CN105816446A - Application of two halophenol compounds in preparation of medicines of resisting type II diabetic nephropathy - Google Patents

Abstract

The invention discloses two halophenol compounds 4,5,2'-trihydroxy-2,5'-dibromobenzophenone and 4,5,2'-tris(4-morpholinoyloxy)- The new medical application of 2,5'-dichlorobenzophenone, specifically in the preparation of anti- Type II diabetic nephropathy drugs, and the present invention also discloses an anti-type II diabetic nephropathy drug using the above two halophenol compounds as active ingredients. Both compounds were effective in improving The renal function of rats with type 2 diabetic nephropathy, its main therapeutic indicators such as uric acid, urinary protein, urinary microalbumin, growth transformation factor TGF-β1, blood sugar, and low-density lipoprotein are better than the current clinical first-line drug captopril, showing Important application prospects in the treatment of diabetic nephropathy.

Description

Application of Two Halophenol Compounds in the Preparation of Drugs Against Type Ⅱ Diabetic Nephropathy

technical field

The invention belongs to the field of new application of compounds, and relates to two halophenol compounds capable of preventing and treating type II diabetic nephropathy, specifically the compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49 ) and compound 4,5,2'-tris(4-morpholineformyloxy)-2,5'-dichlorobenzophenone (A8) in the preparation of anti-Ⅱ diabetic nephropathy drugs.

Background technique

Diabetic nephropathy (diabetic nephropathy, DN) is one of the main complications of diabetes, which eventually leads to end-stage renal disease (ESRD). At present, there are 114 million diabetic patients and 140 million prediabetic patients in China. With the continuous increase of diabetic patients worldwide, the prevalence of DN is also increasing. DN is the primary cause of ESRD in western countries, accounting for 25%-42%, and it is also the main cause of ESRD in my country. The prevalence of DN in type II diabetes is about 20%-25%. DN can range from the initial appearance of proteinuria to hypertension, nephrotic syndrome, and finally to renal failure and death.

At present, the treatment of DN is mainly to control blood sugar, lower blood lipid, inhibit inflammation, and resist oxidative stress. The main function of ACEI/ARB is to reduce blood pressure and urinary microalbumin, reduce glomerular osmotic pressure, and improve insulin utilization. However, for most patients with DN, especially those after DN stage IV, after the application of ACEI/ARB drugs, electrolyte disturbances often occur, and hyperkalemia is easily complicated, and side effects such as dry cough and orthostatic hypotension may occur at the same time. Therefore, currently there is no particularly effective and specific drug for this disease clinically. In view of the danger of DN, finding a suitable drug for the treatment has become a hot research direction in the current medical field.

The pathogenesis of DN mainly includes high glucose, abnormal renal hemodynamics, dyslipidemia, microvascular inflammation, oxidative stress and genetic factors. The halophenol compound 2',4,5-trihydroxy-2,5'-dibromobenzophenone (LM49) and its pharmaceutically acceptable salts and 2',4,5-tris(4-morpholine formyl Oxy)-2,5'-dichlorobenzophenone (A8) and its pharmaceutically acceptable salts were independently designed and synthesized by the research group, which has strong hypoglycemic, hypolipidemic, antioxidative, anti-inflammatory and other effects. Its activity diversity, especially with multiple fits with the pathogenesis of DN, has a good development and application prospect.

But so far, for compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone and compound 4,5,2'-tris(4-morpholinoyloxy)-2 ,5'-Dichlorobenzophenone's anti-type Ⅱ diabetic nephropathy pharmacological effect has not been reported at home and abroad.

Contents of the invention

The object of the present invention is to provide compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) and compound 4,5,2'-three (4-morpholine formyloxy The new pharmacological application of )-2,5'-dichlorobenzophenone (A8) is specifically the application in the preparation of anti-type II diabetic nephropathy drugs.

Another object of the present invention is a new drug against type Ⅱ diabetic nephropathy, which uses compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone, or compound 4, 5,2'-tris(4-morpholinoyloxy)-2,5'-dichlorobenzophenone is the active ingredient, and the drug also includes a common pharmaceutical carrier used in combination with the active ingredient or excipients. The medicine can be made into common preparations such as injections, tablets, dropping pills, solid dispersions or capsules according to different requirements.

The present invention is the first discovery of two halophenol compounds 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) and 4,5,2'-tris(4-morpholine formyl Oxy)-2,5'-dichlorobenzophenone (A8) has significant pharmacological activity against type Ⅱ diabetic nephropathy, and has good development and application value.

Among them, the structural formula of the compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) is as follows:

The structural formula of the compound 4,5,2'-tris(4-morpholinoyloxy)-2,5'-dichlorobenzophenone (A8) is as follows:

The compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) and compound 4,5,2'-tris(4-morpholinoyloxy)-2 , 5'-Dichlorobenzophenone (A8) is two kinds of halophenol compounds well known to those skilled in the art, and its preparation method is also easy to realize.

The present invention establishes SD rat type II diabetic nephropathy model, administers solid dispersions of the two compounds in different doses by intragastric administration, during which various indicators related to blood sugar, blood lipids, urine output, urine, renal function, and inflammatory factors are tested. According to the monitoring, the two compounds have strong renal protective effect on rats with II diabetic nephropathy, showing an important application prospect in the prevention and treatment of diabetic nephropathy.

The main innovations of the present invention are: (1) halophenol compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone (LM49) and its pharmaceutically acceptable salts and 4,5,2 '-Tris(4-morpholineformyloxy)-2,5'-dichlorobenzophenone (A8) and its pharmaceutically acceptable salts are independently created by the applicant, and have strong blood sugar-lowering, blood-lipid-lowering, Antioxidant, anti-inflammatory and other effects, its activity diversity fits multiple times with the pathogenesis of DN. (2) The two compounds can effectively improve the renal function of rats with type Ⅱ diabetic nephropathy when administered in solid dispersion. Density lipoprotein, etc. are superior to the current clinical first-line drug captopril.

Description of drawings

Fig. 1 is hematoxylin-eosin (HE) staining, observation under the electron microscope, each group rat kidney pathological comparison (HE × 200);

Fig. 2 shows Masson staining, observation under electron microscope, pathological comparison of rat kidneys in each group (Masson×400).

detailed description

Compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone and compound 4,5,2'-tris(4-morpholinoyloxy)-2,5'-dichloro Application of benzophenone in the preparation of anti-Ⅱ diabetic nephropathy drugs.

A drug against type II diabetic nephropathy, the active ingredient of which is compound 4,5,2'-trihydroxy-2,5'-dibromobenzophenone, or compound 4,5,2'-tri(4 -morpholinoyloxy)-2,5'-dichlorobenzophenone. The drug also includes common pharmaceutical carriers or excipients used in combination with the active ingredients. The medicine can be made into common dosage forms such as injections, tablets, dropping pills, solid dispersions or capsules according to specific requirements.

The following specific experimental examples will further illustrate the anti-type II diabetic nephropathy function of the two halophenol compounds in the present invention. It should be noted that the following examples are all illustrative and do not make any contribution to the present invention. limited.

Material

Experimental animals:

Male SD rats, weighing 180-220g, source of animal: China National Institutes for Food and Drug Control, license number: SCXK-(Beijing) 2014-0013, certificate number: 11400500008465, the animals used in this test and the related disposal are in line with animal welfare regulations It is required that the experiment must be reviewed by the animal welfare committee of the institution before the experiment is carried out.

feed:

High-sugar and high-fat feed formula: 10% lard, 20% sucrose, 2.5% cholesterol, 0.5% sodium cholate, 67% base material. Feed source: Beijing Keao Xieli Feed Co., Ltd., license number: SCXK-(Beijing) 2014-0010, certificate number: 11002900016120.

Drugs and reagents:

LM49 (prepared by Shanxi Medical University Pharmaceutical Chemistry Laboratory, purity ≥ 99.5%, batch number 20150320); A8 (prepared by Shanxi Medical University Pharmaceutical Chemistry Laboratory, purity ≥ 99.5%, batch number 20150112); polyvinylpyrrolidone (PVP K30 ) (USP26, Jiangyin Jiafeng Chemical Co., Ltd.); Tween 80 (pharmaceutical grade, Sichuan Jinshan Pharmaceutical Co., Ltd.); absolute ethanol (analytical grade, Beijing Chemical Reagent Company); captopril (Shanghai Xudong Haipu Pharmaceutical Co., Ltd. Co., Ltd., production batch number 140603); streptozotocin (Sigma, production batch number 1126C038); rat interleukin-1β ELISA kit (Nanjing Jiancheng, batch number 1501261); rat interleukin-6 ELISA kit (Nanjing Jiancheng, batch number 1412271); rat microalbumin (Alb) ELISA kit (Nanjing Jiancheng, batch number 1506201); rat soluble intercellular adhesion molecule (sICAM-1) detection reagent Box (Nanjing Jiancheng, batch number 1501201); Rat white tumor necrosis factor (TNF-α) ELISA kit (Nanjing Jiancheng, batch number 1507281); Rat white blood vessel endothelial cell adhesion molecule (sVCAM-1) ELISA Detection kit (Nanjing Jiancheng, batch number 1508221); total antioxidant capacity (T-AOC) kit (Nanjing Jiancheng, batch number 20151010); BCA protein quantification kit (Boster Bio, batch number 10K12B46); urea (BUN) kit (Shanghai Fosun Long March Medical Science Co., Ltd., batch number P1311021); Uric acid (UA) kit (Shanghai Fosun Long March Medical Science Co., Ltd., batch number PF1405011); creatinine (CREA) kit (Shanghai Fosun Long March Medical Science Co., Ltd., Batch number D1404063); clinical chemistry comprehensive quality control serum (Shanghai Fosun Changzheng Medical Science Co., Ltd., batch number 842UN); malondialdehyde (MDA) test kit (Nanjing Jiancheng, batch number 20150126); rat nuclear transcription factor (NF-κB) Enzyme immunoassay kit (Nanjing Jiancheng, batch number 1507251); SOD kit (WST-1 method, Nanjing Jiancheng, batch number 20150127); Masson staining solution (Nanjing Jiancheng, batch number 20150915).

instrument:

Constant temperature water bath (Shanghai Hecheng Instrument Manufacturing Co., Ltd.); BP-121S electronic balance (Shanghai Precision Scientific Instrument Company); TU-1810 UV-Vis spectrophotometer (Beijing General Instrument Co., Ltd.); blood glucose meter (Johnson & Johnson, USA) ); microplate reader (Rayto, RT-6100); low-speed automatic balancing centrifuge (Anxin Baiyang Centrifuge Factory, Hebei Province); ultra-low temperature refrigerator (Haier); Konelab Prime 30 automatic biochemical analyzer (Thermo, USA).

Preparation of solid dispersion:

Prepared by solvent method: according to the mass ratio of LM49, PVPK30, tween80 is 1:5:1 and the mass ratio of A8, PVPK30, tween80 is 1:4:1, each drug is accurately weighed, dissolved with appropriate amount of absolute ethanol, Constantly stir and mix evenly, evaporate and remove absolute ethanol on a 60°C water bath to obtain a viscous substance, freeze in a -20°C refrigerator for 2 hours, take it out and dry it in a vacuum drying oven for 24 hours, and crush it for 80 hours after embrittlement Mesh sieve and store in a desiccator for later use.

Preparation of animal models of diabetic nephropathy:

Healthy adult male SD rats (180-220g) were fed with high-sugar and high-fat diet for 4 weeks, fasted overnight, intraperitoneally injected with streptozotocin (STZ) 40mg/kg, and the normal group was injected with the same volume of sodium citrate buffer, After 72 hours, fast for 12 hours, measure the fasting blood glucose of the model rats, if the fasting blood glucose > 16.7mmol/L for 3 consecutive times, it is considered that the diabetes modeling is successful. After successful modeling, the rats continued to be fed with high-fat diet during the administration period, and the normal group was fed with normal diet.

After 6 weeks of administration, urine was collected for 6 hours with metabolic cages.

After fasting overnight, intraperitoneal anesthesia, abdominal aortic blood was drawn to detect relevant indicators. Wash the kidneys with normal saline, record the weight of both kidneys, and calculate the kidney coefficient (weight of both kidneys/body weight). Renal tissue (including glomeruli and renal tubules) from the same part of the left kidney was taken longitudinally and immersed in 10% neutral formalin for pathological analysis. The other part was used for detection of biochemical indicators, and the right kidney was used for mechanism research and stored in a -80°C refrigerator.

Group and dose design:

After successful modeling, except the normal group (10 animals), other animals were randomly divided into 8 groups, 12 animals in each group. Divided into model group, LM49 solid dispersion low dose group (drug content is 3mg/kg), LM49 solid dispersion middle dose group (drug content is 9mg/kg), LM49 solid dispersion high dose group (drug content is 27mg/kg), A8 solid dispersion low dose group (drug content is 3mg/kg), A8 solid dispersion middle dose group (drug content is 9mg/kg), A8 solid dispersion high dose group (drug content is 27mg/kg), captopril control group (drug content is 15mg/kg).

Administration method:

Administration route: gavage.

Dosing volume: normal control group and model group were given normal saline 1.0ml/100g, positive control, LM49 solid dispersion high, medium and low dose groups, A8 solid dispersion high, medium and low dose groups were given different concentrations of drugs, The drug volume is 1.0ml/100g.

Drug preparation: the solid dispersion is dissolved in physiological saline, and the positive control is administered in suspension with 0.5% CMC-Na.

Dosing time: Dosing starts at the end of modeling.

Dosing frequency: 7 days a week for 6 weeks.

Statistical method:

Using IBM SPSS Statistics 20 software for data processing, data in One-way analysis of variance (ANOVA) was used for comparison between groups. According to the results of homogeneity of variance test, LSD test was used if the variance was homogeneous, and Dunnett's T3 test was used if the variance was not homogeneous. P<0.05 was considered statistically significant.

results and analysis:

1. Effects of LM49 and A8 on renal function in rats with diabetic nephropathy

Table 1 Effects of LM49 and A8 on renal function in rats with diabetic nephropathy ( n=6)

Note: Compared with normal note, ^## P<0.01, ^## P<0.05; compared with model group, ^□□ P<0.01, ^□ P<0.05

Table 2 Effects of LM49 and A8 on uric acid and growth transformation factors in rats with diabetic nephropathy ( n=6)

Note: Compared with normal injection, ^## P<0.01, ^## P<0.05; compared with model group, ^□□ P<0.01, ^□ P<0.05; compared with low dose group, ^▲▲ P<0.01, ^▲ P<0.05; compared with the middle dose group, ^○○ P<0.01, ^○ P<0.05; compared with the carpril group, ^●● P<0.01, ^● P<0.05

Table 3 Effects of LM49 and A8 on urinary protein and trace albumin in the urine of diabetic nephropathy rats ( n=6)

Note: Compared with normal injection, ^## P<0.01, ^## P<0.05; compared with model group, ^□□ P<0.01, ^□ P<0.05; compared with low dose group, ^▲▲ P<0.01, ^▲ P<0.05; compared with the middle dose group, ^○○ P<0.01, ^○ P<0.05; compared with the carpril group, ^●● P<0.01, ^● P<0.05

2. Effects of LM49 and A8 on serum blood glucose levels in rats with diabetic nephropathy

Table 4 Effects of LM49 and A8 on serum blood glucose in rats with diabetic nephropathy ( n=6)

Note: Compared with normal injection, ^## P<0.01, ^## P<0.05; compared with model group, ^□□ P<0.01, ^□ P<0.05; compared with low dose group, ^▲▲ P<0.01, ^▲ P<0.05; compared with the middle dose group, ^○○ P<0.01, ^○ P<0.05; compared with the carpril group, ^●● P<0.01, ^● P<0.05

3. Effects of LM49 and A8 on serum cholesterol (TCHO), triglyceride (TG), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) levels in rats with diabetic nephropathy

Table 5 Effects of LM49 and A8 on cholesterol and triglyceride levels in serum of rats with diabetic nephropathy ( n=6)

Note: Compared with normal injection, ^## P<0.01, ^## P<0.05; compared with model group, ^□□ P<0.01, ^□ P<0.05; compared with low dose group, ^▲▲ P<0.01, ^▲ P<0.05; compared with the middle dose group, ^○○ P<0.01, ^○ P<0.05; compared with the carpril group, ^●● P<0.01, ^● P<0.05

Table 6 Effects of LM49 and A8 on serum levels of HDL-C and LDL-C in rats with diabetic nephropathy ( n=6)

Note: Compared with normal injection, ^## P<0.01, ^## P<0.05; compared with model group, ^□□ P<0.01, ^□ P<0.05; compared with low dose group, ^▲▲ P<0.01, ^▲ P<0.05; compared with the middle dose group, ^○○ P<0.01, ^○ P<0.05; compared with the carpril group, ^●● P<0.01, ^● P<0.05

4. Effects of LM49 and A8 on inflammatory factors in kidney tissue of rats with diabetic nephropathy

Table 7 Effects of LM49 and A8 on inflammatory factors TNF-α, NF-κB and adhesion molecule ICAM-1 in kidney tissue of rats with diabetic nephropathy ( n=6)

Note: Compared with normal injection, ^## P<0.01, ^## P<0.05; compared with model group, ^□□ P<0.01, ^□ P<0.05; compared with low dose group, ^▲▲ P<0.01, ^▲ P<0.05; compared with the middle dose group, ^○○ P<0.01, ^○ P<0.05; compared with the carpril group, ^●● P<0.01, ^● P<0.05

Table 8 Effects of LM49 and A8 on inflammatory factors IL-1β, IL-6 and adhesion molecule VCAM-1 in kidney tissue of rats with diabetic nephropathy ( n=6)

Note: Compared with normal injection, ^## P<0.01, ^## P<0.05; compared with model group, ^□□ P<0.01, ^□ P<0.05; compared with low dose group, ^▲▲ P<0.01, ^▲ P<0.05; compared with the middle dose group, ^○○ P<0.01, ^○ P<0.05; compared with the carpril group, ^●● P<0.01, ^● P<0.05

5. Effects of LM49 and A8 on oxidative factors in kidney tissue of rats with diabetic nephropathy

Table 9 Effects of LM49 and A8 on oxidative factors in kidney tissue of rats with diabetic nephropathy ( n=6)

The above studies have shown that LM49 has a clear anti-diabetic nephropathy effect in rats, can effectively reduce renal tissue lesions, and can significantly reduce the levels of uric acid, blood sugar, cholesterol, triglyceride, and low-density lipoprotein in rat serum, and the growth and transformation of kidney tissue. Factors, urinary protein and urinary microalbumin levels, significantly inhibited the expression of TNF-α, NF-κB, IL-6, IL-1β and other inflammatory factors in renal tissue, and reduced the level of renal tubular watery lesions and glomerular fibrosis . At the same time, the above results also show that LM49 has a strong anti-diabetic nephropathy effect in rats, and its main therapeutic indicators, such as uric acid, urinary protein, urinary microalbumin, growth transformation factor TGF-β1, blood sugar, and low-density lipoprotein, are superior to those of current clinical trials. The first-line drug captopril has shown important application prospects.

A8 has a stronger anti-diabetic nephropathy effect in rats, can effectively reduce renal tissue lesions, and can significantly reduce the levels of uric acid, glycosylated hemoglobin, blood sugar, cholesterol, triglyceride, low-density lipoprotein in rat serum and growth transformation in renal tissue Factors, urinary protein and urinary microalbumin levels, significantly inhibited the expression of TNF-α, NF-κB, IL-6, IL-1β and other inflammatory factors in renal tissue, and reduced the level of renal tubular watery lesions and glomerular fibrosis . At the same time, the above results show that A8 has a strong anti-diabetic nephropathy effect in rats, and its main therapeutic indicators, renal index, uric acid, urinary protein, urinary microalbumin, growth transformation factor TGF-β1, blood sugar, low-density lipoprotein, etc. In the current clinical first-line drug captopril, it shows an important application prospect.

in conclusion:

Through the above experiments, it can be shown that compounds LM49 and A8 have definite anti-type II diabetic nephropathy function. In particular, the effect of the high-dose group is more significant, and compound A8 has higher anti-type Ⅱ diabetic nephropathy activity than LM49, showing a good prospect for application and development.

Claims (4)

1. compound 4,5,2'-trihydroxy-2,5'-dibromobenzo-phenone and compound 4,5,2'-tri-(4-morpholine formyloxy)-2,5'-dichloro benzophenone application in preparing type II diabetes resisting nephropathy medicine.

2. the medicine of type II diabetes resisting nephropathy, it is characterised in that: the active component of described medicine is compound 4,5,2'-trihydroxy-2,5'-dibromobenzo-phenone, or compound 4,5,2'-tri-(4-morpholine formyloxy)-2,5'-dichloro benzophenone.

The medicine of type II diabetes resisting nephropathy the most according to claim 2, it is characterised in that: this medicine also includes common pharmaceutical carrier or the excipient used with described active ingredient combinations.

4. according to the medicine of the type II diabetes resisting nephropathy described in Claims 2 or 3, it is characterised in that: described medicine is injection, tablet, drop pill, solid dispersion or capsule.