Discovery of γ-Lactam alkaloid derivatives as potential fungicidal agents targeting steroid biosynthesis

Article abstract of DOI:10.1021/acs.jafc.0c05823

Biological control of plant pathogens is considered as one of the green and effective technologies using beneficial microorganisms or microbial secondary metabolites against plant diseases, and so microbial natural products have played important roles in the research and development of new and green agrochemicals. To explore the potential applications for natural γ-lactam alkaloids and their derivatives, 26 γ-lactams that have flexible substituent patterns were synthesized and characterized, and their in vitro antifungal activities against eight kinds of plant pathogens belonging to oomycetes, basidiomycetes, and deuteromycetes were fully evaluated. In addition, the high potential compounds were further tested using an in vivo assay against Phytophthora blight of pepper to verify a practical application for controlling oomycete diseases. The potential modes of action for compound D1 against Phytophthora capsici were also investigated using microscopic technology (optical microscopy, scanning electron microscopy, and transmission electron microscopy) and label-free quantitative proteomics analysis. The results demonstrated that compound D1 may be a potential novel fungicidal agent against oomycete diseases (EC₅₀ = 4.9748 μg·mL^-1 for P. capsici and EC₅₀ = 5.1602 μg·mL^-1 for Pythium aphanidermatum) that can act on steroid biosynthesis, which can provide a certain theoretical basis for the development of natural lactam derivatives as potential antifungal agents.

Full text of DOI:10.1021/acs.jafc.0c05823

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Article
Discovery of γ‑Lactam Alkaloid Derivatives as Potential Fungicidal
Agents Targeting Steroid Biosynthesis
Daye Huang, Shuangshuang Wang, Di Song, Xiufang Cao,* Wenbo Huang, and Shaoyong Ke*
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ABSTRACT: Biological control of plant pathogens is considered as one of the green and effective technologies using beneficial
microorganisms or microbial secondary metabolites against plant diseases, and so microbial natural products have played important
roles in the research and development of new and green agrochemicals. To explore the potential applications for natural γ-lactam
alkaloids and their derivatives, 26 γ-lactams that have flexible substituent patterns were synthesized and characterized, and their in
vitro antifungal activities against eight kinds of plant pathogens belonging to oomycetes, basidiomycetes, and deuteromycetes were
fully evaluated. In addition, the high potential compounds were further tested using an in vivo assay against Phytophthora blight of
pepper to verify a practical application for controlling oomycete diseases. The potential modes of action for compound D1 against
Phytophthora capsici were also investigated using microscopic technology (optical microscopy, scanning electron microscopy, and
transmission electron microscopy) and label-free quantitative proteomics analysis. The results demonstrated that compound D1 may
be a potential novel fungicidal agent against oomycete diseases (EC50 = 4.9748 μg·mL−1 for P. capsici and EC50 = 5.1602 μg·mL−1 for
Pythium aphanidermatum) that can act on steroid biosynthesis, which can provide a certain theoretical basis for the development of
natural lactam derivatives as potential antifungal agents.
KEYWORDS: γ-lactams, synthesis, bioassay, oomycetes, P. capsici, P. aphanidermatum, proteomic profile
1. INTRODUCTION
Plant diseases are one of the main hazards of crop production
at present, and, among all, plant pathogens are the main source
of crop infections.1−4 Oomycetes are a large group of plant
pathogens that mainly include Phytophthora and Pythium
species, which are the source of major diseases in vegetables;
cucumbers; and all kinds of melons, potatoes, grapes, and
litchi, and cause downy mildew, Phytophthora, late blight, and
other diseases.5−7 Especially, the genus of Phytophthora causes
serious diseases in many crops worldwide. For example,
Phytophthora blight caused by Phytophthora capsici is one of
the most devastating soilborne diseases in vegetable crops
worldwide. Potato late blight caused by Phytophthora infestans
was responsible for the Irish potato famine.8,9 Therefore,
various fungicides have been developed and used to control
these diseases; however, long-term use of fungicides can lead to
increasing resistance, environmental pollution, food safety
threats, etc.10,11 Nowadays, biological control of various plant
pathogens is considered as one of the green and effective
technologies using beneficial microorganisms or microbial
secondary metabolites against plant diseases, and so microbial
natural products have played important roles in research and
development of new and green agrochemicals.12−16 Many
commercial pesticides such as strobilurin fungicides and
pyrethroid insecticides were discovered and developed based
on natural products from plants or microbial metabolites.
As a class of important physiologically active compounds,
azaheterocyclic alkaloids with γ-lactam scaffolds from many
plants or microorganisms (marine and terrestrial organisms)
present diverse structures (Figure 1) and bioactivities, and the
Figure 1. Some typical lactam alkaloids from natural sources.
special cyclic keto-enol structure feature leads to their
extensive biological functions including antiviral, antitumor,
antifungal, antibacterial, insecticidal, herbicidal, and plant
growth promotion activities.17−24 For example, we noticed
Received: September 9, 2020
Accepted: November 10, 2020
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A

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Figure 2. Research strategies for multisubstituted lactam alkaloid derivatives.
Scheme 1. General Synthetic Route for Target Molecules A1−8, B1−4, C1−7, and D1−7
that their analogue, isoharzianic acid (Figure 1), which was
isolated from a T. harzianum strain,22 has been tested for its in
vitro fungicidal activity against S. sclerotiorum and Rhophitulus
solani and in vivo in terms of plant growth promotion and
induction of disease resistance. Many kinds of lactam
analogues (such as tenuazonic acid, reutericyclin, penicillenol
B1, macrocidin A, etc.) were also isolated from various
microorganisms or plant extracts and demonstrated to exhibit a
broad range of biological activity. Thus, it is a more challenging
and application-promising project to find new analogues with
significant biological applications by utilizing natural γ-lactam
scaffolds as a precursor. Recently, during the course of our
research for functional molecules as potential novel agro-
chemicals based on natural lactam scaffolds, several series of
lactam-guided derivatives were synthesized and proved to
possess potential fungicidal activities,25−28 and the change of
five-position substituents of the γ-lactam unit can affect the
activity.
extensive secondary metabolites in organisms. Moreover, many
AAs have been extensively used as typical synthons for the
syntheses of a variety of heterocyclic systems due to their
special difunctional features,29,30 and many AA derivatives have
been demonstrated to exhibit a broad range of pharmacological
properties or agrochemical activities.31−36 Therefore, AAs as
easily available substrates have proven to be a natural choice
for construction of structural diversity heterocycles with
promising bioactivity.
Based on this investigation, a series of extended AA-guided
lactam alkaloid derivatives were constructed for exploring
prospective novel fungicidal agents with good activity and
unique modes of action, as shown in Figure 2. Therefore, to
explore the potential applications for these heterocyclic
derivatives, we report herein the synthesis and characterization
of 26 γ-lactam derivatives that have flexible substituent
patterns, and their in vitro antifungal activities against several
plant pathogens including P. capsici, Pythium aphanidermatum,
Rhizoctonia solani, Botrytis cinerea, Alternaria solani, Pestalo-
tiopsis theae, Corynespora cassiicola, and Fusarium oxysporum
were fully investigated. In addition, active compounds with a
In addition, amino acids (AAs) are a class of important
natural molecules for construction of diverse peptides and
proteins, which can also participate in the biosynthesis of
B
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Table 1. In Vitro Fungicidal Activity of Target Lactam Alkaloid Derivatives at 20 μg·mL−1
a
P.C, P. capsici; P.A, P. aphanidermatum; R.S, R. solani; B.C, B. cinerea; A.S, A. solani; P.T, P. theae; C.C, C. cassiicola; and F.O, F. oxysporum.
b
c
Metalaxyl and dimethomorph: used as the positive control. CK: used as the negative control.
high inhibitory effect were further tested in vivo to verify a
practical application for controlling oomycete diseases. Finally,
the potential mechanisms of action for compound D1 against
P. capsici were also investigated using microscopic technology
(scanning electron microscopy, (SEM) and transmission
electron microscopy (TEM)) and label-free quantitative
proteomics analysis. The results demonstrated that compound
D1 may be a potential fungicidal agent against oomycete
disease that can act on steroid biosynthesis, which can provide
a certain theoretical basis for the development of natural
lactam derivatives as potential antifungal agents.
2. RESULTS AND DISCUSSION
2.1. Synthesis. The key natural or non-natural α-amino
acids were utilized as the basic building blocks to construct the
extended lactam molecular library. The various polysubstituted
heterocyclic lactam derivatives A1−8, B1−4, C1−7, and D1−
7 were conveniently prepared as described in Scheme 1, which
include four or five steps such as esterification, nucleophilic
substitution, chlorination, amidation, and heterocyclization
reactions.
First, the N-unsubstituted α-amino acids including phenyl-
alanine, methionine, alanine, and phenylglycine were utilized as
C
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1
Figure 3. H NMR spectra analyses of compound D1.
building blocks to construct N-unsubstituted lactams A1−8,
which were transformed to various amino acid esters in the
presence of thionyl chloride and methanol. Various substituted
aryl acetic acids were transformed to the corresponding acyl
chlorides and then reacted with the prepared amino acid esters
to obtain the key intermediate aryl acetamides. N-unsub-
stituted lactams A1−8 were conveniently obtained via an
intramolecular heterocyclization reaction of the above aryl
acetamides. Second, four N-substituted lactams B1−4 were
synthesized using a method similar to that for N-unsubstituted
lactams A1−8, except that the N-substituted α-amino acids
were used as substances. All four N-substituted α-amino acids
were prepared using two different methods, as described in
Scheme 1. After this, two kinds of easily obtained cyclic amino
acids including proline and pipecolinic acid were explored to
construct bicycle lactams C1−7 and D1−7. The structures of
all of the synthesized compounds in this work were
characterized on the basis of satisfying spectral analysis, and
the formulae of the tested compounds are presented in Table
1. All of the newly prepared lactam derivatives were confirmed
by NMR and electrospray ionization-mass spectrometry (ESI-
MS) analyses (Supporting Information), and their chemical
structures and basic physicochemical properties are summar-
ized in the Materials and Methods section.
lactam heterocycle. The ESI-MS spectrum indicated that all of
these lactams displayed an obvious [M + H]+ addition ion
fragmentation peak.
2.3. Biological Activity. Several typical plant pathogens
that belong to oomycetes, basidiomycetes, and deuteromycetes
were selected to screen potential fungicidal activities of target
lactam compounds A1−8, B1−4, C1−7, and D1−7 in vitro
using the mycelium growth rate method,26,37 and the results of
antifungal activities of these compounds in vitro are listed in
Table 1, and metalaxyl and dimethomorph were used as the
positive control.
As we can see from Table 1, some of these lactam alkaloid
analogues (such as B1, B2, B3, B4, C7, and D1) present
moderate to good antifungal activities (>50%) against
selectively tested fungi at a lower concentration of 20 μg·
mL−1. First, some easily available N-unsubstituted α-amino
acids were selected directly to construct diverse N-
unsubstituted lactams A1−8 (R1H), and simple aromatic
substrates including phenylacetic acid, p-fluorophenylacetic
acid, p-methoxyphenylacetic acid, and 2,4-dichlorophenylacetic
acid were explored. The bioassay results indicated that most of
these analogues (A1−6) exhibited moderate inhibitory activity
against C. cassiicola belonging to deuteromycetes, and the
inhibition rate is in the range of 28.39−43.93% at a
concentration of 20 μg·mL−1. However, all compounds A1−
8 have no obvious inhibitory activity on pathogens belonging
to oomycetes and basidiomycetes.
2.2. Spectroscopy Studies. The structures of all target
lactams A1−8, B1−4, C1−7, and D1−7 were confirmed by 1H
NMR and mass spectrometry. The 1H NMR of these
compounds exhibited different and unique proton signal
As far as we know, the substituents attached to the nitrogen
atom of the amide linkage usually have an obvious influence on
the activity. Therefore, to investigate the possible effect of this
unit on the activity of final molecules, several N-substituted
lactams B1−4 were designed and synthesized, and their
antifungal activity was screened. From Table 1, what is
interesting is that all N-substituted lactams B1−4 present
1
peaks for different substituents. A representative H NMR
spectrum of compound D1 (Figure 3) demonstrated that all
1H NMR spectral characteristic peaks of these compounds
present an obvious low field signal of aromatic protons linked
to the 3-position in the lactam ring and unique high field signal
peaks for typical alkyl groups attached to the 5-position of the
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Table 2. Inhibition Activity of Compound D1 on Plant Pathogens
entry
treatment
phytopathogens
regression equation
EC50 (μg·mL−1
)
correlation relation r
1
2
3
4
compd. D1
metalaxyl
compd. D1
pyraclostrobin
P. capsici
P. capsici
P. aphanidermatum
P. aphanidermatum
y = 2.0325x + 3.5838
y = 1.5528x + 4.3993
y = 1.0514x + 4.2507
y = 0.84x + 5.3232
4.9748
2.4370
5.1602
0.4123
0.9851
0.9275
0.9929
0.9873
a
a
a
Metalaxyl and pyraclostrobin: used as the positive control.
Figure 4. In vitro antifungal assay for the high potential compound D1 against P. capsici and P. aphanidermatum.
moderate to good activity for deuteromycete pathogens
including B. cinerea, P. theae, C. cassiicola, and F. oxysporum;
especially, these compounds also exhibited moderate activity
against R. solani (basidiomycetes). These results demonstrated
that the introduction of appropriate substituents on the
nitrogen atom of the amide linkage could dramatically change
the biological effects on pathogens.
With these results in hand, we think that the nitrogen
substituents on the lactam ring need to be further optimized. It
can be seen from the literature that bicyclic or polycyclic
scaffolds often appear in a large number of natural
alkaloids,38−40 and so some lactams containing bicycles
would be constructed according to the optimization pathway
in Scheme 1. Natural L-proline was first selected to prepare
pyrrolizinone derivatives containing bicycles C1−7, and some
active substrates such as piperacetic acid and indole-3-acetic
acid have also been used as substances to extend the
investigation. However, only the analogues C5 and C7
exhibited moderate to good activity, and the inhibition rate
for these two derivatives against C. cassiicola was up to 40.03
and 54.35%, respectively.
Then, bicyclic lactams bearing a six-member ring D1−7
were further designed and synthesized by replacement of
normal amino acids with piperidine acid. The bioassay results
indicated that most of this series of analogues present broad-
spectrum bactericidal activity, and compounds D1−4 exhibited
moderate to good antifungal activity against oomycetes,
basidiomycetes, and deuteromycete pathogens. Notably,
compound D1 showed high potential activity against P. capsici,
P. aphanidermatum, R. solani, and B. cinerea with an inhibition
rate that varied from 42.59 to 87.50% at 20 μg·mL−1. In
addition, the inhibition rate of most of the compounds was less
than 60% at the concentration of 20 μg·mL−1, but compound
D1 showed excellent fungicidal activity against oomycetes P.
capsici and P. aphanidermatum, and the inhibition rate was
87.50 and 72.10%, respectively. Metalaxyl could completely
inhibit the growth of oomycetes P. capsici and P.
aphanidermatum at the concentration of 20 μg·mL−1 but was
ineffective against other plant pathogens. However, dimetho-
morph can only inhibit the growth of oomycete P. capsici, and
so the inhibition spectrum of compound D1 was similar to that
of metalaxyl.
According to previous reports,41 oomycetes are sensitive to
metalaxyl, but fungi (basidiomycetes and deuteromycetes) are
tolerant to metalaxyl. The plant pathogens that belong to
oomycetes peronosporales are sensitive to dimethomorph;
other oomycetes and fungi (basidiomycetes and deuteromy-
cetes) are tolerant to dimethomorph. P. capsici and P.
aphanidermatum belong to oomycetes peronosporales and
oomycetes pythiales, respectively; therefore, P. aphaniderma-
tum is tolerant to dimethomorph.
Combining the above analysis, all screening results
demonstrated that the antifungal effect for lactam analogues
can be significantly affected by various factors including the
different aryl substituents at the 3-postion of the lactam core
and the substituents on the N-atom in the lactam ring, which
indicated that the high potential compound D1 could be used
as a promising lead molecule to design new compounds for
development of antifungal agents derived from lactam
alkaloids.
Based on the preliminary screening results, some of the
target compounds with good activities were chosen for further
bioassay investigation at different concentrations (20, 10, 5,
2.5, 1.25 μg·mL−1) to better compare the potential fungicidal
activity, and the EC50 values were calculated using SPSS 22.0
software. All of the EC50 values for highly active lactam
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derivatives and the control are summarized in Table 2. The
results indicated that compound D1 had high fungicidal
activity against P. capsici and P. aphanidermatum with EC50
values of 4.9748 and 5.1602 μg·mL−1, respectively, which was
similar to that of metalaxyl but higher than that of
pyraclostrobin.
The high potential compound D1 exhibited good antifungal
activity against P. capsici and P. aphanidermatum, and the
corresponding inhibitor effects are also indicated in Figure 4,
which further confirmed their good inhibitory activity.
2.4. In Vivo Fungicidal Activity. According to the above
in vitro assay, compound D1 had good inhibitory effects on
oomycete diseases, including those caused by P. capsici and P.
aphanidermatum. To further pursue its high potential
inhibitory effects, an in vivo experiment for compound D1
against Phytophthora blight of pepper was carried out, and the
control efficacy of compound D1 on Phytophthora blight of
pepper is listed in Table 3 and Figure 5. As shown in Table 3
this class of molecular scaffold could be further investigated as
agricultural agents to fight against oomycete diseases.
At present, the prevention and control of the diseases caused
by Phytophthora spp. usually rely on fungicides, such as
metalaxyl, pyraclostrobin, dimethomorph, etc. However, the
increasing use of these fungicides leads to the appearance of
resistant isolates.42 Therefore, the discovery of novel fungicides
with new modes of action is urgently needed, and compound
D1 has a new chemical structure and high potential fungicidal
activity against Phytophthora blight of pepper, which could be
used for management of resistant populations.
2.5. Effect of Compound D1 on the Morphology and
Ultrastructure of P. capsici. First, the effect of compound
D1 on the morphological changes of P. capsici hyphae was
studied by optical microscopy (OM) and scanning electron
microscopy (SEM). As shown in Figure 6, OM observations of
P. capsici treated with 10 μg·mL−1 compound D1 revealed
hyphal deformation and enlargement and increased branching
of mycelium compared with the control. SEM observation of P.
capsici indicated that compound D1 caused some hyphae to
have a thinner and wrinkled surface and some others to have a
swollen and bursting appearance.
In addition, transmission electron microscopy (TEM)
images treated with compound D1 demonstrated the effects
on the ultrastructure of P. capsici, and the images revealed that
the cell wall and membrane were damaged (Figure 7), which
was the reason for the thinner and wrinkled surface of hyphae
observed in SEM. The number of vacuoles increased and they
became distorted and filled the inner volume of the cell. Other
organelles were squeezed by vacuoles. This evidence further
confirmed that these compounds had versatile capabilities to
influence and disturb the normal life of P. capsici and
consequently led to its death. Therefore, changing the
morphology of mycelia might be one of the fungicidal
mechanisms of compound D1 on P. capsici.
2.6. Proteogenomic Analysis. In this study, the statistic
result showed that 34417 unique peptides corresponding to
4155 quantifiable proteins were identified using iTRAQ-
labeling liquid chromatography-mass spectrometry (LC-MS)/
MS analysis. Comparative proteomic analysis (compound D1/
CK) found that 415 proteins were differentially expressed after
Table 3. Control Efficacy of Compound D1 on Phytophthora
Blight of Pepper In Vivo
concentration
disease
index
control efficacy
entry treatment
(μg·mL−1
)
(%)
b
1
compd. D1
2000
1000
1000
12.50 b
18.06 b
6.67 c
79.91 a
70.98 a
89.28 a
-
2
3
metalaxyl
a
CK
62.22 a
a
b
CK - used as the negative control. Different lower-case letters
means significant difference (P < 0.05).
and Figure 5, the tested compound showed an excellent
control effect on Phytophthora blight of pepper with the
control efficacies of 70.97 and 79.91% at concentrations of
1000 and 2000 μg·mL−1, respectively, which was similar to that
of the positive control metalaxyl.
In addition, we can also find that the growth of pepper
treated with compound D1 was obviously better than that of
the control group, and the leaf state of pepper treated with
compound D1 was also superior to that of the group treated
with metalaxyl (Figure 5). This obtained result confirmed that
Figure 5. In vivo fungicidal activity for compound D1 and metalaxyl against P. capsici, and the pictures in the circles represent the local amplification
of the in vivo evaluation results.
F
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Figure 6. Microscopic observation of the hyphal morphology of P. capsici of the blank control ((A) optical microscopy and (C) scanning electron
microscopy images) and the cultures treated with 10 μg·mL−1 compound D1 for 7 days ((B) optical microscopy and (D) scanning electron
microscopy images).
being treated with compound D1, including 135 upregulated
proteins and 280 downregulated proteins, based on standard
criteria: fold changes > 2.0 and P values < 0.05 (Figure S1).
GO enrichment analysis revealed differentially expressed
proteins (DEPs, P < 0.05) associated with 30 functional
groups, including eight associating with molecular functions,
eight with cellular components, and 14 with biological
processes (Figure 8). The DEPs involved in ligase activity,
forming carbon−carbon bonds, biotin binding, palmitoyl-CoA
9-desaturase activity, acyl-CoA desaturase activity, stearoyl-
CoA 9-desaturase activity, amide binding, cofactor binding,
and DBD domain binding, were grouped according to their
CC (Figure 8B). The DEPs in the plasmodesma, cell−cell
junction, mating-type region heterochromatin, chromatid
body, nuclear pore cytoplasmic filaments, cell wall, external
encapsulating structure, and fibrillar center were classified on
the basis of their MF (Figure 8A). The DEPs involved in
triglyceride metabolic process, long-chain fatty acid biosyn-
thetic process, regulation of cholesterol metabolic process,
monocarboxylic acid biosynthetic process, regulation of steroid
metabolic process, fatty-acyl-CoA biosynthetic process, C-
terminal protein amino acid modification, C-terminal protein
lipidation, negative regulation of fatty acid β-oxidation,
negative regulation of fatty acid oxidation, anion trans-
membrane transport, ribonucleoside bisphosphate biosynthetic
process, ribosomal protein import into the nucleus, and white
fat cell differentiation were categorized on the basis of their BP
(Figure 8C).
ones; lipid transport and metabolism; secondary metabolite
biosynthesis, transport, and catabolism; energy production and
conversion; amino acid transport and metabolism; signal
transduction mechanisms; intracellular trafficking, secretion,
and vesicular transport; RNA processing and modification;
translation, ribosomal structure, and biogenesis; and inorganic
ion transport and metabolism, reflecting different molecular
functions, as indicated in Figure S2. This observation suggests
that compound D1 can remarkably trigger and influence a
series of physiological actions (Figure S2).
KEEG analysis was used to determine the mode of action of
compound D1 and the stress response of P. capsici. As shown
in Figure 9 and Table 4, nine pathways were affected by
compound D1 at fold enrichment >1.5, P < 0.05. Steroid
biosynthesis and pentose and glucuronate interconversions
were downregulated by compound D1, which were associated
with 2 and 5 proteins, respectively. The pathways that were
upregulated by compound D1 included biotin metabolism,
ubiquinone and other terpenoid-quinone biosyntheses, arach-
idonic acid metabolism, biosynthesis of unsaturated fatty acids,
fatty acid biosynthesis, fatty acid elongation, and pyruvate
metabolism, which were associated with 2, 5, 6, 7, 9, 6, and 14
proteins, respectively.
Unsaturated fatty acids are one of the components of cell
membranes, which are important for the cell to cope with
adversities, such as low temperature and chemical substances.43
Arachidonic acid, a kind of unsaturated fatty acid, is also a
component of cell membranes in microbiology. Therefore,
these two pathways could be the response to chemical stress of
compound D1 for P. capsici.
The COG analysis revealed that differentially expressed
proteins were associated with 21 functional groups, such as
post-translational modification, protein turnover, and chaper-
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Figure 7. Transmission electron micrographs of P. capsici hyphae. (A) and (B) represent the blank control and (C) and (D) represent the hyphae
exposure to compound D1 at a concentration of 10 μg·mL−1.
It has been reported that energy release can protect P. capsici
against novel fungicide SYP-14288 damage.44 Similar to this
finding, biotin mechanism, ubiquinone and other terpenoid-
quinone biosyntheses, fatty acid biosynthesis, fatty acid
elongation, pentose and glucuronate interconversions, and
pyruvate metabolism were associated with energy metabo-
lism.45−48 Therefore, these six pathways could be a way of
coping with chemical stress for compound D1.
The major function of steroids is related to hormonal
functions for microorganisms. It has been reported that
steroidal hormones act as pheromones in the oomycetes of
Achlya ambisexualis. These compounds control sexual morpho-
genesis.49 Cholesterol ester converting into cholesterol was
inhibited by downregulation of lipase, which affects the
biosynthesis of steroid hormone (Figure 10). This finding
indicated that compound D1 can affect the expression of lipase
associated with steroids, resulting in growth inhibition of P.
capsici. As far as we know, this is a new antimicrobial
mechanism.
pounds had significant in vitro fungicidal activity against
oomycete diseases at a concentration of 20 μg·mL−1. The high
potential compounds were further tested in vivo to verify a
potential application for controlling oomycete diseases. The
potential mechanisms of action for typical compound D1 were
also investigated using SEM/TEM and label-free quantitative
proteomics analysis. SEM and TEM images demonstrated that
this class of compounds had the obvious ability to affect and
change the mycelial morphologies. Label-free quantitative
proteomics indicated that compound D1 may be a potential
fungicidal agent against oomycete diseases that can act on
steroid biosynthesis, which can provide a certain theoretical
basis for the development of natural lactam derivatives as
potential antifungal agents.
4. MATERIALS AND METHODS
4.1. Chemistry. All starting materials and reagents were obtained
from commercial suppliers and used without further purification
unless otherwise specified. NMR spectra were recorded on a Bruker
spectrometer at 600 MHz with dimethyl sulfoxide (DMSO)-d6 as the
solvent and TMS as the internal standard. Mass spectra were obtained
on a Waters ACQUITY UPLC H-CLASS PDA (Waters) instrument.
Optical rotations were measured on an Olympus CX21 microscope
(Tokyo, Japan), SEM images were visualized and obtained using a
Hitachi SU8100 scanning electron microscope (Japan), and TEM
images were obtained using a Hitachi HT7700 transmission electron
microscope (Japan). Analytical thin-layer chromatography was carried
3. CONCLUSIONS
A series of diversity-guided multisubstituted lactams were
synthesized and characterized, and their potential fungicidal
activities against eight pathogenic fungi belonging to
oomycetes, basidiomycetes, and deuteromycetes were fully
evaluated. Preliminary bioassay indicated that some com-
H
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Figure 8. Differential expression proteins between control and treatment groups were classified based on known molecular functions (A), cellular
components (B), and biological processes (C).
Figure 9. KEGG pathway enrichment bubble plot of differentially expressed proteins. The vertical axis shows the KEGG classification enriched
according to fold >1.5, P < 0.05, whereas the horizontal axis presents the value of the fold enrichment converted by Log2. The size of the circular
area in the figure represents the number of differential proteins, and the circular color represents the enrichment P value of the differential protein
under the KEGG classification.
out on precoated plates, and spots were visualized with ultraviolet
light.
4.2. Plant Pathogenic Fungi. P. capsici, P. aphanidermatum, R.
solani, B. cinerea, A. solani, P. theae, C. cassiicola, and F. oxysporum were
obtained from the Hubei Biopesticide Engineering Research Centre.
The strains were retrieved from the storage tube and incubated in
PDA at 25 °C for a week to obtain new mycelia for the antifungal
assay.
4.3. Preparation of Target Lactam Alkaloid Derivatives.
According to the procedures described in our previous reports,25−28 a
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Table 4. KEGG Pathways Significantly Altered in P. capsici Exposed to Compound D1 (P < 0.05)
pathway
protein accession
protein description
ratio
steroid biosynthesis
biotin metabolism
PhycaF7_98581
PhycaF7_37671
PhycaF7_117050
PhycaF7_46737
PhycaF7_71098
PhycaF7_110946
PhycaF7_116439
PhycaF7_104181
PhycaF7_20036
PhycaF7_5899
lipase (triglyceride lipase-cholesterol esterase)
lipase (triglyceride lipase-cholesterol esterase)
VATC domain-containing protein
biotin synthase
flavodoxin-like domain-containing protein
4-hydroxyphenylpyruvate dioxygenase
NAD(P)H:quinone oxidoreductase, type IV
flavodoxin-like protein
NAD(P)H:quinone oxidoreductase, type IV
AB hydrolase−1 domain-containing protein
AB hydrolase−1 domain-containing protein
glutathione S-transferase
hematopoietic prostaglandin D synthase
glutathione peroxidase
0.306
0.279
4.621
2.524
0.662
2.032
5.573
3.817
2.450
2.153
3.613
6.198
1.653
0.386
3.003
0.459
2.255
1.822
0.398
5.679
4.139
3.345
2.048
10.775
6.154
9.617
1.731
0.589
12.737
7.602
1.753
2.255
2.216
1.822
1.987
5.679
4.139
0.235
0.610
0.265
0.406
0.398
1.752
1.929
2.198
0.401
0.462
2.843
10.775
6.154
9.617
0.381
12.737
1.514
0.419
1.602
ubiquinone and other terpenoid-quinone biosynthesis
arachidonic acid metabolism
PhycaF7_51990
PhycaF7_78349
PhycaF7_7873
PhycaF7_82055
PhycaF7_121666
PhycaF7_104257
PhycaF7_114420
PhycaF7_99779
PhycaF7_90838
PhycaF7_39128
PhycaF7_31760
PhycaF7_112188
PhycaF7_87116
PhycaF7_76378
PhycaF7_40349
PhycaF7_112283
PhycaF7_77362
PhycaF7_89078
PhycaF7_77225
PhycaF7_38301
PhycaF7_20861
PhycaF7_114420
PhycaF7_108343
PhycaF7_99779
PhycaF7_27530
PhycaF7_39128
PhycaF7_31760
PhycaF7_114955
PhycaF7_122339
PhycaF7_13297
PhycaF7_121185
PhycaF7_116854
PhycaF7_93601
PhycaF7_39467
PhycaF7_91192
PhycaF7_117052
PhycaF7_115780
PhycaF7_103156
PhycaF7_76378
PhycaF7_40349
PhycaF7_112283
PhycaF7_100447
PhycaF7_77225
PhycaF7_91190
PhycaF7_120908
PhycaF7_94497
bifunctional epoxide hydrolase 2
acyl-CoA desaturase 1
biosynthesis of unsaturated fatty acids
3-oxo-5-α-steroid 4-dehydrogenase
estradiol 17-β-dehydrogenase, putative
FA_desaturase domain-containing protein
elongation of fatty acid protein
elongation of fatty acid protein
FA_desaturase domain-containing protein
fatty acid synthase subunit α (Fragment)
acetyl-co-A carboxylase
fatty acid biosynthesis
acetyl-co-A carboxylase
acetyl-CoA carboxylase, putative
long-chain acyl-CoA synthetase 7, peroxisomal
AMP-binding domain-containing protein
acetyl-CoA carboxylase
holo-[acyl-carrier-protein] synthase (Fragment)
long-chain acyl-CoA synthetase 7, peroxisomal
3-oxo-5-α-steroid 4-dehydrogenase
lysosomal thioesterase PPT2-B
estradiol 17-β-dehydrogenase, putative
palmitoyl-protein thioesterase 1, putative
elongation of fatty acid protein
elongation of fatty acid protein
chlorophyll synthesis pathway protein BchC
xylose isomerase
xylose isomerase
polygalacturonase
pectinesterase
pyruvate kinase
malic domain-containing protein (Fragment)
acetyl-coenzyme A synthetase
pyruvate carboxylase
malate dehydrogenase
malic enzyme
fatty acid elongation
pentose and glucuronate interconversions
pyruvate metabolism
acetyl-co-A carboxylase
acetyl-co-A carboxylase
acetyl-CoA carboxylase, putative
pyruvate, phosphate dikinase
acetyl-CoA carboxylase
acetyl-coenzyme A synthetase
malate dehydrogenase
fumarate hydratase
series of polysubstituted lactam alkaloid derivatives A1−8, B1−4,
C1−7, and D1−7 were prepared in our laboratory. The structures of
all compounds were confirmed by their spectral analysis, and all of the
data for newly synthesized derivatives are as follows.
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9.0 Hz, 2H), 4.12 (dd, J = 13.1, 5.0 Hz, 1H), 3.85 (dd, J = 11.5, 3.9
Hz, 1H), 2.73 (td, J = 12.9, 3.5 Hz, 1H), 2.38−2.27 (m, 1H), 1.89−
1.78 (m, 1H), 1.65 (d, J = 13.1 Hz, 1H), 1.53−1.43 (m, 1H), 1.17
(ddt, J = 13.0, 8.7, 4.5 Hz, 1H), 0.99 (qd, J = 12.5, 3.4 Hz, 1H); MS
(ESI) m/z 248.4 (M + H)+, calcd for C22H19NO5 m/z = 247.1.
4.3.7. 1-Hydroxy-2-phenyl-6,7,8,8a-tetrahydroindolizin-3(5H)-1-
1
D5. White solid, yield 80%. H NMR (600 MHz, DMSO-d6): δ =
11.37 (s, 1H), 7.98−7.95 (m, 2H), 7.32−7.28 (m, 2H), 7.16−7.13
(m, 1H), 4.13 (dd, J = 13.0, 4.9 Hz, 1H), 3.84 (dd, J = 11.5, 3.9 Hz,
1H), 2.73 (td, J = 12.9, 3.5 Hz, 1H), 2.35 (dd, J = 12.7, 3.9 Hz, 1H),
1.85 (d, J = 13.5 Hz, 1H), 1.65 (d, J = 13.1 Hz, 1H), 1.52−1.44 (m,
1H), 1.21−1.12 (m, 1H), 1.00 (qd, J = 12.5, 3.4 Hz, 1H); MS (ESI)
m/z 230.4 (M + H)+, calcd for C22H19NO5 m/z = 229.1.
4.3.8. 2-(Benzo[d]1,3dioxol-5-yl)-1-hydroxy-6,7,8,8a-tetrahy-
droindolizin-3(5H)-1-D6. White solid, yield 76%. 1H NMR (600
MHz, DMSO-d6) δ 11.29 (s, 1H), 7.59 (d, J = 1.2 Hz, 1H),7.54 (dd, J
= 1.8, 8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 5.97 (s, 2H), 4.11 (dd, J
= 4.2, 12 Hz, 1H), 3.81 (dd, J = 3.6, 11.4 Hz, 1H), 2.75−2.70 (m,
1H), 2.33 (d, J = 9.0 Hz, 1H), 1.84 (d, J = 12.6 Hz, 1H), 1.64 (d, J =
13.2 Hz, 1H), 1.47 (d, J = 14.4 Hz, 1H), 1.15 (d, J = 13.2 Hz, 1H),
0.97 (d, J = 9.6 Hz, 1H); MS (ESI) m/z 274.4 (M + H)+, calcd for
C15H15NO4 m/z = 273.1.
4.3.9. 1-Hydroxy-2-(1-methyl-1H-indol-3-yl)-6,7,8,8a-tetrahy-
droindolizin-3(5H)-1-D7. White solid, yield 65%. 1H NMR (600
MHz, DMSO-d6) δ 10.67 (s, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.59 (s,
1H), 7.38 (d, J = 8.4 Hz, 1H), 7.12 (t, J = 8.4 Hz, 1H), 6.99 (t, J = 7.8
Hz, 1H), 4.13 (dd, J = 12.6, 4.2 Hz, 1H), 3.85 (dd, J = 11.4, 4.2 Hz,
1H), 3.78 (s, 3H), 2.79−2.74 (m, 1H), 2.35 (d, J = 10.2 Hz, 1H),
1.86 (d, J = 13.2 Hz, 1H), 1.66 (d, J = 15.6 Hz, 1H), 1.49 (d, J = 13.2
Hz, 1H), 1.20−1.17 (m, 1H), 1.06−1.00 (m, 1H); MS (ESI) m/z
283.5 (M + H)+, calcd for C17H18N2O2 m/z = 282.1.
4.4. In Vitro Fungicidal Evaluation. In this research, all obtained
lactam alkaloid derivatives were evaluated for their antifungal activities
in vitro against eight common plant pathogens (oomycetes P. capsici
and P. aphanidermatum; basidiomycetes R. solani; and deuteromycetes
B. cinerea, A. solani, P. theae, C. cassiicola, and F. oxysporum) at a
concentration of 20 μg·mL−1 on the basis of pesticide guidelines for
laboratory bioactivity tests. Any compound with an inhibition rate of
70% or more at this concentration was further evaluated, and the
median effective concentration (EC50) values were calculated
following the method described by Guan et al.50 The preliminary
screening and further evaluation results are outlined in Tables 1−3.
4.5. Effect of Compound D1 on Phytophthora Blight of
Pepper in the Greenhouse Test. P. capsici was cultivated in V8
juice agar medium at 28 °C for 7 days. Then, the cultures were
flooded with double-distilled water and incubated under continuous
light for 10 days to induce zoosporangia. To encourage the release of
zoospores, they were transferred into a refrigerator at 4 °C for 30 min
and then at room temperature for 30 min.51,52 Then, the
concentration of the zoospores was adjusted to 2 × 104 zoospore·
mL−1.
Figure 10. Steroid biosynthesis pathway.
4.3.1. 6-(Benzo[d]1,3dioxol-5-yl)-7-hydroxy-2,3-dihydro-1H-pyr-
rolizin-5(7aH)-1-C6. White solid, yield 58%. H NMR (600 MHz,
1
DMSO-d6) δ 11.85 (s, 1H), 7.54 (d, J = 1.8 Hz, 1H), 7.51 (dd, J =
7.8, 1.8 Hz, 1H), 6.88 (d, J = 7.8 Hz, 1H), 5.97 (d, J = 1.2 Hz, 2H),
4.13−4.10 (m, 1H), 3.40−3.37 (m, 1H), 3.04−3.00 (m, 1H), 2.19−
2.15 (m, 1H), 2.06−2.03 (m, 2H), 1.34−1.31 (m, 1H); MS (ESI) m/
z 260.5 (M + H)+, calcd for C14H13NO4 m/z = 259.1.
4.3.2. 7-Hydroxy-6-(1-methyl-1H-indol-3-yl)-2,3-dihydro-1H-pyr-
rolizin-5(7aH)-1-C7. White solid, yield 62%. H NMR (600 MHz,
1
DMSO-d6) δ 11.22 (s, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.58 (s, 1H),
7.38 (d, J = 8.4 Hz, 1H), 7.14−7.11 (m, 1H), 7.00−6.98 (m, 1H),
4.18 (dd, J = 9.0, 6.6 Hz, 1H), 3.78 (s, 3H), 3.43−3.40 (m, 1H),
3.07−3.03 (m, 1H), 2.21−2.18 (m, 1H), 2.09−2.05 (m, 2H), 1.39−
1.35 (m, 1H); MS (ESI) m/z 269.6 (M + H)+, calcd for C16H16N2O2
m/z = 268.1.
4.3.3. 1-Hydroxy-2-(o-tolyl)-6,7,8,8a-tetrahydroindolizin-3(5H)-
1-D1. White solid, yield 77%.1H NMR (600 MHz, DMSO-d6): δ =
10.87 (s, 1H), 7.20−7.08 (m, 4H), 4.08 (dd, J = 13.1, 5.0 Hz, 1H),
3.83 (dd, J = 11.5, 3.9 Hz, 1H), 2.75 (td, J = 12.9, 3.4 Hz, 1H), 2.29
(dd, J = 12.6, 3.2 Hz, 1H), 2.16 (s, 3H), 1.85 (d, J = 12.7 Hz, 1H),
1.65 (d, J = 13.0 Hz, 1H), 1.53−1.44 (m, 1H), 1.23−1.13 (m, 1H),
1.08−1.00 (m, 1H); MS (ESI) m/z 244.5 (M + H)+, calcd for
C22H19NO5 m/z = 243.1.
4.3.4. 2-(2,4-Dichlorophenyl)-1-hydroxy-6,7,8,8a-tetrahydroin-
dolizin-3(5H)-1-D2. White solid, yield 52%. H NMR (600 MHz,
For the seedling assay, pepper seeds were sowed in 72 hole trays (4
cm × 4 cm × 4.5 cm) filled with potting mixtures (turf soil/
vermiculite/perlite = 3:1:1, v/v/v). The plants were ready for the
experiment after being grown in a greenhouse for 5 weeks.
1
DMSO-d6): δ = 11.26 (s, 1H), 7.60 (d, J = 2.2 Hz, 1H), 7.40 (dd, J =
8.3, 2.2 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1H), 4.06 (dd, J = 13.1, 5.0 Hz,
1H), 3.86 (dd, J = 11.6, 3.9 Hz, 1H), 2.75 (td, J = 12.9, 3.5 Hz, 1H),
2.25 (dd, J = 12.6, 3.7 Hz, 1H), 1.88−1.80 (m, 1H), 1.65 (d, J = 13.1
Hz, 1H), 1.55−1.44 (m, 1H), 1.23−1.12 (m, 1H), 1.10−1.01 (m,
1H); MS (ESI) m/z 298.3 (M + H)+, calcd for C22H19NO5 m/z =
297.0.
4.3.5. 1-Hydroxy-2-(4-methoxyphenyl)-6,7,8,8a-tetrahydroindo-
lizin-3(5H)-1-D3. White solid, yield 74%. 1H NMR (600 MHz,
DMSO-d6): δ = 11.14 (s, 1H), 7.93−7.89 (m, 2H), 6.90−6.86 (m,
2H), 4.12 (dd, J = 13.0, 5.0 Hz, 1H), 3.80 (dd, J = 11.5, 3.9 Hz, 1H),
3.74 (s, 3H), 2.72 (td, J = 12.8, 3.5 Hz, 1H), 2.36−2.29 (m, 1H), 1.84
(d, J = 12.9 Hz, 1H), 1.64 (d, J = 12.9 Hz, 1H), 1.47 (q, J = 13.2 Hz,
1H), 1.22−1.11 (m, 1H), 1.03−0.93 (m, 1H); MS (ESI) m/z 260.4
(M + H)+, calcd for C22H19NO5 m/z = 259.1.
Compound D1 (4 g) was dissolved in 5mL of DMSO, and then
1995 mL of distilled water containing 0.1% Tween 80 was added to
obtain a solution of a concentration of 2000 μg·mL−1. A solution with
a concentration of 1000 μg·mL−1 was prepared by diluting the above
solution. The concentration of 1000 μg·mL−1 metalaxyl was used as a
positive control at the same conditions. Further, 10 mL of the solution
was poured on the soil near the roots of each seedling; after 24 h, 2 ×
104 zoospores of P. capsici in 200 μL volume was poured into three
holes (0.5 cm × 2.0 cm) near the rhizosphere of the bell pepper. The
seedling was incubated in a greenhouse at 28 °C.
Disease severity was evaluated after inoculation for 12 days and
assessed using a 0−5 scale: 0, no visible disease symptoms; 1, leaves
slightly wilted and the stem with brownish lesions; 2, 30−50% of the
entire plant diseased; 3, 50−70% of the entire plant diseased; 4, 70−
90% of the entire plant diseased; and 5, the plant dead.53
4.3.6. 2-(4-Fluorophenyl)-1-hydroxy-6,7,8,8a-tetrahydroindoli-
zin-3(5H)-1-D4. Orange-yellow solid, yield 71%. 1H NMR (600
MHz, DMSO-d6): δ = 11.44 (s, 1H), 8.07−8.01 (m, 2H), 7.14 (t, J =
K
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Statistical analyses were performed using SPSS version 22.0,
significant differences between the treatments of mean values were
determined by least-significant difference (LSD), which was obtained
by Tukey’s test at P < 0.05 level.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the support of this work by
the National Key R&D Program of China (2017YFD0200502)
and the Program for Leading Talents of Hubei Academy of
Agricultural Sciences (L2018031). The authors also gratefully
acknowledge partial support from the Natural Science
Foundation of Hubei Province (2020CFB717) and Hubei
Agricultural Science Innovation Centre (2019-620-000-001-
27). The authors are indebted to Dr. Yuan Xiao of the Institute
of Hydrobiology, Chinese Academy of Sciences, for her
assistance with ultrastructure observation.
4.6. Effect of Compound D1 on the Morphology and
Ultrastructure of P. capsici. After being treated with distilled water
(negative control) or 10 μg·mL−1 compound D1, the mycelium of P.
capsici were visualized using optical (Olympus CX21, Tokyo, Japan),
scanning electron (Hitachi SU8100, Japan), and transmission electron
microscopies (Hitachi HT7700, Japan), as previously reported.54−56
4.7. Label-Free Quantitative Proteomics Analysis.
4.7.1. Strains and Growth Conditions. The strain used in this
study was P. capsici, which is the causative pathogen of Phytophthora
blight of pepper. The mycelium (3 g) of P. capsici was cultivated in
100 mL of potato dextrose broth (PDB) medium amended with 10
μg·mL−1 compound D1. Control samples were prepared in the
absence of compound D1. Mycelia were cultured at 28 °C for 48 h in
a shaker (180 rpm), collected, and dried under vacuum before being
stored at −80 °C until required.
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AUTHOR INFORMATION
■
Corresponding Authors
Xiufang Cao − Department of Chemistry, College of Science,
Huazhong Agricultural University, Wuhan 430070, China;
Email: caoxiufang@mail.hzau.edu.cn
Shaoyong Ke − National Biopesticide Engineering Research
Centre, Hubei Biopesticide Engineering Research Centre,
Hubei Academy of Agricultural Science, Wuhan 430064,
China; orcid.org/0000-0002-2715-4005;
Email: shaoyong.ke@nberc.com
Authors
(9) Zhou, T.; Yang, X.; Qiu, D.; Zeng, H. Inhibitory effects of
xenocoumacin 1 on the different stages of Phytophthora capsici and its
control effect on Phytophthora blight of pepper. BioControl 2017, 62,
151−160.
Daye Huang − National Biopesticide Engineering Research
Centre, Hubei Biopesticide Engineering Research Centre,
Hubei Academy of Agricultural Science, Wuhan 430064,
China
Shuangshuang Wang − Department of Chemistry, College of
Science, Huazhong Agricultural University, Wuhan 430070,
China
Di Song − Department of Chemistry, College of Science,
Huazhong Agricultural University, Wuhan 430070, China
Wenbo Huang − National Biopesticide Engineering Research
Centre, Hubei Biopesticide Engineering Research Centre,
Hubei Academy of Agricultural Science, Wuhan 430064,
China
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Rey, P. Transcriptional analysis of the interaction between the
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(13) Guo, Y.; Wang, X.; Qu, L.; Xu, S.; Zhao, Y.; Xie, R.; Huang, M.;
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Complete contact information is available at:
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Notes
The authors declare no competing financial interest.
L
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