Novel Ferulic Amide Ac6c Derivatives: Design, Synthesis, and Their Antipest Activity

Article abstract of DOI:10.1021/acs.jafc.1c03892

Thirty-eight novel ferulic amide 1-aminocyclohexane carboxylic acid (Ac6c) derivativesD1-D19andE1-E19were designed and synthesized, and their antibacterial, antifungal, and insecticidal activities were tested. Most of the synthesized compounds displayed e

Full text of DOI:10.1021/acs.jafc.1c03892

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Novel Ferulic Amide Ac6c Derivatives: Design, Synthesis, and Their
Antipest Activity
Renfeng Zhang,† Shengxin Guo,† Peng Deng, Ya Wang, Ali Dai, and Jian Wu*
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ABSTRACT: Thirty-eight novel ferulic amide 1-aminocyclohexane carboxylic acid (Ac6c) derivatives D1−D19 and E1−E19 were
designed and synthesized, and their antibacterial, antifungal, and insecticidal activities were tested. Most of the synthesized
compounds displayed excellent activity againstXanthomonas oryzae pv. oryzae (Xoo), with EC50 values ranging from 11.6 to 83.1 μg/
mL better than that of commercial bismerthiazol (BMT, EC50 = 84.3 μg/mL), as well as much better performance compared to that
of thiediazole copper (TDC, EC50 = 137.8 μg/mL). D6 (EC50 = 17.3 μg/mL), D19 (EC50 = 29.4 μg/mL), E3 (EC50 = 29.7 μg/
mL), E9 (EC50 = 27.0 μg/mL), E10 (EC50 = 18.6 μg/mL), and E18 (EC50 = 20.8 μg/mL) showed much higher activity on
Xanthomonas oryzae pv. oryzicola compared with BMT (EC50 = 80.1 μg/mL) and TDC (EC50 = 124.7 μg/mL). In relation to
controlling the fungus, Rhizoctonia solani, E1, E10, and E13 had much lower EC50 values of 0.005, 0.140, and 0.159 μg/mL
compared to hymexazol at 74.8 μg/mL. Further in vivo experiments demonstrated that E6 and E12 controlled rice bacterial leaf
blight disease better than BMT and TDC did. Scanning electron microscopy (SEM) studies revealed that E12 induced the Xoo cell
membrane collapse. Moreover, D13 (73.7%), E5 (80.6%), and E10 (73.4%) also showed moderate activity against Plutella xylostella.
These results indicated that the synthesized ferulic amide Ac6c derivatives showed promise as candidates for treating crop diseases.
KEYWORDS: ferulic amide, Ac6c derivatives, synthesis, antibacterial activity, antifungal activity, insecticidal activity
and developing novel agrochemicals due to their unique
properties including biocompatibility with the environment
and their novel modes of action.14−16 As an important natural
product, the ferulic acid skeleton is the most widely used today
as it is the lead compound in NP-based drug design.1 Many
molecules derived from ferulic acid have shown various
physiological functions such as antioxidant,17 antibacterial,18
anticancer,19 anticoagulant,20 and anti-inflammatory actions.21
In particular, ferulic acid based compounds have also been
developed for inhibiting plant diseases in recent years. Some
novel ferulic acid skeleton-based compounds, such as amide
derivatives,22 chalcone derivatives,23 α-amino phosphonates
derivatives,24 acylhydrazone derivatives,25 and others, have
reportedly shown promising activity in relation to plant disease
control. In particular, combining an amino group with ferulic
acid could lead to ferulic amides, which can potentially be used
as fungicides in agriculture.24−28
INTRODUCTION
■
Crop pests and pathogens often result in quality and yield losses
in agricultural production.1−3 About 16−20% of the major food
crop production is lost worldwide each year due to preharvest
diseases.4 Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv.
oryzicola (Xoc) are good examples, as they can cause devastating
leaf blight and leaf stripe in rice crops and often lead to serious
disease outbreaks, which reduce crop yield by 20%, rising to 50%
under severe infection.5−8 In addition, as one of the most
aggressive pathogens, Xanthomonas axonopodis pv citri (Xac) in
citrus can cause serious canker and cut down citrus production
worldwide; also, as it is difficult to control once plants are
infected, it has a serious impact on the entire citrus industry.9−11
Currently, the use of chemical pesticides is still the main
measurement taken to control crop diseases and pests. However,
there is a severe shortage of available control agents and a limited
number of actions to take against these diseases. This is
especially true given the continuous increase in resistance or
cross-resistance by the pests and pathogens to the existing
pesticides;12,13 therefore, the prevention and control of crop
pests are becoming more and more difficult. Hence, exploration
of new active molecules with novel modes of action that exhibit
high activity yet possess low-risk profiles for plant pests and
pathogens is urgently needed.
1-Aminocyclohexane carboxylic acid (Ac6c) is also an
important non-proteinogenic quaternary α-amino acid, which
plays an important role in synthetic antimicrobial peptide and
drug discovery.29 The compounds or peptide analogues
containing Ac6c have shown excellent bioactivities including
anticonvulsant,30 antiproliferative,31 antitumor,32 antibacteri-
Received: June 29, 2021
Revised: August 3, 2021
Accepted: August 11, 2021
Natural products (NPs) play a significant role in exploring
novel pesticides. So far, more than 50% of the available
commercial pesticides have been derived from NPs, these
include conventional neonicotinoids, strobilurins, pyrethroids,
ethylicin, fenpiclonil, and fludioxonil, all of which mimic NPs.1
Nowadays, NP-based products are still hot topics for researching
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Figure 1. Design strategy for the target molecules.
Scheme 1. Synthetic Route of the Title Compounds D1−D19 and E1−E19
al,33 insecticidal,34 herbicidal,35 antifungal,36 and plant growth
regulator activities toward improving drought resistance in
plants.27,28 Some Ac6c derivatives also display excellent activity
levels against Phytophthora infestans in tomato plants.37 So far,
about ∼150 Ac6c pharmacophore-containing compounds have
been discovered.38 Moreover, the Ac6c structure displays two
superiorities: (1) The cyclic ring and quaternary α-carbon atom
in Ac6c could restrict its ability to become an intrinsically stable
conformation. This feature often results in a more efficient and
selective ligand than a nonrestricted one when they act upon the
target.39,40 (2) Ac6c possesses a saturated cyclohexane core,
ensuring that Ac6c derivatives are more lipophilic.41 These
factors have ensured that interest in Ac6c derivatives has grown.
The method of “substructure combination” is often used in
pesticide design.42 The rationale of this method is integrated
with different substructures in some active compounds via a
functional linker (such as amide, ether, etc);23,43 based on this
method, a potentially active compound might be ob-
tained.23,24,42,43 Hence, encouraged by the qualities described
above, we sought to combine the ferulic acid core with Ac6c via a
functional amide, then achieve structural diversity through the
reaction site at the hydroxyl on ferulic acid to produce novel
ferulic amide Ac6c derivatives (Figure 1). Antibacterial activities
of these derivatives were then tested on Xoo, Xoc, and Xac, and
antifungal activities were evaluated against Cytospora mandshur-
ica, Colletotrichum gloeosporioides, Gibberella zeae, Sclerotinia
sclerotiorum, Fusarium oxysporum, and Rhizoctonia solani.
Insecticidal activity against diamondback moth (Plutella.
xylostella) was also investigated. Successful results showed that
most of the designed compounds showed outstanding
antibacterial activity. In vivo experiments and the morphological
influence on Xoo were also considered.
MATERIALS AND METHODS
■
Instruments and Chemicals. 1H, 13C, and 19F NMR spectra were
detected on a JEOL ECX 500 NMR (JEOL Ltd., Tokyo, Japan) or an
AVANCE III HD 400M NMR (Bruker Corporation, Fallanden,
Switzerland) spectrometer using dimethyl sulfoxide (DMSO) as the
solvent. The melting points of the ferulic amide Ac6c derivatives D1−
D19 and E1−E19 were measured by a binocular microscope melting
point apparatus (XT-4, Beijing Tech Instrument Co., Beijing, China).
High-resolution mass spectrometry (HR-MS) was given by an Orbitrap
liquid chromatography−mass spectrometry (LC-MS) (Q-Exative,
Thermo Scientific). Scanning electron microscopy (SEM) images
were obtained and visualized using Nova NanoSEM 450 (Thermo
Fisher Scientific, Massachusetts). Ferulic acid methyl ester was bought
from TCI (Tokyo, Japan) and the substituted halogenated hydro-
carbons and 1-aminocyclohexane carboxylic acid were supplied by
Accela (Shanghai, China). All of the reactions were monitored by TLC.
General Procedure for Preparing D1−D19 and E1−E19.
Scheme 1 depicts the synthetic route for the title compounds D1−D19
and E1−E19. They were provided via several steps that started from
B
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Table 1. In Vitro Antibacterial Activity of the Target Compounds against Xoo, Xac, and Xoc
Xoo
Xoc
Xac
compd.
inhibition rate (%)
inhibition rate (%)
inhibition rate (%)
100 μg/mL
50 μg/mL
100 μg/mL
50 μg/mL
100 μg/mL
50 μg/mL
D1
D2
D3
D4
D5
D6
D7
D8
71.4 1.4
56.0 2.9
87.9 1.7
40.4 3.8
90.3 3.6
70.5 1.4
67.7 2.7
57.8 2.8
95.9 1.0
41.1 0.6
46.5 0.0
71.8 1.6
68.0 3.7
28.9 2.4
69.3 1.6
65.1 1.9
38.2 1.7
21.1 5.0
74.1 4.9
61.4 1.4
67.4 3.7
55.9 3.2
86.9 0.5
72.9 3.5
97.8 0.8
96.9 0.4
93.5 0.1
97.4 0.1
67.9 1.7
50.3 3.1
90.1 0.6
94.3 1.2
88.3 3.5
97.9 1.0
97.2 1.7
88.7 4.1
68.5 1.4
74.9 4.9
67.8 2.6
56.2 0.6
36.9 2.6
62.6 2.3
30.5 3.0
82.4 2.6
63.0 0.7
62.1 2.1
42.7 4.1
90.0 0.2
35.0 0.2
23.3 3.4
57.2 2.0
61.6 0.1
26.2 2.5
73.7 3.1
52.3 1.1
21.6 1.9
19.5 3.8
63.1 4.3
44.2 0.6
59.1 0.3
47.0 0.4
55.2 0.9
58.2 0.2
87.0 4.8
95.1 1.3
66.8 1.3
91.8 0.1
63.6 3.2
30.7 2.7
88.7 3.7
81.2 3.1
69.5 1.3
76.1 1.8
79.7 4.6
84.7 3.1
50.4 3.4
63.0 4.3
47.3 2.7
34.6 1.9
33.2 4.6
40.9 1.2
41.8 1.4
45.1 2.1
41.3 3.3
64.8 2.2
45.0 4.7
56.3 4.5
59.6 1.7
35.1 4.3
43.9 3.7
68.5 4.0
70.2 3.6
39.7 2.9
40.8 1.3
34.5 2.2
32.6 0.2
58.5 0.7
70.7 2.6
55.0 4.9
46.2 3.7
76.4 2.4
75.2 3.9
42.8 2.0
55.9 2.1
37.7 2.7
53.9 3.7
74.1 0.7
80.8 0.7
56.2 2.9
54.5 4.4
54.6 1.6
37.0 2.5
32.9 1.8
65.2 3.1
62.3 2.9
69.6 0.4
67.4 4.1
59.0 0.7
60.8 3.4
32.3 2.3
38.1 4.7
39.3 0.2
40.2 2.4
29.1 1.3
55.9 1.1
36.3 3.0
41.5 0.2
50.5 5.0
28.0 4.9
40.6 0.6
60.6 2.9
68.7 4.7
36.9 1.6
38.1 3.6
21.9 1.2
19.1 3.7
48.6 1.1
53.2 1.0
43.8 1.3
28.4 1.3
61.1 4.1
54.4 1.5
40.1 0.7
39.3 3.5
34.4 4.7
41.3 4.0
63.3 1.2
76.4 2.3
19.2 0.9
43.1 1.1
47.4 4.4
28.8 2.4
25.9 2.4
54.0 0.6
58.2 1.3
60.6 3.4
43.5 0.7
50.8 2.8
42.0 2.2
64.2 0.2
47.3 1.2
66.4 4.0
30.2 4.5
75.2 3.3
79.7 1.1
47.8 2.8
74.8 4.5
53.5 4.2
22.8 1.4
50.8 1.8
89.1 3.2
45.4 4.8
34.8 2.8
36.7 3.6
20.4 4.7
35.8 4.1
8.9 1.9
59.2 4.8
54.6 4.9
50.7 2.1
71.1 3.9
20.0 2.5
56.8 0.6
58.1 4.3
74.4 3.3
53.9 3.0
69.9 0.1
54.7 0.6
53.1 4.2
47.6 4.6
81.8 1.9
28.5 4.3
27.2 4.7
72.1 4.7
70.5 1.7
65.2 1.1
65.9 1.3
62.8 1.7
51.0 1.2
27.5 2.9
45.2 4.8
30.5 0.7
24.9 1.3
42.2 0.2
45.0 2.7
48.7 4.6
39.3 4.5
12.5 2.4
8.5 1.0
33.1 4.8
77.3 0.7
25.3 1.6
24.9 1.8
23.4 0.6
17.1 1.1
10.0 0.4
2.7 1.5
42.8 4.1
47.8 3.3
14.3 0.5
54.2 4.5
7.6 2.6
33.6 2.1
41.1 4.3
51.5 3.8
37.2 0.5
46.4 2.0
35.9 1.8
41.1 1.6
40.4 1.0
67.5 3.7
72.6 2.1
7.1 3.2
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
E1
E2
E3
E4
E5
E6
E7
E8
E9
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
50.5 3.4
38.1 1.4
51.5 4.5
39.4 3.1
44.8 5.0
22.0 2.4
b
BMT
TDC
b
65.8 0.5
a
b
Average of three replicates. Commercialized bactericides bismerthiazol (BMT) and thiediazole copper (TDC).
ferulic acid methyl ester as the starting materials, involving two
intermediates B and C.43−45 First, the intermediates B and C were
prepared using a classical literature method with ferulic acid methyl
ester used as the starting material.43,44 Then, the target compounds
D1−D19 were synthesized in accordance with previous reports.45
Finally, due to the conjugate effect of a double bond in α,β-unsaturated
amide, the target compounds D1−D19 were selectively hydrolyzed to
prepare the target compounds E1−E19.44 The details of this synthesis
process are shown in the Supporting Information (SI).
In Vitro Antibacterial Activity Test. Using the turbidimetric
method in refs 46−48, in vitro antibacterial activities of D1−D19 and
E1−E19 against Xoo, Xac, and Xoc were tested. BMT and TDC were
used as positive controls. The test method utilized is given in the
Supporting Information.
modification.49 This test method is provided in the Supporting
Information (SI).
Scanning Electron Microscopy (SEM) Characterization. The
influence of the most active compound E12 on Xoo was observed using
SEM in accordance with methods previously published,48,50 as also
shown in the SI.
In Vitro Antifungal Bioassay. The antifungal activities of D1−
D19 and E1−E19 against F. oxysporum (F. o.), C. mandshurica (C. m.),
C. gloeosporioides (C. g.), G. zeae (G. z.), S. sclerotiorum (S. s.), and R.
solani (R. s.) were evaluated using a previously reported method,51 in
which fungicides hymexazol (HM) and carbendazim were put to
positive control use. The test particulars can be found in the SI.
Insecticidal Activity Test on Plutella Xylostella (P. xylostella).
The insecticidal activities of D1−D19 and E1−E19 were obtained
using previously published methods.52,53 The insecticide chlorpyrifos
was employed as a positive control, and distilled water containing 1%
DMSO was used as a blank control.
In Vivo Antibacterial Activity Test. The curative and protective
activities of compounds E6 and E12 for controlling rice leaf blight were
tested in vivo by employing a previously reported method with little
C
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Table 2. EC50 Values of Target Compounds against Xoo
compd.
toxic regression equation
r
EC50 (μg/mL)
compd.
toxic regression equation
r
EC50(μg/mL)
D1
y = 1.23x + 3.26
0.92
25.6 1.1
E1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
E2
E3
E4
E5
E6
E7
E8
E9
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
y = 0.41x + 4.10
y = 0.31x + 4.41
y = 1.42x + 2.60
y = 1.13x + 2.96
y = 1.71x + 2.83
y = 1.34x + 2.73
y = 2.02x + 2.09
y = 1.65x + 2.42
y = 2.42x + 1.37
y = 0.46x + 4.19
y = 1.00x + 3.93
y = 1.53x + 2.95
y = 1.25x + 2.97
y = 2.04x + 2.04
y = 1.59x + 2.96
y = 1.21x + 3.26
y = 0.53x + 3.89
0.92
0.95
0.92
0.99
0.98
0.93
0.97
0.93
0.97
0.99
0.98
0.92
0.94
0.93
0.91
0.96
0.97
77.7 10.0
83.1 8.9
49.3 5.2
62.9 9.8
14.6 1.5
49.5 8.8
27.2 0.5
24.0 0.3
31.1 1.4
55.3 6.5
11.6 3.6
14.8 1.5
18.4 5.2
28.1 + 1.1
19.1 5.0
27.3 4.3
128.2 5.2
y = 0.63x + 4.11
0.92
26.8 8.9
y = 1.81x + 2.12
y = 0.71x + 3.77
y = 0.42x + 4.20
0.9
0.91
0.97
39.1 2.1
53.5 1.0
78.2 6.1
y = 1.57x + 2.58
0.91
34.6 8.1
y = 0.88x + 3.38
y = 2.07x + 1.47
0.93
0.97
67.8 5.7
49.6 1.1
y = 0.81x + 3.94
y = 0.60x + 3.94
0.92
0.94
19.9 1.9
59.6 4.3
y = 1.14x + 3.02
y = 0.83x + 3.39
0.95
0.91
54.4 6.4
84.3 9.1
b
b
BMT
TDC
y = 1.30x + 2.21
0.94
137.8 4.3
a
b
Average of three replicates. Commercialized bactericides bismerthiazol (BMT) and thiediazole copper (TDC).
a
Table 3. EC50 Values of Target Compounds against Xoc
compd.
toxic regression equation
r
EC50 (μg/mL)
compd.
toxic regression equation
r
EC50(μg/mL)
D1
E1
D2
E2
D3
D4
E3
E4
y = 0.75x + 3.92
y = 0.84x + 3.58
0.93
0.91
29.7 2.3
47.7 4.7
D5
E5
D6
D7
D8
D9
y = 0.21x + 4.74
y = 0.72x + 3.59
1.00
1.00
17.3 8.2
91.5 5.2
E6
E7
E8
E9
y = 0.68x + 4.02
y = 0.82x + 3.96
0.96
0.94
27.0 3.0
18.6 1.9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
y = 0.59x + 3.93
y = 0.85x + 3.72
0.95
0.91
63.4 9.6
31.9 4.1
y = 0.29x + 4.40
0.92
119.0 2.8
y = 0.32x + 4.50
y = 0.35x + 4.46
y = 0.49x + 4.35
y = 1.22x + 2.50
y = 0.87x + 3.17
0.95
0.96
0.92
0.96
0.97
38.7 4.9
32.7 2.3
20.8 6.1
109.9 6.5
124.7 5.0
y = 0.84x + 3.24
y = 0.59x + 4.14
y = 0.47x + 4.10
0.93
0.96
0.98
122.8 3.2
29.4 7.4
80.1 4.3
b
b
BMT
TDC
a
b
Average of three replicates. Commercialized bactericides bismerthiazol (BMT) and thiediazole copper (TDC).
(HRMS), and the spectrum information is listed in the
Supporting Information.
RESULTS AND DISCUSSION
■
Chemistry. As exhibited in the synthetic route (Scheme 1).
Analysis of In Vitro Activity toward Xoo, Xoc, and Xac.
In Vitro Activity Analysis on Xoo. The preliminary activity in
vitro against bacterium Xoo is listed in Table 1, and the
concentration for 50% of maximal effect (EC50) is listed in Table
2. As shown in Table 1, most of the title compounds exhibited
good activity at both 100 and 50 μg/mL. Particularly, the
activities of compounds D9, E6, E7, E8, E9, E13, E15, and E16
were close to 100% at 100 μg/mL, and compounds D3, D5, E4,
E12, E4, E12, E14, and E17 recorded more than 88% activities
at 100 μg/mL, which were much higher than that of BMT
(67.8%) and TDC (65.8%). While the concentration was set to
The intermediate B was easily prepared via a reaction of starting
material A with benzyl chloride or benzyl bromine in the
presence of KI and K2CO3 using N,N-dimethylformamide as a
solvent. Intermediate B was hydrolyzed to obtain intermediate
C, which underwent a reaction with methyl 1-aminocyclohex-
ane-1-carboxylate to obtain the title compounds D1−D19.43−45
After the hydrolysis of the title compounds D1−D19, the title
compounds E1−E19 were prepared.44 The compounds D1−
D19 and E1−E19 were characterized by 1H, 13C, and 19F NMR,
and high-resolution liquid chromatography−mass spectroscopy
D
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Table 4. Protection and Curative Activities of Compound E6 and E12 against Rice Bacterial Leaf Blight under Greenhouse
Conditions at 200 μg/mL In Vivo
protection activity (14 days after spraying)
curative activity (14 days after spraying)
a
a
treatment
morbidity (%)
disease index (%)
control efficiency (%)
morbidity (%)
disease index (%)
control efficiency (%)
E6
100
100
100
100
100
44.6
46.7
56.9
60.0
88.1
49.4
47.0
35.3
31.9
100
100
100
100
100
51.5
46.7
56.0
60.0
83.9
38.6
44.4
33.2
28.5
E12
BMT
TDC
b
CK
a
Statistical analysis was conducted by ANOVA method under the condition of assumed equal variances (P > 0.05) and not assumed equal
variances (P < 0.05). Different uppercase letters indicate the values of protection activity with significant difference among different treatment
b
groups at P < 0.05. Blank control.
Figure 2. Protective activities of compounds E6 and E12 against rice leaf blight at 200 μg/mL.
50 μg/mL, the activities of compounds D5, D9, E7, E9, E12,
and E13 were also higher than 80%. As shown in Table 2, the
EC50 values of the active title compounds ranged from 11.6 to
128.2 μg/mL. Most of their EC50 values were much lower than
these of BMT (84.3 μg/mL) and TDC (137.8 μg/mL).
In Vitro Activity Analysis on Xoc. The preliminary activity in
vitro against bacterium Xoc is listed in Table 1, and the EC50 is
listed in Table 3. Compared with the activity toward Xoo, the
activity toward Xoc was relatively lower, but there were also 15
compounds that had lower EC50 values ranging from 17.3 to
122.8 μg/mL, compared to that of TDC (124.7 μg/mL), and
eleven compounds that had lower EC50 values than that of BMT
(80.1 μg/mL).
In Vitro Activity Analysis on Xac. With regards to controlling
Xac bacterium, most of the title compounds did not exhibit good
activity, but compounds D12 and E13 showed higher activities,
beyond 80% higher, than both BMT (62.8%) and TDC (51.0%)
(Table 1).
Structure−Activity Relationship Analysis of Com-
pounds D1−D19 and E1−E19. Based on the results of
Tables 1−3, the structure−activity relationship (SAR) was
deduced, which concluded the following points: (i) With the
same R group, the antibacterial activities of compounds D1−
D19 and their hydrolysates E1−E19 are quite different.
Generally, most of the hydrolysate E with group −COOH had
higher activity in vitro than that of compound D. For example,
the active compounds E6 (R = 2-F-3-Cl-Ph; EC50 14.6 μg/mL),
E
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Figure 3. Curative activities of compounds E6 and E12 against rice leaf blight at 200 μg/mL.
E12 (R = 3,4-diF-Ph; EC50 11.6 μg/mL), and E13 (R = 3,5-diF-
Ph; EC50 14.8) had much lower EC50 values against Xoo than
compounds D6 (R = 2-F-3-Cl-Ph; EC50 53.5 μg/mL), D12 (R =
3,4-diF−Ph; EC50 67.8 μg/mL), and D13 (R = 3,5-diF-Ph; EC50
49.6 μg/mL). The carboxyl strengthening the hydrophobic
reaction could be a potential reason for these increases in
activity.36 (ii) The R group including a weak electron-donating
group −CH3, strong electron-withdrawing groups −CF3, −F,
and −OCF3, and moderate electron-withdrawing groups −Cl
and −Br could lead to significant differences in antibacterial
activity at different positions of the phenyl group. In terms of
Xoo, the location effect is listed as follows: D1 (R = 2-CH3-Ph) >
D2 (R = 3-CH3-Ph); D4 (R = 4-Cl-Ph) > D11 (R = 2-Cl-Ph);
D10 (R = 3-F-Ph) > D19 (R = 4-F-Ph); D12 (R = 3,4-diF-Ph) >
D13 (R = 3,5-diF-Ph) > D17 (R = 2,5-diF-Ph); E2 (R = 3-CH3-
Ph) > E1 (R = 2-CH3-Ph); E4 (R = 4-Cl-Ph) > E11 (R = 2-Cl-
Ph); E19 (R = 4-F-Ph) > E10 (R = 3-F-Ph); and E13 (R = 3,5-
diF-Ph) > E12 (R = 3,4-diF-Ph) > E17 (R = 2,5-diF-Ph). The
location effect also existed when controlling Xoc and Xac. (iii)
Halogen atoms substituted in phenyl significantly affected the
antibacterial activity. When one or two halogen atoms are
substituted in phenyl, the title compounds could have a higher
antibacterial activity against both Xoc and Xac to some degree
when compared with D15 (R = Ph) and E15 (R = Ph).
Moreover D18, in which the phenyl is substituted by the Br
atom, or in other compounds where the phenyl is substituted by
Cl atom, showed relatively lower activity compared with the
compounds where phenyl was substituted by an F atom
including D3 and E3 (R = 4-F-5-CF3-Ph); D6 and E6 (R = 2-F-
3-Cl-Ph); D7 and E7 (R = 2-F-6-F-Ph); D8 and E8 (R = 2-Cl-4-
F-Ph); D9 and E9 (R = 4-OCF3-Ph); D10 and E10 (R = 3-F-
Ph); D13 and E13 (R = 3,5-diF-Ph); and D19 and E19 (R = 4-
F-Ph). The potential factor is that the introduction of fluorine
atoms could improve the lipophilicity and bioavailability of these
compounds.54,55
In Vivo Antibacterial Activity Analysis. According to the
results in Tables 1−3, it was clear that the title compounds of
hydrolysates E6 and E12 showed the lowest EC50 values against
Xoo, reaching 14.6 and 11.6 μg/mL. Therefore, E6 and E12 were
selected to verify their applications within rice crops on rice
blight at 200 μg/mL in a greenhouse. As shown in Table 4 and
Figures 2 and 3, the protective activities of both E6 and E12
were 49.4 and 47.0%, which were both higher than BMT
(35.3%) and TDC (31.9%). In addition, the curative activity of
both E6 and E12 were measured at 38.6 and 44.4%, which were
also higher than those of BMT (33.2%) and TDC (28.5%).
These results showed that compounds E6 and E12 had an
effective efficiency on rice bacterial blight in rice crops and could
therefore be considered as antibacterial agent candidates.
F
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Figure 4. SEM images for the influence of E12 on Xoo at (A) 0 μg/mL, (B) 25.0 μg/mL, and (C) 50.0 μg/mL.
a
Table 5. Antifungal Activities of the Target Compounds D1−D19 and E1−E19 at 50 μg/mL In Vitro
inhibition rate (%)
b
c
d
e
f
g
compd.
D1
D2
D3
D4
D5
D6
D7
D8
F. o.
C. m.
C. g.
G. z.
S. s.
R.s.
7.8 2.6
0
9.1 1.0
7.0 0.8
8.2 1.3
10.5 0.9
9.9 2.5
17.3 3.5
14.3 4.0
11.4 0.9
7.0 1.8
8.5 0.5
14.3 0.5
13.7 3.3
14.6 1.0
13.5 4.8
8.5 1.0
9.4 1.3
12.9 4.0
15.8 1.8
14.6 1.8
8.9 0.3
5.9 2.5
13.0 2.8
32.6 3.7
0
20.9 2.4
22.1 3.7
24.1 3.2
32.1 1.0
21.5 3.7
22.4 1.4
19.1 0.9
24.2 0.5
21.8 0.0
20.6 2.3
22.4 3.7
13.6 1.8
28.6 3.2
29.8 2.9
10.6 0.5
19.7 5.0
20.3 1.0
13.6 4.5
7.3 1.6
10.7 0.4
11.5 1.0
24.2 2.5
12.5 5.0
44.2 0.5
4.3 1.7
6.8 0.9
32.5 3.6
20.6 3.9
10.0 2.6
23.9 3.7
19.2 2.1
20.1 1.3
22.2 3.1
0
49.6 0.9
16.2 0.9
38.7 2.5
0
15.9 2.3
27.7 3.2
22.7 0.9
39.9 5.0
0
28.1 1.2
28.1 3.9.
23.9 1.6
16.3 1.2
36.0 0.9
23.4 0.9
29.1 0.0
17.1 1.2
20.7 0.5
19.7 0.0
29.1 1.6
23.6 1.6
13.9 0.6
25.7 2.5
20.7 1.6
35.8 2.8
24.4 2.8
34.1 3.0
33.4 0.4
71.5 2.8
26.9 2.1
31.1 1.8
47.3 0.5
0
54.0 1.3
54.9 2.2
53.6 2.7
99.4 1.0
12.8 3.4
98.2 0.0
57.6 2.2
44.6 0.0
49.5 0.4
15.2 4.5
45.1 0.4
32.1 2.7
41.1 3.6
48.6 1.3
30.4 0.9
47.7 3.1
38.1 4.1
79.9 2.4
50.6 2.9
100.0 1.0
36.2 3.5
56.4 4.8
41.3 4.8
0
5.7 1.0
6.3 2.0
15.0 3.3
12.1 1.7
16.4 4.0
11.8 3.6
8.9 2.2
11.2 2.6
12.9 1.7
13.5 3.0
10.9 3.9
8.6 1.7
5.7 2.2
12.5 1.3
14.4 3.0
26.4 1.8
13.5 0.5
12.0 0.9
3.4 2.6
50.1 2.2
33.7 3.9
0
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
E1
7.4 0.4
E2
E3
E4
E5
12.3 0.0
12.7 0.4
18.0 3.1
0
E6
E7
E8
E9
33.0 0.3
21.4 0.9
43.4 1.1
41.4 0.4
36.4 4.6
42.0 1.5
24.3 1.2
22.3 1.8
22.6 1.7
16.9 2.0
36.0 0.9
31.7 3.8
27.4 4.4
18.0 3.7
37.2 3.7
23.4 3.3
5.7 1.5
8.1 2.4
30.7 4.1
5.5 + 1.9
23.6 4.0
0
26.3 0.9
31.9 1.0
43.9 0.9
37.1 4.1
19.3 3.5
30.1 3.7
23.7 2.3
12.0 0.5
29.5 1.0
34.7 3.9
38.3 3.7
32.5 1.8
39.2 3.1
21.3 3.3
61.2 2.3
8.6 1.4
12.1 4.2
3.6 0.9
49.7 2.3
12.4 3.0
50.9 3.6
0
26.4 4.8
0
0
50.9 3.6
0.6 4.2
29.7 4.2
0
58.5 4.3
98.3 0.0
34.2 2.3
24.8 3.0
24.8 0.0
67.1 0.4
1.1 1.7
33.6 3.5
24.2 3.0
25.9 2.7
17.7 1.3
20.8 1.3
42.1 4.9
31.6 1.7
38.5 2.6
30.5 2.0
48.6 1.9
4.2 2.0
83.6 2.9
41.3 1.0
84.0 1.6
95.6 1.4
22.3 4.0
4.8 1.0
81.2 0.5
82.2 2.4
82.2 2.4
56.9 4.0
42.3 1.9
28.1 3.1
57.7 0.0
65.7 1.4
100.0 0.0
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
13.2 2.2
0
5.9 1.1
12.2 0.3
38.2 2.6
29.8 1.6
10.0 1.7
43.0 3.1
h
HM
carbendazim
98.3 0.1
100.0 0.0
99.4 1.0
97.0 2.1
a
b
c
d
e
f
g
The average of three trials. F. o. = FF. oxysporum. C. m. = C. mandshurica. C. g. = C. gloeosporioides. G. z. = G. zeae. S. s. = S. sclerotiorum. R. s.
h
= R. solani. Commercialized agrofungicide hymexazol (HM).
Scanning Electron Microscopy (SEM) Characterization
Analysis. With the help of SEM, morphological observation of
bacterium Xoo was carried out and morphological differences
following treatment with the most active compound E12 were
G
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a
Table 6. EC50 values of Active Compounds against Fungi S. s., and R. s. In Vitro
comp.
pathogens
regression equation
correlation coefficient (r)
EC50(μg/ mL)
E1
E9
S. s.
S. s.
S. s.
S. s.
R. s.
R. s.
R. s.
R. s.
R. s.
R. s.
R. s.
R. s.
R. s.
R. s.
R. s.
R. s.
y = 0.41x + 4.27
y = 1.12x + 3.16
y = 0.99x + 3.03
y = 1.31x + 4.86
y = 1.89x + 3.58
y = 2.27x + 2.57
y = 0.66x + 4.33
y = 0.41x + 5.93
y = 0..51x + 4.78
y = 0..47x + 4.91
y = 0.64x + 5.28
y = 0.29x + 5.23
y =0.53x + 4.74
y =0.44x + 4.85
y = 0.76x + 3.57
y = 1.21x + 5.09
0.94
0.94
0.94
0.92
0.95
0.90
0.98
0.94
0.96
0.99
0.91
0.95
0.91
0.96
0.92
0.90
60.6 4.6
43.8 4.5
96.3 3.6
1.3 0.7
b
HM
carbendazim
D4
D6
D18
E1
E7
E9
E10
E13
E14
E15
5.6 1.1
11.7 0.7
10.1 1.3
0.005 0.004
2.6 1.0
1.6 1.0
0.370 0.117
0.159 0.002
3.1 0.3
2.1 0.4
74.8 0.7
0.8 0.5
b
HM
carbendazim
a
b
Average of three replicates. The agricultural fungicide hymexazol (HM) was used to compare the antifungal activities of the title compounds.
analyzed in more depth. As shown in Figure 4 A, the bacterium
Xoo grown without the addition of the compound solution
treatment had slender cells with a hard surface, thus classified as
a good appearance. After the treatment of E12, the cell
membranes of Xoo collapsed to a varying degree; as the E12
concentrations increased, cell membrane damage severity also
increased (Figure 4B,C). From the SEM characterization of Xoo,
it was clear that E12 resulted in cell membrane deformation or
rupture, which could be the potential mechanism to inhibit
bacterial growth.
mL). Among them, the most active compound E1 (R = 2-CH3−
Ph) had the lowest EC50 value, which reached 0.005 μg/mL and
effectively controlled the growth of the fungus R. s. at a range of
concentrations (Figure S1). From Tables 5 and 6, it can be
observed that the synthesized compounds had the highest
activity against R. s. but no activity against C. m., C. g., or G. z. It
should be noted that most of the hydrolysates E obtained via
hydrolysis of D also exhibited a higher activity than the title
compound D itself. These results indicated that synthesized
novel ferulic amide Ac6c derivatives could also be considered as
antifungal agents for further structural modification.
In Vitro Antifungal Activity Analysis. The preliminary
antifungal activities of D1−D19 and E1−E19 against six fungi,
including F. o., C. m., C. g., G. z., S. s., and R. s. are concluded in
Table 5, and the EC50 values of the active compounds are shown
in Table 6. The data revealed that the compounds displayed
good to excellent antifungal activity against F. o., S. s., and R. s. at
50 μg/mL, particularly E3 and E8, which had good activities at
50.1 and 43.4% against F. o., E9, which had good activities at
41.4, 67.1, and 84.0% against F. o., S. s., and R. s., respectively,
E11, which had good activity at 42.0% against F. o., E1, which
had excellent activity at 71.5% and 100.0% against S. s. and R. s.,
respectively, D4, which showed significantly high activity at
99.4% against S. s., D6, which showed significantly high activity
at 98.2% against S. s., D18, which showed excellent activity at
79.9% against S. s., E7, which showed excellent activity at 83.6%
against S. s., E10, which showed significantly high activity at
95.6% against S. s., E13, which showed excellent activity at 81.2%
against S. s., E14, which showed excellent activity 82.2% against
S. s., and E15, which showed excellent activity at 82.2% against S.
s. All of the antifungal activity levels above were higher than
those of HM (37.2, 48.6, and 65.7% against F. o., S. s., and R. s.,
respectively), and antifungal activity against R. s. of compounds
D4, D6, E1, and E10 were also similar to that of carbendazim
100%. As shown in Table 6, the active compounds E1 and E9
provided impressive actions toward S. s., with the EC50 values of
60.4 and 43.8 μg/mL, respectively, which were evidently higher
than that of HM (96.3 μg/mL). Moreover, the active
compounds D4, D6, D18, E1, E7, E9, E10, E13, E14, and
E15 had the EC50 values of 5.6, 11.7, 10.1, 0.005, 2.6, 1.6, 0.37,
0.159, 3.1, and 2.1 μg/mL, respectively, much better than the
HM levels (74.8 μg/mL). The EC50 values of compounds E1,
E10, and E13 were also lower than that of carbendazim (0.8 μg/
Insecticidal Activity Analysis. Table 7 lists the results of
the insecticidal activities for D1−D19 and E1−E19 against P.
Table 7. Insecticidal Activities of the Title Compounds
against P. xylostella
P. xylostella
P. xylostella
500 μg/mL mortality
500 μg/mL mortality
a
a
compd.
rate (%)
compd.
rate (%)
D1
D2
D3
D4
D5
D6
D7
D8
50.3 1.0
40.2 1.0
40.6 2.5
55.1 0.5
53.9 1.6
37.3 1.2
45.4 0.5
6.7 1.0
67.9 0.5
57.8 0.5
37.0 0.5
60.3 0.5
73.7 2.0
57.3 2.0
63.1 0.5
67.5 1.0
40.9 1.0
47.4 0.5
50.6 2.3
0
E1
E2
E3
E4
E5
E6
E7
E8
33.3 1.5
37.1 3.0
47.4 3.8
57.6 1.7
80.6 0.6
23.8 1.2
63.0 0.6
33.4 1.0
20.7 2.1
73.4 1.5
50.7 4.2
20.9 0.6
47.3 1.0
5.4 3.2
33.7 1.7
50.9 2.0
50.2 2.0
30.8 1.5
30.1 4.3
100.0 0.0
D9
E9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
chlorpyrifos
blank
control
a
average of three replicates.
H
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Article
xylostella at 500 μg/mL. Some compounds showed moderate
insecticidal activity, such as compounds D13 (R = 3.5-diF−Ph),
E5 (R = CH2CH2CH2Cl), and E10 (R = 3-F−Ph) showed
activities of 73.7, 80.6, and 73.4%, respectively. Obviously, the
synthesized compounds did not exhibit good insecticidal activity
toward P. xylostella, but the structural modification of these
structures for exploring the insecticidal molecules is ongoing in
our laboratory.
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Guiyang 550025, China
Ali Dai − State Key Laboratory Breeding Base of Green Pesticide
and Agricultural Bioengineering, Key Laboratory of Green
Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Guiyang 550025, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.1c03892
In conclusion, novel ferulic amide Ac6c derivatives were
synthesized in the present work, and bioassays targeting three
kinds of bacteria (Xoo, Xoc, and Xac), six kinds of fungi (F.
oxysporum, C. mandshurica, C. gloeosporioides, G. zeae, S.
sclerotiorum, and R. solani), and the pest P. xylostella were
carried out. Some more active synthesized compounds were
compared against commercial pesticides, and screening
indicated that they should be considered as agrochemical
candidates, such as E6 and E12, which showed significantly
higher activity against Xoo and also performed well in the pot
experiment and SEM of in vivo studies. E1 whose EC50 value
reached 0.005 toward the fungus R. solani was an example.
Through the structure−activity relationship, it was shown that in
most cases, the hydrolysis of the title compound D resulted in
the more active title compound E, which could be an inspiration
for the discovery of agrochemical agents.
Author Contributions
†R.Z. and S.G. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to express our thanks for the financial support
from NSFC (Natural Science Foundation of China) (Nos.
21762012, 32072445, and 21562012), the Program of
Introducing Talents to Chinese Universities (111 Program,
D20023), and the S & T Planning Project of Guizhou Province
(Nos. [2017] 1402 and [2017] 5788).
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
■
Corresponding Author
Jian Wu − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Guiyang 550025, China;
orcid.org/0000-0002-9173-6608; Phone: +86-851-
88292090; Email: jwu6@gzu.edu.cn, wujian2691@
126.com
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A. B.; Garcia, C. C. M.; Setubal, J. C.; Ferro, J. A.; Oliveira, J. C. F.
Proteomics-based identification of differentially abundant proteins
Authors
Renfeng Zhang − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Guiyang 550025, China
Shengxin Guo − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Guiyang 550025, China
Peng Deng − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Guiyang 550025, China
Ya Wang − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
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https://doi.org/10.1021/acs.jafc.1c03892
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
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https://doi.org/10.1021/acs.jafc.1c03892
J. Agric. Food Chem. XXXX, XXX, XXX−XXX