CN118986974A

CN118986974A - New application of 4- (4-isoxazole) benzenesulfonamide compound

Info

Publication number: CN118986974A
Application number: CN202411162145.1A
Authority: CN
Inventors: 胡长鹏; 李国兵; 张蓉; 秦铭; 刘吴毅; 赖文静; 周胡悦; 刘亚峰
Original assignee: Second Affiliated Hospital Army Medical University
Current assignee: Second Affiliated Hospital Army Medical University
Priority date: 2024-08-22
Filing date: 2024-08-22
Publication date: 2024-11-22

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a novel application of a 4- (4-isoxazole) benzenesulfonamide compound. The invention firstly provides application of a 4- (4-isoxazole) benzenesulfonamide compound in preparing a cell PCSK9 transcription inhibition reagent. Experiments prove that the loss of PCSK9 does not affect the normal growth of cells, and the 4- (4-isoxazole) benzenesulfonamide compound can play a role in inhibiting PCSK9 transcription in the in-vitro culture of cells, compared with the traditional gene knockout method, the PCSK9 transcription inhibitor has more convenience and more similar effect to the traditional gene knockout method, so that the PCSK9 transcription inhibitor has a larger scientific research application prospect. The invention also provides application of the 4- (4-isoxazole) benzenesulfonamide compound in reducing blood fat.

Description

New application of 4- (4-isoxazole) benzenesulfonamide compound

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to a novel application of a 4- (4-isoxazole) benzenesulfonamide compound.

Background

Cardiovascular and cerebrovascular diseases are the leading cause of death worldwide, and atherosclerosis and hyperlipidemia are the central factors responsible for the occurrence of cardiovascular and cerebrovascular diseases. In primary and secondary prevention measures of cardiovascular and cerebrovascular diseases, lowering serum low density lipoprotein cholesterol (LDL-C) can effectively control hyperlipidemia and atherosclerosis, thereby reducing the incidence risk of cardiovascular events. Under normal physiological conditions, low Density Lipoprotein Receptor (LDLR) on the surface of hepatocytes will bind LDL-C and transport LDL-C to intracellular degradation as blood passes through the liver, after which LDLR will return to the surface of hepatocytes to bind more circulating LDL-C particles, thereby lowering plasma LDL-C levels.

Proprotein convertase subtilisin 9 (Proprotein Convertase Subtilisin/kexin Type 9, pcsk 9) is a serine protease secreted by hepatocytes and has been reported in recent years to be an important molecule leading to elevated LDL-C in the blood. PCSK9 is mainly combined with LDLR to prevent the LDLR from being recycled to the surface of liver cells, so that atherosclerosis and cardiovascular and cerebrovascular diseases caused by the fact that LDL-C in serum cannot be degraded and LDL-C is accumulated are avoided. Therefore, the development of PCSK9 inhibitors has important significance for controlling cardiovascular and cerebrovascular diseases. The PCSK9 inhibitor which is currently marketed comprises two injection preparations of the Al Li Xiyou monoclonal antibody and the Elol You Shan, and is used for treating primary hypercholesterolemia, mixed dyslipidemia and atherosclerosis cardiovascular diseases. Other marketed PCSK9 inhibitors also include the RNA interference drug, england; the oral preparation enters clinical trial stage and has antisense oligonucleotide AZD8233 for targeted inhibition of PCSK9 mRNA translation and protein synthesis in liver cells and small molecule polypeptides NNC0385-0434 and MK-0616 which can be taken orally.

The PCSK9 inhibitors are biological medicines, and have the problems of complex purification process, high production cost, high selling price, high transportation and storage requirements and the like; the injection preparation has the problems of inconvenient use and poor patient compliance; the monoclonal antibodies and the polypeptide drugs are easy to cause organisms to generate drug antibodies and increase the risk of side effects, and the antisense oligonucleotide is extremely unstable and has large individual difference of curative effects. Compared with the biological medicines, the micromolecular medicine has the advantages of no immunogenicity, higher oral bioavailability, low development cost, low transportation and storage requirements and the like, so that the development of the micromolecular inhibitor of PCSK9 has great clinical application and industrial popularization value. However, since the binding region of PCSK9 and LDLR is flat and smooth, there is no effective domain for drug binding, the development of a small molecule inhibitor that blocks the binding of PCSK9 and LDLR has been an industrial problem, and no small molecule inhibitor of PCSK9 is currently marketed.

In view of the foregoing, there is a need for developing novel PCSK9 small molecule inhibitors that complement the deficiencies of the prior art.

Disclosure of Invention

In view of the above, the invention aims to provide an application of a compound with a 4- (4-isoxazole) benzenesulfonamide structure in preparing a PCSK9 inhibitor, and the specific technical scheme is as follows.

Application of 4- (4-isoxazole) benzenesulfonamide compounds in preparing cell PCSK9 transcription repressing reagent, wherein the 4- (4-isoxazole) benzenesulfonamide compounds contain 4- (4-isoxazole) benzenesulfonamide parent ring structure; the 4- (4-isoxazole) benzenesulfonamide parent ring structure is shown as a compound in a formula (1):

Further, the 4- (4-isoxazole) benzenesulfonamide compound includes a benzenesulfonamide selected from the group consisting of 3- (5-methyl-4-phenylisoxazol-3-yl) benzenesulfonamide, 4- (3-methyl-5-phenylisoxazol-4-yl) benzenesulfonamide, valdecoxib, 4- (5-methyl-3-phenylisoxazol-4-yl) benzenesulfonic acid, 3-phenyl-4- (4-aminosulfonyl benzyl) -5-methylisoxazole, benzenesulfonamide, 4- (3- (4-fluorophenyl) -5-methylisoxazol-4-yl) benzenesulfonamide, 2-fluoro-4- (5-methyl-3-phenylisoxazol-4-yl) benzenesulfonamide, 3- (5-methyl-3-phenylisoxazol-4-yl) benzene-1-sulfonyl chloride, valdecoxib 3' -sulfonyl chloride, 4- (5-methyl-3-phenylisoxazol-4-yl) benzenesulfonamide, 4- (5-methyl-4-phenylisoxazol-4-yl) benzenesulfonyl ethyl ester, 4-phenylisoxazol-4-yl) benzenesulfonamide, and 4-phenylisoxazol-4-yl ethyl ester, 3-phenyl-4- (4-chlorosulfonylbenzyl) -5-methylisoxazole, N- ((4- (5-methyl-3-phenylisoxazol-4-yl) phenyl) sulfonyl) acetamide, N- (4- (3-methyl-5-phenylisoxazol-4-yl) phenyl) sulfonyl) propanamide, N- ((3- (5-methyl-3-phenylisoxazol-4-yl) phenyl) sulfonyl) propanamide, parecoxib, benzenesulfonamide, 4- (5- (nitroxy) methyl) -3-phenyl-4-isoxazolyl), N- ((4- (5-methyl-3-phenylisoxazol-4-yl) phenyl) sulfonyl) isobutyramide, N- ((4- (5-methyl-3-phenylisoxazol-4-yl) phenyl) sulfonyl) butanamide, 3- (5-methyl-4- (4-sulfamoylphenyl) isoxazol-3-yl) benzenesulfonamide, N- [ [4- (5-methyl-3-phenyl) isoxazol-4-yl) phenylsulfonyl ] benzamide, 3- [4- (4-chlorosulfonylphenyl) -5-methyl-1, 2-oxazol-3-yl ] benzenesulfonyl chloride and/or N- [4- [ 5-methyl-3- [3- (propionylsulfamoyl) phenyl ] -1, 2-oxazol-4-yl ] phenyl ] sulfonylpropionamide.

Further, the 4- (4-isoxazole) benzenesulfonamide compound is used for adding to a cell culture medium to incubate cells.

The application of parecoxib in preparing hypolipidemic drugs.

Further, the parecoxib is used for lowering serum cholesterol.

Further, the parecoxib is used for reducing serum low density lipoproteins.

Further, the medicament also comprises other pharmaceutically acceptable carriers and/or auxiliary agents.

The present examples demonstrate the effect of parecoxib on lowering serum cholesterol levels and serum low density lipoprotein levels. From a clinical perspective, serum cholesterol and serum low density lipoprotein are two representative detection indicators of hyperlipidemia. Therefore, experiments prove that the parecoxib has a reducing effect on serum cholesterol level and serum low-density lipoprotein level, and can be considered to have a good application value in preparing hypolipidemic drugs.

Use of POU2F2 as a molecular marker for the preparation of a parecoxib efficacy assessment reagent for assessing the inhibition of PCSK9 transcription by parecoxib.

Furthermore, the parecoxib curative effect evaluation reagent also comprises a PCSK9 promoter plasmid, and the sequence of the PCSK9 promoter is shown as SEQ ID NO. 1.

Further, the POU2F2 is used as a molecular marker of a PCSK9 transcription factor; the application is to evaluate the expression of parecoxib inhibition transcription factor POU2F2 (further evaluate the inhibition effect of parecoxib on PCSK9 transcription).

Beneficial technical effects

The invention firstly provides application of a 4- (4-isoxazole) benzenesulfonamide compound in preparing a cell PCSK9 transcription inhibition reagent. Experiments prove that the deletion of PCSK9 does not affect the normal growth of cells, and parecoxib can play a role in inhibiting the transcription of PCSK9 in the in-vitro culture of cells, compared with the traditional gene knockout method, the method is more convenient and faster, and the effect is close to that of the traditional gene knockout method, so that the method has a larger scientific research application prospect.

Secondly, the invention provides application of the compound parecoxib with the 4- (4-isoxazole) benzenesulfonamide parent ring structure in preparation of hypolipidemic drugs, and related verification is carried out.

Finally, the invention also provides a novel molecular marker (transcription factor POU2F 2) for evaluating the transcription inhibition effect of parecoxib on PCSK9, and the novel molecular marker has wide scientific research application and clinical popularization prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.

FIG. 1 is a schematic diagram of a PCSK9 promoter fusion tdTomato expression plasmid;

FIG. 2 is a PCSK9 small molecule inhibitor screening procedure;

FIG. 3 is a diagram showing that PCSK9 deletion does not affect normal cell growth;

FIG. 4 is the results of experiments in which parecoxib inhibits transcription and expression of PCSK9, but does not affect cell growth;

FIG. 5 is a parecoxib-reduced low density lipoprotein assay;

FIG. 6 is an in vivo evaluation experiment of parecoxib inhibiting PCSK 9;

FIG. 7 is an in vivo evaluation experiment of parecoxib cholesterol reduction;

fig. 8 is a safety evaluation result after parecoxib administration;

fig. 9 is a parecoxib-inhibited PCSK9 transcription pathway study.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Herein, "and/or" includes any and all combinations of one or more of the associated listed items.

Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.

In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, a rangeThe description of (c) should be taken as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.

Detailed description of some of the drawings:

Fig. 3: a is the expression of PCSK9 protein after WB detection PCSK9 knockout. B is CCK8 to detect the cell activity of PCSK9 knockdown. C is the cell growth after the PCSK9 knockout is detected by plate cloning. D is Ki67 and EDU staining to detect cell proliferation following PCSK9 knockout. E is apoptosis after PCSK9 knockout in flow apoptosis detection.

Fig. 4: a is qPCR detection of expression of PCSK9 mRNA after dosing. B is the expression condition of PCSK9 protein after WB detection and drug addition. C is CCK8 to detect the cell activity after drug addition. D is the cell growth condition after the plate clone detection and drug addition. E is apoptosis after drug addition in flow apoptosis detection. F is Ki67 and EDU staining to detect proliferation of cells after drug administration.

Fig. 8: a is plasma ALT levels. B is plasma AST level. C is plasma CREA level. D is plasma UREA levels. E is plasma LDH levels. F is plasma CK levels. G is tissue HE staining of heart, liver, spleen, lung, kidney after parecoxib administration.

Fig. 9: a is a predicted PCSK9 potential transcription factor, and intersection analysis is carried out on the predicted PCSK9 potential transcription factor and a differential expression gene obtained by sequencing after parecoxib dosing. B is a gene sequencing analysis of 63 differential expressions obtained by intersection, and POU2F2 is the gene with the most obvious expression reduction after chemical compound dosing. C is the mRNA expression of POU2F2 after qPCR detection of parecoxib drug administration. D is the protein expression condition of the transcription factor POU2F2 after WB detection parecoxib drug addition.

Example 1

From the standpoint of regulating PCSK9 expression, a PCSK9 promoter fusion tdTomato expression plasmid is designed and constructed, and the plasmid construction is shown in figure 1. And further packaging the plasmids with lentiviruses to infect human liver cancer HepG2 cells, and obtaining a cell line for stably expressing the PCSK9 promoter and tdTomato. Further, the cell line was inoculated into 96-well plates for culture, 99. Mu.L/well, various small molecule compounds in a commercial compound library were used as test drugs, and 1mM/L of a stock solution of the drug was dissolved in dimethyl sulfoxide, and added to each well plate at 1. Mu.L/well. After 3 minutes of slow mixing by a horizontal shaking table, the mixture is put back into an incubator for 24 hours. Further, microscopic observations record cell wells with reduced red light (tdbitmap), excluding wells with unchanged or increased red light; at the same time, cells were observed for reduced red light and morphologically abnormal wells were considered to be drug toxic leading to cell death. The concentration of drug was reduced 10-fold to a final concentration of 1. Mu.M/L, and further verification was performed, and red-light reduced cell wells were recorded, and the operation schematic is shown in FIG. 2.

Through multiple rounds of screening, the compound with the 4- (4-isoxazole) benzenesulfonamide parent structure can effectively reduce red light expression, and the compound can reduce the starting efficiency of the PCSK9 promoter, namely inhibit transcription of PCSK 9. Therefore, further verification is carried out on different compounds (a compound list is shown in table 1) with a 4- (4-isoxazole) benzenesulfonamide parent structure, and the compounds can be found to reduce red light expression and are considered as small molecule transcription inhibitors of PCSK 9.

Table 1 compounds validated for detection

TABLE 2 sequence information PCSK9 promoter sequence

Example 2

According to the screening results of example 1, qPCR assays were further performed on the compounds listed in table 1 above to investigate their actual effect on PCSK9 mRNA transcription. Human liver cancer HepG2 cells (5×10 ⁵) were inoculated into 6-well plates, cultured overnight, the compounds of Table 1 (final concentration 10. Mu.M/L) were added respectively, the control wells were added with the same volume of solvent (dimethyl sulfoxide), after 48 hours of treatment, the cells were collected, washed 2 times with PBS, and cell mass was used for extracting cellular mRNA using RNAiso PLUS/trizol (takara #9109).

Adding 1mL RNAiso Plus,1mL gun heads into the cell mass, blowing until no obvious mass exists, and standing for 5min at 15-30 ℃; adding chloroform according to 200 mu L/tube, covering a centrifugal tube cover, shaking for 15s until the solution is milky white, and standing for 5min at room temperature; centrifuge at 12,000g at 4℃for 15 min. After centrifugation, the samples were divided into three layers: colorless supernatant (containing RNA), intermediate albumin layer (mostly DNA) and colored lower organic phase.

Transfer the supernatant to another fresh centrifuge tube (no aspiration of white middle layer); adding 500 μl of isopropanol into the supernatant, gently turning over for several times (shaking without severe shaking to avoid RNA cleavage), and standing at room temperature for 10min; centrifuging at 12,000g and 4deg.C for 10min, and collecting precipitate as RNA, carefully sucking and discarding supernatant, and removing precipitate.

1ML of 75% ethanol was added, and the bottom of the centrifuge tube or flick tube was gently washed upside down to suspend the pellet; the supernatant was carefully discarded after centrifugation at 7,500g for 5min at 4 ℃. The centrifuge tube lid was opened and the pellet was dried at room temperature for 5-10min. After the precipitate was dried, 30. Mu.L of RNase-free water was added to dissolve the precipitate; the mRNA concentration was determined by Nanodrop, GDNA ERASER and matched buffer were added to the same amount of mRNA and incubated at room temperature for 5min to remove the remaining DNA fragments. Reverse transcription and qPCR assays were then performed using HiScript IIOne Step qRT-PCR SYBR Green Kit kit (Vazyme #Q221-01), to determine the effect of the compounds in Table 1 on PCSK9 gene transcription at a concentration of 10. Mu.M/L. The experimental results are shown in Table 3.

TABLE 3 inhibition of PCSK9 transcription by 4- (4-isoxazole) benzenesulfonamide compounds explicitly screened by qPCR

Example 3

In this example, a PCSK 9-deleted HepG2 cell line was constructed and subjected to various growth-related cell experiments to verify the effect of PCSK9 deletion on cell growth, and the method adopted in this example was to synthesize PCSK9 gene in sgRNA knockout cells and then to verify cell growth.

A: construction of PCSK9 knockout cell by Western blot detection

The procedure was performed according to the standard experimental procedure in the art and the results are shown in fig. 3A.

B: CCK8 detection of cellular Activity after PCSK9 knockout

(1) When the cell state grows in the logarithmic phase, the cells are collected, and the cell concentration is adjusted to 5X 10 ³/mL according to the cell count result.

(2) Absorbance values were measured in 96-well plates at 500 cells per well using CCK8 reacted with cells for 1h at 0 hour (day 0), 48 hours (day 2) and 96 hours (day 4), respectively, and the results of the analysis are shown in fig. 3B.

C, detecting the growth condition of cells after PCSK9 knockout by plate cloning

(1) When the cell state grows in the logarithmic phase, the cells are collected.

(2) Adding the uniformly mixed cell liquid into a 12-hole plate, and placing into a cell culture box.

(3) The growth of the cell monoclonal was observed, and the well plate was stained with crystal violet solution for a suitable period of time (about 14 days) by changing fresh complete medium every 3 days.

(4) The medium in the well plate was aspirated, washed with PBS, and fixed for 10 minutes with immunostaining fixative solution added to each well.

(5) The immunostaining fixative was recovered, washed with PBS, and stained with 0.1% crystal violet solution for 10min per well.

(6) The crystal violet solution was recovered, washed with PBS, the well plate was buckled dry, photographed after air drying, and analyzed for results, as shown in fig. 3C.

D: detection of cell proliferation following PCSK9 knockout by Ki67 and EDU staining

The operation is carried out according to the standard experimental procedure in the field, and after the dyeing is completed, the result is shown in fig. 3D after photographing and observing under a fluorescence microscope.

E: flow detection of apoptosis after PCSK9 knockout

(1) Cells were washed 2 times with pre-chilled PBS and resuspended in calcium ion-containing Buffer (1×binding Buffer) at a concentration of 1×10 ⁶ cells/ml.

(2) Transferring 100 mu L of the solution into a flow tube, adding Annexin V and nucleic acid dye, shaking gently, mixing well, and incubating for 15min at room temperature in a dark place.

(3) To each tube, 400. Mu.L of calcium ion-containing buffer was added for resuspension and analysis was performed on the machine, as shown in FIG. 3E.

The above experimental results show that the absence of PCSK9 does not affect the normal growth of cells.

Further qPCR and western blot detection were performed on compound 19 (parecoxib), and it was confirmed that compound 19 has a dose-effect relationship with respect to inhibition of mRNA and protein expression of PCSK9 in HepG2 cells, as follows.

HepG2 cells (5X 10 ⁵) were seeded in 6-well plates, incubated overnight, and added with different concentrations of compound 19 (final concentrations 0.01, 0.05, 0.1, 0.5, 1, 10. Mu.M/L, respectively) and control wells were treated with the same volume of solvent (dimethyl sulfoxide) for 24 hours or 48 hours (24-hour and 48-hour groups, respectively).

After reaching the time point, the cells were scraped, centrifuged at 2000rpm, the medium was discarded, washed 2 times with PBS, and the cells were qPCR-detected by the method of example 2 to verify the effect of compound 19 on PCSK9 mRNA expression. CCK8, plate clone, KI67 and EDU staining procedures were as described above to examine the effect of compound 19 on cell growth, and the experimental results are shown in FIG. 4. Parixib was able to inhibit transcription and expression of PCSK9, but did not affect cell growth. The experiment proves that the parecoxib is developed as the cell PCSK9 transcription inhibition reagent, compared with the traditional gene knockout method, the method is more convenient and faster, and the effect is close to that of the traditional gene knockout method, so that the method has a larger scientific research application prospect.

Example 4

Further, compound 19 was subjected to an in vivo evaluation test for the effect of lowering serum low density lipoprotein cholesterol (LDL-C).

Preparing mother liquor by dissolving a compound 19 in DMSO in advance, wherein the concentration of the mother liquor is 20mg/mL; before use, the mother solution is as follows: corn oil = 1:19 after preparing the solvent the mother liquor was diluted to 1mg/mL.

10 7-Week-old Balb/c mice were randomly divided into 2 groups, and the administration group was intraperitoneally injected daily with compound 19 (5 mg/kg/day), and the control group was injected with the same volume of solvent.

After one month of continuous administration, the mice were anesthetized, sampled and biochemically tested, and the LDL-C content in the serum was clarified. The results of the experiment are shown in FIG. 5, and it can be seen that compound 19 was effective in reducing LDL-C concentration in mouse serum, with statistical differences, and specific results are shown in Table 4.

TABLE 4 serum LDL-C concentration in mice (mmol/L)

NO.	Control group	Compound 19 treatment group
			1	0.53	0.39
2	0.55	0.34
			3	0.47	0.37
4	0.45	0.39
			5	0.51	0.39

Compound 19 was further subjected to an in vivo evaluation experiment for PCSK 9-lowering effect, specifically as follows.

(1) Preparing mother liquor by dissolving a compound 19 in DMSO in advance, wherein the concentration of the mother liquor is 20mg/mL; before use, the mother solution is as follows: corn oil = 1:19 after preparing the solvent the mother liquor was diluted to 1mg/mL.

(2) 10 7-Week-old Balb/c mice were randomly divided into 2 groups, and the administration group was intraperitoneally injected daily with compound 19 (5 mg/kg/day), and the control group was injected with the same volume of solvent.

(3) After one month of continuous administration, blood is taken from the orbit every 7 days, blood is sampled and sample blood is sent for biochemical detection, and the PCSK9 content in serum is clear. The results of the experiment are shown in fig. 6, and the results show that the compound 19 can effectively reduce the concentration of PCSK9 in serum of mice, and has statistical difference.

Further, an in vivo evaluation experiment of cholesterol lowering effect was performed on compound 19.

(3) After continuous administration for one month, blood is taken from the orbit every 7 days, blood is sampled and sample blood is sent for biochemical detection, and the PCSK9 and cholesterol content in serum is clear. The results of the experiments are shown in fig. 6-7, and the results show that the compound 19 can effectively reduce the concentration of PCSK9 and cholesterol in serum of mice, and has statistical difference.

Example 5

Safety evaluation of Compound 19.

14 7 Week old Balb/c mice were randomly assigned to 2 groups and a mother liquor was prepared by dissolving compound 19 in DMSO according to the procedure of example 4, and the mother liquor was prepared as follows: corn oil = 1:19 to 1mg/mL. The mice were then injected intraperitoneally at a dose of 5mg/kg/day, and the control group was intraperitoneally injected with the same volume of solvent (corn oil). After one month of continuous administration, mice in the anesthesia experiment group and the control group were subjected to blood sampling and sample feeding biochemical examination, and serum hepatotoxicity markers were detected: glutamic-pyruvic transaminase (AST), glutamic-pyruvic transaminase (ALT); renal toxicity markers: creatinine (CREA), UREA (UREA); cardiac toxicity markers: lactate Dehydrogenase (LDH), creatine Kinase (CK); after blood collection, heart, liver, spleen, lung and kidney were harvested, fixed with 10% formalin and sectioned for HE staining.

As shown in FIG. 8, the 5mg/kg/day dose of compound 19 did not cause an increase in each of the toxicity indexes, but rather had potential hepatorenal protection, and the experimental results showed that it significantly reduced the serum ALT, AST, CERA and UREA levels without significant substantial changes in each organ. The experiment of the embodiment shows that the development of parecoxib as a hypolipidemic drug has a great clinical application value.

Example 6

Use of POU2F2 as a transcription factor of PCSK9 for the preparation of a parecoxib efficacy assessment agent for assessing the inhibition of PCSK9 transcription by parecoxib through POU2F 2. The present example provides the following experimental verification.

(1) The cells to which compound 19 was added (experimental group) and the cells to which no drug was added (control group) were subjected to RNA-Seq sequencing, and differential gene screening was performed, as a result, 1773 core differential genes were found in the experimental group and the control group.

(2) And (2) carrying out transcription factor prediction by using a PCSK9 promoter sequence shown in SEQ ID NO.1, and carrying out Wen analysis on the potential transcription factors of the PCSK9 obtained by prediction and the core differential genes screened in the step (1), wherein the result shows that 63 transcription factors are obviously changed.

(3) Further ranking the 63 transcription factors with a LogFC score showed that the significant downregulation of POU2F2 was most pronounced.

(4) Detecting the mRNA level of POU2F2 by qPCR; and detecting the protein expression level condition of POU2F2 by using Western blot. The results showed that the mRNA expression level and the protein expression level of POU2F2 decreased after the addition of compound 19 to the cells.

The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.

Claims

1. The use of 4-(4-isoxazole)benzenesulfonamide compounds in the preparation of cell PCSK9 transcription inhibition agents, characterized in that the 4-(4-isoxazole)benzenesulfonamide compounds contain a 4-(4-isoxazole)benzenesulfonamide parent ring structure; the 4-(4-isoxazole)benzenesulfonamide parent ring structure is as shown in the compound of formula (1):

2. The use according to claim 1, characterized in that the 4-(4-isoxazole)benzenesulfonamide compound includes 3-(5-methyl-4-phenylisoxazole-3-yl)benzenesulfonamide, 4-(5-methyl-4-phenylisoxazole-3-yl)benzenesulfonamide, 4-(3-methyl-5-phenylisoxazole-4-yl)benzenesulfonamide, valdecoxib, 4-(5-methyl-3-phenylisoxazole-4-yl)benzenesulfonic acid, 3-phenyl-4-(4-aminosulfonylbenzyl)-5-methylisoxazole, benzenesulfonamide, 4-(3-methyl-5-phenylisoxazole-4-yl)benzenesulfonamide, (4-fluorophenyl)-5-methylisoxazol-4-yl)benzenesulfonamide, 2-fluoro-4-(5-methyl-3-phenylisoxazol-4-yl)benzenesulfonamide, 3-(5-methyl-3-phenylisoxazol-4-yl)benzene-1-sulfonyl chloride, valdecoxib 3'-sulfonyl chloride, 4-(5-methyl-3-phenylisoxazol-4-yl)benzene-1-sulfonyl chloride, 4-(5-methyl-3-phenylisoxazol-4-yl)benzenesulfonic acid ethyl ester, 3-phenyl-4-(4-chlorosulfonylbenzyl)-5-methylisoxazole, N-((4-(5-methyl)-3-phenylisoxazol-4-yl)benzenesulfonate 4-(5-(nitrooxy)methyl)-3-phenyl-4-isoxazolyl), N-((4-(5-methyl-3-phenylisoxazolyl)phenyl)sulfonyl)acetamide, N-(4-(3-methyl-5-phenylisoxazol-4-yl)phenyl)sulfonyl)propionamide, N-((3-(5-methyl-3-phenylisoxazol-4-yl)phenyl)sulfonyl)propionamide, parecoxib, benzenesulfonamide, 4-(5-(nitrooxy)methyl)-3-phenyl-4-isoxazolyl), N-((4-(5-methyl-3-phenylisoxazol-4-yl)phenyl)sulfonyl)isobutyramide, N-((4-(5-methyl-3-phenylisoxazol-4-yl)phenyl)sulfonyl)propionamide one or more of 3-(5-methyl-4-(4-sulfamoylphenyl)isoxazol-3-yl)benzenesulfonamide, N-[[4-(5-methyl-3-phenyl-4-isoxazol-3-yl)phenyl]sulfonyl]benzamide, 3-[4-(4-chlorosulfonylphenyl)-5-methyl-1,2-oxazol-3-yl]benzenesulfonyl chloride and/or N-[4-[5-methyl-3-[3-(propionylsulfamoyl)phenyl]-1,2-oxazol-4-yl]phenyl]sulfonylpropionamide.

3. The use according to claim 1, characterized in that the 4-(4-isoxazole)benzenesulfonamide compound is used to be added to a cell culture medium to incubate cells.

4. Application of parecoxib in the preparation of lipid-lowering drugs.

5. The use according to claim 4, characterized in that the parecoxib is used to lower serum cholesterol.

6. The use according to claim 4, characterized in that the parecoxib is used to reduce serum low-density lipoprotein.

7. The use according to any one of claims 4 to 6, characterized in that the drug further comprises other pharmaceutically acceptable carriers and/or adjuvants.

8. Application of POU2F2 as a molecular marker in the preparation of a reagent for evaluating the efficacy of parecoxib, characterized in that the application is to evaluate the inhibitory effect of parecoxib on PCSK9 transcription.

9. The use according to claim 8, characterized in that the parecoxib efficacy evaluation reagent also includes a PCSK9 promoter plasmid, and the sequence of the PCSK9 promoter is shown in SEQ ID NO.1.

10. The use according to claim 8, characterized in that the POU2F2 is used as a molecular marker of the PCSK9 transcription factor; and the use is to evaluate the effect of parecoxib on inhibiting the expression of the transcription factor POU2F2.