CN113499340A

CN113499340A - Application of 1, 3-thiazole phenyl furan thioformate compound in preparation of beta-glucuronidase inhibitor

Info

Publication number: CN113499340A
Application number: CN202110631611.6A
Authority: CN
Inventors: 周镇燚; 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: 2021-06-07
Filing date: 2021-06-07
Publication date: 2021-10-15

Abstract

The invention discloses an application of 1, 3-thiazole phenyl furan thiocarbamate compounds in preparation of beta-glucuronidase inhibitors, wherein the 1, 3-thiazole phenyl furan thiocarbamate compounds have very obvious inhibitory activity on Escherichia coli beta-glucuronidase (EcGUS), and IC (integrated Circuit) is₅₀The value range is 0.25 mu M-2.13 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 1, 3-thiazole phenyl furan thioformate compound in preparation of beta-glucuronidase inhibitor

Technical Field

The invention relates to an application of 1, 3-thiazole phenyl furan thiocarbamate compounds, in particular to a new application of the compounds 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.

Background

Irinotecan (CPT-11), a commonly used first-line chemotherapeutic for the treatment of colon cancer. After entering the human body, the polypeptide is firstly metabolized into an inactive SN-38 glucuronide (SN-38G) compound in the liver, then the compound is excreted into the intestinal tract through the bile duct and is hydrolyzed into an active metabolite SN-38 by intestinal bacteria beta-glucuronidase, and after the SN-38 is accumulated in the intestinal tract in a large amount, the serious delayed diarrhea and intestinal injury are caused, so that the chemotherapy process and the drug dosage are influenced. In addition, the administration of non-steroidal anti-inflammatory drugs containing carboxylic acid such as ketoprofen, diclofenac and indomethacin causes similar intestinal toxicity, thereby causing severe drug-induced diarrhea and seriously affecting the therapeutic effect.

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 intestinal tract is not limited to escherichia coli, escherichia coli β -glucuronidase (EcGUS) is widely distributed in intestinal tracts of humans and animals and is easily expressed heterologously, so EcGUS is often used as a common target for research on intestinal bacteria β -glucuronidase inhibitors.

The 1, 3-thiazole phenyl furan thioformate compound is a derivative containing 5-phenyl-2-furan and thiazole ring structures. The furan ring and the thiazole ring are 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 and thiazole ring structure has various biological activities, such as antibiosis, tumor resistance, anti-inflammation, insect resistance and the like. All the compounds listed in the patent are generously 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 Invention

The invention discovers that a 1, 3-thiazole phenyl furan thiocarbamate compound has good inhibitory activity on EcGUS in vitro by adopting a microplate screening technology (protein level). The novel application of the compound in preparation of the beta-glucuronidase inhibitor is facilitated, so that the compound has great potential to be applied 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 1, 3-thiazole phenyl furan thioformate compounds shown in a formula (I) in preparing a beta-glucuronidase (EcGUS) inhibitor,

in the formula (I), R represents one or more substituents, and is hydrogen, halogen, nitro, C1-C4 alkyl or C1-C4 alkoxy.

Further, the R groups specifically include the following two: the monosubstituent 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 disubstituted group is 2,4-Di-F and 2, 6-Di-F.

Further, IC of the 1, 3-thiazole phenyl furan thiocarbamate compound on EcGUS inhibition₅₀The value ranges from 0.25. mu.M to 2.13. mu.M.

Further, the beta-glucuronidase is escherichia coli beta-glucuronidase.

The invention also provides application of the 1, 3-thiazole phenyl furan thiocarbamate compound shown in the formula (I) in preparing a medicament for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs.

The inhibitor can well explain the good inhibitory activity of the inhibitor on EcGUS in an in vitro protein layer, and is favorable for the development of drugs for treating drug-induced 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-thiazole phenyl furan thiocarbamate compound has the first reported EcGUS inhibitory activity, the inhibitory activity is very obvious, and IC₅₀The value ranged from 0.25. mu.M to 2.13. mu.M (much less than the positive control-IC for D-glucarate-1, 4-lactone (DSL))₅₀The value of 65.69 mu M), has wide application prospect in the aspect of the research and development of drugs for treating drug-induced diarrhea caused by irinotecan or non-steroidal anti-inflammatory drugs.

Drawings

FIG. 1 is a graph showing the inhibitory activity of 1, 3-thiazolylphenylfuran thiocarbamate compound and DSL (final concentration 10. mu.M) on EcGUS.

FIG. 2 is a graph showing the concentration-dependent inhibition curves of 1, 3-thiazolylphenylfuran thiocarbamate compounds and DSL against EcGUS.

FIG. 3 is a graph showing the inhibition types of DSL (A) and Compound 12(B) against EcGUS.

FIG. 4 is a diagram of DSL, compound 12 docking to EcGUS, A is a diagram of DSL docking 2D with EcGUS, and C is a diagram of DSL docking 3D with EcGUS; b is a 2D map of compound 12 in docking with EcGUS, and D is a 3D map of compound 12 in docking with EcGUS.

Detailed Description

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 E.coli beta-glucuronidase (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 15mL, 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 5mL of Ni-NTA agarose resin column (purchased from GE healthcare) 2-3 times with 15mL each of purified water and NTA-0 buffer (20mM Tris-HCl, 0.5M sodium chloride, volume solubility 10% glycerol, pH 7.9), chelating the supernatant with the Ni-NTA agarose resin column at 4 ℃ for 3h, then eluting with 15mL 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) in a gradient manner, collecting one tube per 5mL of eluate, collecting 9 tubes of eluate respectively, subjecting the eluate to SDS-PAGE, and showing that 4 tubes of eluate collected by NTA-20 buffer contain GUS, and the molecular weight of EcGUS is about 71kD, then combining the 4-tube eluent, finally centrifuging for 15min (1/3 with the molecular weight cutoff not larger than the molecular weight of the target protein) by using a Millipore protein ultrafiltration tube with the molecular weight cutoff of 10kD at 7000rpm and 4 ℃ for ultrafiltration, collecting the trapped fluid which is enzyme fluid, and obtaining 7mL of EcGUS proenzyme fluid;

(2) preparation of a p-nitrophenol (PNP) standard: preparing 0.1mM PNP solution (PBS is dissolved), operating in a 96-well plate, adding 0, 10, 20, 40, 60 and 80 muL of 0.1mM PNP solution in each group, supplementing the solution to 100 muL with PBS, incubating for 30min at 37 ℃, measuring absorbances of 0min and 30min at 405nm wavelength respectively by using a microplate reader, then making a scatter diagram by using Excel to obtain a relation formula of y to 3.2617x +0.0547 of the absorbances and the PNP concentration under the mM concentration, wherein y is the absorbance and x is the PNP concentration, and dividing both sides of the above formula by 1000 to convert the relation formula of y to 0.003262x +0.0000547 of the absorbances and the PNP concentration under the muM concentration into y to 0.003262x of the PNP concentration after 0.0000547 is removed.

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

inhibitor (B): 1, 3-thiazole phenyl furan thiocarbamate compounds (compound 1-compound 15 in Table 1) are respectively prepared into 0.1mM solution by using dimethyl sulfoxide (DMSO) to be used as an inhibitor for standby.

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 DMSO and used as a positive control.

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 at 405nm and 30min were measured respectively (period incubation at 37 ℃), using a microplate reader, the relative activity value of each compound against EcGUS at a final concentration of 10 μ M was calculated, and a histogram of the relative activity was plotted using Graphad Prism 6.0 software (see fig. 1), and the inhibition rate of each compound against EcGUS at a final concentration of 10 μ M was further calculated (see table 1 for specific values), and the inhibition activity of 15 1, 3-thiazolylphenylfuranthiocarbamate compounds (compound 1-compound 15 in table 1) was all greater than that of the positive control compound DSL, and the inhibition rate was between 22.0% and 90.2%.

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: since

compounds

2,4 and 10 have a poor inhibition rate of less than 50% at a final concentration of 10. mu.M, we chose the remaining 12 1, 3-thiazolylphenylfuran thiocarboxylates to determine their IC₅₀Values, 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 shown in Table 1), IC of all 12 1, 3-thiazolylphenylfuran thiocarbamate compounds on EcGUS₅₀Are all smaller than the positive control compound DSL, and reach 0.25-2.13 mu M.

TABLE 11, 3-Thiazolophenylfuran thioformate inhibition and IC₅₀Value of

ND represents not detected

Example 2: studies of inhibition types of DSL and inhibitor 12 on EcGUS

Select IC₅₀Inhibitor 12 and positive control DSL, with the smallest values, further explored both their inhibition types and K_iValues, DSL and inhibitor 12 were formulated in a series of concentration gradients in PBS, and substrate was also formulated in PBS at 2, 3, 5, 10mM concentrations, i.e. final concentrations of 200, 300, 500, 1000 μ M. The DSL preparation concentration is 0, 0.5, 1, 2mM, namely the final concentration is 0, 50, 100, 200 μ M; the inhibitor 12 is prepared at the concentration of 0, 2, 5 and 10 mu M, namely the final concentration of 0, 0.2, 0.5 and 1 mu M; solubility combinations of substrate and inhibitor were prepared, using inhibitor 12 as an example, table 2.

TABLE 2 permutation and combination of various concentration points of substrate and inhibitor 12

Note that: c_PNPGDenotes the final solubility of the substrate, C_InThe final solubility of inhibitor (inhibitor 12) is shown and one experimental group (corresponding to one well of a 96-well plate) is shown, with 3 replicates for each concentration.

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 a microplate reader (incubating at 37 ℃), calculating the concentration difference of PNP corresponding to the different concentration 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 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 is drawn by utilizing Graphad Prism 6.0 software linear regression, and K is calculated_iThe values (see figure 3, DSL and inhibitor 12 correspond to A, B in figure 3, respectively) determine the type of inhibition based on the curve intersection point, from figure 3 it can be seen that the positive control DSL image intersects at one point and the intersection point is in the second quadrant, indicating that the DSL is a mixed inhibitor, and K is_iThe value was 32.16. mu.M; the inhibitor 12 images are all parallel lines, indicating that inhibitor 12 is a non-competitive inhibitor, K_iThe value is 0.14. mu.M, by K_iThe comparison of the values shows that the inhibiting activity of the inhibitor 12 on EcGUS is obviously stronger than that of a positive control DSL.

Example 3: molecular docking simulation

The inhibitor 12 with the best inhibitory activity and the interaction strength of the positive control DSL and EcoGUS are analyzed and compared by adopting a Molecular docking method.

The MOE-2014.0901 software is used for molecular docking research, the interaction of the inhibitor 12, DSL and EcoGUS molecular level is researched, and the interaction is visualized by the MOE software. The method comprises the steps of firstly downloading an EcoGUS protein crystal structure (PDB ID:3LPF) from a PDB database, then processing the crystal structure by using MOE (the specific steps are: structure preparation-structure correction-protonation-energy minimization) to obtain the EcoGUS protein which can be further used for butt joint, then preparing small molecules, drawing the structural formula of the small molecules by using the drawing function of the MOE, and carrying out rapid preparation processing to obtain the butt-joint small molecule inhibitors 12 and DSL. Finally, molecular docking is carried out, 3LPF protein downloaded from a PDB database is combined with an inhibitor micromolecule with a quinoline thiourea structure, so that the docking position of the inhibitor micromolecule can be directly selected as a docking pocket when the docking pocket is selected, then the docking is carried out by utilizing a triangular docking method, software can generate 30 conformations of ligand-protein complexes according to the scoring condition, the conformation with the best scoring is selected,i.e. the optimal molecular conformation for docking with the protein pocket. Fig. 4 (a), (B), (C) and (D) show 2D and 3D simulations of DSL and inhibitor 12 docking with EcoGUS, respectively. From (A), it can be found that positive control DSL and Arg562 of B chain

，Glu413

，Glu504

，Asn566

，Trp549

And Lys 568

Strong hydrogen bonding forces are formed. From (C), it was found that inhibitor 12 was only bound to Arg562 of the B chain

，Glu413

Hydrogen bonding forces are formed, more hydrophobic forces are formed with Phe448, Tyr472, Leu361 of the B chain and Phe365 of the a chain. The 2 key amino acids Glu504 and Glu413 are present in the active pocket of the EcGUS protein (PDB ID:3LPF), but in addition to these 2 key amino acid sites of action, the active pocket has the structure of 2 specific loops from chain A, B (Ser360-Glu367), a specific structure that is distinct from other sources of β -glucuronidase. Although DSL forms strong hydrogen bonding with various amino acid residues, it can be found from (C) that DSL itself forms strong hydrogen bondingThe structure of (1) is limited, only the upper half of the active pocket is occupied, and the acting force of the fungus ring structure of the lower half is almost not exerted. Inhibitor 12 has 5-phenyl-2-furan and 1, 3-thiazole structures, and from (D), it can be seen that inhibitor 12 occupies the entire active pocket well, and that inhibitor 12 forms a more hydrophobic interaction with amino acid residues of the fungal ring structure in the lower half in addition to forming a hydrogen bond structure with amino acids in the upper half of the active pocket. Thus, combining the above assays, the difference in vitro activity of inhibitor 12 and the positive control DSL was explained at a molecular level.

Claims

1. An application of 1, 3-thiazole phenyl furan thioformate compounds shown in a formula (I) in preparing a beta-glucuronidase inhibitor,

2. Use according to claim 1, characterized in that the R group is one of the following: 2-Cl, 3-Cl, 4-Cl, 2-F, 3-F, 4-F, 2-NO₂、3-NO₂、4-NO₂4-Br, 4-Me, 4-MeO, H, 2,4-Di-F or 2, 6-Di-F.

3. The use as claimed in claim 1, wherein the 1, 3-thiazolylphenylfuran thiocarbamate compound has IC for inhibiting β -glucuronidase₅₀The value ranges from 0.25. mu.M to 2.13. mu.M.

4. Use according to claim 1, characterized in that the β -glucuronidase is escherichia coli β -glucuronidase.

5. The use according to claim 1, wherein the inhibitor is a medicament for the treatment of drug-induced diarrhea associated with irinotecan.

6. The use according to claim 1, wherein the inhibitor is a medicament for the treatment of non-steroidal anti-inflammatory drug induced drug-induced diarrhea.