pubs.acs.

org/JAFC Article

Antibacterial Functions and Proposed Modes of Action of Novel

1,2,3,4-Tetrahydro-β-carboline Derivatives that Possess an
Attractive 1,3-Diaminopropan-2-ol Pattern against Rice Bacterial
Blight, Kiwifruit Bacterial Canker, and Citrus Bacterial Canker
Hong-Wu Liu, Qing-Tian Ji, Gang-Gang Ren, Fang Wang, Fen Su, Pei-Yi Wang,* Xiang Zhou,
Zhi-Bing Wu, Zhong Li, and Song Yang*
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sı Supporting Information

ABSTRACT: In recent years, naturally occurring tetrahydro-β-carboline (THC) alkaloids and their derivatives have been of
biological interest. However, few studies and developments have reported the use of such structures in managing plant bacterial
diseases. Herein, an array of novel THC derivatives containing an attractive 1,3-diaminopropan-2-ol pattern were prepared to
evaluate the antiphytopathogen activity in vitro and in vivo and explore innovative antibacterial frameworks. Notably, target
compounds exhibited excellent activities against three rebellious phytopathogens, namely, Pseudomonas syringae pv. actinidiae (Psa),
Xanthomonas axonopodis pv. citri, and Xanthomonas oryzae pv. oryzae, at related optimal EC50 values of 2.39 (II9), 2.06 (I23), and
1.69 (II9) μg/mL, respectively. These effects were superior to those of the parent structure 1,2,3,4-THC and positive controls. In
vivo assays showed that II9 exhibited excellent control efficiencies of 51.89 and 65.45% at 200 μg/mL against rice bacterial blight and
kiwifruit bacterial canker, respectively, and I23 substantially relieved the citrus canker on the leaves. Antibacterial mechanisms
indicated that these THC compounds could induce the increment of reactive oxygen species and subsequently endow the tested
bacteria with distinct apoptotic behavior. In addition, II9 could alleviate the hypersensitive response and pathogenicity of Psa.
Overall, these simple THC derivatives can be further developed as versatile antibacterial agents.
KEYWORDS: tetrahydro-β-carboline derivatives, antiphytopathogen, in vitro and in vivo bioassays, ROS, apoptosis,
hypersensitive response

1. INTRODUCTION poses a huge challenge in regulating bacterial infections in

Plant bacterial infections are progressively worsening diseases1
that can produce diverse symptoms, including leaf blight, leaf
spots, green wilt, canker, decay, and deformity, on various
plants and fruit species.2−6 It is estimated that more than 650
kinds of bacterial diseases have been identified in the world.
These diseases can occur and spread on food crops (such as
rice, corn, wheat, and potato),7−10 economic crops (such as
tobacco, peanut, and soybean),11−13 fruit crops (such as citrus,
kiwifruit, mango, peach, apricot),14−17 vegetable crops (such as
cucumber, tomato, pepper, onion),18−21 medicinal crops,22
flower crops,23 and forest crops.24 This occurrence inevitably
affects crops’ quality and yields, thereby damaging the global
agricultural production. Based on recent incomplete statistics,
the occurrence area of bacterial diseases in China increases to
120 million mu each year, thereby rendering bacterial diseases
as the second major disease in agriculture. Given the lack of
effective and interchangeable bactericidal varieties, the
development of resistant plant pathogens has been accelerated
and eventually led to the isolation of resistant strains, such as
bismerthiazol (BT)-resistant pathogens.25 To date, no effective
prevention and eradication methods are available to control
certain intractable diseases, particularly for kiwifruit bacterial
canker, citrus bacterial canker, rice bacterial leaf blight, fruit
tree root cancer, and bacterial leaf streak.26−30 This finding
agriculture. Therefore, developing novel frameworks targeting
bacterial diseases with multiple modes of action to disable
bacterial resistance can promote the management of these
diseases.
Natural tetrahydro-β-carboline (THC) alkaloids are the
predominant class of secondary metabolites that have wide
distributions and sources, including vegetables, fruit or fruit
juices, medicinal plants, cocoa, chocolate, wines, beers, soy
sauces, microbial species, and animals.31 Biological studies
have found that THC alkaloids (Figure 1) display remarkable
pharmacological properties, such as antitumor, anti-inflamma-
tory, antifungal, antioxidant, and antiviral activities.31−39 These
identified biological effects have made THC alkaloids the
recognized platforms for the novel drug discovery process.
Hence, numerous studies that focused on the exploration of
novel bioactive substrates based on the natural THC alkaloids
Received: April 21, 2020
Revised: September 11, 2020
Accepted: October 22, 2020

© XXXX American Chemical Society https://dx.doi.org/10.1021/acs.jafc.0c02528

A J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Figure 1. Natural tetrahydro-β-carboline alkaloids and their derivatives with identified biological activities.
were frequently reported, which provided abundant redeco-
rated compounds with distinct and improved biological
properties and could be potentially applied in many fields,
particularly in the medical field (such as reserpine and tadalafil,
Figure 1).40,41 However, few studies have reported the use of
such THC structures in managing plant bacterial dis-
eases.31,42,43 Herein, novel and simple THC derivatives
containing a typical 1,3-diaminopropan-2-ol pattern were
prepared to evaluate the antiphytopathogen activity in vitro
and in vivo and expand the biological windows of THC
alkaloids and explore innovative antibacterial frameworks.
Moreover, the possible antibacterial mechanism was explored.
2. MATERIALS AND METHODS
2.1. Instruments and Chemicals. NMR detection was
performed on a Bruker Biospin-AG-400/500 instrument (BRUKER
OPTICS, Switzerland). Mass spectrometry was conducted using a
high-resolution mass spectrometer (UItiMate 3000, Thermo
Scientific). Apoptosis analysis was done using a Gallios flow
cytometer (BD-Accuri-C6). Fluorescence detection was made with
a Fluoromax-4cp fluorescence spectrophotometer. 1,2,3,4-Tetrahy-
dro-β-carboline and 1-bromo-2,3-epoxypropane were obtained from
Energy Chemical of Saen Chemical Technology (Shanghai) Co., Ltd.
2.2. Experimental Section. The in vitro antibacterial activities
against three rebellious phytopathogens Pseudomonas syringae pv.
actinidiae (Psa), Xanthomonas axonopodis pv. citri (Xac), and
Xanthomonas oryzae pv. oryzae (Xoo), the in vivo antibacterial
activity for reducing rice bacterial leaf blight of compound II9, and
apoptosis detection experiments followed our reported methods.44,45
2.3. In Vivo Assay against Kiwifruit Bacterial Canker. The
protective and curative assays against kiwifruit bacterial canker
followed the reported method,46 with some modifications. Compound
II9, thiodiazole copper (TC, 20% suspending agent, the positive
control), and an equivalent dimethyl sulfoxide (DMSO) solvent were
used in this experiment. Psa-free healthy kiwifruit twigs with a smooth
surface, uniform size, and a length of about 15 cm were used. Then,
the wound on each twig with 1 mm width was made by a sterilized
knife. For the protective assay, 10 μL of compound II9 or TC solution
at a dose of 200 μg/mL, equivalent DMSO solution, and distilled
B https://dx.doi.org/10.1021/acs.jafc.0c02528
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water were uniformly added on the wound. After 24 h inoculation, 10
μL of Psa bacterial suspension (OD595 = 0.1) was inoculated on every
wound. For the curative assay, 10 μL of Psa bacterial suspension
(OD595 = 0.1) was inoculated on every wound first, and after 24 h
inoculation, 10 μL of related agents were uniformly added on the
wound. All of the treatments were cultured in a climate chamber (95%
RH) with 14 h lighting at 18 °C and 10 h darkness at 16 °C. For each
treatment, at least 10 kiwifruit twigs were used. The length of the
lesion was measured after 28 days’ inoculation. In this study, three
independent experiments were carried out. The control efficiencies I
for the protective and curative activities were calculated using the
following equation
control efficiency I(%) = (C − T )/C × 100
In the equation, C and T are the average lengths of the lesion of the
negative control and the treatment group, respectively.
2.4. In Vivo Assay against Citrus Bacterial Canker. The
protective and curative assays against citrus bacterial canker were
performed according to the reported methods,47−53 with some
modifications. Compound I23, thiodiazole copper (TC, 20%
suspending agent, the positive control), and an equivalent DMSO
solvent were used in this experiment. The citrus leaves of a 2-year-old
citrus tree were cleaned by sterile water and dried in air. Two 3 × 3
rectangle-shaped wounds were punctured on the left and right sides of
the citrus leaf using a disposable sterilized syringe, respectively. For
the protective assay, a filter paper soaked in the solution of compound
I23 or TC solution at 200 μg/mL for 1 h was uniformly stuck to the
wounds. After 24 h treatment, this drug-containing filter paper was
thrown away and a new filter paper soaked in Xac suspensions (OD595
= 0.5) was stuck to the above-mentioned wounds for 24 h. For the
curative assay, the related operations were reversed, wherein
inoculation with Xac was done before application of agents. All of
the treatments were cultured in a climate chamber (95% RH) with 16
h lighting at 28 °C and 8 h darkness at 25 °C. The lesion was
observed after 21 days’ inoculation. Seven independent experiments
were carried out.
2.5. Reactive Oxygen Species (ROS) Accumulation Trig-
gered by II9 (or I23). The content of ROS in the pathogens Xoo (or
Xac) triggered by II9 (or I23) was measured using ROS assay kits
(GENMED Scientifics Inc.). Briefly, Xoo (or Xac) cells (OD595 is
0.5−0.6) were collected by centrifugation (6000 rpm, 5 min, 4 °C).
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Figure 2. Synthetic route for target compounds I1−I25.
These cells were washed by phosphate-buffered saline (PBS) buffer
(pH 7.2, 10 mM) and resuspended in the same PBS buffer. Then,
Xoo (or Xac) cells were incubated with different doses of II9 (I23, for
Xac) for 14 h. Finally, 0.6 mL cells’ solution was mixed with the
GENMED dyeing solution (5 μL) for 20 min and then was detected
by a fluorescence spectrophotometer.
2.6. Hypersensitive Response (HR) and Pathogenicity
Assays. Psa cells were grown in nutrient bouillon medium overnight
at 28 °C and then suspended in sterile distilled water. The bacterial
suspensions were adjusted to an OD595 of 0.1. Nicotiana benthamiana
plants were used for HR assays. To the adult plants, each flag leaf was
inoculated with Psa suspensions with 15−20 independent individuals,
respectively. Before infiltration on tobacco leaves, the bacterial germ
suspensions were incubated with II9 at 4.78 μg/mL (2 × EC50, based
on the growth curve) or DMSO for 2 h. Plants were scored and the
symptoms were checked at 48 h after inoculation in N. benthamiana.
During the experiments, plants were maintained under greenhouse
conditions (16 h of light at 18 °C and 8 h of darkness at 16 °C).
Photographs were taken using a BIO-RAD Gel imaging system. Three
independent experiments were performed.
2.7. Synthesis of the Intermediates and Target Com-
pounds. 2.7.1. Synthesis of the Intermediate 2-(Oxiran-2-
ylmethyl)-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole (2). 1,2,3,4-
Tetrahydro-β-carboline (5.0 g, 28.4 mmol) and anhydrous K2CO3
(4.77 g, 34.1 mmol) in 30 mL of dry dimethylformamide (DMF)
were stirred on an ice bath for 10 min; then, bromo-2,3-epoxypropane
(5.23 g, 37.0 mmol) was added and stirred overnight. After that, 50
mL of ethyl acetate was added into the mixture. The organic layer was
washed by a saturated NH4Cl solution, dried with anhydrous Na2SO4,
and evaporated in vacuum. The pure intermediate 2 was obtained by
column chromatography (CH2Cl2/CH3OH = 100:1, V/V). A yellow
solid, yield 37.9%, mp 123.1−124.7 °C; 1H NMR (500 MHz, CDCl3)
δ 7.98 (s, 1H, −NH), 7.46 (d, J = 7.7 Hz, 1H, Ar-H), 7.26 (d, J = 7.9
Hz, 1H, Ar-H), 7.12 (dt, J = 7.1, 1.3 Hz, 1H, Ar-H), 7.07 (dt, J = 7.8,
1.1 Hz, 1H, Ar-H), 3.88−3.84 (m, 1H, −N-CH2−), 3.66 (d, J = 14.7
Hz, 1H, −N-CH2−), 3.24−3.17 (m, 1H, −O-CH−), 3.05 (dd, J =
13.4, 2.9 Hz, 1H, −N-CH2−), 2.96 (t, J = 5.8 Hz, 2H, −CH2CH2−),
2.88−2.78 (m, 3H, −N-CH2−), 2.54 (dd, J = 5.0, 2.7 Hz, 1H, −O-
CH2−), 2.46 (dd, J = 13.4, 7.1 Hz, 1H, −O-CH2−); 13C NMR (101
MHz, CDCl3) δ 136.1, 131.6, 127.2, 121.4, 119.3, 118.0, 110.8, 108.1,
C https://dx.doi.org/10.1021/acs.jafc.0c02528
pubs.acs.org/JAFC Article
60.2, 51.6, 50.9, 50.7, 44.7, 21.2; HRMS (ESI) [M + H]+ calcd for
C14H17ON2: 229.1335, found: 229.1329.
The synthesis of intermediates 3 and 4 was carried out following
the above synthetic protocols.
2.7.1.1. (R)-2-(Oxiran-2-ylmethyl)-2,3,4,9-tetrahydro-1H-pyrido-
[3,4-b]indole (3). A yellow solid, yield 58.0%, mp 99.5−100.7 °C;
1
H NMR (500 MHz, CDCl3) δ 7.98 (s, 1H, NH), 7.47 (d, J = 7.6 Hz,
1H, Ar-H), 7.28 (d, J = 8.0 Hz, 1H, Ar-H), 7.15−7.11 (m, 1H, Ar-H),
7.11−7.05 (m, 1H, Ar-H), 3.87 (d, J = 14.7 Hz, 1H, −N-CH2−), 3.68
(d, J = 14.7 Hz, 1H, −N-CH2−), 3.22 (tt, J = 5.5, 2.8 Hz, 1H, −O-
CH2−), 3.07 (dd, J = 13.4, 2.8 Hz, 1H, −N-CH2−), 2.97 (t, J = 5.9
Hz, 2H, −CH2CH2−), 2.90−2.79 (m, 3H, −N-CH2−), 2.55 (dd, J =
4.9, 2.7 Hz, 1H, −O-CH2−), 2.46 (dd, J = 13.4, 7.2 Hz, 1H, −O-
CH2−); HRMS (ESI) [M + H]+ calcd for C14H17ON2: 229.1335,
found: 229.1326.
2.7.1.2. (S)-2-(Oxiran-2-ylmethyl)-2,3,4,9-tetrahydro-1H-pyrido-
[3,4-b]indole (4). A yellow solid, yield 58.0%, mp 117.5−119.2 °C;
1
H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H, NH), 7.47 (d, J = 7.4 Hz,
1H, Ar-H), 7.30 (d, J = 7.8 Hz, 1H, Ar-H), 7.13 (t, J = 7.3 Hz, 1H, Ar-
H), 7.09 (t, J = 7.1 Hz, 1H, Ar-H), 3.92 (d, J = 14.7 Hz, 2H, −N-
CH2−), 3.72 (d, J = 14.7 Hz, 2H, −N-CH2−), 3.23 (d, J = 2.9 Hz,
1H, −O-CH2−), 3.08 (dd, J = 13.3, 2.4 Hz, 1H, −N-CH2−), 2.98 (d,
J = 5.5 Hz, 2H, −CH2CH2−), 2.88−2.80 (m, 3H, −N-CH2−), 2.55
(d, J = 4.5 Hz, 1H, −O-CH2−), 2.49 (dd, J = 13.3, 7.1 Hz, 1H, −O-
CH2−); HRMS (ESI) [M + H]+ calcd for C14H17ON2: 229.1335,
found: 229.1325.
2.7.2. General Synthetic Procedures for the Target Compounds.
Intermediate 2 (3 or 4) (0.23 g, 1.0 mmol), amines R1-H (1.1 mmol),
K2CO3 (0.17 g, 1.2 mmol), and isopropanol (6.0 mL) were added to a
15 mL pressure bottle and stirred at 60 °C until intermediate 2 (3 or
4) had reacted completely. After that, the reaction mixture was
quenched with water (20 mL) and extracted with CH2Cl2 (30 mL ×
2). The organic layer was dried with anhydrous Na2SO4 and
evaporated under vacuum. The target compounds were purified by
column chromatography with CH2Cl2 and CH3OH (10:1; V/V) as
eluents.
2.7.2.1. 1-(Piperidin-1-yl)-3-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-
indol-2-yl)propan-2-ol (I1). A white solid, yield 95.7%, mp 136.9−
138.5 °C; 1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H, NH), 7.47 (d, J
= 7.5 Hz, 1H, Ar-H), 7.30 (d, J = 7.7 Hz, 1H, Ar-H), 7.16−7.11 (m,
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

Table 1. In Vitro Bioassay Results of 1, 2, and I1−I25 against Pathogens Xoo, Xac, and Psa
Xoo Xac Psa
comp. regression equation EC50 (μg/mL) regression equation EC50 (μg/mL) regression equation EC50 (μg/mL)
1 y = 5.689x − 1.891 16.27 ± 0.34 y = 1.727x + 2.866 17.22 ± 1.07 y = 2.408x + 2.137 15.43 ± 0.79
2 y = 4.299x − 0.915 23.75 ± 0.21 y = 1.600x + 2.423 40.80 ± 1.17 y = 2.318x + 1.879 24.86 ± 2.19
I1 y = 3.860x + 0.198 17.54 ± 1.69 y = 3.614x − 0.349 30.18 ± 1.25 y = 3.321x + 0.348 25.15 ± 2.25
I2 y = 1.485x + 2.768 31.86 ± 0.22 y = 2.060x + 2.010 28.28 ± 1.45 y = 3.218x − 0.211 41.60 ± 1.55
I3 y = 4.750x − 0.858 17.11 ± 0.21 y = 1.908x + 2.681 16.42 ± 0.41 y = 1.724x + 2.185 42.91 ± 0.33
I4 y = 5.331x − 2.165 22.08 ± 0.13 y = 1.940x + 2.029 33.99 ± 0.68 y = 3.784x − 1.125 41.54 ± 1.52
I5 y = 13.03x − 17.03 48.97 ± 0.08 y = 3.278x − 0.368 43.44 ± 1.26 y = 2.536x + 0.883 42.00 ± 1.85
I6 y = 3.485x + 0.665 17.53 ± 0.37 y = 2.246x + 0.602 90.78 ± 0.66 >100
I7 y = 2.758x + 1.090 26.17 ± 0.70 y = 1.949x + 2.595 17.12 ± 0.69 y = 2.493x + 0.866 45.51 ± 0.10
I8 y = 0.955x + 3.225 72.37 ± 0.20 y = 3.574x + 0.003 25.01 ± 1.40 >100
I9 y = 3.803x − 1.329 46.13 ± 0.57 y = 1.908x + 2.681 28.28 ± 0.80 >100
I10 y = 2.383x + 1.392 32.64 ± 0.22 y = 4.076x − 0.744 25.77 ± 2.84 >100
I11 y = 15.22x − 20.24 45.58 ±0.04 y = 6.025x − 5.781 61.57 ± 0.84 >100
I12 y = 9.032x − 3.130 7.95 ± 0.12 y = 1.886x + 3.371 7.30 ± 0.99 >100
I13 y = 6.810x − 5.847 39.15 ± 2.05 y = 1.816x + 1.987 45.62 ± 0.94 y = 2.149x + 1.034 70.02 ± 0.88
I14 y = 6.552x − 1.247 8.98 ± 0.60 y = 1.333x + 3.990 5.72 ± 0.13 y = 3.228x + 3.134 3.79 ± 0.29
I15 y = 4.454x + 1.651 5.65 ± 0.14 y = 3.664x + 2.943 3.64 ± 0.03 y = 3.811x + 1.928 6.40 ± 0.62
I16 y = 3.435x + 2.051 7.22 ± 0.27 y = 2.153x + 3.544 4.74 ± 0.70 y = 1.822x + 3.576 6.05 ± 0.17
I17 y = 8.425x − 1.236 5.50 ± 0.24 y = 1.453x + 4.541 2.07 ± 0.11 y = 3.655x + 3.075 3.36 ± 0.27
I18 y = 4.706x + 1.987 4.37 ± 0.34 y = 1.017x + 4.530 2.90 ± 0.46 y = 2.427x + 3.710 3.40 ± 0.69
I19 y = 4.598x + 2.265 2.93 ± 0.03 y = 1.559x + 4.290 2.86 ± 0.14 y = 0.988x + 4.306 5.04 ± 0.52
I20 y = 1.848x + 3.803 4.44 ± 0.31 y = 1.451x + 4.380 2.67 ± 0.14 y = 2.186x + 3.196 6.69 ± 0.52
I21 y = 1.417x + 3.603 9.67 ± 0.42 y = 2.084x + 3.238 7.01 ± 0.37 y = 6.654x − 4.635 28.05 ± 1.57
I22 y = 6.412x + 2.746 2.25 ± 0.03 y = 4.687x + 2.752 3.02 ± 0.18 y = 1.311x + 3.999 5.80 ± 0.80
I23 y = 4.034x + 3.303 2.63 ± 0.35 y = 2.039x + 4.361 2.06 ± 0.10 y = 3.466x + 2.829 4.23 ± 0.36
I24 y = 4.808x − 0.863 7.25 ± 0.32 y = 2.885x + 3.934 2.34 ± 0.13 y = 1.112x + 4.231 4.92 ± 0.40
I25 y = 7.346x + 6.640 38.4 ± 0. 20 y = 4.137x + 0.061 15.63 ± 1.17 y = 1.679x + 2.070 55.60 ± 0.06
TC y = 4.105x − 2.740 76.81 ± 2.22 y = 1.706x + 1.885 66.98 ± 0.49 y = 3.576x − 1.704 74.98 ± 3.49
BT y = 5.069x − 2.625 31.94 ± 3.59 y = 1.893x + 1.776 50.51 ± 2.08 y = 4.318x − 3.893 114.76 ± 3.93

Figure 3. Synthesis of target compounds II1−II17.

1H, Ar-H), 7.09 (td, J = 7.4, 1.1 Hz, 1H, Ar-H), 5.50 (s, 1H, −OH),
4.02−3.95 (m, 1H, −O-CH−), 3.81 (s, 2H, −N-CH2−), 3.04−2.88
(m, 2H, −N-CH2−), 2.82 (t, J = 5.5 Hz, 2H, −CH2CH2−), 2.70−
2.48 (m, 4H, −N-CH2−), 2.46−2.27 (m, 4H, −N-CH2−), 1.67−1.53
(m, 4H, −CH2CH2CH2−), 1.52−1.38 (m, 2H, −CH2CH2CH2−);
13
C NMR (101 MHz, CDCl3) δ 136.0, 131.8, 127.2, 121.4, 119.4,
118.0, 110.7, 108.4, 65.1, 63.3, 61.8, 54.9, 51.5, 50.8, 29.7, 26.1, 24.3,
21.2; HRMS (ESI) [M + H]+ calcd for C19H28ON3: 314.2227, found:
314.2220.
For the characterization data of other target compounds, please see
the Supporting Information.
D https://dx.doi.org/10.1021/acs.jafc.0c02528
3. RESULTS AND DISCUSSION
3.1. Synthesis and Antibacterial Activity of Target
Compounds I1−I25. For agrochemicals, a simple structure
bearing efficient bioactivity and short synthetic procedures is
ideal. Thus, a feasible synthetic route containing a facile two-
step reaction is recommended for the preparation of THC
derivatives (Figure 2). In brief, a glycidyl group was introduced
into the 2-position of 1,2,3,4-THC (1) via a simple alkylation
reaction to provide a key intermediate 2. Then, a variety of
target compounds I1−I25 were prepared via a ring-opening
reaction between diverse NH-containing scaffolds and
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Table 2. In Vitro Bioassay Results of II1−II17 against Pathogens Xoo, Xac, and Psa
Xoo Xac Psa
comp. regression equation EC50 (μg/mL) regression equation EC50 (μg/mL) regression equation EC50 (μg/mL)
II1 y = 5.546x + 3.686 1.73 ± 0.03 y = 7.731x + 0.448 3.88 ± 0.06 y = 2.029x + 3.449 5.81 ± 0.65
II2 y = 5.653x + 1.733 3.78 ± 0.25 y = 2.529x + 3.525 3.83 ± 0.38 y = 2.657x + 3.552 3.51 ± 0.51
II3 y = 1.756x + 2.925 15.20 ± 1.51 y = 4.078x + 1.244 8.33 ± 0.25 y = 1.195x + 3.647 13.54 ± 0.26
II4 y = 4.407x + 3.335 2.39 ± 0.17 y = 4.622x + 3.504 2.11 ± 0.21 y = 1.698x + 3.821 4.74 ± 0.71
II5 y = 2.264x + 2.919 8.30 ± 0.73 y = 2.846x + 2.590 7.02 ± 0.35 y = 1.663x + 3.465 4.23 ± 0.43
II6 y = 7.852x + 0.601 3.63 ± 0.28 y = 2.258x + 3.953 2.91 ± 0.03 y = 3.377x + 2.681 4.86 ± 0.27
II7 y = 1.512x + 4.333 2.76 ± 0.17 y = 4.147x + 1.690 6.28 ± 0.29 y = 3.316x + 2.309 6.48 ± 0.96
II8 y = 5.268x + 2.944 2.46 ± 0.15 y = 2.673x + 4.100 2.17 ± 0.04 y = 1.698x + 3.821 4.94 ± 0.64
II9 y = 6.690x + 3.470 1.69 ± 0.06 y = 1.986x + 3.794 4.05 ± 0.15 y = 3.069x + 3.837 2.39 ± 0.07
II10 y = 6.231x − 3.112 20.04 ± 0.19 y = 1.797x + 2.848 15.75 ± 0.57 y = 2.093x + 3.216 3.50 ± 0.73
II11 y = 1.457x + 4.172 3.67 ± 0.89 y = 2.720x + 3.583 3.32 ± 0.10 y = 1.931x + 3.618 5.20 ± 0.63
II12 y = 3.104x + 2.671 5.63 ± 0.49 y = 2.355x + 3.829 4.47 ± 0.12 y = 1.634x + 3.444 8.96 ± 1.31
II13 y = 2.743x + 3.402 3.83 ± 0.14 y = 2.351x + 3.406 4.76 ± 0.38 y = 1.752x + 3.347 8.78 ± 0.48
II14 y = 12.354x − 4.052 5.41 ± 0.53 y = 2.065x + 3.402 5.94 ± 0.83 y = 3.298x + 0.219 28.16 ± 2.97
II15 y = 2.629x + 2.861 6.51 ± 0.39 y = 4.448x + 2.048 4.61 ± 0.58 y = 1.099x + 4.258 4.73 ± 0.36
II16 y = 8.027x + 0.197 3.97 ± 0.24 y = 2.370x + 2.984 7.09 ± 0.33 y = 4.960x + 0.534 7.05 ± 0.66
II17 >100 y = 1.261x + 3.961 6.66 ± 1.03 y = 1.452x + 2.630 42.90 ± 0.19
TC y = 4.105x − 2.740 76.81 ± 2.22 y = 1.706x + 1.885 66.98 ± 0.49 y = 3.576x − 1.704 74.98 ± 3.49
BT y = 5.069x − 2.625 31.94 ± 3.59 y = 1.893x + 1.776 50.51 ± 2.08 y = 4.318x − 3.893 114.76 ± 3.93

Figure 4. Synthesis of target compounds III1−III4.

Table 3. In Vitro Bioassay Results of III1−III4 against Pathogens Xoo, Xac, and Psa
Xoo Xac Psa
comp. regression equation EC50 (μg/mL) regression equation EC50 (μg/mL) regression equation EC50 (μg/mL)
III1 (S) y = 5.402x + 4.428 1.28 ± 0.17 y = 2.274x + 4.140 2.39 ± 0.11 y = 9.164x + 1.831 2.22 ± 0.18
III2 (R) y = 8.996x + 3.734 28.28 ± 1.45 y = 2.620x + 3.261 4.61 ± 0.21 y = 1.645x + 3.760 5.76 ± 1.86
II9 (R/S) y = 6.690x + 3.470 1.69 ± 0.06 y = 1.986x + 3.794 4.05 ± 0.15 y = 3.069x + 3.837 2.39 ± 0.07
III3 (S) y = 5.769x + 1.881 3.47 ± 0.10 y = 3.475x + 3.724 2.33 ± 0.10 y = 0.818x + 5.068 0.82 ± 0.76
III4 (R) y = 4.212x + 3.018 2.95 ± 0.30 y = 3.104x + 3.520 2.99 ± 0.06 y = 1.765x + 4.437 2.09 ± 0.41
I23 (R/S) y = 4.034x + 3.303 2.63 ± 0.35 y = 2.039x + 4.361 2.06 ± 0.10 y = 3.466x + 2.829 4.23 ± 0.36
TC y = 4.105x − 2.740 76.81 ± 2.22 y = 1.706x + 1.885 66.98 ± 0.49 y = 3.576x − 1.704 74.98 ± 3.49
BT y = 5.069x − 2.625 31.94 ± 3.59 y = 1.893x + 1.776 50.51 ± 2.08 y = 4.318x − 3.893 114.76 ± 3.93

intermediate 2 under the condition of K2CO3 in isopropanol at antibacterial effects. The designed THC derivatives displayed
60 °C. The final frameworks of the synthesized compounds identified biological functions against the growth of pathogens:
were validated using the relevant nuclear magnetic resonance Xoo, Xac, and Psa (Table 1). However, introducing either the
and high-resolution mass spectra (Supporting Information). substituted piperidinyl (I1−I5) or morpholinyl (I6) patterns
The common turbidimetric test was used to screen the in vitro into the parent structure 1 cannot promote antibacterial
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Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

Table 4. In Vivo Control Efficiency (14 Days after Spraying) of II9 against Rice Bacterial Blight at 200 μg/mL
protective activity curative activity
chemicals morbidity (%) disease index (%) control efficiency (%)b morbidity (%) disease index (%) control efficiency (%)b
II9 100 40.47 51.89A 100 41.61 50.53A
TC 100 45.71 45.66B 100 46.15 45.14B
BT 100 43.21 48.62A 100 52.94 37.07B
CKa 100 84.13 100 84.13
a
Negative control. bStatistical analysis was conducted using the analysis of variance (ANOVA) method under the condition of equal variances
assumed (P > 0.05) and equal variances not assumed (P < 0.05). Different uppercase letters indicate the control efficiencies with significant
differences among different treatment groups at p < 0.05.

Figure 5. In vivo antibacterial activity of compound II9 against rice bacterial leaf blight at 200 μg/mL. BT and TC were the positive controls.

Table 5. In Vivo Control Efficiency (28 Days after Inoculation) of Compound II9 against Kiwifruit Bacterial Canker at 200 or
500 μg/mL on Kiwi Twigs
protective efficiency (%)a curative efficiency (%)a
chemicals 200 μg/mL 500 μg/mL 200 μg/mL 500 μg/mL
II9 65.45 ± 3.07A 76.77 ± 4.21A 30.35 ± 4.00A 56.12 ± 3.99A
TC 40.91 ± 3.02B 62.20 ± 5.43B 14.91 ± 1.93B 38.10 ± 2.19B
a
Statistical analysis was conducted using the ANOVA method under the condition of equal variances assumed (P > 0.05) and equal variances not
assumed (P < 0.05). Different uppercase letters indicate the control efficiencies with significant differences among different treatment groups at p <
0.05.

competence. By contrast, the ring-opening epoxy group of 2
with substituted piperazines presented differentiated effects on
bioactivity. When the substituents −H, −CH3, −CH2CH3,
−CH(CH3)2, and −COCH3 were located at the piperazine
ring, the antibacterial activity of compounds I7−I11 toward the
three pathogens significantly decreased compared with that of
the parent structure 1. However, only compound I7 presented
an EC50 value of 17.12 μg/mL against Xac. By changing the
substituent into a rigid phenyl group on the piperazine ring,
compound I12 exerted improved anti-Xoo and anti-Xac
activities, with EC50 values of 7.95 and 7.30 μg/mL,
respectively. The EC50 values of compound I12 were superior
to those of 1, I1−I11, TC, and BT, thereby suggesting that
careful optimization of the THC structure was required to
achieve highly bioactive compounds. Conversely, replacing the
benzene ring (I 12 ) into the pyrimidinyl group (I 13)
substantially reduced the anti-Xoo and anti-Xac effects and
presented high EC50 values of 39.15 and 45.62 μg/mL,
respectively, indicating that an aromatic N-heterocyclic group
was unacceptable to improve the bioactivity. Further
modification of compound I12 was performed. Inserting a
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methylene group (I14) between the benzene ring and the
piperazinyl group strongly enhanced the anti-Psa activity from
>100 μg/mL (I12) to 3.79 μg/mL and simultaneously
maintained the anti-Xoo and anti-Xac functions. Thus, the
contribution of substituted groups on the benzene ring toward
bioactivity should be investigated. Notably, compounds I15−I18
bearing the relevant 4-CH3, 2-CH3, 4-Cl, or 2-Cl patterns
presented comprehensive bioactivity toward the three
pathogens at EC50 values within 4.37−7.22, 2.07−4.74, and
3.36−6.40 μg/mL against Xoo, Xac, and Psa, respectively.
Compounds I19−I23 bearing various N-methylbenzylamines
were synthesized to study the effect of breakage of a rigid
piperazine ring on bioactivity. The bioassay results showed that
these compounds also exerted admirable biological effects. In
particular, compounds I22 (2,4-diCl) and I23 (4-CF3) provided
the lowest EC50 values of 2.25 and 2.06 μg/mL, respectively,
against Xoo and Xac. After changing the methyl group of I19
into a sterically hindered (S)-2-phenylethyl group, compound
I24 presented workable bioactivities against Xoo, Xac, and Psa
at 7.25, 2.34, and 4.92 μg/mL, respectively. Compound I25
bearing an N,N-diallylamino group showed a low antipathogen
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Figure 6. In vivo antibacterial activities of compound II9 against kiwifruit bacterial canker at 200 or 500 μg/mL on kiwi twigs. TC was the positive
control under the same conditions.
Figure 7. Curative and protective activities (21 days after inoculation)
of compound I23 against citrus bacterial canker on citrus leaves at 200
μg/mL.
ability. Overall, notable EC50 values of 2.25, 2.06, and 3.36 μg/
mL against Xoo, Xac, and Psa, respectively, were obtained by
elaborately optimizing the parent THC structure.
3.2. Synthesis and Antibacterial Activity of Further
Optimized Compounds II1−II17. Further modification based
on compounds I19, I20, I22, and I23 bearing various N-
methylbenzylamines was carried out. Consequently, com-
pounds II1−II17 bearing various secondary amine patterns
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pubs.acs.org/JAFC Article
were constructed (Figure 3). The biological results (Table 2)
showed that the removal of a methyl group from compounds
I19−I23 can also endow the modified compounds II1−II8 with
excellent bioactivity. In particular, II1 (4-CF3, NH) presented a
low EC50 value of 1.73 μg/mL against Xoo, and II5 (4-F, NH,
4.23 μg/mL) displayed improved anti-Psa activity. In addition,
the substituent position greatly influenced bioactivity, as
evidenced by the following bioactive order: meta-position (3-
F, II4, 2.39 and 2.11 μg/mL) > para-position (4-F, II5, 8.30
and 7.02 μg/mL) > ortho-position (2-F, II3, 15.20 and 8.33
μg/mL) against Xoo and Xac, para-position (4-F, II5, 4.23 μg/
mL) > meta-position (3-F, II4, 4.74 μg/mL) > ortho-position
(2-F, II3, 13.54 μg/mL) against Psa. These results indicated
that a substituent at the ortho-position of the benzene ring
blocked the antibacterial function. A strong bioactive molecule
against Xoo and Psa emerged when the ring-opening substitute
was replaced by 2-thienylmethylamine (II9), presenting EC50
values of 1.69 and 2.39 μg/mL, respectively. By contrast, the
anti-Xoo and anti-Xac abilities evidently decreased when the
ring-opening substitute was replaced by 2-furylmethylamine
(II10). Six molecules containing (S or R)-1-phenylethan-1-
amine substructures (II11−II16) were designed and synthesized
to investigate the effect of chiral amines on bioactivity. The
bioassay results revealed that the S-form configurations showed
better antibacterial capacity than those of R-form config-
urations (except for II15 and II16 against Xoo). Furthermore, a
control molecule II17 containing a phenylamino motif was
synthesized to explore the role of the methylene moiety in
bioactivity. However, removing the methylene pattern sharply
decreased the antibacterial ability compared with II6,
suggesting that this soft methylene linker played an important
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Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article

Figure 8. Apoptotic diagrams of Xoo (a−d) and Xac (e−h) triggered by elevated concentrations of II9 and I23, respectively. The related pathogens
were stained with propidium iodide and Annexin V−FITC.

Figure 9. Accumulation of reactive oxygen species in Xoo (a) and Xac (b) monitored using the ROS detection kit after treatment with elevated
dosages of II9 and I23, respectively.

Figure 10. Hypersensitive response (HR) induced by pretreated Psa with compound II9 (dosage = 4.78 μg/mL) on N. benthamiana.

role in ensuring the antibacterial potency. On the basis of the compounds II9 and I23 against Psa, Xac, and Xoo, the effect of
above-mentioned optimizations, low EC50 values of 1.69 and a single enantiomer on bioactivity must be explored. Thus, four
2.39 μg/mL (II9) against Xoo and Xac, respectively, were absolutely fixed configuration compounds III1−III4 were
discovered. synthesized and are illustrated in Figure 4. The bioassay
3.3. Fixed Chiral Compounds (III1 −III 4) toward results indicated that the fixed configuration had a remarkable
Bioactivity. Given the excellent performance of the racemic influence on the bioactivity (Table 3). For the compound
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Journal of Agricultural and Food Chemistry
containing a 2-thiophenemethylamine pattern, the EC50 values
followed the order: S-forms (III1: 1.28 μg/mL, Xoo; 2.39 μg/
mL, Xac; 2.22 μg/mL, Psa) < R/S-forms (II9: 1.69 μg/mL,
Xoo; 4.05 μg/mL, Xac; 2.39 μg/mL, Psa) < R-forms (III2:
28.28 μg/mL, Xoo; 4.61 μg/mL, Xac; 5.76 μg/mL, Psa),
suggesting that the title compound with an S-form positively
affected the antibacterial power. When the substituted group
was N-methyl-4-(trifluoromethyl)benzylamine, no significant
difference was observed in the anti-Xoo and anti-Xac activities.
By contrast, for the anti-Psa activity, the S-form (III3, 0.82 μg/
mL) exerted the best competence, and the R-form (III4, 2.09
μg/mL) was better than the R/S-form (I23, 4.23 μg/mL).
These results indicated that the combination of the two single
configurations was undesirable to bioactivity. Overall, the
compound with an S-form displayed good activity.
3.4. In Vivo Antibacterial Activity against Three
Bacterial Diseases. Given that compounds II9 and I23
showed excellent activities against Xoo, Psa, and Xac at low
EC50 values of 1.69 (II9), 2.39 (II9), and 2.06 (I23) μg/mL,
respectively, the in vivo bioactivity against the related three
bacterial diseases should be investigated. As shown in Table 4
and Figure 5, compound II9 presented good protective
(51.89%) and curative (50.53%) efficiencies toward rice
bacterial blight at 200 μg/mL, which exceeded those of TC
(45.66 and 45.14%, respectively) and BT (48.62 and 37.07%,
respectively). Moreover, compound II9 exhibited excellent in
vivo efficacy against kiwifruit bacterial canker (Table 5 and
Figure 6), providing protective effects of 65.45% (200 μg/mL)
and 76.77% (500 μg/mL), which were better than that of TC
(40.91%, 200 μg/mL; 62.20%, 500 μg/mL). Compound II9
also presented curative abilities of 30.35% (200 μg/mL) and
56.12% (500 μg/mL), which exceeded those of TC (14.91%,
200 μg/mL; 38.10%, 500 μg/mL). Clearly, compared with
those of the negative control and TC, compound I23 displayed
good curative and protective efficacies in reducing lesion
development of citrus bacterial canker (Figure 7). Almost no
lesion developed on perforation in the curative groups after
treatment with I23. These results offered promising valuable
references for managing the three bacterial diseases by using
these versatile THC derivatives.
3.5. Investigation of Antibacterial Mechanisms Using
Flow Cytometry. Flow cytometry was used to study the
apoptotic effects on Xoo and Xac triggered by the
corresponding bioactive compounds II9 and I23 in a dose-
dependent manner. The result showed that an evident
apoptotic behavior appeared on tested pathogens after
treatment with the designed compounds (Figure 8).
Compared with those of the untreated samples (0.2 and
1.2%, Figure 8a,e), the apoptotic Xoo and Xac cells of the
treated samples were 5.8 and 27.5%, respectively, at 12.5 μg/
mL (Figure 8b,f), 17.2 and 36.6%, respectively, at 25.0 μg/mL
(Figure 8c,g), and 46.1 and 55.9%, respectively, at 50.0 μg/mL
(Figure 8d,h). Moreover, the designed compounds could
strongly induce the generation of late apoptotic cells, with the
proportion changing from 0.1 to 43.7% for Xoo cells and from
0.9 to 34.4% for Xac cells. The reactive oxygen species (ROS)
in tested pathogens must be monitored using the ROS
detection kit because excessive ROS is a key marker that can
activate an apoptotic response.54 Notably, the amount of ROS
in pathogens triggered by THC compounds gradually
increased with the elevation of drug dosages (Figure 9). This
finding indicated that these THC compounds can break the
balance of redox systems of pathogens and subsequently
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pubs.acs.org/JAFC Article
induce the increment of ROS, eventually endowing the tested
bacteria with distinct apoptotic behaviors.
3.6. Growth Curve and Hypersensitive Response (HR)
Assays. Given that compound II9 had good protective efficacy
against the kiwifruit bacterial canker, we wondered if this
compound can alleviate the HR and pathogenicity of Psa. On
the basis of the growth curve of Psa (Figure S1), a final
concentration of 4.78 μg/mL (2 × EC50), which cannot affect
the normal growth of Psa, was selected to study HR. Evidently,
complete loss of HR and pathogenicity was observed after
pretreating Psa with compound II9 (Figure 10), suggesting that
THC compounds might target the type III secretion systems
(T3SS)55 of Psa. Further research on this hypothesis will be
carried out in our upcoming work.
In conclusion, novel THC derivatives containing a typical
1,3-diaminopropan-2-ol pattern were prepared to explore
innovative antibacterial frameworks. In vitro antibacterial
evaluations indicated that target compounds exhibited
excellent activities against three destructive phytopathogens,
namely, Psa, Xac, and Xoo, at corresponding optimal EC50
values of 2.39 (II9), 2.06 (I23), and 1.69 (II9) μg/mL,
respectively. These effects were remarkably superior to those of
the parent structure 1,2,3,4-THC, TC, and BT. In vivo assays
found that II9 exhibited excellent control efficiencies against
rice bacterial blight and kiwifruit bacterial canker, and I23
substantially relieved the citrus canker on the leaves.
Antibacterial mechanisms indicated that these THC com-
pounds could induce distinct apoptotic behaviors on
pathogens Xoo and Xac via probably breaking the balance of
their redox systems. Additionally, II9 could relieve the HR and
pathogenicity of Psa, which indicated that these THC
compounds might target the T3SS of Psa. On the basis of
the above promising results, these simple THC derivatives can
be explored in depth as workable antibacterial alternatives.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c02528.
Synthesis and characterization data (Figure S1); thermal
storage experiments (Figures S2−S4, Table S1); and
NMR and HRMS spectra of the intermediates and title
compounds (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
Pei-Yi Wang − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural 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; orcid.org/0000-0002-
9904-0664; Email: pywang888@126.com
Song Yang − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural 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; College of Pharmacy, East
China University of Science & Technology, Shanghai 200237,
China; orcid.org/0000-0003-1301-3030;
Email: jhzx.msm@gmail.com
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Authors
Hong-Wu Liu − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural 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
Qing-Tian Ji − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural 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
Gang-Gang Ren − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural 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
Fang Wang − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural 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
Fen Su − State Key Laboratory Breeding Base of Green Pesticide
and Agricultural 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
Xiang Zhou − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural 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
Zhi-Bing Wu − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural 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; orcid.org/0000-0002-
2724-9725
Zhong Li − College of Pharmacy, East China University of
Science & Technology, Shanghai 200237, China; orcid.org/
0000-0001-6665-5040
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c02528
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We acknowledge funds from the National Natural Science
Foundation of China (21662009, 21702037, 21877021,
31860516), the Research Project of Ministry of Education
(20135201110005, 213033A), the Guizhou Provincial S&T
Program (LH [2017]7259, [2017]5788), and the Key
Technologies R&D Program (2014BAD23B01).
■
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