.97 (s, 1H, CH), 7.58–7.92 (m, 5H, Ph-H).
2
3852 observed reflections with I > 2r(I ), where W = 1 ⁄ [r2(FO ) +
2
(0.0554P )2 + 0.2627P ] with P = (FO + 2Fc2) ⁄ 3, S = 1.046, (D ⁄ r)max
< 0.0001, (Dq)max = 0.252 e ⁄ ꢀ3 and (Dq)min = )0.312 e ⁄ ꢀ3.
3b (R3=2-F): white crystals, yield 82%, mp 192–193 ꢀC; 1H NMR
(DMSO-d6, 400 MHz) d 14.93 (s, 1H, SH), 10.44 (s, 1H, CH), 7.41–
8.03 (m, 4H, Ph-H).
The herbicidal activity
In vivo herbicidal activity of compounds 4a-r was determined by
rape root and barnyardgrass cup tests according to the reported
method (24).
3c (R3=4-MeO): white crystals, yield 71%, mp 195–196 ꢀC; 1H
NMR (DMSO-d6, 400 MHz) d 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).
Inhibition of the root growth of Rape (Brassica
campestris)
3d (R3=3,4-Me2): white crystals, yield 73%, mp 205–206 ꢀC; 1H
NMR (DMSO-d6, 400 MHz) d 14.86 (s, 1H, SH), 9.79 (s, 1H, CH),
7.66 (s, 1H, Ph-H), 7.62 (d, J = 8.0 Hz, 1H, Ph-H), 7.35 (d,
J = 8.0 Hz, 1H, Ph-H), 2.31 (ds, 6H, CH3).
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 only in dis-
tilled water. The inhibitive rates were calculated from the root
length using the following equation:
1
3e (R3=4-Cl): white crystals, yield 72%, mp 208–209 ꢀC; H NMR
(DMSO-d6, 400 MHz) d 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).
1
3f (R3=2-NO2): yellow crystals, yield 89%, mp 201–202 ꢀC; H NMR
(DMSO-d6, 400 MHz) d 14.96 (s, 1H, SH), 10.88 (s, 1H, CH), 7.87–
8.23 (m, 4H, Ph-H).
Relative inhibition rate ð%Þ ¼ ½ðCK ꢀ PTÞ=CKꢁ ꢂ 100%
where CK is the average root length during the blank assay and PT
is the average root length after treatment during testing.
General synthetic procedures for 1-[(4-
substituted-benzylpiperazin-1-yl)methyl]-4-
(substituted)benzylideneamino-3-
trifluoromethyl-1H-1,2,4-triazole-5(4H)-thione
(4)
Inhibition of the seedling growth of
Barnyardgrass (Echinochloa crusgalli)
Schiff base 3 (1 mmol) and 40% formalin (1.2 mmol) were dissolved
in 15 mL of ethanol, and the mixture was stirred at room tempera-
ture for 10 min. A solution of substituted benzylpiperazine 1
(1 mmol) in 2 mL ethanol was slowly added to. Then, the mixture
was stirred for 2–3 h and placed in a refrigerator overnight. The
resulting precipitate was filtered and recrystallized from ethanol to
give 1,2,4-triazole Mannich base 4.
The evaluated compounds were dissolved in water and emulsified
if necessary. Ten barnyardgrass seeds were placed 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 the
above-ground plant parts from each cup were measured and the
means were calculated. The check test was carried out only in dis-
tilled water. The inhibitive rates were calculated from the plant
heights using the following equation:
Crystal structure determination
Compound 4r was dissolved in hot ethyl alcohol, and the resulting
solution was allowed to stand in air at room temperature to give
single crystal of 4r. A yellow crystal of 4r suitable for X-ray dif-
fraction with dimensions of 0.30 · 0.28 · 0.16 mm was mounted
on a Rigaku MM-07 Saturn 724 CCD diffractometer with Mo-Ka
radiation (k = 0.71073 ꢀ) for data collection. A total of 13449
reflections were collected in the range of 2.01 < h < 25.02 by using
a phi and scan modes at 113(2) K, of which 4345 were independent
with Rint = 0.0277. 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.
Relative inhibition rate ð%Þ ¼ ½ðCK ꢀ PTÞ=CKꢁ ꢂ 100%
where CK is the average plant height during the blank assay and
PT is the average plant height after treatment during testing.
KARI activity test
The cloning of rice KARI has been described previously (25), and
enzyme expression and purification followed that protocol. Protein
concentrations were estimated via the bicinchoninic acid method
44
Chem Biol Drug Des 2011; 78: 42–49

Synthesis, Structure and Biological Activity of Novel 1,2,4-Triazole Mannich Bases
and protein purity was assessed by SDS-PAGE (26,27). KARI activity
24 ꢀC in a dark room for 3 days. The rhizogenesis numbers of every
10 pieces of hypocotyls were measured. Each treatment was per-
formed three times. In contrast, the distilled water was used as a
control. The relative ratios of cucumber cotyledon rhizogenesis were
calculated according to the following formula:
was measured with a continuous assay method (25), following the
consumption of NADPH at 340 nm and 30 ꢀC. Assay solutions con-
tained 0.2 mM NADPH, 1 mM MgCl2, 0.1 mM substrate (2-acetolac-
tate), and inhibitors (4a-r and IpOHA), in 0.1 M phosphate buffer
(pH 8.0). Inhibitors were preincubated with the enzyme, NADPH,
and MgCl2 in phosphate buffer at 30 ꢀC for 10 min. The reaction
was then started by adding the substrate. The percentage of the
inhibition was calculated using the following equation:
Relative ratio % ¼ ðNS ꢀ NC Þ=NC ꢂ 100%
where NS and NC are the numbers of cucumber cotyledon rhizogen-
esis of tested compound and control experiment, respectively.
Relativeinhibitionrateð%Þ ¼ ½ðAbsblank ꢀAbssampleÞ=Absblankꢁꢂ100%
Results and Discussion
where Absblank and Abssample are the absorption curve slopes of
NADPH at 340 nm with blank and inhibitor in testing solution,
respectively.
Synthesis
The synthesis procedures for compound
4 were shown in
Scheme 1. Based on an acetic acid–solvent method reported by Wu
et al. for the condensation of amine and aldehyde (30), Schiff base
3, namely 4-amino-5-trifluoromethyl-4H-1,2,4-trizole-3-thiol, was pre-
pared successfully with high yield and short reaction time. The
Mannich reaction of 3 with formaldehyde and substituted benzyl-
piperazine 1 in ethanol at room temperature led to novel trifluorom-
ethyl-substituted 1,2,4-triazole Mannich base 4 in 44–83% yield.
Compounds 4 were identified by melting point, 1H NMR and IR
spectra. The measured elemental analyses were also consistent
with the corresponding calculated ones (see Supporting Informa-
tion).
The fungicidal activity
Fungicidal activity of 4a-r against G. zeae Petch, Phytophthora infe-
stans (Mont.) de Bary, Cercospora arachidicola, Botryosphaeria ber-
engeriana f. sp. piricola (Nose) koganezawa et Sakuma, and
Fusarium oxysporum f.sp. cucumerinum were evaluated using the
mycelium growth rate test (28).
The method for testing the primary biological activity was per-
formed in an isolated culture. Under a sterile condition, 1 mL of
sample was added to the culture plates, followed by the addition
of 9 mL of culture medium. The final mass concentration was
50 lg ⁄ mL. The blank assay was performed with 1 mL of sterile
water. Circle mycelium with a diameter of 4 mm was cut using a
drill. The culture plates were cultivated at 24 € 1 ꢀC. The extended
diameters of the circle mycelium were measured after 72 h. The
relative inhibition rate of the circle mycelium compared to blank
assay was calculated via the following equation:
Proton magnetic resonance spectra
The proton magnetic resonance spectra of the Mannich bases
have been recorded in CDCl3 and those of the Schiff bases in
DMSO-d6. A comparison of the spectra of the products with the
intermediates leads to the following conclusions: The –CH=N pro-
ton appeared at d 9.73–10.88 as a singlet in the Schiff base 3,
which shifted downfield to d 10.07–11.21 in Mannich bases 4.
The Schiff bases 3 can exist either as a thione or the thiol tauto-
meric forms or as an equilibrium mixture of both forms, because
they have a thioamide, -NH-C(=S) function. The chemical shift at
d 14.84–14.96 as a singlet in 3 may be because of SH proton,
indicating that 3 existed not as thione but as the thiol tautomeric
forms in solution (31). In the Mannich bases 4, neither a NH sig-
nal nor a thiol SH signal is visible. The signal of CH2 protons
neighboring to the triazole ring was observed at d approximately
5.22 as a singlet, and the substituted benzyl CH2 proton appeared
at d 3.45–3.56 as a singlet in Mannich bases 4. The chemical
shifts at 2.86–2.88 ppm and 2.46–2.54 ppm may be because of
piperazine ring protons, which were appeared as two broad sing-
lets, respectively. The signal because of benzene ring protons
appears in the region d 6.98–8.23 in the spectra of all products
and intermediates.
Relative inhibition rate ð%Þ ¼ ½ðdex ꢀ dexÞ=de0xꢁ ꢂ 100%
where dex is the extend0ed diameter of the circle mycelium during
the blank assay; and dex is the extended diameter of the circle
mycelium during testing.
The blank test was made using acetone. Three replicates were per-
formed. The fungicidal tests results were given in Table 1.
The plant growth regulatory activity
The PGR activity of compounds 4a-r was evaluated by means of
cucumber cotyledon test according to a reported procedure (29).
The cucumber seeds (JINKE, No. 4) were supplied by the Biological
Assay Center, Nankai University, China. These seeds were incubated
at 24 ꢀC in a dark room for 3 days and 10 pieces of cotyledons of
the same size were selected. The test samples were dissolved in
N,N-dimethylformamide (DMF) at a concentration of 10 lg ⁄ mL. A
sample solution (0.3 mL) was sprayed over a filter paper (6 cm
diameter), and solvent was volatilized to dryness on air. The filter
paper thus prepared was placed into an incubation vessel (6 cm
diameter) and soaked with distilled water (3 mL). Finally, 10 pieces
of cotyledons were added. These cotyledons were incubated at
Infrared spectra
The spectra of Mannich bases 4a, 4g, and 4m showed bands at
1601–1600 per cm for C=N stretching. The characteristic stretching
vibrations m (C-F) and m (C=S) appears at 1320–1318 per cm, 1197–
1194 per cm and 1166–1154 per cm, respectively.
Chem Biol Drug Des 2011; 78: 42–49
45

Wang et al.
Table 1: The fungicidal and plant growth regulatory activity of compounds 4a-r
Growth inhibition (%, 50 lg ⁄ mL)
Botryosphaeria
berengeriana
f. sp. piricola
(Nose) koganezawa
et Sakuma
Phytophthora
infestans
(Mont.) de Bary
Fusarium
oxysporum f.sp.
cucumerinum
Cercospora
arachidicola
Rhizogenesis
(%, 10 lg ⁄ mL)
Compound
G. zeae Petch
4a
4b
33.2 € 2.7
50.1 € 2.1
0
21.0 € 1.8
60.4 € 1.2
0
70.3 € 1.9
70.4 € 2.4
0
10.8 € 2.4
70.1 € 1.4
0
10.1 € 1.1
80.4 € 2.8
18.7 € 2.2
0
4.6 € 1.8
68.6 € 3.2
10.4 € 2.0
109.3 € 3.4
97.6 € 1.7
62.7 € 2.8
-12.7 € 1.1
51.1 € 2.3
16.2 € 1.7
27.9 € 2.8
39.5 € 2.6
16.4 € 1.3
39.5 € 2.3
45.3 € 1.6
22.0 € 1.8
62.9 € 3.0
10.2 € 0.7
45.3 € 3.3
50.2 € 3.4
4c
4d
0
0
0
19.5 € 1.3
50.3 € 2.7
72.3 € 3.2
50.4 € 2.3
70.6 € 3.4
50.7 € 2.9
44.5 € 3.3
60.7 € 3.0
70.2 € 3.4
70.4 € 4.1
60.2 € 2.9
50.2 € 3.3
80.8 € 2.0
70.4 € 3.4
63.6 € 3.8
73.4 € 2.3
0
0
4e
10.3 € 0.8
53.1 € 3.5
24.3 € 2.0
51.7 € 2.1
19.7 € 2.3
19.6 € 2.0
30.1 € 3.2
19.8 € 2.1
51.8 € 4.5
50.4 € 2.3
36.0 € 1.3
50.9 € 3.9
50.5 € 2.8
30.2 € 2.4
66.1 € 2.7
0
4f
4g
50.2 € 4.1
0
50.3 € 2.9
11.2 € 2.0
0
30.6 € 2.5
18.8 € 2.3
50.3 € 2.6
50.0 € 2.7
51.8 € 2.4
50.7 € 3.6
30.0 € 2.1
30.5 € 1.8
72.6 € 3.6
20.1 € 3.3
19.6 € 1.4
52.6 € 4.0
10.0 € 0.9
0
40.2 € 1.6
50.9 € 3.2
71.9 € 3.1
60.4 € 3.4
40.1 € 3.1
30.3 € 1.8
50.2 € 2.4
70.8 € 3.1
82.6 € 1.8
50.4 € 3.0
70.5 € 2.8
60.7 € 2.9
60.8 € 3.1
81.5 € 2.6
4h
4i
4j
4k
0
4l
4m
30.0 € 1.9
30.5 € 2.2
41.5 € 1.2
0
70.3 € 3.2
70.6 € 3.7
41.6 € 2.8
86.7 € 3.2
4n
4o
4p
4q
4r
Triadimefon
we reported before during our search for novel KARI inhibitors (12),
N-hydroxy-N-isopropyloxamate (IpOHA) (32), a potent inhibitor of
KARI in vitro, was used as a control. As shown in Table 2, com-
pounds 4m and 4r showed higher inhibition abilities of rape root at
a concentration of 100 lg ⁄ mL. Compound 4a showed obvious inhib-
itory activity to the rice KARI enzyme with an inhibition of 76.6%.
Except that, most of compounds exhibited weak herbicidal activity
in vivo and in vitro against rape and barnyardgrass and KARI
enzyme.
Figure 2: Molecular structure of compound 4r.
Fungicidal activity
The in vitro fungicidal results of the Mannich bases 4a-r in inhib-
iting the mycelial growth of five test fungi were listed in Table 1.
The results of preliminary bioassays were compared with that of
a commercial fungicide Triadimefon. As indicated in Table 1, some
compounds exhibited potential fungicidal activity. For example,
compounds 4b, 4f, 4h, and 4m-4p possess approximately 50%
inhibitory rates against G. zeae Petch. Also compound 4b held a
60.4% inhibitory rate against Phytophthora infestans (Mont.) de
Bary. It was observed that most of the compounds exhibited sig-
nificant inhibition effect against Cercospora arachidicola (inhibition
rates of 50.2–80.8%) and the inhibitory effect of 4p (80.8%) on
Cercospora arachidicola was better than that of the control Tria-
dimefon (73.4%). For Botryosphaeria berengeriana f. sp. piricola
(Nose) koganezawa et Sakuma, compounds 4b, 4h, 4p, and 4q
showed favorable activity and held 70.1, 52.6, 70.3, and 70.6%
inhibitory rates, respectively. In addition, almost half of the com-
pounds exhibited good inhibition effect against Fusarium oxyspo-
rum f. sp. cucumerinum (inhibition rates of 60.4–82.6%).
Especially, compound 4b and 4n showed 80.4 and 82.6% inhibi-
Crystal structure
The structure of compound 4r was further confirmed by single-crys-
tal X-ray diffraction analysis (Figure 2). From the molecular struc-
ture, it can be seen that both groups on the N atoms of piperazine
ring (triazole-CH2 and 2,4-dichlorophenyl-CH2) are in the e-bond
positions of chair conformation in the six-member ring. The dihedral
angel between 2-nitrobenzene ring and triazole ring is 2.6ꢀ, which
indicates the two rings are almost coplanar in the molecular struc-
ture. The X-ray analysis also reveals that in this typical compounds
4r, the substituted benzene ring and triazole ring are on the oppo-
site sides of the C=N double bond (Figure 2). The torsion angle of
C(6)-C(7)-N(2)-N(3) is 177.33ꢀ, which indicates that the C=N double
bond is in the (E)-configuration.
Herbicidal activity
To investigate the KARI inhibitory activity and herbicidal activity of
the compounds referring to those of benzotriazoles Mannich bases,
46
Chem Biol Drug Des 2011; 78: 42–49

Synthesis, Structure and Biological Activity of Novel 1,2,4-Triazole Mannich Bases
Table 2: Herbicidal activities data of title compounds (% inhibition)
Rape root
test (Brassica
campestris)
Barnyardgrass cup
test (Echinochloa
crusgalli)
KARI test
100 lg ⁄ mL
(10 lg ⁄ mL)
100 lg ⁄ mL
(10 lg ⁄ mL)
100 lg ⁄ mL
Compound
R1
R2
R3
4a
4b
4c
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
2-F
27.8
10.7
17.0
0
10.4
15.2
5.3
5.0
30.2
10.1
15.2 (5.1)
20.4
30.2
10.1 (5.0)
15.3 (5.2)
15.4
5.1
15.4
20.4 (10.2)
25.4 (5.3)
15.1
20.4 (5.2)
14.4
76.6
21.3
9.7
0
1.1
1.9
0
0
–
21.6
15.9
17.5
22.8
12.1
9.57
28.8
28.0
30.0
99.5
4-MeO
3,4-Me2
4-Cl
2-NO2
H
4d
4e
4f
2.2
17.4
31.4
30.1 (10.3)
0.9
4g
4h
4i
2-F
4-MeO
3,4-Me2
4-Cl
2-NO2
H
4j
7.6
4k
4l
39.3
27.8
50.2
38.1
33.4
13.9
7.2
4m
4n
4o
4p
4q
4r
2-F
4-MeO
3,4-Me2
4-Cl
2-NO2
48.0
71.6 (50.2)
IpOHA
–, Indicates the compound cannot be dissolved in our test system, so no data obtained.
tory activity against Fusarium oxysporum f.sp. cucumerinum,
respectively, and the effects of which were comparable to that of
the control (81.5%). It was worthy of note that in these Mannich
bases, compounds with two chlorine atoms on benzene ring of
benzyl group (at 4-position of piperazine ring), exhibited more obvi-
ous activity than others.
It was found that compounds without substituent in the benzene
ring of the benzyl group (R1=R2=H) almost have a higher level of
promoting activity for plant growth than others (R1=H, R2=Cl and
R1=R2=Cl). For herbicidal activity of the lead compound, compounds
with one or two chloro group(s) in the benzene ring of the benzyl
group showed relatively favorable activity than others; however, the
extent of the herbicidal effect was varying with concentration. On
the whole, the promoting activity of the lead compounds is mainly
for plant growth. Among these compounds, 1-[(4-benzylpiperazin-1-
yl)methyl]-4-(3,4-dimethyl)benzylideneamino-3-trifluoromethyl-1H-1,2,
4-triazole-5(4H)-thione (4d) was the most effective and least inhibi-
tory to plant and could be a favorable activator of plants growth at
a low dose of concentration.
Plant growth regulatory activity
Plant growth regulatory (PGR) activities are defined here as those
effects produced by the compound that leaves the numbers of
cucumber cotyledon rhizogenesis indistinguishable from the
untreated control in the same testing condition. The positive value
of activity data means the compound exhibits potential promoting
activity and can be an activator for the plant growth, while the
negative value means the compound exhibits potential inhibitory
activity and can be an inhibitor for the plant growth. The PGR
activity of the lead compounds 4a-r was screened by cucumber
cotyledon test at a concentration of 10 lg ⁄ mL. As shown in
Table 1, some compounds, such as 4b, 4d, 4e, 4f, 4h, and 4p,
exhibited better promoting activity than Triadimefon, with values of
68.6, 109.3, 97.6, 62.7, 51.1, and 62.9%, respectively. These results
were consistent with those of their low in vivo herbicidal activity
based on Brassica campestris and Echinochloa crusgalli tests at a
dose of 10 lg ⁄ mL (almost no activity), referring to the herbicidal
activity data listed in Table 2. Compound 4g displayed weak inhib-
itory activity for the plant growth, with values of )12.7%, while
the same effect could also be observed in its herbicidal activity
assays.
Based on these preliminary studies, the fungicidal activity was out-
standing for this series of compounds. It appeared prior to the syn-
thesis and testing of substituted derivatives that the fungicidal
activity might be influenced by steric factors. Other novel Mannich
bases derive from Schiff bases containing various alkyl substituents,
or other aryl groups, such as heterocycles, may result in novel fun-
gicides. Anyhow, it is important for their chemical structure that
both two chloro groups are in the benzene ring of the benzyl group
of such kind of compounds.
In summary, we have conveniently synthesized a series of trifluo-
romethyl-substituted 1,2,4-triazole Mannich bases containing substi-
tuted benzylpiperazine ring via Mannich reaction in good yields. The
preliminary bioassays showed that most of the compounds had low
herbicidal activity against Brassica campestris, Echinochloa crusgalli,
Chem Biol Drug Des 2011; 78: 42–49
47

Wang et al.
and KARI enzyme. However, most of them exhibited significant fun-
gicidal activity at the dosage of 50 lg ⁄ mL toward five test fungi.
Among the 18 novel compounds, several showed superiority over
the commercial fungicide Triadimefon against Cercospora arachidico-
la and Fusarium oxysporum f.sp. cucumerinum during this study and
could be further developed as fungicides. Meanwhile, some com-
pounds displayed PGR activity at the dosage of 10 lg ⁄ mL in the
preliminary studies.
Baver W.A., Witkowski D.A., Peters G.R., Gravelle W.D. (1992)
Synthesis and activity optimization of herbicidal substituted 4-
aryl-1,2,4-triazole-5(1H)-thiones. J Agric Food Chem;40:297–305.
11. Li X.-Z., Si Z.-X. (2003) Research advances on triazole com-
pounds bearing biological activities. Pesticides;42:4–5.
12. He F.Q., Liu X.H., Wang B.L., Li Z.M. (2006) Synthesis and bio-
logical activity of new 1-[4-(substituted)-piperazin-1-ylmethyl]-1H-
benzotriazole. J Chem Res;12:809–811.
13. Chaudhary P., Kumar R., Verma A. K., Singh D., Yadav V., Chhillar
A. K., Sharmab G.L., Chandra R. (2006) Synthesis and antimicro-
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Med Chem;14:1819–1826.
Acknowledgments
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antifungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-
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This work was supported by National Basic Research Key Program
of China (No. 2010CB126106), the Fundamental Research Funds for
the Central Universities, Specialized and Research Fund for the Doc-
toral Program of Higher Education (No. 20070055044), Tianjin Natu-
ral Science Foundation (No. 08JCYBJC00800). We thank the
teachers of the Biological Assay Center, Nankai University, for kind
bioassay assistance of compounds.
16. Satyanarayana D., Kalluraya B., George N. (2002) Synthesis and
biological evaluation of some Mannich bases derived from 1,2,4-
triazole derivatives. J Saudi Chem Soc;6:459–464.
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