Synthesis, biological activities and SAR studies of new 3-substitutedphenyl-4-substitutedbenzylideneamino-1,2,4-triazole Mannich bases and bis-Mannich bases as ketol-acid reductoisomerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.10.065

A series of new 3-substitutedphenyl-4-substitutedbenzylideneamino-1,2,4-triazole Mannich bases and bis-Mannich bases were synthesized through Mannich reaction with high yields. Their structures were confirmed by means of IR, ¹H NMR, ¹³C NMR and elemental analysis. The preliminary bioassay indicated that compounds 7g, 7h and 7l exhibited potent in vitro inhibitory activities against ketol-acid reductoisomerase (KARI) with K_i value of (0.38 ± 0.25), (6.59 ± 2.75) and (8.46 ± 3.99) μmol/L, respectively, and were comparable with IpOHA. They could be new KARI inhibitors for follow-up research. Some of the title compounds also exhibited obvious herbicidal activities against Echinochloa crusgalli and remarkable in vitro fungicidal activities against Physalospora piricola and Rhizoctonia cerealis. The SAR of the compounds were analyzed, in which the molecular docking revealed the binding mode of 7g with the KARI, and the 3D-QSAR results provided useful information for guiding further optimization of this kind of structures to discover new fungicidal agents towards Rhizoctonia cerealis.

Full text of DOI:10.1016/j.bmcl.2017.10.065

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological activities and SAR studies of new
3-substitutedphenyl-4-substitutedbenzylideneamino-1,2,4-triazole
Mannich bases and bis-Mannich bases as ketol-acid
reductoisomerase inhibitors
a
b
a
a
a,
a
Bao-Lei Wang a, , Li-Yuan Zhang , Xing-Hai Liu , Yi Ma , Yan Zhang , Zheng-Ming Li , Xiao Zhang
⇑
⇑
a State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Enginnering(Tianjin), College of Chemistry, Nankai University,
Tianjian 300071, PR China
b College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 3-substitutedphenyl-4-substitutedbenzylideneamino-1,2,4-triazole Mannich bases and
bis-Mannich bases were synthesized through Mannich reaction with high yields. Their structures were
confirmed by means of IR, 1H NMR, 13C NMR and elemental analysis. The preliminary bioassay indicated
that compounds 7g, 7h and 7l exhibited potent in vitro inhibitory activities against ketol-acid reductoi-
Received 15 August 2017
Revised 20 October 2017
Accepted 25 October 2017
Available online xxxx
somerase (KARI) with Ki value of (0.38 0.25), (6.59 2.75) and (8.46 3.99) lmol/L, respectively, and
were comparable with IpOHA. They could be new KARI inhibitors for follow-up research. Some of the title
compounds also exhibited obvious herbicidal activities against Echinochloa crusgalli and remarkable
in vitro fungicidal activities against Physalospora piricola and Rhizoctonia cerealis. The SAR of the com-
pounds were analyzed, in which the molecular docking revealed the binding mode of 7g with the
KARI, and the 3D-QSAR results provided useful information for guiding further optimization of this kind
of structures to discover new fungicidal agents towards Rhizoctonia cerealis.
Keywords:
1,2,4-Triazole thione
Mannich base
Ketol-acid reductoisomerase inhibitors
Fungicidal activity
Structure-activity relationship
Ó 2017 Elsevier Ltd. All rights reserved.
Agrochemicals play important role in the crop protection and
increase the yield of crop, which undoubtedly make positive influ-
ence on almost all aspects of our life. Whereas the conventional
agrochemicals are confronted with such problems as environmen-
tal pollution and the resistance of weeds, insects, fungi, etc. There-
fore, the discovery of novel agrochemicals with super active, high
selective and eco-benign characteristics is urgently need, which
also is a permanent subject for agrochemical researchers.1–3 Fur-
thermore, it is known that the vast potential of heterocyclic deriva-
tives has long been recognized in pesticide chemistry and some
other areas.4,5 Among all kinds of agrochemicals which have been
introduced to the market within the last 20 years, approximately
70% of them contain at least one heterocyclic ring.6 For examples,
Pyribencarb, discovered by KUMIAI chemical industry Co., Ltd. is a
pyridine-containing heterocyclic fungicide.7 Cafenstrole, which is
widely used as pre-emergence herbicide for controlling grass in
rice and some other crop fields, is a kind of carbamoyl triazole
agrochemical.8 Diniconazole and Triadimefon are important com-
mercial fungicides that both contain a triazole motif.9 In addition,
among various bioactive heterocyclic derivatives, heterocyclic
Mannich bases which have long been focusing on their uses in
medicinal chemistry area such as antituberculous,10 anticancer,11
and antimicrobial12 agents, have drawn promising attention for
their synthesis and pesticidal activities in recent years.13–16
One important way for the design and discovery of novel agro-
chemicals is to develop new bioactive molecules with potent bio-
logical activity towards their target of action. There are a lot of
enzymes existed in plants and microorganisms that could be used
as potential targets for designing bioactive agrochemicals. For
example, plants, bacteria and fungi have the biosynthetic pathway
for branched chain amino acids (BCAA) – leucine, valine and isoleu-
cine, whereas such pathway does not exist in human and other
mammals.17 Therefore, developing new herbicidal and fungicidal
agents targeting on any enzymes involved in BCAA pathway could
be an ideal way for the design of green agrochemicals. Among
some enzymes in the BCAA pathway, ketol-acid reductoisomerase
(KARI, EC 1.1.1.86) catalyzes the second step reaction of the
pathway via an alkyl migration of the substrate followed by a
⇑
Corresponding authors.
E-mail addresses: nkwbl@nankai.edu.cn (B.-L. Wang), nkzml@vip.163.com
(Z.-M. Li).
https://doi.org/10.1016/j.bmcl.2017.10.065
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang B.-L., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.10.065

2
B.-L. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
NADPH-dependent reduction, and has been used as a promising
target for the design of herbicides and fungicides for many years.
Hoe 704,18 IpOHA19 and CPD20 are several of few traditional KARI
inhibitors with potent inhibitory activities in vitro, however, their
activities in vivo as herbicides were reported rather weak. Thus,
more KARI inhibitors with new structures and strong biological
activities keep as a potential source for discovering novel herbici-
dal and fungicidal compounds.
It was found some novel 5-substituted-1,3,4-oxadiazole Man-
nich bases and bis-Mannich bases containing aryl heterocycle
(furan, pyridine) and piperazine motifs possessed potent KARI inhi-
bitory activities in our recent work.21 Those inhibitors not only
showed herbicidal activities against Brassica campestris, but also
displayed favorable fungicidal activities against several plant fungi.
Moreover, some trifluoromethyl-containing 1,2,4-triazole Mannich
base were also found to have significant herbicidal and fungicidal
activities.22 Taking into account all those clues mentioned above
and with the aim of discovering novel heterocyclic compounds as
KARI inhibitors and agrochemical agents, a series of novel 1,2,4-tri-
azole Mannich bases and bis-Mannich bases bearing substituted
phenyl and piperazine groups were synthesized in this paper.
Meanwhile, the structure-activity relationship (SAR) of the com-
pounds was studied.
respectively with 4-substituted benzaldehyde in ethanol under
glacial acetic acid catalysis and refluxing conditions for 6 h to effi-
ciently give the key intermediates Schiff base 5a-h. It is known that
the thiol structures of 5a-h may undergo some reactions in their
corresponding thione form 50a-h owing to the tautomerism equi-
librium system between ‘‘AN@C(SH)A” and ‘‘AHNAC(@S)A”. For
the following Mannich reaction of these Schiff base intermediates,
formaldehyde and various 4-substituted piperazine, it was found
that the Mannich base products were successfully obtained under
mild conditions (ethanol solvent, room temperature) in short time
and high yield (75%–99%), also the bis-Mannich base products with
symmetric di-substituted structures were conveniently achieved
(yield 83%–00%) using anhydrous piperzaine as amine reactant.
These results indicate that the Schiff base intermediates undertook
the Mannich reaction in thione form (50a-h) through the neighbor-
ing reactive site NAH of thicarbonyl (C@S) group.
The structures of the title compounds were confirmed by IR, 1H
NMR, and 13C NMR spectra. There was good consistency between
the measured elemental analyses data and the corresponding cal-
culated ones. The IR spectra of the title compounds showed bands
at 1568–1668 cmꢀ1 for C@N and 1116–1173 cmꢀ1 for C@S stretch-
ing. In 1H NMR, the chemical shift at d 14.27–14.48 in the solvent
DMSO d6 (or ꢁ11.06 in CDCl3) for intermediates 5a-h as a singlet
indicates that the compounds existed as the triazole thiol form.
For the title compounds, the proton signal of CH2 group which links
the triazole and piperazine rings was observed at d 5.21–5.33 as a
singlet. The piperazine ring proton (CH2) in triazole Mannich base
products appeared at d 3.78–3.96 and d 2.78–3.06 as multiplets,
respectively. Whereas in the case of bis-Mannich base products,
the piperazine ring proton appeared at d ꢁ2.95 as a singlet due
The synthetic routes for several intermediates and the final title
compounds 6, 7 and 8 are shown in Scheme 1. Using the method in
literatures,23–25 intermediates 4-amino-5-substitutedphenyl-4H-
1,2,4-triazole-3-thiol (4a-c) were prepared with ethyl substituted
benzoate as starting material via hydrazinolysis, CS2-nucleophilic
addition and cyclization, and hydrazine-based nucleophilic addi-
tion-elimination, successively. These intermediates further reacted
Scheme 1. The synthetic route of the title compounds 6, 7 and 8.
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B.-L. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
to their symmetric structure. In the 13C NMR spectra of the title
Table 2
Inhibitory constant Ki of the compounds against rice KARI.
compounds, the typical carbon signal at d 163.5–167.6 was derived
from the resonance of thiocarbonyl group (C@S), the piperazine
carbon signals showed up two peaks at d ꢁ50.6 and d ꢁ43.6, and
singlet at d ꢁ50.5 for Mannich base and bis-Mannich base com-
pounds, respectively.
Compd
Ki (mmol/L)
Compd
Ki (mmol/L)
7d
7g
7h
7l
19.75 17.32
0.38 0.25
6.59 2.75
8.46 3.99
8d
35.19 9.02
2.75 0.72
0.0903 0.0133
58.5 13.7
IpOHA21
CPD20
1-cyanoCPC20
Through a continuous assay procedure,20 the in vitro inhibitory
activity test of the title compounds against rice KARI was carried
out, and the results are listed in Table 1. It was found that most
of the compounds showed apparent KARI inhibitory activities at
a test concentration of 200 lg/mL, especially, compounds 6d, 6h,
6l, 7d, 7g, 7h, 7l, 8d and 8h held inhibition rates of 60.45%–
82.04%. The further investigation on these bioactive compounds
revealed that 7d, 7g, 7h, 7l and 8d could be novel KARI inhibitors
from the Ki value determination results (Table 2) (the Ki test for
other compounds didn’t give satisfactory data). Lee and Duggleby
ever reported the CPD derivative 1-cyanoCPC was a kind of KARI
inhibitor with Ki value of (58.5 13.7)
l
mol/L,20 by comparison,
those five compounds in Table 2 were new inhibitors of the
enzyme with lower Ki values. In particular, 7g was the most potent
one [Ki = (0.38 0.25)
was even effective than the control IpOHA [Ki = (2.75 0.72)
L] at the present study. Noticeable, the bis-Mannich bases 7h and
7l whose Ki values were (6.59 2.75) mol/L and (8.46 3.99)
mol/L, respectively, also showed favorable KARI inhibitory
activities.
l
mol/L] among the five KARI inhibitors, and
lmol/
l
l
In view of good KARI inhibitory activities of the compounds,
further investigation on the binding mode of the best active com-
pound 7g with KARI (PDB ID: 1YVE) was carried out via classical
molecular docking procedure using Discovery Studio software.21
As shown in Fig. 1, there are strong H bond interactions between
the S atom of triazole-thione moiety of 7g and KARI amino acid
residue THR519, and the F atom of (4-fluorobenzylidene)amino
moiety and residue ARG522, respectively. Meanwhile, there
Fig. 1. The docking mode of compound 7g with KARI.
The herbicidal activity data of the title compounds from the
barnyard grass (Echinochloa crusgalli) cup test26 are shown in
Table 3. It was found that some of the compounds exhibited obvi-
ous herbicidal activities against Echinochloa crusgalli at either 100
formed a
p-
p
stacking interaction between the benzene ring of
lg/mL or 10 lg/mL concentration. For examples, compounds 6e-g,
7g and residue ARG522. In addition, the figure also shows that this
compound could tightly combine KARI by means of most of the
molecule (including triazole-methylene part, pyrimidine ring and
piperazine ring) fitting into the active pocket of the enzyme. All
of those interactions may contribute the stability of the complex
between compound 7g and KARI enzyme. Maybe in this way, 7g
could inhibit KARI and exert biological activity. Furthermore, for
the cases of 6g, 7e and 7f, the binding modes of KARI with each
of them were found to be different from that of KARI with 7g
(see Supporting Information), which probably resulted in the weak
inhibitory activities of them towards KARI.
7f, 7g and 7h possessed inhibition rates of 25%–45% (10%–25%)
towards the seedlings growth of monocotyledonous barnyard
grass. However, they showed very weak herbicidal activities
against dicotyledonous rape (Brassica campestris) (data not pre-
sented), which were different with those of oxadiazole Mannich
bases and bis-oxadiazole Mannich bases that have significant her-
bicidal activities against Brassica campestris we reported before.21
This may be owing to the structural difference between the
1,3,4-oxadiazole ring and 1,2,4-triazole ring, and the substituents
beared on the rings. It was also found when R1 is 2-F, R2 is F or
Cl, the corresponding compound held better herbicidal activities.
Although most of the compounds displayed a slight superiority
as compared to the potent KARI inhibitors IpOHA and CPD (Table 3),
the herbicidal activities of them were unfavorable on the whole.
Surprisingly, when R1 is fixed as 2-NO2-3,4,5-(MeO)3, the corre-
sponding compounds 8a-h showed no herbicidal activities.
Table 1
In vitro KARI inhibitory activity of the compounds at the concentration of 200 mg/mL.
Compd
%, inhibition
Compd
%, inhibition
As shown in Table 3, the in vitro fungicidal results indicate that
most of the title compounds displayed remarkable activities
against Cercospora arachidicola, Physalospora piricola and Rhizocto-
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
7a
7b
7c
7d
7e
7.40
28.54
0
60.45
10.14
10.34
8.19
72.01
0
7f
7g
7h
7i
7j
7k
7l
8a
8b
8c
8d
8e
8f
8g
8h
IpOHA21
CPD
9.89
75.66
82.04
6.68
1.17
1.35
69.30
30.59
51.84
19.65
81.81
13.65
7.72
6.22
78.38
100
nia cerealis at the concentration of 50 lg/mL, especially for inhibit-
ing the mycelial growth of the latter two. Compounds 6f and 8f
against Cercospora arachidicola possessed inhibition rates of 45.5%
and 70.0%, respectively, and were more effective than the controls
Triadimefon and Carbendazim. Almost half of the compounds
exhibited good inhibitory activities towards Physalospora piricola
with inhibition rates >65%, which were higher than that of Chlor-
othalonil. In particular, 6f, 6k, 7b, 7e and 7f whose fungicidal activ-
ities were 78.3%, 80.4%, 76.1%, 76.1% and 80.4%, respectively, were
superior to both Triadimefon (75.0%) and Chlorothalonil (63.6%).
Noticeably, most of the compounds were found to be able to
effectively inhibit the mycelial growth of Rhizoctonia cerealis with
12.27
0
61.99
14.91
11.94
23.95
74.80
11.75
100
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4
B.-L. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 3
Herbicidal and fungicidal activities of the compounds (%, inhibition).
R2
R2
R1
N
N
N
S
S
N
N
N
N
N
N
R3 6a-c 6e-g 6i-k
6d 6h 6l
, , ,
N
S
,
,
,
N
N
N
7a-c, 7e-g, 7i-k,
8a-c 8e-g
7d, 7h, 7l,
8d 8h
N
R1
,
,
N
R2
R1
N
Compd
R1
R2
R3
Herbicidal test at 100
(10 g/mL)
l
g/mL
Fungicidal test at 50 lg/mL
l
Echinochloa crusgalli
Cercospora
arachidicola
Physalospora
piricola
Rhizoctonia
cerealis
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
8a
8b
8c
8d
8e
8f
8g
8h
H
H
H
H
H
H
H
H
H
H
H
H
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
Cl
Cl
Cl
Cl
F
F
F
F
CF3
CF3
CF3
CF3
Cl
Cl
Cl
Cl
F
2-pyrimidyl
4-Me-2-pyrimidyl
4,6-Me2-2-pyrimidyl
–
2-pyrimidyl
4-Me-2-pyrimidyl
4,6-Me2-2-pyrimidyl
–
2-pyrimidyl
4-Me-2-pyrimidyl
4,6-Me2-2-pyrimidyl
–
2-pyrimidyl
4-Me-2-pyrimidyl
4,6-Me2-2-pyrimidyl
–
2-pyrimidyl
4-Me-2-pyrimidyl
4,6-Me2-2-pyrimidyl
–
2-pyrimidyl
4-Me-2-pyrimidyl
4,6-Me2-2-pyrimidyl
–
2-pyrimidyl
4-Me-2-pyrimidyl
4,6-Me2-2-pyrimidyl
–
2-pyrimidyl
4-Me-2-pyrimidyl
4,6-Me2-2-pyrimidyl
–
0
13.3
36.7
13.3
13.3
36.4
45.5
27.3
4.5
14.3
61.9
26.2
14.3
65.2
78.3
67.4
26.1
43.5
69.6
80.4
41.3
54.3
76.1
43.5
15.2
76.1
80.4
69.6
15.2
39.1
54.3
58.7
43.5
26.2
42.9
21.4
4.8
47.5
78.8
48.8
80.0
91.9
85.1
75.7
44.6
71.6
81.1
87.8
67.6
71.6
87.8
56.8
41.9
45.9
71.6
51.4
63.5
64.9
78.4
83.8
52.7
73.8
56.3
42.5
23.8
76.3
71.3
71.3
25.0
5.0 (3.0)
10.0 (5.0)
15.0 (5.0)
25.0 (10.0)
30.0 (20.0)
30.0 (20.0)
18.0 (3.0)
20.0 (10.0)
15.0 (5.0)
15.0 (10.0)
20.0 (8.0)
10.0 (5.0)
20.0 (15.0)
10.0 (5.0)
30.0 (15.0)
20.0 (10.0)
45.0 (20.0)
20.0 (15.0)
30.0 (25.0)
10.0 (5.0)
5.0
4.5
18.2
31.8
13.6
22.7
27.3
4.5
4.5
13.6
18.2
13.6
9.1
F
F
F
CF3
CF3
CF3
CF3
Cl
Cl
Cl
Cl
F
9.1
18.2
27.3
13.6
20.0
23.3
13.3
10.0
33.3
70.0
30.0
10.0
0
0
0
0
0
0
0
0
2-NO2-3,4,5-(MeO)3
2-NO2-3,4,5-(MeO)3
2-NO2-3,4,5-(MeO)3
2-NO2-3,4,5-(MeO)3
2-NO2-3,4,5-(MeO)3
2-NO2-3,4,5-(MeO)3
2-NO2-3,4,5-(MeO)3
2-NO2-3,4,5-(MeO)3
38.1
14.3
19.0
38.1
F
F
F
0
0
Chlorsulfuron
CPD
IpOHA
Triadimefon
Chlorothalonil
Carbendazim
67.6 (42.1)
7.0
14.4
25.0
30.2
26.8
100
40.0
88.5
8.3
75.0
63.6
97.4
inhibition rates >70%, such as 6e, 6f, 6k, 7b and 7k had 83.8%–
91.9% activities towards this fungus. Interestingly, compound 7g
which has potent KARI inhibitory activity with the lowest Ki in this
series just exhibited moderate activities both in herbicidal and
fungicidal bioassays. This probably may be owing to the existence
of competation from the natural substrate of the KARI reaction in
plants or microorganisms20, and/or some unknown metabolic
pathway that could influence on its in vivo biological activities.
In view of the favorable fungicidal activities of this type of com-
pounds towards Rhizoctonia cerealis, CoMFA was subsequently per-
formed for further exploring the comprehensive structure-activity
relationship.
Due to the great potential that CoMFA could rapidly generate
predictive information of QSAR on the basis of the biological activ-
ity of newly designed molecules,27 it is widely applied to the
design of drug or agrochemicals. Starting from the structural
alignment based on the asterisked atoms of template molecule
6e which is the most active one against Rhizoctonia cerealis in
Fig. 2, CoMFA analyses for the triazole Mannich bases 6, 7 and 8
(excluding the bis-Mannich base compounds among them) were
performed. The cross-validated results were assessed by their q2
value (see the Supporting Information), 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.22 As listed in Table 4,
a predictive CoMFA model was established with the cross-vali-
dated coefficient q2 = 0.513 and the conventional correlation coef-
ficient r2 = 0.726. The contributions of steric and electrostatic fields
are 41.7% and 58.3% as shown in panels a and b of Fig. 3,
respectively.
For the steric field contour map from the generated CoMFA
model, generally the green displays mainly one position, 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 Fig. 3(a), the CoMFA
steric contour plots obviously indicated that the green region is
mainly located around the 3, 4 and 5 position of the benzene ring
that links at the 5-position of triazole ring. This means that the
bulky substituents at the 3, 4 and 5 positions (i. e. R1) will increase
the fungicidal activity. For example, those compounds bearing
3,4,5-trimethoxyl groups in the benzene ring, such as 8a and 8e
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B.-L. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Fig. 2. Superimposition of the compounds for 3D QSAR study.
Table 4
Summary of CoMFA analysis.
q2
r2
F
Contribution (%) (6e)
Steric
41.7
Electrostatic
58.3
CoMFA
0.513
0.726
34.406
the benzylideneamino part were small groups, the fungicidal activ-
ity of the corresponding compounds were comparatively better,
which can be well reflected by the cases of compounds 6e (R1 =
H, R2 = F, 91.9%), 6f (R1 = H, R2 = F, 85.1%) and 6k (R1 = H, R2 = F,
87.8%). Furthermore, introducing small groups in the piperazine
ring (R3) could be favorable for the fungicidal activity of the com-
pounds, e. g. the compounds bearing a 4-methyl-2-pyrimidyl
group were more effective than those bearing 4,6-dimethyl-2-
pyrimidyl group (6b > 6c, 7b > 7c, 7f > 7g, 8b > 8c). For the electro-
static field contour map, the blue contour usually 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
Fig. 3(b), the title compounds bearing electron-withdrawing or
non-electron donating groups at the corresponding positions of
the two benzene rings illustrated in the figure (main effect), and
4 or 6 position of the pyrimidine ring (a little effect), such as 6e,
7b and 8f, exhibited higher activity. These results provide useful
information for further optimization of the compounds.
In summary, a series of new 3-substitutedphenyl-4-substitut-
edbenzylideneamino-1,2,4-triazole Mannich bases and bis-Man-
nich bases were synthesized through Mannich reaction with high
yields and their structures were confirmed by means of organic
spectroscopy and elemental analysis. Among 32 title new com-
pounds, some of them could effectively inhibit the activity of rice
KARI at present study. Particularly, compounds 7g, 7h and 7l
exhibited potent in vitro inhibitory activities against KARI enzyme
with Ki value of (0.38 0.25), (6.59 2.75) and (8.46 3.99) lmol/
L, respectively, and were comparable with IpOHA. These com-
pounds could be new KARI inhibitors for follow-up research. The
molecular docking calculation revealed that there exist strong H
bond and p-p stacking interactions between the best active inhibi-
tor 7g and residues THR519 and ARG522 of KARI, which probably
make great contributions to its biological activity. Furthermore,
the preliminary bioassay also showed that some of the compounds
displayed obvious herbicidal activities against Echinochloa crusgalli
Fig. 3. Contour maps of steric field (a) and electrostatic field (b) from CoMFA model.
at concentration of 100 lg/mL. It was worth noting that some of
the title compounds exhibited remarkable fungicidal activities
in vitro against Physalospora piricola and Rhizoctonia cerealis at 50
showed higher fungicidal activity. However, for those compounds
without bearing 2-nitro-3,4,5-trimethoxyl group, when R1 at the
ortho position of the benzene ring and R2 at the para position in
lg/mL concentration. In addition, the SAR of the compounds were
analyzed, in which the 3D-QSAR results provided useful informa-
Please cite this article in press as: Wang B.-L., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.10.065

6
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This work was supported by the National Natural Science Foun-
dation of China (No. 21372133), Zhejiang Province National Natu-
ral Science Foundation of China (No. LY16C140007) and the
National Key Research and Development Program of China (No.
2017YFD0200505). We thank Dr. Cong-Wei Niu and Dr. Yong-Hong
Li of Nankai University (China) for kind biological test assistance.
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.10.065.
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Please cite this article in press as: Wang B.-L., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.10.065