Pharmaceutical composition for treating diabetic osteoporosis and preparation method thereof

Abstract

The invention belongs to the technical field of specific therapeutic activity of compounds, and particularly relates to a pharmaceutical composition for treating diabetic osteoporosis and a preparation method thereof. The pharmaceutical composition for treating the diabetic osteoporosis is prepared from a GLP-1 / GIP dual-receptor stimulant DA3-CH, a synergist, a pH (Potential of Hydrogen) regulator, an isoosmotic adjusting agent and a preservative. The GLP-1 / GIP dual-receptor stimulant DA3-CH and the synergist both have the effects of reducing blood sugar and improving the bone mineral density reduction symptom caused by diabetes, and due to the synergistic effect of the GLP-1 / GIP dual-receptor stimulant DA3-CH and the synergist, the diabetes can be treated while the bone mineral density reduction symptom caused by the diabetes is improved, and the osteoporosis symptom delayed by the diabetes is relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of specific therapeutic activity of compounds, and in particular relates to a pharmaceutical composition for treating diabetic osteoporosis and a preparation method thereof. Background Art

[0002] Diabetic osteoporosis (DOP) is a chronic complication of diabetes in the skeletal system. It is specifically defined as a metabolic bone disease that occurs on the basis of diabetes, with decreased bone mass per unit volume, namely bone density, increased bone brittleness, and increased risk of fractures. Epidemiological surveys and studies have shown that nearly one-third of patients with type 2 diabetes in my country suffer from osteoporosis. Therefore, as one of the chronic complications of diabetes, DOP has received more and more widespread attention, and early prevention and treatment of DOP has become an important part of the management of chronic diseases of diabetes.

[0003] Liraglutide has a GLP-1 receptor agonist effect, and is similar to GLP-1 in terms of molecular structure, biological activity, target and immunogenicity. Liraglutide belongs to a new generation of incretin hypoglycemic drugs, has a long duration of action, and may have a positive effect on a variety of cardiovascular risk factors. Patent CN106456647A- discloses the application of liraglutide in lowering blood sugar and in alleviating osteoporosis. The novel GLP-1 / GIP dual receptor agonist (DA3-CH) has a dual receptor agonist effect of GLP-1 and GIP. The article The novel GLP-1-GIP dual agonist DA3-CH improves rat type2diabetes through activatingAMPK-ACC signalingpathway discloses that the GLP-1 / GIP dual receptor agonist (DA3-CH) can also lower blood sugar. The prior art has not studied the application of GLP-1 / GIP dual receptor agonist (DA3-CH) in the treatment of diabetic osteoporosis, which limits the application of GLP-1 / GIP dual receptor agonist (DA3-CH) in the treatment of diabetes.

[0004] Therefore, there is an urgent need to develop a pharmaceutical composition that can treat diabetes and delay the symptoms of osteoporosis. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a pharmaceutical composition for treating diabetic osteoporosis, which can treat diabetes and delay the symptoms of osteoporosis at the same time.

[0006] One of the purposes of the present invention is achieved by the following technical solution:

[0007] A pharmaceutical composition for treating diabetic osteoporosis, comprising a GLP-1 / GIP dual receptor agonist DA3-CH, a synergist, a pH regulator, an isotonicity regulator and a preservative; the synergist has the structure of:

[0008]

[0009] Furthermore, the preparation process of the synergist comprises the following steps:

[0010] (1) 3-((3-carboxy-2-hydroxy-4-methoxy-6-methylbenzoyl)oxy)-5-formyl-4,6-dihydroxy-2-methylbenzoic acid, an inorganic base, and benzyl bromide are added to N,N-dimethylformamide, reacted at 50-60° C. for 90-120 min, and then treated to obtain a crude product 1; the crude product 1 and lithium hydroxide are added to methanol, reacted at 70-80° C. for 3-5 h, and then treated to obtain an intermediate 1;

[0011] (2) adding diphenylphosphoryl azide to a mixed system of intermediate 1, triethylamine, toluene and tert-butyl alcohol, reacting at 80-90° C. for 3-5 hours and then treating to obtain crude product 2; reacting crude product 2 and diethylenetriamine at 120-140° C. for 10-15 hours and then treating to obtain intermediate 2;

[0012] (3) adding ethyl 2-bromoacetate to a mixture of intermediate 2, potassium carbonate, N,N-dimethylformamide and water, reacting at 60-80° C. for 2-4 hours, and then treating to obtain intermediate 3;

[0013] (4) adding tert-butyl alcohol to a mixture of chlorosulfonyl isocyanate and dichloromethane at 5 to 8°C, reacting at -2 to 0°C for 30 to 40 minutes, adding a mixed solution of intermediate 3, triethylamine and dichloromethane to the mixture at 5 to 8°C, reacting at -2 to 0°C for 30 to 40 minutes, warming to room temperature for reaction for 1 hour, and treating to obtain a crude product 4; adding the crude product 4 and trifluoroacetic acid to dichloromethane, reacting at room temperature for 30 to 50 minutes, and treating to obtain an intermediate 4;

[0014] (5) Sodium methoxide is added to a mixture of intermediate 4 and tetrahydrofuran, and the mixture is reacted at room temperature for 20 to 40 minutes, followed by treatment to obtain a crude product 5; the crude product 5, pentamethylbenzene, and trichloroborane are reacted at -78 to -75°C for 20 to 30 minutes, followed by treatment to obtain a synergist.

[0015] The reaction principle of the invention for preparing the synergist is as follows: 3-((3-carboxyl-2-hydroxy-4-methoxy-6-methylbenzoyl)oxy)-5-formyl-4,6-dihydroxy-2-methylbenzoic acid and benzyl bromide undergo nucleophilic substitution to achieve the purpose of benzyl protecting phenolic hydroxyl groups, and then hydrolyze under alkaline conditions to obtain intermediate 1; intermediate 1 and diphenylphosphoryl azide undergo Curtius rearrangement reaction to achieve the purpose of introducing amino groups on intermediate 1, and then hydrolyze with diethylenetriamine to obtain intermediate 2; intermediate 2 and ethyl bromoacetate undergo nucleophilic substitution reaction to obtain intermediate 3; intermediate 3 and chlorosulfonyl isocyanate undergo nucleophilic substitution and undergo acidic hydrolysis to obtain intermediate 4; intermediate 4 undergoes amine ester exchange between the amine group on the sulfonamide group and the carbonyl group on the ester group under alkaline conditions to achieve the purpose of preparing a five-membered ring, and then the benzyl group is removed by the joint action of Lewis acid trichloroborane and pentamethylbenzene to obtain the synergist.

[0016] Furthermore, in step (1), the molar ratio of 3-((3-carboxy-2-hydroxy-4-methoxy-6-methylbenzoyl)oxy)-5-formyl-4,6-dihydroxy-2-methylbenzoic acid, inorganic base, benzyl bromide and lithium hydroxide is 1:(5.5-6.5):(3-4):(3-3.5); and the inorganic base is one of potassium carbonate and cesium carbonate.

[0017] Furthermore, in step (2), the molar ratio of the intermediate 1, diphenylphosphoryl azide, triethylamine and diethylenetriamine is 1:(2.2-2.5):(2.2-2.5):(0.08-0.1).

[0018] Furthermore, in step (3), the molar ratio of the intermediate 2, potassium carbonate and ethyl 2-bromoacetate is 1:(5-6):(2.2-2.5).

[0019] Furthermore, in step (4), the molar ratio of the intermediate 3, triethylamine, tert-butyl alcohol, chlorosulfonyl isocyanate and trifluoroacetic acid is 1:(4-5):(2.4-3):(2.4-3):(40-50).

[0020] Furthermore, in step (5), the molar ratio of the intermediate 4, sodium methoxide, trichloroborane and pentamethylbenzene is 1:(3-3.5):(6-7.5):(6-7.5).

[0021] Furthermore, the pH regulator is one of sodium carbonate and sodium hydroxide; the isotonicity regulator is one of mannitol and propylene glycol; and the preservative is chlorobutanol.

[0022] Furthermore, the pharmaceutical composition also includes water for injection, a pH regulator, an isotonic regulator and a preservative. In terms of weight, the GLP-1 / GIP dual receptor agonist DA3-CH is 0.6 to 1.08 parts, the synergist is 0.75 to 1.26 parts, the preservative is 1.2 to 1.5 parts, the isotonic regulator is 1.5 to 3 parts, and the water for injection is 300 parts. The pH range of the pharmaceutical composition is 8.0 to 8.4.

[0023] Furthermore, the amino acid sequence of the GLP-1 / GIP dual receptor agonist DA3-CH is YXEGTFTSDYSIYLDKQAAXEFVNWLLAGGPSSGAPPPSK-NH2, where X=aminoisobutyric acid.

[0024] A second object of the present invention is to provide a method for preparing a pharmaceutical composition for treating diabetic osteoporosis.

[0025] The second object of the present invention is achieved by adopting the following technical solution:

[0026] The preparation method of the pharmaceutical composition for treating diabetic osteoporosis comprises the following steps: S1: weighing a preservative and an isotonicity adjusting agent, adding an appropriate amount of water for injection to completely dissolve them, and obtaining a mixed solution; S2: taking an appropriate amount of water for injection to completely dissolve the GLP-1 / GIP dual receptor agonist DA3-CH enhancer, adding the mixture to the mixed solution of step S1, adjusting the pH value to the target value with a pH adjusting agent, filtering, and sterilizing to obtain the mixture.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The GLP-1 / GIP dual receptor agonist DA3-CH and the synergist of the present invention both have the effects of lowering blood sugar and improving the symptoms of decreased bone density caused by diabetes. The synergistic effect of the two can improve the symptoms of decreased bone density caused by diabetes while treating diabetes, and alleviate the symptoms of diabetes and delay osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a flow chart of the preparation of the synergist of the present invention;

[0030] Figure 2 It is a schematic diagram of the cytotoxicity test of the synergist of the present invention;

[0031] Figure 3 This is a schematic diagram showing the effect of the pharmaceutical composition for treating diabetic osteoporosis of the present invention on the bone resorption function of osteoclasts;

[0032] Figure 4 This is a schematic diagram of the effect of the pharmaceutical composition for treating diabetic osteoporosis of the present invention on blood sugar;

[0033] Figure 5 This is a schematic diagram showing the effect of the pharmaceutical composition for treating diabetic osteoporosis of the present invention on bone density. DETAILED DESCRIPTION

[0034] Below, in conjunction with the accompanying drawings and specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0035] Example 1

[0036] A pharmaceutical composition for treating diabetic osteoporosis comprises 0.9 parts of a GLP-1 / GIP dual receptor agonist DA3-CH, 0.96 parts of a synergist, 1.5 parts of mannitol, 1.2 parts of chlorobutanol and 300 parts of water for injection, wherein the pH value of the pharmaceutical composition is 8.0.

[0037] The preparation of the above-mentioned synergist is as follows Figure 1 As shown, the specific process includes the following:

[0038] (1) 3-((3-carboxyl-2-hydroxy-4-methoxy-6-methylbenzoyl)oxy)-5-formyl-4,6-dihydroxy-2-methylbenzoic acid (CAS: 484-55-9, 0.1 mol) and cesium carbonate (0.6 mol) were added to 200 mL of N,N-dimethylformamide in sequence, stirred evenly at room temperature, and then benzyl bromide (BnBr, 0.35 mol) was added. The temperature was raised to 55° C. and the reaction was allowed to react for 105 min. After cooling to room temperature and quenching the reaction with water, the reaction solution was filtered to collect the filter cake. The filter cake was slurried with water (1 g of filter cake: 5 mL of water) and filtered, and then filtrated with a tert-butyl methyl ether / n-heptane mixed solvent (V 叔丁基甲基醚 :V 正庚烷 =1:2, 1 g filter cake: 4 mL mixed solvent) for slurrying, filtering and drying to obtain crude product 1;

[0039] The crude product 1 was added to 180 mL of methanol, and then 110 mL of 0.07 g / mL lithium hydroxide (LiOH) aqueous solution was added thereto, stirred evenly, and then heated to 75°C for reaction for 4 h; the mixed system was cooled to room temperature and the pH was adjusted to 3 with 2M HCl, the mixed system was filtered to collect the filter cake, the filter cake was slurried with water (1 g filter cake: 5 mL water), filtered, and then slurried with tert-butyl methyl ether (1 g filter cake: 4 mL tert-butyl methyl ether), filtered, and dried to obtain intermediate 1.

[0040] (2) The intermediate 1 (0.1 mol) was placed in a mixed solution of 250 mL toluene and 250 mL tert-butyl alcohol (t-BuOH), and then triethylamine (0.23 mol) was added. Under nitrogen protection, the temperature was raised to 85°C, and diphenylphosphoryl azide (DPPA, 0.23 mol) was added dropwise to the reaction system. After the addition was completed, the reaction was carried out at 85°C for 4 hours. The reaction solution was cooled to room temperature, quenched with water, and separated. The aqueous phase was extracted with ethyl acetate, and the ethyl acetate phases were combined. The ethyl acetate phase was washed with water and then dried over anhydrous sodium sulfate. The ethyl acetate phase was filtered and concentrated to obtain a crude product 2.

[0041] Add diethylenetriamine (NH(C2H4NH 2)2 , 0.09mol), under nitrogen protection, the reaction solution was heated to 130°C for 13h; the mixed system was cooled to room temperature, and then water was slowly added to quench the reaction. After the quenched reaction solution was cooled to room temperature, the reaction system was dispersed with dichloromethane and then separated, the aqueous phase was extracted with dichloromethane, and the dichloromethane phase was washed with saturated brine. The dichloromethane phase was dried and concentrated, and then slurried with isopropanol (1g solid: 3.5mL isopropanol), filtered and dried to obtain intermediate 2. MS results of intermediate 2: [M+H]+m / z: 630.25, ESI-MS (m / z): 630.31.

[0042] (3) The intermediate 2 (0.1 mol) and potassium carbonate (0.55 mol) were added to 200 mL N, N-dimethylformamide and 100 mL deionized water in sequence and stirred evenly. Then, ethyl 2-bromoacetate (BrCH2COOC2H5, 0.23 mol) was added thereto, stirred evenly at room temperature, and then heated to 70°C for reaction for 3 h. The reaction solution was cooled to room temperature, and water and ethyl acetate were added thereto until the organic phase and the aqueous phase were clearly separated. After separation, the aqueous layer was extracted with ethyl acetate, and the organic phases were combined and washed with saturated ammonium chloride solution. After separation, the combined organic phases were dried over sodium sulfate, filtered, and concentrated to obtain the crude product 3. The crude product 3 was slurried with n-heptane (1 g crude product 3: 10 mL n-heptane), filtered, and dried to obtain the intermediate 3.

[0043] (4) Add chlorosulfonyl isocyanate (NCOSO2Cl, 0.13 mol) to 200 mL of dichloromethane, cool the reaction solution to 0°C, then slowly add tert-butyl alcohol (0.13 mol), keep the temperature of the reaction solution at 7°C during the addition process, and react at 0°C for 30 min after the addition is complete; add intermediate 3 (0.05 mol) and triethylamine (0.23 mol) to 100 mL of dichloromethane to prepare a solution, add it dropwise to the mixed system at 0°C, keep the temperature of the reaction solution at 7°C during the addition process, and react at 0°C for 35 min after the addition is complete, then heat to room temperature and react for 1.5 h; quench the reaction solution with water, separate the layers, extract the aqueous phase with dichloromethane, combine the organic layers, wash with 1 M sodium bisulfate solution, wash the aqueous phase with dichloromethane, combine the organic phases, dry with sodium sulfate, filter, and concentrate to obtain crude product 4;

[0044] The crude product 4 was added to 150 mL of dichloromethane and then trifluoroacetic acid (TFA, 1.3 mol) was added, and the mixture was reacted at room temperature for 35 min; a saturated sodium bicarbonate aqueous solution was slowly added to the mixed system to quench the reaction, the reaction solution was separated and the aqueous phase was extracted with dichloromethane, the combined dichloromethane phase was concentrated, the concentrated solution was evenly dispersed with 30 mL of dichloromethane, and then 50 mL of n-heptane was added for slurrying, and the intermediate 4 was obtained after filtration and drying. The MS results of the intermediate 4: [M+H]+m / z: 960.27, ESI-MS (m / z): 960.27.

[0045] (5) At room temperature, intermediate 4 (0.02 mmol) was added to 200 mL of tetrahydrofuran, and then sodium methoxide (CH3ONa, 0.06 mol) was added and reacted at room temperature for 30 min; the pH of the reaction solution was adjusted to 7 with 1 M hydrochloric acid, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined and then quenched with a mixed solution of 1 M hydrochloric acid and saturated brine (V 盐酸 :V 饱和食盐水 =4:1), then dried with sodium sulfate, concentrated and then washed with a mixed solvent of dichloromethane and n-heptane (V 二氯甲烷 :V 正庚烷 =2:1, 1 g solid: 3 mL mixed solvent) slurry, filter and dry to obtain crude product 5;

[0046] The crude product 5 and pentamethylbenzene (Pentamethylbenzene, 0.13 mol) were dispersed in 700 mL of dichloromethane. After the reaction system was cooled to -78°C under a nitrogen atmosphere, a 1 M dichloroborane (BCl3) dichloromethane solution (0.13 mol) was added dropwise to the reaction system and reacted at -78°C for 25 min. The reaction was quenched with dichloromethane:ethanol = 9:1 (200 mL) at -78°C, and then slowly heated to room temperature. The reaction solution was concentrated and purified by column (eluent, n-heptane:ethyl acetate = 1:1) to obtain the enhancer.

[0047] The MS results of the above-mentioned enhancer: [M+H]+m / z: 599.05, ESI-MS (m / z): 599.10; The NMR results of the above-mentioned enhancer: 1 H NMR (C 21 H 20 N4O 13 S2, 400MHz, DMSO-d6): δ19.51 (s, 1H), 15.78 (s, 2H), 10.36 (s, 1H), 7.02 (s, 2H), 6.48 (s, 1H), 3.96-3.85 (d, 7H), 2.49 (s, 3H), 2.25 (s, 3H).

[0048] The preparation process of the above-mentioned pharmaceutical composition comprises the following steps:

[0049] S1: According to the above weight portions, on a sterile workbench, weigh chlorobutanol and mannitol, add 150 parts of water for injection, stir to dissolve, and then add to a sterile dispensing tank; S2: Weigh the GLP-1 / GIP dual receptor agonist DA3-CH and the synergist, dissolve them in 50 parts of water for injection, then add to the above sterile dispensing tank, and stir until the solution is clear; add 0.1M sodium hydroxide to adjust the pH value of the solution to 8.0, supplement the remaining injection water in the formula to make up the volume, and filter with a 0.22μm filter membrane for sterilization.

[0050] Example 2

[0051] A pharmaceutical composition for treating diabetic osteoporosis comprises 0.6 parts of a GLP-1 / GIP dual receptor agonist DA3-CH, 0.75 parts of a synergist, 2.5 parts of propylene glycol, 1.35 parts of chlorobutanol and 300 parts of water for injection, wherein the pH value of the pharmaceutical composition is 8.2.

[0052] The preparation of the above-mentioned synergist comprises the following process:

[0053] (1) 3-((3-carboxyl-2-hydroxy-4-methoxy-6-methylbenzoyl)oxy)-5-formyl-4,6-dihydroxy-2-methylbenzoic acid (0.1 mol) and cesium carbonate (0.65 mol) were added to 200 mL of N,N-dimethylformamide in sequence, stirred evenly at room temperature, and then benzyl bromide (0.4 mol) was added. The temperature was raised to 50° C. and reacted for 2 h. After cooling to room temperature and quenching the reaction with water, the reaction liquid was filtered to collect the filter cake. The filter cake was slurried with water (1 g of filter cake: 5 mL of water) and filtered, and then the mixture was washed with a mixed solvent (V 叔丁基甲基醚 :V 正庚烷 =1:2, 1 g filter cake: 4 mL mixed solvent) for slurrying, filtering and drying to obtain crude product 1;

[0054] The crude product 1 was added to 180 mL of methanol, and then 120 mL of 0.07 g / mL of lithium hydroxide aqueous solution was added thereto. After stirring evenly, the temperature was raised to 70°C and the reaction was carried out for 5 h. After the mixed system was cooled to room temperature, the pH was adjusted to 3 with 2M HCl, the mixed system was filtered to collect the filter cake, the filter cake was slurried with water (1 g of filter cake: 5 mL of water), filtered, and then slurried with tert-butyl methyl ether (1 g of filter cake: 4 mL of tert-butyl methyl ether), filtered, and dried to obtain intermediate 1.

[0055] (2) The intermediate 1 (0.1 mol) was placed in a mixed solution of 250 mL toluene and 250 mL tert-butanol, and triethylamine (0.25 mol) was added. Under nitrogen protection, the temperature was raised to 80°C, and diphenylphosphoryl azide (DPPA, 0.25 mol) was added dropwise to the reaction system. After the addition was completed, the reaction was carried out at 80°C for 5 h. The reaction solution was cooled to room temperature, quenched with water, and separated. The aqueous phase was extracted with ethyl acetate, and the ethyl acetate phases were combined. The ethyl acetate phase was washed with water and then dried over anhydrous sodium sulfate. The ethyl acetate phase was filtered and concentrated to obtain a crude product 2.

[0056] Diethylenetriamine (0.1 mol) was added to the crude product 2, and the reaction solution was heated to 120°C for 15 h under nitrogen protection; the mixed system was cooled to room temperature, and then water was slowly added to quench the reaction, and the quenched reaction solution was cooled to room temperature, the reaction system was dispersed with dichloromethane and then separated, the aqueous phase was extracted with dichloromethane, and the dichloromethane phase was washed with saturated brine, the dichloromethane phase was dried and concentrated, and then slurried with isopropanol (1 g solid: 3.5 mL isopropanol), filtered and dried to obtain intermediate 2. The MS results of the intermediate 2 were consistent with those shown in Example 1.

[0057] (3) The intermediate 2 (0.1 mol) and potassium carbonate (0.6 mol) were added to 200 mL N, N-dimethylformamide and 100 mL deionized water in sequence and stirred evenly. Then, ethyl 2-bromoacetate (0.23 mol) was added thereto, stirred evenly at room temperature, and then heated to 70°C for reaction for 3 h. The reaction solution was cooled to room temperature, and water and ethyl acetate were added thereto until the organic phase and the aqueous phase were clearly separated. After separation, the aqueous layer was extracted with ethyl acetate, and the organic phases were combined and washed with saturated ammonium chloride solution. After separation, the combined organic phases were dried over sodium sulfate, filtered, and concentrated to obtain the crude product 3. The crude product 3 was slurried with n-heptane (1 g crude product 3: 10 mL n-heptane), filtered, and dried to obtain the intermediate 3.

[0058] (4) Add chlorosulfonyl isocyanate (0.15 mol) to 200 mL of dichloromethane, cool the reaction solution to 0°C, then slowly add tert-butyl alcohol (0.15 mol), keep the temperature of the reaction solution at 5°C during the addition process, and react at 0°C for 30 min after the addition is complete. Add intermediate 3 (0.05 mol) and triethylamine (0.25 mol) to 100 mL of dichloromethane to prepare a solution, and add it dropwise to the mixed system at 0°C, keep the temperature of the reaction solution at 5°C during the addition process, and react at 0°C for 30 min after the addition is complete. Then warm it to room temperature and react for 2 h. After quenching the reaction solution with water, separate the layers, extract the aqueous phase with dichloromethane, wash the combined organic layers with 1M sodium bisulfate, wash the aqueous phase with dichloromethane, dry the combined organic phases with sodium sulfate, filter, and concentrate to obtain a crude product 4.

[0059] The crude product 4 was added to 150 mL of dichloromethane and then trifluoroacetic acid (1.5 mol) was added, and the mixture was reacted at room temperature for 40 min; a saturated sodium bicarbonate aqueous solution was slowly added to the mixed system to quench the reaction, the reaction solution was separated and the aqueous phase was extracted with dichloromethane, the combined dichloromethane phase was concentrated, the concentrated solution was evenly dispersed with 30 mL of dichloromethane, 50 mL of n-heptane was added for slurrying, and the intermediate 4 was obtained after filtration and drying. The MS results of the intermediate 4 were consistent with those shown in Example 1.

[0060] (5) At room temperature, intermediate 4 (0.02 mol) was added to 200 mL of tetrahydrofuran, and then sodium methoxide (0.07 mol) was added and reacted at room temperature for 50 min. The reaction solution was adjusted to pH 7 with 1 M hydrochloric acid, and then separated. The aqueous phase was extracted with ethyl acetate. The organic phases were combined and then quenched with a mixed solution of 1 M hydrochloric acid and saturated brine (V 盐酸 :V 饱和食盐水 =4:1), then dried with sodium sulfate, concentrated and then washed with a mixed solvent of dichloromethane and n-heptane (V 二氯甲烷 :V 正庚烷 =2:1, 1 g solid: 3 mL mixed solvent) slurry, filter and dry to obtain crude product 5;

[0061] The crude product 5 and pentamethylbenzene (0.15 mol) were dispersed in 700 mL of dichloromethane, and the reaction system was cooled to -78°C under a nitrogen atmosphere. A 1 M dichloroborane solution (0.15 mol) was added dropwise to the reaction system and reacted at -78°C for 20 min. The reaction was quenched with dichloromethane:ethanol = 9:1 (200 mL) at -78°C, and then slowly heated to room temperature. The reaction solution was concentrated and purified by column (eluent, n-heptane:ethyl acetate = 1:1) to obtain the synergist. The MS and NMR results of the synergist were consistent with those shown in Example 1.

[0062] The preparation process of the pharmaceutical composition comprises the following steps:

[0063] S1: According to the above weight portions, weigh chlorobutanol and propylene glycol on a sterile workbench, add 150 parts of water for injection, stir to dissolve, and add to a sterile compounding tank; S2: Weigh the GLP-1 / GIP dual receptor agonist DA3-CH and the synergist, dissolve until 50 parts of water for injection are added, add to the above-mentioned liquid preparation tank, and stir until the solution is clear; add 10wt% sodium carbonate to adjust the pH value of the solution to 8.2, supplement the remaining injection water in the formula to make up the volume, and filter with a 0.22μm filter membrane for sterilization.

[0064] Example 3

[0065] A pharmaceutical composition for treating diabetic osteoporosis comprises 1.08 parts of a GLP-1 / GIP dual receptor agonist DA3-CH, 1.26 parts of a synergist, 3 parts of propylene glycol, 1.5 parts of chlorobutanol and 300 parts of water for injection, wherein the pH value of the pharmaceutical composition is 8.2.

[0066] The preparation of the above-mentioned synergist includes the following process:

[0067] (1) 3-((3-carboxyl-2-hydroxy-4-methoxy-6-methylbenzoyl)oxy)-5-formyl-4,6-dihydroxy-2-methylbenzoic acid (0.1 mol) and cesium carbonate (0.55 mol) were added to 200 mL of N,N-dimethylformamide in sequence, stirred evenly at room temperature, and then benzyl bromide (0.3 mol) was added. The temperature was raised to 50° C. and reacted for 2 h. After cooling to room temperature and quenching the reaction with water, the reaction liquid was filtered to collect the filter cake. The filter cake was slurried with water (1 g of filter cake: 5 mL of water) and filtered, and then the mixture was washed with a mixed solvent (V 叔丁基甲基醚 :V 正庚烷 =1:2, 1 g filter cake: 4 mL mixed solvent) for slurrying, filtering and drying to obtain crude product 1;

[0068] The crude product 1 was added to 180 mL of methanol, and then 120 mL of 0.06 g / mL of lithium hydroxide aqueous solution was added thereto. After stirring evenly, the temperature was raised to 80° C. and the reaction was carried out for 3 h. After the mixed system was cooled to room temperature, the pH was adjusted to 4 with 2M HCl, the mixed system was filtered to collect the filter cake, the filter cake was slurried with water (1 g of filter cake: 5 mL of water), filtered, and then slurried with tert-butyl methyl ether (1 g of filter cake: 4 mL of tert-butyl methyl ether), filtered, and dried to obtain intermediate 1.

[0069] (2) The intermediate 1 (0.1 mol) was placed in a mixed solution of 250 mL toluene and 250 mL tert-butanol, and then triethylamine (0.22 mol) was added. Under nitrogen protection, the temperature was raised to 80°C, and diphenylphosphoryl azide (DPPA, 0.22 mol) was added dropwise to the reaction system. After the addition was completed, the reaction was carried out at 80°C for 5 h. The reaction solution was cooled to room temperature, quenched with water, and separated. The aqueous phase was extracted with ethyl acetate, and the ethyl acetate phases were combined. The ethyl acetate phase was washed with water and then dried over anhydrous sodium sulfate. The ethyl acetate phase was filtered and concentrated to obtain a crude product 2.

[0070] Diethylenetriamine (0.08 mol) was added to the crude product 2, and the reaction solution was heated to 120°C for 15 h under nitrogen protection; the mixed system was cooled to room temperature, and then water was slowly added to quench the reaction, and the quenched reaction solution was cooled to room temperature, the reaction system was dispersed with dichloromethane and then separated, the aqueous phase was extracted with dichloromethane, and the dichloromethane phase was washed with saturated brine, the dichloromethane phase was dried and concentrated, and then slurried with isopropanol (1 g solid: 3.5 mL isopropanol), filtered and dried to obtain intermediate 2. The MS results of the intermediate 2 were consistent with those shown in Example 1.

[0071] (3) The intermediate 2 (0.1 mol) and potassium carbonate (0.5 mol) were added to 200 mL N, N-dimethylformamide and 100 mL deionized water in sequence and stirred evenly. Then, ethyl 2-bromoacetate (0.22 mol) was added thereto. After stirring evenly at room temperature, the temperature was raised to 80°C for reaction for 2 h. The reaction solution was cooled to room temperature, and water and ethyl acetate were added thereto until the organic phase and the aqueous phase were clearly separated. After separation, the aqueous layer was extracted with ethyl acetate, and the organic phases were combined and washed with saturated ammonium chloride solution. After separation, the combined organic phases were dried over sodium sulfate, filtered, and concentrated to obtain the crude product 3. The crude product 3 was slurried with n-heptane (1 g crude product 3: 10 mL n-heptane), filtered, and dried to obtain the intermediate 3.

[0072] (4) Add chlorosulfonyl isocyanate (0.12 mol) to 200 mL of dichloromethane, cool the reaction solution to 0°C, then slowly add tert-butyl alcohol (0.12 mol), keep the temperature of the reaction solution at 5°C during the addition process, and react at 0°C for 30 min after the addition is complete. Add intermediate 3 (0.05 mol) and triethylamine (0.2 mol) to 100 mL of dichloromethane to prepare a solution, and add it dropwise to the mixed system at 0°C, keep the temperature of the reaction solution at 5°C during the addition process, and react at 0°C for 30 min after the addition is complete. Then warm it to room temperature and react for 1 h. Quench the reaction solution with water, separate the layers, extract the aqueous phase with dichloromethane, wash the combined organic layers with 1 M sodium bisulfate, wash the aqueous phase with dichloromethane, dry the combined organic phases with sodium sulfate, filter, and concentrate to obtain a crude product 4.

[0073] The crude product 4 was added to 150 mL of dichloromethane and then trifluoroacetic acid (1.2 mol) was added, and the mixture was reacted at room temperature for 40 min; a saturated sodium bicarbonate aqueous solution was slowly added to the mixed system to quench the reaction, the reaction solution was separated and the aqueous phase was extracted with dichloromethane, the combined dichloromethane phase was concentrated, the concentrated solution was evenly dispersed with 30 mL of dichloromethane, 50 mL of n-heptane was added for slurrying, and the intermediate 4 was obtained after filtration and drying. The MS results of the intermediate 4 were consistent with those shown in Example 1.

[0074] (5) At room temperature, intermediate 4 (0.02 mol) was added to 200 mL of tetrahydrofuran, and sodium methoxide (0.06 mol) was added, and the mixture was reacted at room temperature for 20 min. The pH of the reaction solution was adjusted to 7 with 1 M hydrochloric acid, and then the layers were separated. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined and then quenched with a mixed solution of 1 M hydrochloric acid and saturated brine (V 盐酸 :V 饱和食盐水 =4:1), then dried with sodium sulfate, concentrated and then washed with a mixed solvent of dichloromethane and n-heptane (V 二氯甲烷 :V 正庚烷 =2:1, 1 g solid: 3 mL mixed solvent) slurry, filter and dry to obtain crude product 5;

[0075] The crude product 5 and pentamethylbenzene (0.12 mol) were dispersed in 700 mL of dichloromethane. After the reaction system was cooled to -75°C under a nitrogen atmosphere, a 1 M dichloroborane solution (0.12 mol) was added dropwise to the reaction system and reacted at -75°C for 30 min. The reaction was quenched at -75°C with dichloromethane: ethanol = 9:1 (200 mL), and then slowly heated to room temperature. After the reaction solution was concentrated, it was purified by column (eluent, n-heptane: ethyl acetate = 1:1) to obtain the synergist. The MS and NMR results of the synergist were consistent with those shown in Example 1. The MS and NMR results of the synergist were consistent with those shown in Example 1.

[0076] The preparation process of the pharmaceutical composition comprises the following steps:

[0077] S1: According to the above weight portions, weigh chlorobutanol and propylene glycol on a sterile workbench, add 150 parts of water for injection, stir to dissolve, and add to a sterile compounding tank; S2: Weigh the GLP-1 / GIP dual receptor agonist DA3-CH and the synergist, dissolve until 50 parts of water for injection are added to the above-mentioned liquid preparation tank, and stir until the solution is clear; add 10wt% sodium carbonate to adjust the pH value of the solution to 8.4, supplement the remaining injection water in the formula to make up the volume, and filter with a 0.22μm filter membrane for sterilization.

[0078] Comparative Example 1

[0079] A pharmaceutical composition for treating diabetic osteoporosis, which differs from Example 1 in that the GLP-1 / GIP dual receptor agonist DA3-CH is omitted.

[0080] Comparative Example 2

[0081] A pharmaceutical composition for treating diabetic osteoporosis, which differs from Example 1 in that a synergist is omitted.

[0082] Test Example 1

[0083] Toxicity of the potentiators of the present invention to bone marrow-derived macrophages (BMMs)

[0084] (1) Cell isolation experiment: 8-week-old female C57BL / 6J mice were selected and killed by cervical dislocation. The intact femur and tibia of the mice were aseptically separated, and the proximal femur bone was cut off. Sterile PBS was aspirated with a syringe, and a needle was inserted from the distal bone cavity to flush out bone marrow cells to prepare a bone marrow single cell suspension. The bone marrow single cell suspension was centrifuged to collect and wash the cell pellet, which was resuspended in α-MEM culture medium containing 10% fetal bovine serum and 20 ng / mL M-CSF and added to a 10 cm culture dish. It was cultured in an incubator at 37°C and 5% CO2 for 24 h. The non-adherent cells were collected, centrifuged, and resuspended in α-MEM culture medium containing 10% fetal bovine serum, 1% double antibody and 20 ng / mL M-CSF. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for 3 days, and the suspended cells were removed to obtain bone marrow-derived macrophages (BMMs).

[0085] (2) Cytotoxicity test

[0086] 5×10 4 BMMs were inoculated into 96-well plates at a cell density of 100 cells / well, and non-adherent cells were washed off and cultured for 24 hours. According to the experimental requirements, BMMs cells were divided into a RANKL control group and a test compound group. Different concentrations (20 μM, 10 μM, 5 μM, 2.5 μM) of the test compound (i.e., the enhancer prepared in Example 1) were added to each group of cells in the test compound group. After incubation for 30 minutes, RANKL (20 ng / mL) was added for stimulation. After incubation for 24 hours at 37°C and 5% CO2, MTT solution was added and cultured for 4 hours. The formed MTT-formazan crystals were dissolved with 100 μL 0.04M hydrochloric acid-isopropanol and the absorbance was measured at 570 nm. The results are shown in Figure 2. Figure 2 shown.

[0087] Figure 2The toxicity of the potentiator to BMMs at different concentrations (20μM, 10μM, 5μM, 2.5μM) is shown. The ordinate represents the cell activity, where the cell activity % = the absorbance value of each test compound group / the absorbance of the RANKL control group × 100%; the abscissa represents the concentration of the potentiator to be tested. Figure 2 It can be seen that the enhancer has no significant cytotoxicity to BMMs at a concentration of 10 μM.

[0088] Test Example 2

[0089] Effect of the pharmaceutical composition of the present invention on the bone resorption function of osteoclasts

[0090] BMMs are divided into 1x10 5 The cells were inoculated into a 24-well hydroxyapatite plate containing α-MEM medium (10% fetal bovine serum, 20 ng / mL M-CSF, 20 ng / mL RANKL) and incubated in an incubator at 37°C and 5% CO2 for 6 days; the experimental groups were added with the pharmaceutical compositions of Examples 1 to 3 at 5 uM and 10 uM synergist concentrations, and the blank control group was added with the same volume of PBS. After 6 days of incubation, the culture medium was removed and the cells were treated with 5wt% sodium hypochlorite solution for 5 minutes to remove the cells; the cells were rinsed with distilled water 5 times and stained with a modified Von Kossa staining method as follows: after removing the cells in the culture plate with sodium hypochlorite, 300μL 5wt% silver nitrate was added to each well of the cell plate and incubated at room temperature in the dark for 30 minutes, then rinsed with distilled water 5 times, and then incubated with a mixed solution of 5wt% sodium carbonate solution and 10% formalin at room temperature for 5 minutes, rinsed with distilled water 5 times, dried, air-dried, observed with an inverted optical microscope, and the bone resorption area of ​​each well was calculated using Image J analysis software. The specific results are shown in Figure 2. Figure 3 shown.

[0091] Figure 3 The effect of Examples 1 to 3 on the area of ​​bone lacuna is shown, and the ordinate represents the percentage of the area of ​​bone lacuna of each group relative to the area of ​​bone lacuna of the control group (only RANKL stimulation), and the abscissa represents the serial number of Examples 1 to 3. Figure 3 It can be seen that Examples 1 to 3 all inhibit the number and area of ​​bone resorption pits formed by osteoclasts mediated by RANKL in a concentration-dependent manner, which indicates that Examples 1 to 3 can inhibit the bone resorption activity of osteoclasts in a concentration-dependent manner.

[0092] Test Example 3

[0093] (1) Animal model: 160 healthy male 6-week-old SPF Kunming mice weighing 20±2g. The mice were kept in an animal room at a constant temperature of 24±1℃, 60±5% humidity, 12h / d light, and the bedding was changed once a day. During the feeding process, the growth of the mice was observed every day, and feed and drinking water were supplemented.

[0094] After the mice were fed for 3 days of adaptation, blood was collected from the tail vein to detect the initial blood glucose content, and the initial body weight was weighed. 20 mice were randomly selected as the blank control group and fed with ordinary feed. The remaining mice were all fed with high-fat and high-sugar feed to establish a diabetes model. After feeding for 21 days, the mice were fasted for 12 hours and injected intraperitoneally with 50 mg / kg STZ (pH 4.5 sodium citrate buffer). The blank group mice were injected with an equal amount of sodium citrate buffer and continued to be fed for 3 days. After fasting for 12 hours but not water, blood was collected from the tail vein, and the blood glucose content (FBG) after modeling was measured with a blood glucose meter. The model was successfully established when the random blood glucose was ≥11mmol / L.

[0095] The mice with successful modeling were randomly divided into 7 groups, namely the model group (MC), the example 1-3 group, the comparative example 1-2 group and the positive control group, with 20 mice in each group. The drug administration was carried out as follows: the blank control group: the same amount of normal saline was administered subcutaneously; the model group: the same amount of normal saline was administered subcutaneously; the example 1-3 group: the pharmaceutical composition of example 1-3 was administered, the dosage was 0.2 mg (the total mass of the GLP-1 / GIP dual receptor agonist DA3-CH and the synergist, the same below) / kg, subcutaneously, once a day; the comparative example 1-2 group: 0.2 mg / kg of the pharmaceutical composition of comparative example 1-2 was administered subcutaneously, once a day; the positive control group: 0.2 mg / kg of liraglutide was administered subcutaneously, once a day;

[0096] (2) Biochemical tests (blood sugar, bone density) were performed on each group of animals:

[0097] On the 21st day after administration, blood glucose level (FBG) was measured with a blood glucose meter. Figure 4 As shown;

[0098] After the blood sugar was measured, the mice were killed by cervical dislocation, and the left femur and left pelvis were taken out and baked at 60℃ for 24h. The bone density was measured by solid density meter. The results are as follows: Figure 5 shown.

[0099] Figure 4 The effect of each group of drug compositions on the blood glucose level of mice is shown. The ordinate represents the blood glucose level of each group of mice, and the abscissa represents the serial number of each group of animal models: blank control group, model group, positive control group, Example 1 to 3 groups, and Comparative Example 1 to 2 groups. Figure 4It can be seen that: compared with the blank control group, the blood glucose content in the serum of the mice in the model group was ≥11mmol / L, indicating that the mouse diabetes model was successfully constructed; compared with the model group, the blood glucose content of the mice in each group was reduced, among which, the blood glucose reduction range of Comparative Example 1 replacing an equal amount of the synergist with the GLP-1 / GIP dual receptor agonist DA3-CH and Comparative Example 2 replacing an equal amount of the GLP-1 / GIP dual receptor agonist DA3-CH with the synergist was slightly lower than that of the positive control group, but the blood glucose reduction range of Examples 1 to 3 was higher than that of the positive control group, which shows that the GLP-1 / GIP dual receptor agonist DA3-CH and the synergist of the present invention have the effect of lowering blood glucose themselves, and the two act synergistically, and the level of blood glucose reduction is significantly reduced.

[0100] Figure 5 The effect of each group of drug compositions on the bone density of mice is shown, the ordinate represents the bone density of each group, and the abscissa represents the serial number of each animal model group: blank control group, model group, positive control group, examples 1 to 3 and comparative examples 1 to 2. Figure 5 It can be seen that compared with the blank control group, the bone density of mice in the model group was significantly reduced, which indicates that diabetes can reduce the bone density of mice.

[0101] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A pharmaceutical composition for treating diabetic osteoporosis, characterized in that: It includes a GLP-1 / GIP dual receptor agonist DA3-CH and a synergist; the structure of the synergist is:

2. The pharmaceutical composition for treating diabetic osteoporosis according to claim 1, characterized in that: The preparation process of the synergist comprises the following steps: (1) 3-((3-carboxy-2-hydroxy-4-methoxy-6-methylbenzoyl)oxy)-5-formyl-4,6-dihydroxy-2-methylbenzoic acid, an inorganic base, and benzyl bromide are added to N,N-dimethylformamide, reacted at 50-60° C. for 90-120 min, and then treated to obtain a crude product 1; the crude product 1 and lithium hydroxide are added to methanol, reacted at 70-80° C. for 3-5 h, and then treated to obtain an intermediate 1; (2) adding diphenylphosphoryl azide to a mixed system of intermediate 1, triethylamine, toluene and tert-butyl alcohol, reacting at 80-90° C. for 3-5 hours and then treating to obtain crude product 2; reacting crude product 2 and diethylenetriamine at 120-140° C. for 10-15 hours and then treating to obtain intermediate 2; (3) adding ethyl 2-bromoacetate to a mixture of intermediate 2, potassium carbonate, N,N-dimethylformamide and water, reacting at 60-80° C. for 2-4 hours, and then treating to obtain intermediate 3; (4) adding tert-butyl alcohol to a mixture of chlorosulfonyl isocyanate and dichloromethane at 5 to 8°C, and reacting at -2 to 0°C for 30 to 40 minutes; adding a mixed solution of intermediate 3, triethylamine and dichloromethane to the mixture at 5 to 8°C, reacting at -2 to 0°C for 30 to 40 minutes, warming to room temperature for reaction for 1 hour, and treating to obtain a crude product 4; adding the crude product 4 and trifluoroacetic acid to dichloromethane, reacting at room temperature for 30 to 50 minutes, and treating to obtain an intermediate 4; (5) Sodium methoxide is added to a mixture of intermediate 4 and tetrahydrofuran, and the mixture is reacted at room temperature for 20 to 40 minutes, and then treated to obtain a crude product 5; the crude product 5, pentamethylbenzene, and trichloroborane are reacted at -78 to -75°C for 20 to 30 minutes, and then treated to obtain the synergist.

3. The pharmaceutical composition for treating diabetic osteoporosis according to claim 2, characterized in that: In step (1), the molar ratio of 3-((3-carboxy-2-hydroxy-4-methoxy-6-methylbenzoyl)oxy)-5-formyl-4,6-dihydroxy-2-methylbenzoic acid, inorganic base, benzyl bromide and lithium hydroxide is 1:(5.5-6.5):(3-4):(3-3.5); the inorganic base is one of potassium carbonate and cesium carbonate.

4. The pharmaceutical composition for treating diabetic osteoporosis according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate 1, diphenylphosphoryl azide, triethylamine and diethylenetriamine is 1:(2.2-2.5):(2.2-2.5):(0.08-0.1).

5. The pharmaceutical composition for treating diabetic osteoporosis according to claim 2, characterized in that: In step (3), the molar ratio of the intermediate 2, potassium carbonate and ethyl 2-bromoacetate is 1:(5-6):(2.2-2.5).

6. The pharmaceutical composition for treating diabetic osteoporosis according to claim 2, characterized in that: In step (4), the molar ratio of the intermediate 3, triethylamine, tert-butyl alcohol, chlorosulfonyl isocyanate and trifluoroacetic acid is 1:(4-5):(2.4-3):(2.4-3):(40-50).

7. The pharmaceutical composition for treating diabetic osteoporosis according to claim 2, characterized in that: In step (5), the molar ratio of the intermediate 4, sodium methoxide, trichloroborane and pentamethylbenzene is 1:(3-3.5):(6-7.5):(6-7.5).

8. The pharmaceutical composition for treating diabetic osteoporosis according to claim 1, characterized in that: The pH regulator is one of sodium carbonate and sodium hydroxide; the isotonicity regulator is one of mannitol and propylene glycol; and the preservative is chlorobutanol.

9. The pharmaceutical composition for treating diabetic osteoporosis according to claim 1, characterized in that: The pharmaceutical composition also includes water for injection, a pH regulator, an isotonic regulator and a preservative. By weight, the GLP-1 / GIP dual receptor agonist DA3-CH is 0.6 to 1.08 parts, the synergist is 0.75 to 1.26 parts, the preservative is 1.2 to 1.5 parts, the isotonic regulator is 1.5 to 3 parts, and the water for injection is 300 parts. The pH range of the pharmaceutical composition is 8.0 to 8.

4.

10. The method for preparing the pharmaceutical composition for treating diabetic osteoporosis according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: S1: weighing a preservative and an isotonicity adjusting agent, adding an appropriate amount of water for injection to completely dissolve them, and obtaining a mixed solution; S2: taking an appropriate amount of water for injection to completely dissolve a GLP-1 / GIP dual receptor agonist DA3-CH enhancer, adding the mixture to the mixed solution of step S1, adjusting the pH value to a target value with a pH adjusting agent, filtering, and sterilizing to obtain the mixture.

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