CN112047882A

CN112047882A - New application of isoquinoline and quinoline derivatives in preparation of blood fat reducing drugs

Info

Publication number: CN112047882A
Application number: CN202011100324.4A
Authority: CN
Inventors: 孙华; 郝思雨; 张梦迪
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2020-12-08

Abstract

The invention provides a new application of isoquinoline and quinoline derivatives in the aspect of preparing a blood fat reducing medicine. The invention discovers that the derivative has good lipase inhibition activity for the first time, can reduce the lipid content of HepG2 cells and can regulate the expression level of AMPK pathway-related protein, and the compound has wide prospects in the aspects of development and application of medicaments for treating hyperlipidemia diseases.

Description

New application of isoquinoline and quinoline derivatives in preparation of blood fat reducing drugs

Technical Field

The invention belongs to the technical field of medicine application, and particularly relates to a new application of 3-aryl isoquinoline and 4-aryl quinoline derivatives.

Background

Quinoline and isoquinoline are two isomers of benzopyridine, both of which are present in coal tar. The quinoline compound can be used for preparing medicines, dyes, photosensitive materials, rubber, solvents, chemical reagents and the like. Quinoline is mainly used for preparing nicotinic acid series, 8-hydroxyquinoline series and quinine series medicaments in medicine. The nicotinic acid series medicine comprises nicotinamide, cardiotonic, stimulant and tapeworm disease treating medicine; the 8-hydroxyquinoline can be used for preparing medicines for treating amebiasis, wound disinfectants, mildewproof agents, textile auxiliaries and the like; bergenine, quinine chloride and hydroxyamiinine all belong to synthetic specific drugs for treating malaria. Isoquinoline can be used for manufacturing pesticides, antimalarial drugs, rubber vulcanization accelerators and chemical reagents for measuring rare metals. The methylquinoline can be used for manufacturing color film sensitizing agents and dyes, and can also be used as solvents, impregnants, corrosion inhibitors, quinine drugs, pesticides and the like.

Papaverine is a natural compound having an isoquinoline ring structure and having antifungal and antiviral activities, and has been an important spasmolytic until now. In recent years, isoquinoline backbones have been commonly used to design active structural groups in drug molecules, such as atracurium, nomifensine, moxaviline, dimoxiline, isovacaine, and the like. Tiwari et al synthesized a series of new 1-aryl tetrahydroisoquinoline derivatives, determined by in vitro antibacterial test that 1-aryl-6, 7-dimethoxy-1, 2, 3, 4-tetrahydroisoquinoline has antibacterial activity; iwasa et al analyze the antibacterial activity structure-activity relationship of dozens of simple isoquinoline bases and benzyl isoquinoline bases, and most compounds have an antibacterial effect; makhey et al synthesized a series of compounds using coralyne as a lead compound and studied anti-tumor.

The research team completes the synthesis of the 3-aryl isoquinoline and 4-aryl quinoline derivatives in the invention for the first time, and finds that the compounds have good alpha-glucosidase inhibition activity and have the potential to be developed into hypoglycemic drugs (see application No. 201910085622.1, an isoquinoline derivative with hypoglycemic activity and application). Although the present invention is structurally consistent with the above patent, the recent research found that the compounds have unexpected hypolipidemic activity, especially activity of inhibiting lipase and activity of regulating AMPK pathway protein expression level.

In conclusion, the activities of the natural medicines and the synthetic isoquinoline or quinoline derivatives are different from the activities of the compound, the compound is synthesized by the research team for the first time, and the compound is found to have the blood fat reducing activity for the first time.

Disclosure of Invention

The invention aims to discover the new application of the existing compound, and provides the new application of 3-aryl isoquinoline and 4-aryl quinoline derivatives in the aspect of reducing blood fat.

The technical scheme adopted by the invention for solving the technical problems is as follows:

isoquinoline and quinoline derivatives having hypolipidemic activity, said isoquinoline and quinoline being a 3-arylisoquinoline derivative and a 4-arylquinoline derivative;

wherein, the structural general formulas of the 3-arylisoquinoline derivative and the 4-arylquinoline derivative are as follows:

wherein, R in the general formula I₁Is hydrogen, 6, 8-dimethoxy, 6, 8-dihydroxy, 6-hydroxy-8-methoxy, 6-methoxy-8-hydroxy, R₂Substituted or unsubstituted furan, pyrrole, pyrimidine and phenyl; r in the general formula II₁Is 6, 7-dimethoxy, 6, 7-dihydroxy, 6-hydroxy-8-methoxy, 6-methoxy-8-hydroxy, R₂Substituted or unsubstituted furan, pyrrole, pyrimidine and phenyl.

Furthermore, the 3-arylisoquinoline derivative is a 3-phenylisoquinoline derivative, and the 4-arylquinoline derivative is a 4-phenylquinoline derivative;

the structural general formulas III and IV of the 3-phenylisoquinoline derivative and the 4-phenylquinoline derivative are as follows:

wherein R in the general formula III₁Is hydrogen, 6, 8-dimethoxy, 6, 8-dihydroxy, 6-hydroxy-8-methoxy, 6-methoxy-8-hydroxy, R₂Is alkyl, trifluoromethyl, methoxy, halogen, hydroxy, nitro or cyano; r in the general formula IV₁Is 6, 7-dimethoxy, 6, 7-dihydroxy, 6-hydroxy-8-methoxy, 6-methoxy-8-hydroxy, R₂Is alkyl, trifluoromethyl, methoxy, halogen, hydroxy, nitro or cyano.

And, the 3-arylisoquinoline derivative is a 3-substituted phenylhydroisoquinoline derivative;

the structural general formula V of the 3-substituted phenyl hydrogenated isoquinoline derivative is as follows:

wherein R is₁Is 6, 8-dimethoxy, 6, 8-dihydroxy, 6-hydroxy-8-methoxy, 6-methoxy-8-hydroxy; r₂Is alkyl, trifluoromethyl, methoxy, halogen, hydroxy, nitro or cyano.

The isoquinoline and quinoline derivatives with hypolipidemic activity are applied to inhibiting the activity of lipase.

Use of isoquinoline and quinoline derivatives having hypolipidemic activity as described above for the modulation of AMPK pathway activity.

The isoquinoline and quinoline derivatives are applied to the preparation of anti-hyperlipidemia drugs.

The invention has the advantages and positive effects that:

the invention discovers for the first time that isoquinoline and quinoline derivatives with the hypolipidemic activity have good lipase inhibition activity and the activity of regulating an AMPK pathway, can be applied to the preparation of anti-hyperlipidemia medicaments, and develops the research direction of a novel medicament for treating hyperlipidemia.

Drawings

Figure 1 is a graph of the effect of compound 6 on the AMPK pathway in HepG2 cells. (A) The effect of compounds on p-AMPK, AMPK, p-ACC, ACC, β -action protein was examined for Western blot analysis.

FIG. 2 is a gray scale analysis of the levels of the relevant proteins of FIG. 1.

Detailed Description

For understanding the present invention, the present invention will be further described with reference to the following examples: the following examples are illustrative and not intended to be limiting, and are not intended to limit the scope of the invention.

The relevant examples are as follows:

example 1

Evaluation of lipase inhibition by Compounds of the invention:

(1) preparation of Tris-HCl buffer solution and reaction stop solution

157mg of Tris (hydroxymethyl) aminomethane powder was precisely weighed with an analytical balance, added with 246mL of distilled water to dissolve it sufficiently, and added with hydrochloric acid to adjust pH to 8.0 to obtain a final concentration of 13mM Tris-HCl buffer. 10.505g of citric acid powder and 14.705g of sodium citrate powder are precisely weighed by a balance; adding the citric acid into 246mL of distilled water for dissolving, adding the sodium citrate into 154mL of distilled water for dissolving, slowly and dropwise adding a citric acid solution into the sodium citrate solution, and adjusting the pH to 4.2 to obtain a sodium citrate reaction stop solution with the final concentration of 0.1 mM.

(2) Preparation of pancreatic lipase

1mg of pancrelipase powder is weighed by a balance and added with 1mL of Tris-HCl buffer solution to prepare an enzyme solution with the concentration of 1mg/mL, and the enzyme solution is put on ice for standby.

(3) Preparing the solution of the desired substrate or test compound

Dissolving the substrate 4-methylumbelliferyl allyl oleate in Tris-HCl buffer solution, and diluting to 0.1mM substrate solution for storage in the dark. Orlistat was selected as a positive control in this experiment, dissolved in DMSO to prepare a 10mM solution to be tested. All test compounds were dissolved in DMSO and prepared as 10mM test solutions.

(4) Pancreatic lipase inhibition activity test system

The test systems were specifically grouped as blank (a1), blank background (a2), sample (A3) and sample background (a4), respectively:

blank group (a 1): Tris-HCl buffer (25. mu.L) + substrate (50. mu.L) + pancrelipase (25. mu.L) + sodium citrate (100. mu.L);

blank background group (a 2): Tris-HCl buffer (50. mu.L) + substrate (50. mu.L) + sodium citrate (100. mu.L);

sample set (a 3): substrate (50 μ L) + pancrelipase (25 μ L) + test compound (25 μ L) + sodium citrate (100 μ L);

sample background group (a 4): Tris-HCl buffer (25 μ L), substrate (50 μ L), test compound (25 μ L), and sodium citrate (100 μ L);

in the experiment process, buffer solution and the compound to be detected are sequentially added into a black 96-well plate and fully mixed, 1mg/mL enzyme (20 mu L) is added to initiate enzyme reaction, and after incubation in a constant-temperature incubator at 25 ℃ of a microporous plate for 30min, 100 mu L sodium citrate is added into each well to terminate the reaction. The amount of 4-methylumbelliferone was determined at excitation of 360 + -20 nm and emission wavelength of 460 + -20 nm, and the pancreatic lipase inhibition was calculated as follows:

each experiment is carried out in parallel for three times, 2 multiple holes are arranged for each concentration, and the error value SD between each group of data is obtained by calculating the average value.

Pancreatic lipase inhibitory Activity of the Compounds of Table 1 and Positive control orlistat

Note:^ano active NA;^bthe inhibition rates of orlistat were 0.08 μ M and 0.008 μ M, respectively.

The results in table 1 show that the compounds have certain inhibitory activity on lipase, and especially the compounds 6 and 17-19 have higher activity.

Example 2

Evaluation of the influence of the cell lipid content of the compound HepG2 of the invention:

respectively diluting palmitic acid, oleic acid, linoleic acid and arachidonic acid with 0.1mol/LNaOH to prepare 100mM stock solution, shaking the oleic acid, the palmitic acid, the linoleic acid and the arachidonic acid in a water bath at 70 ℃ to uniformly dissolve the oleic acid, the palmitic acid, the linoleic acid and the arachidonic acid, preparing the stock solution according to the proportion of 29: 47: 18: 6, adding 1% BSA culture medium with the same volume to the stock solution to prepare an inducer with the concentration of 50mM, dissolving the inducer in a water bath at 55 ℃ for 30min, filtering the stock solution of the 50mM inducer by using a sterile 0.45 mu M pore membrane filter, subpackaging, storing at minus 20 ℃ and keeping the light away, and keeping the inducer stable within 3-4 weeks. HepG2 cells were cultured in 6-well plates at a density of 1X 105 for 24h, and then replaced with serum-free high-sugar medium for 24h, and 0.75mM of formulated inducer was added to HepG2 cell medium to stimulate lipid accumulation in HepG2 cells. Before the cells are stained by the oil red O, the cells are washed three times by 1 XPBS to remove residual lipid components outside the cells, so that the oil red O only stains intracellular lipids. Fixing different groups of cells with 4% paraformaldehyde for 30min, washing the cells with 1 XPBS for 3 times, acting the cells with 60% isopropanol for 5min, then staining the cells with oil red O solution for 1h under dark room temperature condition, and washing the cells with distilled water for 4 times after 1h of oil red O staining is finished. Adding 1mL of 100% isopropanol into each well, combining with intracellular lipid oil red O, dissolving in isopropanol, shaking for 10min, and measuring the absorbance of the oil red O at 492nm by a microplate reader to quantify the intracellular lipid content. Nile red and DAPI staining are another method of evaluating lipid content in HepG2 cells. The 1mg/mL nile red stock solution is prepared by dissolving nile red in acetone in a dark place, and the 10mg/mL DAPI stock solution is prepared by dissolving DAPI in DMSO in a dark place, and is stored at the temperature of minus 20 ℃. Before staining, cells in a 6-well plate were fixed with 4% paraformaldehyde for 30min, and then a final concentration of 10. mu.g/mL nile red and 1. mu.g/mL DAPI solution was added to stain the cells for 15min at room temperature in the dark. After the staining is finished, Nile red and DAPI staining solution is sucked out, the cells are washed for 3 times with 1 XPBS for 5min each time, and then the observation of the state of the captured lipid content is carried out by inverting a fluorescence microscope.

TABLE 2 Activity of Compounds and Positive control lovastatin to reduce lipid content in HepG2 cells

As can be seen from table 2, this class of compounds has good lipid content activity at the cytostatic level, especially compounds 6, 16, 19 and 21.

Example 3

The compound 6 of the invention was used for evaluating the influence of the related proteins by immunoblotting:

in view of the fact that the compound 6 has good lipase inhibition activity and cell-level lipid content inhibition activity in the above experiments, the influence of the cell-level AMPK pathway-related protein expression level is further evaluated. The specific evaluation method is as follows:

(1) extraction of cell proteins:

HepG2 cells at 1X 10⁵Inoculating the cells in a small dish at a certain density, culturing in an incubator, treating the compound, and culturing in the incubator for a sample collection time. Collecting cells with different drug concentration into 15mL centrifuge bucket, centrifuging at 2500rpm for 5min, discarding supernatant culture medium, blowing with 1 XPBS buffer solution to obtain cell suspension, adding 6mL 1 XPBS buffer solutionWashing was performed again at 2500rpm, centrifugation was performed for 5min, and 1 XPBS buffer was discarded and repeated twice. The cells with different dosing concentrations were transferred to centrifuge tubes, 100. mu.L of protein lysate containing 1. mu.L of phosphatase inhibitor and 1. mu.L of protease inhibitor was added, the mixture was blown up evenly, lysed on ice for 1h, centrifuged at 13500rpm at 4 ℃ for 20 min. After centrifugation, the supernatant protein extract is taken and stored at-20 ℃ for later use.

(2) Protein concentration determination:

the BSA Protein Assay was performed by using BSA Protein Assay, preparing 2mg/mL bovine serum albumin BSA with protease lysate, sequentially diluting to concentrations of 1, 0.5, 0.25, and 0.125mg/mL, sequentially adding 2. mu.L of each concentration to 100. mu.L of Quick Start Bradford Dye Reagent, mixing well without generating bubbles, reacting at room temperature for 2min, and measuring the OD value at 600nm in an ELISA reader to prepare a standard curve of Protein concentration. And adding 2 mu L of the protein extracting solution to be detected into 100 mu L of Quick Start Bradford Dye Reagent, measuring the OD (optical density) value of 600, and calculating the concentration of the protein sample according to a drawn standard curve.

(3) SDS-PAGE electrophoresis:

the electrophoresis glass plate, the electrophoresis tank, and the like are washed, and the electrophoresis apparatus is mounted according to the mounting instructions. Preparing separation gel and concentrated gel according to a formula, pouring 6-12% of separation gel to two thirds of the position of an electrophoresis glass plate, adding 1mL of isopropanol, flattening the separation gel, standing at room temperature for 30min, separating the gel, solidifying, pouring out the isopropanol, sucking the isopropanol by using filter paper, pouring 5% of concentrated gel, inserting a comb according to the specification requirement, standing at room temperature for 10min, solidifying the concentrated gel, and slightly pulling out the comb for later use. mu.L of protein extract was taken, added to 1/5 volumes of 24. mu.L of 6 XSB loading buffer, boiled on a 100 ℃ hot block for 5min, and cooled on ice for further use. Putting the solidified device into an electrophoresis tank, adding tris (hydroxymethyl) aminomethane-glycine buffer solution, cleaning a sample adding hole by using a capillary vessel, adding 20 mu L protein extract sample and 5 mu L Marker into the sample adding hole, switching on a power supply, adjusting the voltage to 80V when the current starts, fully compressing the protein in the concentrated gel, adjusting the voltage to 100V when the bromophenol blue passes through the boundary between the concentrated gel and the separation gel, and stopping electrophoresis when the bromophenol blue migrates to the bottom of the electrophoresis tank.

(4) Gel film transfer:

after the proteins were separated by electrophoresis, the glass plate was opened to remove the gel, and the gel was added to the membrane transfer buffer. One PVDF membrane and six filter papers were cut out. Three pieces of filter paper are taken from six pieces of filter paper to be soaked in a membrane transferring buffer solution, then the filter paper is transferred to a membrane transferring instrument, a PVDF membrane is placed in anhydrous methanol to be soaked for 15s and then transferred to the three pieces of filter paper of the membrane transferring instrument, finally gel is placed on the PVDF membrane, then the three pieces of filter paper soaked in the buffer solution are placed on the gel to keep the filter paper moist, electrodes are connected according to the sequence of placing an anode on the membrane and placing a cathode on the gel, current is switched on, membrane transferring is carried out by using 250-300mA, and the membrane transferring time is 90-120 min.

(5) And (3) sealing:

after the gel transfer, the PVDF membrane is immersed in PBST solution containing 5% skimmed milk powder and sealed at room temperature. After 1h, the membranes were washed 3 times with 1 × PBS, 10min each.

(6) Primary anti-reaction:

the blocked PVDF membrane is added into the diluted primary antibody and blocked overnight at 4 ℃ for more than 12 ℃.

(7) Secondary antibody reaction:

the following day, membranes were washed 3 times with 1 × PBS for 10min each time. Adding a secondary antibody anti-mouse/anti-Rabbit HRP in the dark, sealing for 2h at room temperature, finishing sealing the secondary antibody, and then washing the membrane for 3 times with 1 × PBS, wherein each time is 10 min.

(8) Sweeping:

adding color developing agent of ECL, allowing it to develop color, and observing the analysis result by gel phase system.

(9) And (3) data statistics:

all data results are expressed as mean ± standard deviation, single-factor analysis of variance and significance are performed using Prism 5.0 software and SPSS statistical software, and comparison of data results is performed using independent sample T-test and analysis of variance (ANOVA).

The effect of compound 6 on the AMPK pathway-related protein expression levels of HepG2 cells is shown in fig. 1, and the corresponding grayscale analysis is shown in fig. 2. As can be seen from fig. 1 and 2, the AMPK activator AICAR group significantly enhanced the phosphorylation levels of AMPK and ACC proteins, resulting in elevated p-AMPK/AMPK and p-ACC/ACC levels, as compared to the model group, and as a result, compound 6 increased p-AMPK/AMPK expression by 43%, 46%, and 57% and p-ACC/ACC levels by 88%, 76%, and 191%, respectively, after 1 μ M, 7.5 μ M, and 15 μ M treatments, in the compound 6-treated administration group.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Claims

1. Isoquinoline and quinoline derivatives having hypolipidemic activity, characterized by: the isoquinoline and quinoline are 3-aryl isoquinoline derivatives and 4-aryl quinoline derivatives;

2. Isoquinoline and quinoline derivatives with hypolipidemic activity according to claim 1 characterized in that: the 3-arylisoquinoline derivative is a 3-phenylisoquinoline derivative, and the 4-arylquinoline derivative is a 4-phenylquinoline derivative;

wherein R in the general formula III₁Is hydrogen, 6, 8-dimethoxy, 6, 8-dihydroxy, 6-hydroxy-8-methoxy, 6-methoxy-8-hydroxy, R₂Is alkyl, trifluoromethyl, methoxy, halogen, hydroxy, amino, nitro or cyano; r in the general formula IV₁Is 6, 7-dimethoxy, 6, 7-dihydroxy, 6-hydroxy-8-methoxy, 6-methoxy-8-hydroxy, R₂Is alkyl, halogen, amino or cyano.

3. Isoquinoline derivative with hypolipidemic activity according to claim 1 characterized in that: the 3-aryl isoquinoline derivative is a 3-substituted phenyl hydrogenated isoquinoline derivative;

4. Use of isoquinoline and quinoline derivatives with hypolipidemic activity according to claims 1 to 3 for the preparation of hypolipidemic medicaments.

5. Use of isoquinoline and quinoline derivatives having hypolipidemic activity according to any one of claims 1 to 3 for the inhibition of lipase activity.

6. Use of isoquinoline and quinoline derivatives having hypolipidemic activity according to any one of claims 1 to 3 for the modulation of AMPK pathway activity.