Antifungal Application of Rosin Derivatives from Renewable Pine Resin in Crop Protection

Article abstract of DOI:10.1021/acs.jafc.0c00562

In the current work, we synthesized two series of dehydroabietyl amide derivatives from natural product rosin and evaluated their antifungal effects on Valsa mali, Phytophthora capsici, Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium oxysporum. In vitro and in vivo antifungal activities results indicated that rosin-based amide compounds containing thiophene heterocycles had better inhibitory effects on B. cinerea. In particular, compound 5b (5-fluoro-2-thiophene dehydroabietyl amide) exhibited the excellent antifungal properties against B. cinerea with an EC₅₀ of 0.490 mg/L, which was lower compared to the positive control penthiopyrad (0.562 mg/L). Physiological and biochemical studies showed that the primary action mechanism of compound 5b on B. cinerea changes mycelial morphology, increases cell membrane permeability, and inhibits the TCA pathway in respiratory metabolism. Furthermore, QSAR and SAR studies revealed that charge distribution of rosin-based amides derivatives have a key role in the antifungal activity through the hydrogen bonding, conjugation, and electrostatic interaction between the compounds and the receptors of the target. To sum up, this study contributes to the development of rosin-based antifungal agents with a novel structure and preferable biological activity.

Full text of DOI:10.1021/acs.jafc.0c00562

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Agricultural and Environmental Chemistry
Antifungal Application of Rosin Derivatives
from Renewable Pine Resin in Crop Protection
Pan Tao, Chengyu Wu, Jin Hao, Yanqing Gao, Xiaohua
He, Jian Li, Shibin Shang, Zhanqian Song, and Jie Song
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c00562 • Publication Date (Web): 19 Mar 2020
Downloaded from pubs.acs.org on March 20, 2020
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Antifungal Application of Rosin Derivatives from Renewable Pine Resin in Crop
Protection
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Pan Tao,† Chengyu Wu,† Jin Hao,‡ Yanqing Gao,‡ Xiaohua He,‡ Jian Li,*,† Shibin
Shang,§ Zhanqian Song,§ and Jie Song*,||
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†College of Forestry, Northwest A&F University, Yangling, Shaanxi 712100,
People’s Republic of China
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‡College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100,
People’s Republic of China
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§Institute of Chemical Industry of Forest Products, Chinese Academy of Forestry,
Nanjing, Jiangsu 210042, People’s Republic of China
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||Department of Chemistry and Biochemistry, University of Michigan-Flint, Flint, MI
48502, United States
Corresponding Authors:
*E-mail: ericlee99@nwsuaf.edu.cn. Telephone: +86-29-87082392 (Jian Li).
*E-mail: jiesong@umich.edu. Telephone: (810) 762-3275 (Jie Song).
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ABSTRACT ： In the current work, we synthesized two series of dehydroabietyl
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amide derivatives from natural product rosin and evaluated their antifungal effects on
Valsa mali, Phytophthora capsici, Botrytis cinerea, Sclerotinia sclerotiorum and
Fusarium oxysporum. In vitro and in vivo antifungal activities results indicated that
rosin-based amide compounds containing thiophene heterocycles had better inhibitory
effects on B. cinerea. In particular, compound 5b (5-fluoro-2-thiophene
dehydroabietyl amide) exhibited the excellent antifungal properties against B. cinerea
with EC50 of 0.490 mg/L, which was lower compared to the positive control
penthiopyrad (0.562 mg/L). Physiological and biochemical studies showed that the
primary action mechanism of compound 5b on B. cinerea changes mycelial
morphology, increases cell membrane permeability and inhibits TCA pathway in
respiratory metabolism. Furthermore, QSAR and SAR studies revealed that charge
distribution of rosin-based amides derivatives have a key role in the antifungal
activity through the hydrogen bonding, conjugation and electrostatic interaction
between the compounds and the receptors of target. To sum up, this study contributes
to the development of rosin-based antifungal agents with novel structure and
preferable biological activity.
KEYWORDS: rosin, amide, thiophene heterocycle, antifungal activity, action
mechanism, QSAR
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INTRODUCTION
Pesticides have an important role in the agricultural production, especially in
reducing labor intensity, avoiding pests and diseases, cultivating new crop varieties,
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and similar . Moreover, their use brings enormous economic and social benefits for
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human development . Fungicides, which are among the most important types of
pesticides, can control plant diseases caused by various pathogenic microorganisms
by killing or inhibiting the growth of pathogens 3. In fact, the usage of fungicides has
become one of the most economical and effective ways to reduce the huge losses in
agriculture caused by plant diseases 4.
Over recent years, the new fungicides have become the research hot spot,
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resulting in development of fungicides with novel mechanisms . Among them,
succinate dehydrogenase inhibitors (SDHIs) are the most innovative ones because of
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their high efficiency, broad spectrum and novel mechanism . As important amide
fungicides, SDHIs can inhibit the growth of pathogens, eventually killing them
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through their respiratory chain electron transport systems . Nevertheless, the
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widespread use of fungicides has produced more and more resistance problems
.
Meanwhile, their residual toxicity and negative impact on the environment still
haven’t been resolved effectively 10. The research and development of new fungicides
with natural products as the lead compounds may be an effective way to overcome
these problems 11.
There are many kinds of natural products, and they have various biological
activities, unique action mechanism, easy degradation and good environmental
compatibility, which are important alternatives for development of green fungicides
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12. At present, natural products not only provide a large number of novel lead
compounds for the development of fungicides, but also probes that can lead to
discovery of new targets 13. Especially, the active ingredients derived from plants
(secondary metabolism, are synthesized in the process of plant co-evolution to resist
the invasion of outer pathogens) have become the significant part of developing green
bio pesticides 14.
Rosin is a kind of natural resin that is secreted from pine trees. Rosin and its
derivatives have important research and development values as pesticides, herbicides
and bacteriostats in the field of agrochemicals. However, few studies have
investigated its application in antifungal activities 15. Specially, the specific action
mode and regulation response mechanism of ternary phenanthrene ring structure and
chiral centers of rosin and/or its derivatives on fungi target site have not received
adequate attention 16, 17
.
Based on our previous research results, in the present study we designed and
synthesized series of rosin-based amide antifungal agents. Inspired by penthiopyrad
(one of the typical SDHIs), and with the aim to further improve their antifungal
effects, thiophene heterocycles were introduced into one of series of rosin-based
amide derivatives. In order to explore the preliminary mechanisms of action of
rosin-based amide derivatives, the morphological changes of mycelia cell wall and the
antifungal effects on the permeability of mycelia cell membrane and respiratory
metabolism of fungi were examined. Meanwhile, the relationship between the
molecular physicochemical characteristics of rosin-based amide compounds and
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antifungal activity were investigated by SAR and QSAR analysis. The obtained
results provide a new insight for the development of sustainable pine-based fungicide
in crop protection.
MATERIALS AND METHODS
Materials and Structural Characterization. All chemical reagents and solvents
were purchased from commercial company. The melting point was determined by
Hannon MP-450 melting point apparatus (Hanon Subsidiary Company, Jinan, China).
Infrared spectroscopy (IR) analyses of the dehydroabietyl amide derivatives were
measured on a Nicolet IS10 infrared spectrophotometer (Thermo Nicolet Co.,
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Madison, USA). H NMR spectra were recorded on a Bruker AVANCE-III (500
MHz) spectrometer and ESI mass spectra were detected on a Bruker Q-TOF mass
spectrometer (Bruker Co., Karlsruhe, Germany). Elemental analyses were determined
on a Vario EL-III (Elementar Co., Hanau, Germany). Potato dextrose agar (PDA)
culture medium was prepared with potatoes, agar, glucose and distilled water. The
preparation method of potato glucose broth (PDB) culture medium is similar to that of
PDA, except agar was added.
Synthesis of Dehydroabietyl Amide Compounds (3a-p). Dehydroabietic acid
(1, DHAA) and dehydroabietyl chloride (2) were prepared according to previously
reported methods 15. Dehydroabietic amide compounds (3a-p) were obtained by
compound 2 and aromatic amines or ammonium hydroxide by nucleophilic reaction.
In short, 10 mmol amine, 40 mL dichloromethane (DCM) and 2 mL triethylamine
(Et3N) were added to 250 mL flask and evenly stirred. Compound 2 (3.19 g; 10
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mmol) was added to the above solution and completed in 30 minutes. After 12 h of
reaction, the mixture solution was washed to neutral in turn with acid and water.
Finally, the crude product was obtained by drying with anhydrous magnesium sulfate
and removing the solvent under vacuum. The title compounds (3a-o) were purified by
recrystallization with ethanol. Compound 3p was prepared with the similar method as
above-mentioned, except that in compound 2 the 10 mL ammonia water (80%) was
added to 40 mL tetrahydrofuran (THF) at low temperature (0 °C). The synthetic
routes of compounds 3a-p are shown in Scheme 1.
Synthesis of Dehydroabietyl Amides Containing the Thiophene Heterocyclic
Group. The preparation of series of compounds (5a-n) was similar to that of 3a-p.
Briefly, 2-thenoylchloride and 5-substituted analogs were prepared by reaction of
thionyl chloride (1.79 g, 15 mmol) on the corresponding thiophene-2 carboxylic acids
(10 mmol) in DCM under reflux. Then, above acyl chloride was added to compound 4
(prepared by reduction of 3n with lithium aluminium hydride) with the same molar
amount at room temperature. After 12 h reaction, the target compounds 5a-n were
obtained by acid washing, water washing and recrystallization with ethanol. The
synthetic routes of the compounds (5a-n) are shown in Scheme 2.
In Vitro Antifungal Activity. According to the literature, the growth rate method
was used to determine the in vitro antifungal activity of synthesized compounds 18. A
total of 50 mg of tested compounds (including positive control, penthiopyrad) were
added to 5 mL DMSO to prepare initial solution with a concentration of 10 mg/mL.
Then, the above solution was diluted to 500.00, 200.00, 100.00, 50.00, 25.00, 12.50,
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6.25, 3.13, 1.56, and 0.78 mg/L by PDA medium. Equal amount of DMSO without
tested compounds was used as negative control. Activated pathogens were punched
with a 5 mm diameter puncher, and were inoculated in the center of the medicated
medium in a 25 °C incubator. Each experiment was run in triplicate and the statistical
results were averaged. The antifungal activity results were expressed by the inhibition
rate of mycelium growth (IR) in Eq. (1). Linear regression equation was established
by SPSS statistics software, and correlation analysis was carried out to determine the
inhibitory activity of the dehydroabietyl amide derivatives against different
pathogens.
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IR (%) = (B-T)/ B × 100
(1)
where B and T are colony growth diameter of blank control and treated compounds,
respectively.
In Vivo Antifungal Activity. The protective efficacy of test compounds on
strawberry leaves inoculated with B. cinerea was determined by in vivo tissue method
19. The test compounds were dissolved in DMSO, distilled water and Triton-100
(0.2%) and prepared into three concentrations of 100, 50 and 25 mg/L, respectively.
The negative control was DMSO solution containing 0.2% Triton-100 and the
positive control was penthiopyrad. First, the strawberry leaves similar in size and
growth status and without being infected by any of other pathogens were selected and
washed with clean water. Then, the fresh strawberry leaves were evenly sprayed with
test solution and naturally dried. In the tray, the strawberry leaves were inoculated
with B. cinerea and small holes on the tested leaves were pricked with inoculation
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needles. The inoculated strawberry leaves were cultured in the incubator (25 °C and
80% relative humidity) for 48-72 h. The plaque diameters of B. cinerea on strawberry
leaves were measured by cross-over method. Each experiment was run in triplicate.
The protective efficacy (PE) was used to assess the antifungal effect on B. cinerea in
Eq. (2):
PE (%) = (C - T)/ (C-5) × 100
(2)
where C and T are colony expansion sums of control and treated strawberry leaves,
respectively.
Effect of dehydroabietyl amide (5b) on mycelial morphology of B. cinerea. B.
cinerea was cultured in a 90 mm culture dish. When the mycelia grew to 70 mm in
diameter, fresh fungus dishes (5 mm in diameter) were made from the edge of the
colonies. The mycelia were inoculated on PDA medium plates containing no
compound (negative control) and amide 5b with a concentration of 20 mg/L,
respectively. Then, the mycelia were cultured at 25 °C for 2 days. The mycelia cell
wall structure of B. cinerea at the top of each treated colonies were selected and
observed under an S-3400N scanning electron microscope (SEM) (Hitachi, Ltd.,
Tokyo, Japan).
Effect of dehydroabietyl amide (5b) on cell membrane permeability of B.
cinerea. The tested strains were cultured on PDA plate at 25 °C for 3 days and made
into a 7 mm diameter disc. Then, the strains were placed in PDB culture medium and
cultured by shaking (25 °C, 120 rpm) for 3 days. The mycelia were filtered and
washed with distilled water and put into centrifugal tube, and then 10 mL of
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compound 5b (25 mg/L) and distilled water were added into the centrifugal tube,
respectively. Finally, the mycelia were oscillated (120 rpm) in water bath at constant
temperature of 28 °C at different times. The conductivity was measured by a CON510
Eutech/Oakton conductometer (OAKTON Instruments, Waltham, USA). The
negative control was mycelia with distilled water. The conductivity of the compound
5b was determined at 0, 30, 60, 90, 120, 180, 240, 300, 360, 420, 480, 540 min, and
finally boiled (dead treatment) to determine the conductivity. The relative
permeability was calculated for each measurement, and then the permeability of cell
membranes was compared according to the conductivity. Each experiment was run in
triplicate.
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Effect of dehydroabietyl amide (5b) on the respiratory metabolism of B.
cinerea. The respiratory oxygen consumption rate of B. cinerea was measured by
oxygen electrode method 20. A total of 50 mg fresh mycelia were added into 1 mL of
medium solution (0.9 mL of phosphate buffer solution with concentration of 0.1
mol/L, pH = 7.2 and 0.1 mL of glucose solution with concentration of 2%). After
stirring for 5 mins, dissolved oxygen was measured by a JPB-607A dissolved oxygen
meter (INESA Scientific Instrument Co., Shanghai, China). Then, three typical
respiratory inhibitors, namely, malonic acid, iodoacetic acid and sodium phosphate
(inhibiting tricarboxylic acid cycle (TCA) pathway, Embden-Meyerhof-Parnas (EMP)
pathway and hexose monophophate pathway (HMP), respectively) and/or test agents
(25 mg/L) were added. Respiration rate of mycelia (O2, μmol/g·min) was calculated
from the change of oxygen content in medium. The inhibition rate of mycelia
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respiration (IRr) was calculated according to the respiration rate of mycelia before and
after adding test agents in Eq. (3):
IRr (%) = (R0-R1)/ R0 × 100
(3)
where R0 and R1 are respiration rate of mycelia before and after adding tested agents.
According to the respiration rate of mycelia before and after the addition of
typical inhibitors, the superposition rate of tested agents with typical inhibitors (RR)
was calculated in Eq. (4):
RR (%) = (R1-R1')/ R1 × 100
(4)
where R1 is respiration rate of mycelia after adding test agents and R1' is the
respiratory rate of mycelia after adding test agents and typical inhibitors.
QSAR. The density functional theory (DFT) method of Gaussian 16 (Gaussian,
Inc., Wallingford, USA) was used to optimize the molecular structures and calculate
the minimum energy of synthesized compounds 21. Some important molecular
structure properties were obtained, such as molecular energy, dipole, atomic charge
distribution, molecular orbitals and orbital energies, electron density and electrostatic
potential. Then, the molecular structures and antifungal activities were linearly
regressed by CODESSA software 2.7.15 (Semichem, Inc., Shawnee, USA), and the
quantitative relationship models were constructed. Finally, the models were validated
and the antifungal activities were predicted by the built best model. The lowest
unoccupied molecular orbital (LUMO) of compound 5b was viewed, plotted and
visualized by MO editor in GaussView 6.0. The charge distribution of compound 5b
was obtained by viewing the optimized compound molecule result. The molecular
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electrostatic potential (MEP) plot and contour map of compound 5b were generated
by surface and contour action in Surface/Contour of GaussView 6.0, respectively.
RESULTS AND DISCUSSION
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The preparation of dehydroabietyl amide derivatives. The reactivity of
carboxyl or amino groups on compound 1 (DHAA) or 4 (dehydroabietic amine) are
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low because of the steric hindrance of tricyclic phenanthrene skeleton of rosin
.
Therefore, the preparation of rosin-based amides requires more harsh conditions, such
as high temperature, high pressure and the addition of catalysts. However, at higher
temperatures, rosin is prone to decarboxylation or oxidation, resulting in darker color
and more by-products 24. Nonetheless the reactivity can be greatly improved by
converting carboxylic acid into active intermediates acyl chloride. In the present
study, we used thionyl chloride as the acylation reagent so as to easily obtain acyl
chloride. Because the by-product is gas, the crude product can be easily purified,
obtaining the yield of acyl chloride as high as 95-98%. Moreover, in the process of
reaction, amides were formed and hydrochloric acid (HCl) was produced. We used
triethylamine (Et3N) as acid binding agent to react with HCl and to form
corresponding salt precipitation, thus promoting the reaction with higher yield and
less by-products. All reaction processes were monitored by thin layer chromatography
(TLC).
In vitro antifungal activity against B. cinerea and structure-activity
relationship. The antifungal results showed that two series of rosin-based amide
derivatives had certain inhibitory effect on fungi V. mali, P. capsici, B. cinerea, S.
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sclerotiorum and F. oxysporum. However, most of the synthesized compounds
showed lower antifungal activity against V. mali, P. capsici, S. sclerotiorum and F.
oxysporum, but showed better inhibitory effect on B. cinerea (Figure 1a).
According to the statistical results of the structure and activity of the synthesized
compounds, the inhibitory effect on B. cinerea of the rosin-based amide derivatives
containing thiophene heterocyclic structure (5a-n) was better than that of the amide
derivatives (3a-p) without heterocyclic structure (Table 1). The EC50 value of amide
derivatives containing thiophene heterocyclic structure (5a-n) ranged from 0.490 to
78.091 mg/L, while the EC50 value of compounds without heterocyclic structure
(3a-p) ranged were 98.425-188.246 mg/L. Particularly, the activity of compounds
5b-g was generally better, and the inhibitory effect on B. cinerea was comparable to
that of the positive control compound (penthiopyrad). At a concentration of 12.50
mg/L, the inhibition rates of B. cinerea were higher than 90%. Even at 3.13 mg/L,
their inhibition rates were higher than 50%. Among them, compound 5b had the best
antifungal effect on B. cinerea. Its EC50 value was 0.490 mg/L, which was lower than
the EC50 value of penthiopyrad (0.562 mg/L). Combined with the previous reports, we
speculated that this result may be due to the introduction of thiophene heterocyclic
structure, which changes the electron cloud density of rosin-based amide compounds
at the molecular level 25, 26. These results led to the enhanced membrane permeability
of compounds or hydrophobic interaction with specific sites of target protein.
Furthermore, the absorption capacity of drug molecules and interaction by receptors
were improved at the physiological level 27, 28
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In addition, there was certain regularity between the structure of rosin-based
amide derivatives and the inhibitory activity on B. cinerea. Among the compounds
3a-p, antifungal agents with electron-withdrawing groups on the benzene ring had
higher inhibitory effect on B. cinerea than compounds with electron-donating groups
(3k-o > 3b-j). Especially, the compounds with halogen structures on benzene ring had
the highest activity, while as 5a-n, compounds with aromatic ring structure had the
lower activity. According to the existing literature, we speculated that rosin-based
amide derivatives would have larger molecular space structure due to the introduction
of aromatic ring, which may result in the weakening of penetration ability of drug
molecules to membranes or tissues and the absorption and transmission of drugs in
fungi 29, 30. All of these factors can block the binding of the drugs to the receptors, and
in turn affect the antifungal effect of compounds 31.
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In vivo antifungal activity against B. cinerea. The in vivo antifungal activity
against B. cinerea of compound 5b showed good effects comparable to penthiopyrad.
The PE values were 90.00%, 78.50%, and 62.00% at a concentration of 100, 50 and
25 mg/L, respectively. The compound 5b possessed good protective efficacy against
B. cinerea as shown in Figure 1c.
Effect of dehydroabietyl amide (5b) on mycelial morphology of B. cinerea.
After treatment with compound 5b at 20 mg/L, the surface of mycelia cell wall
showed a certain degree of damage (Figure 2). The surface was rough and uneven
and a few small holes appeared. This showed that rosin-based amide derivatives had a
certain degree of damage to the cell wall of B. cinerea.
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Effect of dehydroabietyl amide (5b) on cell membrane permeability of B.
cinerea. The change of relative conductivity reflects the variation of cell membrane
permeability. After treatement with the antifungal agents, the mycelial relative
conductivity of B. cinerea increased with time, but the relative conductivity of the
control did not change much at different time, indicating that compound 5b could
enhance the mycelial cell membrane permeability of B. cinerea. Yet, the change of
relative conductivity caused by compound 5b was much larger compared to control
(Figure 1b). According to previous reports, the cell membrane of fungi are the key
action sites of the drugs, which can destroy the synthesis of phospholipid bilayer or
protein and lead to the leakage of internal electrolytes 32, 33. Combined with the
change of mycelial morphology after treatment with 5b, it can also be inferred that
compound 5b could manifest antifungal properties by destroying cell membrane
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structure . The above reported findings revealed that the action target site of 5b on
B. cinerea could be cell membrane.
Effect of dehydroabietyl amide (5b) on the respiratory metabolism of B.
cinerea. The respiratory inhibition effect of compound 5b and typical inhibitors on B.
cinerea mycelia are shown in Table 2. Compound 5b had a strong inhibitory effect on
the respiration rate of B.cinerea mycelia, and the inhibition rate was 48.59% when the
concentration was 25 mg/L. In addition, by comparing the superposition rate of
typical respiratory inhibitors and tested agents, the main inhibition pathway of tested
agents on respiratory metabolism of B. cinerea can be inferred. The smaller
superposition rate showed that there was no synergistic effect between them, which
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indicated that they had the same inhibiting pathway. The superposition rates of
compound 5b with iodoacetic acid and sodium phosphate were 44.38% and 55.63%,
respectively, which indicated that compound 5b had some inhibitory effects on EMP
and HMP pathways, but the superposition rates of compound 5b with malonic acid
were relatively lower (34.69%), indicating that 5b had more obvious inhibitory effects
on TCA pathway. It has been reported that TCA pathway is related to electron
transport 35, 36. Therefore, compound 5b may inhibit the electron transport by binding
to Qo site of Cyt bc complex, thus inhibiting the respiratory function of B. cinerea
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and achieving the antifungal effect 37, 38
.
QSAR. The quantitative structure-activity relationship of rosin-based amide
derivatives (3a-p and 5a-n) and antifungal activity against B. cinerea were studied by
QSAR. The molecular descriptors of rosin-based amide derivatives were screened by
heuristic regression method in CODESSSA, which revealed that they were
significantly related to the antifungal activity (Table S1). By using these molecular
descriptors as independent variables and antifungal activity as dependent variables, a
series of linear QSAR models with the different number of descriptors were
constructed. In order to reduce the "over-parameterization" problem and elevate the
prediction ability of the model, the "breaking point" rule was used to select the best
number of parameters. The best QSAR model with four descriptors was obtained
through linear regression analysis (Figure 3a). The specific results of the model are
shown in Table 3 (R2 = 0.9587, F = 144.94, s2 = 0.0352). The obtained best QSAR
15
model was validated by triple internal and “leave-one-out” test . The training and
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test results are listed in Table S2 and the average value of training set Rtraining was
2
0.9568 and the testing set Rtest was 0.9541. By comparing the values from the three
models, it could be seen that the difference between them was less than 5%. These
results showed that the best QSAR model had good stability and prediction ability. In
addition, the RLOO2 = 0.9496 (obtained from the “leave-one-out” test) was similar to
the above values, which also confirmed the stability of the QSAR model. The
antifungal activity against B. cinerea of 30 rosin-based amide compounds involved in
the constructed model was predicted. The predicted antifungal activity values and the
difference between the experimental and the predicted values are listed in Table S3.
Figure 3b shows a comparison between the experimental and predicted values of
rosin-based amide derivatives.
According to the best QSAR model, there were four descriptors, namely
maximum net charge (qmax), maximum net atomic charge for an N atom (qNmax), the
LUMO and dipole moment of the molecule (DM), closely related to the antifungal
activity. These descriptors contain abundant information about the relationship
between rosin-based amide compounds structure and their fungicidal activity, which
can provide guidance for designing novel rosin derivatives. qmax and qN
are
max
descriptors related to the charge distribution of compounds, in which N and/or S atom
can act as receptors for hydrogen bonding interaction and form abundant
39
intermolecular hydrogen bonds in fungi (Figure 3c) . At the same time, the charge
distribution is also closely related to the electrostatic interaction between molecules 40,
41. In addition, there are conjugated systems in amide structure of rosin-based
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derivatives (3a-p and 5a-n). Among compounds 5a-n, thiophene, an important
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sulfur-containing five-membered heterocyclic compound, has a high charge density
25
and is more prone to π-π conjugation in fungi . Therefore, the antifungal effects of
rosin-based amides mainly occurred through the hydrogen bonding, conjugation and
electrostatic interaction between the compounds and the receptors of target. In vitro
antifungal activity study showed that the rosin-based compounds containing
thiophene heterocycles exhibit good inhibitory activity against B. cinerea, which was
well corroborated with the results of the model. LUMO is an important indicator of
42
electronic acceptance of compounds . In the process of antifungal action, thiophene
ring accepts electrons and forms complexes, which is beneficial to increase the sites
where derivatives can participate in hydrogen bonding interaction, thus promoting the
interaction between compounds and targets and achieving the antifungal effect
(Figure 3d) 43. DM, similar to the first and second descriptor of the model, reflects the
polar interaction between molecules 44. It is also a descriptor related to the charge
distribution of compounds, which can lead to changes in hydrophobicity or
lipophilicity of molecules, thereby enhancing the hydrogen bonding between
compounds and target sites, and thus improving the antifungal effect 45, 46
.
We synthesized two series of dehydroabietyl amide derivatives from rosin and
then examined their inhibitory effects on fungi V. mali, P. capsici, B. cinerea, S.
sclerotiorum and F. oxysporum. In vitro and in vivo antifungal activities showed that
rosin-based amide compounds containing thiophene heterocycles exhibited better
inhibitory effects. In particular, compound 5b with fluorine atom had the best
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antifungal effect on B. cinerea, and its EC50 was lower compared to the positive
control (penthiopyrad). Physiological and biochemical studies revealed that the
primary action mechanism of 5b on B. cinerea can change mycelial morphology by
destroying cell wall structure, increasing cell membrane permeability and inhibiting
TCA pathway in respiratory metabolism. Furthermore, QSAR and SAR studies
revealed that charge distribution of rosin-based amides derivatives has a key role in
the antifungal activity through the hydrogen bonding, conjugation and electrostatic
interaction between the compounds and the target receptors.
SUPPORTING INFORMATION
The structural characterization data of dehydroabietyl amide derivatives (3a-p
and 5a-n) and values of descriptor related to dehydroabietyl amide derivatives
structure and their fungicidal activity against B. cinerea.
NOTES
The authors declare no competing financial interest.
FUNDING
This study was supported by the National Natural Science Foundation of China
(31870555) and the Fundamental Research Funds for the Central Universities
(2452018031).
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FIGURE CAPTIONS
Scheme 1. Synthesis route of rosin-based amide (3a-p)
Scheme 2. Synthesis route of rosin-based amide with thiophene heterocyclic structure
(5a-n)
Figure 1. (a) In vitro antifungal effects of compound 5b on B. cinerea; (b) Mycelial
relative conductivity of B. cinerea in the presence or absence of 5b; (c) The protective
efficacy of compound 5b and penthiopyrad (at a concentration of 100, 50 and 25
mg/L, respectively) on strawberry leaves inoculated with B. cinerea by in vivo tissue
method
Figure 2. Effect of compound 5b on mycelial morphology in B. cinerea. (a, b): 1.00K
times, plates untreated and treated with the concentration of EC50 values of 5b; (c, d):
3.00K times, plates untreated and treated with the concentration of EC50 values of 5b
Figure 3. (a) The "breaking point" rule was used to confirm the number of descriptors
of the QSAR model. (b) The experimental and predicted values of antifungal activity
against B. cinerea of rosin-based amide derivatives using the built QSAR model. (c)
The charge distribution, MEP plot and contour map of compound 5b from DFT
calculation of Gaussian 16W. (d) LUMO energy map of compound 5b
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Table 1 Antifungal activities of rosin-based derivatives (3a-p and 5a-n) against B. cinerea
IR (%) at a concentration of (mg/L) ± standard deviation (SD; n ≥ 3)
compound
EC50
y=a+bx
R2
log EC50
500.00
200.00
100.00
50.00
25.00
12.50
6.25
3.13
1.56
0.78
3a
3b
3c
3d
3e
3f
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
98.00 ± 1.92 a
98.00 ± 1.31 a
98.00 ± 2.10 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
80.00 ± 1.00 b
73.00 ± 1.14 b
75.00 ± 0.80 b
76.00 ± 0.92 b
75.00 ± 0.94 b
69.00 ± 0.95 b
70.00 ± 0.88 b
54.00 ± 0.98 c
54.00 ± 0.69 c
54.00 ± 0.85 c
94.00 ± 0.91 a
94.00 ± 1.29 a
90.00 ± 1.10 a
95.00 ± 1.48 a
88.00 ± 0.92 b
83.00 ± 1.83 b
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
40.00 ± 0.90 c
38.00 ± 0.93 c
39.40 ± 0.99 c
39.80 ± 1.00 c
39.50 ± 1.10 c
34.00 ± 0.92 d
34.00 ± 0.95 d
28.00 ± 0.98 d
27.00 ± 1.03 d
27.50 ± 0.80 d
42.30 ± 1.41 c
42.30 ± 1.20 c
42.00 ± 0.90 c
43.00 ± 0.75 c
42.00 ± 0.92 c
40.00 ± 1.00 c
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
90.00 ± 2.20 a
80.00 ± 2.10 b
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
22.00 ± 0.50 e
19.50 ± 0.61e
20.00 ± 0.55 e
20.00 ± 0.62 e
20.10 ± 0.43 e
15.20 ± 0.24 e
15.00 ± 0.08 e
12.20 ± 0.15 e
12.00 ± 0.18 e
12.10 ± 0.07 e
30.25 ± 0.39 d
30.25 ± 0.25 d
28.00 ± 0.40 d
30.00 ± 0.25 d
28.00 ± 0.50 d
25.00 ± 0.80 d
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
65.00 ± 1.10 b
48.55 ± 0.96 c
96.00 ± 2.00 a
97.00 ± 0.98 a
81.20 ± 1.50 b
82.30 ± 1.38 b
12.50 ± 0.05 e
11.90 ± 0.08 e
12.50 ± 0.06 e
12.80 ± 0.05 e
12.60 ± 0.03 e
9.50 ± 0.02 f
9.50 ± 0.04 f
6.50 ± 0.01 f
6.30 ± 0.03 f
6.50 ± 0.02 f
16.15 ± 0.25 e
16.30 ± 0.54 e
15.30 ± 0.17 e
16.30 ± 0.30 e
15.30 ± 0.28 e
14.00 ± 0.09 e
98.00 ± 2.50 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
67.88 ± 1.00 b
27.15 ± 0.56 d
67.00 ± 1.12 b
67.00 ± 0.90 b
52.40 ± 0.82 c
51.80 ± 1.13 c
5.60 ± 0.03 f
5.50 ± 0.07 f
6.00 ± 0.03 f
5.99 ± 0.02 f
6.00 ± 0.08 f
3.89 ± 0.05 f
4.00 ± 0.02 f
/
/
/
/
/
125.944
138.152
133.897
132.159
133.754
149.358
148.001
187.390
188.246
187.813
100.061
100.074
106.456
98.425
109.005
119.679
5.614
y=-2.751+0.022x
y=-2.746+0.020x
y=-2.713+0.020x
y=-2.715+0.021x
y=-2.707+0.020x
y=-2.989+0.020x
y=-3.003+0.020x
y=-3.129+0.017x
y=-3.164+0.017x
y=-3.142+0.017x
y=-2.491+0.025x
y=-2.495+0.025x
y=-2.453+0.023x
y=-2.524+0.026x
y=-2.414+0.022x
y=-2.603+0.022x
y=-1.952+0.348x
y=-0.213+0.435x
y=-0.271+0.416x
y=-0.374+0.408x
y=-0.364+0.421x
y=-0.484+0.360x
y=-0.778+0.308x
y=-2.181+0.054x
y=-2.320+0.046x
y=-1.420+0.095x
y=-1.448+0.098x
y=-2.484+0.089x
y=-2.503+0.091x
0.963
0.956
0.963
0.964
0.962
0.948
0.955
0.989
0.989
0.990
0.968
0.967
0.967
0.970
0.962
0.985
0.964
0.999
0.998
0.997
0.998
0.958
0.956
0.984
0.966
0.987
0.984
0.967
0.967
2.100
2.140
2.127
2.121
2.126
2.174
2.170
2.273
2.275
2.274
2.000
2.000
2.027
1.993
2.037
2.078
0.749
-0.310
-0.186
-0.038
-0.063
0.128
0.402
1.607
1.703
1.176
1.169
1.444
1.441
1.893
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
3g
3h
3i
/
/
/
/
/
/
/
/
/
/
/
/
/
3j
/
/
/
/
/
3k
3l
9.20 ± 0.09 f
9.00 ± 0.14 f
9.60 ± 0.13 f
9.00 ± 0.18 f
9.60 ± 0.12 f
8.60 ± 0.17 f
89.00 ± 1.95 a
99.44 ± 2.21 a
99.10 ± 1.93 a
98.67 ± 2.90 a
99.03 ± 2.21 a
96.10 ± 2.00 a
93.00 ± 1.80 a
41.60 ± 0.90 c
18.20 ± 0.20 e
50.18 ± 1.51 c
50.50 ± 1.20 c
25.90 ± 0.35 e
24.90 ± 0.40 e
5.07 ± 0.03 f
4.99 ± 0.04 f
5.00 ± 0.06 f
4.99 ± 0.02 f
5.00 ± 0.03 f
/
/
/
/
/
/
/
3m
3n
3o
3p
5a
5b
5c
5d
5e
5f
/
/
/
/
/
/
/
/
/
/
/
/
65.00 ± 1.02 b
92.78 ± 1.91 a
91.60 ± 2.00 a
90.12 ± 1.86 a
90.70 ± 1.50 a
88.12 ± 1.38 a
82.52 ± 1.57 b
35.50 ± 1.04 d
9.30 ± 0.20 f
40.40 ± 0.95 c
40.40 ± 1.20 c
21.00 ± 0.80 e
22.00 ± 0.72 e
40.00 ± 1.12 b
75.30 ± 1.20 a
73.21 ± 0.95 a
72.02 ± 1.42 a
72.31 ± 1.90 a
70.32 ± 1.66 a
51.08 ± 1.24 b
22.00 ± 0.60 d
/
26.00 ± 0.90 b
61.46 ± 1.02 a
59.78 ± 0.85 a
58.14 ± 1.10 a
59.28 ± 1.23 a
50.30 ± 0.90 a
42.30 ± 1.00 b
/
/
53.48 ± 1.04 a
0.490
51.03 ± 1.12 a
0.652
46.22 ± 1.20 a
0.917
47.03 ± 0.95 a
0.865
41.28 ± 0.90 b
1.343
5g
5h
5i
36.58 ± 0.76 b
2.526
/
/
/
/
/
/
40.443
50.498
14.986
14.770
27.794
27.597
/
5j
31.81 ± 0.97 c
31.25 ± 1.24 c
11.00 ± 0.06 d
10.80 ± 0.05 d
24.80 ± 0.80 b
25.00 ± 0.76 b
/
5k
5l
5m
/
5n
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
100.00 ± 0.00 a
64.00 ± 1.23 b
100.00 ± 0.00 a
32.55 ± 0.25 d
100.00 ± 0.00 a
16.15 ± 0.33 e
100.00 ± 0.00 a
9.20 ± 0.09 f
99.30 ± 2.90 a
4.67 ± 0.03 f
91.80 ± 2.21 a
/
/
/
78.091
0.562
y=-3.277+0.042x
y=-0.237+0.422x
0.951
0.999
penthiopyrad
74.60 ± 1.08 a
60.46 ± 1.02 a
52.23 ± 1.20 a
553
Values in columns followed by similar letters were not significantly different according to Fisher’s protected LSD test (P < 0.05)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 35
554
555
Table 2 The respiratory inhibition rate and superposition rate of compound 5b
and typical inhibitors on B. cinerea mycelia
R0
R1 (R1')
IRr
RR
inhibitors
(O2, μmol/g·min)
12.45 ± 0.30 a
12.45 ± 0.28 a
12.45 ± 0.26 a
12.45 ± 0.29 a
12.45 ± 0.27 a
12.45 ± 0.28 a
12.45 ± 0.29 a
(O2, μmol/g·min)
8.30 ± 0.06 a
7.85 ± 0.03 a
7.05 ± 0.04 b
6.40 ± 0.06 c
4.18 ± 0.03 d
3.56 ± 0.04 e
2.84 ± 0.03 f
(%)
(%)
malonic acid
33.33
36.95
43.37
48.59
66.43
71.41
77.19
/
iodoacetic acid
/
sodium phosphate
5b
/
/
5b and malonic acid
5b and iodoacetic acid
5b and sodium phosphate
34.69
44.38
55.63
556
557
558
559
R0, R1 and R1'are respiration rate of mycelia before adding tested agents, after adding tested agents
and after adding tested agents with typical inhibitors; IRr is the inhibition rate of mycelia
respiration; RR is the superposition rate of tested agents with typical inhibitors. All results are
presented as the mean ± standard deviation (SD; n ≥ 3).
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Page 29 of 35
Journal of Agricultural and Food Chemistry
560
Table 3 The constructed model with four optimum descriptors
descriptor No.
X
± ΔX
t-test
descriptor
Intercept
0
1
2
3
4
1.2767e+01
3.3232e-01
-3.9091e+00
8.2097e-02
-8.1504e+00
3.2808e+00
6.4745e-02
2.5005e-01
7.3287e-03
1.7601e+00
3.8914
5.1327
-15.6331
11.2021
-4.6306
a
qmax
b
qN
max
LUMO c
d
DM
a
b
561
562
Maximum net charge. Maximum net atomic charge for an N atom. c The lowest unoccupied
molecular orbital. d Dipole moment of the molecule.
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