CN1170851C - Oral drug fatty diacylamino acid insulin for treating diabetes and its synthesis method - Google Patents

Abstract

The present invention uses the method of chemical synthesis to synthesize oral medicine for treating diabetes, that is, to synthesize a class of fatty diacylamino acid insulin through chemical modification of insulin, and the general formula of the compound is: insulin-OC(CH ₂ )nCONHCHRCOOH, where n= 6-18; R is the side chain of several amino acids. The present invention also relates to a specific synthesis method of the above-mentioned one class of compounds and a specific synthesis method of sebacylglycine insulin and its hypoglycemic effect.

Description

Oral drug fatty diacylamino acid insulin for treating diabetes and its synthesis method

technical field

The invention belongs to chemically synthesized medical medicine, that is, the oral medicine for treating diabetes by synthesizing a class of aliphatic diacylamino acid insulin through chemical modification of insulin.

technical background

Diabetes is a common metabolic disease, and its incidence is on the rise both at home and abroad. With the aging of the world population, diabetes has become a common and frequently-occurring disease. According to incomplete statistics, there are more than 30 million people with diabetes in the country, and the incidence of diabetes has reached 3.2%. Diabetes is the third leading cause of human death. Insulin is the most effective drug for treating diabetes. However, insulin is a kind of protein, which is easily degraded and inactivated by acid and various proteases in the gastrointestinal tract, so it cannot be directly taken orally, but can only be administered by injection, which brings great pain and inconvenience to patients who need life-long medication. Therefore, the research and development of safe, convenient and effective non-injection drug delivery route of insulin has become an important topic concerned by the medical circles all over the world. There are many ways of non-injection administration of insulin currently being studied, such as direct use of insulin in combination with some absorption enhancers through the oral, eye, nose, lung, rectal mucosa or skin absorption, or insulin through liposomes or polymers Oral administration directly after packaging. Among these ways, direct oral administration is the most convenient and easy to be accepted by patients. However, in these studies, no successful reports have been seen except that the insulin oral spray (Chinese invention patent, patent No. 00114318.2) has made important progress. Even the insulin oral spray also has the disadvantages that the bioavailability needs to be improved and the patient's use is not very convenient. In the prior art, it is hoped to develop oral insulin preparations by chemically modifying insulin. Kuraray Co., Ltd.’s patent EP 0 511600 A2 synthesized derivatives of [protein][Z]n, wherein protein is a polypeptide protein including insulin, and [Z] is -CO-W-COOH, where W is a long chain A carbon water group that may contain heteroatoms. The patent WO 95/07931 applied by Novo Nordisk A/S and the two patents EP 0 712861 A2 and EP 0 712862 A2 of Eli Lilly and Company also respectively introduce the synthesis method of chemically modified insulin. The present invention aims at synthesizing a class of chemically modified insulin that can resist intestinal trypsin hydrolysis for the preparation of direct oral insulin preparations, which is characterized in that the chemically modified group of insulin contains amino acid residues and introduces peptide bonds. The chemically modified insulin (insulin-OC(CH ₂ )nCONHCHRCOOH) of the present invention has not been reported yet. This chemical modification group not only protects the site where trypsin attacks insulin, but also introduces a peptide bond that can compete with the site where trypsin attacks insulin, thereby better protecting insulin from trypsin’s hydrolysis and promoting The role of insulin absorption.

Contents of the invention

The key of the present invention is to chemically modify the appropriate part of the insulin to covalently bind a group containing a peptide bond to protect the site where trypsin attacks the insulin, so that the insulin is prevented from being hydrolyzed by trypsin. The general chemical formula of the substance is as follows:

Insulin-OC(CH ₂ )nCONHCHRCOOH

In the formula, n=6～18; R is one of the side chains of lysine, glycine, alanine, valine, leucine, isoleucine and phenylalanine.

The synthetic route is as follows:

or

In the formula, X=F, Cl, Br, I; n=6～18; R is the side chain of lysine, glycine, alanine, valine, leucine, isoleucine, phenylalanine kind of.

The synthetic method of this kind of compound comprises the following steps: a, react with fatty diacyl halide after amino acid is made into the solution of pH7-11 with sodium hydroxide, wherein the molar ratio of glycine and fatty diacyl halide is 1 to 1, reacts in Stir in an ice bath for 0.2-1 hour to generate fatty diacyl halide amino acid: XOC(CH ₂ )nCONHCHRCOOH;

b, benzotriazole (50 g) is dissolved in tert-butyl methyl ether (800-1000 ml) in a solution containing triethylamine (40-60 g), keep the temperature at 15-30°C, add fatty diacyl halide amino acid dropwise, The molar ratio of benzotriazole to fatty diacyl halide amino acid is 1:1, the reaction precipitate is transferred by filtration, the filtrate is evaporated to dryness at 30-80°C under reduced pressure, and the residue is recrystallized with acetone to obtain fatty diacylamino acid Benzotriazoles:

Benzotriazole-OC(CH ₂ )nCONHCHRCOOH

c, Dissolve insulin (0.8mmol) in a solution of N-methyl-pyrrolidone (5-10ml) and triethylamine (0.2-0.6ml), and at the same time add fatty diacylamino acid benzotriazole (1.2-2mmol) Dissolve in N-methyl-pyrrolidone solution (1.5ml), take the fatty diacylamino acid benzotriazole solution and slowly add it to the above insulin solution, wherein the molar ratio of fatty diacylamino acid benzotriazole to insulin is 1 Ratio 1 to 2 to 1, stirring at 0-20°C for 1-3 hours, adding acetone to the obtained reaction mixture until a precipitate forms, centrifuging, removing the supernatant, and freeze-drying the precipitate to constant weight. The crude drug "fatty diacylamino acid insulin" is made into enteric-coated tablets or capsules, which is an oral insulin medicine for treating diabetes.

Get a kind of this type of compound: sebacylglycine insulin (n=8, R is the side chain of glycine) for animal experiments, the results are as follows: the sebacylglycine insulin and insulin were respectively made into phosphate buffer solution of 5IU/ml (pH=7), get 12 mice fasted for 18 hours, divide into three groups randomly (respectively two groups of sebacylglycine insulin and insulin group), inject 0.15ml/only subcutaneously respectively, take blood sample from tail vein to measure For blood sugar, blood samples were taken every 40 minutes until 2 hours, and blood sugar values at different times were measured. As shown in Figure 2, the results proved that the hypoglycemic effect of sebacylglycine insulin was not different from that of insulin. In sebacylglycine insulin and insulin phosphate buffer solution (5IU/ml), add a certain amount of trypsin respectively, after incubating at 37°C for 8 hours, fast for 12 hours, and randomly divide them into three groups (sebacylglycine Insulin two groups and insulin group, each group of 6) mice were injected with sebacylglycine insulin and insulin phosphate buffer solution 0.15ml/only, and measured the blood glucose value of the mice from 0 to 160min after injection at the same time, the results are shown in Figure 3, insulin Because trypsin hydrolysis has no hypoglycemic effect at all, but sebacylglycine insulin still maintains good biological activity, it can be seen that sebacylglycine insulin has a good ability to resist trypsin degradation.

Description of drawings:

Fig. 1 Sebacylglycine insulin RP-HPLC diagram: Fig. 2 Comparison of insulin and sebacylglycine insulin hypoglycemic ability, its abscissa is: time (minute), ordinate: blood glucose value (mmol/L), Insulin, ● modified insulin-1, ▲ modified insulin-2; Fig. 3 The comparison of insulin and sebacylglycine insulin against trypsin degradation, its abscissa is: time (minute), ordinate: blood sugar value (mmol/ l) ■ insulin, ● modified insulin-2, ▲ modified insulin-1.

Detailed ways

Example: Synthesis of sebacylglycine insulin (n=8, R is the side chain of glycine)

Sebacic acid reacts with thionyl chloride to generate sebacoyl chloride (ClOC(CH ₂ ) ₈ COCl), dissolves glycine in sodium hydroxide solution and reacts with sebacoyl chloride (molar ratio: 1:1), in ice bath After stirring for 0.5 hours, sebacoyl chloride glycine (ClOC(CH ₂ ) ₈ CONHCH ₂ COOH) was generated. Dissolve benzotriazole in tert-butyl methyl ether and triethylamine, keep the temperature at 20-30°C, add sebacoyl chloride glycine dropwise, the reaction precipitate is transferred by filtration, and the filtrate is evaporated to dryness at 60°C under reduced pressure, the residue The compound was recrystallized from acetone to obtain benzotriazole sebacoylglycine (benzotriazole-OC(CH ₂ ) ₈ CONHCH ₂ COOH). Dissolve insulin in N-methyl-pyrrolidone and triethylamine solution, simultaneously dissolve sebacylglycine benzotriazole in N-methyl-pyrrolidone solution, take the sebacylglycine benzotriazole solution for a certain amount Add an appropriate amount to the above insulin solution, stir for 2-3 hours in an ice bath, add acetone to the obtained reaction mixture until a precipitate forms, centrifuge, remove the supernatant, freeze-dry the precipitate to constant weight, and obtain chemically modified insulin—— Insulin sebacoylglycine (insulin-OC(CH ₂ ) ₈ CONHCH ₂ COOH), yield 89%. The synthetic route is as follows:

Sebacylglycine insulin adopts the retention time of RP-HPLC to identify, and the result shows that in the RP-HPLC collection of spectra (as shown in Figure 1), the retention time is that the main peak area of 3.96min accounts for 96.6%, and the retention time is the secondary peak of 4.72min It is 3.4%. Under the same conditions, the retention time of simple insulin is 4.71min, and the main peak with a retention time of 3.96min is sebacylglycine insulin.

Claims (3)

1. A class of medicine for the treatment of diabetes, the medicine is the compound fatty diacylamino acid insulin, the general chemical formula of the compound is as follows: insulin-OC(CH ₂ )nCONHCHRCOOH; In the formula, n=6-18; R is one of the side chains of lysine, glycine, alanine, valine, leucine, isoleucine, and phenylalanine. 2. The medicine for treating diabetes as claimed in claim 1, characterized in that the medicine is an oral medicine for treating diabetes. 3. A method for preparing the product of claim 1, the method comprising the following steps: a, amino acid is made into a solution of pH7-11 with sodium hydroxide and reacted with fatty diacyl halide, wherein the mole of amino acid and fatty diacyl halide The ratio is 1 to 1, and the reaction is stirred in an ice bath for 0.2 to 1 hour to generate fatty diacyl halide amino acid: XOC(CH ₂ )nCONHCHRCOOH; b. Dissolve 50 grams of benzotriazole in 800-1000 ml of tert-butyl methyl ether containing 40-60 grams of triethylamine, keep the temperature at 15-30°C, add fatty diacyl halide amino acid dropwise, in which benzotriazole and The molar ratio of the fatty diacyl halide amino acid is 1:1, the reaction precipitate is transferred by filtration, the filtrate is evaporated to dryness at 30-80°C under reduced pressure, and the residue is recrystallized with acetone to obtain the fatty diacylamino acid benzotriazole: benzene And triazole-OC (CH ₂ ) nCONHCHRCOOH; c. Dissolve 0.8mmol of insulin in 5-10ml of N-methyl-pyrrolidone and 0.2-0.6ml of triethylamine, and dissolve 1.2-2mmol of fatty diacylamino acid benzotriazole in N-methyl-pyrrolidone In 1.5ml of the solution, take the fatty diacylamino acid benzotriazole solution and slowly add it to the above insulin solution, wherein the molar ratio of the fatty diacylamino acid benzotriazole to insulin is 1:1 to 2:1. Stir at 20°C for 1-3 hours, add acetone to the obtained reaction mixture until a precipitate forms, centrifuge, remove the supernatant, and freeze-dry the precipitate to constant weight.

Images