Design, Synthesis, and Antifungal Evaluation of Novel Quinoline Derivatives Inspired from Natural Quinine Alkaloids

Article abstract of DOI:10.1021/acs.jafc.9b04224

Inspired by quinine and its analogues, we designed, synthesized, and evaluated two series of quinoline small molecular compounds (a and 2a) and six series of quinoline derivatives (3a-f) for their antifungal activities. The results showed that compounds 3e and 3f series exhibited significant fungicidal activities. Significantly, compounds 3f-4 (EC₅₀ = 0.41 μg/mL) and 3f-28 (EC₅₀ = 0.55 μg/mL) displayed the superior in vitro fungicidal activity and the potent in vivo curative effect against Sclerotinia sclerotiorum. Preliminary mechanism studies showed that compounds 3f-4 and 3f-28 could cause changes in the cell membrane permeability, accumulation of reactive oxygen species, loss of mitochondrial membrane potential, and effective inhibition of germination and formation of S. sclerotiorum sclerotia. These results indicate that compounds 3f-4 and 3f-28 are novel potential fungicidal candidates against S. sclerotiorum derived from natural products.

Full text of DOI:10.1021/acs.jafc.9b04224

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Agricultural and Environmental Chemistry
Design, Synthesis and Antifungal Evaluation of Novel
Quinoline Derivatives Inspired from Natural Quinine Alkaloids
Guan-zhou Yang, Jia-kai Zhu, xiaodan Yin, Yin-fang Yan, Yu-ling Wang, Xiao-
Fei Shang, Ying-Qian Liu, Zhong-min Zhao, Jing-wen Peng, and Hua Liu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04224 • Publication Date (Web): 18 Sep 2019
Downloaded from pubs.acs.org on September 19, 2019
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Design, Synthesis and Antifungal Evaluation of Novel
Quinoline Derivatives Inspired from Natural Quinine Alkaloids
Guan-Zhou Yang†, Jia-Kai Zhu†, Xiao-Dan Yin†, Yin-Fang Yan†, Yu-Ling Wang§,
Xiao-Fei Shang†, Ying-Qian Liu†*, Zhong-Min Zhao†, Jing-Wen Peng†, Hua Liu†
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†School of Pharmacy, Lanzhou University, Lanzhou 730000, People’s Republic of
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China
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§Gansu Academy of Agricultural Sciences, 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
Inspired by quinine and its analogues, two series of quinoline small molecular
compounds (a and 2a) and six series of quinoline derivatives (3a ~ 3f) were designed,
synthesized and evaluated for their antifungal activities. The results showed that
compounds 3e and 3f series exhibited significant fungicidal activities. Significantly,
compounds 3f-4 (EC50 = 0.41 μg/mL) and 3f-28 (EC50 = 0.55 μg/mL) displayed the
superior in vitro fungicidal activity and the potent in vivo curative effect against
Sclerotinia sclerotiorum. Preliminary mechanism studies showed that compounds
3f-4 and 3f-28 could cause changes in cell membrane permeability, accumulation of
reactive oxygen species, loss of mitochondrial membrane potential, and effectively
inhibition of germination and formation of S. sclerotiorum sclerotia. These results
indicate that compounds 3f-4 and 3f-28 are novel potential fungicidal candidates
against S. sclerotiorum derived from natural products.
Keywords: Quinine; simplified structure; antifungal activity; Sclerotinia sclerotiorum
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INTRODUCTION
Plant pathogenic fungi are one of the most damaging plant parasitic organisms,
and can cause serious diseases and adverse yield losses in crops.1 In addition, fungi
can produce various toxins that can seriously compromise food safety.2 Although
fungus resistant crop varieties and other biological methods have been employed to
solve the fungus problem, fungicidal chemicals remain the principal control tool3.
However, the use of a large number of fungicides has produced resistance to crop
fungi, making the efficacy of conventional fungicides decline.4 Therefore, it is
necessary to develop novel and effective fungicidal agents to control those
agricultural diseases.
Due to the huge diversity in chemical structures, natural products have been rich
sources, which are inspirations for a substantial fraction of bioactive optimization and
have played a significant role in drug discovery.5,6 Nonetheless, biologically active
natural product leads are often victims of several deficiencies such as paucity or
difficulty to obtain from natural sources, some natural product leads possess rather
large, complex, or labile chemical structures which are difficult to obtain
quantitatively by chemical synthesis, most are not optimally ideal for direct use
because these weak activities need to be improved by modifying and optimizing the
molecule comprehensively to pursue the structure–activity relationships.7 The
development and application of the natural product strobilurin A in agricultural
disease control is an extremely successful model, even forming a class of strobilurin
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fungicides which is the first in the market share of agricultural fungicides, including
azoxystrobin, pyraclostrobin, trifloxystrobin, picoxystrobin, fluoxastrobin and so on.8
Among them, azoxystrobin has become the world's best-selling strobilurin fungicides
because of its extensive antimicrobial spectrum, excellent control effect, safety of
crops and growth promotion of crops. Hence, it was used as a positive control drug in
this research.
Quinine, a well-known quinoline natural alkaloid, is isolated from the bark of
several species of Cinchona and Remijia trees,9 which has for decades been a stalwart
of antimalarial chemotherapy and chemoprophylaxis,10 and it is an excellent
representative of quinoline compounds and the origin of quinoline name.11 Quinoline
compounds12 possess a wide variety of pharmacological activities, such as antifungal
activity,13,14 antimalarial activity,15 anticancer activity,16,17,18 antitubercular
activity19,20 and antioxidants activity.21 However, there are few reports on the
antifungal activity of quinine and its optimized structures in agriculture.
In this study, in order to develop novel natural fungicides, quinine and its
analogues (quinidine, cinchonidine and cinchonine) were applied as lead compounds.
Quininone and quinotoxine were obtained by structural simplification and the above
six compounds were evaluated for their activities against eight plant pathogenic fungi
(Table 1). Furthermore, compound 2a-4 with potent antifungal activity were screened
out from a and 2a series compounds, and a total of 68 compounds from 3a, 3b, 3c,
3d, 3e and 3f series were designed, synthesized and evaluated their activities against
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Sclerotinia sclerotiorum, Rhizoctonia solani, Fusarium graminearum and
Magnaporthe oryzae. Among them, compounds 3a-3, 3e-1, 3f-1, 3f-4, 3f-14 and
3f-28, with relatively stronger antifungal activity, were evaluated for their in vivo
protective and curative effects against S. sclerotiorum. Furthermore, scanning electron
microscopy showed that compounds 3f-4 and 3f-28 affected the permeability of the
cell membrane of S. sclerotiorum, and the relative electric conductivity experiments
and fluorescent staining experiments were further verified. Moreover, compounds
3f-4 and 3f-28 effectively inhibited the sclerotia germination and affected the sclerotia
formation process of S. sclerotiorum.
MATERIALS AND METHODS
General. All reactions were performed with commercially available reagents
without further purification. All the compounds of quinine, quinidine, cinchonidine,
cinchonine and series
a
were purchased from commercial companies
(energy-chemical Co., Ltd.) (Table 1) and they were of analytical grade. All reactions
were monitored by thin-layer chromatography (TLC) and preparative thin-layer
chromatography (PTLC) were performed with silica gel plates using silica gel 60
GF254 (Qingdao Haiyang Chemical Co., Ltd.). Melting points were determined in an
open capillary using WRS-2U melting point apparatus (Shanghai Precision
Instrument Co., Ltd.) and are uncorrected. Mass spectra were recorded on a Bruker
Daltonics APEXII49e spectrometer (Bruker Daltonics Inc. U.S.A.) with ESI source as
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13
ionization. H and C NMR spectra were recorded at 400 and 100 MHz on a Bruker
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AM-400 (Bruker Company, U.S.A.) spectrometer using TMS as reference. The
commercial fungicides azoxystrobin and chlorothalonil (analytical grade, 98% purity)
(Jiangsu Bailing Agrochemical Co., Ltd., China) was used as a positive control in
vitro experiment.
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Plant Pathogenic Fungi. Phyllosticta zeae, Rhizoctonia solani, Botrytis cinerea,
Fusarium graminearum, Mycosphaerlla melonis, Magnaporthe oryzae, Fusarium
oxysporum f.sp .vesinfectum and Sclerotinia sclerotiorum were obtained from Institute
of Plant Protection, Gansu Academy of Agricultural Science. The strains were
retrieved from the storage tube and incubated in PDA at 25 °C for a week to get new
mycelia for the antifungal assay.
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Synthesis. The synthetic routes of the quininone, quinotoxine, 2a, 3a, 3b, 3c,
3d, 3e and 3f series compounds were outlined in Figure 2 and 3.
Synthetic procedure for the target compound quininone.22 In a dry 250 mL round
bottom flask, potassium tert-butoxide (0.25 mol), dry quinine (0.1 mol) and
benzophenone anhydrous (0.5 mol) were added, followed by dissolution with an
appropriate amount of anhydrous toluene. The reaction was refluxed for 15 to 18
hours under nitrogen atmosphere, and detected by thin layer chromatography (TLC)
using petroleum ether/ethyl acetate (3:1) as the mobile phase. When the reaction was
completed, it was poured into ice and extracted with 10% hydrochloric acid until it
was colorless. The aqueous phase was collected and then an excess of aqueous
ammonia was added dropwise. A precipitate formed in the solution, which was
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filtered and washed with water several times to give a pale yellow solid. The solid
was purified by chromatography on a silica gel using petroleum ether and ethyl
acetate (1:5) as the eluent to afford compound quininone.
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Synthetic procedure for the target compound quinotoxine.23 Quinine (0.01 mol)
was added to a 250 mL round bottom flask, then a mixture of glacial acetic acid and
water (1:13) was added (56 mL), and the reaction was refluxed at 100 ° C for 35
hours, and detected by TLC using dichloromethane/methanol (30:1). After the
reaction was completed, an excess of NaOH solution was added to the round bottom
flask, extracted with dichloromethane. The combined organic layer was washed with
saturated brine, dried with anhydrous sodium sulfate, and then concentrated under
reduced pressure. The residue was purified by silica gel using petroleum ether and
ethyl acetate (1:3) as the eluent column chromatography to give the target compound
quinotoxine.
General Synthetic procedure for the 2a compounds.24 To a mixture containing
the different aniline 1 (8.12 mmol) and anhydrous magnesium sulfate (MgSO4, 10
mmol) in ethanol (10 mL) was added acetic acid (6.3 mmol) and different substituted
ethyl acetoacetate 2 (16.24 mmol). The mixture was heated at 90 ℃ for 16 h.
Afterward, drying agent (MgSO4) was filter off and the residue was dried under
vacuum. For subsequent cyclocondensation reaction, 15 mL of Dowtherm A was
added and the formed solution was heated at 230-250 ℃ for 30 min. After cooling to
room temperature, the solid formed was filtered and washed with hexane and diethyl
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ether. Lastly, the product was dried under vacuum. The residue was purified by silica
gel as the eluent column chromatography to give the target compounds 2a.
General Synthetic procedure for the 3a and 3c compounds. Anhydrous
2,8-bis(trifluoromethyl)-4-quinolinol (1.0 mmol) was dissolved in an appropriate
amount of PCl3 (10 mL), and stirred at room temperature for 3 hours. After the
reaction was completed, the mixture was concentrated under reduced pressure. The
resulting mixture was added dichloromethane and ice water, the organic phase was
separated, and the aqueous phase was extracted by dichloromethane (2 ×20 mL). The
combined organic layer was washed with saturated brine, dried with anhydrous
sodium sulfate, and then filtered. The solvent was removed in vacuo, and the obtained
product did not require further purification to proceed directly to the next step.
To a solution of 2,8-bis(trifluoromethyl)-4-chloroquinoline (1.5 mmol) in 15 mL
ethanol, different amine (2.0 mmol) and trace amounts of Et3N was added dropwise at
room temperature. Then the reaction mixture was refluxed and stirred continuously
until the raw material disappeared (monitored by TLC). After the reaction was
completed, the reaction mixture was concentrated under reduced pressure. The
resulting mixture was added dichloromethane and water, the organic phase was
separated, and the aqueous phase was extracted by dichloromethane (2 ×20 mL). The
combined organic layer was washed with saturated brine, dried with anhydrous
sodium sulfate, and then filtered. The solvent was removed in vacuo, and the residue
was purified by silica gel column chromatography to give the target compounds
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3a-1~3a-8. The synthesis of compounds 3a-9~3a-14 were similar to above, except
that the starting compound was replaced by 3a-8. In addition, the synthesis of the 3c
series of compounds was also the same, except that the substituted amines were
different.
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General Synthetic procedure for the 3b compounds. To a solution of NaH (1.0
mmol)
in
anhydrous
THF
(15
mL)
was
added
2,8-bis(trifluoromethyl)-4-chloroquinoline (1.0 mmol) and the reaction mixture was
monitored by TLC. After the reaction is completed, ice water was added dropwise to
the mixture, and the resulting mixture was extracted by dichloromethane (3 ×20 mL).
The combined organic layer was washed with saturated brine, dried with anhydrous
sodium sulfate, and then filtered. The solvent was removed in vacuo, and the residue
was purified by silica gel column chromatography to give the target compounds
3b-1~3b-6.
General Synthetic procedure for the 3d compounds. To a solution of
2,8-bis(trifluoromethyl)-4-quinolinol (1.0 mmol) in absolute acetone (15 mL) was
added anhydrous K2CO3 (1.5 mmol) and different iodoalkane (1.0 mmol), and the
reaction mixture was heated to reflux for 12 h and the reaction mixture was monitored
by TLC. After removal of the solvent, the water was added and the mixture was
acidified with dilute hydrochloric acid. After filtration, the product was obtained by
recrystallization from ethanol. After the reaction was completed, the reaction mixture
was concentrated under reduced pressure. The resulting mixture was added
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dichloromethane and water, the organic phase was separated, and the aqueous phase
was extracted by dichloromethane (3×20 mL). The combined organic layer was
washed with saturated brine, dried with anhydrous sodium sulfate, and then filtered.
The solvent was removed in vacuo, and the residue was purified by silica gel column
chromatography to give the target compounds 3d-1~3d-7.
The synthesis of compounds 3d-8~3d-11 was the same as that of compounds
3a-1~3a-8, except that the starting compound was replaced by 3d-7.
General Synthetic procedure for the 3e and 3f compounds. To a solution of
2,8-bis(trifluoromethyl)quinolin-4-ol (1.0 mmol) and K2CO3 (1.5 mmol) in acetone
(15 mL), the substituted acid chloride (3.10 mmol) was added dropwise. The reaction
was monitored by TLC using petroleum ether /ethyl acetate (3:1) as the mobile phase.
After the reaction was completed, the mixture was filtered and evaporated. The target
compound was purified by chromatography on a silica gel column to afford
compounds 3e-1~3e-5 and 3f-1~3f-29.
All compounds physical data in Figure 2 and 3 are provided in the Supporting
Information.
Biological Assay. Quinine (QN), quinidine (QD), cinchonidine (CD), cinchonine
(CN), quininone (QO) and quinotoxine (QT) were screened for their in vitro
antifungal activities against eight fungi, including P. zeae, R. solani, B. cinerea, F.
graminearum, M. melonis, M. oryzae, F. oxysporum f.sp .vesinfectum and S.
sclerotiorum by the mycelium growth rate method.25 The antifungal activities of the
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target compounds a, 2a, 3a, 3b, 3c, 3d, 3e and 3f in detail were included in the
Supporting Information. The protective and curative effects of compounds 3a-3, 3e-1,
3f-1, 3f-4, 3f-14 and 3f-28 against S. sclerotiorum on leaves of rapeseed in detail were
also included in the Supporting Information.
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Mode of Action Studies. Morphological Observation by scanning electron
microscopy (SEM).26 Mycelia plugs were cut from the fungal colony treated with 0.5%
DMSO or the compounds 3f-4 and 3f-28 at 10 μg/mL, fixed in 2.5% glutaraldehyde at
4 °C overnight, and then post fixed for 2 h at 4 °C in 1% OsO4. Finally, the mycelial
were dehydrated in a graded ethanol series, critical point dried and gold coated. They
were observed using a scanning electron microscope (Hitachi, S-3400N, Japan) at an
accelerating voltage of 15.0 kV.
Permeability of fungal cell membrane.27 The fungal cell membrane permeability
of S. sclerotiorum was evaluated by measuring the relative electric conductivity of the
solution. S. sclerotiorum mycelia were placed in 80 mL of PD broth and shaken at
140 rpm for 3 days at 25 °C. After the mycelium grows completely, the mycelia (100
mg) were filtered and then added into a solution containing different concentrations of
target compounds solution. The conductivity was determined with a conductivity
detector (DDSJ-308A, INESA Scientific, China) within 24 h.
Effect on endogenous reactive oxygen species (ROS) production.26 S.
sclerotiorum mycelia tips (0.5 cm) were treated with 1.0 μg/mL of compounds 3f-4
and 3f-28 for 48 h and then placed on a sterile slide in a culture dish. After the
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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 PBS. A coverslip was placed on the hyphae, and the samples were
observed and photographed using LSM-800 with Airyscan (Carl Zeiss Microscopy
GmbH, German).
Effect on mitochondrial membrane potential (MMP).26 After staining with
rhodamine 123 solution (Beyotime, China), the mycelia were incubated at 37 °C for
30 min in darkness and washed with PBS. Finally, the samples were photographed
using an LSM-800 with Airyscan.
Effect on sclerotia germination inhibition.28 S. sclerotiorum were cultured for 21
days to obtain sclerotia. 10.0, 5.0, 2.5 and 1 μg/mL concentrations of compounds 3f-4,
3f-28 and azoxystrobin were prepared, then 15 sclerotia were placed on the culture
and each concentration consisted of three replicates. Azoxystrobin and distilled water
was served as the positive control and blank control, respectively. These dishes were
incubated at 25 ℃ for 3 days, then the inhibition rate of the compounds on the
germination of sclerotia was calculated.
Effect on sclerotia formation Inhibition.29 S. sclerotiorum mycelial block (0.5 cm)
were transferred the PDA plates containing different concentrations of target
compounds and cultured at 25 °C for 21 days (Figure 9). Azoxystrobin served as the
positive control. The sclerotia formed were collected and dried at 60 °C for 24 h. The
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number and weight of sclerotia were calculated.
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Statistical Analyses. Data were presented as means and standard deviations.
Differences among different treatments were analyzed using SAS version 24. Means
and standard deviations were obtained using the raw data, and the EC50 values were
obtained from the parameters in the regression curves.
RESULTS AND DISCUSSION
Chemistry. The synthesis of the compounds quininone, quinotoxine, 2a, 3a, 3b,
3c, 3d, 3e and 3f series were outlined in Figure 2 and Figure 3. They were obtained by
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13
the reactions a - j, and H NMR, C NMR and MS of the target compounds were
applied to confirm their structures.
Briefly, compounds quininone,22 quinotoxine23 and 2a series synthesized
according
to
previously
reported
procedure.24
Intermediate
2,8-bis(trifluoromethyl)-4-quinolinol (a-4) was reacted with PCl3 to obtain
2,8-bis(trifluoromethyl)-4-chloroquinoline, then it continued to react with different
amines in ethanol to give compounds 3a-1~3a-8. Compound 3a-8 then further was
reacted with corresponding amines in ethanol and catalyzed by triethylamine to obtain
target compounds 3a-9~3a-13. Compounds 3c series were synthesized by using the
same method as above described.
Intermediate 2,8-bis(trifluoromethyl)-4-chloroquinoline was reacted with
corresponding amines in THF and catalyzed by NaH to obtain 3b-1~3b-6. Similarly,
compound a-4 was used as the starting material to obtain compounds 3d, 3e, and 3f
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series with different iodoalkane and acyl chloride in acetone and catalyzed by
potassium carbonate. The synthetic routes are simple, efficient, economical,
inexpensive and have high yields, and can be used for mass production.
Biological Activity. In vitro antifungal activities of quinine and its analogues.
The antifungal activities of quinine and its analogues against eight plant pathogenic
fungi were shown in Tables 1. Quine, quinidine, cinchonidine and cinchonine showed
weak antifungal activities against eight plant pathogenic fungi, and the inhibition rate
ranged from 0 to 55.7% at 100 μg/mL. Interestingly, there was no significant
reduction in the antifungal activities of the simplified compounds quininone and
quinotoxine, most of which exceeded quinine. This indicates that the stereo
configuration of quinine and quinuclidine have the few effects on antifungal activity,
and it is feasible to verify the simplification of the structure of quinine.
In order to further improve the antifungal activity, we divided the quininexin into
two parts for structural modification and optimization. One (compounds a and 2a
series) is to comprehensively evaluate the activity of quinoline ring with different
substituents, and the other (compounds 3a, 3b, 3c, 3d, 3e and 3f series) is to modify
the 4-position of quinoline with different structural segments to explore its
structure-activity relationship.
In vitro antifungal activities of compounds a and 2a series. As shown in Table 2
and Table 3, the antifungal activities of compounds a and 2a series against S.
sclerotiorum, R. solani, F. graminearum and M. oryzae were demonstrated at 100 and
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50 μg/mL. Compounds a-1~a-32 are commercially available quinoline
pharmaceutical intermediate, among which compounds a-4, a-9, a-18 and a-25
showed strong antifungal activities against four fungi, and the most of inhibition rates
were more than 50% at 50 μg/mL. Compound a-4 showed the strongest antifungal
activities of compounds a-1~a-32, completely inhibiting the growth of hyphae at 50
μg/mL against S. sclerotiorum, R. solani and F. graminearum, and the inhibition rate
reached 96.08% at 50 μg/mL against M. oryzae, so it was further modified and
optimized as a privileged structure. Compound a-9 (8-hydroxyquinoline) also has
potent antifungal activities close to compound a-4, which has been a research hotspot.
8-hydroxyquinoline and its derivatives have a wide variety of biological effects, such
as neuroprotection,30 anticancer,31 anti-HIV,32 anti-Alzheimer's disease33 and
antifungal.34 It was even used as an agricultural fungicide, but is now off the market.
Compounds a-18 and a-25 also exhibited excellent antifungal activities, but no further
structural optimization was carried out due to the lack of modification sites. Other
compounds have low or no antifungal activities, so compounds 2a-1~2a-16 was
designed and synthesized with a-4 as the privileged structure.
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The antifungal activities of compounds 2a-1~2a-16 against S. sclerotiorum, R.
solani, F. graminearum and M. oryzae were listed in Table 3. The comparison of the
antifungal activities data revealed that the activity of compounds 2a-1~2a-16 with
different substituents was quite different. Among them, the compounds 2a-4, 2a-5,
2a-6, 2a-14 and 2a-15 showed significant antifungal activity against S. sclerotiorum,
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with inhibition rate of 85 to 100% at 50 μg/mL. It can be seen from the
structure-activity relationship analysis that the substituents at positions 2, 6, and 8
were a key factor affecting antifungal activity. The substitution of fluorine (2a-4 and
2a-14), bromine (2a-6), trifluoromethyl （ 2a-5 ） and trifluoromethoxy (2a-15) in
quinoline 6 or 8 can significantly improve the antifungal activity if 2-position
trifluoromethyl group was kept. This may be due to the strong electron-attracting
induction effect and lipophilic property of trifluoromethyl which introduces into drugs
can effectively prolong its role in vivo and enhance metabolic stability. Unfortunately,
antifungal activities of compounds 2a-1~2a-16 did not exceed compound a-4, so we
continued to use compound a-4 as a privileged structure for further optimization.
In vitro antifungal activities of compounds 3a, 3b, 3c, 3d, 3e and 3f series. The
antifungal activities of compounds 3a, 3b, 3c, 3d, 3e and 3f series against S.
sclerotiorum, R. solani, F. graminearum and M. oryzae were listed in Tables 4-6. As
shown in Tables, most compounds exhibited more or less antifungal activities at the
preliminary screening concentration. The antifungal activities of 3e and 3f series were
superior to 3a, 3b, 3c, and 3d series. This indicated that ester groups were favorable
for antifungal activities at the quinoline 4 position, while the amino and ether
substituents were confined or even reduced antifungal activities.
Table 4 shows the data for 3a, 3b, 3c and 3d series of compounds against S.
sclerotiorum, R. solani, F. graminearum and M. oryzae at 50 μg/mL, with a focus on
varying amino and ether substituents at the 4 position of the privileged structure a-4.
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Unfortunately, their antifungal activities were lower than that of the modified
compound a-4, and only compound 3a-3 showed general activity against S.
sclerotiorum with an inhibition rate of 90.00 %. Further, the privileged structure a-4
was modified by different ester groups.
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The antifungal activities of 3e and 3f series against S. sclerotiorum, R. solani, F.
graminearum and M. oryzae at 50 μg/mL were shown in Table 5. Surprisingly, the
compounds 3e and 3f series exhibit potent antifungal activities, especially the
antifungal activities of compounds 3f series against S. sclerotiorum and M. oryzae.
For a clearer comparison of the antifungal activities, the EC50 values of compounds
3f-1 to 3f-28 against S. sclerotiorum, R. solani, F. graminearum and M. oryzae was
evaluated and shown in Table 6.
Except for 3f-22, most of compounds 3f-1~3f-28 exhibited potent fungicidal
activity against S. sclerotiorum, with EC50 values ranging from 0.41 to 15.79 μg/mL.
Compounds 3f-4, 3f-9, 3f-11, 3f-14, 3f-18, 3f-23, 3f-24 and 3f-28 especially showed
significant fungicidal activities against S. sclerotiorum, with EC50 values of 0.41,
0.59, 0.74, 0.64, 0.75, 0.74, 0.84, 0.67 and 0.55 μg/mL, respectively, which were
20-fold more potent than azoxystrobin (EC50 = 16.96 μg/mL) and were slightly better
than chlorothalonil (EC50 = 1.78 μg/mL). Through structure-activity relationships, it
can be seen that changing ester substituents could lead to significant changes in
activity. Among all linear alkyl ester-substituted compounds (3f-1~3f-7, 3f-16 and
3f-17), butyl ester-substituted compound 3f-4 led a much better efficacy than others
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groups and exhibited the strongest antifungal activity. This indicated that the n-butyl
ester group was the optimal active-substituted fragment, and the shortening or
increase of the carbon chain or even the isomer cannot effectively improve the
antifungal activity against S. sclerotiorum. The aromatic ester-substituted compound
3f-8 (EC50 = 15.79 μg/mL) and 4-morpholinecarboxylic ester-substituted compound
3f-22 (EC50 > 50.00 μg/mL) showed the worst antifungal activity against S.
sclerotiorum. Among the halogenated alkane-substituted compounds (3f-9~3f-15),
chloroacetic esters (3f-9), 2-chloropropionic esters (3f-11) and 5-chlorovaleric esters
(3f-14) substituted compounds showed strong antifungal activity against S.
sclerotiorum. It can be seen that the substitution of a halogen atom for a chain alkyl
ester had a limited improvement and even reduces antifungal activity. In all
compounds containing cycloalkane or 6-membered heterocycloalkane substituents
(3f-18~3f-28), cyclopropanecarboxylic ester (3f-18), 2-(4-morpholinyl)acetic ester
(3f-23), 3-morpholin-4-yl-propionic ester (3f-24), 3-(diethylamino)propanoate ester
(3f-26) and 1-piperidinepropionic (3f-28) substituted compounds show outstanding
antifungal activity. Taken together, these results implied that the length of carbon
chain and electronegativity of substituents at the 4 position on the privileged structure
a-4 were crucial to enhance the antifungal activity against S. sclerotiorum.
Significantly, among the compounds 3f-1~3f-28, the most potent activities
against R. solani, F. graminearum and M. oryzae were compounds 3f-26 (EC50 =8.62
μg/mL), 3f-28 (EC50 = 10.48 μg/mL) and 3f-1 (EC50 = 3.80 μg/mL), respectively, and
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more potent than azoxystrobin (EC50>50, =34.43 and > 50 μg/mL), but weaker than
chlorothalonil (EC50=1.14, 0.98 and 3.16 μg/mL). This suggested that privileged
structure a-4 with different substituents were specific to different fungi, which further
led us to find compounds with highly selective inhibition of different fungi. Overall,
considering the discussion above, these findings further underlined that the fungicidal
differences of quinoline compounds modified at four position could be ascribed to a
combination of factors, such as the nature of the substituents, which may depend upon
the length, size, electronic characteristics of substituents, differences in target of
different fungi or other factors.
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In vivo protective and curative effects against S. sclerotiorum. Compounds 3a-3,
3e-1, 3f-1, 3f-4, 3f-14 and 3f-28 displayed superior fungicidal activity against S.
sclerotiorum in different series of compounds and, therefore, were selected to further
evaluate their in vivo protective and curative effects against S. sclerotiorum using the
leaves of rapeseed. As illustrated in Table 7 and Figure 4, most of the tested
compounds displayed strong in vivo curative effect against S. sclerotiorum at 80 and
40 μg/mL, and curative effect were more potent than protective effect. For example,
Compounds 3f-4 and 3f-28 showed outstanding curative effect, and control efficacy
reached 52.15 and 61.50 % at 20 μg/mL, respectively, but still slightly worse than
azoxystrobin (62.51 %). It is worth further exploration that the in vitro antifungal
activity of 3f-4 and 3f-28 is stronger than that of azoxystrobin, but the protective and
curative effects are slightly worse, which makes us more interested in studying their
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mechanism of action. However, it is undoubted that 3f-4 and 3f-28 have positive and
potential value for the control of S. sclerotiorum.
Preliminary mode of action of compounds 3f-4 and 3f-28 against S.
sclerotiorum. Effect of compounds 3f-4 and 3f-28 on the hyphae morphology. The
morphological changes of S. sclerotiorum hyphae were observed under SEM. As
shown in Figure 5, after treatment with 1 μg/mL of 3f-4 and 3f-28 (B and C), the
mycelia of S. sclerotiorum were collapsed, shriveled and shrunken in a
dose-dependent manner relative to the regular, smooth and homogeneous hyphae of
the control group (A). It is speculated that 3f-4 and 3f-28 may affect the permeability
of the cell membrane, resulting in extracellular leakage of the cytoplasm and collapse
and shrinkage of mycelia. Therefore, further verification was carried out.
Effect of compounds 3f-4 and 3f-28 on the Cell Membrane Permeability. In order
to determine whether compounds 3f-4 and 3f-28 acted on the membrane of S.
sclerotiorum, the conductivity changes of hyphae exposed to compounds 3f-4 and
3f-28 were evaluated, which could reflect the changes in the cell membrane
permeability. From Figure 6, after treatment with compounds 3f-4 and 3f-28 (10 and
5 µg/mL), the relative electric conductivities (%) of the mycelia were rapidly raised
within 12 h. From 12 to 24 h, the electrical conductivity of each group increased
slightly. These results indicated that compounds 3f-4 and 3f-28 could damage the cell
membrane of hyphal in a dose-dependent manner, resulting in electrolyte leakage,
thereby increasing conductivity.
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Effect of compounds 3f-4 and 3f-28 on the ROS and MMP production. A
previous study showed that the production and accumulation of ROS led to a decrease
in MMP , an increase in cell membrane permeability and inhibition of fungal spore
germination,35 even resulting to cell death.25 The effect of compounds 3f-4 and 3f-28
on ROS production was studied by DCFH-DA as ROS index (Figure 7). The results
showed that compounds 3f-4 and 3f-28 (B and C) treated hyphae had obvious
fluorescence effect compared with untreated hyphae. The effect of compounds 3f-4
and 3f-28 on MMP was subsequently evaluated using Rhodamine 123. The results
showed that the fluorescence intensity of the compounds 3f-4 and 3f-28 treated
hyphae (E, F) were significantly lower than that of the untreated hyphae (D),
indicating that compounds 3f-4 and 3f-28 could damage the MMP, leading to
mitochondrial dysfunction in S. sclerotiorum mycelia, thereby accelerating cell death.
Sclerotia germination and formation inhibition of compounds 3f-4 and 3f-28.
Sclerotia is the principal survival structure of S. sclerotiorum, playing a major role in
the life cycle of pathogens. The presence of sclerotia makes it difficult to control S.
sclerotiorum mainly due to its ability to survive for a long time under extreme
conditions and the huge transmission ability of ascospores produced by the
germination of sclerotia.36 Therefore, inhibiting the germination and production of
sclerotia is the key to controlling S. sclerotiorum.37 As shown in Figure 8,
Compounds 3f-4 and 3f-28 showed inhibitory activity on the sclerotial germination of
S. sclerotiorum at 10 (100 %, 80 %), 5 (95.56 %, 48.89 %), and 2.5 μg/mL(82.22 %,
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28.89 %) and the inhibitory activity of azoxystrobin (10 ug/mL, 15.56 %) was poor.
This indicated that compounds 3f-4 and 3f-28 had positive value for the transmission
of S. sclerotiorum. The effect of compounds 3f-4, 3f-28 and azoxystrobin on the
sclerotia formation of S. sclerotiorum, was showed Figure 9, and in order to more
accurately describe the experimental results, the number and weight of the formed
sclerotia were counted as shown in Figure 10 and Figure 11. Surprisingly, as the
concentration of compounds 3f-4 and 3f-28 increased, the number of formed sclerotia
continued to decrease, but the weight of sclerotia increased. By carefully observing
the formed sclerotia, it was found that this was caused by the enlargement of the
volume and mass of a single sclerotium, and the internal morphology of the sclerotia
also changed significantly. Overall, these results demonstrate that compounds 3f-4
and 3f-28 can inhibit the germination of sclerotia of S. sclerotiorum and affect the
formation process of sclerotia, thereby reducing the production and spread of the
primary infection sources and controlling the disease.
In summary, by structural simplification of quinine and its analogs and screening
of privileged structures, a series of quinoline small molecule compounds and six
series of sixty-eight quinoline molecules were designed, synthesized and evaluated for
the antifungal activities. The bioassay results showed that compounds 3e and 3f series
showed obvious fungicidal activities in vitro against S. sclerotiorum and M. oryzae,
especially compounds 3f-4 and 3f-28 showed excellent in vivo curative effect against
S. sclerotiorum in rapeseed. Further studies on the mechanism of action of compounds
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3f-4 and 3f-28 found that they could cause changes in cell membrane permeability,
production and accumulation of reactive oxygen species and effect on mitochondrial
membrane potential, and effectively inhibit the germination and formation process of
sclerotia of S. sclerotiorum, thereby effectively control the development of S.
sclerotiorum and be helpful for managing fungal resistance. Further studies on
structural optimization and a detailed mechanism for quinine and its optimized
structures 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:
Corresponding Author
*Phone: +86-898-66969260; fax: +86-898-66969211. Ying-qian Liu, e-mail:
yqliu@lzu.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
This work was supported financially by the National Natural Science Foundation of
China (31371975, 21672092), and the National Key Research and Development
Program of China (2017YFD0201404). Support was also supplied by the
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Fundamental Research Funds for the Central Universities (lzujbky-2016-147,
lzujbky-2017-k23).
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