Design and Discovery of Novel Antifungal Quinoline Derivatives with Acylhydrazide as a Promising Pharmacophore

Article abstract of DOI:10.1021/acs.jafc.1c00670

Inspired by natural 2-quinolinecarboxylic acid derivatives, a series of quinoline compounds containing acylhydrazine, acylhydrazone, sulfonylhydrazine, oxadiazole, thiadiazole, or triazole moieties were synthesized and evaluated for their fungicidal activity. Most of these compounds exhibited excellent fungicidal activity in vitro. Significantly, compound 2e displayed the superior in vitro antifungal activity against Sclerotinia sclerotiorum, Rhizoctonia solani, Botrytis cinerea, and Fusarium graminearum with the EC50 values of 0.39, 0.46, 0.19, and 0.18 μg/mL, respectively, and were more potent than those of carbendazim (EC50, 0.68, 0.14, >100, and 0.65 μg/mL, respectively). Moreover, compound 2e could inhibit spore germination of F. graminearum. Preliminary mechanistic studies showed that compound 2e could cause abnormal morphology of cell walls and vacuoles, loss of mitochondrion, increases in membrane permeability, and release of cellular contents. These results indicate that compound 2e displayed superior fungicidal activities and could be a potential fungicidal candidate against plant fungal diseases.

Full text of DOI:10.1021/acs.jafc.1c00670

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Article
Design and Discovery of Novel Antifungal Quinoline Derivatives
with Acylhydrazide as a Promising Pharmacophore
Yu-Dong Yang,§ Ying-Hui He,§ Kun-Yuan Ma, Hu Li, Zhi-Jun Zhang,* Yu Sun, Yu-Ling Wang,
Guan-Fang Hu, Ren-Xuan Wang, and Ying-Qian Liu*
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ABSTRACT: Inspired by natural 2-quinolinecarboxylic acid derivatives, a series of quinoline compounds containing acylhydrazine,
acylhydrazone, sulfonylhydrazine, oxadiazole, thiadiazole, or triazole moieties were synthesized and evaluated for their fungicidal
activity. Most of these compounds exhibited excellent fungicidal activity in vitro. Significantly, compound 2e displayed the superior
in vitro antifungal activity against Sclerotinia sclerotiorum, Rhizoctonia solani, Botrytis cinerea, and Fusarium graminearum with the EC50
values of 0.39, 0.46, 0.19, and 0.18 μg/mL, respectively, and were more potent than those of carbendazim (EC50, 0.68, 0.14, >100,
and 0.65 μg/mL, respectively). Moreover, compound 2e could inhibit spore germination of F. graminearum. Preliminary mechanistic
studies showed that compound 2e could cause abnormal morphology of cell walls and vacuoles, loss of mitochondrion, increases in
membrane permeability, and release of cellular contents. These results indicate that compound 2e displayed superior fungicidal
activities and could be a potential fungicidal candidate against plant fungal diseases.
KEYWORDS: quinoline, acylhydrazine, oxadiazole, fungicidal activity, pathogenic fungi
Our group has been committed to the discovery of fungicidal
agents based on quinoline alkaloids. In our previous work, a
series of quinoline derivatives substituted at the 4-position with
significant antifungal activity were obtained through structural
simplification of quinine alkaloids.15 Therefore, continuing our
efforts on the discovery of fungicidal quinoline compounds for
the control of plant diseases and considering that acylhydrazine
and oxadiazole moieties, as privileged structures with a broad-range
pharmacological potential,16−20 are widely used in pesticidal and
medicinal chemistry, here, we introduced acylhydrazine and
oxadiazole groups into the 2-position of quinoline ring to synthesize
a series of quinoline derivatives and systematically evaluated their
in vitro fungicidal activities against four pathogenic fungi. Moreover,
the preliminary mechanism of action of the most effective fungicidal
candidate against F. graminearum was also evaluated.
INTRODUCTION
■
Phytopathogenic fungi cause enormous damage to food crops,
resulting in diminishing economic quality and quantity of agri-
cultural products.1,2 Each year, at least 10% decrease of global
food production has been attributed to phytopathogenic fungi.3
For example, Fusarium graminearum not only causes loss of
germinability, reduced emergence, and post emergence blight of
seedlings, but also is the main causal agent of fusarium head
blight in adult plants, which is the most important and destructive
fungal diseases in wheat worldwide.4 At present, chemical
fungicides are still the most effective method to control fungal
infections because of their low cost, high convenience, and
fast effect.5 Unfortunately, the extensive and continuous use of
synthetic agrochemicals has resulted in the augmented frequency
of severe drug resistance, which drastically reduced the effec-
tiveness of these compounds.6,7 The above-cited problems have
highlighted an urgent need for the development of novel agents to
combat these phytopathogenic fungi.
MATERIALS AND METHODS
■
General. All reactions were performed with commercially available
reagents and solvents without further purification. All reactions were
monitored by thin-layer chromatography (TLC), and preparative TLC
was performed with silica gel plates using silica gel 60 GF254 (Qingdao
Haiyang Chemical Co., Ltd., China). Melting points were determined
in an open capillary using a WRS-2U melting point apparatus (Shanghai
Precision Instrument Co., Ltd., China) and were uncorrected. 1H and
13C NMR spectra were recorded on a Bruker AVANCE-III HD
2-Quinolinecarboxylic acid derivatives, distributed in plants,8,9
insects,10 marine organisms,11,12 and bacteria,13 have attracted much
attention as their multiple biological activities. Kynurenic acid,
6-hydroxykynurenic acid, and transtorine were isolated from Ephedra
species.8,9 The isomer of kynurenic acid, transtorine, showed
moderate antibacterial activity against Staphylococcus aureus,
Pseudomonas aeruginosa, and Enterobacter cloacae.9 Perspicamide
A was first isolated from the Australian ascidian Botrylloides
persipicuum in 2005 by Quinnet al.,11 and its synthesized analogues
exhibited potent activity against Leishmania donovani.14 However,
multifarious activities of 2-quinolinecarboxylic acid derivatives
have been reported except fungicidal evaluation against plant
pathogenic fungi.
Received: February 1, 2021
Revised: June 12, 2021
Accepted: July 7, 2021
Published: July 21, 2021
https://doi.org/10.1021/acs.jafc.1c00670
J. Agric. Food Chem. 2021, 69, 8347−8357
© 2021 American Chemical Society
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Figure 1. (A) Natural 2-quinolinecarboxylic acid derivatives. (B) Bioactive drugs containing carbohydrazide or oxadiazole moieties. (C) Design of
target compounds.
400 MHz or a NEO 500 MHz (Bruker Daltonics Inc., Germany)
spectrometer using tetramethylsilane as the reference. Mass spectra
were recorded on a Bruker APEXII49e spectrometer (Bruker Daltonics
Inc., Germany) with the electrospray ionization (ESI) source as
ionization. The commercial fungicide carbendazim (analytical grade,
98% purity) (Shanghai Titan Scientific Co., Ltd., China) was used as a
positive control in the in vitro experiment.
Fungi. Four pathogenic fungi strains, Rhizoctonia solani, Botrytis
cinerea, F. graminearum, and Sclerotinia sclerotiorum, were obtained from
the Gansu Academy of Agricultural Sciences, Gansu Province of China,
and maintained during the experiments on potato dextrose agar
medium at 25 °C.
Synthesis of Compound 1. To a solution of quinaldic acid
(23.10 mmol) in N,N-dimethylformamide (DMF, 30 mL), K2CO3
(23.10 mmol) and CH3I (46.20 mmol) were added. The reaction
mixture was stirred at room temperature for 24 h. The solution was
poured into 300 mL of water. The solid was filtered, washed with water,
and dried. The resulting solid was dissolved in 50 mL of methanol, and
hydrazine hydrate (92.40 mmol) was added dropwise. The reaction
mixture was refluxed for 8 h. Then, the solid was filtered, washed with
methanol, and recrystallized from methanol to afford the desired
compound 1 as a white solid (yield 68%). mp 141−142 °C. 1H NMR
(400 MHz, DMSO-d6): δ 10.03 (s, 1H), 8.55 (d, J = 8.5 Hz, 1H), 8.09−
8.05 (m, 3H), 7.86 (t, J = 8.4 Hz, 1H), 7.75−7.66 (t, J = 8.2 Hz, 1H);
13C NMR (100 MHz, DMSO-d6): δ 163.37, 150.53, 146.54, 138.16,
130.89, 129.69, 129.12, 128.52, 128.38, 119.10; ESI-MS m/z: 188.13
[M + H]+.
room temperature. The organic layer was washed with water and brine
(3 × 50 mL), dried, and concentrated. The residue was purified by
column chromatography on silica gel using DCM/acetone mixture as
the eluent to afford the target compounds 2a−2h.
Data for Compound 2a. Yellow solid; mp 90−92 °C; yield: 28%;
1H NMR (400 MHz, CDCl3): δ 8.27−8.16 (m, 2H), 8.03 (d, J = 8.5 Hz,
1H), 7.79 (d, J = 8.2 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.54 (t, J =
7.4 Hz, 1H), 1.15 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 162.10,
148.07, 145.54, 136.41, 129.09, 128.79, 128.32, 126.88, 126.71, 117.82,
54.55, 26.32; ESI-MS m/z: 244.21 [M + H]+.
General Synthetic Procedure for Compounds 3a−3v. To a
solution of compound 1 (2.14 mmol) in DCM (30 mL), substituted
acid (2.14 mmol), EDCI (4.27 mmol), HOBt (3.21 mmol), and NMM
(4.91 mmol) were added. The reaction mixture was stirred at room
temperature for 24 h. After completing the reaction, 30 mL of DCM was
added in the mixture. The organic layer was washed with water, 1 M
HCl aqueous solution, saturated NaHCO3 aqueous solution, and brine
(3 × 50 mL). The organic layer was dried over anhydrous Na2SO4 and
removed under vacuum. The residue was purified by column chro-
matography on silica gel using DCM/acetone mixture as the eluent to
afford the target compounds 3a−3v.
Data for Compound 3a. White solid; mp 209−211 °C; yield: 42%;
1H NMR (400 MHz, DMSO-d6): δ 11.08 (s, 1H), 10.71 (s, 1H), 10.22
(s, 1H), 8.60 (d, J = 8.4 Hz, 1H), 8.18−8.07 (m, 3H), 7.91 (t, J = 7.6 Hz,
1H), 7.76 (t, J = 7.3 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ
163.30, 160.16, 149.54, 146.49, 138.44, 131.16, 129.72, 129.39, 128.84,
128.63, 119.29; ESI-MS m/z: 216.14 [M + H]+.
General Synthetic Procedure for Compounds 2a−2h. Quinoline-
2-carboxylic acid (2.31 mmol) was dissolved in SOCl2 (5 mL), and the
reaction mixture was refluxed for 4 h and then concentrated to obtain
quinoline-2-carbonyl chloride. To a solution of substituted hydrazine
hydrochloride (4.62 mmol) in dichloromethane (DCM, 25 mL) was
dropwise added a solution of NaOH (6.93 mmol) in water (5 mL)
below 5 °C. After 20 min of stirring, quinoline-2-carbonyl chloride in
dry DCM (10 mL) was added dropwise below 5 °C. After stirring in the
ice−water bath for 30 min, the mixture was permitted to stand for 3 h at
General Synthetic Procedure for Compounds 4a−4h. To a
solution of compound 1 (1.60 mmol) in pyridine (10 mL), the
substituted sulfonylchloride (1.60 mmol) was added below 5 °C. After
stirring at room temperature for 3 h, the mixture was removed under
vacuum, and the residue was dissolved in 40 mL of DCM. The organic
layer was washed with saturated NaHCO3 aqueous solution and brine
(3 × 50 mL), dried, and removed under vacuum. The residue was
purified by column chromatography on silica gel using DCM/acetone
mixture as the eluent to afford the target compounds 4a−4h.
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Figure 2. Synthesis of target compounds 2a−2h, 3a−3v, 4a−4h, 5a−5o, 6a−6y, 7a−7f, 8a−8b, 9a−9m, 10a−10q, and 11a−11m. Reagents and
conditions: (a) (i) CH3I, K2CO3, DMF, rt, and 24 h; (ii) N2H4·H2O, MeOH, reflux, and 8 h; (b) (i) SOCl2, reflux, and 4 h and (ii) RNHNH2·HCl,
DCM, NaOH, H2O, 0 °C, 0.5 h then rt, and 3 h; (c) RCOOH, EDCI, HOBt, NMM, DCM, rt, and 24 h; (d) POCl3, reflux, and 12 h; (e) Lawesson’s
reagent, THF, reflux, and 12 h; (f) RSO2Cl, Et3N, DCM, rt, and 3 h; (g) RCHO, EtOH, AcOH, reflux, and 8 h; (h) for compound 9a: (i) KSCN, HCl,
EtOH, reflux, and 48 h and (ii) 2N NaOH, reflux, and 48 h; (i) for compound 9b: (i) CS2, KOH, EtOH, reflux, and 48 h and (ii) N2H4·H2O, reflux, and
4 days; (j) for compound 6w: CNBr, NaHCO3, dioxane, H2O, rt, and 8 h; (k) for compound 6x: triphosgene, DCM, reflux, and 2 h; (l) for compound
6y: (i) CS2, KOH, EtOH, reflux, and 8 h and (ii) 2 N HCl; (m) for compound 8m: (i) CS2, KOH, EtOH, and 8 h and (ii) conc. H2SO4 and 2 h; and
(n) RX, K2CO3, DMF, rt, and 24 h.
Data for Compound 4a. White solid; mp 203−205 °C; yield: 64%;
1H NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 9.69 (s, 1H), 8.61 (d,
J = 8.5 Hz, 1H), 8.22−8.07 (m, 3H), 7.91 (t, J = 8.4 Hz, 1H), 7.76 (t, J =
8.1 Hz, 1H), 3.10 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 164.56,
149.53, 146.49, 138.41, 131.16, 129.78, 129.42, 128.92, 128.61, 119.45,
41.22; ESI-MS m/z: 266.11 [M + H]+.
Data for Compound 6a. White solid; mp 163−165 °C; yield: 40%;
1H NMR (400 MHz, CDCl3): δ 8.64 (s, 1H), 8.37 (s, 2H), 8.27 (d, J =
8.5 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.68
(t, J = 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 153.78, 147.89,
142.97, 137.69, 130.91, 130.76, 130.13, 128.83, 128.53, 127.83, 119.85;
ESI-MS m/z: 198.13 [M + H]+.
General Synthetic Procedure for Compounds 5a−5o. To a
solution of compound 1 (1.34 mmol) in ethanol (10 mL) were added
the substituted benzaldehyde (2.00 mmol) and acetic acid (0.1 mL), and
the reaction mixture was refluxed for 8 h. The target compounds were
filtered, washed, and recrystallized from ethanol.
Data for Compound 5a. White solid; mp 136−137 °C; yield: 69%;
1H NMR (400 MHz, CDCl3): δ 11.06 (s, 1H), 8.33−8.26 (m, 2H),
8.22 (d, J = 8.5 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 8.2 Hz,
1H), 7.71−7.65 (m, 3H), 7.54 (t, J = 7.5 Hz, 1H), 7.19−7.12 (m, 2H),
2.57 (q, J = 7.4 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz,
CDCl3): δ 159.12, 148.07, 148.04, 146.16, 145.28, 136.67, 130.14,
129.28, 128.53, 128.45, 127.19, 127.16, 126.91, 126.82, 118.10, 27.83,
14.25; ESI-MS m/z: 304.20 [M + H]+.
General Synthetic Procedure for Compounds 6a−6v. Compounds
3a−3v (0.93 mmol) were dissolved in POCl3, and the mixture was
refluxed for 12 h. After cooling to room temperature, the reaction
mixture was poured into 100 g of crushed ice. The aqueous layer was
extracted with DCM (3 × 40 mL). The combined organic layer was
washed with saturated NaHCO3 aqueous solution (3 × 50 mL), dried
over anhydrous Na2SO4, and removed under vacuum. The target com-
pounds were purified by column chromatography on silica.
General Synthetic Procedure for Compounds 7a−7f. To a solution
of 6y (1.31 mmol) in DMF (5 mL) were added K2CO3 (1.31 mmol)
and haloalkane (2.62 mmol). The reaction mixture was stirred at room
temperature for 24 h. The solution was poured into water (100 mL).
The solid was filtered, washed with water, dried, and purified by column
chromatography on silica gel using petroleum ether/ethyl acetate mixture
as the eluent to afford the target compounds.
Data for Compound 7a. White solid; mp 129−130 °C; yield: 96%;
1H NMR (400 MHz, CDCl3): δ 8.25−8.22 (m, 3H), 7.88 (d, J = 8.1 Hz,
1H), 7.80 (t, J = 8.5 Hz, 1H), 7.64 (t, J = 8.1 Hz, 1H), 2.83 (s, 3H);
13C NMR (100 MHz, CDCl3): δ 167.21, 165.30, 147.83, 143.11,
137.47, 130.58, 130.04, 128.64, 128.25, 127.77, 119.54, 14.73; ESI-MS
m/z: 244.12 [M + H]+.
General Synthetic Procedure for Compounds 8a−8l. To a solution
of compounds 3a−3v (1.60 mmol) in tetrahydrofuran (THF) (10 mL)
was added Lawesson’s reagent (1.60 mmol). The reaction mixture was
refluxed for 12 h and then distilled under vacuum. DCM (50 mL) was
added into the residue. The organic layer was washed with saturated
NaHCO3 aqueous solution and brine (3 × 50 mL), dried, and removed
under vacuum. The residue was purified by column chromatography on
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Table 1. Fungicidal Activities of Kynurenic Acid, Quinaldic Acid, and Compounds 1, 2a−2h, and 3a−3v against Four Pathogenic
Fungi
inhibition rate SD (%) at 100 μg/mL
compound
R
S. sclerotiorum
R. solani
B. cinerea
F. graminearum
kynurenic acid
quinaldic acid
1
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
23.93 2.65
30.47 0.31
49.23 0.33
95.50 0.57
99.08 0.48
99.13 0.27
99.80 0.10
99.86 0.10
99.35 0.20
90.83 0.12
73.31 1.23
23.93 0.65
23.12 0.93
37.49 1.33
30.30 0.81
43.31 0.25
55.32 1.23
96.12 0.08
33.85 1.03
79.48 0.64
91.24 0.46
13.00 0.41
37.20 0.79
66.32 0.75
48.09 2.66
96.37 0.11
75.79 1.78
81.60 2.05
87.82 0.90
35.30 0.46
73.79 1.58
75.12 1.72
56.63 0.52
100 0.00
15.71 1.20
22.45 1.33
34.63 1.73
64.58 1.37
96.25 0.92
94.43 0.46
98.60 0.58
98.85 0.20
100 0.00
37.72 0.09
33.62 1.75
83.03 1.70
18.13 0.52
90.71 0.17
85.38 0.49
89.80 0.96
91.30 0.61
99.40 0.06
89.45 0.18
81.05 0.45
20.13 1.49
19.60 1.93
13.41 0.80
15.31 1.00
20.02 0.80
68.82 1.10
89.20 0.30
17.14 2.99
37.63 0.71
98.88 0.40
11.58 0.42
26.55 0.50
52.53 0.60
27.93 1.19
98.06 0.49
17.40 1.60
96.93 0.06
74.73 0.91
50.35 0.66
37.72 0.09
22.23 0.32
18.33 1.11
0.00 0.00
26.05 1.48
23.02 2.10
39.07 0.95
17.72 1.97
82.08 1.77
85.12 0.95
68.54 0.21
100 0.00
t-Bu
Ph
4-CH3-Ph
2-F-Ph
4-F-Ph
2,4-F-Ph
4-Cl-Ph
4-CF3-Ph
H
74.38 0.34
80.64 1.70
69.15 0.25
15.10 1.36
22.12 1.32
36.10 1.21
57.95 0.27
11.59 0.28
46.60 0.27
75.00 0.36
11.10 0.36
35.08 0.85
85.11 0.81
17.93 0.44
49.47 0.38
66.03 2.59
42.03 1.71
81.66 1.50
18.33 0.96
70.54 0.49
65.86 0.18
63.54 1.08
57.49 3.11
39.08 1.96
22.85 0.65
100 0.00
85.82 0.51
61.49 0.65
12.50 1.87
12.42 2.58
49.03 1.10
52.65 2.63
38.38 1.68
34.53 1.02
87.87 0.56
28.49 1.17
52.40 2.28
91.18 0.42
25.30 0.79
69.32 0.75
76.29 1.96
57.19 0.86
94.20 0.48
40.46 0.35
91.88 0.32
83.40 0.57
88.40 3.95
80.12 0.80
14.39 0.54
65.38 1.07
99.91 0.05
CH3
CH2CH3
cyclopropyl
Ph
4-CH3-Ph
2-OCH3-Ph
3-OCH3-Ph
4-OCH3-Ph
2-F-Ph
3-F-Ph
4-F-Ph
2,6-F-Ph
2,3,4,5,6-F-Ph
2-Cl-Ph
4-Cl-Ph
2,6-Cl-Ph
2-Br-Ph
furan-2-yl
thiophene-2-yl
pyridine-3-yl
CH2-4-OCH3-Ph
3v
carbendazim
silica gel using petroleum ether/ethyl acetate mixture as the eluent to
afford the target compounds.
Fungicidal Activity Assay. The in vitro fungicidal activity of the
target compounds against four pathogenic fungi was tested by the
mycelium growth rate method.21,22
Data for Compound 8a. White solid; mp 124−125 °C; yield: 75%;
1H NMR (500 MHz, CDCl3): δ 9.15 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H),
Spore Germination Inhibition Assay. The inhibitory activity
of compound 2e on spore germination of F. graminearum was assessed
by microscopic observation.23 Spore suspensions (1 × 105 to 5 ×
105 spores/mL) were prepared to inoculate conidia in sterile water.
Compound 2e in DMSO was added to the conidial suspension to
obtain different concentrations of 25, 10, and 5 μg/mL. Then, 150 μL of
these mixtures were removed, put on concave slides, and incubated in a
biochemical incubator at 25 °C. Conidial suspension with 0.5% DMSO
in water (v/v) was treated as a blank control. After incubation for 12 h,
the number of germinated spores was measured by approximately
counting 80 conidia in the blood counting chamber under a biological
microscope photographic system at 200× magnification. The experi-
ment was repeated three times, and the inhibitory rates were calculated
according to the following formula
8.23 (d, J = 8.5 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.1 Hz,
1H), 7.69 (t, J = 7.8 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H); 13C NMR
(125 MHz, CDCl3): δ 170.84, 153.90, 148.75, 147.96, 137.41, 130.36,
129.59, 128.89, 127.91, 127.84, 118.59; ESI-MS m/z: 214.10 [M + H]+.
General Synthetic Procedure for Compounds 10a−10q and
11a−11m. Compounds 10a−10q and 11a−11m were synthesized using
a similar procedure to compounds 2a−2h.
Data for Compound 10a. Yellow solid; mp 210−212 °C; yield:
54%; 1H NMR (400 MHz, DMSO-d6): δ 10.49 (s, 1H), 8.90 (s, 1H),
8.12 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.89 (t, J = 7.3 Hz,
1H), 7.75 (t, J = 7.5 Hz, 1H), 7.09−6.91 (m, 4H); 13C NMR (100 MHz,
DMSO-d6): δ 166.30, 157.62, 155.30, 153.35, 145.90, 145.38, 140.56,
131.39, 129.36, 129.30, 129.08, 127.80, 115.80, 115.58, 114.05, 113.63;
ESI-MS m/z: 361.97 [M + H]+.
Inhibition of spore germination (%) = [(N0 − N )/N0] × 100
1
Synthetic details and data of the other quinoline derivatives shown in
where N0 and N1 are the average values of the spore germination rates of
Figure 1 are provided in the Supporting Information.
black control and treatment, respectively.
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Table 2. Fungicidal Activities of Compounds 4a−4h and 5a−5o against Four Pathogenic Fungi
inhibition rate SD (%) at 100 μg/mL
compound
R
S. sclerotiorum
R. solani
B. cinerea
F. graminearum
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
CH3
CH2CH3
2-F-Ph
4-F-Ph
2-Cl-Ph
11.47 0.28
0.00 0.00
85.29 0.35
91.54 0.12
29.58 0.65
84.76 0.22
30.47 0.31
66.70 1.20
72.49 1.12
0.00 0.00
0.00 0.00
0.00 0.00
12.26 1.29
29.83 0.91
9.65 0.12
45.56 1.72
51.98 1.64
0.00 0.00
5.68 0.41
5.40 1.25
0.00 0.00
12.69 0.93
0.00 0.00
13.80 0.48
20.27 0.48
56.43 0.75
70.99 0.86
64.35 1.33
71.53 1.59
53.50 1.41
32.08 0.94
65.68 3.08
3.61 1.50
10.69 2.05
15.71 1.20
8.83 0.66
14.54 0.35
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
11.57 3.30
6.74 0.48
0.00 0.00
2.92 1.31
0.00 0.00
6.74 0.61
20.55 0.21
27.90 0.85
55.46 2.25
13.47 0.82
39.90 0.31
34.20 0.92
17.36 2.03
39.57 1.40
10.55 1.33
16.90 0.31
19.15 0.41
19.45 0.43
14.44 1.08
6.54 1.37
23.49 1.08
21.59 0.93
16.80 0.63
18.15 1.37
11.86 1.20
16.16 0.77
6.69 1.43
7.31 0.19
16.30 1.55
7.13 1.09
55.59 1.88
75.99 1.23
23.60 0.88
48.38 5.72
42.13 0.69
32.62 0.32
32.87 1.02
0.00 0.00
0.00 0.00
0.00 0.00
11.17 1.40
18.54 1.96
13.31 3.22
13.03 0.82
12.15 1.81
0.00 0.00
0.00 0.00
17.90 0.19
11.21 1.11
0.00 0.00
5.25 0.39
4-Cl-Ph
4-CF3-Ph
4-OCF3-Ph
4-CH2CH3-Ph
4-CN-Ph
2-NO2-Ph
4-NO2-Ph
4-OCH3-Ph
2-F-Ph
4-F-Ph
2-Cl-Ph
4-Cl-Ph
2,6-Cl-Ph
4-Br-Ph
2-CF3-Ph
4-CF3-Ph
2-OCF3-Ph
4-OCF3-Ph
5m
5n
5o
Scanning Electron Microscopy Observations. Scanning elec-
tron microscopy (SEM) observations were performed according to
the described methods to examine the effects of compound 2e on the
microstructure of F. graminearum.24 After treatment with compound 2e
at a concentration of 1 μg/mL, mycelial blocks (5.0 mm × 4.0 mm)
were cut from the culture medium. The samples were treated by 4%
glutaraldehyde for 4 h at 4 °C and then fixed with 1% osmium tetraoxide
solution (w/v) for 2 h. The samples were washed with 0.01 M
phosphate-buffered saline and then dehydrated with a series of ethanol
solutions. After drying the samples at a critical point and gold-spraying,
they were observed with a scanning electron microscope (Hitachi,
S-3400N, Japan).
Determination of the Leakage of Cellular Contents. The
release of cellular content was determined according to the method
with some modifications.26 The mycelia (400 mg) of F. graminearum
were added into compound 2e solutions (25, 10, 5, and 2.5 μg/mL) to
obtain mycelia suspension. Then, the samples were incubated at 25 °C
for 24 h and then centrifuged. Finally, the absorbance values of the
supernatants were measured at 260 and 280 nm with a UV−vis spec-
trophotometer (Shimadzu, UV-1700, Japan).
Statistical Analysis. All assays were at least performed in triplicate
by conventional methods, and the results were presented as means
standard deviations. Statistical analysis was carried out by SPSS 24.0.
Transmission Electron Microscopy Observations. The
dehydrated mycelial blocks were embedded in resin at 70 °C for 24 h
and then cut into thin sections. After double-staining the samples with
uranyl acetate and lead citrate, they were observed with a transmission
electron microscope (Hitachi, HT7700, Japan).
Determination of Cell Membrane Permeability. The cell
membrane relative permeability rate of F. graminearum was evaluated
according to the method with some modifications.25 The mycelial disk
of F. graminearum (5 mm) was placed in 60 mL of PD broth medium
with 140 rpm shaking at 25 °C for 48 h. Then, the mycelia (500 mg)
were filtered and added into compound 2e solution with different
concentrations (25, 10, 5, and 2.5 μg/mL). Finally, the conductivity
values were determined with a conductivity detector (0 h was marked as
L0 and 2, 4, 6, 8, 10, 12, 24, and 48 h were marked as L1, HORIBA,
EC1100, Japan). The conductivities of samples treated by boiling water
for 0.5 h were marked as L2. The relative permeability rate of the cell
membrane was calculated by the following formula
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of all intermediates and target
compounds was performed as shown in Figure 2. Briefly, inter-
mediate 1 and compounds 2a−2h, 3a−3v, 4a−4h, 5a−5o, 6a−6y,
7a−7f, 8a−8b, 9a−9m, 10a−10q, and11a−11m were synthesized
according to our previously reported procedure.20 Quinoline-2-
carbonyl chloride was reacted with various substituted hydrazine
hydrochlorides and NaOH to obtain target compounds 2a−2h.
Intermediate 1 was reacted with various substituted acids to afford
compounds 3a−3v and then further cyclized in POCl3 to obtain
target compounds 6a−6v or reacted with Lawesson’s reagent in
THF to obtain target compounds 8a−8l. Intermediate 1 was
reacted with various substituted sulfuryl chlorides in pyridine
to obtain target compounds 4a−4h or reacted with various
substituted aldehydes in ethanol to afford compounds 5a−5o.
Compound 6y was reacted with various substituted haloalkanes
in DMF to obtain target compounds 7a−7f. Compounds
10a−10q and 11a−11m were synthesized with substituted
quinoline-2-carboxylic acid27 or heteroaromatic carboxylic acid
Relative electric conductivity (%)
= [(L1 − L0)/(L2 − L0)] × 100
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Table 3. Fungicidal Activities of Compounds 6a−6y and 7a−7f against Four Pathogenic Fungi
inhibition rate SD (%) at 100 μg/mL
compound
R
S. sclerotiorum
R. solani
B. cinerea
F. graminearum
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
H
CH3
58.34 0.16
16.82 3.24
28.79 1.33
50.32 4.71
23.70 0.57
15.19 0.28
14.10 1.10
7.90 0.72
36.56 0.71
11.95 1.52
7.39 1.54
10.99 0.64
25.49 0.72
86.85 0.89
0.00 0.00
6.18 0.80
0.00 0.00
0.00 0.00
27.08 0.33
23.17 0.81
29.49 6.18
30.50 2.16
14.26 2.64
86.40 0.58
90.72 1.52
64.39 0.72
89.65 0.66
93.41 0.32
91.56 0.81
74.24 0.26
77.18 0.54
42.57 1.35
28.26 0.79
47.73 0.89
53.58 1.19
18.59 1.07
13.45 1.31
27.68 0.68
38.02 0.38
41.47 1.62
38.13 1.19
22.45 3.76
22.61 3.05
12.73 1.13
48.36 2.68
15.03 0.92
23.86 0.35
11.25 1.40
16.18 0.57
52.98 1.46
16.56 0.78
18.83 0.93
22.63 0.91
0.00 0.00
58.35 0.22
45.63 0.77
100 0.00
66.77 0.97
27.27 0.24
29.58 3.48
47.61 2.10
4.25 0.57
9.08 0.70
28.11 0.99
18.43 0.37
56.85 0.77
21.25 0.15
21.03 2.19
22.68 4.49
7.67 0.56
32.34 2.37
18.05 0.13
20.08 0.75
0.00 0.00
0.00 0.00
36.19 0.13
5.04 0.62
15.69 1.52
46.19 0.90
19.08 1.68
74.03 0.64
65.35 0.17
51.23 1.83
99.40 0.44
91.26 0.73
79.54 0.19
27.39 0.92
90.08 0.63
47.61 0.52
23.02 2.10
39.98 1.39
53.54 0.33
0.00 0.00
7.33 1.13
9.73 0.22
7.00 0.27
14.95 0.63
0.00 0.00
0.00 0.00
0.00 0.00
15.21 1.04
38.59 0.05
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
20.64 1.02
0.00 0.00
15.52 0.93
39.99 1.37
13.33 2.56
76.48 0.95
27.28 0.30
43.43 3.63
82.61 1.44
70.68 1.01
32.96 1.42
35.91 7.88
51.11 1.15
CH2CH3
cyclopropyl
Ph
4-CH3-Ph
2-OCH3-Ph
3-OCH3-Ph
4-OCH3-Ph
2-F-Ph
3-F-Ph
4-F-Ph
2,6-F-Ph
2,3,4,5,6-F-Ph
2-Cl-Ph
4-Cl-Ph
2,6-Cl-Ph
2-Br-Ph
furan-2-yl
thiophene-2-yl
pyridine-3-yl-
4-OCH3-Ph-CH2
NH2
6s
6t
6u
6v
6w
6x
6y
7a
7b
7c
7d
7e
7f
OH
SH
CH3
CH2CH3
CH2CH2F
CH2CH2Cl
CH2CH2Br
CH2CH2CH3
100 0.00
99.21 0.34
95.70 0.07
91.70 0.82
94.54 0.85
with hydrazine hydrochlorides using a similar procedure to
compounds2a−2h. All target compounds were purified by recrys-
tallization or column chromatography, and their structures were
confirmed on the basis of 1H NMR, 13C NMR, and MS data.
Structure−Activity Relationship Studies. With 132 com-
pounds in hand, we evaluated their fungicidal activity against
four pathogenic fungi, S. sclerotiorum, R. solani, B. cinerea, and
F. graminearum, to probe their structure−activity relationship
(SAR). The biological data for all quinoline derivatives are
shown in Tables 1−7.
As shown in Table 1, the natural 2-quinolinecarboxylic acid,
kynurenic acid, displayed marginal to weak fungicidal activity
with the inhibitory rate of <50% against four fungi at a con-
centration of 100 μg/mL. The dehydroxylated compound,
quinaldic acid, showed a similar activity. However, interestingly,
quinoline-2-acylhydrazine 1 exhibited significant activity and
was more potent than kynurenic acid. Subsequently, the effects
of quinoline compounds 2a−2h and 3a−3v with a focus on
varying substituents at the acylhydrazine and diacylhydrazine
groups were investigated. Compared with compound 1, sub-
stitution for a tert-butyl group (2a) resulted in a 2-fold increase
in activity against S. sclerotiorum and R. solani but diminished
potency against B. cinerea and F. graminearum. Substitution for
phenyl ring (2b−2h) was favorable for activity that depended
significantly on the nature of substituents and their position at
the phenyl ring. Of particular note, compound 2e (p-F-C6H4)
displayed the most effective inhibition among this series
with inhibitory rates of 99.86, 98.85, 91.30, and 100% against
S. sclerotiorum, R. solani, B. cinerea, and F. graminearum, respec-
tively, at a concentration of 100 μg/mL. A possible explanation
is that the introduction of a benzene ring and an electron-
withdrawing group enhanced the induction and conjugation
effect of quinoline acylhydrazine molecules and enhanced the
antifungal effect.28
In the diacylhydrazine series (3a−3v), substitution of alkyl
groups (3a−3d) led to a substantially reduced fungicidal
potency, but in contrast, good activity accompanied substitution
with a range of aromatic groups (3e−3v); particularly, the sub-
stituents on the ortho-position of aromatic ring (3g, 3j, and 3o)
were recommended for the enhancement of activity than the
para- (3i, 3l, and 3p) and meta-counterparts (3h and 3k). The
fungicidal activity of ortho-substituted compounds increased
following 2-F (3j) ≈ 2-Cl (3o) > 2-OCH3 (3g) > 2-Br (3r).
Furthermore, replacement of phenyl ring with furan-2-yl (3s) or
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Table 4. Fungicidal Activities of Compounds 8a−8b and 9a−9m against Four Pathogenic Fungi
inhibition rate SD (%) at 100 μg/mL
compound
R
S. sclerotiorum
R. solani
B. cinerea
F. graminearum
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
9a
9b
H
CH3
cyclopropyl
Ph
2-OCH3-Ph
2-F-Ph
85.83 0.69
0.00 0.00
17.63 0.33
0.00 0.00
0.00 0.00
21.45 0.69
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
96.51 0.20
33.90 1.82
13.85 0.56
80.28 0.67
25.81 0.33
61.13 2.13
5.40 0.48
7.98 1.25
10.80 2.21
4.69 1.94
8.24 0.23
20.06 0.11
0.00 0.00
13.44 5.82
24.90 0.73
56.93 3.27
32.54 0.88
8.48 1.30
98.80 0.52
11.78 1.19
33.62 1.75
0.00 0.00
0.00 0.00
6.29 2.12
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
25.80 4.54
63.08 1.76
20.94 1.00
13.11 1.43
83.01 1.66
10.38 1.07
44.43 1.84
8.43 0.82
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
58.96 1.32
23.60 0.30
26.05 1.48
4-F-Ph
2,6-F-Ph
2-Cl-Ph
4-Cl-Ph
2-Br-Ph
4-OCH3-Ph-CH2
SH
H
NH2
Table 5. Fungicidal Activities of Compounds 10a−10q against Four Pathogenic Fungi
inhibition rate SD (%) at 25 μg/mL
compound
R
S. sclerotiorum
R. solani
B. cinerea
F. graminearum
2e
H
99.34 0.06
73.15 0.80
86.00 0.24
0.00 0.00
95.41 0.64
82.90 0.32
87.22 1.65
18.88 1.67
83.61 2.66
83.92 1.06
67.18 1.62
83.12 0.68
88.26 0.41
86.20 0.80
82.42 1.56
82.19 0.90
81.61 1.53
87.05 0.39
89.10 0.38
48.03 1.04
86.82 0.12
40.51 0.14
86.11 0.18
74.97 1.03
79.34 0.96
13.90 1.35
83.48 0.33
73.99 0.51
63.48 0.78
85.06 0.38
88.29 0.39
92.55 0.43
82.16 0.71
85.61 1.10
78.35 1.44
80.90 0.78
85.61 0.41
14.80 1.38
81.97 0.47
80.68 1.07
100 0.00
66.90 0.73
79.35 0.61
18.69 0.22
81.44 0.77
66.54 0.50
53.21 0.63
99.85 0.06
99.75 0.03
88.70 0.63
96.20 0.86
90.53 0.35
98.10 0.18
91.07 0.33
99.60 0.07
42.91 0.65
85.70 0.96
56.28 0.58
10a
10b
10c
10d
10e
10f
3-Br
4-Cl
4,7-Cl
4-Br
87.45 0.24
80.26 0.76
62.67 0.54
97.70 0.33
98.53 0.23
91.06 0.36
87.42 0.35
86.73 0.80
90.75 0.38
86.11 0.36
98.05 0.23
86.81 0.47
91.33 0.50
35.95 0.59
5-Cl
5-Br
10g
10h
10i
6-OCH3
6-F
6-Cl
10j
6-Br
10k
10l
6,8-Br
7-Br
10m
10n
10o
10p
10q
8-OCH3
8-F
thiophene-2-yl (3t) resulted in a slight increase in activity. The
SAR for the sulfonylhydrazine (4a−4h) and acylhydrazone
(5a−5o) derivatives was probed next, and the results are shown
in Table 2. It turned out that extension of diacylhydrazine to
sulfonylhydrazine or acylhydrazone led to a substantially reduced
fungicidal potency. Taken together, these results implied that the
electronegativity and size of substituents at the hydrazine scaffold
were crucial.
In synthetic methodology, acylhydrazine is used as an important
precursor for building the skeletons in diverse heterocyclic com-
pounds, such as 1,3,4-oxadiazole and 1,3,4-thiadiazole. In addition
to acylhydrazines, diacylhydrazines, acylhydrazones, and sulfo-
nylhydrazines, the effects of different five-membered heterocycles
at the 2-position on the quinoline scaffold were investigated.
As shown in Table 3, cyclization of diacylhydrazines 3a−3v to
obtain 1,3,4-oxadiazole derivatives 6a−6v bearing alkyl and
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Table 6. Fungicidal Activities of Compounds 11a−11m against Four Pathogenic Fungi
Table 7. Fungicidal Activities of Selected Compounds against Four Pathogenic Fungi
EC50 (μg/mL)
compound
2b
S. sclerotiorum
R. solani
B. cinerea
F. graminearum
1.38 0.01
0.71 0.02
1.39 0.04
0.39 0.01
1.04 0.02
0.82 0.01
2.16 0.04
7.73 0.10
21.96 0.51
18.69 0.56
39.91 0.47
36.78 0.06
32.41 0.21
33.85 1.37
47.15 0.19
0.38 0.01
0.36 0.01
0.45 0.02
0.24 0.01
0.34 0.01
1.07 0.03
0.38 0.01
0.36 0.01
0.78 0.02
0.68 0.01
1.22 0.01
4.22 0.23
1.00 0.02
0.46 0.01
0.87 0.03
0.79 0.01
1.88 0.04
10.20 0.11
29.42 0.28
14.82 0.58
5.73 0.14
5.11 0.03
4.92 0.06
4.96 0.03
36.24 0.63
1.21 0.02
0.97 0.02
0.96 0.05
0.69 0.01
1.07 0.01
1.48 0.03
0.94 0.02
0.71 0.01
1.56 0.02
0.14 0.01
0.50 0.02
0.39 0.02
0.51 0.01
0.19 0.01
0.14 0.03
0.29 0.06
0.51 0.01
31.20 0.60
38.74 0.51
48.90 0.08
21.85 0.10
22.73 0.38
19.33 0.13
28.86 0.63
43.52 0.51
1.39 0.05
1.01 0.05
1.01 0.01
0.87 0.05
0.74 0.01
1.05 0.02
0.83 0.02
0.97 0.02
1.19 0.03
>100
2.00 0.13
2.69 0.07
23.58 1.02
0.18 0.01
0.46 0.03
0.39 0.03
4.70 0.14
12.40 0.37
31.79 0.11
53.63 1.38
65.96 2.23
70.22 2.67
>100
2c
2d
2e
2f
2g
2h
3g
3j
3o
7b
7c
7d
7f
91.40 3.47
56.79 0.64
0.76 0.01
0.68 0.02
0.89 0.01
0.57 0.01
0.90 0.02
1.39 0.03
0.86 0.02
0.71 0.01
1.04 0.05
0.65 0.02
9a
10g
10h
10n
11d
11g
11h
11i
11j
11m
carbendazim
aromatic groups resulted in substantially reduced fungicidal
potency. Extension of 2-H (6a) to 2-oxo (6x) or 2-thioxo (6y)
led to a similar activity, while 2-amino (6w) showed inferior
activity. However, interestingly, alkylation of compound 6y was
beneficial for the improvement of the fungicidal activity. For
example, compounds 7a−7f exhibited excellent fungicidal
activity against R. solani with the inhibitory rates of >90% at
100 μg/mL, which were more potent than that of 6y (45.63%).
Likewise, it can be seen that the structural changes in general
were detrimental to activity, and no favorable substitution could
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Figure 3. Spore germination inhibition of F. graminearum in the untreated control (A) and 5, 10, and 25 μg/mL of compound 2e-treated control
(B−D). The inhibitory rate of 2e on the spore germination (E). *P < 0.05 compared to the control group. **P < 0.01 compared to the control group.
Figure 4. Scanning electron micrographs of F. graminearum hyphae
in untreated control (A) and 1.0 μg/mL of compounds 2e-treated
control (B).
be found for substitution of 1,3,4-thiadiazole (8b−8l) and 1,2,4-
triazole (9a and 9b), except for 8a and 8m (Table 4). Overall,
from the available fungicidal activities, we could obviously find
that the compounds containing the acylhydrazine moiety
Figure 5. TEM observations of cell structure of F. graminearum.
(2a−h) showed higher fungicidal activities than the other seven
Ultrastructure of the hyphae in the untreated control (A,B) and 1 μg/mL
series derivatives (3a−3v, 4a−4h, 5a−5o, 6a−6y, 7a−7f, 8a−8m,
of 2e-treated treated control (C,D). Cell wall (CW), mitochondrion
(M), plasma membrane (PM), rough endoplasmic reticulum (RER),
Finally, the effects of compounds 10a−10q and 11a−11m
and vacuole (V).
and 9a−9b).
with a focus on varying substituents on the quinoline ring
or heterocycle replacement were evaluated, and the biological
data are shown in Tables 5 and 6. Compound 2e, the potent
compound against four pathogenic fungi, was utilized as the
reference point for relative comparison of substituted quinoline
or other heterocycle. Compared with the unsubstituted com-
pound 2e, substitution of electron-withdrawing groups or
electron-donating groups at various positions on the quinoline
ring led to a slight decrease in activity against four pathogenic
fungi at a concentration of 25 μg/mL (Table 5). Furthermore,
replacement of quinoline with various heterocycles, such as iso-
quinoline, quinoxaline, pyrazine, and pyridine, obtained compounds
11a−11m, which displayed similar activity relative to 2e (Table 6).
Overall, these findings further underlined that the fungicidal
differences of quinoline compounds could be ascribed to a com-
bination of factors, such as a different interaction at the site or
the nature of the substituents, which may depend on the size,
electronic characteristics of substituents, or other factors.20
The EC50 values of potent compounds 2b−2h, 3g, 3j, 3o, 7b−
7d, 7f, 8a, 10g, 10h, 10n, 11d, 11g−11j, and 11m were further
evaluated, and the results are presented in Table 7. Most of these
Figure 6. Effect of compound 2e on the cell membrane permeability of
F. graminearum mycelia.
compounds exhibited significant fungicidal activities against
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Figure 7. Effect of compound 2e on release of cellular content F. graminearum mycelia. (A) Absorbance value of nucleic acids (OD260) and
(B) absorbance value of protein (OD280). *P < 0.05 compared to control group. **P < 0.01 compared to control group.
S. sclerotiorum, R. solani, B. cinerea, and F. graminearum, with
EC50 values ranging from 0.24−47.15, 0.46−36.24, 0.14−48.90,
and 0.18−91.40 μg/mL, respectively. Especially, compound 2e
exhibited the greatest inhibitory activity against S. sclerotiorum,
R. solani, B. cinerea, and F. graminearum with the EC50 values of
0.39, 0.46, 0.19, and 0.18 μg/mL, respectively, and were more
potent than those of carbendazim (EC50, 0.68, 0.14, >100, and
0.65 μg/mL, respectively). Therefore, compound 2e was selected
as the candidate compound to study its preliminary mode of
action against F. graminearum.
Preliminary Mode of Action of Compound 2e against
F. graminearum. Effect of Compound 2e on the Spore
Germination Inhibition. In Figure 3, the results of spore
germination inhibition assay showed that compound 2e could
suppress spore germination of F. graminearum in a concen-
tration-dependent manner, with an inhibition rate of 59.82% at a
concentration of 25 μg/mL.
relative electric conductivity. Moreover, the cellular content
leakage assay of F. graminearum mycelia such as nucleic acids
and proteins also supported the above observation. As shown in
Figure 7, after treatment with compound 2e, macromolecular
nucleic acids and proteins were released from the mycelial cells
of F. graminearum in a concentration-dependent manner. Taken
together, compound 2e could cause damage to the cell membranes
of the mycelia and lead to the electrolyte and cellular content leakage.
In summary, a series of quinoline compounds containing acyl-
hydrazine, acylhydrazone, sulfonylhydrazine, oxadiazole, thia-
diazole, and triazole moieties were synthesized and evaluated
for their fungicidal activity. The results showed that most of
these compounds exhibited excellent fungicidal activity in vitro.
Significantly, compound 2e displayed the superior in vitro
antifungal activity against S. sclerotiorum, R. solani, B. cinerea, and
F. graminearum and were more potent than carbendazim. Moreover,
compound 2e could inhibit spore germination of F. graminearum.
Although the preliminary mechanistic studies revealed that
compound 2e might exert its antifungal effect through acting on
the cell membrane, and influencing the normal functions of PMs,
the specific biological target or targets of 2e remain unknown.
Further studies on structural optimization and target identification
are in progress.
Effect of Compound 2e on the Hyphae Morphology.
As shown in Figure 4A, untreated mycelia grew well with a
smooth surface and an intact structure. However, after treatment
with compound 2e at a concentration of 1 μg/mL, treated mycelia
grew abnormally with the phenomenon of rough and incomplete
surface (Figure 4B).
The ultrastructural features of the hyphae treated with
compound 2e at a concentration of 1 μg/mL were observed
using transmission electron microscopy. The untreated mycelia
showed cellular morphology with normal and uniform cell walls
(CWs), plasma membranes (PMs), rough endoplasmic reticulum
(RER), and cytoplasmic organelles including mitochondria and
vacuoles (Figure 5A,B). In contrast, after treatment with compound
2e, the CWs of mycelia were irregularly thickened, and protrusions
existed at the outer layer of CWs. Besides, organelles were partially
missing, and no distinct mitochondrion was observed. Moreover, a
large number of vacuoles existed in the cytoplasm, which were
irregularly shaped and filled most of the cytoplasm (Figure 5C,D).
Effects of Compound 2e on the Cell Membrane. To con-
firm whether compound 2e acted on the cell membrane of
F. graminearum, we next investigated membrane permeability
by measuring the change in relative electric conductivity of
mycelia exposed to 2e, and the results are shown in Figure 6.
The conductivity of four groups treated with compound 2e at
concentrations of 2.5, 5, 10, and 25 μg/mL and the conductivity
of all treated groups were higher than that of the untreated
control group and increased in a time- and concentration-
dependent manner. Therefore, it indicated that compound 2e
might cause an increase in membrane permeability, result in the
release of charged ions such as K+ and Na+, and the increase of
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.1c00670.
■
sı
Melting points, 1H and 13C NMR, and MS spectra for the
target compounds and spectra of the representative
compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Zhi-Jun Zhang − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China; orcid.org/
0000-0003-3055-3426; Email: zhangzhijun198803@
163.com
Ying-Qian Liu − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China; orcid.org/
0000-0001-5040-2516; Email: yqliu@lzu.edu.cn
Authors
Yu-Dong Yang − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China
Ying-Hui He − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China
8356
https://doi.org/10.1021/acs.jafc.1c00670
J. Agric. Food Chem. 2021, 69, 8347−8357

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
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Hu Li − School of Pharmacy, Lanzhou University, Lanzhou
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Yu Sun − School of Pharmacy, Lanzhou University, Lanzhou
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Yu-Ling Wang − Gansu Academy of Agricultural Sciences,
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Guan-Fang Hu − Gansu Academy of Agricultural Sciences,
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Ren-Xuan Wang − School of Pharmacy, Lanzhou University,
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.1c00670
Author Contributions
§Y.-D.Y. and Y.-H.H. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported financially by the National Natural
Science Foundation of China (21877056 and 21672092) and
the National Key Research and Development Program of China
(2017YFD0201404). Support was also supplied by the Key
Program for international S&T cooperation projects of China
Gansu Province (18YF1WA115), the Natural Science Foundation
of China Gansu Province (20JR5RA216), and the Fundamental
Research Funds for the Central Universities (lzujbky-2019-27).
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