Design, Synthesis, and Structure-Activity Relationship of Quinazolinone Derivatives as Potential Fungicides

Quinazolinone Derivatives as Potential Fungicides

Article

Design, Synthesis, and Structure−Activity Relationship of

Jing-Wen Peng, Xiao-Dan Yin, Hu Li, Kun-Yuan Ma, Zhi-Jun Zhang, Rui Zhou, Yu-Ling Wang,

Guan-Fang Hu, and Ying-Qian Liu *

Cite This: J. Agric. Food Chem. 2021, 69, 4604 −4614

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ABSTRACT: Plant diseases caused by phytopathogenic fungi reduce the yield and quality of crops. To develop novel antifungal

agents, we designed and synthesized eight series of quinazolinone derivatives and evaluated their anti-phytopathogenic fungal

activity. The bioassay results revealed that compounds KZL-15, KZL-22, 5b, 6b, 6c, 8e, and 8f exhibited remarkable antifungal

activity in vitro. Especially, compound 6c displayed the highest bioactivity against Sclerotinia sclerotiorum, Pellicularia sasakii, Fusarium

graminearum, and Fusarium oxysporum, displaying appreciable IC₅₀values (50% inhibitory concentration) of 2.46, 2.94, 6.03, and

1.9 μg/mL, respectively. A further mechanism interrogation revealed abnormal mycelia, damaged organelles, and changed

permeability of cell membranes in S. sclerotiorum treated with compound 6c. In addition, the in vivo bioassay indicated that

compound 6c possessed comparable curative and protective eﬀects (87.3 and 90.7%, respectively) to the positive control

azoxystrobin (89.5 and 91.2%, respectively) at 100 μg/mL concentration against S. sclerotiorum. This work validated the potential of

compound 6c as a new and promising fungicide candidate, contributing to the exploration of potent antifungal agents.

KEYWORDS: phytopathogenic fungi, quinazolinone, Sclerotinia sclerotiorum

INTRODUCTION

thrin, evodiamine, rutaecarpine, quinazolinone, and 2-

methylquinazolinone (Figure 1). Its derivatives display various

■

Plant diseases caused by phytopathogenic fungi reduce crop

yields and dampen the quality and safety of agricultural

products, causing severe production losses of agricultural and

horticultural crops worldwide and threatening global food

pharmacological properties, such as antifungal, antibacteri-

al, antimalarial, anticancer, and antiviral activities. In

addition, the backbone of 4(3H)-quinazolinone is also critical

for the prevention and treatment of agricultural diseases;

commercial fungicides ﬂuquinconazole and proquinazid are

two representative drugs (Figure 1). Because of the signiﬁcant

value in pharmaceuticals, the synthesis and bioactivity of

4(3H)-quinazolinone and its derivatives have drawn consid-

erable interest. On the other hand, hydrazide has been widely

used as a functional fragment in drug design owing to a lot of

its derivatives exhibiting various bioactivities, such as

anticancer, antibacterial, antifungal, insecticidal, and

herbicidal activities. Tebufenozide and chromafenozide

Figure 1) are two compounds bearing a hydrazide group,

which have been launched as commercial insecticides.

Based on the above considerations and our consistent eﬀorts

in developing antimicrobial natural alkaloid derivatives for

agricultural applications,

activity relationship (SAR) obtained from screening the

antifungal activities of the six natural products containing the

(3H)-quinazolinone skeleton in Figure 1 to guide the design

and synthesis of eight series of 4(3H)-quinazolinone

security and public health.

For instance, Sclerotinia

sclerotiorum, a phytopathogenic fungus distributed globally,

invades hundreds of crop species and is responsible for

Sclerotinia stem rot of oilseed rape (Brassica napus L.), which is

−5

most devastating for its production.

The immense loss

caused by phytopathogenic fungi led to the wide application of

various antifungal agents. However, the misuse and overuse of

many traditional fungicides for a long time have caused

toxicity, altered pharmacokinetics, and ever-rising resistance.

Thus, it is urgently needed to discover novel fungicides

featuring a unique acting mechanism, optimized targeting

(

ability, and safety to the environment.

Natural products are highly eﬃcient, low-toxic, environ-

mentally friendly, and meet the requirements of the Environ-

mental Action Plan (EAP). For example, validamycin isolated

from Streptomyces shows signiﬁcant activity against some

26−28

herein, we used the structure−

phytopathogenic fungi. Nevertheless, many of these products

are diﬃcult to obtain, poorly soluble, and instable during

metabolism, which severely hinders their further application,

availability, solubility, and instability; these problems can be

addressed by structural optimization of natural products, which

is an eﬀective strategy for discovering new antifungal agents.

The 4(3H)-quinazolinone skeleton belongs to the N-

containing heterocyclic building block and is composed of a

Received: August 26, 2020

Revised: February 22, 2021

Accepted: March 10, 2021

Published: April 19, 2021

benzene ring and a pyrimidine ring, This skeleton is widely

present in natural products, including luotonin A, tryptan-

2021 American Chemical Society

Journal of Agricultural and Food Chemistry

J. Agric. Food Chem. 2021, 69, 4604−4614

604

Article

Figure 1. Chemical structures of several natural products and commercial drugs containing the quinazolinone backbone, hydrazide.

derivatives. Subsequently, these synthesized compounds were

tested against S. sclerotiorum, Rhizoctonia solani, Botrytis cinerea,

Fusarium graminearum, Pellicularia sasakii, and Fusarium

oxysporum. Afterward, a pot experiment was performed with

S. sclerotiorum to further evaluate the prospective application of

developing 4(3H)-quinazolinone derivatives as agricultural

fungicides. Furthermore, preliminary action mechanism

investigations were conducted for evaluating morphological

changes, relative electric conductivity, and cytoplasmic content

leakage.

product was washed with water and dried to obtain KZL-2−KZL-

16.

General Synthetic Procedure for Compounds KZL-17−KZL-34.

Hydrazine hydrate (0.0600 mL, 1.27 mmol) was added dropwise to a

stirred suspension of intermediate A (0.300 g, 1.06 mmol) in ethanol

(5 mL). The solution was reﬂuxed and monitored by thin-layer

chromatography (TLC). After cooling and in vacuo concentration,

H O (20 mL) was added and ethyl acetate (EtOAc, 3 × 20 mL) was

extracted, which was dried under MgSO and concentrated in vacuo

to obtain a crude product. Puriﬁcation by column chromatography

(with 9:1 petroleum ether/EtOAc) aﬀorded KZL-17−KZL-34.

Synthesis of Key Intermediate 2. In a 50 mL ﬂask, an appropriate

amount of triethylamine was added dropwise to a mixture solvent of

glycine methyl ester hydrochloride (0.310 g, 2.47 mmol) and

intermediate 1 in benzene (20 mL). After reﬂuxing for 10 h, water

was added and extracted with EtOAc (3 × 20 mL), dried with

MATERIALS AND METHODS

■

Instruments and Chemicals. A WRS-2U melting point

apparatus (Shanghai Precision Instrument Co., Ltd., China) was

used to determine the uncorrected melting points (mp). A Bruker

AM-400 spectrometer (Bruker Company, Billerica, MA, US.) was

MgSO , and concentrated. Target compounds were harvested after

puriﬁcation by ﬂash chromatography (using 8:1 petroleum ether/

used to obtain the H and C NMR spectra of compounds (with

tetramethylsilane as the internal standard). Mass spectra were

recorded using a Bruker Daltonics APEXII49e spectrometer (Bruker

Daltonics Inc., Billerica, MA, USA) with an ESI or EI ionization

source. A scanning electron microscope (Hitachi, S-3400N, Japan)

and a transmission electron microscope (Hitachi, HT7700, Japan)

were used for morphological observation. Hypha relative conductivity

and cytoplasmic content leakage were obtained with a conductivity

meter (Leici DDS-307, China) and a UV−vis spectrophotometer

EtOAc).

Synthesis of Key Intermediate 3. In a 50 mL ﬂask, a solution of

lithium hydroxide (0.190 g, 5.00 mmol) in H O was dropped into

intermediate 2 (0.700 g, 2.47 mmol) in tetrahydrofuran (THF) (20

mL). The solution was maintained at room temperature for 3 h and

monitored by TLC, followed by reduced pressure evaporation. The

residue was diluted with 2 N hydrochloric acid and extracted with

dichloromethane (DCM). Target compounds were ﬁnally harvested

after drying with MgSO₄and evaporated and puriﬁed by ﬂash

chromatography (using 10:1 DCM /methanol).

(

UV1902 PC, China). Commercial solvents and reagents used were

of reagent grade, and the commercial fungicide azoxystrobin was used

as positive control in the assay.

Fungi. S. sclerotiorum, R. solani, B. cinerea, F. graminearum, and F.

oxysporum were provided by Gansu Academy of Agricultural Sciences

and P. sasakii was obtained from Huazhong Agricultural University.

These fungi were maintained in a potato dextrose agar medium at 4

General Methods for the Preparation of Compounds 1a−1g,

a. At 0 °C, to a suspension of intermediate 2 (5.00 g, 28.4 mmol) in

dry DCM, the corresponding amine (0.210 mL, 2.00 mmol),

hydroxybenzotriazole (0.270 g, 2.00 mmol), and 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (0.380 g, 2.00 mmol) were

added successively. The reaction mixture was stirred for 24 h at

room temperature. After conﬁrmation of the completion of the

reaction by TLC, the residue was washed with brine, dried over

°C.

Synthetic Procedures. The synthetic route of target compounds

KZL-1−KZL-34 and 1a−8i is outlined in Schemes 1−3 (Figure 1).

Synthesis of Key Intermediate 1(A). A stirred solution of

substituted aminobenzoic acid (0.500 g, 3.65 mmol) in triﬂuoroacetic

anhydride or acetic anhydride (6 mL) was reﬂuxed for 4 h. The

anhydrous MgSO , and after removing the solvent was puriﬁed by

silica gel column chromatography (6:1 petroleum ether/EtOAc) to

give 1a−1g and 2a.

Synthesis of Key Intermediate 4. In a 50 mL ﬂask, hydrazine

hydrate (0.360 mL, 7.41 mmol) was dropped into intermediate 2

reaction was concentrated under reduced pressure to a brown oil.

Synthesis of Quinazolinone KZL-1. In a 25 mL ﬂask, the mixture

of 2-aminobenzoic acid (0.500 g, 3.65 mmol) and formamide (2.90

mL, 73.0 mmol) was reﬂuxed at 150 °C for 7 h. Then, a white

precipitate was formed, ﬁltered, washed with water, and dried to give

KZL-1.

General Methods for the Preparation of Compounds KZL-2−

KZL-16. In a 50 mL ﬂask, ammonium acetate (1.90 g, 24.7 mmol) was

added to the corresponding intermediate A (0.700 g, 2.47 mmol) and

reﬂuxed for 60 min. Then, the reaction mixture was transferred into

stirred ice-water, ﬁltered, and the precipitate was obtained. The crude

(0.700 g, 2.47 mmol) solution in methanol (20 mL) at room

temperature while stirring and then reﬂuxed for a short time; a white

solid was formed and that was collected by ﬁlteration.

General Synthetic Procedure for Compounds 3a−3g. In a 25 mL

ﬂask, intermediate 4 (0.350 g, 1.00 mmol) was mixed with a

corresponding benzaldehyde (0.110 mL, 1.05 mmol) solution in

ethanol (10 mL) and was reﬂuxed for 2 h. After cooling, the crude

product was collected by ﬁltration, washed, and recrystallized from

hot ethanol to yield 3a−3g.

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Figure 2. Synthesis route of target compounds.

Synthesis of Target Compound 4a. In a 25 mL ﬂask, to a

suspension of KOH (0.170 g, 3.00 mmol) in ethanol (10 mL), 4

compound was harvested after puriﬁcation by column chromatog-

raphy (120:1 DCM /methanol).

(

0.350 g, 1.00 mmol) and CS (0.150 mL, 2.50 mmol) were mixed.

General Synthetic Procedure for Compounds 6a−6i. In a 25 mL

ﬂask, the corresponding substituted phenylhydrazine (0.200 mL, 2.12

mmol) was added to a stirred solution of intermediate A (0.300 g,

After 4 h of reﬂuxing, the solvent was concentrated under reduced

pressure, extracted with DCM (3 × 20 mL), dried with MgSO , and

.06 mmol) in ethanol (5 mL) and monitored by TLC for

concentrated. Puriﬁcation by column chromatography (50:1 DCM

completion. The reaction mixture was reﬂuxed for 7 h and

methanol) gave 4a.

Synthesis of Compounds 4b-4e. In a 25 mL ﬂask, the

concentrated under vacuum. Then, it was poured into H O (20

mL), extracted with EtOAc, and dried over anhydrous MgSO ; the

corresponding alkyl halide (0.0600 mL; 1.00 mmol) was dissolved

in a suspension of compound 4a (0.400 g, 1.00 mmol) and

triethylamine (0.140 mL, 1.00 mmol) in EtOH (15 mL). After

reﬂuxing for 8 h, evaporation was performed, followed by puriﬁcation

of the residue by column chromatography (80:1 DCM /methanol) to

solvent was removed and puriﬁcation of the residue by Column

chromatography (15:1 petroleum ether/EtOAc) gave 6a−6i.

General Synthetic Procedure for Compounds 7a−7h. Com-

pound 22 (0.300 g, 1.01 mmol) was mixed with aromatic aldehydes

(0.100 mL, 1.01 mmol) in ethanol (8 mL) that contained glacial

harvest 4b−4e.

acetic acid (0.580 mL, 10.1 mmol) and reﬂuxed for 7 h with TLC

monitoring. The solution was concentrated, dissolved in EtOAc (10

mL), washed with H O (20 mL), dried under MgSO , and

General Synthetic Procedure for Compounds 5a−5g. In a 25 mL

ﬂask, a solution of 1 (0.300 g, 1.06 mmol) in pyridine (3 mL) was

added to substituted benzenesulfonyl hydrazide (0.200 g, 1.17 mmol),

stirred at 78 °C for 7 h; after that, the solution was concentrated,

poured into ice-cold water, and acidiﬁed with 2 N hydrochloric acid;

then, the solution was extracted with EtOAc (3 × 20 mL), dried with

concentrated. Compounds 7a−7h were obtained after puriﬁcation

of the residue by column chromatography (10:1 petroleum ether/

EtOAc).

General Synthetic Procedure for Compounds 8a−8i. In a 25 mL

ﬂask, a solution of 22 (0.300 g, 1.06 mmol), dry dimethyl formamide

MgSO , and evaporated under reduced pressure. The target

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Table 1. Antifungal Activity of Six Natural Products Containing a Quinazolinone Skeleton

average inhibition rate ± SD (%) (n = 3)

compd.

luotonin A

conc. (μg/mL)

S. s.

R. s.

B. c.

F. g.

P. s.

F. o.

100

56.3 ± 0.3

40.0 ± 0.1

36.7 ± 0.0

0.00 ± 0.00

31.5 ± 0.5

21.7 ± 0.0

9.03 ± 0.02

0.00 ± 0.00

5.90 ± 0.02

0.00 ± 0.00

41.7 ± 0.2

9.67 ± 0.27

32.8 ± 0.3

0.00 ± 0.00

66.7 ± 0.2

34.2 ± 0.5

0.00 ± 0.00

51.5 ± 0.3

23.0 ± 0.4

15.5 ± 0.5

0.00 ± 0.00

2.92 ± 0.05

0.00 ± 0.00

8.97 ± 0.180

8.04 ± 0.26

30.0 ± 0.2

0.00 ± 0.00

44.2 ± 0.4

22.2 ± 0.3

27.9 ± 0.1

19.4 ± 0.2

12.5 ± 0.3

0.00 ± 0.00

35.3 ± 0.3

19.0 ± 0.2

0.00 ± 0.00

4.48 ± 0.21

0.00 ± 0.00

31.7 ± 0.4

12.9 ± 0.1

28.5 ± 0.3

24.2 ± 0.3

tryptanthrin

evodiamine

rutaecarpine

quinazolinone

100

-methyl quinazolinone

100

0.00 ± 0.00

10.6 ± 0.2

Compd.: compound. Conc.: concentration. S. s.: Sclerotinia sclerotiorum. R. s.: Rhizoctonia solani. B. c.: Botrytis cinerea. F. g.: Fusarium

graminearum. P. s.: Pellicularia sasakii. F. o.: Fusarium oxysporum.

(

3 mL), and substituted acid chloride (0.230 mL, 3.18 mmol) was

the curative activity, inoculation was conducted after the leaves were

sprayed with 6c of diﬀerent concentrations with liquid ﬂowing at 24 h.

DMSO and azoxystrobin were used as negative control and positive

control, respectively. After inoculation, the leaves were incubated at

25 °C with a photoperiod of 16 h and a relative humidity of 85%.

After 72 h, the lesion in two perpendicular directions was measured

for calculating the average lesion diameter. The control eﬃcacy was

calculated according to the following formula

stirred at 0 °C for 30 min. Then, after overnight reaction at room

temperature with TLC monitoring, the reaction was transferred into

ice-cold water with stirring. The mixture was then subjected to EtOAc

(

3 × 20 mL) extraction, washed with water, dried with anhydrous

sodium sulfate, and evaporated under reduced pressure. Compounds

a−8i were obtained after puriﬁcation of the residue by column

chromatography (50:1 petroleum ether/EtOAc).

The spectral data of all compounds are provided in the Supporting

Information .

Control efficacy (%) = [(D_C_K− D )/(D − 5)] × 100

Antifungal Bioassay In Vitro. The antifungal activity of six

natural products and synthetic compounds was assayed against six

pathogenic fungi (S. sclerotiorum, R. solani, B.cinerea, F. graminearum,

P. sasakii, and F. oxysporum) using the mycelial growth inhibitory rate

where control eﬃcacy represents the disease control eﬃcacy, DCK

represents the lesion diameter in water control, and DT represents the

lesion diameter in treatment. The experiment was performed twice

with 10 replicates per treatment.

method. The potato dextrose agar (PDA) medium was sterilized at

21 °C for 20 min. Test compounds were dissolved in dimethyl

Scanning Electron Microscopy Observations.

Samples

sulfoxide (DMSO) before mixing with PDA at 40−50 °C, with

compound concentrations in the medium of 100 and 50 μg/mL,

respectively. The mixture was then poured into sterilized Petri dishes.

After cooling, a sterilized inoculation needle was used to pick up test

fungi with a mycelial disk of approximately 5 mm diameter, which

were then inoculated in the center of PDA Petri dishes. 0.5% DMSO

was used as blank control, while diﬀerent concentrations of

azoxystrobin served as positive control. When the Petri dish of

blank control was completely occupied by fungi, mycelium diameters

were measured and used to determine the inhibition rate using the

formula

treated with DMSO control and 5 μg/mL compound 6c were

prepared according to reported methods. Then, a scanning electron

microscope (Hitachi, S-3400N, Japan) was used for observation.

Transmission Electron Microscopy Observations. Myce-

lium plug samples treated with 0.5% DMSO or compound 6c (5 μg/

mL) were prepared according to reported methods. A transmission

electron microscope (Hitachi, HT7700, Japan) was used for

observation.

Determination of Cell Membrane Permeability.

The

permeability of S. sclerotiorum membrane was analyzed by

determining the relative electric conductivity of mycelium suspension.

The PD medium (80 mL) containing S. sclerotiorum mycelial disks (5

mm) was shaken at 150 rpm for 72 h at 25 °C for complete growth of

mycelia. Then, after ﬁltration, mycelia (200 mg) were treated with

compound 6c of diﬀerent concentrations, with deionized water as

blank control. The data were obtained using a conductivity detector

inhibition rate (%) = (C − T)/( C − 5 mm) × 100

where C represents the fungal growth diameter on untreated PDA and

T represents the fungal growth diameter on treated PDA. The colony

diameter of all samples was measured in triplicate and determined by

the cross-bracketing method. Otherwise, anti-bioassay compounds

were prepared in fresh PDA Petri dishes with concentrations of 100,

(

Leici DDS-307, China) within 24 h.

Determination of Cytoplasmic Content Leakage. The

general procedures were followed for the determination of

cytoplasmic content leakage according to cell membrane permeability;

afterward, the absorbance value was determined with a UV

spectrophotometer (UV 1902PC, INESA Scientiﬁc, China) within

0, 25, 10, 5, 2.5, 1, and 0.5 μg/mL, and the IC₅₀values (median

inhibitory concentration) were further determined.

Antifungal Bioassay In Vivo. Based on the in vitro results of

antifungal activity, compound 6c was further tested for protective and

curative activity against S. sclerotiorum according to our previous

0 h.

Statistical Analysis. The statistical analysis of all antifungal

work. Cole leaves with similar shapes were picked for testing.

Afterward, the leaves were soaked in 1% sodium hypochlorite for 2

min for disinfection, which was followed by rinsing with running

water and blotting with sterile ﬁlter paper. The diﬀerence between the

protective activity assay and the curative activity assay was the time of

inoculation. The 5 mm agar disk containing mycelia was placed

carefully at the widest central part of the rapeseed leaf while avoiding

placing it on the main vein. To evaluate the protective activity, the

leaves were sprayed with 6c of diﬀerent concentrations. Inoculation

was conducted when the liquid ﬂowed on the surface at 24 h. To test

activity assays was carried out using SPSS 24.0. The IC values, 95%

CI, regression equation, and R are provided in the Supporting

Information .

RESULTS AND DISCUSSION

■

Chemistry. The syntheses of all compounds are outlined in

schemes 1−3 (Figure 2), and their structures were conﬁrmed

using H NMR, C NMR, and MS analyses.

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Table 2. Antifungal Activity of a Series of Quinazolinone Derivatives at 100 μg/mL

average inhibition rate ± SD (%) (n = 3)

compound

S. s.

R. s.

B. c.

F. g.

P. s.

F. o.

KZL-01

KZL-02

KZL-03

KZL-04

KZL-05

KZL-06

KZL-07

KZL-08

KZL-09

KZL-10

KZL-11

KZL-12

KZL-13

KZL-14

KZL-15

KZL-16

KZL-17

KZL-18

KZL-19

KZL-20

KZL-21

KZL-22

KZL-23

KZL-24

KZL-25

KZL-26

KZL-27

KZL-28

KZL-29

KZL-30

KZL-31

KZL-32

KZL-33

KZL-34

0.00 ± 0.00

88.2 ± 0.2

0.00 ± 0.00

13.7 ± 1.1

93.9 ± 0.1

75.3 ± 0.5

58.4 ± 1.6

95.9 ± 0.1

19.0 ± 2.5

87.1 ± 2.2

95.5 ± 0.6

87.0 ± 0.6

100 ± 0

41.7 ± 1.2

32.8 ± 0.3

84.0 ± 0.4

29.5 ± 0.8

17.6 ± 4.3

41.1 ± 3.7

88.5 ± 1.0

56.5 ± 1.2

52.5 ± 0.3

95.2 ± 0.5

82.0 ± 1.3

75.7 ± 0.7

76.6 ± 1.1

98.7 ± 0.0

98.8 ± 0.1

45.5 ± 1.8

85.9 ± 0.3

79.4 ± 0.8

80.0 ± 0.7

47.8 ± 1.5

58.1 ± 0.0

97.8 ± 0.3

100 ± 0

51.5 ± 0.9

15.5 ± 1.5

82.0 ± 0.7

28.9 ± 0.5

0.00 ± 0.00

32.8 ± 2.0

85.5 ± 1.3

78.7 ± 0.2

59.0 ± 2.8

93.4 ± 0.3

30.7 ± 0.3

67.9 ± 0.5

71.8 ± 0.7

84.7 ± 1.1

99.7 ± 0.1

36.7 ± 1.7

9.07 ± 1.05

32.1 ± 1.0

21.1 ± 1.5

40.0 ± 0.5

42.4 ± 0.7

97.2 ± 0.6

95.2 ± 0.7

45.2 ± 1.3

57.9 ± 0.8

85.3 ± 0.8

94.2 ± 1.06

91.6 ± 0.4

77.5 ± 1.2

74.1 ± 2.0

51.4 ± 0.4

63.4 ± 0.4

72.2 ± 1.5

69.6 ± 4.5

0.00 ± 0.00

48.8 ± 1.9

50.2 ± 1.6

0.00 ± 0.00

34.7 ± 3.4

0.00 ± 0.00

51.0 ± 1.6

100 ± 0

0.00 ± 0.00

70.2 ± 1.3

0.00 ± 0.00

59.0 ± 0.2

60.4 ± 0.2

49.2 ± 0.6

26.3 ± 0.6

49.4 ± 1.6

75.2 ± 0.3

77.9 ± 0.2

73.9 ± 1.1

68.7 ± 1.6

39.2 ± 0.1

8.97 ± 0.88

30.0 ± 0.7

70.1 ± 0.2

21.4 ± 2.0

0.00 ± 0.00

26.0 ± 1.0

50.1 ± 0.1

33.2 ± 0.6

39.0 ± 1.2

71.7 ± 0.8

70.6 ± 0.7

72.8 ± 0.5

67.9 ± 0.5

85.6 ± 1.5

96.2 ± 0.6

29.2 ± 0.0

0.00 ± 0.00

20.4 ± 0.4

8.87 ± 1.33

20.9 ± 0.6

10.6 ± 0.7

94.3 ± 0.6

88.3 ± 1.1

35.8 ± 1.2

32.6 ± 1.3

51.2 ± 1.3

71.9 ± 0.6

60.9 ± 1.0

65.0 ± 2.3

53.4 ± 0.9

19.6 ± 1.1

13.1 ± 0.7

30.8 ± 0.2

29.6 ± 1.8

13.0 ± 1.9

47.9 ± 0.4

50.7 ± 1.6

0.00 ± 0.00

15.6 ± 0.5

51.0 ± 0.5

0.00 ± 0.00

28.9 ± 0.8

58.9 ± 0.3

51.7 ± 0.1

41.6 ± 1.8

53.8 ± 0.1

56.9 ± 0.4

61.9 ± 0.0

69.6 ± 1.0

43.5 ± 0.5

63.3 ± 0.1

44.2 ± 0.7

27.9 ± 0.7

84.6 ± 0.6

41.5 ± 0.6

32.1 ± 0.9

67.1 ± 1.3

87.8 ± 0.4

86.9 ± 0.3

63.5 ± 0.5

98.2 ± 0.5

80.4 ± 0.2

77.5 ± 0.5

75.1 ± 0.7

93.4 ± 0.3

99.9 ± 0.1

51.2 ± 0.0

94.1 ± 0.1

94.3 ± 0.6

81.1 ± 0.0

77.5 ± 0.5

93.1 ± 0.4

100 ± 0

31.7 ± 0.4

28.5 ± 0.3

70.4 ± 0.1

29.1 ± 0.6

30.9 ± 0.5

45.5 ± 0.3

69.0 ± 0.3

56.6 ± 0.6

39.4 ± 0.1

70.4 ± 0.2

44.4 ± 0.6

69.1 ± 0.4

71.2 ± 0.5

63.0 ± 0.6

90.5 ± 0.6

51.0 ± 1.4

18.2 ± 0.5

27.6 ± 0.6

30.4 ± 0.0

29.7 ± 0.2

28.9 ± 1.7

84.3 ± 0.1

79.4 ± 0.1

39.2 ± 0.9

16.5 ± 0.4

33.3 ± 1.3

48.2 ± 0.2

19.6 ± 0.4

95.8 ± 0.5

33.0 ± 0.8

0.00 ± 0.00

15.8 ± 0.1

43.5 ± 0.6

6.2 ± 0.2

0.00 ± 0.00

36.9 ± 0.2

49.2 ± 2.9

0.00 ± 0.00

50.0 ± 2.0

0.00 ± 0.00

6.15 ± 0.21

0.00 ± 0.00

18.0 ± 0.7

32.0 ± 0.7

29.6 ± 0.6

20.3 ± 0.1

34.5 ± 0.1

78.5 ± 0.0

76.5 ± 0.2

71.1 ± 1.0

31.4 ± 0.1

36.9 ± 0.0

11.2 ± 2.4

53.4 ± 1.5

45.0 ± 0.2

31.7 ± 0.1

36.3 ± 0.6

17.6 ± 0.6

72.1 ± 1.0

74.8 ± 0.9

57.1 ± 0.5

54.4 ± 0.0

60.1 ± 1.5

88.7 ± 0.5

66.2 ± 0.0

68.2 ± 0.2

58.8 ± 0.3

49.7 ± 0.4

72.7 ± 2.2

31.8 ± 0.6

31.1 ± 0.2

13.4 ± 1.0

42.6 ± 4.6

43.6 ± 4.8

0.00 ± 0.00

28.2 ± 1.6

73.7 ± 0.9

0.00 ± 0.00

8.84 ± 1.76

27.5 ± 1.3

0.00 ± 0.00

89.7 ± 0.94

43.9 ± 0.0

43.2 ± 0.2

19.0 ± 0.0

30.3 ± 0.0

89.7 ± 0.8

93.0 ± 0.2

96.4 ± 0.1

86.7 ± 0.1

84.3 ± 0.1

100 ± 0

85.0 ± 0.4

100 ± 0

99.7 ± 0.0

100 ± 0

63.9 ± 0.9

61.4 ± 0.8

100 ± 0

97.5 ± 0.8

96.4 ± 0.1

99.3 ± 0.5

96.0 ± 0.8

53.1 ± 0.7

59.9 ± 0.1

77.6 ± 0.2

75.3 ± 0.8

30.7 ± 0.4

57.9 ± 1.4

49.8 ± 0.8

13.7 ± 1.0

24.4 ± 0.8

11.2 ± 0.3

16.5 ± 0.7

99.6 ± 0.0

0.00 ± 0.00

9.47 ± 0.37

5.22 ± 0.35

8.85 ± 0.02

14.0 ± 0.6

75.6 ± 0.1

68.7 ± 1.0

39.6 ± 0.9

49.3 ± 0.2

88.1 ± 0.0

81.8 ± 0.9

94.5 ± 0.4

69.7 ± 0.5

55.9 ± 1.0

100 ± 0

97.6 ± 0.1

88.8 ± 0.9

78.8 ± 0.2

93.6 ± 0.9

77.0 ± 0.2

0.00 ± 0.00

46.6 ± 0.2

40.8 ± 1.8

0.00 ± 0.00

20.6 ± 0.1

40.9 ± 1.7

0.00 ± 0.00

20.3 ± 0.0

0.00 ± 0.00

43.0 ± 0.2

64.0 ± 0.3

67.1 ± 0.1

40.1 ± 1.2

44.7 ± 0.0

57.0 ± 0.0

71.6 ± 1.6

80.2 ± 1.0

55.4 ± 0.0

65.7 ± 0.6

608

J. Agric. Food Chem. 2021, 69, 4604−4614

Journal of Agricultural and Food Chemistry

Article

Table 2. continued

average inhibition rate ± SD (%) (n = 3)

compound

S. s.

R. s.

B. c.

F. g.

P. s.

F. o.

71.5 ± 0.0

96.4 ± 0.0

89.1 ± 0.3

94.4 ± 0.2

95.0 ± 0.6

18.3 ± 0.4

8.55 ± 0.15

37.1 ± 0.0

91.7 ± 0.0

17.0 ± 0.2

7.97 ± 0.30

29.8 ± 0.2

14.3 ± 0.2

14.0 ± 0.1

0.00 ± 0.00

25.4 ± 0.7

8.07 ± 0.34

12.0 ± 0.1

25.3 ± 1.0

79.6 ± 0.7

72.7 ± 3.7

23.8 ± 0.5

16.8 ± 0.2

93.0 ± 0.4

96.2 ± 0.6

33.4 ± 0.4

29.3 ± 0.4

25.9 ± 0.0

55.7 ± 1.0

16.9 ± 0.1

91.3 ± 0.2

53.8 ± 1.0

86.2 ± 0.1

86.8 ± 0.6

0.00 ± 0.00

12.7 ± 0.7

22.2 ± 0.1

77.0 ± 0.4

19.0 ± 0.5

12.9 ± 0.4

53.8 ± 0.8

31.8 ± 0.4

30.0 ± 0.7

16.4 ± 0.1

38.7 ± 0.6

33.8 ± 0.4

18.8 ± 0.4

32.0 ± 0.2

83.1 ± 1.0

49.5 ± 1.1

48.7 ± 0.4

46.0 ± 0.4

83.7 ± 0.5

89.1 ± 0.4

36.6 ± 0.7

39.8 ± 0.2

53.0 ± 0.8

59.1 ± 0.7

10.2 ± 0.0

77.5 ± 0.3

23.0 ± 0.1

81.6 ± 0.4

89.2 ± 0.2

0.00 ± 0.00

10.9 ± 0.9

15.5 ± 0.2

75.4 ± 0.2

20.7 ± 1.0

13.7 ± 1.2

14.8 ± 0.2

57.5 ± 0.8

40.1 ± 0.7

0.00 ± 0.00

46.1 ± 0.4

0.00 ± 0.00

11.8 ± 0.4

31.8 ± 0.2

12.1 ± 0.7

73.6 ± 1.0

51.6 ± 0.8

51.7 ± 0.0

78.3 ± 0.2

85.3 ± 0.3

38.1 ± 0.1

64.6 ± 0.2

67.5 ± 0.2

46.4 ± 0.3

18.8 ± 0.1

64.0 ± 1.0

48.0 ± 0.5

79.0 ± 0.1

78.9 ± 0.1

0.00 ± 0.00

84.8 ± 0.4

0.00 ± 0.00

36.4 ± 0.9

11.1 ± 0.1

21.3 ± 0.6

0.00 ± 0.00

10.8 ± 0.9

0.00 ± 0.00

6.90 ± 0.27

17.9 ± 0.0

44.7 ± 1.8

49.5 ± 1.1

29.1 ± 0.5

46.4 ± 1.2

68.8 ± 0.5

71.9 ± 0.2

35.5 ± 0.2

20.5 ± 0.3

40.7 ± 0.1

71.8 ± 1.4

27.4 ± 0.2

78.0 ± 0.1

67.7 ± 0.6

92.4 ± 0.7

94.2 ± 0.8

24.8 ± 0.4

0.00 ± 0.00

25.9 ± 0.7

89.7 ± 0.2

19.8 ± 0.2

6.18 ± 0.45

47.6 ± 0.4

32.1 ± 0.2

28.0 ± 0.2

28.7 ± 0.3

38.3 ± 1.5

34.6 ± 0.6

19.9 ± 1.0

48.7 ± 2.1

29.2 ± 0.4

35.9 ± 0.2

64.8 ± 0.2

55.6 ± 0.2

75.9 ± 0.0

85.9 ± 0.5

11.9 ± 0.3

28.6 ± 1.2

85.9 ± 0.0

54.8 ± 0.3

0.00 ± 0.00

72.2 ± 0.5

27.3 ± 0.0

65.2 ± 0.2

74.4 ± 0.2

11.0 ± 0.5

0.00 ± 0.00

63.0 ± 0.0

0.00 ± 0.00

15.4 ± 0.0

4.16 ± 0.47

9.83 ± 0.01

0.00 ± 0.00

32.8 ± 0.6

10.1 ± 0.1

22.2 ± 0.4

8.89 ± 0.01

63.8 ± 1.1

73.4 ± 0.5

0.00 ± 0.00

7.15 ± 0.41

10.0 ± 0.9

50.5 ± 0.9

azoxystrobin

S. s.: Sclerotinia sclerotiorum. R. s.: Rhizoctonia solani. B. c.: Botrytis cinerea. F. g.: Fusarium graminearum. P. s.: Pellicularia sasakii. F. o.: Fusarium

oxysporum.

Table 3. IC₅₀of a Series of Quinazolinone Derivatives

Against Six Phytopathogenic Fungi (μg/mL)

IC₅₀

compound

S. s.

6.42

10.7

R. s.

4.06

11.6

14.3

6.21

B. c.

9.99

23.7

F. g.

P. s.

F. o.

KZL-15

KZL-22

12.5

24.3

4.50

3.36

12.7

>25.0

24.0

>25.0

11.9

5.01

3.60

2.46

7.85

4.07

7.76

8.01

7.10

8.66

6.51

>25.0

8.41

>25.0

3.06

2.94

11.1

17.3

5.14

5.38

3.45

6.03

>25.0

20.3

17.6

15.8

S. s.: Sclerotinia sclerotiorum. R. s.: Rhizoctonia solani. B. c.: Botrytis

cinerea. F. g.: Fusarium graminearum. P. s.: Pellicularia sasakii. F. o.:

Fusarium oxysporum.

Figure 3. Antifungal activities of compound 6c against S. sclerotiorum

in vitro.

Intermediate 1 was prepared by the reaction of triﬂuoro-

acetic anhydride (or acetic anhydride) and the corresponding

aminobenzoic acid. Intermediate 2 was obtained by the

reaction of intermediate 1 and glycine methyl ester hydro-

chloride. Intermediate 2 was acidiﬁed by alkalization in the

mixture of THF solution of potassium hydroxide to aﬀord

intermediate 3. Then, intermediate 4 was obtained by the

mixture of intermediate 2 and hydrazine hydrate without

further puriﬁcation.

compounds 1a−2a were obtained through the reaction of

intermediate 3 with the corresponding amine. Compounds

3a−3g were obtained through the reaction of intermediate 4

with the corresponding benzaldehyde, and intermediate 4 was

transformed into oxadiazoles 4a−4e by reacting with

iodobenzene diacetate. The intermediate derivatization

method was often used to ﬁnd active candidate compounds;

compounds 5a−5g, 6a−6i, 7a−7h, and 8a−8i were synthe-

sized by this method, including intermediates 1 and 22.

Compounds KZL-01−KZL-34 were obtained from inter-

mediate 1 through a one-step reaction in good yields, and

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J. Agric. Food Chem. 2021, 69, 4604−4614

Journal of Agricultural and Food Chemistry

Compd.: compound. ASB: azoxystrobin.

Article

Table 4. Protective and Curative Activities of Compound 6c In Vivo

curative eﬀect

protective eﬀect

compd.

concentration (μg/mL)

lesion length (mm ± SD)

control eﬃcacy (%)

lesion length (mm ± SD)

control eﬃcacy (%)

15.4 ± 0.6

10.7 ± 0.5

7.62 ± 0.65

9.30 ± 0.35

7.75 ± 0.28

7.18 ± 0.80

25.7 ± 1.5

49.9

72.3

87.3

79.2

86.7

89.5

8.34 ± 0.70

7.52 ± 0.43

6.15 ± 0.10

7.28 ± 0.41

6.78 ± 0.80

6.09 ± 0.14

17.3 ± 0.6

72.9

79.5

90.7

81.5

85.5

91.2

100

ASB

Control

Figure 4. Curative and protective activities of compound 6c against S. sclerotiorum. (A) Curative eﬀects of compound 6c against S. sclerotiorum. (B)

Protective e ﬀ ects of compound 6c against S. sclerotiorum .

Figure 5. SEM patterns of S. sclerotiorum hyphae treated with DMSO

or compound 6c. (A,B) Control treatment with 0.5% DMSO, ×500,

and ×1000. (C,D) Treatment with compound 6c at 2.46 μg/mL

Figure 6. TEM observation of the cellular structure of S. sclerotiorum

treated with (A,B) 0.5% DMSO control and (C,D) 0.5% DMSO and

compound 6c at 2.46 μg/mL (IC ). CW: cell wall; CM: cell

(

IC ), ×500, and ×1000.

membrane; N: nucleus; V: vacuole.

Antifungal Bioassay In Vitro and SAR. First, we

results show that when the N-2 point of quinazolinone was

evaluated the antifungal eﬃcacy of six natural products

containing a quinazolinone skeleton [luotonin A, tryptanthrin,

evodiamine, rutaecarpine, quinazolinone (KZL-01), and 2-

methylquinazolinone (KZL-02)] at 100 and 50 μg/mL against

S. sclerotiorum, R. solani, B. cinerea, F. graminearum, P. sasakii,

and F. oxysporum, which are critical for agriculture. The data in

Table 1 show that quinazolinone and 2-methylquinazolinone

had a broader antifungal spectrum than luotonin A,

tryptanthrin, evodiamine, and rutaecarpine, which were more

complex in structure. Therefore, we synthesized a series of

quinazolinone derivatives KZL-01−KZL-34 with a single

quinazolinone skeleton and tested their antifungal activity at

substituted by triﬂuoromethyl (−CF ), most of the target

compounds exhibited potent activity against the six phytopa-

thogenic fungi at 100 μg/mL, and compounds KZL-15 and

KZL-22 with strong antifungal activity were selected as the

lead compounds for the next structure derivatization.

The results in Table 2 suggest that when the quinazolinone

nucleus was substituted by an electron-withdrawing group, the

compound possessed stronger antifungal activity. In order to

further investigate the antifungal eﬀect of side-chain-

substituent quinazolinone derivatives against phytopathogenic

fungi, eightseries of quinazolinone 3-substituted derivatives

were designed and synthesized based on compounds KZL-15

and KZL-22 (compounds 1a−4e and 5a−8i). From the data

00 μg/mL, whose results are summarized in Table 2. The

610

Journal of Agricultural and Food Chemistry

J. Agric. Food Chem. 2021, 69, 4604−4614

with compound 6c led to extensive hyphal branching of

Article

Notably, the highly active compounds 5b, 6b(6c), and

e(8f) were ﬂuorobenzenesulfonyl-, ﬂuorophenyl-, and ﬂuo-

robenzoyl-substituted derivatives of KZL-22 at the 3-position,

respectively. Therefore, it was implied that compared with

compound KZL-15, KZL-22 was more suitable for structural

derivatization, and the hydrazine fragment at the 3-position of

quinazolinone was crucial to maintain or improve the

antifungal activity of the derivative. On the other hand, these

compounds contained a common substituent group: ﬂuo-

rophenyl. Therefore, it can be concluded that ﬂuorophenyl is

an important pharmacophore for the derivatization of KZL-22.

Furthermore, compounds 5f, 6h, and 8i were 4-methylbenze-

nesulfonyl-, 4-methylphenyl-, and 4-methylbenzoyl-substituted

derivatives of KZL-22 at 3-position, respectively, and exhibited

poor antifungal activity at 100 μg/mL concentration.

Compared with compounds 5a, 5b, 5c, 6b, 6c, 8e, and 8f, it

was therefore extrapolated that the derivatives possessed better

antifungal activity when the benzene ring was replaced by an

electron-withdrawing group (−F).

Figure 7. Changes in cell membrane permeability of S. sclerotiorum

treated with compound 6c.

in Table 2, we can observe that compound 2a possessed higher

antifungal activity against the six test fungi than compounds

In Vivo Activity. The antifungal activity of the most active

compound 6c was assessed in vitro against S. sclerotiorum

1a−1g; so, we can speculate that the hydrazide fragment had a

(

Figure 3), and compound 6c exhibited excellent curative and

protective eﬀects, as determined using pot experiments in vivo

Table 4). The following conclusions could be drawn from

better antifungal potential than the amide fragment in our

modiﬁcation strategy. Moreover, the antifungal activity of the

derivatives disappeared when the hydrazide fragment was

converted to acylhydrazone (3a−3g), but the activity still

existed when the hydrazide fragment was converted to 1,3,4-

oxadiazole (4a−4e). Interestingly, we observed that com-

pounds 5a, 5b, 5c, 6b, 6c, 8e, and 8f containing the same

substituent group, ﬂuorophenyl, showed signiﬁcant antifungal

activities. Then, we extrapolated that ﬂuorophenyl could

maintain or increase the antifungal activity of the lead

compound KZL-22.

(

Table 4: ﬁrst, compound 6c possessed stronger protective

eﬀects (72.9, 79.5, and 90.7% at 25, 50, and 100 μg/mL,

respectively) than curative eﬀects (49.9, 72.3, and 87.3% at 25,

50, and 100 μg/mL, respectively); second, the curative eﬀect

and the protective eﬀect of compound 6c at 100 μg/mL

concentration (87.3 and 90.7%, respectively) were comparable

to those of azoxystrobin (89.5 and 91.2%, respectively); third,

the curative and protective eﬃcacies of compound 6c were

concentration-dependent. The images in Figure 4 validate the

negligible phytotoxicity of compound 6c on oilseed rape leaves

even at a high concentration of 100 μg/mL, which was benign

to the cole leaves.

For further exploration of the quinazolinone derivatives,

Table 3 outlines the IC₅₀values of target compounds with

remarkable antifungal activity at 100 μg/mL concentration.

From the values shown in Table 3, we can ﬁnd that compound

Preliminary Exploration of Antifungal Mechanism.

Eﬀects of 6c on S. sclerotiorum Morphology. The eﬀects of

compound 6c on the morphology of S. sclerotiorum were

investigated by scanning electron microscopy (SEM) and

transmission electron microscopy (TEM). As shown in Figure

5, the mycelia showed a smooth, uniform, and normal

morphology in the control group. In sharp contrast, treatment

c displayed the highest bioactivity against S. sclerotiorum, P.

sasakii, F. graminearum, and F. oxysporum, displaying

appreciable IC₅₀values of 2.46, 2.94, 6.03, and 11.9 μg/mL,

respectively. Also, compound 8f showed the highest antifungal

activity against R. solani and B. cinerea with IC values of 3.45

and 6.51 μg/mL, respectively. In addition, compounds 5b, 6b,

and 8e also exhibited moderate to signiﬁcant activity against

the six tested fungal strains.

Figure 8. Cellular content release from S. sclerotiorum treated with compound 6c.

611

J. Agric. Food Chem. 2021, 69, 4604−4614

Journal of Agricultural and Food Chemistry

Lanzhou 730000, People’s Republic of China; orcid.org/

Article

mycelia and other abnormalities including bent, shrunk,

collapsed, and deformed structures. The ultrastructural

alterations of S. sclerotiorum were also analyzed by TEM, as

shown in Figure 6 organelles in the control group were

arranged regularly with complete cell walls and obvious

diaphragms. However, after treatment with compound 6c,

the volume of vacuoles increased, the number of organelles

decreased, the nucleus disappeared, the color of the

cytoplasmic matrix became darker, and the cell wall became

thinner. SEM observation proved the mycelium damage of S.

sclerotiorum caused by compound 6c, and TEM results

validated that the compound could destroy the organelles

and nucleus of S. sclerotiorum.

Eﬀect of 6c on Cell Membrane Permeability. In order to

determine whether compound 6c acted on the membrane of S.

sclerotiorum, we measured the cell membrane permeability of

mycelia treated with diﬀerent concentrations of compound 6c.

Treatment with compound 6c signiﬁcantly increased the

relative electric conductivities of the mycelia 12 h post-

treatment (Figure 7). The values of the high-concentration

treatment group were higher than those of the low-

concentration treatment group.

IC₅₀values of quinazolinone derivatives against six

phytopathogenic fungi in detail; physical properties of

the target compounds; and spectra of representative

compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

■

Ying-Qian Liu − School of Pharmacy, Lanzhou University,

000-0001-5040-2516 ; Email: yqliu@lzu.edu.cn

Authors

Jing-Wen Peng − School of Pharmacy, Lanzhou University,

Lanzhou 730000, People’s Republic of China

Xiao-Dan Yin − School of Pharmacy, Lanzhou University,

Lanzhou 730000, People’s Republic of China; orcid.org/

000-0001-9282-5816

Hu Li − School of Pharmacy, Lanzhou University, Lanzhou

30000, People’s Republic of China

Kun-Yuan Ma − School of Pharmacy, Lanzhou University,

Lanzhou 730000, People’s Republic of China

Zhi-Jun Zhang − School of Pharmacy, Lanzhou University,

Lanzhou 730000, People’s Republic of China; orcid.org/

000-0003-3055-3426

Rui Zhou − School of Pharmacy, Lanzhou University,

Lanzhou 730000, People’s Republic of China

Eﬀects of 6c on Cellular Content Release. The

concentrations of nucleic acids and proteins in S. sclerotiorum

mycelial suspensions were evaluated by determining the

characteristic absorbance at 260 and 280 nm. However, there

were no signiﬁcant changes in absorbance after treatment with

compound 6c, according to Figure 8.

Yu-Ling Wang − Gansu Academy of Agricultural Sciences,

Lanzhou 730000, People’s Republic of China

Guan-Fang Hu − Gansu Academy of Agricultural Sciences,

Lanzhou 730000, People’s Republic of China

CONCLUSIONS

■

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jafc.0c05475

In conclusion, compounds KZL-01−KZL-34 were designed

and synthesized based on diversity-oriented synthesis; in vitro

antifungal activity screening found that most of the target

compounds have potent antifungal activity against six

phytopathogenic fungi, and two lead compounds KZL-15

and KZL-22 were obtained. Furthermore, eight novel series of

quinazolinone hybrids were synthesized, and the antifungal

results revealed that compound 6c displayed the highest

antifungal activity among all synthetic compounds against S.

sclerotiorum, P. sasakii, F. graminearum, and F. oxysporum with

IC values of 2.46, 2.94, 6.03, and 11.9 μg/mL respectively. In

Author Contributions

J.-W.P. and X.-D.Y. 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 the Key

Program for International S&T Cooperation Projects of

China Gansu Province (18YF1WA115). Support was also

provided by The Natural Science Foundation of Gansu

Province (20JR5RA311).

addition, an in vivo bioassay was carried out to explore the

potential of compound 6c in practical applications; the results

indicated that compound 6c exhibited comparable curative

eﬀect and protective eﬀect (87.3 and 90.7%, respectively) to

those of azoxystrobin (89.5 and 91.2%, respectively) at 100

μg/mL, respectively. To further explore the application value

of compound 6c in controlling S. sclerotiorum, we have studied

its preliminary mechanism of action. The results showed that

compound 6c could cause obvious hyphal malformation and

organelle damage of S. sclerotiorum and an increase in cell

membrane permeability. Based on the above ﬁndings, we can

say that these quinazoline derivatives with superior antifungal

activity in vivo and in vitro are promising candidate

compounds, and investigation of the speciﬁc action mechanism

of compound 6c is in progress.

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