Journal of Agricultural and Food Chemistry
Article
DCM was extracted and recrystallized with petroleum ether to obtain
intermediate B. Thiosemicarbazide (32.9 mmol), potassium hydroxide
(49.4 mmol), and carbon disulfide (49.4 mmol) were added to absolute
ethanol (60 mL) and reacted for 8−10 h. After the reaction was
completed, the reaction system was stirred in water and adjusted to
acidity with dilute hydrochloric acid. Intermediate C was collected by
suction filtration.31 Intermediate C (4.6 mmol) and potassium
carbonate (6.9 mmol) were stirred in DMF (2 mL) for 20 min. Next,
intermediate B (4.6 mmol) was added to the reaction system and
reacted for 2−6 h. Then, the reaction system was poured into water and
stirred until solid precipitation to obtain intermediate D. Intermediate
D (2.6 mmol) and triethylamine (3.8 mmol) were dissolved in DCM
(20 mL), and the differently substituted benzoyl chlorides (2.6 mmol)
were dropped slowly into the reaction flask and reacted for 1−6 h.
Finally, target compounds 1−4 were obtained by column chromatog-
raphy (Figure 2).
Preparation of Target Compounds 5−8. Differently substituted
aniline (2.6 mmol) and triethylamine (3.8 mmol) were dissolved in
DCM (20 mL), and chloroacetyl chloride (2.6 mmol) was dripped
slowly into the reaction flask and reacted for 2−6 h (Figure 3). Then,
intermediate E was obtained by extraction with DCM and
recrystallization with petroleum ether. Intermediate C (4.6 mmol)
and potassium carbonate (6.9 mmol) were stirred in DMF (2 mL), and
intermediate E (4.6 mmol) was added to the reaction system and
reacted for 2−6 h. After the reaction was completed, an appropriate
amount of water was added to wash the product, and then intermediate
F was obtained by filtration and recrystallization. (E)-2-(Methox-
yimino)-2-(o-tolyl)acetyl (2.6 mmol) was dropped slowly into the
DCM mixture of intermediate F (2.6 mmol) and triethylamine (3.8
mmol) to react for 1−6 h. Finally, crude target compounds 5−8 were
obtained by column chromatography.
Preparation of Target Compounds 9−30. Intermediate C (4.6
mmol) and potassium carbonate (6.9 mmol) were added to DMF (2
mL). Then, different halogenated hydrocarbons or benzyl chloride (4.6
mmol) was added to the above system, and the reaction was completed
after 2−6 h (Figure 4). An appropriate amount of water was added for
washing, and intermediate G was obtained by filtration. (E)-2-
(Methoxyimino)-2-(o-tolyl)acetyl chloride (2.6 mmol) was dropped
slowly into the DCM mixture of intermediate G (2.6 mmol) and
triethylamine (3.8 mmol). The reaction was completed after 1−6 h, and
the target compounds 9−22 were obtained by column chromatog-
raphy. Compounds 9−22 were oxidized in an ethanol mixture of 30%
hydrogen peroxide (8.0 mmol) and ammonium molybdate (0.15
mmol) for 3−6 h under ice bath conditions. Finally, target compounds
23−30 were obtained by column chromatography.
Antibacterial Activity In Vitro Assay. The antibacterial activities
in vitro of compounds 1−30 were tested by the turbidimetric
method.32,33 Thiodiazole copper was used as a positive control, and a
test solution containing no compound was used as a negative control.
All target compounds were dissolved in DMSO, diluted to the required
concentration with 0.1% Tween-20, and then added to a test tube
containing nutrient solution (NB) medium. Then, the test tube was
incubated with shaking for 24−36 h at 180 rpm and 28 °C, and the
absorbance (OD595) was measured at 595 nm. The test was repeated
three times, with three parallel tests each time.
Antibacterial Activity In Vivo Assay. The antibacterial activity in
vivo of compound 30 against BLB was evaluated.35,36 Compound 30
and a positive control were diluted into a test solution of 200 mg/L.
Thiodiazole copper was used as a positive control, and the same
concentration of the solution without the compound was used as a
negative control. The curative activity was tested according to the
following steps. The rice leaves of ″fengyouxiangzhan″ at the six- to
eight-leaf stage were inoculated with Xoo by the leaf-cutting method,
and the test solution was sprayed evenly on the rice leaves 24 h after
inoculation. The protective activity was tested similarly. After the test
solution was sprayed on rice leaves for 24 h, Xoo was inoculated on the
rice leaves. After 14 days of cultivation in the greenhouse, the lengths of
rice leaf lesions were measured, and the antibacterial activity of
compound 30 was calculated and analyzed using the disease index. The
test was repeated three times.
The antibacterial activity in vivo against BLS was evaluated using
previously reported methods.37 The curative activity was tested
according to the following steps. The rice leaves at the six- to eight-
leaf stage were inoculated with Xoc by the pressure penetration method.
When inoculating, the back of the leaf was facing up, 1 mL of the Xoc
solution was aspirated with a sterile syringe without a needle, and then
the bacterial solution was gently pressed into the leaves. After 24 h, the
test solution was sprayed onto the rice leaves, and the protective activity
was tested. After the test solution was sprayed on the rice leaves for 24 h,
Xoc was inoculated on the rice leaves. After 14 days of cultivation in the
greenhouse, the length of rice leaf lesions was measured, and the
equation below was used to calculate the antibacterial activity of
compound 30 against BLS. The test was repeated three times. In the
following equation, C and T mean the length of the rice leaf lesion for
the negative control and treatment groups, respectively.
Antibacterial activity I(%) = (C − T)/C × 100
Morphological Changes. According to previously reported
methods,38,39 the changes in the cell surface morphology after
compound 30 treatment were observed using SEM. The Xoo or Xoc
solution was washed three times and suspended in 1 mL of phosphate-
buffered saline (PBS). Next, the DMSO solution containing compound
30 was diluted to 200, 50, 25, and 5 mg/L using PBS and incubated at
28 °C for 10 h. The test solution lacking the compound was used as a
negative control. Cells were fixed with 2.5% glutaraldehyde for 8 h, and
then anhydrous ethanol with different concentration gradients was used
for dehydration. Finally, all the samples were observed after freeze-
drying and coating with gold.
Defensive Enzyme Activity. The changes in the contents of the
defensive enzymes (phenylalanine ammonia lyase (PAL), catalase
(CAT), superoxide dismutase (SOD), and peroxidase (POD)) were
calculated in rice leaves 1, 3, 5, and 7 days after rice infection with Xoc
under greenhouse conditions using relevant enzyme kits (Suzhou
Keming Biotechnology Co., Ltd.) for testing.
Differentially Expressed Protein Analysis. Preparation of Rice
Samples. Compound 30 was diluted into a test solution (200 mg/L) to
spray on rice leaves at the six- to eight-leaf stage. After the test solution
was sprayed for 24 h, rice were inoculated with Xoc. The negative
control was treated in the same way. Then, the rice samples were quick-
frozen and ground.
3D-QSAR Analysis. The CoMFA model was established on the
submitted. Twenty-one compounds were selected as the training set,
and eight compounds were used as the test set. The pEC50 values of all
compounds were uploaded to the server. Compound 2 was used as a the
template molecule. The partial least squares (PLS) regression approach
was used to perform CoMFA analysis. The cross-validation coefficient
(q2), non-cross-validation coefficient (r2), and corresponding non-
cross-validation coefficient of the prediction (r2pred) were used to
evaluate the CoMFA model. Predicted pEC50 values, steric field, and
electrostatic field models were obtained from the Cloud 3D-QSAR
server. The PyMol software (version 2.1) was used to visualize steric
and electrostatic field models.
Total Protein Extraction and Identification. The extraction and
identification of total rice protein were carried out according to our
previous works.40,41
Bioinformatics Analysis. The differentially expressed proteins
(DEPs) were analyzed and classified by Gene Ontology (GO)
annotations on the Kyoto Encyclopedia of Genes and Genomes
(KEGG) and Uniprot software. GO annotations can be divided into
three categories including cellular components, biological processes,
and molecular functions. Additionally, KEGG annotations were
pathway.html). For target lists with unlabeled proteomics results, the
GO database was downloaded as a backend list. The tags corresponding
to DEPs with differential expression levels >1.5 were marked in the GO
database, and the protein content of each GO term was calculated.
C
J. Agric. Food Chem. XXXX, XXX, XXX−XXX