CN111419843B

CN111419843B - Application of cyanoimine thiazolidine furan carboxamides in the preparation of β-glucuronidase inhibitors

Info

Publication number: CN111419843B
Application number: CN202010405503.2A
Authority: CN
Inventors: 王鸿; 崔紫宁; 魏斌; 周涛顺
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2021-07-27
Anticipated expiration: 2040-05-14
Also published as: CN111419843A

Abstract

The invention discloses an application of a cyanoimine thiazolidine furoylamide compound in preparing a beta-glucuronidase inhibitor, the cyanoimine thiazolidine furoylamide compound has very obvious inhibitory activity on Escherichia coli beta-glucuronidase (EcGUS), and IC (integrated Circuit) is₅₀The value range is 1.2 mu M-23.1 mu M, and the compound has wide application prospect in the aspect of research and development of medicaments for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs.

Description

Application of cyanoimine thiazolidine furan carboxamide compound in preparation of beta-glucuronidase inhibitor

(I) technical field

The invention relates to application of a cyanoimine thiazolidine furoylamide compound, in particular to new application of the cyanoimine thiazolidine furoylamide compound in preparation of a beta-glucuronidase inhibitor, which is beneficial to research and development of medicaments for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs and belongs to the technical field of chemical medicines.

(II) background of the invention

Irinotecan (CPT-11), a commonly used drug for the treatment of colon cancer. After entering human body, it is metabolized into inactive SN-38 glucuronide (SN-38G) in liver, then SN-38G is excreted into intestinal tract through bile duct, and hydrolyzed into SN-38 by intestinal bacteria beta-glucuronidase, after SN-38 is accumulated in intestinal tract, it can cause serious delayed diarrhea and intestinal injury, and seriously affect chemotherapy process. 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 intestinal tracts of human beings and animals, and in 2010, students verify that the inhibition of the intestinal bacteria beta-glucosidase can relieve drug-induced diarrhea caused by CPT-11 for the first time, and then the development and application of the intestinal bacteria beta-glucuronidase inhibitor are paid extensive attention. Although the source of β -glucuronidase in the intestinal tract is not limited to escherichia coli, escherichia coli β -glucuronidase (EcGUS), which is ubiquitous and readily produced in the human and animal intestinal tracts, is often used as a common target for the study of intestinal bacteria β -glucuronidase inhibitors.

The cyanoimine thiazolidine furoylamide compound is a derivative containing a 5-phenyl-2-furan structure. The furan ring is rich in electronic groups and can easily form intermolecular hydrogen bonds with a plurality of biological macromolecules, so that the compound containing the 5-phenyl-2-furan structure has various biological activities, such as antibiosis, antitumor, anti-inflammation, insect resistance and the like. The compounds listed in the invention are fully provided by professor tretinoin university of south China agriculture, but no research has been published on whether the compounds have EcGUS inhibitory activity.

Disclosure of the invention

The invention provides application of a cyanoimine thiazolidine furancarboxamide compound in preparation of a beta-glucuronidase inhibitor, which is beneficial to research and development of drugs for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs.

The technical scheme adopted by the invention is as follows:

the invention provides an application of cyanoimine thiazolidine furan carboxamide compound shown in formula (I) in preparing a beta-glucuronidase (EcGUS) inhibitor,

in the formula (I), R is a substituent group, and the substituent group comprises chlorine, bromine, fluorine, nitro, methyl, methoxy and hydrogen.

Further, R is 2-Cl, 3-Cl, 4-Cl, 2-F, 4-F, 2,4-Di-F, 2-NO₂、4-NO₂、4-Br、4-Me、4-MeO、H。

Further, the cyanoimine thiazolidine furoylamide compound has IC for inhibiting beta-glucuronidase₅₀The value ranges from 1.2. mu.M to 23.1. 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, and can be prepared from E.coli.

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

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

the cyanoimine thiazolidine furoylamide compound is reported to have the EcGUS inhibitory activity for the first time, and the inhibitory activity is very obvious, IC₅₀The values ranged from 1.2. mu.M to 23.1. mu.M (much less than the positive control-IC for D-glucarate-1, 4-lactone₅₀The value is 67.3 mu M), 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.

(IV) description of the drawings

FIG. 1 is a graph showing the inhibitory activity of cyanoimine thiazolidine furoylamides and D-glucaric acid-1, 4-lactone (final concentration: 10. mu.M) against EcGUS.

FIG. 2 is a graph showing the concentration-dependent inhibition curves of cyanoimido thiazolidine furoylamides and D-glucaric acid-1, 4-lactone against EcGUS.

FIG. 3 is a graph showing the type of EcGUS inhibition by compounds 1(A), 2(B) and 3 (C).

(V) detailed description of the preferred embodiments

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. 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.

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 (trypsin 10g/L, yeast extract 5g/L, sodium chloride 10g/L, solvent water, pH 7.0) containing 30. mu.g/mL of kanamycin, cultured at 200rpm and 37 ℃ until OD600 reached 0.5, followed by addition of isopropyl-. beta. -D-thiogalactoside (IPTG) at a final concentration of 100mM, and cultured overnight at 200rpm and 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 is collected, the thalli is washed by PBS (pH 7.4) for 2-3 times, then lysate (20mM 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), 300mM NaCl, 5mM imidazole (imidazole), glycerol (glycerol) with the volume concentration of 10 percent and the solvent being water, pH 7.4) is added into 1/10 of the volume of the original bacterial solution (culture solution before centrifugation) for 20mL, the thalli is ultrasonically broken for 20min (placed on ice) under the conditions of 300W and 10s ultrasonic intervals for 10s, then the thalli is centrifuged for 10min at 8000rpm at 4 ℃ for 10min, and the supernatant is taken. Then washing 15mL of Ni-NTA agarose resin column (purchased from GE healthcare) 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 3h, then eluting with 15mL of each of NTA-0 buffer r, 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), gradient eluting with 15mL of each of the eluents, collecting one tube per 5mL of eluents, collecting 9 tubes of eluents, subjecting each tube of eluents to-PAGE, and showing that the 4 tubes of the eluents collected by the NTA-20 buffer contain GUS, and the molecular weight of EcGUS is about 71kD, then the 4 tube eluents are merged, finally a Millipore protein ultrafiltration tube with 10kD (the cut-off molecular weight is not larger than 1/3 of the molecular weight of the target protein) is used for filtering, the collected cut-off liquid is enzyme liquid, and about 7mL of EcGUS raw enzyme liquid is obtained;

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

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

inhibitor (B): cyanoimine thiazolidine furoylamides (compounds 1 to 13 in Table 1) were each prepared as a 0.1mM solution in dimethyl sulfoxide (DMSO) as inhibitors.

Positive control (DSL): d-glucaric acid-1, 4-lactone (D-Saccharomyces acid 1,4-lactone, DSL, available from Sigma-Aldrich) was prepared as a 0.1mM solution in dimethyl sulfoxide (DMSO) as a positive control for future use.

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

Enzyme: the β -glucuronidase (EcGUS) prepared in step (1) was diluted 500-fold with PBS and then measured to have a concentration of 1. mu.g/mL with a kit, and used as a reaction enzyme solution.

Reaction: the reaction was run in 96-well plates, blank: enzyme 10 μ L + PBS 70 μ L + volume concentration 1% DMSO 10 μ L +2.5mM substrate 10 μ L; 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 treated with 3 replicates, enzyme, PBS, inhibitor/positive control, and substrate were sequentially added, OD values were measured at 405nm for 0min and 30min (period incubation at 37 ℃), respectively, using a microplate reader, the relative activity values of each compound at a final concentration of 10 μ M for EcGUS were calculated, and a histogram of the relative activity was plotted using the Graphad Prism 6.0 software (see fig. 1), and the inhibition rates of each compound at a final concentration of 10 μ M for EcGUS were further calculated (see table 1 for specific values), where the inhibition activity of 13 cyanoimine thiazolidine furoylamides (compound 1-compound 13 in table 1) was greater than that of the positive control compound, and the inhibition rate was between 31.4% and 95.2% DSL.

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 (%) ═ experimental group Δ C_PNPBlank group Δ C_PNP；

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

(4)IC₅₀determination of the value: determination of IC of 13 cyanoimine thiazolidine furoylamides₅₀Values, a series of inhibitor concentration points (e.g., 0.001, 0.01, 0.1, 0.3, 0.5, 3, 5, 20, 50 μ M) were set within a final concentration of 0.001-100 μ M, and the reaction was carried out in a 96-well plate as follows: blank group: enzyme 10 μ L + PBS 70 μ L + volume fraction 1% DMSO 10 μ L +2.5mM substrate 10 μ L; experimental groups: enzyme 10. mu.L + PBS 70. mu.L + inhibitor at different concentrations 10. mu.L +2.5mM substrate 10. mu.L; setting 3 parallels in each group, loading samples according to the sequence of enzyme, PBS, inhibitor/positive control and substrate, respectively measuring OD values of 0min and 30min under the wavelength of 405nM of an enzyme labeling instrument (incubation at 37 ℃), obtaining the relative activity value of each inhibitor to EcGUS under different solubility conditions through calculation, finally converting the concentration point of the inhibitor into nM, taking the derivative with the bottom of 10 to obtain lg value, and drawing IC by using Graphad Prism 6.0 software with lg value as horizontal coordinate and relative activity as vertical coordinate₅₀The IC of each inhibitor to EcGUS is obtained by a curve chart and analysis of the software₅₀Value (each compound IC)₅₀Values are given in Table 1), IC of all 13 cyanoimine thiazolidine furoylamides against EcGUS₅₀Are all smaller than the positive control compound DSL, and reach 1.2-22.3. mu.M.

TABLE 1 thirteen cyanoimine thiazolidinefuransInhibition rate of carboxamides and IC₅₀Value of

Example 2: study on inhibition types of EcGUS by

Compounds

1, 2 and 3

Selecting three compounds with the best re-screening inhibitory activity, namely a compound 1(R is 2-Cl), a compound 2(R is 3-Cl) and a compound 3(R is 4-Cl), wherein the corresponding structures are respectively substituted by chlorine on an ortho-benzene ring, a meta-benzene ring and a para-benzene ring, and the compound 2 has the best inhibitory activity. The inhibitor was formulated in a series of concentration gradients in PBS and the substrate was also formulated in PBS at 2, 3, 5, 10mM concentrations, i.e., 200, 300, 500, 1000. mu.M final concentration. For example, compound 1 is formulated at a concentration of 0, 30, 40, 90 μ M, i.e., a final concentration of 0, 3, 4, 9 μ M; the compound 2 is prepared at the concentration of 0, 5, 12 and 30 mu M, namely the final concentration of 0, 0.5, 1.2 and 3 mu M; the compound 3 is prepared at the concentration of 0, 20, 50 and 80 mu M, namely the final concentration of 0, 2, 5 and 8 mu M; solubility combinations of substrate and inhibitor are prepared using compound 1 as an example, table 2.

TABLE 2 permutation and combination of different concentration points of substrate and Compound 1

Note that: c_PNPGDenotes the final solubility of the substrate, C_InIndicates the final solubility of inhibitor (compound 1), □ indicates an experimental group (corresponding to one well of a 96-well plate), and 3 groups were made for each solubility combination.

Then according to the reaction system: carrying out reaction on 10 mu L of enzyme, 70 mu L of PBS, 10 mu L of inhibitor with different concentrations and 10 mu L of substrate with different concentrations (the combination of the inhibitor and the substrate with different concentrations is shown in a table 2) in a 96-well plate, setting 3 parallel groups for each group, loading according to the sequence of the enzyme, the PBS, the inhibitor/positive control and the substrate, respectively measuring the absorbance of 0min and 30min at the wavelength of 405nm by using an enzyme labeling instrument (incubation at 37 ℃), calculating the concentration difference of PNP corresponding to different solubility groups according to the calculation process of the step (3), and finally calculating the values of 1/V (mu mol/min/mg) and 1/PNPG (mu mol/min/mg), wherein V (mu mol/min/mg) is the catalysis speed of the enzyme and represents the molar quantity of the product catalytically produced per minute per milligram of the enzyme under the conditions of certain temperature, pH value and substrate concentration;

the calculation process is as follows:

1/V(μmol/min/mg)＝1/(ΔC_PNP*100/10/30/1)；1/PNPG＝1/ΔC_PNP；

wherein Δ C_PNPThe difference in PNP concentration between the 0min and 30min systems was shown, 100 was 100. mu.L for the reaction system, 10 was 10. mu.L for the enzyme, 30 was 30min for the reaction time, and 1 was 1. mu.g/mL for the enzyme preparation solubility.

Finally, a graph of inhibition double reciprocal curves (see figure 3, and compounds 1, 2 and 3 correspond to A, B, C in figure 3 respectively) is drawn by using Graphad Prism 6.0 software linear regression, the inhibition type is judged according to curve intersection points, and 3 compound images have an intersection point with the y axis from figure 3, which shows that the inhibition of EcGUS by the

compounds

1, 2 and 3 belongs to competitive inhibition.

Claims

1. An application of a cyanoimine thiazolidine furan carboxamide compound shown in formula (I) in preparing a medicament for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs,

in the formula (I), R is 2-Cl, 3-Cl, 4-Cl, 2-F, 4-F, 2,4-Di-F and 2-NO₂、4-NO₂、4-Br、4-Me、4-MeO、H。

2. The use according to claim 1, wherein the medicament is a β -glucuronidase inhibitor.

3. Use according to claim 2, characterized in that the β -glucuronidase is derived from escherichia coli.