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

Article abstract of DOI:10.1021/acs.jafc.9b06793

Neocryptolepine is an alkaloid isolated from traditional African herbal medicine Cryptolepis sanguinolenta, and its broad spectrum of biological activities has been illuminated in past decades. In this study, neocryptolepine and its derivatives (1-49) were designed and synthesized from economical and readily available starting materials. Their structures were confirmed by proton nuclear magnetic resonance, carbon nuclear magnetic resonance, and mass spectrometry. The synthesized compounds were screened for their antifungal profile against six agriculturally important fungi Rhizoctonia solani, Botrytis cinerea (B. cinerea), Fusarium graminearum, Mycosphaerella melonis, Sclerotinia sclerotiorum, and Magnaporthe oryzae. The results of in vitro assay revealed that compounds 5, 21, 24, 35, 40, 45, and 47 presented remarkable antifungal activity against the fungi tested with EC₅₀ values lower than 1 μg/mL. Significantly, compound 24 displayed the most effective inhibitory potency against B. cinerea (EC₅₀ = 0.07 μg/mL), and the data from in vivo experiments revealed that compound 24 demonstrated comparable protective activity with the positive control boscalid. Preliminary mechanism studies indicated that compound 24 showed impressive spore germination inhibitory effectiveness and lower cytotoxicity than azoxystrobin, imparted on normal function of the cell membrane and cell wall, and arrested the normal function of the nucleus. Besides the excellent inhibitory activity against agriculturally important phytopathogenic fungi tested, the designed assemblage possesses several benefits with a high profile of variation in synthesized molecules, the ease of synthesis, and good cost-effectiveness of commercially available synthetic reagents, all of these have highlighted the potential worth of compound 24 as a new and highly efficient agricultural fungicide.

Full text of DOI:10.1021/acs.jafc.9b06793

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Agricultural and Environmental Chemistry
Design, Synthesis and Antifungal Evaluation of
Neocryptolepine Derivatives against Phytopathogenic Fungi
jiakai zhu, jian mei gao, cheng jie yang, Xiao-Fei Shang, zhong min zhao,
Raymond Kobla Lawoe, rui zhou, yu sun, xiaodan Yin, and Ying-Qian Liu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b06793 • Publication Date (Web): 29 Jan 2020
Downloaded from pubs.acs.org on January 29, 2020
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Design, Synthesis and Antifungal Evaluation of
Neocryptolepine Derivatives against
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Phytopathogenic Fungi
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Jia-Kai Zhu, Jian-Mei Gao, Cheng-Jie Yang, Xiao-Fei Shang, Zhong-Min Zhao,
Raymond Kobla Lawoe, Rui Zhou, Yu Sun, Xiao-Dan Yin, Ying-Qian Liu*
School of Pharmacy, Lanzhou University, Lanzhou 730000, People’s Republic of
China
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E-mail address: yqliu@lzu.edu.cn. (Ying-qian Liu)
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ABSTRACT:
Neocryptolepine is an alkaloid isolated from traditional African herbal medicine
Cryptolepis sanguinolenta and its broad spectrum of biological activities have been
illuminated in past decades. In this study, neocryptolepine and its derivatives (1-49)
were designed and synthesized from economical and readily available starting
materials. Their structures were confirmed by H NMR, 13C NMR and MS. The
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synthesized compounds were screened for their antifungal profile against six
agriculturally important fungi Rhizoctonia solani, Botrytis cinerea, Fusarium
graminearum, Mycosphaerlla melonis, Sclerotinia sclerotiorum and Magnaporthe
oryzae. The results of in vitro assay revealed that compounds 5, 21, 24, 35, 40, 45 and
47 presented remarkable antifungal activity against the fungi tested with EC50 values
lower than 1 μg/mL. Significantly, compound 24 displayed the most effective
inhibitory potency against Botrytis cinerea (EC50 = 0.07 μg/mL), and the data from in
vivo experiments revealed that compound 24 demonstrated comparable protective
activity with the positive control boscalid. Preliminary mechanism studies indicated
that compound 24 showed impressive spore germination inhibitory effectiveness,
lower cytotoxicity than azoxystrobin, imparted on normal function of cell membrane,
cell wall and arrested the normal function of the nucleus. Besides, the excellent
inhibitory activity against agriculturally important phytopathogenic fungi tested, the
designed assemblage possesses several benefits with a high-profile of variation in
synthesized molecules, the ease of synthesis and good cost-effectiveness of
commercially available synthetic reagents, all of these have highlighted the potential
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worth of compound 24 as a new and highly efficient agricultural fungicide.
KEYWORDS: Neocryptolepine, alkaloid, antifungal activity, Botrytis cinerea.
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INTRODUCTION
The agricultural challenge of providing adequate and safe food supply for all
populations is currently threatened due to infestation of food crops by virulent
pathogens, especially fungi.1,2 Crop plants have become the source of nutrients for
phytopathogens at different stages of their development since the dawn of agriculture,
causing a huge loss in economic value of crops thereby compromising global food
security.3 The great discoveries and developments of efficient agrochemicals to
combat these pathogens have steadily succumbed to multi-drug resistance, which
rendered these chemical agents ineffective.4 Additionally, emerging results from
recent investigations have indicated that resistant pathogenic species are multiplying
rapidly within a short time once resistance sets in, giving rise to a new challenge to
address.5 Aside from the problems put forth, potential harm these chemical fungicides
pose to human health, animals, non-target species and the environment has stirred
serious concerns.6 These concerns have continued to drive the design and synthesis
of bioactive chemical agents with high efficiency, de novo modes of action, greater
selectivity, biocompatibility and benign to human health as better alternatives to
traditional synthetic fungicides.7
In this context, natural products have emerged as highly promising sources of
lead candidates for use in small-molecule drug discovery.8 It has been shown that
essential features of natural products are chemical diversity and diversity in biological
functions, making natural product libraries favorable and attractive in drug discovery.9
Nonetheless, the paucity of these biologically active products in native plants and the
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challenges of extracting the target compounds from other metabolites with similar
chemical properties have hindered research interests in this area.10 These problems
have necessitated chemical mimicking of natural products by synthesizing libraries of
structurally simplified natural compounds with improved bioactivities by adopting
simple and ecofriendly synthetic protocols. Since alkaloids are biologically active
compounds and play a vital role in plant defense mechanisms, alkaloids and their
derivatives have often been evaluated and developed as fungicides in the past several
years.11 In the course of our previous screening for novel naturally occurring
phytopesticides from the plants, neocryptolepine, a naturally isomeric indoloquinoline
alkaloid, was found to exhibit superior broad-spectrum antifungal activity compared
with its corresponding analogues cryptolepine and isocryptolepine,.12 This
encouraging results prompted us to further extend our investigation by synthesizing a
series of new neocryptolepine derivatives and structure activity relationships on target
compounds for antifungal activity were probed.(Figure 1).
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Neocryptolepine is a minor alkaloid isolated from the root bark extract of
traditional African herbal medicine Cryptolepis sanguinolenta,13 which has been used
to treat rheumatism, urinary, respiratory infections, fevers due to malaria and stomach
disorders.14 Extensive literature review has revealed the inhibitory activity of
neocryptolepine against Gram-positive bacteria and Gram-negative bacteria,15 and its
antiplasmodial activity has also been extensively investigated.16 The pioneering works
of Laurent D. et al. indicated that neocryptolepine intercalated with DNA and
interfered with the catalytic activity of human topoisomerase II.17 Subsequent
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investigations on cytotoxicity and cell cycle effects of neocryptolepine were well
documented.18 Introduction of methyl group at the C11 position of neocryptolepine by
DIMIQ strongly enhanced the antitumor potential of this metabolite.19 Also,
modification at C2 and C9 positions further improved the antitumor activity.20,21 In
addition, derivatives of neocryptolepine bearing C11-alkylamino-substitution were
extensively evaluated to possess remarkable biological profiles against fungi, bacteria,
malaria and tumors.22-26
To our knowledge, there is no report on applications of neocryptolepine or its
derivatives against agricultural pathogens. Given the broad pharmacological activities
of neocryptolepine, we envisioned that chemically mimicked analogues of this plant
metabolite may effectively suppress activities of phytopathogenic fungi. With the
resurgence in multi-drug resistance, the development of more potent and
biocompatible neocryptolepine derivatives should yield novel antifungal agents to
selectively combat fungal pathogens. Hence, as continuation of our screening
campaign for bioactive agents against phytopathogens, a library of novel
neocryptolepine derivatives was designed and synthesized by employing an efficient
and flexible route under mild condition (Figure 2).27 The impacts of SAR (Structure−
Activity Relationship) of A and D rings substitutions on inhibitory activity against six
phytopathogenic fungi were assessed. Compounds found to be most effective in in
vitro were further subjected to in vivo investigations. The possible mechanism action
of the most active compound 24 against B. cinerea, was preliminarily explored in this
study.
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MATERIALS AND METHODS
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Chemicals. All reagents and solvents were commercially obtained and used directly
without further purification. Quinolines and indoles were purchased from a
commercial source (Shanghai Sun Chemical Technology Co.,LTD).
Instruments. The melting points were determined in an open capillary using
WRS-2U melting point apparatus (Shanghai Precision Instrument Co., Ltd., Shanghai,
China) and are uncorrected. Mass spectra were recorded on a Bruker Daltonics
APEXII49e spectrometer (Bruker Daltonics Inc., Billerica, MA, US.) with ESI source
as ionization. 1H and 13C-NMR spectra were recorded at 400 MHz and 100 MHz on a
Bruker AM-400 (Bruker Company, Billerica, MA, US.) spectrometer using TMS as
reference. Microscopic morphology of fungal hyphae was observed by a Scanning
electron microscopy (Hitachi, S-3400N, Japan). Microscopic internal structure of
fungal cells was investigated by a transmission electron microscopy (FEI Tecnai G2
spirit Bio-Twin (T12)). The relative conductivity of hyphae was performed by a
conductivity meter (Leici DDS-307, China). The absorbance of cytotoxicity assay was
measured with Thermo Scientific Multiskan MK3 microplate reader (USA).
Fungi. Six plant pathogenic fungi species, R. solani, B. cinerea, F. graminearum, M.
melonis, S. sclerotiorum and M. oryzae were isolated from susceptible plants
cultivated in greenhouses and orchards at the Gansu academy of agricultural sciences
in 2010, Gansu Province of China. Then, they were purified and identified by the
Institute of Plant Protection, Gansu Academy of Agricultural Science. The fungi were
maintained during the experiments on potato dextrose agar medium (PDA: potato 200
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g, dextrose 20 g, agar 15 g and distilled water 1000 mL) at 25 °C.
Synthetic Procedures.
General Synthetic Procedure for the Intermediates Ⅰ. Under nitrogen, a mixture
of appropriate quinoline (7.7 mmol) and MeI (11.6 mmol) in isopropanol (1 M) was
heated at 90 °C for 3 h. The reaction was cooled to room temperature, and the
resulting precipitate was isolated by vacuum filtration, washed with a mixture of
isopropanol/ethyl acetate (1:1), dried in vacuo, and obtained yellow solid for the next
step.
General Synthetic Procedure for the Intermediates Ⅱ. To a solution of
potassium hydroxide (0.148 mol) in water (30 mL) and 1,2-dichloroethane (30 mL),
then the mixture was added hydrogen peroxide (6.4 mL, 35%) and
1-methylquinolinium iodide (Ⅰ, 15 mmol in 15 mL water) over 30 min at 0°C. The
resulting mixture was stirred at room temperature for 48 h, then the organic layer was
separated and the aqueous layer was extracted with dichloromethane (30 mL × 3). The
combined organic layer was dried with anhydrous magnesium sulfate. The organic
layer was concentrated under reduced pressure to obtain the title compound as a
yellow oil for the next step.
General Synthetic Procedure for the Target Compounds 1-49. A mixture of
Intermediate Ⅱ (5 mmol), indole (5 mmol) and p-TSA (5 mmol) in absolute ethanol
(10 mL) was stirred open to air in a 50 mL round bottom flask at reflux for 12 h. After
cooling to room temperature, the reaction mixture was washed with 1 M NaOH (50
mL) and the aqueous layer extracted with CH2Cl2 (3 × 80 mL). The combined organic
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layer was then dried with anhydrous magnesium sulfate. The organic layer was
concentrated under reduced pressure, and the residue was purified by column
chromatography on silica gel, removing impurities with petroleum ether/ethyl acetate
(2:1), then eluting with dichloromethane/methanol (40:1) to yield the final compound
as a red solid.
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The physical data of all compounds in detail are provided in Supporting
Information.
Antifungal Activity Assay In Vitro
Neocryptolepine and its derivatives were evaluated by the mycelium growth rate
method28 for their antifungal activity against six fungi, including R. solani, B. cinerea,
F. graminearum, M. melonis, S. sclerotiorum and M. oryzae. All the synthetic
compounds were dissolved in dimethyl sulfoxide (DMSO) and then were added to
potato dextrose agar (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 μg/mL). The blank control was
maintained with 0.5% DMSO (v/v) mixed with PDA and azoxystrobin was used as a
positive control because azoxystrobin possesses a broad - spectrum activity against
phytopathogenic fungi. The mycelial disk (5mm) of phytopathogenic fungi were
inoculated on PDA plates and then were incubated at 25 ℃ in the dark. Each sample
was measured in triplicate, 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:
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Mycelial growth inhibition (%) = [(C − T)/(C − 5 mm)] × 100
Where C and T represent the diameter of fungal growth on untreated PDA and treated
PDA, respectively.
Antifungal Activity Assay In Vivo
The protective activity in vivo. Based on the preceding test of in vitro antifungal
activity, compounds 5 and 24 against B. cinerea were further tested in vivo on tomato.
The synthesized compounds and positive control boscalid, in 0.1 mL of DMSO were
dissolved in 10 mL deionized water at a series of concentrations (200, 100, 50, 25
μg/mL). Boscalid was used as positive control because it is a specific commercial
fungicide for controlling B. cinerea. Each sample measured in quadruplicate was
sprayed evenly onto the tomato, which had been already washed and treated with
water and 75% aqueous ethyl alcohol. After 24 h, each tomato was punctured with an
inoculating needle and then pathogen was inoculated. 1% DMSO in 10 mL water was
set up as the blank control. All the treated samples were then placed into an
illumination incubator in 25 ℃ and 100% relative humidity for 4 days.
Scanning Electron Microscopy (SEM) Observations
Mycelia blocks (5.0mm×4.0 mm) were cut from the fungi after treating 24 at a
concentration of 0.1 μg/mL, all the samples were treated by 4% glutaraldehyde for 4 h
and washed three times with 0.01 M PBS (pH=7.2), then fixed with 1% osmium
tetraoxide solution (w/v) for 2 h. After that, each sample was dehydrated with graded
ethanol series (20%, 50%, 80%, 90%) for 10 min, respectively. Subsequently, the
samples were dried at a critical point and gold-sprayed and observed by using a
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scanning electron microscope.
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Transmission Electron Microscopy (TEM) Observations
The dehydrated mycelial blocks were embedded in resin at 70 °C for 24 h and
then cut into thin sections. After the samples were double-stained with uranyl acetate
and lead citrate, they were observed with a transmission electron microscope.
Spore Germination Inhibition Assay
Spore suspensions (1×105–5×105 spores/mL) were prepared by inoculating
conidia in sterile water. The synthesized compound 24 dissolved in DMSO was added
to the spore suspension at three concentrations (5, 2.5, 1 μg/mL). After that, 100 μL of
the mixtures were put on concave slides and incubated in a biochemical incubator at
27 ℃, respectively. Conidial suspension with 0.5% DMSO (v/v) in water was treated
as a blank control. After incubation for 12 h, the number of germinated spores were
measured by approximately counting 100 conidia in blood counting chamber under a
biological microscope photographic system at 400× magnification.
Determination of Cell Membrane Permeability
According to the described method,29 the influence of candidate compound on
the cell membrane relative permeability rate of B. cinerea was determined. The
mycelial disk of B. cinerea (5 mm) was placed in 60 mL of PD broth medium and
shaking at 140 rpm for 4 days at 27 °C. After that, the mycelia were filtered and
added into the solution of 24 with different concentrations (10, 5, 1, 0.1 μg/mL,
respectively). Eventually, the conductivity values were determined with a
conductivity detector (at 0 h was marked as L0, and 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h,
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12 h 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:
Relative electric conductivity (%) = [(L1 − L0)/(L2 − L0)] × 100
Cytotoxicity of Compound 24 against Two Human Cell Lines
Cytotoxicity of target compound 24 was examined in vitro against two human
cell lines (HL7702 and PC12) respectively following previously published
procedure.12 The cell lines tested were treated with different concentrations (25, 10, 5,
1 μg/mL respectively) of target compound 24 in the growth medium for 24 h, and the
absorbance was measured at 450 nm. Five replicates were performed.
Statistical Analysis
All assays were at least performed in triplicate by conventional methods, and
results were presented as means ± standard deviations. The statistical analysis was
carried out 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 Supporting
Information.
RESULTS AND DISCUSSION
Chemistry
According to the previous work by Matthew K.V. et al.,30 neocryptolepine
derivatives were prepared using neocryptolepine as a precursor and employing an
efficient and flexible method under mild conditions that provided the desired products
with high yields. The detailed synthetic protocol for the preparation of
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neocryptolepine and its analogues was illustrated in Figure 2. Intermediate Ⅰ was
easily obtained with high yield (＞90 %) by the reaction of corresponding quinoline
and methyl iodide in IPA under inert gas. Subjecting Intermediate Ⅰ to hydrogen
peroxide oxidation in the mixture of potassium hydroxide aqueous solution and
1,2-dichloroethane afforded the intermediate Ⅱ without further purification.
Neocryptolepine (1) and its analogues were produced when intermediate Ⅱ and
substituted indoles were refluxed in ethyl alcohol under the catalysis of
p-Toluenesulfonic acid. It is worth noting that the raw materials were obtained
commercially to synthesize the intermediates and the final products with good yield.
The structures of target compounds were confirmed by 1H and 13C NMR and MS.
In Vitro Activity and Structure Activity Relationship (SAR)
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An efficient and simple method was used to produce the target compounds for
investigation of effects of substitutions at rings A and D of the lead compound 1 on
the structure-activity relationship, wherein hydrogen at position C11, methyl at
position C5 and nitrogen at position C6 were not substituted (Figure 1). All the
synthesized compounds (1-49) and the positive control azoxystrobin were screened
against six agriculturally important phytopathogenic fungi, R. solani, B. cinerea, F.
graminearum, M. melonis, S. sclerotiorum and M. oryzae.
Rings A and D of the lead conpound 1 were modified to synthesize a series of
novel compounds to investigate the impact of substitutions on antifungal activity
against the fungi tested. And the results of inhibition rates at 10 μg/mL of all
compounds indicated that some synthetic compounds possessed significant activities
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with 80% higher inhibition against all six phytopathogenic fungi (Supporting
Information). As shown in Tables 1 and 2, in vitro antifungal assay revealed that most
of the tested compounds showed broad-spectrum inhibitory activity against the plant
fungal pathogens tested with the EC50 values ranging from 1.07 to > 30 μg/mL against
Sclerotinia sclerotiorum, 0.75 - >30 μg/mL against Rhizoctonia solani, 0.94 - >30
μg/mL against Fusarium graminearum, 0.07 - >30 μg/mL against Botrytis cinerea (B.
cinerea), 0.62 - >30 μg/mL against Magnaporthe oryzae and 0.11 - >30 μg/mL
against Mycosphaerlla melonis. The results indicated that most of the target
compounds demonstrated bioactivity more potent than the lead compound 1, and
positive control azoxystrobin against fungi tested. Particularly, target compounds 5,
21, 24, 35, 40, 45 and 47 were the most potent and exhibited remarkable antifungal
activity against the fungi tested with EC50 values lower than 1 μg/mL. Notably,
compounds 5 and 24 displayed the best inhibitory potency against B. cinerea with
EC50 values of 0.08 μg/mL and 0.07 μg/mL respectively (Figure 3), which were
superior to EC50 value of 4.44 μg/mL of the lead compound 1 and the EC50 value of
＞30μg/mL of positive control azoxystrobin. Tested compound 24 was also the most
effective against M. melonis with EC50 value of 0.11 μg/mL compared to the EC50
value of 20.40 μg/mL of the positive control azoxystrobin. Therefore, the effect of
substituents on the phenyl rings A and D was further explored. With the exception of
compound 13, compounds 4-6, 9-11 and 15 containing electron withdrawing (Cl, F
and Br) groups on the benzyl ring-A and no substituents (R1= H) on phenyl ring-D,
and compounds 19-20, 23-25, 28-30 and 32-33 containing electron withdrawing (Cl,
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F and Br) groups on the phenyl ring-D with no substituents on the ring-A (R=H)
displayed pronounced inhibitory activity against the fungi tested compared to the lead
compound 1 and standard azoxystrobin.
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Similarly, substitutions at the ring-A and ring-D with electron donating (CH3 and
OCH3) groups enhanced biological profile of the tested compounds 2-3, 7-8, 14,
21-22 and 26-27 against B. cinerea and M. melonis, respectively. The bioassay data
indicated that electronic effect played a crucial role in the activity. The impact of
structural variations due to the substitution pattern of the phenyl ring was also
assessed. Results of activity tests showed the positioning and kind of functionality on
the parent structure 1 was indispensable in antifungal activity of the synthesized
compounds. Substituents at positions C2, C3, C8 and C9 were the most beneficial,
providing the strongest antifungal activity (compounds 4-6, 10-11, 18-25 and 35-49)
against B. cinerea and M. melonis than substituents in the other positions C1, C4, C7
and C10 (compounds 12-15, 26-27 and 30-33). Additionally, the effects of the same
functional group at different positions on the ring-A and ring-D were evaluated. For
example, substitution of methoxy group at position 8 improved suppressive activity of
the title compound 22 (EC50 = 0.11 μg/mL) against B. cinerea than substitution of
methoxy group at positions 2 and 9 of the title compounds 3 (EC50 = 0.20 μg/mL) and
17 (EC50 = 0.76 μg/mL), respectively. Also, compounds 21-22 and 24 showed that
substitution at the position 8 dominates in terms of biological effects. Replacement of
8-F by 8-Br was well tolerated with marginal loss in potency. This influence was
further emphasized in substituent 8-Cl proving to be the most tolerated and active
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compound than 1-Cl, 2-Cl, 3-Cl and 9-Cl, respectively. This new finding that activity
was retained or improved by substitution at the 8-position gave scope for further
structural modification and SAR development.
As substitution at the 8-position had given the most potent compound, the
tolerances to substitution at this position were examined in detail by synthesizing
novel compounds decorated with different functional groups on benzyl rings A and D
to explore structural variations and effects of conformational rigidification on the
antifungal activity of compounds (34-49) in this series as summarized in Table 3.
These experimental data in general indicated that a variety of small groups were
tolerated in these regions and enhanced fungicidal activity of the target compounds
with EC50 values less than l μg/mL which were much lower than the EC50 values of
the lead compound 1 and the positive control azoxystrobin. The extent to which
functionality is tolerated was dependent on the electronic effects of the functional
groups, with Cl at position 2 and Br at position 8 significantly heightened the
fungicidal activity of compound 47 against B. cinerea with EC50 value of 0.09 μg/mL.
Similarly, Cl at position 2 and F at position 8 improved the biological effects of
compound 45 against M. melonis with EC50 value of 0.18 μg/mL. Besides, being the
most potent compound in vitro, good solubility of compound 24 in organic solvent
and water propelled it as a candidate compound.
On the other hand, most of the final products showed excellent potency against
S. sclerotiorum and substitution at position 8 (21, 22, 23, 24, 25) markedly favored
antifungal activity, especially compound 21 (EC50 = 1.07 μg/mL). Inhibitory effects
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improved in the order: Me- < Cl- < Br- < F- < MeO- ( 21, 24, 25, 23 and 22).
Surprisingly, most substitutions were not favorable for bioactivity against R.
solani with EC50 > 30 μg/mL compared with lead compound 1. The unsatisfactory
inhibitory potential of these derivatives might be due to a different internal
mechanism of action exhibited against R. solani. This notwithstanding, title
compound 45 (2-Cl-8-F) was very effective against against R. solani with EC50 value
of 0.75 μg/mL.
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In comparison of the bioactivity of 34, 36-39, 41-44, 46-49 (with substituents on
ring-D), the antifungal activity of compounds 2, 5 and 6 against M. oryzae with EC50
values of 0.81, 0.63 and 0.82 μg/mL, respectively, was significantly enhanced after
introducing weak electron donating (CH3) group and electron withdrawing (Cl and
Br) groups into position 2 of ring-A, suggesting that appreciable bioactivity could be
achieved through introducing substituents at position 2. Nonetheless, compounds 35,
40 and 45 with substituents on rings A and D demonstrated antifungal effects
comparable with compound 5. Meanwhile, this study also offered a strategy to
achieve high-efficient fungicides via manipulating the positions of substituents on the
various rings of neocryptolepine.
In Vivo Activity
Based on the bioassay data of SAR in vitro, compounds 5 and 24 had been
chosen to further investigate their in vivo activity against B. cinerea using boscalid as
a positive control and 1% DMSO in 10 mL as a blank control. As presented in Figure
4, it was observed that the growth of hyphae was disrupted after treatment with the
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target compounds compared with the blank control. The inhibitory rates of
compounds 24, 5 and the positive control to boscalid were 54.91%, 32.45% and
54.61% respectively at concentration of 100 μg/mL. These results revealed that
compound 24 exhibited inhibitory activity similar to the positive control boscalid and
superior to compound 5. And more significantly, this result further demonstrated the
fungicidal potency of compound 24 in crop protection.
Preliminary antifungal mechanism of compound 24 against B. cinerea
SEM of Compound 24 on the Hyphae Morphology. To elucidate the effective
pathway of candidate compound 24 against B. cinerea, the SEM had been treated with
compound 24 at concentration of 0.1 μg/mL to observe the effect of compound 24 on
mycelium morphology. As shown in Figure 5, in the blank control group, the hyphae
grew normally and endosome of the cell appeared orderly arranged with a complete
shape and a smooth surface. In contrast, after treating the SEM with compound 24,
drastic changes in mycelial morphology was observed as mycelial growth appeared
abnormal, the arrangement of the endosome appeared distorted, the surface shrunk
and became rough.
TEM of Compound 24 on the Hyphae Morphology. Ultrastructural changes of
hyphae treated with compound 24 had been observed by using TEM. The natural
conidia comprised an outer layer made of electron opaque mannoproteins and an inner
layer with more electron transparent made of interconnected chitin and glucans. As
presented in Figure 6, the hyphae of blank control revealed that mycelial cells were
normal, cell membrane and cell wall were intact, and cytoplasmic organelles were
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evenly distributed. After treating the TRM with compound 24, the ultractructure of
the fungus was drastically altered, the internal organelles of the cells ruptured and
became disordered. Besides, the outer layer of the cell wall became detached from the
cell membrane compared with blank control. This phenomenon indicated that
compound 24 altered the structure of the cell wall, thereby disrupting the permeability
of the cell membrane. These data indicated the shrinkage of the cell membrane was
due to compound 24 and had therefore imparted the nuclear enlargement.
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Effect of Compound 24 on the Spore Germination Inhibition. The results of spore
germination inhibition assay showed that compound 24 markedly suppressed spore
germination of B. cinerea at four different concentrations as showed in Figure 7. The
data of this experiment indicated that the effect of compound 24 on the spore
germination of B. cinerea was concentration-dependent and completely inhibited
spore germination at concentration of 5 μg/mL. The position of the electron
withdrawing (Cl) group on the ring-D might be the reason for the pronounced activity
compound 24 in vitro and in vivo against B. cinerea.
Effect of Compound 24 on the Cell Membrane Integrity. To verify the conclusion
of TEM about cell membrane integrity, the cell membrane permeability of B. cinerea
was determined by testing the change in relative electric conductivity of mycelia
suspensions with four different concentrations (10, 5, 1, 0.1 μg/mL) of compound 24.
As shown in Figure 8, the conductivity rates of the mycelia suspensions treated with
compound 24 were all higher than blank control group and respectively increased in
time-dependent and dose-dependent manners. Although the conductivity was
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observed to be increasing, the difference between the treatment group and the blank
group at each concentration was too small to elucidate that compound 24 damaged the
integrity of the cell membrane. Therefore, it was reasoned that candidate compound
24 slightly affected the cell membrane as the concentrations increased, but this was
not the effect of the excellent in vitro activity of compound 24 against B. cinerea.
Cytotoxicity Assay
Synthesized compound 24 was probed for cytotoxicity against two human cell
lines HL7702 and PC12 cell lines in vitro by employing azoxystrobin as a positive
control. As shown in Figure 9, the results indicated that compound 24 displayed lower
cytotoxicity compared to positive control azoxystrobin against the two cell lines
tested at different concentrations (25, 10, 5, 1 μg/mL respectively). It is noteworthy
that when the concentration reached 25 μg/mL, compound 24 was equipotent to
azoxystrobin against PC12. Compared with the excellent antifungal activity in vitro
and in vivo, cytotoxicity of compound 24 was not significant.
In summary, neocryptolepine had been used as a lead structure to design and
synthesize a series of novel bioactive derivatives with respect to structure and
chemical diversity. The synthesized compounds were screened for their antifungal
profile against six important phytopathogenic fungi. This study demonstrated that
substitutions at C2 of ring A and C8 of ring D of the lead compound 1 played a key
role to improve susceptibilities of the fungi tested to the final target compounds.
Moreover, the results of in vitro bioassay demonstrated that compounds 5, 21, 24, 35,
40, 45 and 47 exhibited pronounced antifungal activity compared with
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neocryptolepine, and compound 24, in particular, was found to possess the best
activity with EC50 value of 0.07 μg/mL against B. cinerea. The data from in vivo
experiment also showed that compound 24 demonstrated protective performance
comparable to the positive control boscalid. Additionally, the observations of SEM
and TEM indicated that compound 24 caused a slight damage to the cell membrane,
imparted the cell wall of fungus and arrested the normal function of the cell nucleus.
Furthermore, compound 24 completely inhibited the spore germination at
concentration of 5 μg/mL, and displayed lower cytotoxicity than azoxystrobin against
HL7702 and PC12 cell lines in vitro at different concentrations. Compound 24,
therefore, displayed great potency to be used as highly efficient antifungal agent
because it demonstrated an excellent inhibitory effectiveness against the agriculturally
significant fungi tested. Besides, the effective inhibitory activity, the designed library
holds several merits with a high level of diversity in synthesized molecules, the ease
of synthesis and good cost-effectiveness of commercially available synthetic reagents.
Further explorations on structural modification of compound 24 and its specific
mechanisms of action are in progress.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
In vitro antifungal activity against six phytopathogenic fungi at 10 μg/mL; In vitro
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EC50 values of all compounds against six phytopathogenic fungi in detail; Detailed
1
data of all synthesized compounds; H NMR, 13C NMR and Mass spectra of the
representative compounds.
Corresponding Author
E-mail: yqliu@lzu.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
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 supplied by the Key Program for
international S&T cooperation projects of China Gansu Province (18YF1WA115).
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Figure Captions
Figure 1. Strategy of Neocryptolepine derivatives as potent antifungal agents.
Figure 2. Synthetic route of Neocryptolepine and its derivatives.
Figure 3. In vitro antifungal activities of compound 24 against B. cinerea.
Figure 4. In vivo protective antifungal activities of candidate compounds against B.
cinerea.
Figure 5. Scanning electron micrographs of the hyphae from the colony of B. cinerea.
Figure 6. Transmission electron microscopy observations of cell structure of B.
cinerea. Ultrastructure of the hyphae in the untreated control (A and B), treated with
compound 24 at concention of 0.1 μg/mL(C and D).
Figure 7. Spore germination inhibition assays of compound 24 against B. cinerea.
Figure 8. Determination of cell membrane permeability of compound 24 against B.
cinerea.
Figure 9. Cytotoxicity assay of compound 24 against cell lines HL7702 and PC12.
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EC50 (μg/mL)
compound
S.s.
17.65
4.01
3.72
6.21
5.03
2.96
6.59
6.01
＞30
3.16
3.97
＞30
15.59
12.98
＞30
＞30
R.s.
9.00
B.c.
4.44
0.21
0.20
0.23
0.08
0.10
0.55
0.54
0.92
0.20
0.27
1.78
＞30
0.72
4.13
＞30
F.g.
16.31
1.83
M.o.
17.63
0.81
M.m.
1.16
1.77
0.78
1.87
0.98
0.98
1.01
2.22
2.17
0.88
1.56
7.29
7.39
2.91
4.32
20.40
1
2
8.92
3
7.11
2.77
1.30
4
13.95
0.94
17.37
0.63
＞30
11.79
＞30
8.76
5
6
3.77
0.82
7
5.31
8.54
8
12.82
16.79
8.47
11.41
17.98
10.19
10.02
20.41
15.90
6.07
＞30
12.93
6.68
9
10
11
20.87
22.34
22.67
8.27
＞30
＞30
19.29
13.48
＞30
＞30
12
13
14
15
16.57
12.03
＞30
27.43
azoxystrobin
618
619
620
621
622
623
624
625
Table 1. Antifungal Activity of A Ring Substitution Compounds in Vitro.
S.s., Sclerotinia sclerotiorum ; R.s., Rhizoctonia solani; B.c., Botrytis cinerea; F.g., Fusarium
graminearum; M.o., Magnaporthe oryzae; M.m., Mycosphaerlla melonis. All values are the mean
of three replicates.
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