CN112773795B

CN112773795B - Application of 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound in preparation of beta-glucuronidase inhibitor

Info

Publication number: CN112773795B
Application number: CN202011588920.1A
Authority: CN
Inventors: 王鸿; 崔紫宁; 魏斌; 周涛顺; 李亚胜
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2022-07-22
Anticipated expiration: 2040-12-29
Also published as: CN112773795A

Abstract

The invention discloses a method for preparing beta-glucurone by using 1,3, 4-thiadiazole phenyl furan sulfo-methyl ester compoundsThe 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound has very obvious inhibition activity on escherichia coli beta-glucuronidase (EcGUS), and IC (integrated Circuit) is₅₀The value range is 0.1-5.4 mu M, and the compound has wide application prospect in the research and development of medicaments for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs.

Description

Application of 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound in preparation of beta-glucuronidase inhibitor

Technical Field

The invention relates to a 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound, in particular to a new application thereof in preparation of a beta-glucuronidase inhibitor, which is beneficial to further research and development of a drug for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs and belongs to the technical field of chemical medicines.

Background

Irinotecan (CPT-11), a commonly used drug for the treatment of colon cancer. After entering a human body, the SN-38 glucuronide (SN-38G) is metabolized into inactive SN-38 glucuronide in the liver, then the SN-38G is excreted into the intestinal tract through the bile duct, intestinal bacteria can remove glycosyl of the SN-38G by utilizing self-generated beta-glucuronidase to supply self energy demand, and simultaneously the generated SN-38 is accumulated in the intestinal tract to a certain degree to kill epithelial cells of the intestinal tract and cause irreversible damage to the intestinal mucosa, so that delayed diarrhea of a patient is caused, and the chemotherapy process is seriously influenced. In addition, many carboxylic acid-containing non-steroidal anti-inflammatory drugs such as ketoprofen, diclofenac, and indomethacin, when taken, cause similar intestinal toxicity, resulting in severe drug-induced diarrhea.

Beta-glucuronidase is a member of the glycosidase family 2, and is capable of hydrolyzing beta-D-linked glucuronide bonds. Beta-glucuronidase can be produced by a plurality of microorganisms in human and animal intestines, in 2010, students verify that the inhibition of intestinal bacteria beta-glucosidase can relieve drug-induced diarrhea caused by CPT-11 for the first time, and then the development and application of intestinal bacteria beta-glucuronidase inhibitors are widely concerned. Although the source of the beta-glucuronidase in the intestinal tract is not limited to escherichia coli, the escherichia coli beta-glucuronidase (EcGUS) is widely distributed in the intestinal tracts of humans and animals and is easy to prepare, so that the EcGUS is often used as a common target for researching intestinal bacterium beta-glucuronidase inhibitors, selectively inhibits the activity of the EcGUS in the intestinal tract, and can directly reduce the accumulation of SN-38 in the intestinal tract, thereby protecting the intestinal tract of a patient from damage.

The 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound contains a 5-phenyl-2-furan structure, and the 5-phenyl-2-furan structure has various biological activities such as antibiosis, antitumor, anti-inflammation, insect resistance and the like, and is initially applied to research on prevention and treatment of plant bacterial diseases (pesticide, biochem, physiol.160(2019) 87-94). The compounds listed in the invention are all generously provided by professor tretinoin university of south china agriculture (pesticide. biochem. physiol.160(2019)87-94), however, whether the compounds have EcGUS inhibitory activity or not has not been reported.

Disclosure of Invention

The invention aims to provide application of a 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound in preparation of a beta-glucuronidase inhibitor, and is favorable for further research and development of a drug for treating drug-induced diarrhea caused by irinotecan or a non-steroidal anti-inflammatory drug.

The technical scheme adopted by the invention is as follows:

the invention provides an application of 1,3, 4-thiadiazole phenyl furan thio-methyl ester compounds shown as a formula (I) or (II) in preparing a beta-glucuronidase (EcGUS) inhibitor,

in the formula (I), R is mono-substituted or poly-substituted, and is hydrogen, halogen, nitryl, C1-C4 alkyl or C1-C4 alkoxy; in the formula (II), R is the same as the formula (I).

Further, the R single substituent in the formula (I) is: 2-Cl, 3-Cl, 4-Cl, 2-F, 3-F, 4-F, 2-NO₂、3-NO₂、4-NO₂4-Br, 4-Me, 4-MeO, -H; the R double substituent in the formula (I) is: 2,4-di-F, 2, 6-di-F.

Further, the 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound is shown as a formula (II), and R is 3-F.

Further, the 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound has IC (integrated Circuit) of inhibiting effect on beta-glucuronidase₅₀The value ranges from 0.1 to 5.4. mu.M.

Further, the beta-glucuronidase is derived from enterobacteria, preferably escherichia coli; beta-glucuronidase is commercially available from biochemical companies, such as Sigma Aldrich (Shanghai) trade company, and can be prepared from E.coli.

The invention also provides application of the 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound in preparation of a medicament for treating drug-induced diarrhea.

Further, the drug-induced diarrhea drug is a drug for treating diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs.

Compared with the prior art, the invention has the following beneficial effects:

the 1,3, 4-thiadiazole phenyl furan thiomethyl ester compound is reported for the first time on the EcGUS inhibitory activity, the inhibitory activity is very obvious, and IC₅₀The value ranged from 0.1 to 5.4. mu.M (better than the positive control, i.e., IC for D-glucarate-1, 4-lactone)₅₀The value is 67.3 mu M), has wide application prospect in the aspect of developing medicaments for treating the drug-induced diarrhea caused by the irinotecan or the non-steroidal anti-inflammatory drugs, and is expected to be further applied to the development of medicaments for treating the drug-induced diarrhea caused by the irinotecan or the non-steroidal anti-inflammatory drugs.

Drawings

FIG. 1 is an electrophoretogram of the purified EcGUS proenzyme.

FIG. 2 is a standard curve for p-nitrophenol (PNP).

FIG. 3 is a bar graph showing the inhibition rate of compounds of formulae I and II and amoxapine (which is a marketed antidepressant and is also a reference drug with the strongest inhibitory activity against EcGUS) and D-glucaric-1, 4-lactone (both at a final concentration of 10. mu.M) against EcGUS.

FIG. 4 is a diagram showing the docking results of compound II-5 and EcGUS molecules, wherein A is a 2D plane action diagram of the docking of compound II-5 and EcGUS molecules, B is a whole diagram showing the spatial position of the docking of compound II-5 and EcGUS molecules, and C is a local 3D stereo action diagram of the docking of compound II-5 and EcGUS molecules.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of protection of the invention is not limited thereto:

the test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The preparation of the compounds I-1 to I-15 and II-1 to II-15 used in the examples of the present invention is referred to Pestic. biochem. physiol.160(2019) 87-94.

Example 1: screening of EcGUS inhibitors

(1) Preparation of EcGUS: escherichia coli (Escherichia coli BL21(DE3)) stored at-80 ℃ was inoculated into 200mL of LB liquid medium (10 g/L trypsin, 5g/L yeast extract, 10g/L sodium chloride, water as solvent, pH 7.0) containing 30. mu.g/mL kanamycin, cultured at 200rpm at 37 ℃ until OD600 reached 0.5, followed by addition of isopropyl-. beta. -D-thiogalactoside (IPTG) at a final concentration of 100mM, overnight culture at 200rpm at 30 ℃ to induce expression of EcGUS, and expression of the enzyme was detected by SDS-PAGE. After the expression is completed, the culture solution is centrifuged for 5min at 9000rpm at 4 ℃, the thalli are collected, the thalli are washed 2-3 times by PBS (pH 7.4), then lysate (20mM 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), 300mM NaCl, 5mM imidazole (imidazole), glycerol (glycerol) with the volume concentration of 10% and the solvent being water, pH 7.4) is added according to 1/10 of the volume of the original bacterial solution (culture solution before centrifugation), 20mL of the thalli are re-suspended, the thalli are ultrasonically broken for 20min (placed on ice) at intervals of 10s under the conditions of 300W and 10s of ultrasonic, and then the thalli are centrifuged for 10min at 8000rpm at 4 ℃ for 10min, and supernatant is taken. Then washing 15mL of Ni-NTA agarose resin column (purchased from GE medical company) with 15mL of each of purified water and NTA-0 buffer (20mM Tris-HCl, 0.5M sodium chloride, volume solubility 10% glycerol, pH 7.9) for 2-3 times, chelating the supernatant with the Ni-NTA agarose resin column at 4 ℃ for 3 hours, then performing gradient elution with 15mL of each of NTA-0 buffer, NTA-20 buffer (20mM Tris-HCl, 0.5M sodium chloride, volume solubility 10% glycerol, 20mM imidazole, pH 7.9), NTA-250 buffer (20mM Tris-HCl, 0.5M sodium chloride, volume solubility 10% glycerol, 250mM imidazole, pH 7.9), collecting one tube of each 5mL of the eluate, collecting 9 tubes of the eluate, performing SDS-PAGE on each tube of the eluate, and showing that the 4 tubes of the NTA-20 buffer contain the EcU protein E, and the molecular weight of EcGUS is about 71kD, then the 4 tubes of eluent are combined, finally, a Millipore protein ultrafiltration tube with 10kD (the molecular weight cut-off is not larger than 1/3 of the molecular weight of the target protein) is used for filtering, the collected trapped fluid is enzyme fluid, and about 7mL of EcGUS proenzyme fluid (shown in figure 1) is obtained.

(2) Preparation of a p-nitrophenol (PNP) standard curve: preparing 1mM PNP solution (PBS dissolved), operating in a 96-well plate, adding 0, 10, 20, 40, 60, 80 μ L of 1mM PNP solution in each group of 3 parallels, supplementing to 100 μ L with PBS, incubating for 30min at 37 ℃, measuring absorbance at 405nM wavelength for 0min and 30min respectively by using a microplate reader, then making a scatter diagram by using Excel (see figure 2), obtaining the relation of the absorbance and the PNP concentration as y (3.2617 x + 0.0547), wherein y is the absorbance, x is the PNP concentration, converting and removing blank interference by unit, and the relation of the absorbance and the PNP concentration at μ M concentration is y (0.003262 x).

(3) Screening of EcGUS inhibitors (10. mu.M final concentration of inhibitor):

inhibitor (B): compounds shown in formulas I and II in Table 1 were prepared into 0.1mM of mother liquor with dimethyl sulfoxide (DMSO), respectively, for use.

Positive control: d-glucaric acid-1, 4-lactone (D-Saccharomyces acid 1,4-lactone, DSL, available from Sigma-Aldrich) and Amoxapine (Amoxapine, AMX, a positive control with the strongest EcGUS inhibitory activity reported in the literature at present, available from Sigma-Aldrich) were prepared into 0.1mM solution with dimethyl sulfoxide (DMSO) as a positive control for use.

Substrate: 4-Nitrophenyl-. beta. -D-glucopyranosiduronide (. beta. -PNPG, available from Sigma-Aldrich) was made up in PBS to a 2.5mM stock solution for future use.

Enzyme: the beta-glucuronidase (EcGUS) prepared in the step (1) is diluted 500 times with PBS, and the concentration measured by a kit is 1 mu g/mL, and the diluted product is used as a reaction enzyme solution.

Reaction: the reaction was run in 96-well plates (100 μ L) as follows, blank: 10. mu.L of enzyme + 70. mu.L of PBS + 10. mu.L of 1% DMSO-based pure water solution in volume + 10. mu.L of 2.5mM substrate; experimental groups: enzyme 10. mu.L + PBS 70. mu.L +0.1mM inhibitor 10. mu.L +2.5mM substrate 10. mu.L; positive control group: enzyme 10. mu.L + PBS 70. mu.L +0.1mM positive control 10. mu.L +2.5mM substrate 10. mu.L; each group was made into 3 replicates, loading enzyme, PBS, inhibitor/positive control, and substrate in order, then measuring OD values at 405nm (incubation period 37 ℃) for 0min and 30min separately using a microplate reader, obtaining relative activity values of each compound at a final concentration of 10 μ M to EcGUS by calculation, and drawing a relative activity histogram (see fig. 3) with GraphPad Prism 6.0 software, wherein the inhibitory activity of 30 compounds represented by formulas i and ii is greater than that of the positive control compound DSL, and the inhibition rate is 74.6% -104.8%.

The specific calculation process is as follows:

ΔOD＝OD_30min–OD_0min；

ΔC_PNPΔ OD/0.003262(0.003262 is a correlation coefficient between the absorbance obtained in step (2) and the PNP concentration)

Relative Activity (%). DELTA.C of Experimental group_PNPBlank group Δ C_PNP；

Inhibition rate (%) is 1-relative activity (%);

(4)IC₅₀determination of the value: determination of the IC of 30 compounds of the formulae I and II₅₀Values, a series of inhibitor concentration points (e.g., 0.001, 0.01, 0.05, 0.1, 0.3, 0.5, 1,3, 10 μ M) were set at a final concentration of 0.001-100 μ M, and the reaction was carried out in a 96-well plate, and the reaction system (total volume 100 μ L) was as follows: blank group: enzyme 10. mu.L + PBS 70. mu.L + volume fraction 1% DMSO in pure water 10. mu.L +2.5mM substrate 10. mu.L; experimental groups: enzyme 10. mu.L + PBS 70. mu.L + inhibitor at different concentrations 10. mu.L +2.5mM substrate 10. mu.L; each group was provided with 3 replicates per group, enzyme, PBS, inhibitionAdding reagent/positive control and substrate in sequence, measuring OD values of 0min and 30min at 405nM wavelength of an enzyme-labeling instrument (incubation period at 37 ℃), obtaining relative activity values of each inhibitor to EcGUS under different solubility conditions by calculation, converting micromole per liter (mu M) of the concentration point of the inhibitor into nanomole per liter (nM) and obtaining lg value by taking a derivative with 10 as a base, and drawing IC by utilizing GraphPad Prism 6.0 software by taking the lg value as a horizontal coordinate and the relative activity as a vertical coordinate₅₀The IC of each inhibitor to EcGUS is obtained by a curve chart and analysis of the software₅₀Value (each compound IC)₅₀Values in Table 1), IC of all 30 compounds of the formulae I and II on EcGUS₅₀Are all smaller than the positive control compound DSL, and reach 0.08-5.4 mu M, and compound II-5 (IC)₅₀0.08 μ M) achieved AMX (IC)₅₀0.08 μ M) in vitro inhibitory activity.

TABLE 1 half Inhibitory Concentration (IC) of EcGUS by the compounds of formulae I and II₅₀Value)

Example 2: molecular docking simulation

The rule of the interaction between inhibitor II-5 with the best inhibitory activity and EcGUS is analyzed by adopting a Molecular docking method.

MOE-2014.0901 software was used to perform molecular docking studies to understand the interaction of inhibitor II-5 with EcGUS. Firstly, downloading an EcGUS protein crystal structure (PDB ID: 3K4D) from a PDB database, then processing the crystal structure by using MOE-2014.0901 (the specific steps are structure preparation-structure correction-protonation-energy minimization) to obtain a protein capable of being butted, then preparing a small molecule II-5, drawing a small molecule structural formula by using a drawing function carried by MOE-2014.0901, and quickly preparing to obtain the small molecule II-5 capable of being butted. Finally, molecular docking is carried out, and Site Finder automatic recognition is selectedDocking pockets of other proteins, then docking by selecting a triangular docking method, generating 30 conformations of ligand-protein complexes by software according to the scoring condition, and selecting the conformation with the best score, namely the optimal molecular conformation for docking with the protein pockets. The docking simulation of inhibitor II-5 with EcGUS is shown in FIG. 4 (A), (B) and (C). From (A) and (C), the carbonyl oxygen atom of the inhibitor II-5 and the amino acid residue Glu413, the nitrogen atom of thiadiazole and the histidine His330 form strong hydrogen bond acting force, and the hydrogen bond distance is 1.84,

in addition, inhibitor II-5 forms a p-pi conjugated interaction with the amino acid residue tyrosine Tyr 472. Finally, it was also found from (B) that inhibitor II-5 is inserted just into the active pocket of the enzyme interacting with the active site residue.

Claims

1. The application of 1,3, 4-thiadiazole phenyl furan thio-methyl ester compound shown as a formula (I) or a formula (II) in preparing a medicine for treating diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs is characterized in that the compound is used for inhibiting beta-glucuronidase, and the beta-glucuronidase is derived from intestinal bacteria;

the R single substituent in the formula (I) is: 2-Cl, 3-Cl, 4-Cl, 2-F, 3-F, 4-F, 2-NO₂、3-NO₂、4-NO₂4-Br, 4-Me, 4-MeO, -H; the R double substituent in the formula (I) is: 2,4-di-F, 2, 6-di-F;

in the formula (II), R is 3-F.