A guanidine-containing phenylfurancarboxamide compound and its application

Abstract

The present invention belongs to the technical field of plant disease prevention and control, and specifically relates to a guanidine-containing phenylfuran carboxamide compound and its application. Experiments have shown that the guanidine-containing phenylfuran carboxamide compound has a significant inhibitory effect on Xanthomonas such as rice bacterial leaf blight, rice bacterial leaf spot, citrus canker, and crucifer black rot, both in vivo and in vitro. It is further found that the compound has a good binding affinity with histidinol dehydrogenase in bacteria, and can inhibit the degradation of histidine by binding to histidinol dehydrogenase, thereby inhibiting bacterial growth. The experimental results also show that histidinol dehydrogenase can be used as a target for inhibiting the activity of Xanthomonas, and is used to further screen high-activity inhibitors.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease prevention and control, and more specifically, relates to an application of a guanidine-containing phenylfuran carboxamide compound in preventing and controlling plant bacterial diseases and a method for screening target inhibitors thereof. Background Art

[0002] Xanthomonas pathogenic bacteria include Xanthomonas oryzae pv.oryzae, Xoo, Xanthomonas oryzae pv.oryzicola, Xoc, Xanthomonas campestris pv.citri, Xac, Xanthomonas campestris pv.campestris, Xcc, etc. They are the culprits of many plant diseases and can cause very serious economic losses in crop production. At present, the prevention and control of Xanthomonas pathogens in plants mainly include the development of resistant plants and antimicrobial drugs. For example, Chinese patent application CN107881191A discloses a Xanthomonas-resistant cabbage plant, which is modified by genetic technology to make it resistant to Xanthomonas. However, the research and development of resistant plants requires a lot of time and manpower and material resources, and can only be targeted at a certain plant. For other plants, it is necessary to re-develop, which is costly. In contrast, antibiotics can be widely used in most plants, but the widespread use of antibiotics has also caused the generation of Xanthomonas resistance. Therefore, there is an urgent need to provide more new antibiotics. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing high research and development costs of resistant plants and the fact that long-term use of antibiotics can easily cause bacteria to develop drug resistance, and to provide a guanidine-containing phenylfurancarboxamide compound for use in preventing and controlling plant bacterial diseases.

[0004] The purpose of the present invention is to provide a guanidine-containing phenylfurancarboxamide compound.

[0005] Another object of the present invention is to provide a use of a guanidine-containing phenylfurancarboxamide compound in the preparation of a histidinol dehydrogenase inhibitor.

[0006] Another object of the present invention is to provide the use of histidinol dehydrogenase as a target in screening drugs for preventing and controlling plant bacterial diseases.

[0007] The present invention also provides a histidinol dehydrogenase target probe and application of the probe in screening histidinol dehydrogenase inhibitors.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] A guanidine-containing phenyl furan carboxamide compound, wherein the guanidine-containing phenyl furan carboxamide compound has the following structure:

[0010]

[0011] In addition, the present invention also provides the use of guanidine-containing phenylfurancarboxamide compounds in the preparation of a compound for preventing and controlling plant bacterial diseases, wherein the guanidine-containing phenylfurancarboxamide compound has a structure of formula (III):

[0012]

[0013] Wherein, R1 is monosubstituted or polysubstituted, and the substituents are selected from hydrogen, halogen, nitro, hydroxyl, C 1～4 Alkyl, C 1～4 Haloalkyl, C 1～4 Alkoxy, C 1～4 One or more of halogenated alkoxy; R2 is selected from hydrogen, C 1～4 One or two of the alkyl groups.

[0014] Preferably, R1 is selected from one or more of hydrogen, fluorine, chlorine, bromine, nitro, hydroxyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, trifluoromethyl, trifluoromethoxy or trifluoroethyl; R2 is selected from one or more of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0015] More preferably, R1 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, 4-methoxy; and R2 is selected from hydrogen and methyl.

[0016] Specifically, R1 is selected from 2-nitro, 4-chloro or 4-bromo; R2 is selected from hydrogen and methyl.

[0017] Furthermore, the guanidine-containing phenylfurancarboxamide compound may also be a pharmaceutically acceptable salt or solvate thereof.

[0018] Furthermore, the bacteria are pathogenic bacteria of the genus Xanthomonas.

[0019] Furthermore, the Xanthomonas pathogenic bacteria are oryzae bacteria, oxysporum flavus, citrus canker or crucifer black rot.

[0020] The present invention proves through experiments that the guanidine-containing phenylfurancarboxamide compounds have significant inhibitory effects on Xanthomonas such as rice bacterial leaf blight, rice bacterial leaf streak, citrus canker, and crucifer black rot, both in vivo and in vitro, and can be used for preventing and controlling plant diseases related to Xanthomonas.

[0021] Furthermore, the present invention finds that the guanidine-containing phenylfurancarboxamide compound has good binding affinity with histidinol dehydrogenase, and inhibits the degradation of histidine by binding to histidinol dehydrogenase, thereby ultimately achieving an anti-Xanthomonas effect.

[0022] Therefore, the present invention also claims the use of guanidine-containing phenylfurancarboxamide compounds in the preparation of histidinol dehydrogenase inhibitors, wherein the guanidine-containing phenylfurancarboxamide compounds have the structure of formula (III):

[0023]

[0024] Wherein, R1 is monosubstituted or polysubstituted, and the substituents are selected from hydrogen, halogen, nitro, hydroxyl, C 1～4 Alkyl, C 1～4 Haloalkyl, C 1～4 Alkoxy, C 1～4 One or more of halogenated alkoxy; R2 is selected from hydrogen, C 1～4 One or two of the alkyl groups.

[0025] Preferably, R1 is selected from one or more of hydrogen, fluorine, chlorine, bromine, nitro, hydroxyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, trifluoromethyl, trifluoromethoxy or trifluoroethyl; R2 is selected from one or more of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0026] More preferably, R1 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, 4-methoxy; and R2 is selected from hydrogen and methyl.

[0027] Specifically, R1 is selected from 2-nitro, 4-chloro or 4-bromo; R2 is selected from hydrogen and methyl.

[0028] Based on this, the present invention also claims to protect the use of histidinol dehydrogenase as a target in screening drugs for preventing and treating plant bacterial diseases.

[0029] In addition, the present invention also provides a histidinol dehydrogenase target probe, wherein the probe has the following structure:

[0030]

[0031] Furthermore, the present invention also claims to protect the use of the histidinol dehydrogenase target probe in screening histidinol dehydrogenase inhibitors.

[0032] In addition, the preparation method of the guanidine-containing phenylfurancarboxamide compound comprises the following steps:

[0033] S1. In the presence of a diluent, the compound represented by formula I reacts with SOCl2 to prepare a compound represented by formula II;

[0034]

[0035] S2, in the presence of a diluent and an acid-binding agent, reacting the compound of formula II with the compound of formula IV to obtain a compound of formula III after separation;

[0036]

[0037] Furthermore, in step S2, the acid binding agent is first mixed with the compound of formula IV, a portion of the diluent is added, and then the solution of the compound of formula II is slowly added, and the temperature is controlled between 0° C. and 5° C. to react for 1 to 2 hours.

[0038] Furthermore, the diluent is an inert organic solvent, in particular an aliphatic, alicyclic or aromatic optionally halogenated hydrocarbon, specifically 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.

[0039] Further, the presence of an acid binding agent is conducive to the reaction, and the acid binding agent is preferably sodium hydroxide, potassium carbonate, sodium ethoxide, triethylamine, trimethylamine, tributylamine, pyridine, N,N-dimethylaniline, N,N-dimethylbenzylamine, N-methylpiperidine, N-methylmorpholine, N,N-dimethylaminopyridine, diazabicyclooctane, diazabicyclononene or diazabicycloundecene. Preferably, the acid binding agent is sodium hydroxide.

[0040] The present invention has the following beneficial effects:

[0041] The invention provides a guanidine-containing phenylfurancarboxamide compound, and experiments prove that the guanidine-containing phenylfurancarboxamide compound has good binding affinity with histidinol dehydrogenase, inhibits the degradation of histidine by binding to histidinol dehydrogenase, has a significant inhibitory effect on Xanthomonas such as rice bacterial leaf blight pathogen, rice bacterial leaf streak pathogen, citrus canker pathogen, crucifer black rot pathogen, etc., both in vivo and in vitro, and is very suitable for preventing and controlling plant diseases related to Xanthomonas. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a growth curve diagram of the effect of the compounds in Experimental Example 3 on different Xanthomonas (A: Xoo, B: Xoc, C: Xac, D: Xcc).

[0043] Figure 2 This is a graph showing the in vivo activity experimental data of the compounds in Experimental Example 4 against different Xanthomonas (A-B: Xoo, C-D: Xoc, E-F: Xac, G-H: Xcc).

[0044] Figure 3 This is the KEGG enrichment analysis diagram of Xoo's significantly differentially expressed genes after compound stress in Experimental Example 5.

[0045] Figure 4 The data statistics of the histidine metabolic pathway gene expression of Xoc (A), Xac (B) and Xcc (C) after compound stress in Experimental Example 6 are shown.

[0046] Figure 5 The western blot diagrams show the effects of the compound probe on the protein level of Xanthoceras oryzae in Experimental Example 8, wherein A: in vivo labeling of Xoo by probes of different concentrations, and B: in vivo labeling of WT and ΔhisD of Xoo by probes of 200 μg / mL.

[0047] Figure 6 The western blot diagram (A) and dissociation constant statistical diagram (B) of the binding affinity of the compound in Experimental Example 9 to histidinol dehydrogenase. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Example 1: Synthesis of Compound III-1

[0051] 0.44 g of 5-(2-chlorophenyl)-2-furancarboxylic acid (2 mmol) and 5 mL of dithionyl chloride were added to a 50 ml single-mouth bottle in sequence, and the mixture was heated under reflux for 1.5 hours. The heating was stopped, and the reaction solution was cooled to room temperature. The remaining dithionyl chloride was evaporated under reduced pressure to obtain 5-(2-chlorophenyl)-2-furancarboxylic acid chloride intermediate (no purification was required, and it was directly used in the next step);

[0052] In another 50mL three-necked flask, 0.20g (2.0mmol) of guanidine hydrochloride, 0.16g of sodium hydroxide (4.0mmol) and 20mL of acetonitrile were added in sequence, cooled to 0°C in an ice bath, and the acetonitrile solution (≈20mL) of the intermediate was slowly added dropwise. After the addition was complete, the mixture was transferred to room temperature for reaction; monitored by thin layer chromatography (TLC). After the reaction was complete, 20ml of water was added, and the aqueous phase was extracted with ethyl acetate three times. The ethyl acetate layers were combined, dried over anhydrous magnesium sulfate overnight, filtered, and the filtrate was spin-dried to obtain a brown crude product. The crude product was separated and purified by silica gel column chromatography (ethyl acetate / V: ethanol / V: ammonia water / V=40:2:1) to obtain a white solid compound with a yield of 72% (III-1).

[0053] 1 H NMR(600MHz,DMSO-d6)δ7.88–7.86(m,1H),7.58(dd,J=8.0,1.2Hz,1H),7.49(td,J=7.6,1.3Hz,1 H),7.39(td,J=7.7,1.7Hz,1H),7.16(d,J=3.5Hz,1H),7.06(d,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H10ClN3O2(MH + ):262.03778; Found:262.03882.

[0054]

[0055] Example 2: Synthesis of Compound III-2

[0056] 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 a yellow solid compound III-2.

[0057] 1H NMR(600MHz,DMSO-d6)δ7.80(t,J=1.9Hz,1H),7.72(dt,J=8.0,1.3Hz,1H),7.49(t,J=7.9Hz,1H), 7.40(ddd,J=8.0,2.1,1.0Hz,1H),7.14(d,J=3.5Hz,1H),7.02(d,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H10ClN3O2(MH + ):262.03778; Found:262.03888.

[0058]

[0059] Example 3: Synthesis of Compound III-3

[0060] 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 white solid compound III-3.

[0061] 1 H NMR (600MHz, DMSO-d6) δ7.79–7.74(m,2H),7.54–7.50(m,2H),7.05(d,J=3.5Hz,1H),7.01(d,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H10ClN3O2(MH + ):262.03778; Found:262.03870.

[0062]

[0063] Example 4: Synthesis of Compound III-4

[0064] 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 white solid compound III-4.

[0065] 1 H NMR(600MHz,DMSO-d6)δ7.84(td,J=7.7,1.7Hz,1H),7.42(tdd,J=7.0,5.2,1.8Hz,1H ),7.37–7.33(m,2H),7.06(d,J=3.5Hz,1H),6.90(t,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H10FN3O2(MH +):246.06733; Found:246.06821.

[0066]

[0067] Example 5: Synthesis of Compound III-5

[0068] 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 yellow solid compound III-5.

[0069] 1 H NMR(600MHz,DMSO-d6)δ7.60(d,J=7.9Hz,1H),7.58–7.55(m,1H),7.51(td,J=8.0,6.0Hz,1H) ,7.18(td,J=8.4,2.6Hz,1H),7.12(d,J=3.5Hz,1H),7.02(d,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H10FN3O2(MH + ):246.06733; Found:246.06830.

[0070]

[0071] Example 6: Synthesis of Compound III-6

[0072] 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 yellow solid compound III-6.

[0073] 1 H NMR (600MHz, DMSO-d6) δ7.80–7.78(m,2H),7.31–7.28(m,2H),7.00(d,J=3.4Hz,1H),6.97(d,J=3.4Hz,1H).HR-MS(ESI):Calcd for C12H10FN3O2(MH + ):246.06733; Found:246.06833.

[0074]

[0075] Example 7: Synthesis of Compound III-7

[0076] The method is the same as Example 1, except that 5-(2,4-difluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain yellow solid compound III-7.

[0077] 1 H NMR (600MHz, DMSO-d6) δ7.86 (td, J=8.8, 6.5Hz, 1H), 7.40 (ddd, J=11.7, 9.2, 2.6Hz, 1H), 7. 23(td,J=8.3,2.2Hz,1H),7.05(d,J=3.5Hz,1H),6.85(t,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H9F2N3O2(MH + ):264.05790; Found:264.05875.

[0078]

[0079] Example 8: Synthesis of Compound III-8

[0080] The method is the same as Example 1, except that 5-(2,6-difluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride to obtain yellow solid compound III-8.

[0081] 1 H NMR (600MHz, DMSO-d6) δ7.49(tt,J=8.4,6.3Hz,1H),7.24(t,J=8.6Hz,2H),7.07(d,J=3.4Hz,1H),6.88(dt,J=3.3,1.6Hz,1H).HR-MS(ESI):Calcd for C12H9F2N3O2(MH + ):264.05790; Found:264.05899.

[0082]

[0083] Example 9: Synthesis of Compound III-9

[0084] 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 yellow solid compound III-9.

[0085] 1H NMR (600MHz, DMSO-d6) δ7.94 (dd, J=8.2, 1.2Hz, 1H), 7.85 (dd, J=7.8, 1.4Hz, 1H), 7.78 (td, J=7.6, 1.2 Hz,1H),7.63(td,J=7.7,1.4Hz,1H),7.02(d,J=3.5Hz,1H),6.86(d,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H10N4O4(MH + ):273.06183; Found:273.06284.

[0086]

[0087] Example 10: Synthesis of Compound III-10

[0088] 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 yellow solid compound III-10.

[0089] 1 H NMR(600MHz,DMSO-d6)δ8.49(t,J=1.9Hz,1H),8.21–8.17(m,2H),7.76(td,J=8.0,1 .5Hz,1H),7.30(dd,J=3.5,1.2Hz,1H),7.06(d,J=3.5Hz,1H).HR-MS(ESI):Calcdfor C12H10N4O4(MH + ):273.06183; Found:273.06305.

[0090]

[0091] Example 11: Synthesis of Compound III-11

[0092] 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 yellow solid compound III-11.

[0093] 1 H NMR (600MHz, DMSO-d6) δ8.34–8.29(m,2H),8.01–7.98(m,2H),7.34(d,J=3.5Hz,1H),7.09(d,J=3.6Hz,1H).HR-MS(ESI):Calcd for C12H10N4O4(MH +):273.06183; Found:273.06302.

[0094]

[0095] Example 12: Synthesis of Compound III-12

[0096] 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 white solid compound III-12.

[0097] 1 H NMR (600MHz, DMSO-d6) δ7.72–7.69(m,2H),7.66–7.64(m,2H),7.06(d,J=3.5Hz,1H),7.02(d,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H10BrN3O2(MH + ):305.98726; Found:305.98834.

[0098]

[0099] Example 13: Synthesis of Compound III-13

[0100] 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 yellow solid compound III-13.

[0101] 1 H NMR (600MHz, DMSO-d6) δ7.69–7.64(m,2H),7.28(d,J=7.9Hz,2H),7.04(d,J=3.5Hz,1H),6.95(d,J=3.5Hz,1H),2.34(s,3H).HR-MS(ESI):Calcd for C13H13N3O2(MH + ):242.09240; Found:242.09335.

[0102]

[0103] Example 14: Synthesis of Compound III-14

[0104] 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 white solid compound III-14.

[0105] 1 H NMR (600MHz, DMSO-d6) δ7.69–7.67(m,2H),7.03–7.01(m,2H),6.98(d,J=3.4Hz,1H),6.83(d,J=3.4Hz,1H),3.80(s,3H).HR-MS(ESI):Calcd for C13H13N3O3(MH + ):258.08731; Found:258.08817.

[0106]

[0107] Example 15: Synthesis of Compound III-15

[0108] 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 gray solid compound III-15.

[0109] 1 H NMR (600MHz, DMSO-d6) δ7.77–7.75(m,2H),7.48–7.44(m,2H),7.36–7.33(m,1H),7.02(d,J=3.4Hz,1H),7.01(d,J=3.5Hz,1H).HR-MS(ESI):Calcd for C12H11N3O2(MH + ):228.07675; Found:228.07753.

[0110]

[0111] Example 16: Synthesis of Compound III-16

[0112] The method is the same as Example 1, except that 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain yellow solid compound III-16.

[0113] 1 H NMR (600MHz, DMSO-d6) δ8.48(s,1H),7.87(d,J=7.8Hz,1H),7.57(dd,J=8.0,1.3Hz,1H),7.48(td,J=7 .6,1.3Hz,1H),7.38(td,J=7.7,1.7Hz,1H),7.16(s,1H),7.03(s,1H),2.75(s,3H).HR-MS(ESI):Calcd for C13H12ClN3O2(MH +):276.05343; Found:276.05463.

[0114]

[0115] Example 17: Synthesis of Compound III-17

[0116] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain brown solid compound III-17.

[0117] 1 H NMR (600MHz, DMSO-d6) δ8.52(s,1H),7.79(s,1H),7.71(d,J=7.8Hz,1H),7.48(t,J=7.9Hz, 1H),7.38(dd,J=8.1,2.1Hz,1H),7.13(s,1H),7.00(s,1H),2.76(s,3H).HR-MS(ESI):Calcd for C13H12ClN3O2(M-H+):276.05343; Found:276.05399.

[0118]

[0119] Example 18: Synthesis of Compound III-18

[0120] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a yellow solid compound III-18.

[0121] 1 H NMR(600MHz, DMSO-d6)δ8.50(s,1H),7.76(d,J=8.3Hz,2H),7.54–7.50(m,2H),7.05(s,1H),7.00(s,1H),2.75(s,3H).HR-MS(ESI):Calcd for C13H12ClN3O2(M+H + ):278.06908; Found:278.06906.

[0122]

[0123] Example 19: Synthesis of Compound III-19

[0124] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a red solid compound III-19.

[0125] 1 H NMR (600MHz, DMSO-d6) δ12.24(s,1H),9.60(s,1H),9.30(s,1H),8.89(s,1H),8.44(t,J=7.3Hz,1H),7.91(d,J =3.8Hz,1H),7.51(q,J=6.8Hz,1H),7.42–7.34(m,2H),7.13(s,1H),2.96(d,J=5.0Hz,3H).HR-MS(ESI):Calcd for C13H12FN3O2(MH + ):260.08298; Found:260.08401.

[0126]

[0127] Example 20: Synthesis of Compound III-20

[0128] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain yellow solid compound III-20.

[0129] 1 H NMR(600MHz,Chloroform-d)δ8.00(t,J=6.9Hz,1H),7.26–7.23(m,1H),7.21(d,J=3.5Hz,1H),7.19( t,J=7.2Hz,1H),7.11(dd,J=11.3,8.1Hz,1H),6.90(t,J=3.6Hz,1H),2.77(s,3H).HR-MS(ESI):Calcd for C13H12FN3O2(MH + ):260.08298; Found:260.08362.

[0130]

[0131] Example 21: Synthesis of Compound III-21

[0132] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a yellow solid compound III-21.

[0133] 1 H NMR (600MHz, DMSO-d6) δ8.52 (s, 1H), 7.80 (t, J = 6.7Hz, 2H), 7.31 (t, J = 8.7Hz, 2H), 7.05–6.95 (m, 2H), 2.80 (d, J = 42.6Hz, 3H). HR-MS (ESI): Calcd for C13H12FN3O2(MH + ):260.08298; Found:260.08368.

[0134]

[0135] Example 22: Synthesis of Compound III-22

[0136] The method is the same as Example 1, except that 5-(2,4-difluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a red solid compound III-22.

[0137] 1 H NMR(600MHz,DMSO-d6)δ8.53(s,1H),7.86(q,J=8.0Hz,2H),7.43(ddd,J=11.7,9.2,2 .6Hz,2H),7.26(td,J=8.5,2.6Hz,2H),6.86(s,1H),2.77(s,3H).HR-MS(ESI):Calcd for C13H11F2N3O2(M+H + ):280.08920; Found:280.08902.

[0138]

[0139] Example 23: Synthesis of Compound III-23

[0140] The method is the same as Example 1, except that 5-(2,6-difluorophenyl)-2-furoyl chloride is used instead of 5-(2-chlorophenyl)-2-furoyl chloride, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a yellow solid compound III-23.

[0141] 1H NMR(500MHz,DMSO-d6)δ8.53(s,1H),7.52–7.46(m,1H),7.25(t,J=8.6Hz,2H),7.07(s,1H),6.90(s,1H),2.80(d,J=27.2Hz,3H).HR-MS(ESI):Calcd for C13H11F2N3O2(M+H + ):280.08920; Found:280.08896.

[0142]

[0143] Example 24: Synthesis of Compound III-24

[0144] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a yellow solid compound III-24.

[0145] 1 H NMR (500MHz, DMSO-d6) δ8.49(s,1H),7.93(d,J=8.1Hz,1H),7.85(d,J=5.6Hz,1H),7.77(t,J=7.6Hz ,1H),7.65–7.60(m,1H),7.01(s,1H),6.94–6.81(m,1H),2.78(t,J=16.0Hz,3H).HR-MS(ESI):Calcd for C13H12N4O4(MH + ):287.07748; Found:287.07858.

[0146]

[0147] Example 25: Synthesis of Compound III-25

[0148] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a yellow solid compound III-25.

[0149] 1H NMR(600MHz,DMSO-d6)δ8.57(s,1H),8.47(s,1H),8.16(dd,J=15.5,8.0Hz,2H),7.73( t,J=8.0Hz,1H),7.28(s,1H),7.04(s,1H),2.80(d,J=45.7Hz,3H).HR-MS(ESI):Calcd for C13H12N4O4(MH + ):287.07748; Found:287.07861.

[0150]

[0151] Example 26: Synthesis of Compound III-26

[0152] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a yellow solid compound III-26.

[0153] 1 H NMR(600MHz,DMSO-d6)δ8.54(s,1H),8.31(d,J=8.8Hz,2H),7.99(d,J=8.8Hz,2H ),7.06(s,1H),2.80(d,J=47.0Hz,3H),2.50(d,J=1.9Hz,4H).HR-MS(ESI):Calcd forC13H12N4O4(MH + ):287.07748; Found:287.07858.

[0154]

[0155] Example 27: Synthesis of Compound III-27

[0156] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a yellow solid compound III-27.

[0157] 1H NMR (600MHz, DMSO-d6) δ8.59 (s, 1H), 7.72 (d, J = 8.3Hz, 2H), 7.67–7.64 (m, 2H), 7.06 (d, J = 34.5Hz, 2H), 2.81 (d, J = 40.4Hz, 3H). HR-MS (ESI): Calcd for C13H12BrN3O2(MH + ):320.00291; Found:320.00418.

[0158]

[0159] Example 28: Synthesis of Compound III-28

[0160] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain a yellow solid compound III-28.

[0161] 1 H NMR(500MHz,DMSO-d6)δ8.54(s,1H),7.68–7.63(m,2H),7.27(d,J=8.0Hz,2H),7 .01(s,1H),6.93(d,J=3.5Hz,1H),2.78(s,3H),2.33(s,3H).HR-MS(ESI):Calcd forC14H15N3O2(MH + ):256.10805; Found:256.10919.

[0162]

[0163] Example 29: Synthesis of Compound III-29

[0164] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain yellow solid compound III-29.

[0165] 1H NMR (600MHz, DMSO-d6) δ8.62–8.42(m,1H),7.70(d,J=8.3Hz,2H),7.03(d,J=8.8Hz,2 H),6.98(s,1H),6.85(s,1H),3.80(s,3H),2.80(d,J=38.8Hz,3H).HR-MS(ESI):Calcd for C14H15N3O3(MH + ):272.10296; Found:272.10410.

[0166]

[0167] Example 30: Synthesis of Compound III-30

[0168] 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, and 1-methylguanidine hydrochloride is used instead of guanidine hydrochloride to obtain yellow solid compound III-30.

[0169] 1 H NMR (500MHz, DMSO-d6) δ8.59(s,1H),7.79–7.75(m,2H),7.45(t,J=7.8Hz,2H),7.33(t,J=7. 4Hz,1H),7.07(dd,J=9.0,3.5Hz,1H),7.01(d,J=3.4Hz,1H),2.78(s,3H).HR-MS(ESI):Calcd for C13H13N3O2(MH + ):242.09240; Found:242.09300.

[0170]

[0171] Experimental Example 1 Minimum inhibitory concentration of compounds against rice bacterial blight pathogen

[0172] The test pathogen used was Xanthomonas oryzae pv.oryzae (Xoo), which was obtained by sampling from the diseased parts of field crops, greenhouse vegetables and stored fruits, inoculated and purified in the corresponding culture medium, and identified as Xanthomonas oryzae pv.oryzae.

[0173] Compound test concentration: The compound to be tested was dissolved in DMSO and diluted to 50 mg mL -1 .

[0174] The Xoo strain (PXO99 A) were streaked on PSA plates for activation, and after 2 to 3 days, single colonies were picked and placed in M210 liquid medium and grown to OD 600 The cell culture medium was about 2.0, and the dilution ratio was 1:100, and the culture medium was transferred to new M210 medium and grown to OD 600 The minimum inhibitory concentration (MIC) of the compound was determined by 96-well plate method: 200 μL of culture medium containing bacteria was added to the first row, and 100 μL of culture medium containing bacteria was added to the remaining wells. 4 μL of the prepared compound was added to the wells in the first row to make the final concentration reach 200 μg mL -1 , gradient dilution, take out 100μL from the first row of wells and add to the second row of wells and mix well, follow the above method until 100μL is aspirated from the 12th row and discarded, put the 96-well plate into a 28℃, 180rpm incubator for 36-48h, and observe the results (if there is bacterial growth, there will be white precipitation at the bottom of the well plate, and the wells without precipitation in the horizontal row of the 96-well plate correspond to the concentration of the drug MIC value). The results are shown in Table 1.

[0175] Table 1 Minimum inhibitory concentration of compounds against Xoo

[0176]

[0177]

[0178] Note: NT a The compound was poorly soluble in DMSO and was not tested for activity.

[0179] As can be seen from the table, the guanidine-containing phenylfurancarboxamide compounds provided by the present invention have good inhibitory effects on rice bacterial blight pathogens; among them, compounds III-03, III-12, III-24 and III-27 have good antibacterial activity against Xoo.

[0180] Experimental Example 2: Half effective concentration of the compound against Xanthomonas oryzae

[0181] The Xoo strain (PXO99 A ) were streaked on PSA plates for activation, and after 2 to 3 days, single colonies were picked and placed in M210 liquid medium and grown to OD 600 The cell culture medium was about 2.0, and the dilution ratio was 1:100, and the culture medium was transferred to new M210 medium and grown to OD 600 The compound (compounds III-03, III-12, III-24 and III-27 as examples) was prepared into 5 different concentrations of drug-containing culture medium, and the OD of each sterile culture medium was measured with an enzyme marker. 60040 μL of Xoo bacterial solution in the logarithmic phase was added respectively, and the mixture was cultured in a shaking incubator at 28°C and 180 rpm for 48 h. The OD of each bacterial solution was measured with an enzyme-labeled instrument. 600 value.

[0182] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium

[0183] Effectiveness % = (corrected control culture medium bacterial solution OD value - corrected drug-containing culture medium OD value) / corrected control culture medium bacterial solution OD value × 100

[0184] The inhibition rate data were converted into probability values ​​(y), and the drug concentration (μg / mL) was converted into logarithmic values ​​(x). Regression analysis was performed in Excel data processing software to obtain the toxicity regression equation (y=ax+b) and correlation coefficient (R), and the concentration for 50% of maximal effect (EC 50 The results are shown in Table 2.

[0185] Table 2 EC values ​​of compounds for Xoo 50 value

[0186] Compound MIC (μg / mL) Regression equation <![CDATA[r 2 ]]> <![CDATA[EC 50 (μg / mL)]]> III-03 6.25 Y=17.69*X-13.40 0.9987 3.67±0.06 III-12 6.25 Y=17.75*X+4.740 0.9999 3.01±0.14 III-24 6.25 Y=24.68*X-36.78 0.9995 4.02±0.11 III-27 6.25 Y=6.465*X+28.79 0.9981 6.00±0.31 Chlorphenamine 6.25 Y=16.22*X-9.665 0.9995 3.29±0.20

[0187] As can be seen from the table, the antibacterial effect of the guanidine-containing phenylfurancarboxamide compounds provided by the present invention is similar to that of the positive control chlorothalonil, and the antibacterial activity of compound III-12 is even better than that of the positive control chlorothalonil.

[0188] Experimental Example 3 In vitro activity of compounds against different Xanthomonas

[0189] 1. EC of compounds against different Xanthomonas 50

[0190] Different test pathogens were used: Xanthomonas oryzae pv.oryzae, Xoo, Xoc, Xac and Xcc. Compound III-12 was used as an example. The EC values ​​of the compound against different Xanthomonas were determined by referring to the method of Experimental Example 2. 50 The results are shown in Table 3.

[0191] Table 3 In vitro activity of compounds against different Xanthomonas

[0192]

[0193]

[0194] As can be seen from the table, the guanidine-containing phenylfurancarboxamide compound III-12 provided by the present invention has different degrees of inhibitory effects on Xoo, Xoc, Xac and Xcc.

[0195] 2. Growth curves of compounds on different Xanthomonas

[0196] The wild-type strains of Xoo, Xoc, Xac and Xcc were activated by streaking on plates, and single colonies were picked and placed in liquid culture medium after 2 to 3 days. 600 The concentration of 1x MIC and 2x MIC of compound III-12 were added respectively and placed on a 96-well plate. An equal volume of WT was used as a wild-type control, an equal volume of DMSO was used as a solvent control, and an equal volume of bismerthiazol was used as a positive control. Three replicates were set for each group. The temperature was set at 28°C. The growth curve was measured with an automatic growth curve analyzer. The data were read every 1h for a total of 48h.

[0197] Results Figure 1 It can be seen that the guanidine-containing phenylfurancarboxamide compound III-12 provided by the present invention has different degrees of inhibitory effects on Xoo, Xoc, Xac and Xcc.

[0198] Experimental Example 4 In vivo activity of compounds against different Xanthomonas

[0199] The wild-type strains of Xoo, Xoc, Xac and Xcc were activated by streaking on plates, and single colonies were picked and placed in liquid culture medium after 2 to 3 days. 600 The concentration of the compound III-12 was about 2.0, and the cells were collected by centrifugation, washed once with sterile water, and then resuspended in sterile water for later use. Compound III-12 with a concentration of 1 and 2 times the MIC was added, incubated for 4 hours, and inoculated according to the following method:

[0200] Xoo inoculation: leaf cutting inoculation method was used, and the lesions were checked 14 days after inoculation; an equal volume of untreated wild-type strain was used as a blank control group, an equal volume of untreated wild-type strain plus DMSO was used as a solvent control, and an equal volume and concentration of untreated wild-type strain plus bismerthiazol was used as a positive control.

[0201] Xoc inoculation: pressure inoculation method was used, and the lesions were checked 14 days after inoculation; an equal volume of untreated wild-type strain was used as a blank control group, an equal volume of untreated wild-type strain plus DMSO was used as a solvent control, and an equal volume and concentration of untreated wild-type strain plus bismerthiazol was used as a positive control.

[0202] Xac inoculation: pressure inoculation method was used, and the lesions were checked 5 days after inoculation; an equal volume of untreated wild-type strain was used as a blank control group, an equal volume of untreated wild-type strain plus DMSO was used as a solvent control, and an equal volume and concentration of untreated wild-type strain plus bismerthiazol was used as a positive control.

[0203] Xcc inoculation: pressure inoculation method was used, and the lesions were checked 5 days after inoculation; an equal volume of untreated wild-type strain was used as a blank control group, an equal volume of untreated wild-type strain plus DMSO was used as a solvent control, and an equal volume and concentration of untreated wild-type strain plus bismerthiazol was used as a positive control.

[0204] Results Figure 2 As can be seen from the figure, compared with the blank control WT and the solvent control DMSO, compound III-12 has good inhibitory activity against Xoo, Xoc, Xac and Xcc, and its in vivo antibacterial activity at the same concentration is better than that of the positive control chlorothalonil.

[0205] Experimental Example 5 Study on the target of the compound against Xanthomonas oryzae

[0206] The wild-type Xoo strain was activated by streaking on the plate. After 2 to 3 days, a single colony was picked and placed in liquid culture medium and grown to OD 600 The cell density was about 2.0. The cells were collected by centrifugation, washed once with sterile water, and then resuspended in fresh culture medium and adjusted to OD 600 to 0.05, compound III-12 was added to a final concentration of 2 times MIC, and total RNA was extracted after culturing for 16 hours; the extracted RNA was subjected to transcriptome library sequencing, and after obtaining the original sequencing sequence (Sequenced Reads), the gene expression level of the samples was analyzed using DESeq2 Rpackage (v1.28.1); KEGG (Kyoto Encyclopedia of Genes and Genomes) was performed using clusterProfiler R package (v3.16.1).

[0207] Results Figure 3The significantly down-regulated genes of Xoo treated with compound III-12 were mainly enriched in histidine metabolism, metabolic pathways, oxidative phosphorylation, C5-branched dibasic acid metabolism and sulfur metabolism. Among them, the pathway with the highest enrichment rate and Qvalue < 0.001 among the significantly down-regulated genes was histidine metabolism; at the same time, the results also showed that the gene expression of the histidine synthesis pathway of Xoo under the stress of compound III-12 did not change significantly, but the histidine degradation pathway was significantly inhibited, proving that compound III-12 affects bacterial growth by inhibiting the histidine degradation pathway.

[0208] Experimental Example 6: Study on the effects of compounds on the histidine metabolic pathways of different Xanthomonas

[0209] The wild-type strains of Xoc, Xac and Xcc were activated by streaking on plates, and single colonies were picked and placed in liquid culture medium after 2 to 3 days. 600 The cell density was about 2.0. The cells were collected by centrifugation, washed once with sterile water, and then resuspended in fresh culture medium and adjusted to OD 600 to 0.05, compound III-12 was added to a final concentration of 2 times MIC, and total RNA was extracted after culturing for 16 hours. The extracted RNA was subjected to qRT-PCR to verify the expression of genes hisB (responsible for encoding histidine biosynthesis bifunctional protein), hisC (responsible for encoding histidinol-phosphate transaminase), hisD (responsible for encoding histidinol dehydrogenase), hutH (responsible for encoding histidine ammonia lyase), hutU (responsible for encoding urate hydratase), hutI (responsible for encoding imidazoline propionase), hutF (responsible for encoding methylaminoglutarate deaminase) and hutG (responsible for encoding N-formylglutamate amidohydrolase) in the histidine metabolic pathway of Xoc, Xac and Xcc. (Note: in Xac, sedB is homologous to hutF; in Xcc, XC_2378 is homologous to hisB, XC_2379 is homologous to hisC, XC_2380 is homologous to hisD, XC_2355 is homologous to hutH, XC_2357 is homologous to hutU, XC_2353 is homologous to hutI, XC_2354 is homologous to hutF, and XC_2356 is homologous to hutG).

[0210] Results Figure 4Under the stress of compound III-12, there was no obvious change in the gene expression of the histidine synthesis pathways (hisB, hisC, hisD) of Xoc, Xac and Xcc, but the histidine degradation pathway (hutH, hutU, hutI, hutF and hutG) was significantly inhibited, proving that compound III-12 achieves the effect of anti-Xanthomonas pathogens by targeted inhibition of the histidine degradation pathway.

[0211] Experimental Example 7: Verification of the target of the compound at the cellular level against Xanthomonas oryzae

[0212] From Experimental Example 6, it can be seen that in the histidine metabolic pathway, the gene expression of the histidine synthesis pathway is not affected under the stress of compound III-12, while the gene expression of the histidine degradation pathway is significantly inhibited. HisD (responsible for encoding histidinol dehydrogenase) is a key node in the synthesis and metabolism pathway of histidine. Therefore, the antibacterial activity of compound III-12 against the wild-type strain (WT) of Xoo and the hisD-deficient strain (ΔhisD) of Xoo was determined by referring to the method of Experimental Example 1.

[0213] The results showed that the MIC of compound III-12 against WT was 6.25 μg / mL, while the MIC against ΔhisD was 50 μg / mL, proving that the absence of hisD would lead to the ineffectiveness of the antibacterial function of compound III-12. It also proved that the protein encoded by hisD, namely histinol dehydrogenase, is the target of compound III-12.

[0214] Experimental Example 8: Verification of the target of the compound at the protein level in Xanthomonas oryzae

[0215] 1. Synthesis of Compound III-12 Probe

[0216] Compound III-12 was used as the parent to design and synthesize an alkyne-containing probe. This alkyne can be coupled with a fluorescent group such as biotin or rhodamine in the cell through a click reaction, so that the small molecule compound can be detected by the fluorescence imaging system after binding to the target protein. The synthesis route and specific steps are as follows:

[0217]

[0218] S1. Add 0.04 mol of 2-methyl-4-bromoaniline aqueous solution to a three-necked flask equipped with a thermometer, slowly add concentrated hydrochloric acid while stirring with a glass rod, control the reaction temperature within 0-5°C, add sodium nitrite aqueous solution dropwise to the reaction after the temperature is constant, continue stirring for 1 hour, add 1.2 g of copper chloride and 0.04 mol of furancarboxylic acid to the reaction system, and react at room temperature for 3 hours; remove the solvent, and adjust the pH of the aqueous solution of the remaining solid to weak alkalinity with sodium bicarbonate aqueous solution and filter again, extract the filter cake with ethyl acetate, discard the ethyl acetate layer solution after extraction, adjust the pH of the aqueous layer solution to acidity with dilute hydrochloric acid, and recrystallize the solid obtained after filtration with ethanol: water = 1:1. The obtained solid is 5-(4-bromo-2-methylphenyl)furan-2-carboxylic acid with a yield of 60%.

[0219] S2. Add 5-(4-bromo-2-methylphenyl)furan-2-carboxylic acid (10 mmol) and dichlorothionyl (20 mL) into a single-mouth bottle and heat to reflux for 1.5 h. Stop heating and use reduced pressure distillation to remove excess solvent. The obtained reaction intermediate does not need to be purified. Add 20 mL of dichloromethane and slowly add 6 mL of ethanol under low temperature stirring. After the addition is complete, transfer the reaction system to room temperature and react for 2 h. Remove the solvent to obtain a brown oily crude product. The crude product is separated and purified by silica gel column chromatography (EA / V:PE / V=1:20) to obtain the key intermediate I-12-Probe with a yield of 90%.

[0220] S3. Add 5mmol key intermediate I-12-Probe, 6mmol NBS, 0.5mmol AIBN and 20mL CCl4 into a three-necked flask in sequence, and reflux for 48h under nitrogen protection. After the reaction is completed, remove the solvent to obtain a brown oily crude product. The crude product is separated and purified by silica gel column chromatography (EA / V:PE / V=1:20) to obtain an intermediate product containing benzyl bromide substitution, with a yield of 50%.

[0221] S4. Sodium hydride (3 mmol) was placed in a three-necked flask, and ultra-dry tetrahydrofuran (10 mL) was added under nitrogen protection. The mixture was placed on ice and stirred to dissolve. 2.5 mmol of propargyl alcohol was slowly added with a syringe, and the mixture was stirred and reacted at low temperature for 1 hour. Subsequently, 2 mmol of the intermediate product containing benzyl bromide substitution was added with a syringe, and the reaction was carried out at room temperature for 12 hours. After the reaction was completed, the solvent was removed to obtain a crude intermediate product containing alkyne substitution. Aqueous sodium hydroxide solution was added to the crude intermediate product, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added to the reaction system for extraction, and the organic layer was discarded after extraction. The pH of the aqueous layer was adjusted to acidic with dilute hydrochloric acid, and ethyl acetate was added for extraction, and the aqueous layer was discarded. The solvent of the ethyl acetate layer was removed by reduced pressure distillation. The obtained liquid was the key intermediate II-12-Probe, and the yield was 50%.

[0222] S5. Add 2 mmol of II-12-Probe and 5 mL of dichlorothione to a 50 mL single-necked bottle in sequence; heat to reflux for 1.5 h, and use reduced pressure distillation to remove excess solvent to obtain an acyl chloride intermediate; add 20 mL of acetone, 2.0 mmol of guanidine hydrochloride and 4.0 mmol of sodium hydroxide to a three-necked bottle in sequence, wait for the reaction system to cool to 0°C, slowly add the acetone solution of the acyl chloride intermediate, and after the solution is added dropwise, transfer the reaction system to room temperature, monitor the reaction by TLC, and after the reaction is complete, add water and ethyl acetate for extraction, combine the ethyl acetate layers and dry them over anhydrous magnesium sulfate, and remove the solvent from the filtrate obtained by post-treatment by reduced pressure distillation to obtain a brown crude product, which is separated and purified by a silica gel column (EA / V:EtOH / V:NH3·H2O / V=200:10:1) to obtain III-12-Probe in a yield of 55%.

[0223] 1 H NMR (600MHz, DMSO-d6) δ7.82(d,J=2.2Hz,1H),7.71(d,J=8.3Hz,1H),7.64(dd,J=8.3,2.3Hz,1H),7.08(d,J=3 .5Hz,1H),7.03(d,J=3.5Hz,1H),4.63(s,2H),4.35(d,J=2.4Hz,2H),3.55(t,J=2.4Hz,1H).HR-MS(ESI):Calcd for C 16 H 13 O3N3Br[MH + ]:374.01348,Found:374.01395.

[0224] 2. Verification of the target of compound III-12 probe at the protein level against Xanthomonas oryzae

[0225] The wild-type Xoo strain was activated by streaking on the plate. After 2 to 3 days, a single colony was picked and placed in liquid culture medium and grown to OD 600About 0.6, take 200mL of bacterial solution and centrifuge at 8000rpm, 4℃, 20min to collect bacteria, rinse the bacteria three times with PBS and resuspend them in 10mL PBS, add different concentrations of compound III-12 probe respectively, and the blank control is an equal volume of DMSO, incubate at 25℃ for 2h; collect bacteria by centrifugation at 8000rpm, 4℃, 20min, rinse the bacteria three times with PBS and resuspend them in 10mL PBS, break the cells under a high-pressure cell disruptor, collect the supernatant by centrifugation of the cell lysate at 8000rpm, 4℃, 1h, filter the supernatant with a 0.45μm filter membrane, concentrate the protein with a 15mL, 10kDa ultrafiltration tube at 4000rpm, 4℃, and determine the protein concentration after diluting the protein to 1mg / mL with PBS.

[0226] Take 44 μL of protein with a concentration of 1 mg / mL, add 5 μL of click reagent (1 μL of 10 mM BiotinPEG3-N3, 1 μL of 0.25 M NaASC, 3 μL of BTTAA / CuSO4 mixed solution) in sequence, and incubate at 37°C for 2 h; add 20 μL of 5×SDS loading buffer to the mixture, heat in a 95°C water bath for 5-8 min, take 10 μL of sample for polyacrylamide gel electrophoresis separation, transfer to the membrane at 250 mA in an ice bath for 1.5 h, rinse the membrane 3 times with 0.05% PBST, block the membrane with PBST containing 5% skim milk powder at room temperature for 2 h, rinse the membrane 3 times with 0.05% PBST, add HRP-labeled streptavidin to 30 mL of PBST containing 5% skim milk powder at a ratio of 1:1000, incubate at 4°C in the dark for 2 h, and rinse the membrane 3 times with 0.05% PBST. Finally, add chemiluminescent reagent for imaging analysis.

[0227] Results Figure 5 It can be seen that different concentrations (50μg / mL, 100μg / mL and 200μg / mL) of probe III-12-Probe were selected for in vivo labeling of Xoo wild type. The probe with a concentration of 200μg / mL clearly labeled the 35kDa and 44kDa proteins of Xoo wild type under in vivo labeling conditions; while at the same concentration, the proteins here could not be labeled in the hisD knockout ΔhisD, proving that the loss of hisD caused the probe III-12-Probe to lose its binding target, and therefore could not label the protein. These results also prove that the protein encoded by hisD, namely histinol dehydrogenase, is the target of compound III-12.

[0228] Experimental Example 9: Binding affinity of compound III-12 to histidinol dehydrogenase

[0229] Histidinol dehydrogenase was obtained by constructing an in vitro recombinant protein, and the dissociation constant (K) between compound III-12 and histidinol dehydrogenase in vitro was measured by microscale thermophoresis (MST). d ). 10 μM purified histidinol dehydrogenase was labeled with the red fluorescent dye NT-647 N-hydroxysuccinimide using the Monolith NT Protein Labeling Kit RED-NHS. The additional labeling reagent was removed by buffer exchange column chromatography, and the labeled protein was eluted in NTA buffer (300 mM sodium chloride and 50 mM sodium phosphate buffer, pH = 7). The binding reaction was performed on a microthermophoresis instrument using standard treated capillaries. The labeled protein was titrated with compound III-12 (2 mM) and exchanged into 1× MST buffer (50 mM Tris, 150 mM NaCl, 10 mM MgCl2, pH = 6) using a 1:1 serial dilution method. Nano Temper Analysis software was used to fit the curve and determine K d value.

[0230] Results Figure 6 , the dissociation constant K of compound III-12 and histidinol dehydrogenase d =0.12±0.036 μM, which again indicates that histidinol dehydrogenase is the binding target of compound III-12.

[0231] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A guanidine-containing phenylfurancarboxamide compound, characterized in that: The guanidine-containing phenylfurancarboxamide compound has the following structure: 。 2. The use of guanidine-containing phenylfurancarboxamide compounds in preventing and controlling plant bacterial diseases, characterized in that: The guanidine-containing phenylfurancarboxamide compound has a structure of formula (III): Wherein, R1 is monosubstituted or polysubstituted, and the substituents are selected from hydrogen, halogen, nitro, C 1~4 Alkyl, C 1~4 One or more of the alkoxy groups; R2 is selected from hydrogen, C 1~4 One of the alkyl groups; The plant bacteria are pathogenic bacteria of the genus Xanthomonas.

3. The application according to claim 2, characterized in that: The guanidine-containing phenylfurancarboxamide compound is replaced by a pharmaceutically acceptable salt thereof.

4. The application according to claim 2, characterized in that: The Xanthomonas pathogenic bacteria are rice bacterial leaf blight pathogen, rice bacterial leaf streak pathogen, citrus canker pathogen or crucifer black rot pathogen.

5. The application according to claim 2, characterized in that: The guanidine-containing phenylfurancarboxamide compound achieves an antibacterial effect by binding to histidinol dehydrogenase to inhibit the degradation of histidine.

6. Use of guanidine-containing phenylfurancarboxamide compounds in the preparation of histidinol dehydrogenase inhibitors, characterized in that: The guanidine-containing phenylfurancarboxamide compound has a structure of formula (III): Wherein, R1 is monosubstituted or polysubstituted, and the substituents are selected from hydrogen, halogen, nitro, hydroxyl, C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Alkoxy, C 1~4 One or more of halogenated alkoxy; R2 is selected from hydrogen, C 1~4 One of the alkyl groups.

7. A histidinol dehydrogenase target probe, characterized in that: The probe has the following structure: 。

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