CN112028833B - Para-aminosalicylic acid azole derivative and preparation method and application thereof - Google Patents

Para-aminosalicylic acid azole derivative and preparation method and application thereof Download PDF

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CN112028833B
CN112028833B CN202011025656.0A CN202011025656A CN112028833B CN 112028833 B CN112028833 B CN 112028833B CN 202011025656 A CN202011025656 A CN 202011025656A CN 112028833 B CN112028833 B CN 112028833B
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aminosalicylic acid
azole derivative
acid azole
bacteria
molecules
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CN112028833A (en
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杨大成
潘建芳
范莉
唐雪梅
任艳会
徐兴然
胡军华
周围
徐志刚
吴玉珠
李洋
韩海燕
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Southwest University
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Abstract

The invention discloses a p-aminosalicylic acid azole derivative, a preparation method and application thereof, belonging to the technical field of drug synthesis. The structural formula of the p-aminosalicylic acid azole derivative is shown as follows. The antibacterial activity test result shows that the aminosalicylic acid azole derivatives have good inhibition activity on common pathogenic bacteria, the inhibition activity of part of molecules on pichia is stronger than or equal to that of positive drug fluconazole, the inhibition activity of all molecules on citrus colletotrichum anthracnose bacteria is better, the inhibition activity of part of molecules on citrus colletotrichum anthracnose bacteria is equal to or stronger than that of positive control prochloraz, and most of molecules have good inhibition effect on citrus canker bacteria. The result of the activity measurement of the anti-tumor cells shows that the aminosalicylic acid azole derivative has an inhibiting effect on three tumor cells. The p-aminosalicylic acid azole derivative has potential application prospects in the fields of bacteria resistance, fungi resistance, citrus bacteria resistance and tumor resistance.

Description

Para-aminosalicylic acid azole derivative and preparation method and application thereof
Technical Field
The invention relates to the technical field of drug synthesis, in particular to an aminosalicylic acid azole derivative and a preparation method and application thereof.
Background
Para-aminosalicylic acid (PAS), an antibacterial agent for the treatment of tuberculosis. It is also used in the treatment of inflammatory bowel disease, acting by inhibiting NF-. kappa.B and scavenging free radicals. Is mainly used for treating tuberculosis.
PAS amino derivatives fall into three broad categories. The first is a PAS schiff base derivative. Research results show that the antibacterial concentration of the synthesized target compound to mycobacterium smegmatis and mycobacterium bovis is lower than PAS; schiff base derivatives of pyrazinamide and PAS, p H37The Rv inhibitory activity was stronger than PZA (MICs 3.13 and 6 μ g/mL respectively) with sustained release. The second type is PAS hydrazone derivative, INH reacts with PAS to prepare compound containing hydrazone group, and the test result shows that the compound is all compared with positive control (MIC)INH＝1μg/mL、MICCiprofloxacin1.5. mu.g/mL and MICNorfloxacin hydrochloride10. mu.g/mL) is preferred. In the third category, derivatives of amino groups with additional functional groups also exhibit good biological activity.
The compound containing the azole heterocycle is widely applied to the fields of pesticides, medicines, dyes and the like. Many marketed drugs contain different types of azole building blocks, and only in 2018, 16 drug molecules in top 200 contain azole fragments. Azole heterocyclic compounds exhibit various biological activities, such as antibacterial, antimalarial, antifungal, anti-HIV, anti-inflammatory, and anti-tuberculosis, among others. Azole derivatives are of continuous interest in the field of medicinal chemistry, and many of them are not only marketed drugs, but also highly active molecules are emerging, such as antibacterial Tazobactum, anti-HIV TSAO, and anticancer CAI. However, no report is found on the derivative of PAS linked to azole and the biological activity thereof.
Disclosure of Invention
In view of the above, the present invention aims to provide p-aminosalicylic acid azole derivatives, and a preparation method and applications thereof.
Through research, the invention provides the following technical scheme:
1. a p-aminosalicylic acid azole derivative represented by the formula I, an oxynitride compound, a oxysulfide compound or a pharmaceutically acceptable salt thereof:
Figure GDA0003640403630000011
in formula I, R is selected from:
Figure GDA0003640403630000012
Figure GDA0003640403630000021
R1and R2Independently selected from H or C1-C3 alkyl; r3And R4Independently selected from H or C1-C3 alkyl; r5Is H or C1-C3 alkyl; r6Is H or C1-C3 alkyl; r7Is H, C1-C3 alkyl or amino; r8H, C1-C3 alkyl, substituted or unsubstituted phenyl, wherein the substituents on the phenyl are one or more independently selected from halogen, hydroxyl, amino or C1-C3 alkyl; r9And R10Independently selected from H or C1-C3 alkyl; r11is-O-, -S-or-NH-; r12And R13Independently selected from H, halogen or alkanoyl; l is selected from: - (CH)2)n+1-、-(CH2)nCO-or-CO (CH)2)nCO-, n is 1,2, 3 or 4; x is selected from: C1-C6 alkyl.
Preferably, in said formula I, R1And R2Independent of each otherIs selected from H or methyl; r3And R4Independently selected from H or methyl; r5Is H or methyl; r6Is H or methyl; r7Is methyl or amino; r8Is methyl, substituted or unsubstituted phenyl, and the substituent on the phenyl is one or more and is independently selected from halogen, hydroxyl, amino or C1-C3 alkyl; r is9And R10Are both methyl; r11is-O-, -S-or-NH-; r12And R13Independently selected from H, halogen or acetyl; l is selected from: - (CH)2)n+1-or- (CH)2)nCO-, n is 1,2, 3 or 4; x is selected from: C1-C3 alkyl.
Preferably, in the formula I, the compound of formula I,
r is selected from:
Figure GDA0003640403630000022
Figure GDA0003640403630000023
l is selected from: - (CH)2)nCO-, n is 1,2, 3 or 4; x is selected from: methyl or ethyl.
Preferably, the para-aminosalicylic acid azole derivative shown in the formula I is any one of the following compounds:
Figure GDA0003640403630000024
Figure GDA0003640403630000031
Figure GDA0003640403630000041
preferably, the para-aminosalicylic acid azole derivative shown in the formula I is any one of the following compounds: TM1-2, TM1-4, TM1-5, TM1-6, TM1-8, TM1-9, TM1-10, TM1-11, TM1-12, TM1-14, TM4-1, TM4-2, TM4-4, TM4-7, TM4-8, TM4-9, TM4-10, TM4-11, TM4-12, TM4-13, TM4-14, TM4-15 and TM 4-17.
2. The preparation method of the p-aminosalicylic acid azole derivative comprises the following steps:
esterifying carboxyl para-aminosalicylate to prepare an intermediate IM 1;
Figure GDA0003640403630000042
reacting the intermediate IM1 with a linker reagent to prepare an intermediate IM 2;
Figure GDA0003640403630000043
coupling the intermediate IM2 with oxazole to prepare a target molecule p-aminosalicylic acid azole derivative;
Figure GDA0003640403630000044
wherein X, L and R are as defined above for the formula X, L and R in the formula of the p-aminosalicylic acid azole derivative; z in IM2 is halogen.
Preferably, the preparation method of the p-aminosalicylic acid azole derivative comprises the following steps:
A. under the action of acid, p-aminosalicylic acid reacts with alcohol to prepare an intermediate IM 1; the alcohol is methanol or ethanol; the acid is sulfuric acid;
B. subjecting the intermediate IM1 and chloroacetyl chloride to aminoacylation in an organic solvent under the action of alkali to obtain an intermediate IM 2; the organic solvent is dichloromethane, chloroform, acetone, ethyl acetate, tetrahydrofuran or diethyl ether; the alkali is potassium carbonate, triethylamine or sodium bicarbonate;
C. coupling the intermediate IM2 with oxazole in an organic solvent under the action of alkali to prepare a p-aminosalicylic acid azole derivative; the organic solvent is dichloromethane, chloroform, acetonitrile, tetrahydrofuran or N, N-dimethylformamide; the alkali is sodium bicarbonate, triethylamine, sodium hydroxide, sodium methoxide or potassium carbonate.
More preferably, in step B, the organic solvent is dichloromethane or chloroform, and the base is sodium bicarbonate.
More preferably, in the step C, the organic solvent is N, N-dimethylformamide, and the base is potassium carbonate.
3. The application of the p-aminosalicylic acid azole derivative in antibacterial drugs.
4. The application of the p-aminosalicylic acid azole derivative in antifungal medicines.
5. The p-aminosalicylic acid azole derivative is applied to antitumor drugs.
6. The application of the p-aminosalicylic acid azole derivative in the drug for resisting citrus bacteria is provided.
Unless otherwise indicated, the term "nitroxide" as used herein refers to the formation of a tertiary nitrogen with an oxygen atom+N-O-An organic material of a structural unit; "sulfur oxide" refers to a sulfoxide or sulfone resulting from the oxidation of a precursor sulfide; the "pharmaceutically acceptable salt" may be an acidic salt or a basic salt, such as an inorganic acid salt, an organic acid salt, an inorganic base salt or an organic base salt.
The term "C1-C3 alkyl" refers to straight or branched chain saturated monovalent hydrocarbon radicals having 1-3 carbon atoms such as methyl, ethyl, propyl and isopropyl.
The term "halogen" refers to F, Cl, Br and I.
The invention has the beneficial effects that:
1) the p-aminosalicylic acid azole derivatives provided by the invention take p-aminosalicylic acid as a parent nucleus, reasonably modify amino and carboxyl to construct a p-aminosalicylic acid azole derivative with a novel structure, and the chemical structure of the product is shown in the specification1H NMR，13C NMR and HR MS confirmation;
2) the result of in vitro antibacterial activity determination shows that the overall antibacterial activity of the compound is good. Wherein, for staphylococcus aureus, the MIC values of 9 compounds are 32 mug/mL, and the MIC values of the intermediates IM2-1 and IM2-2 are respectively 25.6 mug/mL and 12.8 mug/mL, which are stronger than PAS and methyl ethyl ester thereof (MIC value >256 mug/mL). For E.coli, the MIC values of compounds TM4-11, TM4-12 and TM4-13 were 64. mu.g/mL, 32. mu.g/mL and 64. mu.g/mL, respectively, which are stronger than those of PAS and its intermediates (MIC value ≧ 256. mu.g/mL). For Micrococcus luteus, the MIC values of compounds TM4-9 and TM4-11 were 16 μ g/mL, and the MIC value of intermediate IM2-2 was 3.2 μ g/mL, which is stronger than PAS and its methyl and ethyl esters (MIC value >256 μ g/mL). For Salmonella, the MIC value of intermediate IM2-2 was 0.8. mu.g/mL, which is much stronger than PAS and its methyl ethyl ester (MIC value > 256. mu.g/mL). Thereby proving that the aminosalicylic acid azole derivatives and the intermediates thereof have potential application prospect in the antibacterial field;
3) the p-aminosalicylic acid azole derivative provided by the invention has good bacteriostatic activity on pichia pastoris, wherein MIC of 4 molecules is less than or equal to 4 mug/mL, and the MIC value of the p-aminosalicylic acid azole derivative is the same as that of a positive control drug fluconazole, and the inhibitory activity on pichia pastoris strains is shown to be stronger than or equal to that of the fluconazole; the inhibitory activity of the intermediate IM2-1 and IM2-2 on Pichia pastoris reaches 64 mu g/mL, which is stronger than that of mother nucleus PAS. Thereby proving that the aminosalicylic acid azole derivatives and the intermediates thereof have potential application prospect in the antifungal field;
4) the p-aminosalicylic acid azole derivative provided by the invention has an inhibiting effect on 3 cancer cells at a concentration of 5 mu M. Aiming at HCT116 of colon cancer cells, the inhibition rate of compounds TM1-2, TM4-4, IM2-1 and IM2-2 is over 50 percent, and the inhibition rate of intermediate IM2-2 is up to 60 percent. Most compounds were stronger than the maternal PAS (8%) against the colon cancer cells SW 620. Aiming at the PC3 of the prostate cancer cells, the inhibition rate of compounds TM4-3, TM4-5 and IM2-2 is higher than 50%, the inhibition rate of intermediate IM2-2 reaches 78%, and the inhibition effect of 63% of the compounds on the PC3 of the prostate cancer cells is stronger than that of the PAS (5%). Proves that the aminosalicylic acid azole derivatives and the intermediates thereof have potential application prospect in the anti-tumor field;
5) the p-aminosalicylic acid azole derivatives provided by the invention have 8 and 3 molecules respectively reaching 70% inhibition rate of positive drugs to colletotrichum gloeosporioides at concentrations of 1 mug/mL and 4 mug/mL; for the citrus brown spot germs, the number of molecules reaching the inhibition rate of 70 percent of the positive drug is 1 and 3 respectively; the target compound TM1-2 and the intermediate IM2-2 have good inhibitory activity on the citrus brown spot germ, and are respectively the same as or stronger than the inhibitory activity of positive control prochloraz. Meanwhile, TM1-2, TM1-5, TM4-10 and IM2-2 did not exhibit drug resistance. In addition, most target compounds have good inhibition effect on citrus canker bacteria. Proves that the aminosalicylic acid azole derivatives and the intermediates thereof have potential application prospects in the field of resistance to citrus germs.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
First, main reagent and instrument
P-aminosalicylic acid, chloracetyl chloride, dichloromethane, N-dimethylformamide, imidazole, 2-methylimidazole, 4-methylimidazole, pyrazole, benzimidazole, 1,2, 4-triazole, 2, 4-dimethylpyrazole, 1H-tetrazole, 5-methyltetrazole, 4, 6-dimethyl-2-mercaptopyrimidine, 1-methyl-5-mercaptotetrazole, 1-phenyl-5-mercaptotetrazole, 2-mercapto-5-methyl-1, 3, 4-thiadiazole; concentrated sulfuric acid, methanol, sodium bicarbonate and potassium carbonate; 2-amino-5-mercapto-1, 3, 4-thiadiazole; absolute ethyl alcohol; all the foregoing are analytical reagents. The other reagents are all commercial chemical pure or analytical pure products.
Nuclear magnetic resonance apparatus (AV-600, TMS as internal standard, DMSO-d6 as solvent,1the H NMR was 600MHz and the resulting mixture was,13c NMR 151 MHz); high resolution mass spectrometer (Varian 7.0T); a melting point tester (X-6); an automatic polarimeter (WZZ-2S); ultraviolet-visible spectrophotometer (ZF-1); rotary evaporator (RE-2000).
Preparation of bis, p-aminosalicylic acid azole derivatives
1. Synthesis of intermediate IM1-1
Figure GDA0003640403630000071
To the reaction flask were added 1.53g (10mmol) of PAS and 25mL of methanol, and the mixture was stirred at room temperature. And (3) carrying out ice bath, dropwise adding 1.3mL (25mmol) of concentrated sulfuric acid, and carrying out oil bath reflux reaction after dropwise adding, and monitoring by Thin Layer Chromatography (TLC) until the reaction is finished. Cooling in ice bath, adjusting pH to 7-8 with sodium carbonate solution, refrigerating, filtering, and washing filter cake with ice water. The filtrate was extracted with Dichloromethane (DCM) (3X 30mL) and the organic phase was collected and washed with saturated NaCl solution. Drying with anhydrous sodium sulfate, rotary steaming, combining with a filter cake, vacuum drying, and performing column chromatography to obtain intermediate IM1-1 (white solid) 1.096g with a yield of 65%.
2. Synthesis of intermediate IM2-1
Figure GDA0003640403630000072
To a reaction flask were added IM 1-15 mmol (0.766g), DCM 5mL, NaHCO312.5mmol (1.1g), cooled in an ice bath, and 10mmol (0.75mL) of chloroacetyl chloride was added dropwise. After the dropwise addition, the reaction was continued in ice bath and monitored by TLC until the reaction was completed. Stopping stirring, adding 10mL of ice-cold saturated saline solution, adjusting pH to 4-5 with 2N HCl solution, transferring into separating funnel, extracting with Ethyl Acetate (EA) twice, combining organic phases, washing with saturated saline solution, and adding anhydrous Na2SO4Drying, rotary evaporating to remove solvent, and performing column chromatography (PE (petroleum ether): EA is 10: 1-5:1, v/v) to obtain pure intermediate IM2-1 of 1.03g, with yield of 83%.
3. Synthesis of p-aminosalicylic acid azole derivative TM1
Figure GDA0003640403630000073
Adding oxazole (RH), N-Dimethylformamide (DMF) and K into a reaction bottle in sequence2CO3Stirring at room temperature, adding intermediate IM2-1, transferring to a water bath at 45 ℃ for stirring, and tracking and monitoring by TLC until the reaction is finished. Stirring was stopped and ice-cold saturatedAdjusting pH to 6-7 with NaCl solution and 2N HCl solution, and refrigerating. Suction filtering, washing the filter cake with saturated brine (10mL × 1) and ice water (5mL × 1), vacuum drying to obtain crude product, and performing column chromatography (DCM: CH)3OH & lt 50:1-5:1, v/v), thin layer chromatography and/or solvent dispersion to obtain the target compound TM 1. The experimental conditions and results are shown in table 1.
TABLE 1 Experimental conditions and results for the preparation of TM1
Figure GDA0003640403630000081
4. TM1 product structure characterization data are as follows:
Figure GDA0003640403630000091
TM1-1:Methyl 4-(2-(1H-imidazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.184.0～186.3℃；1H NMRδ10.64(s,1H,H-5),10.60(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.63(s,1H,H-8),7.36(d,J＝1.3Hz,1H,H-4),7.17(s,1H,H-10),7.09(dd,J＝8.7Hz,1.4Hz,1H,H-2),6.90(s,1H,H-9),4.94(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.38,167.06,161.64,145.45,138.83,131.44,128.44,121.20,110.91,108.35,106.75,52.70,49.74.HRMS calcd for C13H13N3O4,[M+H]+276.0979,found 276.0976.
TM1-2:Methyl 2-hydroxy-4-(2-(2-methyl-1H-imidazol-1-yl)acetamido)benzoateWhite solid；m.p.188.6～191.0℃；1H NMRδ10.65(s,1H,H-5),10.61(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.36(d,J＝1.8Hz,1H,H-4),7.08(dd,J＝8.7Hz,1.8Hz,1H,H-2),6.84(s,1H,H-10),6.74(s,1H,H-9),4.84(s,2H,H-7),3.86(s,3H,H-6),2.18(s,3H,H-8).13C NMRδ169.68,168.23,164.36,144.2,143.01,131.52,126.94,119.19,114.11,108.42,106.21,51.69,49.61,13.27.HR MS calcd for C14H15N3O4,[M+H]+290.1135,found 290.1131.
TM1-3:Methyl 2-hydroxy-4-(2-(4-methyl-1H-imidazol-1-yl)acetamido)benzoateWhite solid；m.p.184.8～186.9℃；1H NMRδ10.64(s,1H,H-5),10.56(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.48(s,1H,H-8),7.36(d,J＝1.8Hz,1H,H-4),7.08(dd,J＝8.7Hz,1.8Hz,1H,H-2),6.84(s,1H,H-10),4.84(s,2H,H-7),3.86(s,3H,H-6),2.08(s,3H,H-9).13C NMRδ169.38,167.17,161.64,145.46,138.03,136.78,131.43,117.36,110.91,108.32,106.74,52.70,49.72,14.05.HR MS calcd for C14H15N3O4,[M+H]+290.1135,found 290.1133.
Figure GDA0003640403630000092
TM1-4:Methyl 4-(2-(1H-benzo[d]imidazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.227.5～229.3℃；1H NMRδ10.78(s,2H,H-3 and H-5),8.22(s,1H,H-8),7.67(t,J＝8.5Hz,2H,H-9 and H-12),7.54(d,J＝7.8Hz,1H,H-1),7.24(s,1H,H-4),7.20(d,J＝6.3Hz,2H,H-10 and H-11),6.94(d,J＝8.6Hz,1H,H-2),5.20(s,2H,H-7),3.80(s,3H,H-6).13C NMRδ169.27,166.72,163.01,145.48,145.30,143.66,134.92,131.58,122.87,121.99,119.82,110.81,109.97,108.75,107.52,52.45,47.87.HR MS calcd for C17H15N3O4,[M+H]+326.1135,found 326.1147.
TM1-5:Methyl 4-(2-(1H-pyrazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.203.1.0～206.3℃；1H NMRδ10.69(s,1H,H-5),10.51(s,1H,H-3),7.80(d,J＝8.5Hz,1H,H-1),7.76(s,1H,H-10),7.54(d,J＝1.7Hz,1H,H-4),7.47(s,1H,H-9),7.23(dd,J＝8.7Hz,1.7Hz,1H,H-2),6.29(s,1H,H-9),4.82(s,2H,H-7),3.87(s,3H,H-6),H-8.13C NMRδ169.13,166.78,161.82,145.41,139.56,132.10,131.34,110.93,108.40,106.76,105.83,61.57,54.98.HR MS calcd for C13H13N3O4,[M+H]+276.0979,found 276.0977.
TM1-6:Methyl 4-(2-(3,5-dimethyl-1H-pyrazol-1-yl)acetamido)-2-hydroxybenzoateWhite solid；m.p.260.3～262.4℃；1H NMRδ10.66(s,1H,H-5),10.47(s,1H,H-3),7.80(d,J＝8.5Hz,1H,H-1),7.54(d,J＝1.7Hz,1H,H-4),7.23(dd,J＝8.7Hz,1.7Hz,1H,H-2),5.82(s,1H,H-8),4.82(s,2H,H-7),3.87(s,3H,H-6),2.69(s,3H,H-9),2.17(s,3H,H-10).13C NMRδ169.47,166.92,161.34,145.84,142.59,137.32,129.37,113.56,104.29,104.15,102.84,61.28,54.18,14.71,13.50.HR MS calcd for C15H17N3O4,[M+H]+304.1292,found 304.1294.
Figure GDA0003640403630000101
TM1-7:Methyl 4-(2-(1H-1,2,4-triazol-1-yl)acetamido)-2-hydroxybenzoate White solid；m.p.220.8～222.9℃；1H NMRδ10.71(s,1H,H-5),10.65(s,1H,H-3),8.57(s,1H,H-8),8.01(s,1H,H-9),7.76(d,J＝8.7Hz,1H,H-1),7.36(d,J＝1.5Hz,1H,H-4),7.09(dd,J＝8.7Hz,1.6Hz,1H,H-2),5.19(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.35,165.82,161.62,151.88,146.10,145.19,131.47,110.97,108.57,106.88,52.70,52.36.HR MS calcd for C12H12N4O4,[M+H]+277.0931,found 277.0938.
TM1-8:Methyl 4-(2-(1H-tetrazol-1-yl)acetamido)-2-hydroxybenzoate White solid；m.p.187.8～189.9℃；1H NMRδ10.90(s,1H,H-5),10.64(s,1H,H-3),9.05(s,1H,H-8),7.77(d,J＝8.7Hz,1H,H-1),7.33(s,1H,H-4),7.09(d,J＝8.7Hz,1H,H-2),5.77(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.27,164.14,161.55,153.89,144.88,131.56,111.04,108.87,107.03,55.57,52.74.HR MS calcd for C11H11N5O4,[M+H]+278.0884,found 278.0881.
TM1-9:Methyl 4-(2-(2H-tetrazol-2-yl)acetamido)-2-hydroxybenzoate White solid；m.p.207.3～209.8℃；1H NMRδ10.91(s,1H,H-5),10.63(s,1H,H-3),9.43(s,1H,H-8),7.77(d,J＝8.7Hz,1H,H-1),7.34(s,1H,H-4),7.10(d,J＝8.7Hz,1H,H-2),5.54(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.29,164.76,161.56,145.70,145.00,131.54,110.99,108.76,106.96,52.72,50.66.HR MS calcd for C11H11N5O4,[M+H]+278.0884,found 278.0882.
Figure GDA0003640403630000111
TM1-10:Methyl 2-hydroxy-4-(2-(5-methyl-1H-tetrazol-1-yl)acetamido)benzoate White solid；m.p.189.0～190.8℃；1H NMRδ10.86(s,1H,H-5),10.64(s,1H,H-3),7.77(d,J＝8.7Hz,1H,H-1),7.34(d,J＝1.3Hz,1H,H-4),7.08(dd,J＝8.7Hz,1.4Hz,1H,H-2),5.66(s,2H,H-7),3.87(s,3H,H-6),2.49(s,3H,H-8).13C NMRδ169.29,164.23,162.91,161.56,144.91,131.53,111.03,108.82,107.01,55.45,52.73,10.83.HR MS calcd for C12H13N5O4,[M+H]+292.1040,found 292.1039.
TM1-11:Methyl 4-(2-((4,6-dimethylpyrimidin-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.184.5～186.7℃；1H NMRδ10.70(s,1H,H-5),10.54(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.34(d,J＝1.6Hz,1H,H-4),7.08(dd,J＝8.8Hz,1.6Hz,1H,H-2),6.96(s,1H,H-8),4.05(s,2H,H-7),3.87(s,3H,H-6),2.31(s,6H,H-9 and H-10).13C NMRδ169.65,169.19,167.87,167.43(2C),161.86,145.88,131.23,116.54,110.84,108.04,106.52,61.52,36.13,23.76(2C).HR MS calcd for C16H17N3O4S,[M+H]+348.1013,found 348.1016.
TM1-12:Methyl 2-hydroxy-4-(2-((1-methyl-1H-tetrazol-5-yl)thio)acetamido)benzoateWhite solid；m.p.262.1～264.4℃；1H NMRδ10.66(brs,2H,H-3 and H-5),7.75(d,J＝8.7Hz,1H,H-1),7.34(s,1H,H-4),7.06(d,J＝8.2Hz,1H,H-2),4.33(s,2H,H-7),3.99(s,3H,H-6),3.87(s,3H,H-8).13C NMRδ169.34,166.24,161.81,153.74,145.37,131.44,110.77,108.52,106.87,52.65,38.28,34.15.HR MS calcd for C12H13N5O4S,[M+H]+324.0761,found 324.0758.
Figure GDA0003640403630000112
TM1-13:Methyl 2-hydroxy-4-(2-((1-phenyl-1H-tetrazol-5-yl)thio)acetamido)benzoateWhite solid；m.p.224.9～226.6℃；1H NMRδ10.66(s,2H,H-5 and H-3),7.69(m,6H,H-1,H-8～H-12),7.18(s,1H,H-4),6.90(d,1H,H-2),4.41(s,2H,H-7),3.80(s,3H,H-6).13C NMRδ169.20,165.85,163.79,154.42,145.29,133.54,131.61,131.14,130.55(2C),124.93(2C),109.37,108.92,107.91,52.29,38.44.HR MS calcd for C17H15N5O4S,[M+H]+386.0918,found 386.0921.
TM1-14:Methyl 2-hydroxy-4-(2-((5-methyl-1,3,4-thiadiazol-2-yl)thio)acetamido)benzoate White solid；m.p.191.7～193.0℃；1H NMRδ10.63(s,1H,H-5),8.57(s,1H,H-3),7.67(d,J＝8.4Hz,1H,H-1),7.20(s,1H,H-4),6.93(d,J＝8.4Hz,1H,H-2),4.28(s,2H,H-7),3.80(s,3H,H-6),2.67(s,3H,H-8).13C NMRδ169.32,166.46,166.22,164.68,162.34,145.45,131.46,110.40,108.58,107.12,52.55,38.73,15.63.HR MS calcd for C13H13N3O4S2,[M+H]+340.0420,found 340.0417.
TM1-15:Methyl 4-(2-((5-amino-1,3,4-thiadiazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.200.3～202.9℃；1H NMRδ10.63(s,1H,H-5),10.55(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.36(d,J＝1.5Hz,1H,H-4),7.32(s,2H,H-8),7.06(dd,J＝8.7Hz,1.6Hz,1H,H-2),4.02(s,2H,H-7),3.87(s,3H,H-6).13C NMRδ169.38,167.10,162.79(2C),161.62,145.54,131.38,110.93,108.36,106.74,52.69,36.24.HR MS calcd for C12H12N4O4S2,[M+H]+341.0373,found 341.0362.
5. synthesis of intermediate IM1-2
Figure GDA0003640403630000121
To the reaction flask were added PAS 20mmol and absolute ethanol 25mL, and the mixture was stirred at room temperature. And (4) performing ice bath, slowly adding 50mmol of concentrated sulfuric acid dropwise, performing oil bath reflux reaction at 80 ℃ after the concentrated sulfuric acid is completely added, and monitoring by TLC until the reaction is finished. Cooling in ice bath, adjusting pH to 7-8 with saturated sodium carbonate, refrigerating, filtering, and washing filter cake with ice water. The filtrate was extracted with DCM (3X 30mL), and the organic phase was collected and washed with saturated NaCl solution. Drying with anhydrous sodium sulfate, rotary steaming, combining with a filter cake, vacuum drying, column chromatography, rotary steaming and weighing to obtain 2.32g of intermediate IM1-2 (white solid) with the yield of 64%.
6. Synthesis of intermediate IM2-2
Figure GDA0003640403630000122
Adding IM 1-25 mmol, DCM 5mL, NaHCO into a reaction bottle312.5 mmol; cooled in ice bath, and 10mmol of chloroacetyl chloride is added dropwise. The reaction was continued in an ice bath and monitored by TLC tracking to the end of the reaction. Stopping stirring, adding 10mL of ice-cold saturated saline solution, adjusting pH to 4-5 with 2N HCl solution, stirring, transferring into separating funnel, EA extracting twice, mixing organic phases, washing with saturated saline solution, and anhydrous Na2SO4Drying, removing the solvent by rotary evaporation, performing column chromatography to obtain a pure product, drying, and weighing to obtain 1.16g of intermediate IM2-2 with the yield of 90%.
7. Synthesis of p-aminosalicylic acid azole derivative TM4
Figure GDA0003640403630000131
Adding oxazole (RH), DMF and K into a reaction bottle in sequence2CO3Stirring at room temperature, adding intermediate IM2-2, transferring to a water bath at 45 ℃ for stirring, and tracking and monitoring by TLC until the reaction is finished. Stopping stirring, adding ice-cold saturated NaCl solution, adjusting pH to 6-7 with 2N HCl solution, and refrigerating. Suction filtration was performed, and the filter cake was washed with saturated brine (10mL × 1) and ice water (5mL × 1), and dried in vacuo to give a crude product, which was subjected to column chromatography (PE/EA ═ 10:1-1:3, v/v). When the oxazole is mercaptooxazole, DMF is changed to Tetrahydrofuran (THF) before column chromatographyThe dispersion is first repeated with petroleum ether and the remaining conditions or operations are the same as described above for the non-mercaptooxazole. Drying to obtain the target compound TM 4. The experimental conditions and results are shown in table 2.
TABLE 2 Experimental conditions and results for preparing TM4
Figure GDA0003640403630000132
Figure GDA0003640403630000141
8. TM4 product structure characterization data are as follows:
Figure GDA0003640403630000142
TM4-1:Ethyl 4-(2-(1H-imidazol-1-yl)acetamido)-2-hydroxybenzoate White solid；m.p.206.7～207.9℃；1H NMRδ10.72(s,1H,H-5),10.60(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.63(s,1H,H-11),7.36(d,J＝1.7Hz,1H,H-4),7.17(s,1H,H-9),7.10(dd,J＝8.7Hz,1.8Hz,1H,H-2),6.90(s,1H,H-10),4.94(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.12,167.05,161.82,145.46,138.82,131.35,128.45,121.18,110.91,108.38,106.74,61.56,49.74,14.50.HR MS calcd for C14H15N3O4,[M+H]+290.1135,found 290.1135.
TM4-2:Ethyl 2-hydroxy-4-(2-(2-methyl-1H-imidazol-1-yl)acetamido)benzoate White solid；m.p.203.4～205.7℃；1H NMRδ10.72(s,1H,H-5),10.61(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.36(d,J＝1.7Hz,1H,H-4),7.10(dd,J＝8.7Hz,1.8Hz,1H,H-2),7.05(s,1H,H-9),6.73(s,1H,H-10),4.85(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),2.23(s,3H,H-11),1.33(t,J＝6.9Hz,3H,H-7).13C NMRδ169.12,166.91,161.82,145.41,144.47,131.34,126.51,121.45,110.94,108.41,106.79,61.56,49.18,14.50,13.01.HR MS calcd for C15H17N3O4,[M+H]+304.1292,found 304.1293.
TM4-3:Ethyl 2-hydroxy-4-(2-(4-methyl-1H-imidazol-1-yl)acetamido)benzoate White solid；m.p.207.3～209.9℃；1H NMRδ10.71(s,1H,H-5),10.56(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.48(s,1H,H-11),7.36(d,J＝1.6Hz,1H,H-4),7.09(dd,J＝8.7Hz,1.6Hz,1H,H-2),6.84(s,1H,H-9),4.84(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),2.08(s,3H,H-11),1.33(t,J＝6.9Hz,3H,H-7).13C NMRδ169.13,167.15,161.82,145.47,138.02,136.79,131.33,117.36,110.91,108.35,106.73,61.56,49.74,14.50,14.04.HR MS calcd for C15H17N3O4,[M+H]+304.1292,found 304.1289.
Figure GDA0003640403630000151
TM4-4:Ethyl 4-(2-(1H-benzo[d]imidazol-1-yl)acetamido)-2-hydroxybenzoate White solid；m.p.246.1～248.1℃；1H NMRδ10.77(s,1H,H-5),10.71(s,1H,H-3),8.24(s,1H,H-9),7.77(d,J＝8.7Hz,1H,H-1),7.68(d,J＝7.8Hz,1H,H-13),7.55(d,J＝7.9Hz,1H,H-10),7.36(d,J＝1.8Hz,1H,H-4),7.28–7.17(m,2H,H-11 and H-12),7.12(dd,J＝8.8Hz,1.8Hz,1H,H-2),5.22(s,2H,H-8),4.34(q,J＝7.0Hz,2H,H-6),1.32(t,J＝7.1Hz,3H,H-7).13C NMRδ169.11,166.82,161.82,145.47,145.41,143.67,134.94,131.38,122.88,122.00,119.84,110.95,110.81,108.43,106.78,61.57,47.86,14.50.HR MS calcd for C18H17N3O4,[M+H]+340.1292,found 340.1291.
TM4-5:Ethyl 4-(2-(1H-pyrazol-1-yl)acetamido)-2-hydroxybenzoate White solid；m.p.221.9～223.7℃；1H NMRδ10.72(s,1H,H-5),10.61(s,1H,H-3),7.76(d,J＝8.7Hz,2H,H-1 and H-9),7.47(s,1H,H-11),7.36(d,J＝1.5Hz,1H,H-4),7.09(dd,J＝8.7Hz,J＝1.5Hz,1H,H-2),6.29(s,1H,H-10),5.06(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.13,166.78,161.82,145.41,139.56,132.10,131.34,110.93,108.40,106.76,105.83,61.57,54.98,14.50.HR MS calcd for C14H15N3O4,[M+H]+290.1135,found 290.1133.
TM4-6:Ethyl 4-(2-(1H-1,2,4-triazol-1-yl)acetamido)-2-hydroxybenzoate White solid；m.p.273.5～275.8℃；1H NMRδ10.71(s,2H,H-5 and H-3),8.56(s,1H,H-9),8.01(s,1H,H-10),7.77(d,J＝8.7Hz,1H,H-1),7.35(d,J＝1.5Hz,1H,H-4),7.09(dd,J＝8.7Hz,1.7Hz,1H,H-2),5.18(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.08,165.82,161.79,151.88,146.09,145.20,131.40,110.96,108.59,106.85,61.58,52.35,14.50.HR MS calcd for C13H14N4O4,[M+H]+291.1088,found 291.1087.
Figure GDA0003640403630000161
TM4-7:Ethyl 4-(2-(1H-tetrazol-1-yl)acetamido)-2-hydroxybenzoate White solid；m.p.245.7～247.1℃；1H NMRδ10.90(s,1H,H-5),10.71(s,1H,H-3),9.05(s,1H,H-9),7.78(d,J＝8.7Hz,1H,H-1),7.33(d,J＝1.7Hz,1H,H-4),7.09(d,J＝8.7Hz,1.7Hz,1H,H-2),5.77(s,2H,H-8),4.34(q,J＝7.0Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.02,164.13,161.74,153.88,144.89,131.47,111.02,108.87,107.00,61.61,55.57,14.49.HRMS calcd for C12H13N5O4,[M+H]+292.1040,found 292.1040.
TM4-8:Ethyl 4-(2-(2H-tetrazol-2-yl)acetamido)-2-hydroxybenzoate White solid；m.p.255.1～256.8℃；1H NMRδ10.78(s,2H,H-5 and H-3),9.42(s,1H,H-9),7.78(d,J＝8.7Hz,1H,H-1),7.33(d,J＝1.7Hz,1H,H-4),7.09(d,J＝8.7Hz,1.7Hz,1H,H-2),5.53(s,2H,H-8),4.34(q,J＝7.0Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.03,164.76,161.75,145.70,144.99,131.48,110.97,108.79,106.93,61.60,50.66,14.50.HR MS calcd for C12H13N5O4,[M+H]+292.1040,found 292.1042.
TM4-9:Ethyl 2-hydroxy-4-(2-(5-methyl-1H-tetrazol-1-yl)acetamido)benzoateWhite solid；m.p.203.1～205.8℃；1H NMRδ10.86(s,1H,H-5),10.71(s,1H,H-3),7.78(d,J＝8.7Hz,1H,H-1),7.33(d,J＝1.7Hz,1H,H-4),7.08(d,J＝8.7Hz,1.7Hz,1H,H-2),5.65(s,2H,H-8),4.34(d,J＝7.1Hz,2H,H-6),2.49(s,3H,H-9),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.03,164.22,162.91,161.74,144.91,131.46,111.02,108.85,106.98,61.61,55.45,14.50,10.84.HR MS calcd for C13H15N5O4,[M+H]+306.1197,found 306.1195.
Figure GDA0003640403630000162
TM4-10:Ethyl 2-hydroxy-4-(2-(5-methyl-2H-tetrazol-2-yl)acetamido)benzoateWhite solid；m.p.216.3～218.8℃；1H NMRδ10.85(s,1H,H-5),10.71(s,1H,H-3),7.78(d,J＝8.7Hz,1H,H-1),7.33(d,J＝1.7Hz,1H,H-4),7.09(dd,J＝8.7Hz,1.8Hz,1H,H-2),5.43(s,2H,H-8),4.34(d,J＝7.0Hz,2H,H-6),2.51(s,3H,H-9),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.02,164.64,161.74,153.95,144.91,131.46,111.02,108.83,107.00,61.61,49.78,14.50,8.76.HR MS calcd for C13H15N5O4,[M+H]+306.1197,found 306.1196.
TM4-11:Ethyl 4-(2-((4,6-dimethylpyrimidin-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.279.3～281.2℃；1H NMRδ10.71(s,1H,H-5),10.55(s,1H,H-3),7.74(d,J＝8.7Hz,1H,H-1),7.37(d,J＝1.6Hz,1H,H-4),7.09(dd,J＝8.8Hz,1.6Hz,1H,H-2),6.96(s,1H,H-10),4.33(q,J＝7.1Hz,2H,H-6),4.05(s,2H,H-8),2.31(s,6H,H-9 and H-11),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.65,169.19,167.87,167.43(2C),161.86,145.88,131.23,116.54,110.84,108.04,106.52,61.52,36.13,23.76(2C),14.51.HR MS calcd for C17H19N3O4S,[M+H]+362.1169,found 362.1165.
TM4-12:Ethyl 2-hydroxy-4-(2-((1-methyl-1H-tetrazol-5-yl)thio)acetamido)benzoate White solid；m.p.241.7～243.3℃；1H NMRδ10.78(s,2H,H-5 and H-3),7.74(d,J＝8.7Hz,1H,H-1),7.34(s,1H,H-4),7.05(d,J＝8.3Hz,1H,H-2),4.35～4.31(m,4H,H-8 and H-6),3.99(s,3H,H-9),1.32(t,J＝7.1Hz,3H,H-7).13C NMRδ169.08,166.27,153.75,145.41,131.34(2C),110.73,108.50,106.85,61.51,38.24,34.15,14.51.HR MS calcd for C13H15N5O4S,[M+H]+338.0918,found 338.0920.
Figure GDA0003640403630000171
TM4-13:Ethyl 2-hydroxy-4-(2-((1-phenyl-1H-tetrazol-5-yl)thio)acetamido)benzoateWhite solid；m.p.214.3～215.9℃；1H NMRδ10.74(s,2H,H-5 and H-3),7.75(d,J＝8.7Hz,1H,H-1),7.72–7.67(m,5H,H-9、H-10、H-11、H-12 and H-13),7.34(d,J＝1.5Hz,H-4),7.06(dd,J＝8.6Hz,1.5Hz,1H,H-2),4.43(s,2H,H-8),4.33(q,J＝7.1Hz,2H,H-6),1.32(t,J＝7.1Hz,3H,H-7).13C NMRδ169.09,166.00,162.00,154.38,145.40,133.51,131.36,131.16,130.56(2C),124.90(2C),110.76,108.51,106.84,61.52,38.35,14.51.HR MS calcd for C18H17N5O4S,[M+H]+400.1074,found 400.1074.
TM4-14:Ethyl 2-hydroxy-4-(2-((5-methyl-1,3,4-thiadiazol-2-yl)thio)acetamido)benzoate White solid；m.p.218.1～219.9℃；1H NMRδ10.71(s,1H,H-5),10.65(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.37(d,J＝1.5Hz,H-4),7.08(dd,J＝8.7Hz,1.5Hz,1H,H-2),4.34(q,J＝7.1Hz,2H,H-6),4.30(s,2H,H-8),2.68(s,3H,H-9),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.11,166.51,166.23,164.62,161.81,145.50,131.33,110.88,108.39,106.70,61.57,38.67,15.64,14.51.HR MS calcd for C14H15N3O4S2,[M+H]+353.0504,found 353.0501.
TM4-15:Ethyl 4-(2-((5-amino-1,3,4-thiadiazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.237.1～238.8℃；1H NMRδ10.71(s,1H,H-5),10.54(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.37(d,J＝1.5Hz,H-4),7.32(s,2H,H-9),7.06(dd,J＝8.7Hz,1.5Hz,1H,H-2),4.34(q,J＝7.0Hz,2H,H-6),4.03(s,2H,H-8),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ170.48,169.12,167.09,161.81,149.61,145.55,131.30,110.89,108.35,106.70,61.56,39.29,14.51.HR MS calcd for C13H14N4O4S2,[M+H]+355.0529,found 355.0523.
Figure GDA0003640403630000181
TM4-16:Ethyl 4-(2-((5-chlorobenzo[d]oxazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.249.4～251.7℃；1H NMRδ10.74(s,1H,H-5),10.71(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.74(d,J＝1.8Hz,1H,H-9),7.69(d,J＝8.7Hz,1H,H-11),7.38(d,J＝2.0Hz,1H,H-10),7.36(d,J＝1.5Hz,1H,H-4),7.10(dd,J＝8.7Hz,1.5Hz,1H,H-2),4.44(s,2H,H-8),4.34(q,J＝7.0Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.09,166.30,166.05,161.80,150.63,145.42,142.96,131.37,129.48,124.78,118.52,111.99,110.91,108.49,106.75,61.57,37.46,14.51.HR MS calcd for C18H15ClN2O5S,[M+H]+407.0463,found 407.0462.
TM4-17:Ethyl 2-hydroxy-4-(2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido)benzoate White solid；m.p.245.3～247.2℃；1H NMRδ12.53(s,1H,H-9),10.81(s,1H,H-5),10.72(s,1H,H-3),7.75(d,J＝8.7Hz,1H,H-1),7.38(d,J＝1.8Hz,1H,H-10),7.36(d,J＝1.5Hz,1H,H-4),7.09(dd,J＝8.7Hz,J＝1.5Hz,1H,H-2),6.94(s,1H,H-13),6.79～6.74(m,1H,H-11),4.34(q,J＝7.1Hz,2H,H-6),4.26(s,2H,H-8),3.77(s,3H,H-12),1.33(t,J＝7.1Hz,3H,H-7).HR MS calcd for C19H19N3O5S,[M+H]+402.1118,found 402.1118.
TM4-18:Ethyl 4-(2-((6-chlorobenzo[d]thiazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.253.5～255.9℃；1H NMRδ10.74(s,1H,H-5),10.72(s,1H,H-3),8.18(d,J＝1.9Hz,1H,H-9),7.80(d,J＝8.7Hz,1H,H-11),7.76(d,J＝8.7Hz,1H,H-1),7.49(dd,J＝8.7,2.1Hz,1H,H-10),7.38(d,J＝1.8Hz,1H,H-4),7.11(dd,J＝8.7Hz,1.8Hz,1H,H-2),4.44(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.12,167.72,166.39,161.83,151.78,145.50,136.86,131.32,129.47,127.28,122.58,122.07,110.91,108.42,106.74,61.56,38.41,14.50.HR MS calcd for C18H15ClN2O4S2,[M+H]+423.0235,found 423.0237.
Figure GDA0003640403630000182
TM4-19:Ethyl 4-(2-((6-bromobenzo[d]thiazol-2-yl)thio)acetamido)-2-hydroxybenzoate White solid；m.p.241.7～243.8℃；1H NMRδ10.74(s,1H,H-5),10.72(s,1H,H-3),8.31(d,J＝1.8Hz,1H,H-9),7.76(d,J＝8.8Hz,1H,H-11),7.74(d,J＝8.7Hz,1H,H-1),7.61(dd,J＝8.7,1.8Hz,1H,H-10),7.38(d,J＝1.7Hz,1H,H-4),7.11(dd,J＝8.7Hz,1.8Hz,1H,H-2),4.44(s,2H,H-8),4.34(dd,J＝14.1,7.0Hz,2H,H-6),1.33(t,J＝7.0Hz,3H,H-7).13C NMRδ169.12,167.78,166.38,161.83,152.06,145.50,137.30,131.32,129.97,124.90,122.95,117.47,110.91,108.42,106.74,61.56,38.41,14.51.HR MS calcd for C18H15BrN2O4S2,[M+H]+:466.9729,found 466.9727.
TM4-20:Ethyl 2-hydroxy-4-(2-((6-methoxybenzo[d]thiazol-2-yl)thio)acetamido)benzoate White solid；m.p.236.4～238.8℃；1H NMRδ10.73(s,1H,H-5),10.71(s,1H,H-3),7.76(d,J＝8.7Hz,1H,H-1),7.72(d,J＝8.9Hz,1H,H-9),7.62(d,J＝2.5Hz,1H,H-12),7.39(d,J＝1.9Hz,1H,H-4),7.11(dd,J＝8.7Hz,2.0Hz,1H,H-2),7.06(dd,J＝8.9,2.6Hz,1H,H-11),4.39(s,2H,H-8),4.34(q,J＝7.1Hz,2H,H-6),3.81(s,3H,H-10),1.33(t,J＝7.1Hz,3H,H-7).13C NMRδ169.14,166.62,162.78,161.84,157.29,147.45,145.55,136.79,131.30,122.09,115.63,113.52,110.90,106.72,105.47,61.55,56.18,38.39,14.49.
bioactivity detection of tris-p-aminosalicylic acid azole derivatives
1. In vitro antibacterial Activity assay
The activity (MIC value) of the compounds against staphylococcus aureus (staphylococcus aureus ATCC 25129), Micrococcus luteus (Micrococcus luteus), Escherichia coli (Escherichia coli ATCC 25922), Acinetobacter baumannii (Acinetobacter baumannii ATCC 19606), Salmonella (Salmonella Enteritidis ATCC 13076) and Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 27853) was determined by the broth microdilution method.
A sample of 3.2mg is taken, a DMSO solution with the mass fraction of 5% Tween 80 is prepared into a mother solution with the concentration of 3.2mg/mL, 200 mu L of the mother solution is absorbed, and the mother solution is diluted to 500 mu L by broth with the concentration of 1.28 mu g/mu L to be used as a solution to be detected.
Inoculating the preserved strain into a common liquid culture medium, and placing the strain in a constant temperature shaking table at 37 ℃ for activation culture for 17 h. Respectively diluting with brain heart infusion Broth (BHI) culture medium to 105CFU/mL of bacterial suspension is ready for use.
The operation is as follows: add 60. mu.L of blank broth to well 1 of each column of 96-well plate, and add 50. mu.L of blank broth to the remaining wells; adding 40 mu L of prepared solution to be tested (the concentration is 1.28 mu g/mu L) into the 1 st hole of each row, then diluting the substance to be tested twice, namely adding the solution to be tested into the first hole, fully blowing the solution by using a liquid transfer gun (at least three times or more) to fully and uniformly mix the substance to be tested and broth, then sucking 50 mu L of the solution to be added into the 2 nd hole, fully blowing the solution to be fully and uniformly mixed with the broth, sucking 50 mu L of the solution to be added into the 3 rd hole, repeating the steps until reaching the 8 th hole, sucking 50 mu L of the solution and discarding; at this time, the concentration of the analyte in each well is 512,256,128,64,32,16,8,4 (unit: μ g/mL) from top to bottom. The last two columns of each plate are used as controls, the two columns do not contain the substance to be detected, one column is used as a bacteria growth control and added with bacteria liquid, and the other column is used as a negative control and not added with bacteria liquid. Finally, 50 mu L of diluted bacteria solution is added into each row of 1-8 holes, and a multi-hole test is adopted, wherein each plate tests 5 compounds, and the concentration of the substance to be tested in each hole, namely the final concentration of the substance to be tested, is 256,128,64,32,16,8,4 and 2 (unit: mu g/mL) from top to bottom.
And (3) putting the inoculated 96-well plate into a constant-temperature incubator at 37 ℃ for culturing for 20-24h, and observing the growth condition of bacteria in the hole. And determining that the bacteria in the negative control hole grow normally and the bacteria in the positive control hole do not grow. The concentration of the drug in wells with no bacterial growth was observed as the MIC value of the drug against the bacteria.
The results of the in vitro bacteriostatic activity test on the aminosalicylic acid azole derivatives and the intermediates are shown in table 3.
TABLE 3 inhibitory Activity of Compounds against 6 pathogenic bacteria (MIC values, μ g/mL)
Figure GDA0003640403630000201
Figure GDA0003640403630000211
From the analysis in table 3, it can be seen that: PAS, methyl ester (IM1-1) and ethyl ester (IM1-2) of PAS had little inhibitory activity (MIC > 256. mu.g/mL); the bacteriostatic activity of the target compound, namely the aminosalicylic acid azole derivative is better than that of PAS, IM1-1 and IM1-2 on the whole. The inhibitory activity of the compound on staphylococcus aureus is better than that of other strains, the MIC values of 9 compounds are 32 mu g/mL, wherein 6 ethyl ester derivatives (TM4) and 3 methyl ester derivatives (TM1) are contained; the bacteriostatic activity of the intermediate IM2-1 and IM2-2 on Staphylococcus aureus is as low as 25.6 mu g/mL and 12.8 mu g/mL. For E.coli, the MIC value of compound TM4-12 was 32. mu.g/mL. For Micrococcus luteus, the MIC values for compounds TM4-9 and TM4-11 were 16 μ g/mL. Particularly, the MIC value of the intermediate IM2-2 to Micrococcus luteus is 3.2 mu g/mL, the MIC value to Salmonella is 0.8 mu g/mL, and the antibacterial activity is very strong. The above results show that both the p-aminosalicylic acid chloroacetyl derivative and the p-aminosalicylic acid azole derivative have antibacterial activity. Thereby proving that the aminosalicylic acid azole derivatives and the intermediates thereof have potential application prospects in the antibacterial field.
2. In vitro antifungal Activity assay
The activity of the compounds against pichia pastoris (MIC values) was determined using the NCCLS recommended broth microdilution method with fluconazole as positive control drug.
(1) Preparation of sample solution
Accurately weighing 3.2mg of a sample in a 2mL PE tube by a ten-thousandth balance in a drying chamber, adding 1mL DMSO into the PE tube by a pipette gun, dissolving into clear transparent liquid, preparing into 3.2mg/mL mother solution or stock solution, sealing with a sealing film, and storing in a refrigerator in a dark place. The solvent is DMSO, DMSO/tween-80 (200/1 (v/v)) is used as the solvent for a part of difficultly soluble compounds to increase the solubility, and tween-80 is used as a cosolvent.
(2) Preparation of test solution
According to the effect or content of the object to be detected and the required volume, the required amount of the object to be detected is calculated, various required antibacterial objects to be detected are accurately weighed, and the objects to be detected are diluted to the required concentration by using a proper solvent and a diluent.
Preparing a solution B to be detected: sucking 320 mu L of stock solution with the concentration of 3.2mg/mL, adding 180 mu L of Sabouraud's medium to the total volume of 0.5mL, wherein the concentration of the diluent is 2048 mu g/mL, and the solution B to be detected is obtained.
(3) Preparation of the bacterial suspension
Inoculating the preserved strain into a Sabouraud's agar liquid culture medium, and placing the strain in a constant temperature shaker at 30 ℃ for activation culture for 24 hours. After activation, the surface colonies of agar were washed with distilled water and diluted to 10 with Sabouraud's medium5CFU/mL of bacterial suspension is ready for use.
(4) Sample application operation
Under the aseptic condition, 50 mu L of the Sabouraud's medium is added into each hole of a 96-hole plate; adding 50 mu L of prepared solution to be tested into the first hole and the second hole of the first row, and diluting by twice to obtain 1024 mu g/mL solution; fully blowing and beating the first hole and the second hole by using a pipette gun to fully and uniformly mix the object to be detected with the culture medium, sucking 50 mu L of the liquid to be detected, adding the liquid to the first hole and the second hole of the second row, blowing and beating the liquid to be detected and the culture medium to be fully and uniformly mixed, repeating the steps until the eighth row is reached, sucking 50 mu L of the liquid to be detected in each hole of the eighth row, and discarding the liquid; at the moment, the concentration of the substance to be detected in each hole is 1024,512,256,128,64,32,16 and 8 (unit: mu g/mL) from high to low (from top to bottom); then 50 mu L of diluted bacterial liquid is added into each hole of a 96-hole plate, and the concentration of the substance to be detected in each hole, namely the final substance to be detected, is 512,256,128,64,32,16,8 and 4 (unit: mu g/mL) from high to low (from top to bottom).
(5) Culture and result determination
And putting the inoculated 96-well plate into a 30 ℃ constant temperature incubator for culturing for 30h and 48 h. After the completion of the culture, the 96-well plate was taken out from the incubator, and the growth of bacteria in the well was observed. The result was normalized by determining the normal growth of bacteria in the blank drug-free control (negative control) wells and the no growth of bacteria in the positive control (medium + strain + positive drug) wells. The concentration of the drug in the wells with no bacterial growth was visually observed as the MIC of the drug against the bacteria.
The results of the in vitro antifungal activity test on the aminosalicylic acid azole derivatives and intermediates are shown in table 4.
TABLE 4 MIC value determination results (μ g/mL) of Pichia pastoris
Figure GDA0003640403630000221
Figure GDA0003640403630000231
As can be seen from Table 4, MIC of the mother nucleus PAS, IM1-1 and IM1-2 is more than 256 μ g/mL, and almost has no inhibitory activity to Pichia pastoris; the intermediate IM2-1 and IM2-2 have the inhibiting activity on pichia pastoris up to 64 mug/mL, which is stronger than that of mother nucleus PAS. The inhibiting activity of most target compounds on pichia is good and is stronger than that of PAS, methyl ester of PAS and ethyl ester of PAS of parent bodies; after the culture is carried out for 24h, 30 molecules of 35 target molecules have MIC values of 4-256 mu g/mL, 28 molecules of 4-128 mu g/mL, 16 molecules of 4-64 mu g/mL, 10 molecules of 4-32 mu g/mL and even 4 molecules of 4 mu g/mL, which are the same as the MIC value of a positive control drug fluconazole, show that the inhibitory activity of the 4 molecules on Pichia pastoris strains is stronger than or equal to that of fluconazole, thereby proving that the aminosalicylic acid azole derivatives and intermediates thereof have potential application prospects in the antifungal field.
3. Determination of antitumor biological Activity
The inhibition rate of a series of small molecular compounds in tumor cells is obtained by MTT experiments, and the specific steps are as follows:
(1) tumor cell suspension in logarithmic growth phase was inoculated in 96-well culture plates (3X 10)3Cells/well), 200. mu.L of total medium per well, 5% CO at 37 ℃2Culturing in a cell culture box overnight;
(2) after the cells are completely attached to the wall, replacing the previous culture medium with a complete culture medium containing 10 mu M of small molecular compounds, adding an equal volume of DMSO complete culture medium into a control group, and continuously culturing for 48 hours;
(3) after addition of 20. mu.L of MTT solution, the mixture was left at 37 ℃ with 5% CO2After incubation for 4h in a cell incubator, taking out the culture plate, gently sucking the supernatant to avoid sucking purple crystals as much as possible, and then adding 150 mu L of DMSO solution into each hole;
(4) placing the treated culture plate on a shaking table, and gently shaking for 10min in a dark place at room temperature to ensure that the crystal violet is dissolved uniformly as much as possible;
(5) and (3) placing the culture plate on an enzyme labeling instrument, selecting the wavelength of 570nm, reading and recording the light absorption value (OD value) of the corresponding hole in a 96-well plate, and further carrying out statistics to obtain the corresponding inhibition rate of the small molecular compound in different tumor cells. The results of the measurement at a concentration of 5. mu.M are shown in Table 5.
TABLE 5 results of inhibitory Activity of Compounds on tumor cells
Figure GDA0003640403630000241
The data in Table 5 are analyzed, and the aminosalicylic acid azole derivative has an inhibiting effect on three cancer cells under the concentration of 5 mu M. Aiming at HCT116 of colon cancer cells, the inhibition rate of 4 compounds (TM1-2, TM4-4, IM2-1 and IM2-2) is over 50 percent, and the inhibition rate of an intermediate IM2-2 reaches 60 percent, which is far stronger than that of a parent to aminosalicylic acid, methyl p-aminosalicylate and ethyl ester thereof. The total body of SW620 has a certain inhibiting effect on colon cancer cells, and most compounds are stronger than PAS (8%) of a mother nucleus. Aiming at the PC3 of the prostate cancer cells, the inhibition rate of 3 compounds (TM4-3, TM4-5 and IM2-2) is higher than 50%, the inhibition rate of the intermediate IM2-2 reaches 78%, and the inhibition effect of 63% of the compounds on the PC3 of the prostate cancer cells is stronger than that of the mother nucleus PAS (5%). Proves that the aminosalicylic acid azole derivatives and the intermediates thereof have potential application prospects in the anti-tumor field.
4. Determination of biological activity of citrus fungus germs
1) Preliminary screening
(1) Preparation of mother liquid and diluent of substance to be measured
And diluting the mother liquor of the substance to be detected to the required concentration by using a proper solvent and a diluent. The sample mass is 1.0mg, and 1.0mg/1mL to 1.0mg/mL mother solution of the substance to be detected is prepared; each test article was set at 2 dilutions, 0.001mg/mL (i.e., 1000-fold dilution, 1. mu.g/mL) and 0.004mg/mL (i.e., 250-fold dilution, 4. mu.g/mL).
(2) Operation of
Preparation of a culture medium of a to-be-detected object: preparation of a culture medium of a to-be-detected object diluted by 1000 times: taking 5 mu L of the dilution of the substance to be detected with the concentration of 1 mu g/mL and 5mL of hot PDA culture medium, and fully and uniformly mixing in a 10mL centrifuge tube; preparation of a medicament culture medium diluted by 250 times: 20 μ L of the 4 μ g/mL dilution of the test substance was mixed with 4980 μ L of hot PDA medium in a 10mL centrifuge tube.
Control group: the PDA culture medium without the test substance and the culture medium with prochloraz added (diluted 1000 times and 250 times) are used as controls, and are respectively a blank control and a positive control. Inoculating bacteria: the prepared culture medium of the to-be-detected substance is poured into a 24-well plate, and one well is poured for each concentration of each to-be-detected substance of each strain. Mycelia of the strain cultured at 28 ℃ for 7 days were picked and inoculated into each well. Culturing: the 24-well plate is placed in an incubator with 28 ℃ and 16h of illumination for 48 h. Measurement: the colony diameter was measured using a cross method. And (3) calculating: inhibition [% ] is (CK colony diameter value-measured colony diameter value) × 100%/CK colony diameter value. Screening: and comparing the inhibition rates of different substances to be tested with the inhibition rate of prochloraz to obtain a primary screening result.
2) Double sieve
And (3) carrying out secondary screening on high-activity molecules TM1-5 and TM4-10 obtained by primary screening to inhibit Colletotrichum citricola gloeosporioides strain Co.3 to obtain a virulence equation. TM1-2 and IM2-2 carry out re-screening on the Alternaria alternata strain Al.6 of the citrus limosa to obtain a virulence equation.
The first step is as follows: and (5) diluting the substance to be detected in a gradient manner. 6 dilution gradients were set, namely 0.01, 0.004, 0.002, 0.001, 0.0005, 0.00025 (unit: mg/mL), namely dilutions 100, 250, 500, 1000, 2000, 4000 fold. The second step is that: preparing a culture medium of the object to be tested. 50, 20, 10, 5, 2.5 and 1.25 microliter of the dilution of the analyte to be detected and 5mL of hot PDA culture medium are fully and uniformly mixed in a 10mL centrifuge tube. The test medium was poured into the wells and each gradient repeated 4 times for each test. PDA medium and prochloraz were used as control groups (blank control and positive control). The third step: and (5) inoculating bacteria. Mycelia of the strain cultured at 28 ℃ for 7 days were picked and inoculated into the center of each well. The fourth step: and (5) culturing. The 24-well plate is placed in an incubator with 28 ℃ and 16h of illumination for 48 h. The fifth step: and (6) measuring. The cross method measures the colony diameter. And a sixth step: and (4) calculating. Processing the data by using a pesticide indoor bioassay data processing system (PBT data processing system) to obtain a regression equation and KD50、KD90R, standard error, chi-squared value, and 95% confidence value.
The results of the primary and secondary screening measurements are shown in tables 6, 7, 8 and 9.
TABLE 6 inhibitory Activity of Compounds on Citrus fungal pathogens (preliminary screening results)
Figure GDA0003640403630000251
Figure GDA0003640403630000261
The data in the table 6 are analyzed to find that the tested compound has certain inhibitory activity on both the citrus colletotrichum gloeosporioides strain and the citrus brown spot bacterial strain; under the concentrations of 1 microgram/mL and 4 microgram/mL, the number of molecules reaching the 50% inhibition rate of the positive drug of the colletotrichum citriodorum is respectively 19 and 18, and the number of molecules reaching the 70% inhibition rate of the positive drug is respectively 8 and 3; for the citrus brown spot germs, the number of molecules reaching 50 percent inhibition rate of the positive medicament is respectively 4 and 9, and the number of molecules reaching 70 percent inhibition rate of the positive medicament is respectively 1 and 3. Particularly, the inhibitory activity of the target compound TM1-2 and the intermediate IM2-2 on the citrus brown spot germ is equal to or stronger than that of a positive control prochloraz respectively, and the compound shows good development potential.
TABLE 7 inhibitory Activity of Compounds on Citrus fungal pathogens (preliminary screening results)
Figure GDA0003640403630000271
Table 7 shows that 8 saxacin drugs and PAS had no inhibitory activity against both citrus fungi at the tested concentrations; however, TM1-13, TM4-1 and TM4-8 showed certain inhibitory activity to the Colletotrichum citricola strains, which is stronger than the positive control, and deserves further development.
TABLE 8 inhibitory Activity of highly active molecules against Colletotrichum citricola (rescreening results)
Figure GDA0003640403630000272
TABLE 9 inhibitory Activity of highly active molecules against Phoma citrifolia (rescreened results)
Figure GDA0003640403630000273
Analysis of the data in tables 8 and 9 shows that TM1-2, TM1-5, TM4-10 and IM2-2 do not show drug resistance, and prove that the aminosalicylic acid azole derivatives and intermediates thereof have potential application prospects in the field of resistance to citrus germs.
5. Determination of biological activity of citrus canker resistant bacteria
The determination method comprises the following steps: weighing 1mg of sample, dissolving the sample in 50 mu L of DMSO, and fixing the volume by using ultrapure water to obtain sample mother liquor with different concentrations. 10 mu L of the mother liquor is taken as a sample solution a in 1mL of ultrapure water (0.02% Tween), and then sample solutions b, c, d and the like with different concentrations are prepared in sequence by adopting a multiple dilution method.
Washing the ulcer germs cultured for 3 days on the PDA culture medium with 5mL of LB liquid culture medium, adding into 195mL of LB liquid culture medium, and shaking and mixing for later use. 450 mu L of the bacterial liquid of the citrus canker pathogen and 50 mu L of the sample solutions with different concentrations (a to D) are respectively added into a 2mL centrifuge tube, so that the final concentrations of the samples in the mixed bacterial liquid are respectively A (1.6 mu g/mL), B (0.64 mu g/mL), C (0.5 mu g/mL), D (0.1 mu g/mL), 28 ℃, 200 r.min-1OD determination after constant temperature shaking culture for 14h600The OD value of each mixed bacterial solution was calculated and the inhibition ratio was calculated (inhibition ratio%Blank space-ODSample (I))/ODBlank spaceX 100%). Each sample was replicated three times per concentration. The results of the measurement of citrus canker pathogenic bacteria are shown in tables 10 and 11.
Inhibitory Activity of Compounds of Table 10 against Leptosphaeria citricola
Figure GDA0003640403630000281
Inhibitory Activity of Compounds of Table 11 against Leptosphaeria citricola
Figure GDA0003640403630000282
Figure GDA0003640403630000291
As can be seen from the data in Table 11, the inhibition of the parent PAS was 9.44% and 6.27% at the above concentrations tested, respectively, and the activity was very weak. And the inhibition rate of most compounds is better than that of PAS under the test concentration of the p-aminosalicylic acid azole derivatives. Wherein, under the test concentration of 1.6 mug/mL, the inhibition activity of the target molecule is higher than 7 molecules of 30%, and under the test concentration of 0.64 mug/mL, the inhibition rate is higher than 5 molecules of 30%. Thereby proving that the aminosalicylic acid azole derivatives have good inhibition effect on citrus canker pathogenic bacteria.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (8)

1. A p-aminosalicylic acid azole derivative represented by the formula I:
Figure FDA0003640403620000011
in the formula I, the compound has the following structure,
r is selected from:
Figure FDA0003640403620000012
Figure FDA0003640403620000013
l is-COCH2- ；
X is methyl or ethyl.
2. The para-aminosalicylic acid azole derivative or the pharmaceutically acceptable salt thereof according to claim 1, wherein the para-aminosalicylic acid azole derivative represented by the formula i is any one of the following compounds:
Figure FDA0003640403620000014
Figure FDA0003640403620000021
Figure FDA0003640403620000031
3. the para-aminosalicylic acid azole derivative or the pharmaceutically acceptable salt thereof according to claim 2, wherein the para-aminosalicylic acid azole derivative represented by the formula i is any one of the following compounds:
Figure FDA0003640403620000032
Figure FDA0003640403620000041
4. a process for producing a p-aminosalicylic acid azole derivative of claim 1, which comprises the steps of:
esterifying carboxyl p-aminosalicylate to obtain an intermediate IM 1;
Figure FDA0003640403620000042
reacting the intermediate IM1 with a linker reagent to prepare an intermediate IM 2;
Figure FDA0003640403620000043
coupling the intermediate IM2 with oxazole to prepare a target molecule p-aminosalicylic acid azole derivative;
Figure FDA0003640403620000051
wherein X, L and R are as defined in X, L and R in the structural formula of the para-aminosalicylic acid azole derivative according to claim 1; z in IM2 is halogen.
5. Use of an azole derivative of p-aminosalicylic acid as claimed in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for the manufacture of an antibacterial agent.
6. Use of p-aminosalicylic acid azole derivatives of any one of claims 1 to 3 or pharmaceutically acceptable salts thereof for the preparation of antifungal agents.
7. Use of a p-aminosalicylic acid azole derivative of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for the preparation of a medicament against colon and prostate cancer.
8. Use of a para-aminosalicylic acid azole derivative of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament against citrus bacteria.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5324740A (en) * 1992-06-06 1994-06-28 Kureha Chemical Industry Co., Ltd. Azole derivatives, pharmaceutical compositions containing the same, and method for treating mycosis and estrogen-dependent diseases
CN1185736A (en) * 1995-01-18 1998-06-24 奥尔顿有限公司 Use of thiazolium compounds for preventing and preverting the formation of advanced glycosylation endproducts
DE19943508A1 (en) * 1999-09-10 2001-03-15 Morphochem Ag 3-vinylpyrene derivatives, process for their preparation and their use as medicines
CN1315320A (en) * 2000-01-11 2001-10-03 寿制药株式会社 Ether and amide compounds as medicide for treating glycuresis and preparing process thereof
CN109096278A (en) * 2018-09-26 2018-12-28 西南大学 Fluoquinolone-nitrogen azoles hybrid derivatives, preparation method and its usage

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007059195A1 (en) * 2005-11-14 2007-05-24 University Of Southern California Integrin-binding small molecules
US20090029949A1 (en) * 2006-05-25 2009-01-29 Parrill-Baker Abby L GPCR Ligands Identified by Computational Modeling
WO2014081856A2 (en) * 2012-11-20 2014-05-30 Azzazy Hassan Ligands that target hcv-e2 binding sites on cd81 and therapeutic methods using them
US10647725B2 (en) * 2016-04-21 2020-05-12 The University Of Toledo Anti-infective 2-aminothiophenes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5324740A (en) * 1992-06-06 1994-06-28 Kureha Chemical Industry Co., Ltd. Azole derivatives, pharmaceutical compositions containing the same, and method for treating mycosis and estrogen-dependent diseases
CN1185736A (en) * 1995-01-18 1998-06-24 奥尔顿有限公司 Use of thiazolium compounds for preventing and preverting the formation of advanced glycosylation endproducts
DE19943508A1 (en) * 1999-09-10 2001-03-15 Morphochem Ag 3-vinylpyrene derivatives, process for their preparation and their use as medicines
CN1315320A (en) * 2000-01-11 2001-10-03 寿制药株式会社 Ether and amide compounds as medicide for treating glycuresis and preparing process thereof
CN109096278A (en) * 2018-09-26 2018-12-28 西南大学 Fluoquinolone-nitrogen azoles hybrid derivatives, preparation method and its usage

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
2-羟基-1-萘醛缩对氨基水杨酸希夫碱铬配合物的合成及体外抗肿瘤活性研究;王水英等;《中国药物与临床》;20160815(第08期);第1096-1098页 *
对氨基水杨酸衍生物的设计、合成及其生物活性研究;潘建芳;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》;20201231;摘要 *

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