CN106432026A - Compound with potential therapeutic activity on diabetes - Google Patents

Abstract

The present invention relates to a class of compounds with potential therapeutic activity on diabetes, whose general formula is: Where X can be: hydrogen, hydrocarbon group, halogen, hydroxyl group, hydrocarbon group, acyloxy group, aldehyde group, acyl group, nitro group, amino group, hydrocarbon amino group, carboxyl group, acid halide group, acyloxyacyl group, ester group, amido group, Groups such as cyano group, guanidino group, amidino group, azido group, inorganic acid ester group (wherein n is greater than or equal to 0). Y can be: a heteroatom, 1, 2-vinylene, methylene or no atom, etc. Z: It can be substituted on the 2, 3, and 4 positions of the adamantane ring, and Z can be hydrogen, hydrocarbon group, halogen, hydroxyl group, hydrocarbon oxygen group, acyloxy group, aldehyde group, acyl group, nitro group, amino group, hydrocarbon amino group, carboxyl group , acyl halide, acyloxyacyl, ester, amido, cyano and other common organic groups.

Description

A class of compounds with potential therapeutic activity against diabetes

technical field

The present invention relates to the fields of medicinal chemistry and pharmacotherapeutics, in particular to the treatment and/or prevention of diabetes and related diseases alone or in combination. More specifically, it relates to a series of compounds represented by the following formulas, their preparation methods, pharmaceutically acceptable salts or pharmaceutically acceptable solvates, and the like.

Background technique

Diabetes (Diabetes Mellitus) is a metabolic disease caused by a variety of etiologies, characterized by uncontrolled chronic hyperglycemia and accompanied by a variety of complications, mainly caused by insufficient insulin secretion and/or functional defects . Diabetes is a serious threat to human life and health. At present, there is no cure for diabetes, so diabetic patients can only choose continuous drug treatment to stabilize the blood sugar level in the body, which brings a heavy economic burden to the patient's family and society.

Diabetes can be mainly divided into type I diabetes, type II diabetes, gestational diabetes and other types of diabetes, of which type II diabetes accounts for more than 90%. The current treatment of type II diabetes mainly relies on oral medication in addition to injecting insulin. Traditional oral hypoglycemic drugs include: (1) insulin sensitizers, such as pioglitazone; (2) sulfonylureas, such as glimepiride; (3) biguanides, such as metformin; (4) α-glucosidase Inhibitors, such as acarbose; (5) mealtime blood sugar regulators, such as repaglinide. Due to the many side effects of traditional hypoglycemic drugs, especially hypoglycemia and weight gain, the long-term medication of patients is limited. Through a large number of experiments, people have developed new hypoglycemic drugs with better efficacy and fewer side effects - incretin potentiators, including glucagon-like peptide-1 (Glucagon-1ike peptide 1, GLP-1) analogs and Dipeptidyl peptidase-4 (Dipeptidyl peptidase IV, DPP-4) inhibitor. At present, incretin potentiators have become one of the hotspots in the research and development of drugs for the treatment of type II diabetes in the world.

Based on the above oral antidiabetic drugs, the present invention proposes the following inventions in order to further improve the curative effect, reduce side effects, and search for better drugs.

Contents of the invention

The object of the present invention is the compound represented by the following formula, its preparation method and its therapeutic application in diabetes and related diseases.

In the above structure, where X can be: hydrogen, hydrocarbon group, halogen, hydroxyl group, alkoxyl group, acyloxy group, aldehyde group, acyl group, nitro group, amino group, hydrocarbon amino group, carboxyl group, acid halide group, acyloxyacyl group, Groups such as ester group, amide group, cyano group, guanidino group, amidino group, azido group, inorganic acid ester group (wherein n is greater than or equal to 0). Y can be: heteroatom, 1,2-vinylidene, methylene or no atom, etc. Z: It can be substituted on the 2, 3, and 4 positions of the adamantane ring, and Z can be hydrogen, hydrocarbon group, halogen, hydroxyl group, hydrocarbon oxygen group, acyloxy group, aldehyde group, acyl group, nitro group, amino group, hydrocarbon amino group, carboxyl group , acyl halide, acyloxyacyl, ester, amido, cyano and other common organic groups.

Further, X can specifically be halogen, hydroxyl, alkoxy, acyloxy, acyl, amino, hydrocarbon amino, carboxyl, ester, amide, cyano, azido, inorganic acid ester and other groups. Specifically, Y may be a heteroatom, a methylene group, or the like. Z can be substituted on the 3-position of the adamantane ring, and Z can specifically be halogen, hydroxyl, alkoxy, acyloxy, acyl, nitro, amino, hydrocarbon amino, carboxyl, ester, amide, cyano, etc.

Still further, X is more specifically a group such as a halogen, a hydroxyl group, an amino group, a carboxyl group, a cyano group, an azide group, or an inorganic acid ester group. Specifically, Y may be a heteroatom or the like. Z can be substituted on the 3-position of the adamantane ring, and Z can specifically be halogen, hydroxyl, nitro, amino, carboxyl, cyano and the like.

Furthermore, X is more specifically a group such as halogen, azido or the like. Specifically, Y may be an oxygen atom, a nitrogen atom, or the like. Z can be substituted on the 3-position of the adamantane ring, and Z can specifically be hydroxyl, nitro, carboxyl, etc.

A specific compound example CMD-05-151013 can be described as, but not limited to this example, wherein Z is substituted at the 3-position of the adamantane ring, Z is OH, X is chlorine (wherein n is equal to 3), Y is methylene.

Regarding the preparation method of this type of compound, using amantadine as a starting material, react with (S)-1-chloroacetyl-2-cyanopyrrolidine to prepare amantadine monosubstituted by amino group, and then to adamantane The ring is substituted and then reacted with the corresponding acid chloride or activated ester to prepare the target compound. And other corresponding alternative preparation methods that can be accomplished by the professional knowledge possessed by those skilled in the art. An example of the production method is presented here, but not limited to this example.

Using amantadine hydrochloride as the starting material, using mixed acid and potassium hydroxide as reagents, the 3-position of the amantadine ring is hydroxylated to obtain amantadine alcohol; Acetyl-2-cyanopyrrolidine was reacted to prepare amino monosubstituted adamantane alcohol, and then continued to react with 5-chlorovaleryl chloride to prepare the target compound CMD-05-151013. And other corresponding alternative preparation methods.

The research of the present invention finds that the above compound can be used for the treatment of SD rat type 2 diabetes induced by high-fat diet combined with intraperitoneal injection of STZ.

The mechanism of action may involve multiple targets, including ①inhibiting the activity of DPPIV enzyme, reducing the decomposition of GLP-1 by DPPIV enzyme, and increasing the concentration of plasma GLP-1; ②directly promoting the release of GLP-1, increasing the concentration of plasma GLP-1 ; ③By increasing the concentration of GLP-1, improving the shape and function of islet cells, and promoting the release of insulin, so as to play the role of lowering blood sugar.

The research of the present invention shows that the above-mentioned compound can significantly inhibit the STZ-induced apoptosis of rat islet cell line INS-1 in vitro and promote the proliferation of INS-1 cell, and at the same time, the compound can effectively promote the development of human intestinal endocrine cell line. Release of GLP-1 in NCI-H716.

The research of the present invention shows that the in vivo test of the above-mentioned compound includes: 1. Through the oral glucose tolerance test in rats, the compound can effectively reduce the area under the curve after the oral administration of glucose in rats, and the AUC reduction of the compound at 4.5 mg/kg orally is 51.35%, which is the same as The dose of vildagliptin corresponding to the molar concentration is 3 mg/kg, and the AUC reduction range is 47.59%, which shows that the compound is slightly better than vildagliptin (see Figure 1).

②By detecting the activity of DPPIV in fasting plasma, glucose-loaded plasma and random plasma of rats, the compound can effectively inhibit the activity of DPPIV in plasma. They were 50%, 70%, and 55%, respectively, and the corresponding inhibition rates of 3 mg/kg vildagliptin were 65%, 75%, and 60%, respectively, and there was no statistical difference between them (see Figure 2).

③ By detecting the GLP-1 concentration and insulin concentration in rat fasting plasma, glucose-loaded plasma and random plasma, it can be seen that the compound can increase the GLP-1 content and insulin concentration in plasma, and has statistical significance (see Figure 3 ).

④ By detecting the glycated hemoglobin content in random plasma of rats, it can be known that the compound can reduce the glycated hemoglobin content in rat plasma by 1.5%-2%, and vildagliptin can reduce it by 1%-2% (see Figure 4 ).

5. By detecting total cholesterol in rat plasma, triglyceride, and low-density lipoprotein content, it can be seen that the compound can effectively reduce triglyceride in rat plasma, low-density lipoprotein content, and there is a statistical difference (see Figure 5 ).

⑥ Observed by HE staining, immunofluorescence staining, and Tunnel staining, the compound can significantly improve the destruction of islets in T2DM model mice, repair islet cells, reduce the expression of glucagon, and reduce the apoptosis of islet cells in the model group.

Moreover, the toxicity test showed that when the inhibition rate of the viability of the 293 cell line was 10%, the concentration of CMD ^- 05 was greater than 1* ¹⁰ -4 M, and the concentration of vildagliptin was greater than 1*10 ^-4 M; the viability of the HT22 cell line was inhibited When the rate is 10%, the concentration of CMD-05 is greater than or equal to 3*10 ^-5 M, and the concentration of vildagliptin is greater than 1*10 ^-5 M; when the activity inhibition rate of LO-2 cell line is 10%, CMD- The concentration of 05 is greater than or equal to 1*10 ^-4 M, and the concentration of vildagliptin is greater than 1*10 ^-5 M. The above results suggest that the compound has less cytotoxicity, and compared with vildagliptin, the cytotoxicity is less than that of vildagliptin. After oral administration of the compound at a single dose of 2 g/kg to Kunming mice, the mice had no adverse reaction or death within 15 days.

The above results show that the compounds we found have significant anti-diabetic effects and low toxicity.

The object of the present invention is to provide a kind of medicine and composition thereof characterized in that, contain the above-mentioned compound of effective amount, and the various dosage forms that pharmaceutically acceptable carrier or excipient etc. are made and its combination with other related medicines combination.

Description of drawings

Figure 1 is the area under the OGTT curve of rats in each group on day 1, day 19 and day 29

Figure 2 is a diagram of the inhibition rate of DPPIV in fasting, glucose load and random plasma of rats in each group

Fig. 3 is fasting of each group rat, the content (A) of GLP-1 and the level (B) figure of insulin in the glucose load and random plasma

Figure 4 is a diagram of the glycated hemoglobin content in fasting, glucose load and random plasma of rats in each group

Fig. 5 is the total cholesterol (A) in the random plasma of each group of rats, triglyceride (B), low-density lipoprotein content (C) figure

detailed description

The present invention can be further understood through the following specific examples, but they do not constitute a limitation to the content of the present invention. Under the above-mentioned premise of the present invention, the extension and expansion content that the present invention belongs to the professional knowledge of those skilled in the art should all be within the protection scope of the present invention.

Example 1

Preparation of CMD-05-151013:

38ml of 98% concentrated sulfuric acid and 4ml of 65% concentrated nitric acid were mixed and cooled in an ice bath, and 3.75 grams of amantadine hydrochloride was added in portions to the mixed acid, and stirred and reacted for 3 hours under an ice bath; 60 grams of crushed ice was added and stirred for 0.5 Add an appropriate amount of potassium hydroxide solid to adjust the pH value to 12-13, stir and react in an ice bath for 2 hours; filter with suction, adjust the pH value of the filtrate to 8-9 with concentrated hydrochloric acid, and concentrate to dryness; add 80ml of absolute ethanol Reflux for 1 hour, let cool, filter with suction, and concentrate the filtrate to dryness; then add 10ml of the mixed solution (acetone:ethyl acetate=3:1) and reflux for 1 hour, place it in an ice bath, and filter with suction to obtain 3-aminoadamantanol The solid is 3.2 g, and the melting point is greater than 256°C.

Add 3 g of 3-aminoadamantanol, 0.25 g of potassium iodide, and 10 g of potassium carbonate into 30 ml of tetrahydrofuran and stir to raise the temperature to 40 ° C. Slowly add 3 g of (S)-1-chloroacetyl-2- A solution of cyanopyrrolidine dissolved in 30ml of tetrahydrofuran was dropped in about 1.5 hours. After dropping, keep warm at 40°C for 1 hour, then heat up to reflux for 2 hours. After the reflux was completed, filter while hot, wash the filter cake with a small amount of tetrahydrofuran, combine the filtrates, and concentrate to an oily substance. The oil was dissolved by adding an appropriate amount of methyl ethyl ketone, left to crystallize, filtered, and dried to obtain 4 g of amino monosubstituted adamantane amino alcohol solid, melting point 147-149°C. The purity determined by HPLC normalization method is 98.1%, and the structural characterizations such as IR and NMR meet the requirements.

Add 5 g of the above-mentioned amino monosubstituted adamantine alcohol, 0.2 g of DMAP, 15 g of potassium carbonate, and 100 ml of anhydrous tetrahydrofuran into the reaction flask and stir. Dissolve 5ml of 5-chloropentanoyl chloride in 50ml of tetrahydrofuran, and slowly add it dropwise to the reaction solution under ice-cooling. After the dropwise addition, raise the temperature and stir to reflux, and monitor by TLC (developing solvent: ethyl acetate: methanol = 10:1). After the reaction is completed, filter while hot, wash the filter cake with an appropriate amount of tetrahydrofuran, combine the filtrates, concentrate under reduced pressure to obtain an oily liquid, add methanol to the oily liquid for recrystallization to obtain CMD-05-151013 white solid. mp: 158-161°C. The purity determined by HPLC normalization method is 95%, and the structural characterizations such as IR and NMR meet the requirements.

Example 2

In vitro cell experiment of CMD-05-151013: ①The effect of the compound on the release of GLP-1 from intestinal L cells: culture human intestinal L cell line NCI-716, two days before the experiment, the cells were treated with 1*10 ⁶ cells/ml Spread in Matrigel-coated 12-well plates, add DMEM containing 10% FBS for culture. After 48 hours, aspirate the cell supernatant, wash with KRB buffer containing 0.2% BSA, add 1ml of drug-containing KRB buffer (pH7.2), incubate at 37°C for 2 hours, absorb the supernatant and add 50ug/ml PMSF , stored at -80°C. Use the ELISA kit to detect the concentration of GLP-1 in the medium; ②The effect of the compound on the proliferation of rat islet cells: inoculate rat islet cells INS-1 in a 96-well plate, and the plating density is 1*10 ⁵ cells/ml , after 24 hours of incubation, the medium was replaced, and the final concentration was 3*10 ^-4 , 1*10 ^-4 , 3*10 ^-5 , 1*10 ^-5 , 3*10 ^-6 , 1*10 ^-6 , 3* 10 ^-7 , 1*10 ^-7 , 3*10 ^-8 , 1*10 ^-8 drug-containing medium continued to incubate for 24 hours, then add CCK-810ul to each well, continue to incubate for 2 hours, shake on a horizontal shaker for 10s, at 450nm 3) The anti-apoptotic effect of the compound on STZ-induced rat islet cells: Inoculate INS-1 cells at 3*10 ⁴ cells/ml on a 6-well plate and culture them for 24 hours, then add a final concentration of 10 ^{- 5} , 10 ^-6 , 10 ^-7 M of the compound and 3 mM STZ buffer, continued to culture for 36 hours, collected cells, resuspended in PBS, and analyzed cell apoptosis by flow cytometry.

Example 3

In vivo experiment of CMD-05-151013: ①Effect on normal rats: select 25 SD male rats about 200g, and randomly divide them into 5 groups: vildagliptin group (3mg/kg), CMD-05 (4.5mg /kg), CMD-05 (1.5mg/kg), CMD-05 (0.5mg/kg), solvent control group. When doing oral glucose tolerance test, test the fasting blood sugar and give the drug by gavage, measure the blood sugar 30 minutes later and give 25% glucose aqueous solution by gavage, and measure the blood sugar 30 minutes, 60 minutes, and 120 minutes after giving the glucose solution by gavage. When measuring GLP-1, Insulin, and DPPIV in glucose-loaded plasma, the drugs of each group were administered by intragastric administration for 30 minutes, and then 25% glucose aqueous solution was administered by intragastric administration. After 30 minutes, blood was collected through the venous plexus of the eyes, centrifuged at 3000 rpm for 5 minutes, and plasma was separated, using ELISA reagents The content of GLP-1 and Insulin in the plasma was detected by the kit, and the activity of DPPIV was measured by the fluorescence method. ②Effect on model rats: Select 70 male SD rats with a weight of about 100 g, randomly select 6 as normal controls, and inject small doses of STZ (35 mg/kg) intraperitoneally after feeding the remaining 64 high-fat and high-sugar diets for 30 days, and continue high Lipid feed was fed for 30 days, and 30 rats with fasting blood glucose greater than 16.7mmol/L were selected for three consecutive days and divided into five groups: Vildagliptin group (3mg/kg), CMD-05 (4.5mg/kg), CMD-05 (1.5 mg/kg), CMD-05 (0.5 mg/kg), solvent control group. Continuously administered for one month, oral glucose tolerance test was performed on the first day, the 19th day, and the 29th day of administration (the specific steps were as described above), and the rats were fasted for about 30 days, the glucose load, random plasma, and detected by ELISA Insulin content, GLP-1 content, glycosylated hemoglobin content, DPPIV activity, and kits were used to detect the content of total cholesterol, low-density lipoprotein, and triglyceride in random plasma. After the administration, the pancreatic tissue was separated, stained with HE, observed the shape of the pancreatic tissue, performed immunofluorescent staining, observed the expression of insulin and glucagon in the pancreatic tissue, performed Tunnel staining, observed the apoptosis of the pancreatic islet tissue, and performed transmission electron microscopy. Changes in secretory granules in islet microstructure were observed.

Example 4

Toxicity test of CMD-05-151013: ①Cytotoxicity: culture human embryonic kidney cell 293, normal human liver cell LO-2, mouse hippocampal neuron HT22, and seed the cells in a 96-well plate after the cells grow well. The plate density is 1*10 ⁵ cells/ml. After seeding the plate for 4 hours, add different concentrations of vildagliptin and the compound so that the final concentration is 3*10 ^-4 , 1*10 ^-4 , 3*10 ^-5 , 1*10 ^-5 , 3*10 ^{- 6} , 1*10 ^-6 , 3*10 ^-7 , 1*10 ^-7 , 3*10 ^-8 , 1*10 ^-8 , add 20ul of MTT solution after 24h of culture, continue to culture for 4h, suck out the medium, add 150ul of DMSO , Shake on a horizontal shaker for 5 min, measure the OD value at 450 nm, and calculate the cell inhibition rate. ②Animal toxicity: 10 male and 10 female Kunming mice were randomly selected, fasted and watered overnight, given 2 g/kg of the compound by intragastric administration with a volume of 0.4 ml/10 g, and observed for 15 days after administration.

Claims (9)

1. a class is shown below compound and its prepare with which to diabetes and the therapeutic use of diseases related aspect；

Wherein X can be：Hydrogen, alkyl, halogen, hydroxyl, oxyl, acyloxy, aldehyde radical, acyl group, nitro, amino, hydrocarbon amino, carboxylic (wherein n is big for the groups such as base, acid halide group, acyl-oxygen acyl group, ester group, amide groups, cyano group, guanidine radicals, amidino groups, azido, mineral acid ester group In or be equal to 0)；Y can be：Hetero atom, 1,2- ethenylidene, methylene or no atom etc.；Z：May replace diamantane (obsolete) ring 2, 3rd, on 4, Z can be hydrogen, alkyl, halogen, hydroxyl, oxyl, acyloxy, aldehyde radical, acyl group, nitro, amino, hydrocarbon amino, carboxylic The common organic groups such as base, acid halide group, acyl-oxygen acyl group, ester group, amide groups, cyano group.

2. X can be specifically halogen, hydroxyl, oxyl, acyloxy, acyl group, amino, hydrocarbon amino, carboxylic according to claim 1 The groups such as base, ester group, amide groups, cyano group, azido, mineral acid ester group；Y can be specifically hetero atom, methylene etc.；Z can use Generation on 3 of diamantane (obsolete) ring, Z can be specifically halogen, hydroxyl, oxyl, acyloxy, acyl group, nitro, amino, hydrocarbon amino, Carboxyl, ester group, amide groups, cyano group etc..

3. according to claim 1 or claim 2 further, X more specifically halogen, hydroxyl, amino, carboxyl, cyano group, azido, no The groups such as machine perester radical；Y can be specifically hetero atom etc.；Z is may replace on 3 of diamantane (obsolete) ring, Z can be specifically halogen, Hydroxyl, nitro, amino, carboxyl, cyano group etc..

4. according to claim 1,2 or 3 further, the X more specifically group such as halogen, azido；Y can be specifically oxygen, Nitrogen-atoms etc.；Z is may replace on 3 of diamantane (obsolete) ring, and Z can be specifically hydroxyl, nitro, carboxyl etc..

5., according to claim 1,2,3 or 4, specific examples of compounds CMD-05-151013 can be described as, but simultaneously The example is not confined to, wherein Z is substituted on 3 of diamantane (obsolete) ring, it is chlorine (wherein n is equal to 3) that Z is OH, X, and Y is methylene Base.

6. the compound according to claim 1,2,3,4 or 5 has the treatment to diabetes and diseases related aspect and makees With.

7., according to claim 1,2,3,4 or 5, it is to provide a kind of medicine and combinations thereof characterized in that, having The above-claimed cpd of effect amount, and the various dosage forms made such as pharmaceutically acceptable carrier or excipient and its related drugs group Compound.

8., with amantadine as initiation material, react with (S) -1- chloracetyl -2- Cyanopyrolidine, prepare amino list and take The amantadine in generation, is then replaced and continues to be reacted with corresponding acyl chlorides or Acibenzolar to diamantane (obsolete) ring, prepare target chemical combination Thing；And other corresponding alternative preparation methoies that the Professional knowledge that has of those skilled in the art can be completed.

9. according to claim 8, a specific compound CMD-05-151013 prepares example, but is not limited to The example, with amantadine hydrochloride as initiation material, is reagent with nitration mixture and potassium hydroxide, and 3 of amantadine ring are carried out Hydroxylating, obtains diamantane (obsolete) ammonia alcohol；Diamantane (obsolete) ammonia alcohol is reacted with (S) -1- chloracetyl -2- Cyanopyrolidine again, is prepared The mono-substituted diamantane (obsolete) ammonia alcohol of amino, is then proceeded to be reacted with 5-Chlorovaleryl Chloride, prepares target compound CMD-05- 151013；And other corresponding alternative preparation methoies.