Design, Synthesis, and Antifungal Evaluation of Cryptolepine Derivatives against Phytopathogenic Fungi

Article abstract of DOI:10.1021/acs.jafc.0c06480

Inspired by the widely antiphytopathogenic application of diversified derivatives from natural sources, cryptolepine and its derivatives were subsequently designed, synthesized, and evaluated for their antifungal activities against four agriculturally important fungi Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, and Sclerotinia sclerotiorum. The results obtained from in vitro assay indicated that compounds a1-a24 showed great fungicidal property against B. cinerea (EC50 < 4 μg/mL); especially, a3 presented significantly prominent inhibitory activity with an EC50 of 0.027 μg/mL. In the pursuit of further expanding the antifungal spectrum of cryptolepine, ring-opened compound f1 produced better activity with an EC50 of 3.632 μg/mL against R. solani and an EC50 of 5.599 μg/mL against F. graminearum. Furthermore, a3 was selected to be a candidate to investigate its preliminary antifungal mechanism to B. cinerea, revealing that not only spore germination was effectively inhibited and the normal physiological structure of mycelium was severely undermined but also detrimental reactive oxygen was obviously accumulated and the normal function of the nucleus was fairly disordered. Besides, in vivo curative experiment against B. cinerea found that the therapeutic action of a3 was comparable to that of the positive control azoxystrobin. These results suggested that compound a3 could be regarded as a novel and promising agent against B. cinerea for its valuable potency.

Full text of DOI:10.1021/acs.jafc.0c06480

pubs.acs.org/JAFC
Article
Design, Synthesis, and Antifungal Evaluation of Cryptolepine
Derivatives against Phytopathogenic Fungi
Yong-Jia Chen,§ Hua Liu,§ Shao-Yong Zhang, Hu Li, Kun-Yuan Ma, Ying-Qian Liu,* Xiao-Dan Yin,
Rui Zhou, Yin-Fang Yan, Ren-Xuan Wang, Ying-Hui He, Qing-Ru Chu, and Chen Tang
Cite This: J. Agric. Food Chem. 2021, 69, 1259−1271
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Inspired by the widely antiphytopathogenic application of diversified derivatives from natural sources, cryptolepine
and its derivatives were subsequently designed, synthesized, and evaluated for their antifungal activities against four agriculturally
important fungi Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, and Sclerotinia sclerotiorum. The results obtained from in
vitro assay indicated that compounds a1−a24 showed great fungicidal property against B. cinerea (EC50 < 4 μg/mL); especially, a3
presented significantly prominent inhibitory activity with an EC50 of 0.027 μg/mL. In the pursuit of further expanding the antifungal
spectrum of cryptolepine, ring-opened compound f1 produced better activity with an EC50 of 3.632 μg/mL against R. solani and an
EC50 of 5.599 μg/mL against F. graminearum. Furthermore, a3 was selected to be a candidate to investigate its preliminary antifungal
mechanism to B. cinerea, revealing that not only spore germination was effectively inhibited and the normal physiological structure of
mycelium was severely undermined but also detrimental reactive oxygen was obviously accumulated and the normal function of the
nucleus was fairly disordered. Besides, in vivo curative experiment against B. cinerea found that the therapeutic action of a3 was
comparable to that of the positive control azoxystrobin. These results suggested that compound a3 could be regarded as a novel and
promising agent against B. cinerea for its valuable potency.
KEYWORDS: cryptolepine, Botrytis cinerea, antifungal activity, structure activity relationship, antifungal mechanism
interacting with multiple biological targets in the process of
evolution, which were easily degraded and had low impact to
the environment and nontarget organisms.8 Searching for lead
compounds from natural products by structural modification
and simplification had proven its great potency and success.
For instance, dimethomorph, created in 1988 as a novel
systemic fungicide with curative, protectant, and antifungal
activity, was inspired from the natural cinnamate; by modifying
the structure of strobilurin A, the shortcomings of its poor light
stability and bad volatility were well solved, leading to the
creation of azoxystrobin, trifloxystrobin, fluoxastrobin, and so
on;9 hymexazol was obtained by modifying the ibotenic acid
isolated from higher fungi Amanita ibotengutake.10 Therefore,
searching for active lead compounds from natural alkaloids was
a valid and bright routine to deal with fungal infections of
crops.
INTRODUCTION
■
Epidemic diseases of crops caused by fungi deeply affected the
course of human history and processed a major restriction on
social and economic development. Research revealed that
more than 19,000 different types of fungi could cause plant
diseases and some of them could be dormant in dead plants
until opportunities were conducive to their proliferation.1 The
loss of crops caused by fungi had become a severe issue that
cannot be ignored. For example, rice blast could bring about
10−35% of harvest losses in 85 countries around the world
because of the spread of Magnaporthe oryzae; more than 200
crop species including different fruits and vegetables could be
severely damaged by Botrytis cinerea as it is a necrotrophic,
haploid, heterothallic ascomycete with a unique infection
approach;2,3 Phytophthora could transmit from potatoes and
tomatoes to other important corps, causing symptoms like fruit
rot, trunk ulcers, branch wilting, and serious economic losses.4
Ever since, the most effective way to control harvest
deterioration in crops still remained the common use of
chemical fungicides, although long-term and widespread
utilization of them had led to a developed increase in fungal
resistance. In fact, the disadvantages of using agricultural
chemicals had already surfaced due to their influences on food,
environment, and other side effects on humans like
carcinogenicity and teratogenicity.3,5,6
In our proceeding pursuit of antifungal natural compounds,
a traditional African climbing herbal medicinal plant named
Cryptolepis sanguinolenta caught our greatest interest.11 As a
shrub indigenous to West Africa, C. sanguinolenta had long
been employed by Ghanaian traditional healers in the
treatment of various fevers, including malaria.12 The extraction
Received: October 12, 2020
Revised: January 7, 2021
Accepted: January 13, 2021
Published: January 26, 2021
Since penicillin was accidentally discovered and successfully
used in clinical treatment, natural products provided a clear
direction for the discovery of antimicrobial drugs.7 Natural
alkaloids were the metabolites produced by natural organisms
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271
© 2021 American Chemical Society
1259

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 1. Strategies of structural modification and simplification of cryptolepine.
Figure 2. Synthesis of target compounds a1−a24.
and separation test revealed that it contains cryptolepine,
neocryptolepine, quindoline, hydroxycryptolepine, and other
ingredients. Further research revealed that the main
component cryptolepine, whose total synthesis was prior to
separation and determination, had exhibited unprecedented
pharmacological effects, including antimalarial activity,13−18
antibacterial activity,19−22 anticancer activity,23−26 antifungal
activity,27−29 anti-inflammatory activity,30,31 antidiabetic activ-
ity,32 antituberculosis activity,33 and so on.34−36 Although
fruitful achievements had been widely reported, the exploration
of cryptolepine and its derivatives in fungicide chemistry was
barely conducted. This implied us that more emphasis should
be laid on the study of cryptolepine and its derivatives in the
control of crop fungi.
1260
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 3. Synthesis of target compounds b1−b3 and c1−c3.
Figure 4. Synthesis of target compounds d1−d4, e1−e4, f1−f2, g1−g2, h1−h2, and i1−i2.
Enlightened from our previous study of neocryptolepine37
MATERIALS AND METHODS
■
and structural simplification of natural products,38,39 a library
of derivatives and simplifications of cryptolepine had been
availably designed and synthesized in various ways to evaluate
their antiphytopathogenic activities. As shown in Figure 1, a
succession of substituted A-ring, D-ring, and A, D-ring
derivatives a1−a24 was synthesized to explore their influence
on activity. Meanwhile, some different types of bioisosterisms
b1−b3 and c1−c3 were created by replacing N at 10-position
with O and S to study the results of different ring-forming
atoms. Then, ring-opened simplifications d1−d4, e1−e4, f1−
f2, g1−g2, h1−h2, and i1−i2, and methyl isomer j1 had been
synthesized to further hunt for active compounds. By virtue of
its excellent activity to B. cinerea (EC50 = 0.027 μg/mL),
compound a3 was chosen to be a privileged candidate to
evaluate its mechanisms of action. Preliminary results showed
that spores and hyphae were inhibited and destroyed,
prompting us to study the influence of a3 on the changes of
reaction oxygen species (ROS), cell nuclear morphology, and
mitochondrial membrane potential (MMP) of B. cinerea. Based
on the above research results, in vivo antifungal experiments on
apples had been progressed.
General. All of the compounds of quinolones and indoles were
purchased from commercial companies (Energy Chemical Co., Ltd.)
without further purification. All reactions were performed with
1
commercially available reagents without further purification. H and
13C NMR spectra were recorded at 400 and 100 MHz on a Bruker
AM-400 (Bruker Company) spectrometer using TMS as a reference.
Mass spectra were recorded on a Bruker Daltonics APEXII49e
spectrometer (Bruker Daltonics Inc.) with the ESI source as
ionization. The relative conductivity of hyphae was performed by a
conductivity meter (Leici DDS-307, China). The microscopic
morphology of fungal hyphae was observed by a scanning electron
microscope (Hitachi, S-3400 N, Japan). The commercial fungicide
azoxystrobin (analytical grade, 98% purity) (Jiangsu Bailing Agro-
chemical Co., Ltd., China) was used as a positive control in antifungal
activity assays.
Plant Pathogenic Fungi. Four plant pathogenic fungi species
Rhizoctonia solani, B. cinerea, Fusarium graminearum, and Sclerotinia
sclerotiorum were isolated from susceptible plants cultivated at the
Gansu Academy of Agricultural Sciences, Gansu Province of China.
Then, they were purified and identified by the Institute of Plant
Protection, Gansu Academy of Agricultural Science, and maintained
during the experiments on potato dextrose agar medium (PDA:
potato 200 g, dextrose 20 g, agar 15 g, and distilled water 1000 mL) at
25 °C for the antifungal assay.
1261
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Synthetic Procedures. The synthetic routes of series of a1−a24,
b1−c3, c1−c3, d1−d4, e1−e4, f1−f1, g1−g2, h1−h2, i1−i2, and j1
are outlined in Figures 2−5.
To a round-bottomed flask was added phenoxyacetic acid or 2-
phenylsulfanylacetic acid (2.4 mmol) and thionyl chloride (20 mL).
Then, the mixture was heated at 80 °C for 2 h. When the reaction was
over, excess thionyl chloride was removed under reduced pressure to
obtain F2 (X = O) and F3 (X = S) and directly put into the next step
without purification.
For X = N, a mixture of F1 (90 mmol) and aniline (100 mmol) in
DMF was heated under reflux for 18 h. When the reaction was
completed, a solution of 10% sodium hydroxide was slowly added to
adjust the pH of the mixture to 9. The whole mixture was extracted
with DCM, and the aqueous phase was collected. Then, the pH of
aqueous phase was adjusted to neutral with a solution of 10%
hydrochloric acid to obtain a large amount of precipitate. The
obtained solid was washed with water repeatedly to get G1 without
further purification.
For X = O or S, anthranilic acid (50 mmol) was added to aqueous
solution of sodium hydroxide and stirred until completely dissolved.
Then, a dropping funnel was used to slowly add the prepared F2 or
F3 to the mixture at 0 °C. The reaction would be quickly completed,
and a large amount of white solid formed. The precipitate was filtered
and dried in vacuo to give G2 and G3.
A mixture of G1−G3 (21.5 mmol) and polyphosphoric acid (5 w/
w) was heated at 130 °C for 2 h. Then, the mixture was cooled to
room temperature and poured into ice water (250 mL) and
neutralized with saturated solution of KOH. The precipitated solid
was filtered and dried to give H1−H3 and used for the next step
without further purification.
A suspension of H1−H3 (23.8 mmol) in POCl3 (60 mL) was
refluxed at 110 °C for 3 h. After evaporating excess POCl3, the
reaction mixture was poured into ice water and neutralized with
solution of sodium hydroxide. The precipitated solid was purified by
column chromatography using petroleum ether and ethyl acetate as an
eluent to give I1−I3.
Pd/C (10%, 40 mg) and Et3N (0.32 mmol) were orderly added
into a CH3OH/THF = 1:1 (vol/vol) solution of I1−I3. After
removing the air in the flask and flushing hydrogen for 5 min, the
system was closed and reacted at room temperature for overnight.
When the reaction was completed, Pd/C was removed by filtration,
and the filtrate was concentrated under reduced pressure. The residue
was purified by column chromatography using petroleum ether and
ethyl acetate as eluents to give target compounds b1−b3.
CH3I (0.6 mL) was added to a solution of compounds b1−b3
(0.09 mmol) in sulfolane (1 mL). The mixture was heated at 55 °C
for 8 h until the solid formed. Target compounds c1−c3 could be
obtained by filtering and washing the solid with a small amount of
ethyl acetate.
General Synthetic Procedure for Target Compounds d1−d4 and
e1−e4 (Figure 4, Route 1).42 To a solution of 3-aminoquinoline (10
mmol) in DCM (30 mL) was added substituted phenylboronic acid
(13.36 mmol), Cu(OAc)2 (10.41 mmol), TEA (10.41 mmol), and
molecular sieves (2 g). The mixture was stirred at room temperature
for 24 h. Then, small amounts of ammonium hydroxide was added
into the mixture and continued to stir for 30 min. When the reaction
was completed, the mixture was extracted with DCM. The combined
organic phases were washed with saturated brine and dried with
anhydrous sodium sulfate, and the solvent was removed in vacuo.
Compounds d1−d4 were gained by column chromatography using
petroleum ether and ethyl acetate as eluents.
CH3I (2.0 mmol) was added to a solution of d1−d4 (1.0 mmol) in
1,4-dioxane (15 mL). After stirring at 55 °C for 8 h, the solid was
precipitated and target compounds e1−e4 could be obtained by
washing the solid with a small amount of ethyl acetate without further
purification.
General Synthetic Procedure for Target Compounds f1−f2 and
g1−g2 (Figure 4, Route 2).43 To a sealed tube was added 3-
bromoquinoline (1.0 mmol), CuI (0.1 mmol), substituted phenol
(1.2 mmol), and K3PO4 (2 mmol) and then was filled with nitrogen.
Dimethyl sulfoxide (DMSO) (2 mL) was added to the mixture with a
syringe and heated at 90 °C for 24 h. When the reaction was
completed, ethyl acetate and water were added to the resulting
Figure 5. Synthesis of target compound j1.
General Synthetic Procedure for Target Compounds a1−a24
(Figure 2).40 To a solution of different indoles A1−A12 (15 mmol) in
CH3CN was added 60% NaH (21 mmol) at 0 °C. The mixture was
stirred for 10 min, and TsCl (16.5 mmol) was added. Then, the
mixture was allowed to reach room temperature and stirred for 4 h.
The reaction was quenched with a saturated aqueous solution of
NH4Cl. The resulting solution was extracted with ethyl acetate (3 ×
100 mL). The combined organic phases were washed with brine (100
mL), dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo to provide the corresponding compounds B1−B12.
To a solution of B1−B12 (11 mmol) and H2O (110 mmol) in
acetone (110 mL) was added NBS (12 mmol). The mixture was
stirred at room temperature until the complete disappearance of the
starting material as indicated by TLC. Et3N (12 mmol) was added to
the mixture and stirred further 1 h. The resulting precipitate was
separated by filtration, washed with acetone, and dried in vacuo to
give the corresponding compounds C1−C12.
A mixture of C1−C12 (0.5 mmol), different N-methylanilines
(0.55 mmol), and Et3N (1.0 mmol) in AcOEt (5 mL) was heated for
0.5−6 h. After addition of H2O, the whole was extracted with AcOEt
(3 × 15 mL) and washed with brine (15 mL). The organic layer was
dried over MgSO4 and concentrated under reduced pressure. Then,
the mixture was dissolved in AcOEt (5 mL). To this solution was
added BF3·Et2O (2.5 mmol), and the mixture was heated at 50 °C
with stirring for 3 h. After addition of saturated NaHCO3 aq, the
whole mixture was extracted with AcOEt (3 × 15 mL) and washed
with brine (15 mL). The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography to give the corresponding
compounds D1−D24.
Phosphoryl chloride (2 mmol) was added to dimethylformamide
(DMF) (1 mL) at −16 °C and stirred for 0.5 h. A solution of D1−
D24 (1 mmol) in DMF (1 mL) was added to the mixture and stirred
at room temperature for 1 h. The resultant mixture was added to
saturated NaHCO3 aq (5 mL) at 0 °C, and the whole was extracted
with AcOEt (3 × 10 mL) and washed with saturated NaHCO3 aq (10
mL). The organic layer was dried over MgSO4 and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography to give the corresponding compounds E1−E24.
To a solution of Me2NH·HCl (0.1 mmol) in DMF (1 mL) was
added E1−E24 (0.05 mmol), and the mixture was heated at 150 °C
with stirring for 1.5 h. The mixture was cooled to room temperature
and added with 5% Na2CO3 aq (5 mL). After stirring at room
temperature for 10 min, the resultant mixture was extracted with
AcOEt (3 × 10 mL) and washed with saturated Na2CO3 aq (5 mL).
The organic layer was dried over Na2CO3 and concentrated under
reduced pressure. The residue was purified by alumina column
chromatography to give target compounds a1−a24.
General Synthetic Procedure for Target Compounds b1−b3 and
c1−c3 (Figure 3).41 To a solution of anthranilic acid (96 mmol) in
DMF and tetrahydrofuran (THF) (vol/vol = 1:1) was added
bromoacetyl bromide (96.6 mmol) at 0 °C. The mixture was allowed
to reach room temperature and stirred for overnight. The resulting
solution was added to water (150 mL) to form a light-yellow
precipitate. The precipitate was filtered and washed with water until
neutral pH, and the solid residue was dried in vacuo to give the
corresponding compound F1 (X = N).
1262
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
mixture, the organic phase was separated, and the aqueous phase was
extracted by ethyl acetate. The combined organic phases were washed
with saturated brine and dried with anhydrous sodium sulfate, and the
solvent was removed in vacuo. Target compounds f1−f2 were gained
by column chromatography using petroleum ether and ethyl acetate
as eluents.
CH3I (2.0 mmol) was added to a solution of f1−f2 (1.0 mmol) in
1,4-dioxane (15 mL). After stirring at 55 °C for 8 h, the solid was
precipitated and target compounds g1−g2 could be obtained by
washing the solid with a small amount of ethyl acetate without further
purification.
General Synthetic Procedure for Target Compounds h1−h2 and
i1−i2 (Figure 4, Route 3).44 To a solution of 3-bromoquinoline (47.3
mmol) in 1,4-dioxane was added CuI (2.4 mmol), NaI (94.5 mmol),
and N,N′-dimethyl-1,2-ethanediamine (0.5 mL). The mixture was
heated at 110 °C for 48 h. When the reaction was completed, 30 mL
of 30% ammonium hydroxide was added and stirred at room
temperature for 10 min. Then, water was added to the resulting
mixture, and the whole mixture was extracted with ethyl acetate. The
combined organic phases were washed with saturated brine and dried
with anhydrous sodium sulfate, and the solvent was removed in vacuo.
Target intermediate 3-iodoquinoline was gained by column
chromatography using petroleum ether and ethyl acetate as eluents.
To a round-bottomed flask was added 3-iodoquinoline (1.96
mmol), CuI (0.1 mmol), K2CO3 (3.92 mmol), substituted thiophenol
(1.96 mmol), and 2- isopropoxyethanol (3.92 mmol). Then, the
mixture was heated at 80 °C for 16 h. When the reaction was
completed, a solution of 10% hydrogen peroxide was added and the
whole mixture was stirred at room temperature for 10 min. Then,
water and ethyl acetate were added to the resulting mixture, the
organic phase was separated, and the aqueous phase was extracted by
ethyl acetate. The combined organic phases were washed with
saturated brine and dried with anhydrous sodium sulfate, and the
solvent was removed in vacuo. Target compounds h1−h2 were
gained by column chromatography using petroleum ether and ethyl
acetate as eluents.
where dc and dt were average diameters of the fungal colony of black
control and treatment, respectively.
Spore Germination Inhibition Assay. According to the
described method,46 the phytopathogenic fungi included in the
assay was grown on PDA plates in darkness at 25 °C for 2 weeks. The
spores were harvested from sporulating colonies and suspended in
sterile distilled water containing 0.1% (vol/vol) Tween 80. The
concentrations of spores in the suspension were determined using a
hemocytometer and adjusted to 1 × 106 spores per mL.
The stock solutions of the tested compounds dissolved in DMSO
were diluted 100-fold with sterile distilled water, which were further
prepared as 2-fold dilutions for the assay, in which the final
concentration of DMSO was 1% (vol/vol). Sterile distilled water
with 1% DMSO was used as a negative control. A 30 μL spore
suspension was placed on separate glass slide containing 30 μL of
prepared test solutions for a final volume of 60 μL. The inoculated
glass slides were incubated in a moisture chamber at 25 °C for 12 h. A
spore was considered to be germinated if the germ tube length was
longer than the short radius of the spore, and the number of spores
germinated at each concentration was counted under the microscope.
The percentage of spore germination was calculated, and the half
maximal effective concentration (EC50) values were derived from the
data analysis of the concentration-inhibition rate. The experiment was
independently performed three times under the same conditions. The
experiment was repeated three times, and the inhibition rates were
calculated according to the following formula
inhibition germination of spores (%) = [(N0 − N )/N0] × 100
1
where N0 and N1 were average values of the spore germination rates
of the black control and treatment, respectively.
Scanning Electron Microscopy Observations. (After treating
with a3 at a concentration of 0.05 μg/mL for 72 h, mycelia blocks
(5.0 mm × 4.0 mm) were cut from the fungi.). All the samples were
treated by 4% glutaraldehyde for 4 h, washed three times with 0.01 M
PBS (pH = 7.2), and then fixed with 1% osmium tetraoxide solution
(wt/vol) for 2 h. After that, each sample was dehydrated with graded
ethanol series (20, 50, 80, and 90%) for 10 min. Subsequently, the
samples were dried at a critical point and gold-sprayed and observed
by using a scanning electron microscope (Hitachi, S-3400N, Japan).
Determination of Cell Membrane Permeability. Effects of
compound a3 on the cell membrane permeability were determined by
the conductivity method.47 The mycelial disk of B. cinerea (5 mm)
was placed in 60 mL of PD broth medium and shook at 140 rpm for 4
days at 27 °C. After that, the mycelia were filtered and added into the
solution of a3 with different concentrations (50, 25, 10, and 5 μg/
mL). Eventually, the conductivity values were determined with a
conductivity detector (0 h was marked as L0, and 0.5, 1, 2, 4, 6, 8, 10,
12, and 24 h were marked as L1). The conductivities of samples
treated by boiling water for 30 min were remarked as L2. The relative
permeability rate of the cell membrane was calculated by the following
formula
CH3I (2.0 mmol) was added to a solution of h1−h2 (1.0 mmol) in
1,4-dioxane (15 mL). After stirring at 55 °C for 8 h, the solid was
precipitated and target compounds i1−i2 could be obtained by
washing the solid with a small amount of ethyl acetate without further
purification.
General Synthetic Procedure for Target Compound j1 (Figure 5).
To a solution of compound b1 in acetone was added potassium
hydroxide (1.5 mmol) and CH3I (1.2 mmol). The mixture was
reacted at room temperature for 15 min. When the reaction was over,
the solvent was removed in vacuo, and the residue was purified by
silica gel column chromatography to give target compound j1.
In Vitro Antifungal Assay. Cryptolepine and its derivatives were
tested by the mycelium growth rate method45 for their antifungal
activities against four fungi, including R. solani, B. cinerea, F.
graminearum, and S. sclerotiorum. All the synthetic compounds were
dissolved in DMSO and then were added to PDA medium that was
prepared and sterilized to obtain a series of concentrations (50, 25,
10, 5, 2.5, 1, 0.5, 0.1, 0.05, and 0.01 μg/mL). The antifungal activities
of target compounds a1−a24, b1−c3, c1−c3, d1−d4, e1−e4, f1−f1,
g1−g2, h1−h2, i1−i2, and j1 in detail were included in the
Supporting Information. The blank control was maintained with 0.5%
DMSO (vol/vol) mixed with PDA (The same amount of DMSO was
added to the sterile medium as a blank control.), and azoxystrobin
was used as a positive control. The mycelial disks (5 mm) of
phytopathogenic fungi were inoculated on PDA plates and then were
incubated at 25 °C in the dark. Each sample was measured in
triplicates, and its diameters (mm) of inhibition zones were measured
by the cross-bracketing method. The growth inhibition rates were
calculated when the blank control hyphae grew to the edge of the
Petri dish according to the following formula
relative electric conductivity (%)
= [(L1 − L0)/(L2 − L0)] × 100
ROS Production of B. Cinerea. The accumulation of ROS was
measured by the method previously described.48 Mycelia tips (0.5
cm) were treated with compound a3 (0.05 and 0.1 μg/mL) for 48 h
and then placed on a sterile slide in a culture dish. After the samples
were cultured at 25 °C for 24 h, the PDA medium was removed
carefully and the hyphae were stained with 10 μM DCFH-DA
solution (Beyotime, Shanghai, China). The hyphae were incubated at
37 °C for 30 min in the darkness and washed twice with phosphate-
buffered saline (PBS). A coverslip was placed on the hyphae, and the
samples were observed and photographed using the microscopic
morphology of fungal hyphae that was observed by a scanning
electron microscope (Hitachi, S-3400 N, Japan).
MMP of B. Cinerea. Effects of compound a3 on the MMP of B.
cinerea mycelia were analyzed according to the previously described
method.49 After staining with rhodamine 123 solution (Beyotime,
mycelial growth inhibition (%)
= [(dc − dt)/(dc − 5 mm)] × 100
1263
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
a
Table 1. In Vitro Antifungal Activities of Cryptolepine Derivatives against Four Phytopathogenic Fungi at 50 and 25 μg/mL
inhibition rate SD (%)
compd
concn (μg/mL)
S. s.
B. c.
F. g.
R. s.
a1
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
79.91 0.44
70.56 0.23
89.82 0.12
82.86 0.71
84.34 0.02
75.78 0.04
78.51 0.18
72.72 0.15
86.67 0.56
74.71 0.09
56.37 1.01
40.55 0.01
95.23 0.77
90.15 1.72
92.65 0.71
84.48 0.94
82.74 0.48
74.17 0.48
83.86 0.72
75.93 1.07
76.31 0.25
67.92 0.76
94.35 0.82
89.65 1.01
97.85 0.54
96.69 0.87
86.46 1.54
83.57 0.55
97.03 0.36
95.55 0.39
96.72 0.16
94.13 0.27
94.38 0.44
93.31 0.34
99.11 0.28
97.33 0.49
98.43 0.21
96.26 0.52
99.71 0.11
99.19 0.03
99.21 0.02
99.15 0.76
87.81 0.25
76.48 0.66
99.22 0.05
99.09 0.03
89.63 0.29
77.78 0.85
94.33 0.31
92.13 0.39
89.00 1.35
86.84 1.38
87.88 1.04
83.16 0.68
98.03 0.36
95.76 0.07
88.02 1.83
81.75 1.34
72.15 0.76
51.57 0.33
76.84 0.37
67.79 0.71
71.29 0.35
68.63 0.15
65.83 0.54
35.02 1.09
65.13 0.88
63.18 0.25
75.63 0.74
59.84 0.41
44.25 0.45
35.75 1.69
55.93 0.43
38.95 0.76
34.67 1.11
30.53 1.27
62.85 0.66
50.54 4.87
65.52 0.52
51.49 1.86
56.94 0.99
24.58 1.95
46.84 1.07
27.04 1.04
64.83 2.19
45.59 0.97
42.87 1.55
9.65 1.26
17.62 2.76
26.40 0.71
23.67 0.24
53.98 0.49
47.66 0.02
52.15 0.47
49.35 0.42
10.89 0.05
8.37 0.06
52.86 0.15
49.27 0.23
10.95 0.08
6.49 0.43
53.6 1.68
43.03 0.41
35.12 0.26
20.75 0.44
6.19 0.15
a2
a3
a4
a5
a6
a7
a8
a9
0
0
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
a22
a23
a24
b1
30.74 0.35
8.15 0.69
38.35 1.05
23.83 1.75
29.68 1.74
11.83 0.79
20.78 1.25
4.62 1.62
57.56 0.54
34.79 0.79
47.65 2.27
20.88 0.34
33.78 0.89
20.83 1.32
37.64 1.73
18.79 0.71
47.72 1.26
31.57 0.57
32.07 0.84
2.13 1.44
13.15 4.91
100
100
0
0
99.58 0.12
98.21 0.32
96.45 0.68
95.56 0.46
97.09 0.66
95.41 0.33
95.06 0.32
94.39 0.08
99.30 0.36
98.25 0.05
0
0
14.67 1.27
0
0
100
100
0
0
64.96 2.45
48.12 0.67
48.23 2.46
32.61 0.53
47.27 1.93
28.22 0.48
45.11 0.54
23.43 0.38
54.03 1.64
39.25 0.53
27.71 0.93
18.66 1.24
56.94 0.99
24.58 1.95
66.23 1.56
48.78 0.77
57.89 0.78
44.49 1.27
41.36 1.37
14.63 1.33
52.03 1.65
40.02 0.37
49.98 1.98
25.69 1.61
48.66 0.27
100
100
100
100
100
100
0
0
0
0
0
0
99.33 0.24
98.44 0.44
100
100
100
100
0
0
0
0
0
0
13.15 4.91
0
0
71.69 0.28
59.48 1.31
90.33 0.32
84.91 0.42
94.35 0.82
89.65 1.01
23.45 0.36
8.69 1.48
5.14 1.14
99.40 0.28
98.68 0.21
98.44 0.16
97.68 0.09
53.79 2.52
48.47 1.86
32.53 2.56
16.95 0.99
29.68 1.74
11.83 0.79
40.44 0.26
27.15 0.93
34.44 1.32
21.98 1.11
27.45 0.66
24.11 0.94
39.17 1.46
30.28 0.64
48.01 2.03
30.74 0.94
47.23 0.55
100
100
0
0
38.41 0.53
16.38 0.65
b2
0
0
0
0
0
0
b3
41.64 0.09
14.05 0.02
74.35 3.36
68.99 2.04
34.87 1.82
19.68 1.35
99.36 0.27
96.35 0.21
58.45 1.31
49.57 0.93
50.34 0.34
c1
c2
0
0
0
0
c3
38.47 0.51
1264
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Table 1. continued
inhibition rate SD (%)
compd
concn (μg/mL)
S. s.
B. c.
F. g.
R. s.
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
24.29 0.02
76.22 0.66
43.74 0.77
76.97 0.53
66.81 0.28
76.21 0.46
44.04 1.35
68.33 0.46
47.11 0.26
12.55 0.05
29.21 0.58
92.53 0.16
87.36 1.92
96.15 0.38
95.65 0.52
97.33 0.58
94.38 0.44
92.28 0.33
84.47 1.18
20.39 0.19
12.33 0.63
56.92 0.15
36.57 0.11
58.95 0.93
42.38 0.35
40.95 0.44
20.67 0.11
100
19.43 0.48
66.99 0.54
57.85 0.49
64.95 0.54
50.22 1.15
63.79 1.49
51.30 0.61
56.38 0.35
42.11 0.45
23.21 0.37
14.84 1.03
69.48 0.31
47.68 0.32
69.81 0.37
50.25 0.22
10.83 0.09
31.26 1.62
84.86 1.45
75.01 1.49
82.42 1.82
80.75 0.14
75.11 0.25
68.93 2.13
67.89 0.36
50.11 0.32
d1
d2
d3
d4
e1
e2
e3
e4
f1
0
0
0
0
0
0
15.33 0.26
7.33 2.14
15.65 0.15
17.05 0.79
8.33 0.36
14.9 0.22
7.21 0.20
0
0
8.83 0.12
0
0
0
0
0
0
0
0
93.13 0.01
90.11 0.09
88.23 0.47
83.35 0.16
70.35 0.46
70.05 2.07
65.22 1.17
58.23 0.47
6.55 0.27
89.74 0.40
85.93 0.38
83.63 0.03
77.21 0.15
95.93 0.09
95.44 0.34
92.12 0.41
f2
G1
g2
h1
h2
i1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7.29 0.11
6.89 0.28
0
0
0
0
96.58 0.08
94.33 0.41
86.83 0.55
77.45 0.65
14.21 0.34
7.23 0.35
20.65 0.29
7.67 0.16
71.22 0.69
50.68 0.54
45.57 0.29
30.43 0.98
100
0
64.99 1.80
60.43 0.74
53.11 0.32
48.87 0.26
21.43 0.36
8.20 0.75
10.35 0.28
79.25 0.56
70.58 1.81
63.46 0.62
57.56 0.56
98.06 0.09
95.34 0.28
91.32 0.37
15.12 1.21
6.67 0.45
0
0
0
0
0
0
0
0
i2
0
0
0
0
0
0
j1
53.40 3.25
36.34 0.73
47.88 0.43
27.00 0.23
64.95 2.82
51.93 1.66
61.85 0.06
57.32 0.28
63.18 0.58
32.11 0.59
50.47 0.90
35.27 0.87
ASB
a
S.s., Sclerotinia sclerotiorum; B.c., Botrytis cinerea. F.g., Fusarium graminearum; R.s., Rhizoctonia solani. ASB: azoxystrobin.
China), the mycelia were incubated at 24 °C for 30 min in darkness
and washed with PBS. Finally, the samples were photographed using
an LSM-800 with Airyscan.
Effects on the Nuclear Morphology of B. Cinerea. After
staining with Hoechst 33258 (Beyotime, China), the mycelia were
incubated at 24 °C for 30 min in darkness and washed with PBS.
Finally, the samples were photographed using an LSM-800 with
Airyscan.
In Vivo Curative Activity Bioassay. Based on the preceding test
of in vitro antifungal activity, compound a3 against B. cinerea was
further tested on apple. The synthesized compound and positive
control azoxystrobin in 0.1 mL of DMSO were dissolved in 10 mL of
deionized water at a series of concentrations (200, 100, 50, and 25
μg/mL). Each apple was punctured with an inoculating needle, and
then, the pathogen was inoculated. After 24 h, each sample measured
in quadruplicates was sprayed evenly onto the apple, which had been
already washed and treated with water and 75% aqueous ethyl alcohol.
DMSO (1%) in 10 mL of water was set up as the blank control. All
the treated samples were then placed into an illumination incubator at
25 °C and 100% relative humidity for 6 days. All assays were at least
performed in triplicates by conventional methods, and results were
presented as mean standard deviations.
Statistical Analysis. The statistical analysis was performed by
SPSS 24.0. The EC50 values were obtained from the parameters in the
regression curves, and 95% CI, regression equation, and R2 are
provided in the Supporting Information.
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of the A-ring, D-ring, and A, D-
ring substituted derivatives, bioisosterisms, ring-opened
products, and methyl isomers of cryptolepine is outlined in
1
Figures 2−5. H and 13C NMR and MS of compounds were
applied to confirm their structures.
To investigate the influence of the electronic effects on the
biological activities of cryptolepine derivatives, an available and
flexible routine directed by Abe et al.40 was employed to obtain
target compounds a1−a24 (Figure 2). Furthermore, a series of
bioisosterisms were obtained by using anthranilic acid as the
starting material40 (Figure 3), and ring-opened derivatives
were primarily gained through some enduring synthetic
routes42−44 (Figure 4). The methyl isomer j1 of cryptolepine
1265
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Table 2. EC50 Values of Cryptolepine Derivatives against
a
Four Phytopathogenic Fungi
EC50 (μg/mL)
compd
S. s.
B. c.
F. g.
R. s.
>30
a1
a2
a3
a4
a5
a6
a7
a8
5.507
1.246
1.038
3.136
5.466
>30
0.050
0.037
0.027
3.954
0.034
3.078
0.876
0.066
1.027
0.148
2.115
0.042
0.065
0.068
0.131
0.041
0.087
0.159
0.037
0.034
0.037
0.047
0.056
0.071
>30
>30
>30
0.041
>30
>30
3.970
1.655
1.198
3.303
>30
>30
>30
>30
1.450
1.513
>30
21.838
13.728
10.912
>30
18.920
20.815
>30
>30
>30
26.669
21.970
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
14.069
20.752
24.000
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
6.170
3.821
9.395
22.449
>30
>30
>30
>30
3.632
5.321
>30
>30
9.911
18.649
>30
>30
>30
>30
3.248
3.099
6.306
4.472
12.119
2.445
1.852
2.714
2.520
1.283
1.193
1.353
1.424
1.664
1.148
17.933
2.177
1.476
>30
>30
>30
8.217
>30
>30
24.250
8.682
21.263
25.083
>30
a9
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
a22
a23
a24
b1
b2
b3
c1
Figure 6. Spore germination inhibition assays of untreated control
(A), 10 (B), and 25 μg/mL (C) of compound a3.
c2
c3
d1
d2
d3
d4
e1
e2
e3
e4
f1
f2
g1
g2
h1
h2
i1
i2
j1
>30
>30
>30
>30
3.302
3.226
>30
23.996
21.892
>30
5.599
14.712
>30
>30
16.914
>30
>30
>30
22.188
27.436
Figure 7. Scanning electron micrographs of B. cinerea hyphae in the
untreated control (A,B) and 0.05 μg/mL (C,D) of compound a3.
>30
>30
2.498
6.706
>30
>30
19.421
>30
1.644
1.938
>30
>30
>30
ASB
>30
a
S.s., Sclerotinia sclerotiorum; B.c., Botrytis cinerea. F.g., Fusarium
graminearum; R.s., Rhizoctonia solani. ASB: azoxystrobin.
was easily obtained by adding CH3I to a solution of KOH and
b1 in acetone (Figure 5).
In Vitro Activity and Structure−Activity Relationship.
An efficient and productive method was used to synthesize
target compounds (a1−a24) to investigate the electronic
impact on inhibitory activity. As shown in Tables 1 and 2, the
data of cryptolepine and its ring-substituted derivatives against
Figure 8. Effects of compound a3 on the cell membrane permeability
of B. cinerea.
R. solani, B. cinerea, F. graminearum, and S. sclerotiorum were
demonstrated at 50 and 25 μg/mL. The inhibition activity of
1266
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
range of antifungal spectrum and explore the structure−activity
relationship (SAR).
Statistics showed that inhibitory rate of most A-ring
derivatives (a2−a12) against B. cinerea at a concentration of
50 μg/mL exceeded 90%, which was superior to the positive
control azoxystrobin. Especially compound a3 (EC50 = 0.027
μg/mL) exhibited the unprecedented activity among them.
Besides, the inhibitory rate of the majority of modifications
against S. sclerotiorum was better than cryptolepine. Exper-
imental results revealed that when some atoms or certain
groups like chlorine, trifluoromethyl, methoxyl, and methyl
were introduced into position C2 or position C3 of the A ring,
the inhibition effects to S. sclerotiorum and B. cinerea were
variably strengthened. Comparing a3 (EC50 = 1.038 μg/mL)
with a10 (EC50 = 4.472 μg/mL) against S. sclerotiorum or a7
(EC50 = 0.876 μg/mL) with a11 (EC50 = 2.115 μg/mL)
against B. cinerea, we found that antifungal activities of
compounds modified at position C2 was better than that at
position C3. However, the introduction of some larger atoms
or groups like a6 (bromine at C2) or a11 (tert-butyl at C3)
could evidently reduce the activity against S. sclerotiorum and B.
cinerea. Most of the compounds showed reduced antifungal
activity against F. graminearum, except for a2 (EC50 = 13.728
μg/mL), a3 (EC50 = 10.912 μg/mL), and a5 (EC50 = 18.920
μg/mL) modified with a chlorine atom, methoxyl group, and
methyl group, respectively. Nearly, all modified compounds
did not show good activity against R. solani.
Figure 9. Effects of a3 on the reactive oxygen species, cell nuclear, and
MMP of B. cinerea.
the lead compound cryptolepine to S. sclerotiorum (EC50
=
5.507 μg/mL) and B. cinerea (EC50 = 0.050 μg/mL) was
evidently better than other two fungi, prompting us to execute
some ring-substituted modifications to further expand the
Figure 10. In vivo curative antifungal activities of compound a3 against B. cinerea. ASB: azoxystrobin.
1267
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Subsequently, a series of D-ring derivatives (a13−a21) were
synthesized to further investigate their antifungal activities.
Most of the D-ring compounds exhibited better antifungal
activities with an EC50 of 1.148−2.714 μg/mL against S.
sclerotiorum and an EC50 of 0.034−0.159 μg/mL against B.
cinerea, which were almost equal with compound a3 and prior
to other A-ring derivatives. When the electron-donating group
like methyl or methoxyl was introduced at position C7 or
position C8 of the D-ring, the activity against S. sclerotiorum
was better than compounds with the electron-withdrawing
group. One thing worth noting was that the compound a20
bearing a bromine at 8-position showed excellent activity
against B. cinerea with an EC50 of 0.034 μg/mL, illustrating that
the introduction of a large group into the A-ring might
improve the antifungal effect. Compounds modified at A, D-
rings together showed almost equal activity against S.
sclerotiorum and B. cinereal, but all A-ring, D-ring, and A, D-
ring derivatives showed poor activity against F. graminearum
and R. solani. We speculated that introduction of different
substitution groups at the A-ring or D-ring could be used to
search for compounds with improved antifungal activity, but
other modification methods were needed to expand the
antifungal spectrum of lead cryptolepine.
ition effects would become vastly worse after methylation of
the N of the quinoline ring. Lastly, the methyl isomer j1 of
cryptolepine was synthesized, but its antifungal activity was
weaker than cryptolepine, implying that the introduction of
methyl at N-10 was not a good strategy.
By systematic structural modification and ring-opened
simplification, we have obtained compound a3 (EC50
=
0.027 μg/mL against B. cinerea) with improved activity and
compound f1 (EC50 = 5.599 μg/mL against F. graminearum
and EC50 = 3.632 μg/mL against R. solani) with an expanded
antifungal spectrum, which was of great significance for the
further research and exploration of cryptolepine.
Preliminary Mode of Action of Compound a3. Effects
of Compound a3 on the Spore Germination. B. cinerea is a
saprophytic fungus that infects the host mainly by conidia, so
the harm to the host plant could be reduced by inhibiting
spore germination. As shown in Figure 6, the outcomes of
spore germination inhibition assay indicated that compound a3
could markedly inhibit the spore germination of B. cinerea by
65% and more than half of the spores did not germinate at a
concentration of 10 μg/mL. Furthermore, the length of the
germ tube formed by the germinated spores was shorter than
the untreated group. Meanwhile, the data from the experiment
indicated that effects of compound a3 on the spore
germination of B. cinerea were concentration-dependent.
Scanning Electron Micrographs of Compound a3 on the
Hyphae Morphology. To elucidate the effective pathway of
compound a3 against B. cinerea, the mycelia were treated with
0.05 μg/mL compound a3 (Figure 7C,D) and we observed
structural changes. We found that the tested mycelia were
collapsed, shriveled, and crumpled compared with the regular,
smooth, and homogeneous hyphae (Figure 7A,B). This
indicated that compound a3 might damage the cell membrane
of mycelia, leading to spillage of cellular contents and barriers
to the transport of the cellular material.
Effects of Compounds a3 on the Cell Membrane
Permeability. To determine whether compound a3 acted on
the membrane of B. cinerea, the test of changes in electrical
conductivity of hyphae was executed. As shown in Figure 8,
after treating the hyphae of B. cinerea with compound a3, the
increase of conductivity was evidently rapid within 24 h and
obviously concentration-dependent. These results suggested
that compound a3 could damage the cell membrane of hyphae
in a dose-dependent manner, resulting in electrolyte leakage,
thereby increasing conductivity.
Effects of Compound a3 on ROS Production. From the
fluorescence of Figure 9A−C, it was found that hyphae treated
with a3 at 0.05 and 0.1 μg/mL had an obvious fluorescence
effect compared with untreated hyphae, and fluorescence
intensity enhanced significantly with the increase of the
compound concentration. Cells of B. cinerea were forced to
practice oxidative stress response under the destructive effect
of the compound a3, leading to changes of cell membrane
permeability and hyphae morphology.
Effects of Compound a3 on the Nuclear Morphology. The
influence of a3 at 0.05 and 0.1 μg/mL to the nucleus was
shown at Figure 9D−F. The blue fluorescence signal of the
drug application group was obviously weaker than the normal
growing group, indicating that a3 could cause severe damage
to the cell nucleus of B. cinerea and induce mycelial death.
Effects of Compound a3 on the MMP. The influence of a3
to the MMP was shown at Figure 9G−I. The results showed
that the fluorescence intensity of hyphae treated with a3 was
According to the previous work reported by Ning et al.,19
cryptolepine derivatives were prepared by replacing the
nitrogen atom at position 10 with oxygen or sulfur to search
for antimicrobial agents. Hence, a whole variety of bioisoster-
isms and salt forms of them were synthesized to explore their
activities against plant pathogenic fungi.23 From the activity
data results, we found that the salt form c1 of cryptolepine
exhibited an improved activity with an EC50 of 0.041 μg/mL
against B. cinerea. However, the antifungal activity of
cryptolepine would become worse when the methyl at C5
was lacking. Bioisosterisms b2, b3, and their salt forms showed
poor activity against four pathogenic fungi. Similar to A-ring,
D-ring, and A, D-ring derivatives, most compounds did not
show good activity against F. graminearum and R. solani. This
implied that the integrity of the methyl group at 5-positon was
vitally essential for antifungal activity of lead cryptolepine and
the replacement of the nitrogen atom at position 10 could not
effectively expand the antifungal spectrum.
The C-ring of cryptolepine was opened and then formed a
series of quinolone derivatives decorated by heteroatoms
linking with different substituted benzenes. Pleasantly, we
found that compounds d1, d2, d3, f1, f2, and h1 exhibited
significantly improved activity against F. graminearum and R.
solani; especially, f1 possessed the most potent antifungal
activity with an EC50 of 5.599 μg/mL against F. graminearum
and an EC50 of 3.632 μg/mL against R. solani. Among them,
compound h1 displayed an outstanding inhibition effect
against S. sclerotiorum with an EC50 of 2.498 μg/mL and
compound d3 possessed an EC50 of 1.198 μg/mL against B.
cinerea. When nitrogen was replaced with oxygen (compounds
f1 and f2), the antifungal activity of formed derivatives against
R. solani and F. graminearum was enhanced effectively.
Compounds decorated with sulfur (compounds h1 and h2)
showed moderate enhancement to R. solani. Altogether,
compounds replaced with oxygen performed better inhibition
effects than compounds replaced with sulfur or nitrogen. The
SAR study revealed that compounds bearing an electron-
withdrawing group in the para position of the benzene ring was
worse than the introduction of the electron-donating group or
unmodified compounds. For ring-opened derivatives, inhib-
1268
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
weaker than the untreated hyphae, and compound a3 might
cause mitochondrial dysfunction in mycelia by changing the
MMP.
Yin-Fang Yan − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China
Ren-Xuan Wang − 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
Qing-Ru Chu − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China
In Vivo Curative Effects against B. cinerea. Based on the
bioassay data in vitro, compound a3 was selected to further
investigate in vivo activity against B. cinerea. As presented in
Figure 10, the therapeutic effect of compound a3 was gradually
equal to azoxystrobin with the increase of dosage. The
ulceration range in the administration group was markedly
smaller than the blank control. Besides, the therapeutic effects
at low concentrations of 25 and 50 μg/mL were similar to the
positive control drug azoxystrobin on the 5th day, indicating
that compound a3 might have a longer curative time at low
concentrations.
In conclusion, we synthesized a series of different types of
compounds by structural modification and simplification of
cryptolepine. Bioassay results showed that compound a3
possessed the most potent antifungal activity against B. cinerea
and ring-opened compound f1 possessed an improved
inhibition effect against F. graminearum and R. solani. The
mechanism study of compound a3 found that it could bring
about adverse changes to spore germination, normal form of
mycelium, and cell membrane of B. cinerea. Simultaneously,
reactive oxygen, the normal cell structure, and MMP were
badly produced, disrupted, and affected. In view of the above
study on compound a3, it would be a helpful candidate to
control crop disease. Further studies on the structural
optimization cryptolepine and more detailed mechanisms of
a3 are in progress.
Chen Tang − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c06480
Author Contributions
§Y. -J. C. and H.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported financially by the National Key
Research and Development Program of China
(2017YFD0201404) and the National Natural Science
Foundation of China (21877056, 21672092). Support was
also provided by the Key Program for International S&T
Cooperation Projects of China Gansu Province
(18YF1WA115).
REFERENCES
■
ASSOCIATED CONTENT
(1) Jain, A.; Sarsaiya, S.; Wu, Q.; Lu, Y.; Shi, J. A review of plant leaf
fungal diseases and its environment speciation. Bioengineered 2019,
10, 409−424.
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c06480.
(2) Fisher, M. C.; Henk, D. A.; Briggs, C. J.; Brownstein, J. S.;
Madoff, L. C.; McCraw, S. L.; Gurr, S. J. Emerging fungal threats to
animal, plant and ecosystem health. Nature 2012, 484, 186−194.
In vitro EC50 values; physical properties of target
compounds; and spectrograms (PDF)
́
́
(3) Aqueveque, P.; Cespedes, C. L.; Alarcon, J.; Schmeda-
́
Hirschmann, G.; Canumir, J. A.; Silva, M.; Sterner, O.; Radrigan,
̃
R.; Aranda, M.; Aranda, M. Antifungal activities of extracts produced
by liquid fermentations of Chilean Stereum species against Botrytis
cinerea (grey mould agent). Crop Protect. 2016, 89, 95−100.
(4) Lee, H. B.; Kim, Y.; Kim, J. C.; Choi, G. J.; Park, S.-H.; Kim, C.-
J.; Jung, H. S. Activity of some aminoglycoside antibiotics against true
fungi, Phytophthora and Pythium species. J. Appl. Microbiol. 2005, 99,
836−843.
(5) Wang, L.; Li, C.; Zhang, Y.; Qiao, C.; Ye, Y. Synthesis and
biological evaluation of benzofuroxan derivatives as fungicides against
phytopathogenic fungi. J. Agric. Food Chem. 2013, 61, 8632−8640.
(6) Yang, J.; Guan, A.; Li, Z.; Zhang, P.; Liu, C. Design, synthesis
and structure- activity relationship of novel spiro-pyrimidinamines as
fungicides against Pseudoperonospora cubensis. J. Agric. Food Chem.
2020, 68, 6485−6492.
(7) Rossiter, S. E.; Fletcher, M. H.; Wuest, W. M. Natural products
as platforms to overcome antibiotic resistance. Chem. Rev. 2017, 117,
12415−12474.
AUTHOR INFORMATION
■
Corresponding Author
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
Yong-Jia Chen − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China; orcid.org/
0000-0001-6184-3261
Hua Liu − School of Pharmacy, Lanzhou University, Lanzhou
730000, People’s Republic of China
Shao-Yong Zhang − Key Laboratory of Vector Biology and
Pathogen Control of Zhejiang Province, College of Life
Science, Huzhou University, Huzhou 313000, China
Hu Li − School of Pharmacy, Lanzhou University, Lanzhou
730000, People’s Republic of China
Kun-Yuan Ma − 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/
0000-0001-9282-5816
(8) Hao, Y.; Wang, K.; Wang, Z.; Liu, Y.; Ma, D.; Wang, Q.
Luotonin A and its derivatives as novel antiviral and antiphytopatho-
genic fungus agents. J. Agric. Food Chem. 2020, 68, 8764−8773.
(9) Sauter, H.; Steglich, W.; Anke, T. Strobilurine: Evolution einer
neuen Wirksto -ffklasse. Angew. Chem. 1999, 111, 1416−1438.
(10) Ali-Shtayeh, M.; Salah, A. M. A.; Jamous, R. M. Ecology of
hymexazol- insensitive Pythium species in field soils. Mycopathologia
2003, 156, 333−342.
(11) Vlietinck, A. J.; Pieters, L.; Apers, S.; Cimanga, K.; Mesia, K.;
Tona, L. The value of central-African traditional medicine for lead
finding: Some case studies. J. Ethnopharmacol. 2015, 174, 607−617.
Rui Zhou − School of Pharmacy, Lanzhou University,
Lanzhou 730000, People’s Republic of China
1269
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
(12) Komlaga, G.; Agyare, C.; Dickson, R. A.; Mensah, M. L. K.;
Annan, K.; Loiseau, P. M.; Champy, P. Medicinal plants and finished
marketed herbal products used in the treatment of malaria in the
Ashanti region, Ghana. J. Ethnopharmacol. 2015, 172, 333−346.
(13) Lavrado, J.; Cabal, G. G.; Prudencio, M.; Mota, M. M.; Gut, J.;
Rosenthal, P. J.; Díaz, C.; Guedes, R. C.; dos Santos, D. J. V. A.;
Bichenkova, E.; Douglas, K. T.; Moreira, R.; Paulo, A. Incorporation
of basic side chains into cryptolepine scaffold: Structure- antimalarial
activity relationships and mechanistic studies. J. Med. Chem. 2011, 54,
734−750.
(14) Paulo, A.; Figueiras, M.; Machado, M.; Charneira, C.; Lavrado,
J.; Santos, S. A.; Lopes, D.; Gut, J.; Rosenthal, P. J.; Nogueira, F.;
Moreira, R. Bis-alkylamine indolo[3,2-b]quinolines as hemozoin
ligands: Implications for antimalarial cytostatic and cytocidal
activities. J. Med. Chem. 2014, 57, 3295−3313.
(15) Figueiras, M.; Coelho, L.; Wicht, K. J.; Santos, S. A.; Lavrado,
J.; Gut, J.; Rosenthal, P. J.; Nogueira, F.; Egan, T. J.; Moreira, R.;
Paulo, A. N10, N11-di-alkylamine indolo[3,2-b]quinolines as
hemozoin inhibitors: Design, synthesis and antiplasmodial activity.
Bioorg. Med. Chem. 2015, 23, 1530−1539.
(16) Mudududdla, R.; Mohanakrishnan, D.; Bharate, S. S.;
Vishwakarma, R. A.; Sahal, D.; Bharate, S. B. Orally effective
aminoalkyl 10H-indolo[3,2-b]quinoline-11- carboxamide kills the
malaria parasite by inhibiting host hemoglobin uptake. ChemMed-
Chem 2018, 13, 2581−2598.
(17) Kuntworbe, N.; Ofori, M.; Addo, P.; Tingle, M.; Al-Kassas, R.
Pharmacokinetics and in vivo chemosuppressive activity studies on
cryptolepine hydrochloride and cryptolepine hydrochloride-loaded
gelatine nanoformulation designed for parenteral administration for
the treatment of malaria. Acta Trop. 2013, 127, 165−173.
(18) Wang, N.; Wicht, K. J.; Shaban, E.; Ngoc, T. A.; Wang, M.-Q.;
Hayashi, I.; Hossain, M. I.; Takemasa, Y.; Kaiser, M.; El Tantawy El
Sayed, I.; Egan, T. J.; Inokuchi, T.; Inokuchi, T. Synthesis and
evaluation of artesunate-indoloquinoline hybrids as antimalarial drug
candidates. MedChemComm 2014, 5, 927−931.
(19) Sun, N.; Du, R.-L.; Zheng, Y.-Y.; Huang, B.-H.; Guo, Q.;
Zhang, R.-F.; Wong, K.-Y.; Lu, Y.-J. Antibacterial activity of N-
methylbenzofuro[3,2-b]quinoline and N-methylbenzoindolo[3,2-b]-
quinoline derivatives and study of their mode of action. Eur. J. Med.
Chem. 2017, 135, 1−11.
(20) Zhao, M.; Kamada, T.; Takeuchi, A.; Nishioka, H.; Kuroda, T.;
Takeuchi, Y. Structure-activity relationship of indoloquinoline analogs
anti-MRSA. Bioorg. Med. Chem. Lett. 2015, 25, 5551−5554.
(21) Ntie-Kang, F.; Lifongo, L. L.; Simoben, C. V.; Babiaka, S. B.;
Sippl, W.; Mbaze, L. M. a. The uniqueness and therapeutic value of
natural products from West African medicinal plants. Part I:
uniqueness and chemotaxonomy. RSC Adv. 2014, 4, 28728−28755.
(22) Sawer, I. K.; Berry, M. I.; Ford, J. L. The killing effect of
cryptolepine on Staphylococcus aureus. Lett. Appl. Microbiol. 2005,
40, 24−29.
(27) Ablordeppey, S. Y.; Fan, P.; Li, S.; Clark, A. M.; Hufford, C. D.
Substituted indoloquinolines as new antifungal agents. Bioorg. Med.
Chem. 2002, 10, 1337−1346.
(28) Bolden, S.; Zhu, X. Y.; Etukala, J. R.; Boateng, C.; Mazu, T.;
Flores-Rozas, H.; Jacob, M. R.; Khan, S. I.; Walker, L. A.;
Ablordeppey, S. Y. Structure-activity relationship (SAR) and
preliminary mode of action studies of 3-substituted benzylthioquino-
linium iodide as anti-opportunistic infection agents. Eur. J. Med. Chem.
2013, 70, 130−142.
(29) Boateng, C. A.; Zhu, X. Y.; Jacob, M. R.; Khan, S. I.; Walker, L.
A.; Ablordeppey, S. Y. Optimization of 3-(phenylthio)quinolinium
compounds against opportunistic fungal pathogens. Eur. J. Med. Chem.
2011, 46, 1789−1797.
(30) Olajide, O. A.; Ajayi, A. M.; Wright, C. W. Anti-inflammatory
properties of cryptolepine. Phytother Res. 2009, 23, 1421−1425.
(31) Olajide, O. A.; Bhatia, H. S.; de Oliveira, A. C. P.; Wright, C.
W.; Fiebich, B. L. Anti-neuroin flammatory properties of synthetic
cryptolepine in human neuroblastoma cells: Possible involvement of
NF-kB and p38 MAPK inhibition. Eur. J. Med. Chem. 2013, 63, 333−
339.
́
́
(32) Rasouli, H.; Yarani, R.; Pociot, F.; Popovic-Djordjevic, J. Anti-
diabetic potential of plant alkaloids: Revisiting current findings and
future perspectives. Pharmacol. Res. 2020, 155, 104723.
(33) Tuyiringire, N.; Deyno, S.; Weisheit, A.; Tolo, C. U.; Tusubira,
D.; Munyampundu, J.-P.; Ogwang, P. E.; Muvunyi, C. M.; Heyden, Y.
V. Three promising antimycobacterial medicinal plants reviewed as
potential sources of drug hit candidates against multidrug-resistant
tuberculosis. Tuberculosis 2020, 124, 101987.
(34) Kongkham, B.; Prabakaran, D.; Puttaswamy, H. Opportunities
and challenges in managing antibiotic resistance in bacteria using
plant secondary metabolites. Fitoterapia 2020, 147, 104762.
(35) Stell, J. G. P.; Wheelhouse, R. T.; Wright, C. W. Metabolism of
cryptolepine and 2-fluorocryptolepine by aldehyde oxidase. J. Pharm.
Pharmacol. 2012, 64, 237−243.
(36) Nuthakki, V. K.; Mudududdla, R.; Sharma, A.; Kumar, A.;
Bharate, S. B. Synthesis and biological evaluation of indoloquinoline
alkaloid cryptolepine and its bromo-derivative as dual cholinesterase
inhibitors. Bioorg. Chem. 2019, 90, 103062.
(37) Zhu, J.-K.; Gao, J.-M.; Yang, C.-J.; Shang, X.-F.; Zhao, Z.-M.;
Lawoe, R. K.; Zhou, R.; Sun, Y.; Yin, X.-D.; Liu, Y.-Q. Design,
synthesis, and antifungal evaluation of neocryptolepine derivatives
against phytopathogenic fungi. J. Agric. Food Chem. 2020, 68, 2306−
2315.
(38) Wang, S.; Dong, G.; Sheng, C. Structural simplification of
natural products. Chem. Rev. 2019, 119, 4180−4220.
(39) Yang, G.-Z.; Zhu, J.-K.; Yin, X.-D.; Yan, Y.-F.; Wang, Y.-L.;
Shang, X.-F.; Liu, Y.-Q.; Zhao, Z.-M.; Peng, J.-W.; Liu, H. Design,
synthesis, and antifungal evaluation of novel quinolone derivatives
inspired from natural quinine alkaloids. J. Agric. Food Chem. 2019, 67,
11340−11353.
(23) Le Gresley, A.; Gudivaka, V.; Carrington, S.; Sinclair, A.;
Brown, J. E. Synthesis, analysis and biological evaluation of novel
indolquinonecryptolepine analogues as potential anti-tumour agents.
Org. Biomol. Chem. 2016, 14, 3069−79.
(24) Yuan, J.-M.; Wei, K.; Zhang, G.-H.; Chen, N.-Y.; Wei, X.-W.;
Pan, C.-X.; Mo, D.-L.; Su, G.-F. Cryptolepine and aromathecin based
mimics as potent G-quadruplex binding, DNA-cleavage and
anticancer agents: Design, synthesis and DNA targeting-induced
apoptosis. Eur. J. Med. Chem. 2019, 169, 144−158.
(40) Abe, T.; Suzuki, T.; Anada, M.; Matsunaga, S.; Yamada, K. 2-
Hydroxyindoline- 3-triethylammonium bromide: A reagent for formal
C3-electrophilic reactions of indoles. Org. Lett. 2017, 19, 4275−4278.
(41) Bierer, D. E.; Dubenko, L. G.; Zhang, P.; Lu, Q.; Imbach, P. A.;
Garofalo, A. W.; Phuan, P.-W.; Fort, D. M.; Litvak, J.; Gerber, R. E.;
Sloan, B.; Luo, J.; Cooper, R.; Reaven, G. M. Antihyperglycemic
activities of cryptolepine analogues: An ethnobotanical lead structure
isolated from Cryptolepis sanguinolenta. J. Med. Chem. 1998, 41,
2754−2764.
(25) Mahale, S.; Bharate, S. B.; Manda, S.; Joshi, P.; Bharate, S. S.;
Jenkins, P. R.; Vishwakarma, R. A.; Chaudhuri, B. Biphenyl-4-
carboxylic acid [2-(1H indol-3-yl)-ethyl]-methylamide (CA224), a
nonplanar analogue of fascaplysin, inhibits Cdk4 and tubulin
polymerization: Evaluation of in vitro and in vivo anticancer activity.
J. Med. Chem. 2014, 57, 9658−9672.
(26) Qin, Q.-P.; Wei, Z.-Z.; Wang, Z.-F.; Huang, X.-L.; Tan, M.-X.;
Zou, H.-H.; Liang, H. Imaging and therapeutic applications of Zn(II)-
cryptolepine-curcumin molecular probes in cell apoptosis detection
and photodynamic therapy. Chem. Commun. 2020, 56, 3999−4002.
(42) Mazu, T. K.; Etukala, J. R.; Zhu, X. Y.; Jacob, M. R.; Khan, S. I.;
Walker, L. A.; Ablordeppey, S. Y. Identification of 3-phenyl-
aminoquinolinium and 3-phenylaminopyridinium salts as new agents
against opportunistic fungal pathogens. Bioorg. Med. Chem. 2011, 19,
524−533.
(43) Maiti, D.; Buchwald, S. L. Orthogonal Cu- and Pd-based
catalyst systems for the O- and N-arylation of aminophenols. J. Am.
Chem. Soc. 2009, 131, 17423−17429.
(44) Boateng, C. A.; Zhu, X. Y.; Jacob, M. R.; Khan, S. I.; Walker, L.
A.; Ablordeppey, S. Y. Optimization of 3-(phenylthio)quinolinium
1270
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
compounds against opportunistic fungal pathogens. Eur. J. Med. Chem.
2011, 46, 1789−1797.
(45) Shang, X.-F.; Zhao, Z.-M.; Li, J.-C.; Yang, G.-Z.; Liu, Y.-Q.; Dai,
L.-X.; Zhang, Z.-J.; Yang, Z.-G.; Miao, X.-L.; Yang, C.-J.; Zhang, J.-Y.
Insecticidal and antifungal activities of Rheum palmatum L.
anthraquinones and structurally related compounds. Ind. Crop. Prod.
2019, 137, 508−520.
(46) Zhang, J.; Yan, L.-T.; Yuan, E.-L.; Ding, H.-X.; Ye, H.-C.;
Zhang, Z.-K.; Yan, C.; Liu, Y.-Q.; Feng, G. Antifungal activity of
compounds extracted from cortex pseudolaricis against Colleto-
trichum gloeosporioides. J. Agric. Food Chem. 2014, 62, 4905−4910.
(47) Gao, Y.; He, L.; Li, X.; Lin, J.; Mu, W.; Liu, F. Toxicity and
biochemical action of the antibiotic fungicide tetramycin on
Colletotrichum scovillei. Pestic. Biochem. Physiol. 2018, 147, 51−58.
(48) Wen, S.-Q.; Jeyakkumar, P.; Avula, S. R.; Zhang, L.; Zhou, C.-
H. Discovery of novel berberine imidazoles as safe antimicrobial
agents by down regulating ROS generation. Bioorg. Med. Chem. Lett.
2016, 26, 2768−2773.
(49) Zhang, Z.-J.; Jiang, Z.-Y.; Zhu, Q.; Zhong, G.-H. Discovery of
β-carboline oxadiazole derivatives as fungicidal agents against rice
sheath blight. J. Agric. Food Chem. 2018, 66, 9598−9607.
1271
https://dx.doi.org/10.1021/acs.jafc.0c06480
J. Agric. Food Chem. 2021, 69, 1259−1271