Thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of heterocyclic compounds, and particularly relates to a thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative as well as a preparation method and application thereof. The invention provides a thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative with a novel structure. Researches and experiments prove that the compound has good inhibitory activity on a type III secretion system (T3SS) of rice bacterial blight, and can be used as a rice bacterial blight T3SS activity inhibitor. Part of the compounds can reduce the pathogenicity of Xanthomonas oryzae pv. Oryzae and do not affect the growth of Xanthomonas oryzae pv. Oryzae, so that the drug resistance of pathogenic bacteria can be delayed, and the effect of preventing and / or treating Xanthomonas oryzae pv. Oryzae can be achieved at the same time. Meanwhile, it is proved that the action target of the compound is CRP-like protein, and a basis is provided for further development and application of the compound.

Description

Technical Field

[0001] The present invention belongs to the technical field of heterocyclic compounds and more specifically relates to a thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative and its preparation method and application. Background Art

[0002] Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a global rice disease that poses a serious threat to global rice production. During epidemics, the disease can reduce rice yields by 20% to 30%, and in severe cases, by up to 50%, posing a significant challenge to food security.

[0003] Currently, the prevention and control of rice bacterial blight primarily relies on the cultivation of disease-resistant varieties and the use of chemical agents. While the promotion of resistant varieties is economical and environmentally friendly, the long-term, large-scale cultivation of a single resistant variety can lead to pathogen mutations under environmental selection pressure, resulting in the emergence of new pathogenic species and a gradual loss of resistance. Chemical control, on the other hand, remains the primary means of controlling the disease, with commonly used agents including thiophanate-methyl, sinozolin, and chlorothalonil. However, these traditional fungicides or antibiotics primarily work by inhibiting the growth of pathogens or disrupting their vital activities, and long-term use can easily lead to the emergence of drug-resistant strains. Studies have shown that Xoo strains in some areas have developed resistance to chlorothalonil and certain antibiotics, which has reduced the effectiveness of chemical control and increased the difficulty of disease prevention and control (Xu Ying. Monitoring and study of resistance mechanisms of Xanthomonas oryzae to streptomycin and thiothiazolinone [D]. Nanjing, Doctoral Dissertation of Nanjing Agricultural University, 2010.; Shen Guangbin, Zhou Mingguo. Monitoring of resistance of Xanthomonas oryzae to thiothiazolinone [J]. Plant Protection, 2002, 28(1): 9-11.).

[0004] In recent years, researchers have gradually shifted their focus to intervention strategies targeting pathogen virulence factors, rather than directly killing the pathogen. Compared to traditional methods, interfering with virulence factors (such as the type III secretion system) does not significantly affect pathogen growth, thereby significantly reducing the risk of developing resistant strains. The type III secretion system (T3SS) is a key virulence factor in many Gram-negative pathogens and plays a vital role in the pathogenicity of Xoo, responsible for injecting effector proteins into host cells to suppress plant immune responses. Studies have shown that inhibiting T3SS function can significantly weaken the pathogenicity of Xoo, while reducing the selection pressure on pathogens and delaying the development of drug resistance (Rasko DA, Sperandio V. Anti-virulence strategies to combat bacteria-mediated disease [J]. Nature Reviews Drug Discovery, 2010, 9 (2): 117-128.; Yuan X, Yu M, Yang C H. Innovation and application of the type III secretion system inhibitors in plant pathogenic bacteria [J]. Microorganisms, 2020, 8 (12): 1956.). Therefore, the development of new antimicrobial agents targeting T3SS is expected to provide new ideas for the green prevention and control of rice bacterial blight.

[0005] However, research on T3SS inhibitors for plant pathogens is still in the exploratory stage, and there is an urgent need to develop new compounds or biological agents that are highly efficient, low-toxic and environmentally friendly to meet the needs of sustainable agricultural development. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings that the research on T3SS inhibitors for plant pathogens is still in the exploratory stage and there is an urgent need to develop new compounds or biological agents that are highly efficient, low-toxic and environmentally friendly, and to provide a thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative.

[0007] The purpose of the present invention is to provide a method for preparing the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative.

[0008] Another object of the present invention is to provide the use of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative in the preparation of drugs for preventing or treating plant diseases.

[0009] Another object of the present invention is to provide the use of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative as a virulence factor T3SS inhibitor in rice bacterial blight pathogen.

[0010] Another object of the present invention is to provide a pesticide composition.

[0011] Another object of the present invention is to provide a small molecule probe based on thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivatives.

[0012] Another object of the present invention is to provide applications of the small molecule probe.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] The present invention protects a thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative, the structure of which is shown in formula (III):

[0015]

[0016] Wherein, said R is one or more, and said R is independently selected from hydrogen, halogen, nitro, hydroxyl, C 1～4 Alkyl, C 1～4 Halogenated alkyl, C 1～4 Alkoxy or C 1～4 Halogenated alkoxy.

[0017] Preferably, each of the R groups is independently selected from hydrogen, fluorine, chlorine, bromine, nitro, methyl or methoxy.

[0018] More preferably, R is selected from hydrogen, 2-nitro, 3-nitro, 4-nitro, 2-fluoro, 3-fluoro, 4-fluoro, 4-bromo, 2-chloro, 3-chloro, 4-chloro, 2,4-difluoro, 2,6-difluoro, 4-methyl or 4-methoxy.

[0019] Most preferably, said R is selected from 2-chloro.

[0020] Furthermore, the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative is replaced with a pharmaceutically acceptable salt or solvate thereof. In pharmacology, the activity of a compound is generally determined by the pharmacophore in its molecular structure, i.e., the key atoms or functional groups that are directly involved in the drug-target interaction. When a compound forms a pharmaceutically acceptable salt or solvate, its activity generally remains unchanged.

[0021] The present invention also provides a method for preparing the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative, which is characterized by comprising the following steps:

[0022] S1. In the presence of a diluent, the compound represented by formula Ⅰ is reacted with SOCl2 to prepare a compound of formula Ⅱ:

[0023]

[0024] S2. In the presence of a diluent and an acid binding agent, the compound of formula II is reacted with 2-mercaptothiophene to obtain a compound of formula III:

[0025]

[0026] Preferably, in step S1, the reaction temperature is 30-50°C.

[0027] Preferably, in step S1, the reaction time is 1 to 3 hours.

[0028] Preferably, in step S2, the reaction temperature is 0-5°C.

[0029] Preferably, in step S2, the reaction time is 1 to 3 hours.

[0030] Furthermore, in step S2, the reaction further includes post-treatment, which includes filtration, drying and purification by silica gel chromatography, specifically thin layer chromatography TLC monitoring. After the reaction is completed, the reaction is filtered and excess acid binding agent is removed. The filtrate is spin-dried to obtain a crude product, which is separated and purified by silica gel chromatography (ethyl acetate / V: petroleum ether / V=1:8) to obtain the target product.

[0031] Furthermore, the diluent in step S1 and step S2 is selected from inert organic solvents.

[0032] Preferably, the diluent is selected from at least one of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, petroleum ether, hexane, cyclohexane, dichloromethane, chloroform, carbon tetrachloride, ether, diisopropyl ether, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, acetone, butanone, methyl isobutyl ketone, acetonitrile, propionitrile, butyronitrile, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-formanilide, N-methylpyrrolidone, hexamethylphosphoric triamide, methyl acetate, ethyl acetate, dimethyl sulfoxide, methanol, ethanol, n-propanol, isopropanol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, diethylene glycol monomethyl ether and diethylene glycol monoethyl ether, preferably at least one of benzene, toluene and tetrahydrofuran.

[0033] As a more preferred embodiment, the diluent is selected from acetonitrile, toluene, acetone or tetrahydrofuran.

[0034] As a most preferred solution, the diluent is selected from acetonitrile.

[0035] The presence of an acid binding agent is beneficial to the reaction. Preferably, the acid binding agent is selected from potassium carbonate, sodium hydroxide, sodium ethoxide, triethylamine, trimethylamine, tributylamine, pyridine, N,N-dimethylaniline, N,N-dimethylbenzylamine, N-methylpiperidine, N-methylmorpholine, N,N-dimethylaminopyridine, diazabicyclooctane, diazabicyclononene or diazabicycloundecene.

[0036] More preferably, the acid binding agent is selected from potassium carbonate.

[0037] Furthermore, in step S2, the molar ratio of the compound represented by formula II, 2-mercaptothiophene and the acid binding agent is (1-2):(1-1.5):(1-3), preferably 1:1:2.

[0038] The present invention protects the use of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative in preparing medicines for preventing or treating plant diseases.

[0039] Furthermore, the plant disease is rice bacterial blight.

[0040] The present invention protects the application of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative as a virulence factor T3SS inhibitor in rice bacterial blight pathogen.

[0041] Furthermore, the target of the compound is CRP-like protein (cyclic adenosine monophosphate receptor protein, CRP-like protein, referred to as Clp).

[0042] Furthermore, some of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivatives serve as T3SS inhibitors of the virulence factor in Xanthomonas oryzae, and do not affect the growth of Xanthomonas oryzae.

[0043] The present invention protects a pesticide composition comprising one or more of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivatives.

[0044] The present invention protects a small molecule probe based on a thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative, wherein the small molecule probe has the following structure:

[0045]

[0046] The present invention also protects the use of the small molecule probe in determining the direct action target of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention provides a class of thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivatives with novel structures. Research and experiments have demonstrated that these compounds exhibit excellent inhibitory activity against the type III secretion system (T3SS) of rice bacterial blight and can be used as inhibitors of T3SS activity. Some of these compounds can also reduce the pathogenicity of rice bacterial blight pathogens while not affecting their growth, thereby delaying the development of drug resistance in the pathogens while simultaneously preventing and / or controlling rice bacterial blight. Furthermore, the present invention demonstrates that the target of these compounds is a CRP-like protein, providing a foundation for the further development and application of these compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Figure 2 is the growth curve of Xoo in M210 medium (A-B) and XOM2 medium (C-D) in the presence of compounds.

[0050] Figure 2 This is a statistical graph showing the effect of compound III-1 on the HR of Xoo in tobacco.

[0051] Figure 3 This is a statistical chart of the lesion length of mature rice plants after treatment with compound III-1.

[0052] Figure 4 This is the synthetic route of compound III-1 probe.

[0053] Figure 5 The growth curves of Xoo in M210 medium (left) and XOM2 medium (right) in the presence of probes.

[0054] Figure 6 This is the result of in vivo labeling of Xoo by the probe.

[0055] Figure 7 This is a diagram of the enrichment and purification of 25-35kDa protein in Xoo by probe III-1-Probe-2.

[0056] Figure 8 The electrophoresis diagram of recombinantly expressed Clp in vitro (left) and the statistical diagram of the dissociation constant between the compound and Clp (right). DETAILED DESCRIPTION

[0057] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0058] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0059] Example 1 Synthesis of Compound III-1

[0060] To a 50 mL single-necked flask, 0.44 g of 5-(2-chlorophenyl)-2-furoylcarboxylic acid (2 mmol), 2 mL of dichloromethane, and 5 mL of thionyl chloride were added in sequence; the mixture was heated (40°C) and refluxed for 1.5 hours. Heating was stopped, and the reaction solution was cooled to room temperature. The remaining thionyl chloride was evaporated under reduced pressure to obtain 5-(2-chlorophenyl)-2-furoyl chloride intermediate (2 mmol), which was used directly in the next step without purification.

[0061] In a separate 50mL single-necked flask, 4mmol of KCO was added. An appropriate amount of acetonitrile was added to dissolve the mixture, and 2mmol of 2-mercaptothiophene was drawn up using a syringe and added to the single-necked flask. The mixture was then allowed to react at 0°C for 30 minutes. A solution of 5-(2-chlorophenyl)-2-furoyl chloride in acetonitrile was then added dropwise using a constant-pressure dropping funnel. After the addition was complete, the mixture was allowed to react at room temperature for 2 hours. After the reaction was complete, the mixture was filtered and the excess KCO was removed. The filtrate was then dried to obtain the crude product, which was separated and purified by silica gel column chromatography (ethyl acetate / v:petroleum ether / v = 1:8) to obtain 0.75g of S-2-thiophene-5-(2-chlorophenyl)-2-furoylcarboxylic acid thioester as a pale yellow powder, Compound III-1, in a 78% yield.

[0062] 1 H NMR(600MHz,Chloroform-d)δ8.00(d,J=7.9Hz,1H),7.62(d,J=5.3Hz,1H),7.49(d,J=8.0Hz,1H),7.41 -7.37(m,2H),7.32(t,J=7.6Hz,1H),7.28(dd,J=7.0,3.7Hz,2H),7.18-7.15(m,1H).HR-MS(ESI):Calcd for C 15 H9ClO2S2[M+Na + ]:342.96247,Foμnd:342.96246

[0063]

[0064] Example 2 Synthesis of Compound III-2

[0065] The method is the same as Example 1, except that 5-(3-chlorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain an orange powder, namely compound III-2.

[0066] 1H NMR(600MHz,Chloroform-d)δ7.76(s,1H),7.66(s,1H),7.61(t,J=4.5Hz,1H),7.4 0-7.31(m,4H),7.15(q,J=4.2Hz,1H),6.82(t,J=3.8Hz,1H).HR-MS(ESI):Calcdfor C 15 H9ClO2S2[M+Na + ]:342.96247,Foμnd:342.96249

[0067]

[0068] Example 3 Synthesis of Compound III-3

[0069] The method is the same as Example 1, except that 5-(4-chlorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain a brown solid, namely compound III-3.

[0070] 1 H NMR(600MHz,Chloroform-d)δ7.75-7.73(m,2H),7.62(dd,J=5.3,1.3Hz,1H),7.43(d,J=2.0Hz,2H),7.34(d,J= 3.7Hz,1H),7.27(dd,J=3.6,1.3Hz,1H),7.17(dd,J=5.4,3.6Hz,1H),6.81(d,J=3.7Hz,1H).HR-MS(ESI):Calcd for C 15 H9ClO2S2[M+Na + ]:342.96247,Foμnd:342.96246

[0071]

[0072] Example 4 Synthesis of Compound III-4

[0073] The method is the same as Example 1, except that 5-(2-fluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain a light yellow solid, namely compound III-4.

[0074] 1H NMR(600MHz,Chloroform-d)δ8.00(td,J=7.7,1.8Hz,1H),7.62(dd,J=5.4,1.3Hz,1H),7.39-7.36 (m,2H),7.28(dt,J=5.7,1.8Hz,2H),7.19-7.16(m,2H),7.01(t,J=3.6Hz,1H).HR-MS(ESI):Calcd for C 15 H9FO2S2[M+Na + ]:326.99202,Foμnd:326.99155

[0075]

[0076] Example 5 Synthesis of Compound III-5

[0077] The method is the same as Example 1, except that 5-(3-fluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain light yellow powder III-5.

[0078] 1 H NMR(600MHz,Chloroform-d)δ7.62(dd,J=5.3,1.3Hz,1H),7.59(ddd,J=7.8,1.6,0.9Hz,1H),7.50(ddd,J=9.6,2.6,1.6Hz,1H),7.42(td,J=8.1,5.7Hz,1H), 7.34(d,J=3.8Hz,1H),7.28(dd,J=3.6,1.3Hz,1H),7.17(dd,J=5.4,3.6Hz,1H ),7.09(tdd,J=8.5,2.6,0.9Hz,1H),6.84(d,J=3.7Hz,1H).HR-MS(ESI):Calcd for C 15 H9FO2S2[M+Na + ]:326.99202,Foμnd:326.99191

[0079]

[0080] Example 6 Synthesis of Compound III-6

[0081] The method is the same as Example 1, except that 5-(4-fluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain a light yellow powder, namely compound III-6.

[0082] 1H NMR(600MHz,Chloroform-d)δ7.82-7.76(m,2H),7.62(dd,J=5.4,1.3Hz,1H),7.34(d,J=3.7Hz ,1H),7.27(dd,J=3.6,1.3Hz,1H),7.19-7.12(m,3H),6.76(d,J=3.8Hz,1H).HR-MS(ESI):Calcd for C 15 H9FO2S2[M+Na + ]:326.99202,Foμnd:326.99207

[0083]

[0084] Example 7 Synthesis of Compound III-7

[0085] The method is the same as Example 1, except that 5-(2,4-fluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain a brown solid, namely compound III-7.

[0086] 1 H NMR(600MHz,Chloroform-d)δ7.98(td,J=8.6,6.3Hz,1H),7.62(dd,J=5.4,1.3Hz,1H),7.37(d,J=3.8Hz,1H),7.28(dd,J=3 .6,1.3Hz,1H),7.17(dd,J=5.4,3.6Hz,1H),7.02(dddd,J=8.6,7.6,2.5,1.0Hz,1H),6.98-6.91(m,2H).HR-MS(ESI):Calcd forC 15 H8F2O2S2[M+Na + ]:344.98260,Foμnd:344.98260

[0087]

[0088] Example 8 Synthesis of Compound III-8

[0089] The method is the same as Example 1, except that 5-(2,6-fluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain an orange-red solid, namely compound III-8.

[0090] 1H NMR(600MHz,Chloroform-d)δ7.61(dd,J=5.4,1.3Hz,1H),7.37-7.33(m,2H),7.27(dd,J=3.6,1.3Hz,1 H),7.16(dd,J=5.3,3.6Hz,1H),7.04(t,J=8.7Hz,2H),7.00(dt,J=3.7,1.8Hz,1H).HR-MS(ESI):Calcd for C 15 H8F2O2S2[M+Na + ]:344.98260,Foμnd:344.98230

[0091]

[0092] Example 9 Synthesis of Compound III-9

[0093] The method is the same as Example 1, except that 5-(2-nitrophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain a yellow solid, namely compound III-9.

[0094] 1 H NMR(600MHz,Chloroform-d)δ7.83-7.80(m,2H),7.67(t,J=7.7Hz,1H),7.61(d,J=5.4Hz,1H),7.56(t,J=7.8Hz,1H),7.3 2(dd,J=3.8,1.2Hz,1H),7.25(s,1H),7.15(ddd,J=5.2,3.6,1.3Hz,1H),6.79(dd,J=3.7,1.2Hz,1H).HR-MS(ESI):Calcd forC 15 H9NO4S2[M+Na + ]:353.98652,Foμnd:353.98596

[0095]

[0096] Example 10 Synthesis of Compound III-10

[0097] The method is the same as Example 1, except that 5-(3-nitrophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain a light yellow powder, namely compound III-10.

[0098] 1H NMR(600MHz,Chloroform-d)δ8.60(t,J=2.0Hz,1H),8.23(ddd,J=8.3,2.3,1.0Hz,1H),8.13(ddd,J=7.8,1.7,1.0Hz,1H),7.68-7.6 2(m,2H),7.38(d,J=3.7Hz,1H),7.29(dd,J=3.6,1.3Hz,1H),7.18(dd,J=5.3,3.6Hz,1H),6.99(d,J=3.7Hz,1H).HR-MS(ESI):Calcd for C 15 H9NO4S2[M+H + ]:332.00458,Foμnd:332.00403

[0099]

[0100] Example 11 Synthesis of Compound III-11

[0101] The method is the same as Example 1, except that 5-(4-nitrophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain an orange-yellow solid, namely compound III-11.

[0102] 1 H NMR(600MHz,Chloroform-d)δ8.34-8.30(m,2H),7.97-7.94(m,2H),7.64(dd,J=5.4,1.3Hz,1H),7.38(d,J=3 .8Hz,1H),7.29(dd,J=3.6,1.3Hz,1H),7.18(dd,J=5.4,3.6Hz,1H),7.02(d,J=3.7Hz,1H).HR-MS(ESI):Calcd for C 15 H9NO4S2[M+H + ]:332.00458,Foμnd:332.00436

[0103]

[0104] Example 12 Synthesis of Compound III-12

[0105] The method is the same as Example 1, except that 5-(4-bromophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain an orange-red solid, namely compound III-12.

[0106] 1H NMR(600MHz,Chloroform-d)δ7.66(d,J=8.4Hz,2H),7.62(d,J=5.4Hz,1H),7.58(d,J=8.4Hz,2H),7.33(d, J=3.6Hz,1H),7.27(d,J=3.6Hz,1H),7.16(dd,J=5.4,3.6Hz,1H),6.81(d,J=3.7Hz,1H).HR-MS(ESI):Calcd for C 15 H9BrO2S2[M+Na + ]:386.91195,Foμnd:386.91165

[0107]

[0108] Example 13 Synthesis of Compound III-13

[0109] The method is the same as Example 1, except that 5-(4-methylphenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain a light pink solid, namely compound III-13.

[0110] 1 H NMR(600MHz,Chloroform-d)δ7.72-7.69(m,2H),7.61(dd,J=5.4,1.3Hz,1H),7.34(d,J=3.7Hz,1H),7.28-7 .26(m,2H),7.25(s,1H),7.16(dd,J=5.4,3.6Hz,1H),6.76(d,J=3.7Hz,1H),2.40(s,3H).HR-MS(ESI):Calcd for C 16 H 12 O2S2[M+Na + ]:323.01709,Foμnd:323.01715

[0111]

[0112] Example 14 Synthesis of Compound III-14

[0113] The method is the same as Example 1, except that 5-(4-methoxyphenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain an orange-yellow solid, namely compound III-14.

[0114] 1H NMR(600MHz,Chloroform-d)δ7.76-7.73(m,2H),7.61(dd,J=5.4,1.3Hz,1H),7.34(d,J=3.7Hz,1H),7.27(dd,J=3.6 ,1.3Hz,1H),7.16(dd,J=5.4,3.6Hz,1H),6.98-6.96(m,2H),6.69(d,J=3.7Hz,1H),3.86(s,3H).HR-MS(ESI):Calcd for C 16 H 12 O3S2[M+Na + ]:339.01201,Foμnd:339.01227

[0115]

[0116] Example 15 Synthesis of Compound III-15

[0117] The method is the same as Example 1, except that 5-phenyl-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain a light yellow solid, namely compound III-15.

[0118] 1 H NMR(600MHz,Chloroform-d)δ7.83-7.81(m,2H),7.62(dd,J=5.3,1.3Hz,1H),7.46(t,J=7.6Hz,2H),7.41-7.38(m,1H),7 .35(d,J=3.8Hz,1H),7.28(dd,J=3.6,1.3Hz,1H),7.17(dd,J=5.4,3.6Hz,1H),6.82(d,J=3.8Hz,1H).HR-MS(ESI):Calcd forC 15 H 10 O2S2[M+Na + ]:309.00144,Foμnd:309.00122

[0119]

[0120] Example 16 Inhibitory activity of the compound represented by formula III on the XooT3SS hpa1 gene promoter

[0121] Test pathogen: Hpa1 in Xanthomonas oryzae pv.oryzae PXO99A, test concentration: 10 mg / mL.

[0122] Activate hpa1 in PXO99 on PSA-resistant plates containing ampicillin at a final concentration of 100 μg / mL. A After 3 days, single colonies were placed in M210 rich medium and ampicillin was added. The culture was carried out at 28 °C and 200 rpm until the OD 600 The bacterial solution was transferred into fresh M210 medium at a ratio of 1:50 and cultured until the OD 600 The OD value is about 0.6. The bacterial suspension was centrifuged at 8000 rpm for 10 min, the cells were collected and washed twice with XOM2. The cells were centrifuged at 8000 rpm for 10 min, the supernatant was removed and resuspended in XOM2 medium, and ampicillin was added. 600 Adjust the concentration to 0.3, then add the compound to a final concentration of 10 μg / mL. An equal volume of DMSO was used as a solvent control. The experiment was repeated in triplicate. Induction was carried out overnight at 28°C, 200 rpm, for 16 hours. After induction, 2 mL of bacterial culture was aspirated and centrifuged at 12,000 rpm for 1 minute to remove the supernatant. The cells were washed twice with 1 mL of 0.01 mol / L PBS (pH 7.4) and resuspended in 2 mL of PBS. GFP mean fluorescence intensity (MFI) was measured using a flow cytometer (Beckman Coulter, USA).

[0123] Table 1 Inhibitory activity of compounds on the hpa1 gene promoter

[0124]

[0125]

[0126] Note: Inhibition rate = [1-AvgMFI (compound) / AvgMFI (DMSO)] × 100%.

[0127] The results showed that the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivatives of the present invention all had inhibitory activity on the hpa1 gene promoter. Among them, except for III-2, III-7 and III-12, the inhibitory activity of the other nine compounds on the hpa1 gene promoter was better than that of the positive control TS006 (o-hydroxycinnamic acid).

[0128] Example 17 Growth curve test of Xoo under compound treatment

[0129] Pick Xoo PXO99 A Wild type cells were cultured overnight to OD 600 The cells were collected by centrifugation at 4000 rpm for 10 min and the OD 600Adjust to 0.1 and resuspend in rich medium M210 and induction medium XOM2 respectively. Since Xoo grows very slowly in XOM2, 0.5% sucrose needs to be added exogenously to ensure normal bacterial growth (Tsuge S, Furutani A, Ikawa Y. Regulatory network of hrp gene expression in Xanthomonas oryzaepv.oryzae[J]. Journal of General Plant Pathology, 2014, 80(4): 303-313.). Add the compound at a final concentration of 10 μg / mL respectively, and an equal volume of DMSO as a solvent control. After pipetting and mixing, add the mixture to the growth curve plate, adding 200 μL to each well. Set up 4 parallel replicates for each treatment, and the experiment is repeated more than three times independently.

[0130] The growth of Xoo in M210 rich medium and XOM2 poor medium in the presence of compounds is shown in Figure 1 Compared with the wild-type and solvent controls, although III-11 and III-12 did not affect bacterial growth in rich culture medium, they significantly inhibited bacterial growth in poor culture medium, indicating that they can kill bacteria when they are relatively fragile. Compound III-1 did not affect bacterial growth in either culture medium or at any stage of bacterial growth, which better meets the requirements of a T3SS inhibitor and can therefore be used as a potential T3SS inhibitor for subsequent studies.

[0131] Example 18 Tobacco Allergy

[0132] Pick Xoo PXO99 A Wild type cells were cultured overnight to OD 600 The cells were collected by centrifugation at 12,000 rpm for 2 minutes and resuspended in an equal volume of sterile water. Compound III-1 was added to a final concentration of 10 μg / mL. An equal volume of DMSO was used as a solvent control, and sterile water was used as a blank control. The mixture was incubated in a 28°C incubator for 2 hours. After incubation, 5 μL of the bacterial solution was injected into each tobacco leaf using a 1 mL sterile syringe. Disease development was observed after one week.

[0133] Xoo treated with compound Ⅲ-1 induced allergic reactions to tobacco. Figure 2 Compared to the DMSO control, compound III-1 significantly inhibited the hypersensitive responses (HR) of Xoo on tobacco, further confirming compound III-1 as a T3SS inhibitor. CK represents the blank control, WT represents the wild type, and WT+DMSO represents the solvent control.

[0134] Example 19 Detection of Xoo Pathogenicity to Rice under Compound III-1 Treatment

[0135] The strain culture and compound treatment methods were the same as those in Example 18. After incubation, the leaves were inoculated by cutting them 3-4 cm below the leaf tip using scissors dipped in the bacterial solution. 15 leaves were inoculated for each treatment, and the disease was observed after half a month.

[0136] Depend on Figure 3 It can be seen that compared with the solvent control DMSO, compound III-1 significantly reduced the toxicity of Xoo on rice, and the effect was better than the positive control TS006.

[0137] Example 20 Study on the Target of Test Compounds against Xanthomonas oryzae

[0138] Based on the principles of activity-based protein profiling (ABPP), a series of small molecule probes were generated using compound III-1 as a precursor by adding an alkyne group to the benzene ring of the parent structure. Negative probes (NPs) were also designed and synthesized to serve as negative controls. By testing the inhibitory activity of the probes against the Xoo hpa1 gene promoter, small molecule probes with high inhibitory activity were screened.

[0139] Using a small molecule probe with high inhibitory activity, we conducted in vivo experiments on Xanthomonas oryzae through click reactions, polyacrylamide gel electrophoresis, and Streptavidin blot experiments to analyze the probe's potential targets. We then used pμll down experiments and mass spectrometry to identify the probe's specific targets. The specific experimental process is as follows:

[0140] 20.1 Synthesis of Compound III-1 Probes (III-1-Probe-1, III-1-Probe-2, and III-1-NP)

[0141] The reaction route is as follows Figure 4 Taking III-1-Probe-1 as an example, the specific synthesis steps of compound III-1 probe are described:

[0142] To a 100mL three-necked flask equipped with a thermometer, add 0.04mol of 2-chloro-5-methylaniline in water while stirring continuously with a glass rod to fully dissolve the mixture. Next, slowly add 12mL of concentrated hydrochloric acid. Place the reaction in a cryogenically controlled reaction tank at 0-5°C. Once the temperature stabilizes, slowly add 4mol / L sodium nitrite solution dropwise to the reaction flask. Use pH paper to test until the pH turns blue, then stop adding the solution. Continue stirring for 1 hour, then add 1.2g of copper chloride and 0.04mol of furanic acid. After reacting at room temperature for 3 hours, remove the solvent by filtration. Adjust the pH of the filter cake to a weakly alkaline state with saturated NaHCO₃ solution and filter again. The filter cake was washed and extracted with ethyl acetate. After extraction, the ethyl acetate layer solution was discarded. The remaining solid and the aqueous layer solution after extraction were adjusted to acidic pH with dilute hydrochloric acid and then filtered. The solid was recrystallized from ethanol:water = 1:1 to obtain the intermediate 5-(2-chloro-5-methylphenyl)-2-furancarboxylic acid in a yield of 70%.

[0143] To a 50 mL single-necked flask, 10 mmol of 5-(2-chloro-5-methylphenyl)-2-furancarboxylic acid and an appropriate amount of thionyl chloride were added sequentially, and the mixture was refluxed at 110°C for 3 hours. After the reaction was completed, the remaining thionyl chloride was removed by vacuum distillation, and an appropriate amount of dichloromethane was added. The mixture was placed in an ultra-low temperature constant temperature reaction tank at -10°C. After the temperature stabilized, 5 mL of anhydrous ethanol was slowly added dropwise. After reacting for 1 hour, the solvent was removed by vacuum distillation. The product was separated and purified by silica gel column chromatography (ethyl acetate / V:petroleum ether / V=1:8) to obtain the intermediate 5-(2-chloro-5-methylphenyl)-2-furanethyl ester in a yield of 90%.

[0144] To a 100mL single-necked flask, 18mmol NBS (N-bromosuccinimide), 4.5mmol AIBN (azobisisobutyronitrile), 9mmol 5-(2-chloro-5-methylphenyl)-2-furyl ester, and an appropriate amount of anhydrous carbon tetrachloride solution were added sequentially. The mixture was refluxed at 80°C for 48 hours under a nitrogen atmosphere. After the reaction, the solid was removed by filtration, and the filtrate was distilled under reduced pressure to obtain a crude product. This was then separated and purified by silica gel column chromatography (ethyl acetate / V:petroleum ether / V = 1:14) to obtain the intermediate containing benzyl bromide.

[0145] In a 100mL single-necked flask, add 9mmol of dry sodium hydride and dry tetrahydrofuran, stir evenly on ice, and slowly add 6mmol of propargyl alcohol via syringe under nitrogen protection. After the reaction solution reacts on ice for 0.5h, the benzyl bromide intermediate (4.5mmol) is added and the reaction is allowed to react at room temperature for 24h. After the reaction is complete, vacuum distillation is performed to obtain a crude product containing alkyne. The crude product is basified with aqueous sodium hydroxide solution. After pH test paper is used to check the reaction solution to be alkaline, it is extracted with ethyl acetate. The resulting aqueous layer is acidified with concentrated HCl, pH test paper is used to check the reaction solution to be acidic, and it is extracted with ethyl acetate. After extraction, the organic layer is vacuum distilled to obtain phenylfuran carboxylic acid containing alkyne.

[0146] In a 50 mL single-necked flask, add 2 mmol of alkyne-containing furanoic acid and an appropriate amount of thionyl chloride, followed by reflux at 110°C for 3 h. After the reaction is complete, remove the remaining thionyl chloride by vacuum distillation to obtain the acid chloride intermediate.

[0147] In a separate 50mL single-necked flask, add 4mmol of potassium carbonate, an appropriate amount of acetonitrile, and 2-mercaptothiophene in sequence. After reacting at -10°C for 30 minutes, slowly add the acetonitrile solution of the acid chloride intermediate dropwise using a separatory funnel. Once complete, allow to react at room temperature for 5 hours. After completion of the reaction, filter and evaporate under reduced pressure to obtain the crude product, which is then separated and purified by silica gel column chromatography (ethyl acetate / V:petroleum ether / V = 1:20) to obtain III-1-Probe-1. The synthesis method of III-1-Probe-2 is the same as that of III-1-Probe-1.

[0148] In the synthesis of III-1-NP, after obtaining the alkyne-containing acyl chloride intermediate, an appropriate amount of dichloromethane was added and the reaction was placed in an ultra-low temperature constant temperature reaction tank at -10°C. After the temperature was constant, 2 mL of anhydrous ethanol was slowly added dropwise. After the reaction was continued for 1 hour, the solvent was removed by vacuum distillation and the product was separated and purified by silica gel chromatography (ethyl acetate / V: petroleum ether / V = 1:20).

[0149] III-1-Probe-1:

[0150] 1 H NMR(600MHz,Chloroform-d)δ7.9542(d,J=2.1Hz,1H),7.6267(dd,J=5.3,1.3Hz,1H),7.4787(d,J=8.2Hz,1H),7.3766(d,J=3.7Hz,1H),7.3452 (dd,J=8.2,2.0Hz,1H),7.2971-7.2748(m,2H),7.1709(dd,J=5.3,3.6Hz,1H),4.6631(s,2H),4.2363(d,J=2.4Hz,2H),2.5086(t,J=2.4Hz,1H).

[0151] III-1-Probe-2:

[0152] 1H NMR(600MHz,Chloroform-d)δ7.9826(d,J=8.1Hz,1H),7.6213(dd,J=5.3,1.3Hz,1H),7.5072(s,1H),7.3779-7.3600(m,2H ),7.2928-7.2703(m,2H),7.1660(dd,J=5.4,3.6Hz,1H),4.6327(s,2H),4.2338(d,J=2.4Hz,2H),2.5067(t,J=2.4Hz,1H).

[0153] III-1-Probe-NP:

[0154] 1 H NMR (600MHz, DMSO-d6) δ7.8713(d,J=8.1Hz,1H),7.5593(s,1H),7.4639(d,J=6.3Hz,1H),7.4413(d,J=3.7Hz,1H),7.2671(d,J =3.7Hz,1H),4.5909(s,2H),4.3436-4.3122(m,2H),4.2478(d,J=2.4Hz,2H),3.5272(t,J=2.4Hz,1H),1.3183(t,J=7.1Hz,3H).

[0155] 20.2 Probe inhibition activity on the hpa1 gene promoter and growth curve test

[0156] Specific experimental operations are Example 16 and Example 17.

[0157] As shown in Table 2 and Figure 5 As shown, among the probes synthesized with Ⅲ-1 as the mother structure, the inhibitory activities of Ⅲ-1-Probe-1 and Ⅲ-1-Probe-2 on the hpa1 gene promoter are not much different from those of the mother structure Ⅲ-1 and do not affect the growth of Xoo, indicating that the introduction of the alkyne group has no effect on the mother structure, and the two probes can be used for the next experiment.

[0158] Table 2 Inhibitory activity of compounds and probes on the hpa1 gene promoter

[0159]

[0160]

[0161] Note: Inhibition rate = [1-AvgMFI (compound) / AvgMFI (DMSO)] × 100%.

[0162] 20.3 In vivo labeling experiment of total protein of Xanthomonas oryzae using probe

[0163] Prepare 100 mL of OD 600 The wild-type strain is 0.6-0.8Xoo. Bacteria were collected by centrifugation at 8000 rpm for 20 minutes, washed twice with 0.01 mol / L PBS, and resuspended in 60-70 mL of PBS. For each treatment, 10 mL of bacterial suspension was added to Ⅲ-1-Probe-1, Ⅲ-1-Probe-2, and Ⅲ-1-Probe-NP at a final concentration of 10 μg / mL. An equal volume of DMSO was used as a solvent control. After incubation at 28°C and 220 rpm for 2 hours, the cells were collected by centrifugation at 8000 rpm for 20 minutes at 4°C, resuspended in 10 mL of PBS containing 100 μL of protease inhibitors, and disrupted using an ultrasonic cell disruptor.

[0164] After the disruption, centrifuge at 4°C and 8000 rpm for 1 h to collect the supernatant, filter the supernatant into a 10 kDa ultrafiltration tube using a 0.45 μM filter membrane, and concentrate the protein by centrifugation at 4°C and 8000 rpm to a concentration of approximately 1 mg / mL.

[0165] To 45 μL of 1 mg / mL Xoo protein, add click reagent containing 1 μL of 0.25 M NaASC, 1 μL of 10 mM BiotinPEG3-N3, and 3 μL of a mixture of BTTAA / CuSO4 (50 mM BTTAA / V:50 mM CuSO4 / V = 2:1). Incubate at 37°C in a shaker in the dark for 2 hours. After incubation, add 15 μL of 5× SDS loading buffer to the mixture and heat in a boiling water bath at 100°C for 10 minutes. Then, perform 20 μL of the mixture on a 12% SDS-PAGE separating gel at 100 V for 1.5 hours. After electrophoresis, stain one gel with Coomassie Brilliant Blue and transfer the other gel to a NC membrane at 250 mA on ice for 1.5 hours. After transfer, rinse the membrane three times with PBST containing 0.05% Tween 20 for 15 minutes each. Block the membrane with PBST containing 5% skim milk powder for 2 hours and rinse three times with PBST containing 0.05% Tween 20 for 15 minutes each. Next, add HRP-conjugated streptavidin (1:1000) to 20 mL of PBST containing 5% skim milk powder. Incubate the membrane with this mixture at 4°C in the dark for 2 hours. After incubation, rinse three times with PBST containing 0.05% Tween 20 for 15 minutes each. Finally, add a colorimetric solution for chemiluminescence, and analyze the images using a Bio-Rad imaging system.

[0166] Depend on Figure 6It can be seen that compared to the blank control DMSO, III-1-Probe-2 at a concentration of 10 μg / mL significantly labels proteins in the 25-35 kDa range in Xoo, while the negative probe showed no obvious labeled band at 10 μg / mL. Compared to probe III-1-Probe-1, probe III-1-Probe-2 exhibits bands at 25-35 kDa, indicating that probe III-1-Probe-2 exhibits higher reactivity toward proteins in the 25-35 kDa range. Therefore, III-1-Probe-2 was selected for subsequent target research experiments on Xoo. Here, P1 represents III-1-Probe-1, P2 represents III-1-Probe-2, D represents the solvent control DMSO, and NP represents the negative probe.

[0167] 20.4 In vivo pull-down experiments and mass spectrometry analysis of Xoo

[0168] Prepare 100 mL of OD 600 The wild-type strain of 0.6-0.8Xoo was obtained. Cells were harvested by centrifugation at 8000 rpm for 20 minutes, washed twice with 0.01 mol / L PBS, and resuspended in 60 mL of PBS. 10 mL of each treatment was supplemented with probes III-1-Probe-2 and III-1-Probe-NP at a final concentration of 10 μg / mL, respectively. An equal volume of DMSO was used as a solvent control. After incubation at 28°C and 220 rpm for 2 hours, cells were harvested by centrifugation at 8000 rpm for 20 minutes at 4°C, washed twice with PBS, and resuspended in 10 mL of PBS containing 100 μL of protease inhibitors. Cells were disrupted using an ultrasonic cell disruptor. After disruption, the supernatant was collected by centrifugation at 8000 rpm at 4°C for 1 hour, filtered through a 0.45 μM filter membrane into a 10 kDa ultrafiltration tube, and the protein was concentrated by centrifugation at 8000 rpm at 4°C to a concentration of approximately 1 mg / mL.

[0169] 950 μL of 1 mg / mL Xoo total protein was added to a click reagent containing 10 μL of 0.25 M NaASC, 10 μL of 10 mM BiotinPEG3-N3, and 30 μL of a BTTAA / CuSO4 (50 mM BTTAA / V:50 mM CuSO4 / V = 2:1) mixture. The cells were incubated at 37°C in a shaker in the dark for 2 hours. After incubation, 5 mL of pre-chilled acetone was added and the cells were frozen at -80°C overnight. The next day, the protein was collected by centrifugation at 12,000 rpm for 2 minutes at 4°C. The protein precipitate was washed three times with pre-chilled methanol. After the methanol evaporated, 1 mL of 0.2% SDS / PBS was used to dissolve the protein precipitate. An equal volume of streptavidin affinity magnetic beads was then added and incubated at room temperature in the dark for 2 hours. After incubation, place the protein-magnetic bead mixture on a magnetic rack, remove the supernatant, and then wash the mixture three times with 6M urea, 1% SDS / PBS, and PBS. Finally, add 30μL of PBS to dissolve the mixture. Add 15μL of 5× SDS loading buffer to the mixture, heat in a boiling water bath for 10 minutes, and perform 40μL of electrophoresis on a 12% SDS-PAGE separation gel at 100V for 1.5 hours. Finally, analyze the mixture with Coomassie Brilliant Blue staining.

[0170] To purify the protein 25-35kDa in Xoo, streptavidin beads were used for enrichment and purification. Figure 7 As shown, III-1-Probe-2 was found to enrich and purify proteins in the 25-35 kDa range, while the blank control group failed to enrich proteins in the 25-35 kDa range. This result demonstrates that III-1-Probe-2 specifically interacts with proteins in the 25-35 kDa range. LC-MS / MS analysis of the 25-35 kDa protein bands and search using Proteome Discoverer 2.4 software provided detailed information on potential target proteins (Table 3).

[0171] Table 3 Mass spectrometry analysis results of the 25-35 kDa band in Xoo

[0172]

[0173]

[0174] 20.5 Testing the Binding Affinity of Compound III-1 to Clp

[0175] CRP-like protein (Clp) was obtained by constructing in vitro recombinant protein (see Figure 8Left), the in vitro dissociation constant (K) of compound L1 and Clp protein was measured using surface plasmon resonance (SPR) technology. d ). Compound III-1 was used as the stationary phase and Clp protein was used as the mobile phase. TM An AD1520 chip array printer was mounted on a 3D photocrosslinking chip. PBST (pH 7.4, 0.1% Tween 20) was added to the Clp mobile phase sample stock solution to dilute it to a five-point concentration gradient: 10 nM, 40 nM, 160 nM, 640 nM, and 2560 nM. This concentration gradient was then passed over the chip surface. Based on the real-time detection results from the SPR instrument, the interaction kinetic curve was fitted and the affinity parameters were output.

[0176] The results are as follows Figure 8 (right figure) shows that the dissociation constant K of compound III-1 and Clp is d =3.69E-08 M (mol / L), which again indicates that Clp is the binding target of compound III-1.

[0177] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative, characterized in that: The structure of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative is shown in formula (III): Wherein, said R is one or more, and said R is independently selected from hydrogen, halogen, nitro, hydroxyl, C 1～4 Alkyl, C 1～4 Halogenated alkyl, C 1～4 Alkoxy or C 1～4 Halogenated alkoxy.

2. The thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative according to claim 1, characterized in that: The R groups are each independently selected from hydrogen, fluorine, chlorine, bromine, nitro, methyl or methoxy.

3. The thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative according to claim 2, characterized in that: The R is selected from hydrogen, 2-nitro, 3-nitro, 4-nitro, 2-fluoro, 3-fluoro, 4-fluoro, 4-bromo, 2-chloro, 3-chloro, 4-chloro, 2,4-difluoro, 2,6-difluoro, 4-methyl or 4-methoxy.

4. The thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative according to any one of claims 1 to 3, characterized in that: The thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative is replaced by a pharmaceutically acceptable salt or a solvate thereof.

5. The method for preparing the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative according to any one of claims 1 to 3, characterized in that: The steps include: S1. In the presence of a diluent, the compound represented by formula Ⅰ is reacted with SOCl2 to prepare a compound of formula Ⅱ: S2. In the presence of a diluent and an acid binding agent, the compound of formula II is reacted with 2-mercaptothiophene to obtain a compound of formula III:

6. Use of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative according to any one of claims 1 to 4 in the preparation of a drug for preventing or treating plant diseases.

7. Use of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative according to any one of claims 1 to 4 as an inhibitor of the virulence factor T3SS in Xanthomonas oryzae.

8. A pesticide composition, characterized in that The pesticide composition comprises one or more of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivatives according to any one of claims 1 to 4.

9. A small molecule probe based on a thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative, characterized in that: The small molecule probe has the following structure:

10. The small molecule probe according to claim 9 is used to determine the direct target of the thiophene-containing 5-(substituted phenyl)-2-furancarboxylic acid derivative according to any one of claims 1 to 4.

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