Design, Synthesis, Antibacterial Evaluation, and Induced Apoptotic Behaviors of Epimeric and Chiral 18β-Glycyrrhetinic Acid Ester Derivatives with an Isopropanolamine Bridge against Phytopathogens

Article abstract of DOI:10.1021/acs.jafc.9b06147

Because only a handful of agrochemicals can manage bacterial infections, the discovery and development of innovative, inexpensive, and high-efficiency antibacterial agents targeting these infections are challenging. Herein, a series of novel epimeric and chiral 18β-glycyrrhetinic acid (GA) ester derivatives with various tertiary amine pendants were designed, synthesized, and screened for pharmacological activity. Results showed that some of the title compounds were conferred with significantly enhanced antibacterial activity toward phytopathogens Xanthomonas oryzae pv oryzae (A₂, B₁-B₃, and C₁, EC₅₀ values within 3.81-4.82 μg/mL) and Xanthomonas axonopodis pv citri (B₁, EC₅₀ = 3.18 μg/mL; B₂, EC₅₀ = 2.76 μg/mL). These activities are superior to those of GA (EC₅₀ > 400 μg/mL), thiodiazole copper, and bismerthiazol. Pharmacophore studies revealed that the synergistic combination of GA skeleton and tertiary amine scaffolds contributed to the biological actions. In vivo experiments displayed their promising applications in controlling bacterial infections. Antibacterial mechanism studies revealed that the title compounds could trigger apoptosis in the tested pathogens, evident by bacteria morphological changes observed in scanning electron microscopy images. This outcome should motivate the development of various apoptosis inducers against plant bacterial diseases by a novel mode of action compared to that of existing agricultural chemicals.

Full text of DOI:10.1021/acs.jafc.9b06147

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Agricultural and Environmental Chemistry
Design, Synthesis, Antibacterial Evaluation, and Induced Apoptotic
Behaviors of Novel Epimeric and Chiral 18#-Glycyrrhetinic Acid Ester
Derivatives with an Isopropanolamine Bridge against Phytopathogens
Meng Xiang, Xiang Zhou, Ting-Rong Luo, Peiyi Wang, Li-Wei Liu, Zhong Li, Zhibing Wu, and Song Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b06147 • Publication Date (Web): 08 Nov 2019
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Design, Synthesis, Antibacterial Evaluation, and Induced Apoptotic Behaviors of
Novel Epimeric and Chiral 18β-Glycyrrhetinic Acid Ester Derivatives with an
Isopropanolamine Bridge against Phytopathogens
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Meng Xiang a, Xiang Zhou a, Ting-Rong Luo a, Pei-Yi Wang* a, Li-Wei Liu a, Zhong
Li b, Zhi-Bing Wu a, Song Yang* a,b
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a
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State Key Laboratory Breeding Base of Green Pesticide and Agricultural
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Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Center for R&D of Fine Chemicals of Guizhou University,
Guiyang, 550025, China.
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b
College of Pharmacy, East China University of Science & Technology, Shanghai,
China 200237.
* Corresponding author.
E-mail: jhzx.msm@gmail.com (S. Yang), pywang888@126.com (P.-Y. Wang)
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Abstract
Because only a handful of agrochemicals can manage bacterial infections, thus, the
discovery and development of innovative, inexpensive, and high-efficiency
antibacterial agents targeting these infections are challenging. Herein, a series of
novel epimeric and chiral 18β-glycyrrhetinic acid (GA) ester derivatives with various
tertiary amine pendants was designed, synthesized, and screened for pharmacological
activity. Results showed that some of the title compounds were conferred with
significantly enhanced antibacterial activity toward phytopathogens Xanthomonas
oryzae pv. oryzae (A2, B1–B3, and C1, EC50 values within 3.81–4.82 µg/mL) and
Xanthomonas axonopodis pv. citri (B1, EC50 = 3.18 µg/mL; B2, EC50 = 2.76 µg/mL).
These activities are superior to those of GA (EC50 > 400 µg/mL), thiodiazole copper,
and bismerthiazol. Pharmacophore studies revealed that the synergistic combination
of GA skeleton and tertiary amine scaffolds contributed to the biological actions. In
vivo experiments displayed their promising applications in controlling bacterial
infections. Antibacterial mechanism studies revealed that the title compounds could
trigger apoptosis in the tested pathogens, evident by bacteria morphological changes
observed in scanning electron microscopy images. This outcome should motivate the
development of various apoptosis inducers against plant bacterial diseases by a novel
mode of action compared with existing agricultural chemicals.
Keywords
18β-glycyrrhetinic acid hybrids, antibacterial, in vitro and in vivo bioassays, apoptosis
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1. Introduction
Plant bacterial diseases and induced complications represent a significant threat on
global food security and have become one of the greatest challenges in agriculture that
should be urgently addressed.1-4 Currently, only a handful of agrochemicals, such as
thiodiazole copper (TC), bismerthiazol (BT), kocide, zhongshengmycin, streptomycin
(banned for its potential risk), and Zn thiazole, are used to manage these bacterial
diseases.5,6 However, their limited field efficacies and the ever-increasing resistance
to these common pesticides have made crop protection a difficult task.3,7-10 Therefore,
the discovery and development of innovative, inexpensive, low toxic, and
high-efficiency antibacterial agents targeting plant bacterial diseases have become the
primary task in agriculture.
Natural products are a valuable source that has long been exploited to treat
various diseases in medical and agricultural fields because of their wide range of
pharmacological behaviors.11-16 As in the case of this study, natural products also
serve as major lead compounds for new drug development. Among these compounds,
pentacyclic triterpene ingredients found in many medicinal plants have been strongly
highlighted and extensively investigated for their substantial applications as flavor
sweeteners, food additives, cosmetics, substrate materials, and medical drugs.17–20
18β-Glycyrrhetinic acid (GA), a typical pentacyclic triterpenoid isolated from
Glycyrrhiza sp., has moderate hepatoprotective,21-23 antioxidative,24,25 antitumor,26-28
antipruritic,29,30 and anti-inflammatory31-33 effects. Moreover, it contains a natural
18-β-H-oleanane-type skeleton, a hydroxyl group at the C-3 position, an unsaturated
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ketone at the C11–13 positions, and a carboxylic group at the C-20 position; thus, GA
contains ample functionality to serve as a lead compound.27,34–36 However, GA has
some undesirable physicochemical features, including high hydrophobicity, which
contributes to its inadequate bioavailability, low water solubility and membrane
permeability, which seriously restrict its potential and practical applications.37-39 To
enhance its water solubility, bioavailability, selectivity, and pharmacological effects,
numerous structural modifications based on the GA framework have been attempted,
resulting in abundant redecorated derivatives with broadened biological windows.40-42
Intensive investigations revealed that these modification strategies normally suffer
from a long or/and complicated synthetic route, usage of expensive reaction reagents,
and unsatisfactory biological effects. In contrast to these works, this study involved
the preparation of a series of simple epimeric and chiral GA derivatives with an ester
group and various tertiary amine pendants. These derivatives were prepared through
two facile substitution reactions to modulate the hydrophobicity of GA and explore
antibacterial agents targeting plant bacterial diseases. Within these title molecules, the
newly formed ester group and later introduced tertiary amines were used to potentially
improve biocompatibility and membrane penetrability, regulate molecular
hydrophobic/hydrophilic performances, promote binding affinity, and strengthen
additional interactions with target species. To our knowledge, few studies have used
this type of GA derivatization to evaluate the general antibacterial effect against plant
pathogens. In this study, phytopathogens Xanthomonas oryzae pv. oryzae (Xoo) and
Xanthomonas axonopodis pv. citri (Xac), which represent the most destructive
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bacteria in agriculture, were tested.43,44 The antibacterial mechanism was studied by
flow cytometry and scanning electron microscopy (SEM) after exposing the tested
pathogens to the designed drugs.
2. Materials and methods
2.1 Instruments and Chemicals
Bruker Biospin AG-400 (BRUKER OPTICS, Switzerland) and JEOLECX-500 NMR
(JEOL, Japan) spectrometers were used to confirm structural assignments of the
derivatives. CDCl3 and TMS were used as the solvent and internal standard,
respectively. The related chemical shifts and coupling constants (J) were recorded in
parts per million (ppm) and hertz (Hz), respectively. Thermo Scientific Q Exactive
device (UItiMate 3000, Thermo SCIENTIFIC, United States) was exploited to detect
HRMS of title compounds dissolved in methanol. FEI Nova NanoSEM 450 (FEI,
United States) instrument was used for monitoring the morphology of tested
pathogens. The starting material 18β-glycyrrhetinic acid (purity > 97%) was
purchased from Energy Chemical of Saen Chemical Technology (Shanghai) Co., Ltd.
2.2 Experimental section
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Turbidimeter test for in vitro antibacterial assay, in vivo testing for controlling rice
bacterial blight (compounds B1 and C1 were evaluated), and SEM patterns for tested
pathogens follow previously published protocols.9
2.3 Apoptosis detection by flow cytometry
The apoptotic effect triggered by C1 and B1 was evaluated by flow cytometry.
Meanwhile, the Xoo and Xac cells were double stained using AnnexinV-FITC and
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propidium iodide (PI) under the guidance of the manufacturer’s instruction
(ANNEXIN V- FITC/PI Apoptosis Detection Kit, Solarbio). Briefly, Xoo (or Xac)
was exposed to the related different concentrations of C1 (B1 was used for Xac) in
nutrient broth medium (formula: 20 g glucose, 2 g yeast powders, 10 g peptone and 6
g beef extracts in 2.0 L deionized water) at 28 °C for 18 hours. Then 300 µL of
bacteria liquid was took and incubated with 5 µL of annexin V-FITC for 10 min and 5
µL of PI for 5 min at room temperature in the dark. Samples were analyzed using a
Gallios flow cytometer (BD Accuri C6).
3. Results and Discussion
The efficient preparation of GA ester derivatives is shown in Figure 2. In general, GA
reacts with a racemic epibromohydrin in an N,N-dimethylformamide solution
containing the base K2CO3 to yield the GA-ester 1 bearing the electrophilic epoxy tail.
The epoxy tail was then ring-opened with a diverse set of secondary amines to
provide an array of epimeric title compounds A1-A5, B1-B10, and C1-C12. Their final
molecular frameworks were confirmed through analysis of their corresponding NMR
and HRMS spectra. The in vitro toxicity of the designed compounds were analyzed by
the classical turbidimetric test on the two plant bacterial strains Xoo and Xac. The
bioactivity outcomes are illustrated in Table 1, which demonstrated that some of
target compounds possess potent antibacterial capacity against these two plant
pathogens with EC50 values of 3.81 and 2.76 µg/mL, respectively. Those data were
substantially superior to those of GA (EC50 > 400 µg/mL), BT, and TC, indicating
that this facile modification strategy on the GA framework could efficiently empower
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these title compounds with desirable pharmacological effects. For series A1–A5,
slightly increasing the hydrophobic property of target compounds could lead to
improved antibacterial efficiency, as illustrated by comparing A1 (dimethyl, 10.5 and
9.57 µg/mL against Xoo and Xac, respectively) with A2 (diethyl, 3.81 and 5.10 µg/mL
against Xoo and Xac, respectively). By contrast, introducing the diallyl group (A3),
diethanol group (A4), or propargyl group (A5) significantly quenched the antibacterial
effects. This finding indicated that a rigid and steric unsaturated alkane or a
hydrophilic hydroxyl pattern at the tail of the title compounds was disadvantageous to
bioactivity. When the above non-cyclic amines were modified into cyclic amines,
compounds B1–B3 bearing pyrrolidine rings displayed significant inhibition toward
the tested pathogens, affording the corresponding EC50 values of 4.79, 4.15, and 4.43
µg/mL against Xoo and 3.18, 2.76, and 3.65 µg/mL against Xac. Increasing the ring
size to piperidine led to decreased bioactivity and yielded EC50 values within 6.89–
20.5 and 4.29–6.64 µg/mL for compounds B4–B8 against Xoo and Xac, respectively.
Notably, the position of the methyl substituents on the piperidine ring showed ~3-fold
difference in anti-Xoo behavior, as demonstrated by comparing the EC50 values of
compounds B5 (2-CH3, 10.7 µg/mL), B6 (3-CH3, 7.19 µg/mL), and B7 (4-CH3, 20.5
µg/mL), in which the methyl group at the 3-position was found to be beneficial to
anti-Xoo activity. Surprisingly, switching the methyl group (B6) into ethyl formate
patterns (B9-B10) dramatically reduced antibacterial ability, suggesting that a
relatively bulk and/or dipole interactions from the ester group reduce activity. Further
modifying the piperidine moiety into various substituted piperazines afforded
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different levels of antibacterial potency for compounds C1–C11. The anti-Xoo effect
was gradually reduced with the increment of carbon amounts (C1–C5) on the
piperazine ring and presented the minimal EC50 value of 4.82 µg/mL for C1. Thus, an
additional hydrophobic and sterically hindered group was unfavorable to anti-Xoo
activity. Whereas less than a two-fold difference in activity was observed against Xac
with the same series of compounds. The introduction of an electron-withdrawing
group (acetyl, C6) or substituted benzyl scaffolds (C7–C11) on the piperazine ring
failed to generate substantial bioactivity. Meanwhile, replacing the piperazine ring
into morpholine substructure (for compound C12) provided negligible activity against
the two pathogens. This outcome showed that the nitrogen atom played a crucial role
in strengthening further interactions with the bacterial target species in contrast to the
oxygen atom. The bioassay results revealed that antibacterial functions were
influenced by diverse factors, including the type and size of amine and the
substituents on the N-containing scaffolds. Therefore, the molecular architectures
should be carefully optimized.
Compounds B1 (4.79 and 3.18 µg/mL) and C1 (4.82 and 3.66 µg/mL), which
possess excellent antibacterial competences, were selected as reference substances to
explore possible pharmacophores. Given the poor efficacy of GA, compound D1 was
primarily synthesized (Figure 3) to evaluate whether the bioactivity is ascribed to the
latter introduced motif containing an isopropanolamine bridge. However, D1 provided
weak antibacterial actions with the EC50 exceeding 100 µg/mL (Table 2). This
outcome indicated that the GA skeleton served as a vital ingredient in promoting the
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pharmaceutical effects of final hybrids. Compounds E1 and E2 were prepared to
examine the influence of the introduced hydroxyl group in the compound A to C
series toward bioactivity (Figure 4). When the bioactivity of compound B1 was
compared, the removal of the hydroxyl group significantly reduced the antibacterial
power with EC50 values changing from 4.79 µg/mL (B1, with hydroxyl group) to 53.7
µg/mL (E1, without hydroxyl group) against Xoo and from 3.18 µg/mL (B1) to 13.3
µg/mL (E1) against Xac. An opposite pattern was obtained for E2 bearing the
piperazine group, which afforded improved potency against Xoo with an EC50 value
of 2.76 µg/mL and a similar effectiveness against Xac with an EC50 value of 3.17
µg/mL. This phenomenon indicated that this newly produced hydroxyl group could
affect the biological efficacy at different levels. Prudently, the absolute configuration
toward bioactivity should be investigated. Thus, compounds F1–F4 were constructed
by switching the racemic reagents into the corresponding monomers. However,
anti-Xoo activity was reduced for these absolute single configurations, providing EC50
values within 5.15–5.98 µg/mL. Differently, improved anti-Xac ability was observed
for F1–F4, affording EC50 values of 1.98–3.08 µg/mL. This interesting outcome
showed that the absolute configurations could weaken the interactive effects targeting
bacterial receptors of Xoo and heighten additional interactions with Xac. The above
investigations showed that the GA skeleton was essential for biological actions,
whereas the latter generated hydroxyl group and the absolute configuration could
influence bioactivity at varying degrees.
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Bioactive compounds B1 and C1 bearing different types of N-containing
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fragments were selected to detect potential in vivo effectiveness against rice bacterial
blight. The results showed that B1 and C1 exerted prominently therapeutic and
preventive effects at 200 μg/mL with control efficiencies within 50.19%–52.91%
(Table 3 and Figure 6). These values clearly outperformed those of commercial agents
(BT and TC, within 33.43%–42.39%), validating their potential applications as
antibiotic alternatives.
GA derivatives can induce apoptotic effects on tested cell lines including
SKOV3 and OVCAR3 cell lines, hepatocellular carcinoma cell lines, leukemia HL-60
cell lines, and A253 carcinoma cell lines.27,45-49 Therefore, flow cytometry was
exploited to investigate the apoptotic actions of pathogens induced by the
corresponding bioactive compounds C1 (for Xoo) and B1 (for Xac). As revealed in
Figure 7, C1 could trigger remarkable late apoptotic behavior toward Xoo cells in a
dose-dependent manner, resulting in the percentage increasing from 14.2% (6.25
μg/mL, Figure 7b) to 87.7% (50 μg/mL, Figure 7e) for late apoptotic cells. This
interesting finding indicated that the anti-Xoo behavior of these novel GA hybrids
may be ascribed to the induced apoptotic mechanism of pathogens. Similarly, B1
could trigger the late apoptotic behavior of Xac with increased proportion from 6.5%
(6.25 μg/mL, Figure 8b) to 32.4% (50 μg/mL, Figure 8e). In addition, the ratio of
dead cells increased with increasing drug dosages, affording an increased percentage
of 15.4% at 25 μg/mL (Figure 8d). This intriguing outcome prompted us to further
explore the underlying antibacterial mechanism of GA hybrids. Such work may
benefit the development of various apoptosis inducers against plant bacterial diseases
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by a novel mode of action compared with existing agricultural chemicals.
Topological studies on the pathogens treated with GA hybrids were conducted by
SEM frames in a dose-dependent way. As depicted in Figure 9, the outline of Xoo was
transformed from homogenous rod shapes (Figure 9a) to a mass of rods with
malformed surfaces (Figures 9b-9f). Furthermore, increase of drug concentrations led
to incrementally lysed or/and deformed cells, suggesting that strong interactions
occurred between the designed GA hybrids and pathogens. Similar events were
observed when Xac was treated with compound B1 (Figure 10), further verifying that
GA hybrids presented marked effects on the tested pathogens. This result was
consistent with the discovered apoptosis phenomenon.
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In brief, several epimeric and chiral GA ester derivatives with diverse tertiary
amine pendants were synthesized and screened for antibacterial actions. The results
showed that certain title compounds were conferred with markedly enhanced
antibacterial behaviors toward the phytopathogens Xoo (A2, B1–B3 and C1, EC50
values within 3.81–4.82 µg/mL) and Xac (B1, EC50 = 3.18 µg/mL; B2, EC50 = 2.76
µg/mL). This effect was superior to those of precursor GA (EC50 > 400 µg/mL), TC,
and BT. Pharmacophore studies revealed that the synergistic combination of the GA
skeleton and tertiary amine scaffolds dictated to the remarkable biological actions. In
vivo experimental results showed that B1 and C1 exerted prominent therapeutic and
preventive effects at 200 μg/mL with the control efficiencies in the range of 50.19%–
52.91%. These values clearly outperformed those of commercial agents (BT and TC,
within 33.43%–42.39%), validating their potential applications as antibiotic
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alternatives. Antibacterial mechanism studies revealed that the title compounds could
trigger the apoptosis of the tested pathogens, consequently resulting in changes in the
bacterial morphology as observed in the SEM images. Given this facile structural
modification and the promising biological behaviors, we anticipate that this study can
provide a perceptible approach for modifying the GA framework to provide a series of
antibacterial alternatives to current commercial agents.
Supporting Information
1
Supplementary data including synthesis, characterization data, H NMR, 13C NMR
and 19F NMR spectra for the intermediates and title compounds.
Funding Sources
We thank the Chinese government for support provided by grants NNSF (31860516,
21702037, 21877021, 21662009), Guizhou PSTP ([2012]6012, LH[2017]7259,
[2017]5788), and Research Project of Ministry of Education (20135201110005,
213033A).
Conflict of interest
The authors declare no competing financial interest.
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Figure captions
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Figure 1. Design strategy for 18β-glycyrrhetinic acid ester derivatives with various
tertiary amine pendants.
Figure 2. Synthetic route for A1–A5, B1–B10, and C1–C12.
Figure 3. Molecular structure for the control molecule D1.
Figure 4. Synthetic route for E1–E2.
Figure 5. Synthetic route for F1–F4.
Figure 6. In vivo experimental images of B1 and C1 against rice bacterial blight (drug
dosage: 200 μg/mL). BT and TC served as the positive comparison.
Figure 7. Apoptosis effect of Xoo monitored by flow cytometry after incubation with
escalating concentrations of C1. Cells were stained with annexin V-FITC and
propidium iodide (PI).
Figure 8. Apoptosis effect of Xac monitored by flow cytometry after treatment with
escalating concentrations of B1. Cells were stained with annexin V-FITC and
propidium iodide (PI).
Figure 9. Morphological changes in Xoo after treatment with escalating
concentrations of C1. Scale bars are 1 µm for a–f.
Figure 10. Morphological changes in Xac after treatment with escalating
concentrations of B1. Scale bars are 1 µm for a–f.
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Tables
Table 1. In vitro antibacterial testing of A1–A5, B1–B10, and C1–C12 against plant
bacterial strains Xoo and Xac.
Xoo
Xac
Compounds
EC50
(µg/mL)
EC50
(µg/mL)
Regression equation
R2
Regression equation
R2
GA
1
> 400
> 400
> 400
> 400
A1
A2
A3
A4
A5
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
BT
TC
y=2.946x+1.987
y=1.354x+4.214
0.979
0.969
10.5 ± 0.1
3.81 ± 0.18
> 100
y=1.377x+3.649
y=0.746x+4.472
0.976
0.997
9.57 ± 0.11
5.10 ± 0.08
> 100
> 100
> 100
> 100
> 100
y=3.175x+2.839
y=2.739x+3.307
y=3.297x+2.868
y=1.164x+4.024
y=3.166x+1.741
y=1.247x+3.932
y=1.733x+2.726
y=2.080x+2.564
0.981
0.995
0.988
0.973
0.905
0.980
0.984
0.998
4.79 ± 0.08
4.15 ± 0.11
4.43 ± 0.08
6.89 ± 0.17
10.7 ± 0.1
7.19 ± 0.49
20.5 ± 0.3
14.8 ± 0.3
> 100
y=1.173x+4.410
y=0.898x+4.604
y=0.628x+4.647
y=1.184x+4.051
y=0.961x+4.273
y=0.705x+4.554
y=0.850x+4.462
y=0.839x+4.310
0.999
0.999
0.944
0.998
0.967
0.967
0.957
0.979
3.18 ± 0.02
2.76 ± 0.16
3.65 ± 0.02
6.33 ± 0.28
5.71 ± 0.21
4.29 ± 0.12
4.30 ± 0.11
6.64 ± 0.35
> 100
> 100
> 100
y=2.768x+3.109
y=1.675x+3.571
y=2.660x+2.372
y=1.052x+3.361
y=1.357x+2.936
0.960
0.934
0.915
0.954
0.967
4.82 ± 0.08
7.13 ± 0.21
9.73 ± 0.18
36.1 ± 0.7
33.2 ± 0.6
> 100
y=1.223x+4.311
y=0.719x+4.579
y=0.468x+4.736
y=0.784x+4.519
y=1.015x+4.152
0.894
0.956
0.999
0.999
0.894
3.66 ± 0.17
3.85 ± 0.21
3.67 ± 0.12
4.11 ± 0.08
6.85 ± 0.32
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
y=1.499x+2.052
y=1.540x+1.788
0.963
0.960
92.6 ± 2.1
121.8 ± 3.6
y=2.153x+0.938
0.962
77.0 ± 2.0
443
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Table 2. In vitro antibacterial testing of D1, E1–E2, and F1–F4 against plant bacterial
strains Xoo and Xac.
Xoo
Xac
R2
Compounds
EC50
(µg/mL)
EC50
(µg/mL)
Regression equation
R2
Regression equation
B1
C1
y=3.175x+2.839
y=2.768x+3.109
0.981
0.960
4.79 ± 0.08
4.82 ± 0.08
> 100
y=1.173x+4.410
y=1.223x+4.311
0.999
0.894
3.18 ± 0.02
3.66 ± 0.17
> 100
D1
E1
y=3.790x-1.556
y=4.283x+3.110
y=2.719x+3.047
y=3.669x+2.149
y=3.984x+1.992
y=3.079x+2.808
y=1.499x+2.052
y=1.540x+1.788
0.995
0.998
0.999
0.944
0.998
0.844
0.963
0.960
53.7 ± 0.9
2.76 ± 0.06
5.23 ± 0.23
5.98 ± 0.13
5.69 ± 0.13
5.15 ± 0.06
92.6 ± 2.1
121.8 ± 3.6
y=0.952x+3.930
y=0.845x+4.576
0.974
0.998
0.947
0.886
0.932
0.992
13.3 ± 0.6
3.17 ± 0.11
2.00 ± 0.03
1.98 ± 0.07
3.08 ± 0.11
2.55 ± 0.03
E2
F1 (R)
F2 (S)
F3 (R)
F4 (S)
BT
y=1.6155x+4.5148
y=1.2163x+4.6381
y=1.6148x+4.2118
y=1.5889x+4.3551
TC
y=2.153x+0.938
0.962
77.0 ± 2.0
446
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448
Table 3. In vivo control efficiency (14 days after spraying) of B1 and C1 against rice
bacterial blight at 200 μg/mL.
Curative effect
Protective effect
Chemicals
Morbidity
(%)
Disease index
(%)
Control efficiency
(%)b
Morbidity
(%)
Disease index
(%)
Control efficiency
(%)b
B1
C1
100
100
100
100
100
40.46
41.83
55.92
48.39
84.00
51.83
50.19
33.43
42.39
/
100
100
100
100
100
40.44
39.56
50.52
52.86
84.00
51.85
52.91
39.86
37.07
/
BT
TC
CKa
449
450
a Negative control. b Statistical analysis was conducted by ANOVA under the condition of equal variances assumed (P > 0.05)
and equal variances not assumed (P < 0.05).
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Figures
Figure 1
R1
R2
O
O
O
O
N
*
*
OH
O
O
R1
OH
O
H
H
N
H
O
O
R2
O
Introducing
tertiary amines
Introducing the
ester group
H
H
H
HO
HO
HO
H
H
H
GA-hybrids
GA-ester
GA
Target compounds with good
Lead compound
antibacterial activity ?
453
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Figure 2
O
O
O
R
O
*
OH
OH
O
O
*
O
H
H
H
Br
O
O
O
RH
*
℃
Isopropanol,
K2CO3, DMF, r.t.
60
H
H
H
HO
H
HO
HO
A1-A5, B1-B10, C1-C12
H
H
GA
1
OH
N
N
N
N
N
N
N
S
N
N
N
N
N
R
OH
OH
OH
A4
A2
A3
A5
A1
B1
B2
B6
B3
B4
B5
B7
COOCH3
B8
COOCH2CH3
COOCH2CH3
R =
S
R
N
NH
N
N
N
N
N
N
N
N
N
N
N
F
N
O
N
N
C5
B9
B10
Cl
C3
C4
C6
C2
C1
N
CH2
C7
N
N
CH2
C8
N
N
CH2
C9
Cl
N
N
CH2
C10
N
N
CH2
C11
N
O
C12
455
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Figure 3
O
O
O
COOH
Br
RH
O
O
O
N
OH
℃
Isopropanol 60
K2CO3, DMF, r.t.
2
D1
457
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Figure 4
O
O
O
R
Br
O
O
OH
H
H
H
O
O
O
Br
Br
RH
K2CO3, DMF, r.t.
K2CO3, DMF, r.t.
H
H
H
HO
HO
HO
H
H
H
N
NH
N
E1
R =
3
E2
459
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Figure 5
O
O
R
O
O
O
OH
H
H
O
O
O
Cl
RH
℃
Isopropanol,
60
℃
K2CO3, DMF, 60
H
H
N
N
NH
R =
F1, F3
HO
HO
H
H
4
F1
F3
O
GA
O
R
O
O
O
OH
H
H
O
O
O
Cl
RH
℃
℃
K2CO3, DMF, 60
Isopropanol,
60
H
H
N
N
NH
R =
F2, F4
HO
HO
H
5
H
F2
F4
461
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Figure 6
463
464
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Figure 7
466
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