Bioactivity-Guided Synthesis Accelerates the Discovery of 3-(Iso)quinolinyl-4-chromenones as Potent Fungicide Candidates

Article abstract of DOI:10.1021/acs.jafc.0c06700

Fungal infections could cause tremendous decreases in crop yield and quality. Natural products, including flavonoids and (iso)quinolines, have always been an important source for lead discovery in medicinal and agricultural chemistry. To promote the discovery and development of new fungicides, a series of 3-(iso)quinolinyl-4-chromenone derivatives was designed and synthesized by the active substructure splicing principle and evaluated for their antifungal activities. The lead optimization was guided by bioactivity. The bioassay data revealed that the 3-quinolinyl-4-chromenone 13 showed significant in vitro activities against S. sclerotiorum, V. mali, and B. cinerea with EC50 values of 3.65, 2.61, and 2.32 mg/L, respectively. The 3-isoquinolinyl-4-chromenone 25 exhibited excellent in vitro activity against S. sclerotiorum with an EC50 value of 1.94 mg/L, close to that of commercial fungicide chlorothalonil (EC50 = 1.57 mg/L) but lower than that of boscalid (EC50 = 0.67 mg/L). For V. mali and B. cinerea, 3-isoquinolinyl-4-chromenone 25 (EC50 = 1.56, 1.54 mg/L) showed significantly higher activities than chlorothalonil (EC50 = 11.24, 2.92 mg/L). In addition, in vivo experiments proved that compounds 13 and 25 have excellent protective fungicidal activities with inhibitory rates of 88.24 and 94.12%, respectively, against B. cinerea at 50 mg/L, while the positive controls chlorothalonil and boscalid showed inhibitory rates of 76.47 and 97.06%, respectively. Physiological and biochemical studies showed that the primary action of mechanism of compounds 13 and 25 on S. sclerotiorum and B. cinerea may involve changing mycelial morphology and increasing cell membrane permeability. In addition, compound 13 may also affect the respiratory metabolism of B. cinerea. This study revealed that compounds 13 and 25 could be promising candidates for the development of novel fungicides in crop protection.

Full text of DOI:10.1021/acs.jafc.0c06700

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Bioactivity-Guided Synthesis Accelerates the Discovery of
3‑(Iso)quinolinyl-4-chromenones as Potent Fungicide Candidates
Wei Wang,# Shan Zhang,# Jianhua Wang, Furan Wu, Tao Wang,* and Gong Xu*
Cite This: J. Agric. Food Chem. 2021, 69, 491−500
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ABSTRACT: Fungal infections could cause tremendous decreases in crop yield and quality. Natural products, including flavonoids
and (iso)quinolines, have always been an important source for lead discovery in medicinal and agricultural chemistry. To promote
the discovery and development of new fungicides, a series of 3-(iso)quinolinyl-4-chromenone derivatives was designed and
synthesized by the active substructure splicing principle and evaluated for their antifungal activities. The lead optimization was
guided by bioactivity. The bioassay data revealed that the 3-quinolinyl-4-chromenone 13 showed significant in vitro activities against
S. sclerotiorum, V. mali, and B. cinerea with EC50 values of 3.65, 2.61, and 2.32 mg/L, respectively. The 3-isoquinolinyl-4-chromenone
25 exhibited excellent in vitro activity against S. sclerotiorum with an EC50 value of 1.94 mg/L, close to that of commercial fungicide
chlorothalonil (EC50 = 1.57 mg/L) but lower than that of boscalid (EC50 = 0.67 mg/L). For V. mali and B. cinerea, 3-isoquinolinyl-4-
chromenone 25 (EC50 = 1.56, 1.54 mg/L) showed significantly higher activities than chlorothalonil (EC50 = 11.24, 2.92 mg/L). In
addition, in vivo experiments proved that compounds 13 and 25 have excellent protective fungicidal activities with inhibitory rates of
88.24 and 94.12%, respectively, against B. cinerea at 50 mg/L, while the positive controls chlorothalonil and boscalid showed
inhibitory rates of 76.47 and 97.06%, respectively. Physiological and biochemical studies showed that the primary action of
mechanism of compounds 13 and 25 on S. sclerotiorum and B. cinerea may involve changing mycelial morphology and increasing cell
membrane permeability. In addition, compound 13 may also affect the respiratory metabolism of B. cinerea. This study revealed that
compounds 13 and 25 could be promising candidates for the development of novel fungicides in crop protection.
KEYWORDS: natural product, flavonoid, (iso)quinoline, fungicidal activity, structure−activity relationship
the Cinchona tree in 1820.24 To date, there are more than 150
INTRODUCTION
■
(iso)quinoline alkaloids isolated,25 which show a wide range of
Fungal infection could cause tremendous decreases in crop
biological effects, such as antitumor,26 antimalarial,27 anti-
yield and quality, which threatens worldwide food security.1,2
bacterial,28 and antifungal activities.29 Also, (iso)quinolines
Treating crops with fungicides has been one of the most
have been identified as a pharmacophore in many complex
effective ways against plant diseases. Nevertheless, widespread
natural products, which exhibit highly antifungal activities.
use and misuse of chemical fungicides have caused more and
They have been considered as potential active groups and a
more resistance in the fungi.3,4 Meanwhile, green fungicides
functional scaffold reasonably in agrochemical discovery. For
with high efficiency and low toxicity are needed to reduce the
instance, the quinoline skeleton has been imbedded in the
negative impact on the environment. Using natural products as
marketed fungicides tebufloquin30 and quinofumelin (Figure
lead compounds to develop new fungicides is one of effective
1).31,32
solutions to these problems.5,6 Natural products with novel
Recently, We have developed a general approach for the
structures, as a source for new pesticides, not only can provide
synthesis of 3-(iso)quinolinyl 4-chromenones,33 which dis-
unique modes of action7 but also have good environmental
played obvious antifungal activities during our preliminary
compatibility, which is important for development of green
biological screening. Motived by this, in the present study, we
fungicides.8
attempted to further investigate flavonoid-based antifungal
Natural flavonoids have been found in many plants, such as
agents. Quercetin was taken as the lead compound for
tea,9 ginkgo,10 Hippophae rhamnoides,11 Flos Sophorae
systematic structural optimization. Attracted by the known
Immaturus, etc.9,12 Furthermore, flavonoids exhibit a variety
fungicidal activities of (iso)quinoline, we expected that the
of biological activities including antitumor,13,14 anti-inflamma-
antifungal effects of the lead compound could be further
tory,15 antioxidation,16 anticarcinogenic,17 anti-HIV,18 insecti-
cidal,19 and fungicidal effects (Figure 1).20,21 Containing a
valuable structural core, flavonoids have always been used as
Received: October 22, 2020
Revised: December 16, 2020
Accepted: December 18, 2020
Published: December 31, 2020
lead compounds in the development of medicines and
pesticides.22
Meanwhile, (iso)quinolines, one of the largest classes of
alkaloids, take a great proportion of the active natural
products.23 The first quinoline was isolated from the bark of
https://dx.doi.org/10.1021/acs.jafc.0c06700
© 2020 American Chemical Society
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Figure 1. Chemical structures of flavonoids (A) and (iso)quinolines (B) with biological activities.
Figure 2. Design of the target compounds.
on a WRS-1B melting point apparatus (Jingsong, Shanghai, China)
and were measured without correction. High-resolution mass
spectrometry (HRMS) was recorded using an electrospray ionization
(ESI) technique. Thin-layer chromatography (TLC) was performed
on a silica gel 60 GF254 plate. The silica gel (size of 200−300 mesh)
used for the column chromatography was purchased from Qingdao
Haiyang Chemistry Plant (China). Microscopic morphology of fungal
hyphae was observed by a scanning electron microscope (Hitachi, S-
3400 N, Tokyo, Japan). The conductivity was measured by a
CON510 Eutech/Oakton conductometer (OAKTON Instruments,
Waltham). Dissolved oxygen was measured by a JPB-607A dissolved
oxygen meter (INESA Scientific Instrument Co., Shanghai, China).
Fungi. Colletotrichum orbiculare (C. orbiculare), Fusarium oxy-
sporum (F. oxysporum), Sclerotinia sclerotiorum (S. sclerotiorum),
Physalospora piricola (P. piricola), Valsa mali (V. mali), Altenaria
alternariae (A. alternariae), and Botrytis cinerea (B. cinerea) were
provided by the College of Plant Protection, Northwest A&F
University.
improved by introducing (iso)quinoline to the flavonoid
skeleton through an active substructure splicing strategy.
With this in mind, series of novel 3-(iso)quinolinyl-4-
chromenone derivatives were designed and synthesized
(Figure 2). The fungicidal activities of these novel compounds
were evaluated to validate the idea, and the structure−activity
relationships (SARs) were analyzed. The results of biological
activity assays indicate that many of target compounds exhibit
excellent fungicidal activities, which initially verified the
rationality of our design strategy. To explore the preliminary
mechanisms of the action of 3-(iso)quinolinyl-4-chromenone
derivatives, the morphological changes of the mycelia cell wall
and the antifungal effects on the permeability of the mycelia
cell membrane and respiratory metabolism of fungi were
examined.
MATERIALS AND METHODS
Instruments and Chemicals. All chemicals (reagent grade) were
Synthesis. General Procedure for the Preparation of Inter-
mediates 1a−h. n-Butyllithium (2.0 mL, 5.0 mmol, 2.5 M in hexane)
was added dropwise to the solution of alkyne (5 mmol) in dry THF
(30 mL) at −78 °C. The solution was stirred at this temperature
under argon for 1 h. After that, salicylaldehyde (2.27 mmol) was
added using a syringe at the same temperature; the solution was
stirred for 2 h at −78 °C. Then, the reaction was quenched by the
■
purchased from commercial sources (Energy, Shanghai, China). All
1
the H and 13C NMR spectra were measured on a Bruker AV-400,
AV-500, or AV-600 spectrometer with CDCl3, CD2Cl2, or DMSO-d6
as the solvent and tetramethylsilane as the internal standard. Chemical
shifts were reported in ppm (δ). Melting points (mp) were recorded
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Figure 3. (A) General synthesis of 1a-h. Reagents and conditions: (a) n-BuLi, THF, −78 °C; MnO2, acetone. (B) General synthesis of 3−22.
Reagents and conditions: (b) quinoline N-oxide, DMF, 140 °C, HCl. (C) General synthesis of 23−33. Reagents and conditions: (c) isoquinoline
N-oxide, DMF, 140 °C, HCl.
addition of aqueous saturated NH4Cl (20 mL). The phases were
separated and the aqueous phase was extracted with EtOAc (3 × 10
mL). The combined organic layers were washed with brine (30 mL)
and dried over Na2SO4. The resulting liquid was concentrated under a
reduced pressure. Then, the crude material was dissolved in 30 mL of
acetone, and activated MnO2 (15.0 g, 172.5 mmol) was added to the
mixture. Then, the reaction mixture was stirred for 12 h at room
temperature, filtered through a pad of celite, concentrated under a
reduced pressure, and purified by flash chromatography on silica gel
(petroleum ether:ethyl acetate = 60:1) to afford the product.
General Procedure for the Preparation of Compounds 3−22.
Intermediate 1 (1 mmol) and quinoline N-oxide (1.5 mmol) are
dissolved in 1.5 mL of DMF in a 20 mL sealed tube; then, 25 μL of 12
M HCl solution was added into the mixture quickly. The reaction
mixture was stirred at 140 °C in a dry block heater for 2 h. After that,
the solvent was removed, and the crude product was purified via
column chromatography on silica gel (Gradient elution: petrol
ether:ethyl acetate = 15:1 to dichloromethane:methanol = 100:1) to
give the corresponding product.
the solvent was removed, and the crude product was purified via
column chromatography on silica gel (Gradient elution: petrol
ether:ethyl acetate = 15:1 to dichloromethane:methanol = 100:1) to
give the corresponding product.
Biological Assay. In Vitro Fungicidal Activities. All synthesized
compounds were screened for their in vitro antifungal activities against
C. orbiculare, F. oxysporum, S. sclerotiorum, P. piricola, V. mali, A.
alternariae, and B. cinerea at the concentration of 50 mg/L for the
preliminary screening according to a mycelia growth inhibition
method, with quercetin, chlorothalonil, tebuconazole, procymidone,
and boscalid as positive controls.
In Vivo Fungicidal Activities against B. cinerea. The Cucumis
sativus Linn. leaves of rape were collected from the Key Laboratory of
Botanical Pesticide R&D in Northwest A&F University. For the
protective activity assay, healthy leaves of Cucumis sativus Linn. were
sprayed with the target compounds (50 mg/L), respectively, and then
cultivated at 25 °C for 24 h before inoculation with B. cinerea.
Chlorothalonil and boscalid were used as the positive controls.
Effects of Compounds 13 and 25 on Mycelial Morphology of S.
sclerotiorum and B. cinerea. S. sclerotiorum and B. cinerea were
cultured in a 90 mm culture dish. When the mycelia grew to 70 mm in
diameter, fresh fungus dishes (5 mm in diameter) were made from the
edge of the colonies. The mycelia were inoculated on potato dextrose
agar (PDA) medium plates containing no compound (negative
control) and compounds 13 and 25 with a concentration of 5 mg/L,
General Procedure for the Preparation of Compounds 23−33.
Intermediate 1 (1 mmol) and isoquinoline N-oxide (1.5 mmol) are
dissolved in 1.5 mL of DMF in a 20 mL sealed tube; then, 25 μL of 12
M HCl solution was added into the mixture quickly. The reaction
mixture was stirred at 140 °C in a dry block heater for 2 h. After that,
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respectively. Then, the mycelia were cultured at 25 °C for 2 days. The
mycelia cell wall structure of S. sclerotiorum at the top of each treated
colonies were selected and observed under an S-3400 N scanning
electron microscope (SEM) (Hitachi, Ltd., Tokyo, Japan).
Effects of Compounds 13 and 25 on Cell Membrane
Permeability of S. sclerotiorum, V. mali, and B. cinerea. The tested
strains were cultured on a PDA plate at 25 °C for 72 h. Then, the
strains were placed in a potato dextrose broth (PDB) culture medium
and cultured by shaking (25 °C, 120 rpm) for 72 h. The mycelia were
filtered and washed with distilled water and put into a centrifugal
tube, and then 10 mL of compounds 13 and 25 (25 mg/L) and
distilled water were added into the centrifugal tube, respectively.
Finally, the mycelia were oscillated (120 rpm) in a water bath at a
constant temperature of 28 °C at different times. The conductivity
was measured by a CON510 Eutech/Oakton conductometer
(OAKTON Instruments, Waltham). The negative control was
mycelia with distilled water. The conductivity of compounds 13
and 25 were determined at 0, 30, 60, 120, 180, 240, and 300 min and
finally boiled (dead treatment) to determine the conductivity. The
relative permeability was calculated for each measurement, and then
the permeability of cell membranes was compared according to the
conductivity. Each experiment was run in triplicate.
Effects of Compounds 13 and 25 on the Respiratory
Metabolism of B. cinerea. The respiratory oxygen consumption
rate of B. cinerea was measured by the oxygen electrode method.34
A total of 500 mg of fresh mycelia were added into 10 mL of
medium solution (9 mL of phosphate buffer solution with a
concentration of 1 M, pH = 7.2 and 0.2 mL of glucose solution
with a concentration of 2%). After stirring for 10 min, dissolved
oxygen was measured by a JPB-607A dissolved oxygen meter (INESA
Scientific Instrument Co., Shanghai, China).
Figure 4. Bioactivity guided synthesis of compounds 3−22.
To confirm this, the compounds 15 (R2 = 7-OCH3), 16 (R2 =
8-OCH3), and 17 (R2 = 3-OCH3) were synthesized.
Interestingly, they all show good biological activities against
S. sclerotiorum, V.mali, and B.cinerea, which further proved that
the methoxy group is a factor determining the biological
activities of the target compounds. Comparing the biological
activities of compounds 8, 13, 15, 16, and 17, we found that
the compounds with a methoxy group at the C5/C6 position
of quinoline exhibited relatively higher activities. However,
because the cost of the starting material 6-methoxyquinoline ($
201/100 g, Energy) is much lower than that of 5-
methoxyquinoline ($ 857/100 g, Energy), we chose compound
13 (R2 = 6-OCH3) as the lead compound for further
optimization. Therefore, after R2 is fixed as the 6-OCH3
group, the influence of R1 substituents at the 4-position of a
phenyl ring on the antifungal activities was then investigated.
The compounds 18, 19, 20, 21, and 22 were synthesized. The
results show that compound 13 (R1 = H) exhibited the best
antifungal activity compared with the corresponding com-
pounds.
Then, compounds 13 and 25, and boscalid (20 mg/L) were added.
Respiration rates of mycelia (O2, μmol/g min) were calculated from
the change of oxygen content in the medium. The inhibition rates of
mycelia respiration (IRr) were calculated according to the respiration
rates of mycelia before and after adding test agents with the following
equation:
In order to investigate whether compounds 3−22 possess a
broad spectrum of bioactivity, we selected four more fungi (C.
orbiculare, F. oxysporum, P. piricola, and A. alternariae) to test
their antifungal activities. For C. orbiculare, compound 17 (R2
= 3-OCH3, 80.81%) showed an excellent inhibitory rate (about
80%), which is better than that of other compounds. In
addition, compounds 6 (R2 = 5-Cl, 72.22%) and 11 (R2 = 6-
Cl, 76.77%) displayed good antifungal activities against C.
orbiculare (inhibition rate > 70%). For F. oxysporum, although
the inhibition rates of all compounds are below 70%,
compound 17 with a methoxy group (R2 = 3-OCH3) still
showed the highest inhibition rate (67.62%), and compounds
15 (R2 = 7-OCH3, 57.90%) and 16 (R2 = 8-OCH3, 63.81%)
showed moderate fungicidal activity compared with other
compounds. For P.piricola, unfortunately, only compounds 3
(R2 = H, 52.22%), 13 (R2 = 6-OCH3, 50.00%), and 21 (R2 =
6-OCH3, 55.21%) gave the inhibition rates over 50%.
To further optimize the leads, compounds 23−33 were
designed by bioisosterism and evaluated for their antifungal
effects against S. sclerotiorum, V.mail, and B. cinerea at 50 mg/L.
As shown in Table 2, some of them displayed good fungicidal
activity against specific fungi. Among them, compounds 23, 24,
25, 28, and 29 displayed good antifungal activities against S.
sclerotiorum (inhibition rate > 80%) at 50 mg/L, which showed
inhibition rates of 98.33, 98.06, 100, 84.44, and 98.33%,
respectively. For the V. mali, compounds 23, 24, and 25
exhibited >90% inhibition rates. Notably, compounds 23−33
displayed good antifungal activities against B. cinerea (>80%
inhibition rates). Of note, in the 3-isoquinolinyl-4-chromenone
series, compound 25 showed the best antifungal activities
against the seven fungi.
IRr(%) = (R0 − R1)/R0
where R0 and R1 are respiration rates of mycelia before and after
adding tested agents.
RESULTS AND DISCUSSION
■
Chemistry Synthesis. The synthetic route for compounds
1a−h was described in Figure 3A. The target compounds can
be divided into two series: (1) 3-quinolinyl-4-chromenones
(Figure 3B); (2) 3-isoquinolinyl-4-chromenones (Figure 3C).
The chemical structures of all prepared compounds were
1
confirmed by H NMR, 13C NMR, and HRMS.
Fungicidal Activities and SAR Discussion. A bioactivity-
guided synthesis was applied for the discovery of antifungal
candidates. All compounds were evaluated against seven fungi
including C. orbiculare, F. oxysporum, S. sclerotiorum, P. piricola,
V. mali, A. alternariae, and B. cinerea. This biologically
indicating method is handleable and intuitionistic. The results
generated from this tactic are easy to interpret with a
significant value (Figure 4).
Initially, 3-quinolinyl-4-chromenones 3−14 were synthe-
sized and evaluated for their antifungal activities. As can be
seen from Table 1, most of the synthesized compounds
exhibited fair to good antifungal activities against S.
sclerotiorum, V. mali, and B. cinerea. Among them, compounds
8 (R2 = 5-OCH3) and 13 (R2 = 6-OCH3) displayed over 96%
inhibitory activity, which indicated that the methoxy group at
the quinoline moiety may be crucial for their significant in vitro
antifungal bioactivity. The higher biological activity of the
methoxy group may be related to its electron-donating effect.35
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a b c d e f g h i
, , , , , , , ,
Table 1. In Vitro Fungicidal Activities (Inhibition Rate) of the Compounds 3−22 (50 mg/L)
a
b
c
d
e
f
g
h
compd
C.o
F.o
S.c
P.p
V.m
A.a
B.c
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
50.56 1.10
49.49 0.88
61.11 1.75
72.22 0.87
51.01 4.63
48.33 1.65
47.98 0.87
51.52 0.81
76.77 1.51
35.86 1.75
48.48 1.52
45.57 3.2
66.16 0.68
59.60 2.31
80.81 0.88
45.96 2.31
36.36 1.51
46.46 0.89
51.52 1.52
46.46 0.72
33.33 2.15
70.73 1.45
97.62 0.48
34.44 2.86
30.48 2.18
52.38 0.83
38.10 0.83
31.43 1.43
26.11 3.44
40.48 1.65
56.19 4.12
50.95 2.98
36.19 0.83
33.33 0.72
15.31 0.90
57.90 1.32
63.81 0.83
67.62 0.82
11.09 2.72
10.95 0.83
28.10 1.69
45.71 2.47
19.05 3.86
<5
50.56 1.22
44.12 2.25
93.14 1.30
61.27 0.98
47.06 2.94
100
81.25 0.64
91.67 0.28
72.22 0.33
42.08 3.11
100
52.22 1.01
<5
65.64 1.12
72.92 1.38
93.75 0.52
92.19 0.90
88.02 1.04
100
65.63 0.53
59.90 1.57
60.94 1.56
59.38 1.27
64.58 1.80
50.00 1.57
70.31 0.90
91.67 0.12
31.77 0.50
61.46 1.21
63.02 0.98
27.02 1.56
85.42 0.89
90.63 1.57
75.00 0.91
41.33 1.42
39.33 2.58
52.00 2.02
75.33 1.15
48.00 1.65
<5
90.01 1.04
34.09 2.23
37.12 1.52
59.85 0.76
37.12 1.52
95.5 0.55
71.67 1.45
88.33 1.22
73.91 2.13
71.11 1.03
96.97 0.31
48.48 0.62
98.48 0.25
100
42.53 3.77
35.06 1.15
32.18 1.52
25.19 3.92
<5
75.00 0.26
92.22 0.13
54.36 2.25
53.33 2.12
97.92 0.22
39.49 0.51
93.75 1.10
91.67 0.65
88.54 0.75
37.84 2.53
39.19 2.47
59.46 1.28
90.99 0.42
55.41 1.37
73.96 1.80
71.67 1.65
62.72 0.52
<5
31.48 0.37
19.35 4.52
50.00 2.63
30.30 1.61
48.85 2.17
27.01 3.59
43.68 2.51
7.29 5.11
18.75 2.53
28.65 4.88
55.21 2.62
24.48 4.55
8.62 0.99
86.15 0.64
66.67 0.60
46.67 0.59
100
75.00 1.20
84.17 0.85
38.33 3.52
22.00 4.25
34.67 3.10
75.00 1.02
41.67 3.05
100
91.67 1.52
<5
26.34 3.55
32.80 2.38
80.65 0.66
23.66 4.21
9.09 4.32
91.6 0.58
quercetin
C
boscalid
i
76.19 0.55
50.00 0.78
100
100
66.67 2.08
44.59 0.78
85.76 0.61
a
b
c
d
e
Data are given as the mean of triplicate experiments. C.o: Colletotrichum orbiculare. F.o: Fusarium oxysporum. S.s: Sclerotinia sclerotiorum. P.p:
f
g
h
i
Physalospora piricola. V.m: Valsa mali. A.a: Altenaria alternariae. B.c: Botrytis cinerea. C: chlorothalonil.
a b c d e f g h i
, , , , , , , ,
Table 2. In Vitro Fungicidal Activities (Inhibition Rate) of the Compounds 23−33 (50 mg/L)
a
b
c
d
e
f
g
h
compd
C.o
F.o
S.c
P.p
V.m
A.a
B.c
23
24
25
26
27
28
29
30
31
32
33
56.67 2.14
58.33 1.65
65.00 1.32
54.30 0.69
52.22 1.25
65.00 1.05
60.00 0.45
45.00 2.61
37.78 2.78
40.00 2.65
53.33 0.88
33.33 2.15
70.73 1.45
97.62 0.48
62.22 2.04
68.44 1.03
69.33 0.28
32.78 2.57
41.11 1.10
62.78 1.42
20.00 3.23
16.67 3.57
40.00 1.65
38.33 2.41
62.78 1.58
<5
98.33 0.13
98.06 0.20
100
57.04 0.37
52.22 0.98
63.33 0.63
67.41 0.97
41.11 2.30
72.22 0.63
3.33 0.63
54.44 0.42
42.59 0.74
69.63 0.98
42.96 0.37
8.62 0.99
86.15 0.64
66.67 0.60
97.95 0.58
95.38 0.14
100
78.13 0.90
76.56 0.90
79.85 0.14
73.44 1.82
75.00 1.57
62.50 0.14
34.90 0.50
46.35 0.53
59.90 0.57
67.71 0.53
66.15 0.53
<5
96.14 0.30
97.67 0.13
100
72.22 1.41
50.00 2.44
84.44 1.58
98.33 0.22
42.78 3.11
75.56 2.02
46.67 3.44
30.56 4.20
100
82.05 0.95
86.67 0.83
87.69 1.32
38.97 2.75
47.18 2.35
85.13 0.88
63.59 1.22
49.74 1.38
73.96 1.80
71.67 1.65
62.72 0.52
88.56 0.22
85.55 0.55
90.34 0.30
81.23 0.79
86.71 1.85
88.21 0.52
92.12 0.79
88.86 0.60
9.09 4.32
91.6 0.58
quercetin
i
C
76.19 0.55
50.00 0.78
100
100
66.67 2.08
44.59 0.78
boscalid
85.76 0.61
a
b
c
d
e
Data are given as the mean of triplicate experiments. C.o: Colletotrichum orbiculare. F.o: Fusarium oxysporum. S.s: Sclerotinia sclerotiorum. P.p:
f
g
h
i
Physalospora piricola. V.m: Valsa mali. A.a: Altenaria alternariae. B.c: Botrytis cinerea. C: chlorothalonil.
In order to further determine the fungicidal potency and
probe the SARs of these novel 3-(iso)quinolinyl-4-chrome-
none derivatives, several compounds with superior fungicidal
activities were selected for further study. Their EC50 values
against S. sclerotiorum, V. mali, and B. cinerea are shown in
Table 3. In general, the compounds bearing methoxy groups
showed better fungicidal activities than that with other
substituents. For example, compound 8 (R2 = 5-OCH3, EC50
= 3.79 mg/L) was more active than compound 5 (R2 = 5-F,
mali and B. cinerea, respectively. Meanwhile, the compounds
with substitution at the 5/6-position of the quinoline ring
showed better fungicidal activities than that with substitution
at other positions. For instance, compounds 8 (R2 = 5-OCH3,
EC50 = 3.79, 2.37 mg/L) and 13 (R2 = 6-OCH3, EC50 = 3.65,
2.32 mg/L) were more active than compounds 15 (R2 = 7-
OCH3, EC50 = 5.27, 2.47 mg/L), 16 (R2 = 8-OCH3, EC50
=
8.55, 6.54 mg/L), and 17 (R2 = 3-OCH3, EC50 = 18.18, 13.54
mg/L), against S. sclerotiorum and B. cinerea. For V. mali,
compound 8 (EC50 = 1.65 mg/L) was more active than
compounds 13 (EC50 = 2.61 mg/L), 17 (EC50 = 4.97 mg/L),
15 (EC50 = 3.33 g/L), and 16 (EC50 = 3.43 mg/L).
EC50 = 4.60 mg/L) and compound 13 (R2 = 6-OCH3, EC50
=
3.65 mg/L) was more active than compound 10 (R2 = 6-F,
EC50 = 7.50 mg/L) against S. sclerotiorum. Compound 13 (R2
= 6-OCH3, EC50 = 2.61, 2.32 mg/L) was more active than
compound 10 (R2 = 6-F, EC50 = 3.47, 4.72 mg/L) against V.
The above SARs of the 3-quinolinyl-4-chromenones 3−22
against S. sclerotiorum, V. mali, and B. cinerea could be
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a
Table 3. Fungicidal Activities of the Target Compounds with EC50 Values against S. sclerotiorum, V. mali, and B. cinerea
a
fungi
compd
R1
R2
5-F
5-OCH3
6-CH3
6-F
6-OCH3
7-OCH3
8-OCH3
3-OCH3
6-OCH3
H
EC50
95% CI
regression equation
R2
S. sclerotiorum
5
8
9
H
H
H
H
H
H
H
H
4.60
3.79
23.49
7.50
3.65
5.27
8.55
18.18
7.82
4.20
4.42
1.94
2.17
4.37
3.56
1.57
0.67
3.55
2.97
5.79
1.65
3.38
3.47
2.61
3.33
3.43
4.97
6.81
3.52
3.18
1.56
3.02
3.57
3.98
11.24
0.27
8.20
2.37
25.87
4.72
2.32
2.47
6.54
13.54
4.78
3.57
4.52
1.54
7.65
6.45
5.28
38.48
4.90
2.92
1.26
0.10
2.34−4.87
1.03−6.35
14.63−51.40
5.94−9.14
2.74−4.51
4.45−6.22
6.55−10.75
15.72−21.29
5.73−10.01
3.54−4.96
2.18−7.15
1.61−2.29
1.72−2.64
3.58−5.28
2.35−4.74
0.95−2.22
0.47−0.89
0.03−8.13
1.83−4.09
4.10−7.50
1.36−1.95
2.54−4.32
2.77−4.20
2.16−3.10
2.47−4.24
2.72−4.21
3.87−6.18
5.43−8.41
2.83−4.23
2.48−3.92
1.23−1.89
2.37−3.69
2.52−4.58
3.21−4.80
8.20−15.12
0.16−0.40
6.69−9.84
1.64−3.17
19.42−38.46
3.59−5.83
1.38−3.31
1.92−3.03
5.53−7.58
9.52−19.66
3.70−5.82
2.80−4.38
3.66−5.44
1.12−1.98
6.24−9.13
4.69−8.24
3.68−6.86
25.11−82.53
3.50−6.27
1.61−4.22
0.61−2.04
0.07−0.14
y = −0.673 + 1.205x
y = −0.908 + 1.571x
y = −2.528 + 1.844x
y = −1.237 + 1.414x
y = −1.001 + 1.781x
y = −1.066 + 1.477x
y = −1.088 + 1.167x
y = −2.507 + 1.989x
y = −0.946 + 1.059x
y = −0.928 + 1.489x
y = −0.892 + 1.382x
y = −0.487 + 1.692x
y = −0.449 + 1.338x
y = −0.796 + 1.243x
y = −0.696 + 1.262x
y = −0.241 + 0.954x
y = 0.183 + 1.042x
y = −0.184 + 0.334x
y = −0.583 + 1.231x
y = −0.859 + 1.126x
y = −0.386 + 1.769x
y = −0.493 + 0.934x
y = −0.809 + 1.498x
y = −0.630 + 1.511x
y = −0.610 + 1.167x
y = −0.619 + 1.156x
y = −0.810 + 1.163x
y = −0.971 + 1.165x
y = −0.842 + 1.543x
y = −0.705 + 1.401x
y = −0.286 + 1.490x
y = −0.711 + 1.481x
y = −0.820 + 1.486x
y = −0.883 + 1.471x
y = −0.941 + 0.895x
y = 0.528 + 0.937x
y = −1.396 + 1.528x
y = −0.558 + 1.493x
y = −1.388 + 0.982x
y = −1.049 + 1.558x
y = −0.311 + 0.852x
y = −0.619 + 1.575x
y = −1.693 + 2.076x
y = −0.862 + 0.761x
y = −1.121 + 1.651x
y = −0.754 + 1.364x
y = −0.933 + 1.425x
y = −0.209 + 1.125x
y = −1.410 + 1.597x
y = −0.928 + 1.147x
y = −0.829 + 1.147x
y = −1.160 + 0.732x
y = −0.874 + 1.268x
y = −0.485 + 1.043x
y = −0.059 + 0.581x
y = 0.891 + 0.905x
0.992
0.985
0.992
0.970
0.986
0.952
0.990
0.991
0.948
0.941
0.993
0.990
0.980
0.973
0.997
0.960
0.979
0.993
0.989
0.977
0.987
0.987
0.968
0.977
0.976
0.959
0.958
0.983
0.982
0.954
0.968
0.985
0.994
0.950
0.991
0.962
0.977
0.953
0.951
0.987
0.988
0.986
0.999
0.978
0.986
0.958
0.959
0.988
0.990
0.988
0.974
0.988
0.982
0.986
0.967
0.970
10
13
15
16
17
21
23
24
25
26
28
29
CH3
4-CH3
4-F
H
H
H
H
4-Cl
4-OCH3
3-CH3
Ph
H
chlorothalonil
boscalid
V. mali
4
6
7
8
9
10
13
15
16
17
21
23
24
25
26
27
H
H
H
H
H
H
H
H
5-CH3
5-Cl
5-Br
5-OCH3
6-CH3
6-F
6-OCH3
7-OCH3
8-OCH3
3-OCH3
6-OCH3
H
H
H
H
H
H
H
CH3
4-CH3
4-F
4-Cl
4-OCH3
4-CF3
3-CH3
28
H
chlorothalonil
tebuconazole
B. cinerea
3
8
9
10
13
15
16
17
21
23
24
25
26
27
28
29
H
H
H
H
H
H
H
H
H
5-OCH3
6-CH3
6-F
6-OCH3
7-OCH3
8-OCH3
3-OCH3
6-OCH3
H
H
H
H
H
CH3
4-CH3
4-F
4-Cl
4-OCH3
4-CF3
3-CH3
Ph
H
H
6-CH3
32
H
chlorothalonil
boscalid
procymidone
a
Confidence interval.
summarized as follows: (1) The compounds bearing methoxy
groups in the quinoline moiety show better fungicidal activities
than that with other groups (H, CH3, F, Cl, Br, and OCF3).
(2) The compounds with a 5/6-methoxy group in the
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quinoline part exhibit similar antifungal activities, which are
relatively higher than that with other groups. (3) The
compounds including 6-methoxy quinoline were chosen for
further lead optimization due to the availability of the starting
material and cost considerations. When R2 is fixed as a 6-
methoxy group, the compound 13 with R1 = H at the 4-
position of the phenyl ring displays the best antifungal activity.
As for the 3-isoquinolinyl-4-chromenones series, we initially
explored the effect of the substituents on the phenyl ring. The
results indicated that introduction of a chlorine atom on the
phenyl ring (compound 25) was favorable for increased
fungicidal activities (Figure 5). Also, compound 25 displayed
the most potent fungicidal activities (EC50 = 1.94, 1.56, 1.54
mg/L) against S. sclerotiorum, V. mali, and B. cinerea,
respectively.
Cucumis sativus Linn. leaves. The efficacy of the antifungal
protection is shown in Figure 6A. Compounds 13 and 25
showed promising protective activities of 88.24 and 94.12%,
respectively. Notably, compounds 13 and 25 showed better
antifungal effects than chlorothalonil (76.47%) but slightly
worse than boscalid (97.06%) at the concentration of 50 mg/
L. (Table 4).
Table 4. In Vivo Activity of the Target Compounds 13 and
25 against B. cinerea on Cucumis sativus Linn. Leaves (50
mg/L)
protective activity
compd
diameter of lesions (cm)
control efficacy (%)
13
25
0.7 0.1
0.6 0.1
0.9 0.2
0.5 0.1
2.2 0.2
88.24%
94.12%
76.47%
97.06%
chlorothalonil
boscalid
CK
Effects of Compounds 13 and 25 on Cell Membrane
Permeability of S. sclerotiorum, V. mali, and B. cinerea.
The change of relative conductivity indicates the variation of
cell membrane permeability. According to literature,34,36 the
cell membrane of fungi is an important target for fungicides,
which can inhibit the synthesis of a phospholipid bilayer or
protein and cause the leakage of internal electrolytes.37,38 As
shown in Figure 6B, after treatment with compounds 13 and
25, the mycelial relative conductivities of S. sclerotiorum, V.
mali, and B. cinerea increased with time, and the increase extent
is much greater than that with the control, which indicates that
Figure 5. Summarized SARs of the target compounds against S.
sclerotiorum, V. mali, and B. cinerea.
In Vivo Fungicidal Activities. In vivo fungicidal activity of
the target compounds against B. cinerea was carried out on
Figure 6. (A) In vivo fungicidal activity of the target compounds against B. cinerea was carried out on Cucumis sativus Linn. leaves. (B) Mycelial
relative conductivity of S. sclerotiorum, V. mali, and B. cinerea in the presence or absence of compounds 13 and 25.
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compounds 13 and 25 could enhance the mycelial cell
membrane permeability of S. sclerotiorum, V. mali, and B.
cinerea. Combined with the change of mycelial morphology
after treatment with compounds 13 and 25, it can also be
inferred that compounds 13 and 25 could manifest antifungal
properties by destroying the cell membrane structure. The
above reported findings revealed that the action target site of
compounds 13 and 25 on S. sclerotiorum, V. mali, and B. cinerea
could be the cell membrane.
In summary, series of 3-(iso)quinolinyl-4-chromenone
derivatives were designed and synthesized by the active
substructure splicing principle and evaluated for their
antifungal activities. The lead optimization was guided by
bioactivity. The antifungal bioassay discovered the highly
active compounds 13 and 25 with a broad spectrum of
excellent in vitro fungicidal activities and better in vivo efficacy
against B. cinerea. Physiological and biochemical studies
showed that the primary action of mechanism of compounds
13 and 25 on S. sclerotiorum and B. cinerea may involve
changing mycelial morphology and increasing cell membrane
permeability. In addition, compound 13 may also affect the
respiratory metabolism of B. cinerea. This study reveals that
compounds 13 and 25 could be promising antifungal
candidates and provide a valuable reference for further
development of 3-(iso)quinolinyl-4-chromenones in crop
protection.
Scanning Electron Microscopy (SEM) Analysis. SEM of
S. sclerotiorum and B. cinerea revealed morphological changes in
the fungal cell surface. As shown in Figure 7, the surfaces of S.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c06700.
■
sı
The physical and spectroscopic data of 1a−h, 3−33
(PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Tao Wang − Key Laboratory of Applied Surface and Colloid
Chemistry, Ministry of Education, School of Chemistry and
Chemical Engineering, Shaanxi Normal University, Xi’an
710119, Shaanxi Province, China; orcid.org/0000-0002-
1494-5297; Email: chemtao@snnu.edu.cn
Gong Xu − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China; State Key
Laboratory of Crop Stress Biology for Arid Areas, Key
Laboratory of Botanical Pesticide R&D in Shaanxi Province,
Shaanxi Key Laboratory of Natural Products & Chemical
Biology, Yangling 712100, Shaanxi, China; orcid.org/
0000-0002-4305-4370; Email: gongxu@nwafu.edu.cn
Figure 7. Surfaces of S. sclerotiorum (A, E) and B. cinerea (I, M) cells
in the untreated groups were relatively smooth and regular, whereas
when treated with compounds 13 (B, F, J, N), 25 (C, G, K, O), and
Boscalid (D, H, L, P) at 5 mg/L there were significant shrinkage.
sclerotiorum and B. cinerea cells in the untreated groups (A, E, I,
and M) were relatively smooth and regular, whereas when
treated with compounds 13 (B, F, J, and N) and 25 (C, G, K,
and O), and boscalid (D, H, L, and P) at 5 mg/L, there were
significant shrinkage. Of note, for the B. cinerea, the extent of
mycelial shrinkage with compound 25 (Figure 7, K and O) is
greater than that with compound 13 (Figure 7, J and N). This
showed that compounds 13 and 25 have a certain degree of
damage to the cell wall of B. cinerea, and the damage intensity
with compound 25 is greater.
Effects of Compounds 13 and 25 on the Respiratory
Metabolism of B. cinerea. The respiratory inhibition effects
of compounds 13 and 25, and boscalid on B.cinerea mycelia are
shown in Table 5. Compounds 13 and 25 have inhibitory
Authors
Wei Wang − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China
Shan Zhang − Key Laboratory of Applied Surface and Colloid
Chemistry, Ministry of Education, School of Chemistry and
Chemical Engineering, Shaanxi Normal University, Xi’an
710119, Shaanxi Province, China
Jianhua Wang − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China
Furan Wu − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China
Table 5. Respiratory Inhibition Rate of Compounds 13 and
25 and Boscalid on B. cinerea Mycelia
compd
R0 (O2, μmol/g min) R1 (O2, μmol/g min)
IR (%)
13
25
boscalid
3.83 0.02
3.67 0.03
3.73 0.03
2.63 0.03
2.87 0.09
2.53 0.02
32.16 0.58
19.06 1.43
33.02 0.59
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c06700
Author Contributions
#W.W. and S.Z. contributed equally.
Notes
effects on the respiration rate of B. cinerea mycelia, and their
inhibition rates were 32.16 and 19.06%, respectively. The
inhibition rate of compound 13 is close to that of boscalid
(33.02%). We speculate that the antifungal mechanism of
compound 13 may be similar to that of boscalid, which
involves the inhibition of fungal respiration.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
G.X. acknowledges support from the National Natural Science
Foundation of China (21801207), the Program for Science &
■
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Technology Innovation Team of Shaanxi Province (2020TD-
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A&F University. T.W. acknowledges support from the
National Natural Science Foundation of China (21502110),
the Natural Science Foundation of Shaanxi Province (2019JQ-
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(B14041), and the Fundamental Research Funds for the
Central Universities (GK201903039). Partial instrumentation
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We are appreciative to Fu-zhen Guo (Northwest A&F
University) for scanning electron microscopy (SEM) analysis.
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