CN103626837B - Glycogen phosphorylase inhibitors cholic acid derivative, its preparation method and medical usage containing bio-cleavable dipeptide - Google Patents

Abstract

The invention relates to a kind of glycogen phosphorylase inhibitor cholic acid derivatives containing biocleavable dipeptides, their preparation method and their drug combination. The derivatives are liver-targeting prodrugs of glycogen phosphorylase, and the biocleavable dipeptide can be cleaved by enzymes in vivo to release glycogen phosphorylase inhibitors. Such compounds can be used to prevent and treat diabetes and its complications, hyperlipidemia, obesity, hyperglucagonism, insulin resistance, fasting hyperglycemia, hypertension and its complications, atherosclerosis, metabolic Syndrome or tumor.

Description

Glycogen phosphorylase inhibitor bile acid derivatives containing biocleavable dipeptide, its Preparation method and medicinal use

technical field

The invention relates to a kind of glycogen phosphorylase inhibitor cholic acid derivatives containing biocleavable dipeptides, their preparation method and their drug combination. Glycogen phosphorylase inhibitors Bile acid derivatives are liver-targeting prodrugs of glycogen phosphorylase, and compared with glycogen phosphorylase inhibitors, oral administration of glycogen phosphorylase inhibitors can increase The concentration can be used as the preferred drug for lowering blood sugar, especially for the treatment of fasting hyperglycemia. Such compounds can be used to prevent and treat diabetes and its complications, hyperlipidemia, obesity, hyperglucagonism, insulin resistance, fasting hyperglycemia, hypertension and its complications, atherosclerosis, metabolic Syndrome or tumor.

Background technique

Type 2 diabetes is a group of metabolic syndromes characterized by fasting and postprandial hyperglycemia caused by genetic defects and acquired environmental factors. Sustained hyperglycemia can lead to complications in blood vessels, kidneys, nervous system, and retina. Therefore, it is very important to maintain normal blood sugar levels. Clinical studies have shown that the significant increase in glucose output from the liver in diabetic patients is one of the important causes of fasting hyperglycemia. Studies on glucose labeled with ³ H have shown that in healthy individuals, the rate of glucose output by the liver is 1.8-2.0 mg·kg ^-1 ·min ^-1 in the basic state; while type 2 diabetic patients with moderate fasting hyperglycemia, The glucose output rate of the liver increased by about 0.5mg·kg ^-1 ·min ^-1 , which was significantly higher than that of normal people.

Hepatic glucose production mainly comes from two aspects: 1) gluconeogenesis; 2) enzymatic degradation of glycogen. Studies have shown that gluconeogenesis and glycogen degradation contribute equally to hepatic glucose production in diabetic patients. It is worth noting that metformin is one of the first-choice hypoglycemic drugs in clinical practice, and its mechanism of action is considered to lower blood sugar mainly by inhibiting hepatic gluconeogenesis. On the other hand, there is currently no effective drug to inhibit the excessive degradation of hepatic glycogen clinically.

On an empty stomach, liver glycogen generates glucose-1-phosphate through the action of glycogen phosphorylase, and then converts it into glucose-6-phosphate under the catalysis of phosphoglucomutase, and finally converts it into glucose-6-phosphate under the action of glucose-6-phosphatase dephosphorylation to export glycogen. Or directly enter the anaerobic and aerobic metabolic pathways to participate in energy supply. Since glycogen phosphorylase is a key factor in glycogen metabolism, its pharmacological inhibition may be used to treat diseases related to excessive glycogen degradation, such as diabetes, ischemic myocardial injury and tumors ( Curr. Protein. Pept. Sci., 2002, 3, 561; Am. J. Physiol. Heart. Girc. Physiol., 2004, 286, H1177). In addition, hypertension and its associated pathological changes such as atherosclerosis, hyperlipidemia, and hypercholesterolemia are all associated with elevated insulin levels. Glycogen phosphorylase inhibitors can effectively reduce insulin levels, so they can be used to treat hypercholesterolemia, hyperinsulinemia, hyperlipidemia, atherosclerosis and myocardial ischemia.

In recent years, the field of developing novel glycogen phosphorylase inhibitors has received considerable attention. For example, U.S. Patent Application No. 6,297,269 and European Patent Application No. EP0832066 describe substituted N-(indole-2-carbonyl) amides and their derivatives as glycogen phosphorylase inhibitors, U.S. Patent Application No. 6,107,329 Substituted N-(indole-2-carbonyl)glycinamides and their derivatives are described as glycogen phosphorylase inhibitors, and European Patent Application No. WO2006059163 describes pyrrolophosphorylases as glycogen phosphorylase inhibitors. Pyridine-2-carboxylic acid amide derivatives. However, there are three glycogen phosphorylase isoenzymes in human tissues, which are named muscle glycogen phosphorylase, brain glycogen phosphorylase, and liver glycogen phosphorylase according to the tissues with their dominant expression. , the three have a high degree of homology. Muscle-type glycogen phosphorylase is mainly found in muscle tissue, and its function is to supply energy for muscle contraction. Brain-type glycogen phosphorylase is mainly expressed in the brain and heart of adults, and can provide glucose during hypoxia or severe hypoglycemia The emergency supply of hepatic glycogen phosphorylase affects systemic blood glucose by regulating glucose from hepatic glycogen stores. Due to the high homology of the three isoenzymes of this enzyme, the currently reported inhibitors of this target generally lack selectivity to liver glycogen phosphorylase, resulting in myotoxic reactions to muscle tissue, and the clinical application is limited. In order to reduce the myotoxicity of glycogen phosphorylase inhibitors, improve their hypoglycemic activity, and increase their bioavailability, the reported glycogen phosphorylase inhibitors have been modified to search for alternative effects on liver glycogen phosphate. new derivatives of enzymes.

Cholic acid is currently the only oral liver-targeting drug carrier, which has a special transport system in the body. Bile acid can be absorbed specifically by the liver through the Na ⁺ dependent transport system (NTCP) and the Na ⁺ independent transport system (OTAP) on the liver cell membrane. Bile acid is an endogenous hepatocyte-specific natural ligand with a high degree of organ specificity. Bile acid is biosynthesized from cholesterol in liver cells, then combined with glycine or taurine, discharged into the small intestine with bile, and then absorbed into the liver, and enterohepatic circulation continues in the adult body. Repeat 6-15 times a day, and the total amount of bile acid involved in the circulation reaches 17-40 grams. Therefore, it has a high transport capacity. As an endogenous natural ligand, cholic acid has good biocompatibility and is suitable as a carrier for targeted drugs. Using cholic acid as a targeting carrier can not only achieve liver targeting of drugs, reduce toxic and side effects, but also Improve the bioavailability of drugs in the body.

The spacer between the targeting carrier and the small molecule drug plays an important role. The spacer group enables the low-molecular drug to form a stable or temporary combination with the targeting carrier, which can maintain a certain stability in the blood circulation. When it is broken again, it has certain stability, hydrolysis and enzymolysis. A suitable spacer group can control the release rate and site of drug release from the polymer. Peptide spacer is a kind of spacer that has been widely studied in recent years. Since the oligopeptide formed by the amino acid sequence can be hydrolyzed by enzymes in the lysosome, the specially designed oligopeptide can be used as a spacer to connect the drug to the targeting carrier. Since different enzymes act differently on different peptides, the length and composition of the peptide spacer can affect the release rate of the drug. De Marre et al. studied the prodrug of mitomycin C-poly[N-(2-hydroxyethyl)-L-glutamine] linked to oligopeptides. After hydrolysis with tritosomes for 3 hours at pH 5.5, With Ala-Leu-Ala-Leu as the spacer prodrug, the release of mitomycin C can reach 81.0%, while with Gly-Phe-Leu as the spacer prodrug, the release of mitomycin C It only reaches 2.4%, indicating that the oligopeptide structure and composition of the spacer have a great influence on the drug release (J. Control Release, 1994, 31, 89-97).

Contents of the invention

The present invention discloses for the first time the cholic acid derivatives containing biocleavable dipeptide-containing glycogen phosphorylase inhibitors with medicinal value represented by formula (I), their preparation method and medical application, including in the preparation of antidiabetic and Uses of the complication medicine, blood lipid-lowering medicine, weight-loss medicine, anti-atherosclerosis medicine, metabolic syndrome medicine and antitumor medicine. In particular, the compound represented by formula (I) is a liver-targeting prodrug of a glycogen phosphorylase inhibitor, so it can be used to treat diseases related to abnormal glycogen metabolism in the liver, these diseases include: diabetes and its concurrent hyperlipidemia, obesity, hyperglucagonism, insulin resistance, fasting hyperglycemia, hypertension and its complications, atherosclerosis, metabolic syndrome or tumors. In addition, the present invention also provides a pharmaceutical preparation containing the compound represented by formula (I).

The present invention relates to compounds represented by formula (I) and pharmaceutically acceptable salts or esters thereof:

in:

X ₁ , X ₂ , X ₃ and X ₄ are all C or one of X ₁ , X ₂ , X ₃ and X ₄ is N and the others must be C;

R ₁ and R ₁ ' independently represent H, halogen, hydroxyl, cyano, C _0-4 alkyl, C _1-4 alkoxy, fluoromethyl, difluoromethyl, trifluoromethyl, vinyl , ethynyl;

R ₂ represents CH ₃ -, (CH ₃ ) ₂ CHCH ₂ -, CH ₃ CH ₂ (CH ₃ ) CH-, H ₂ NCH ₂ CH ₂ CH ₂ CH ₂ -;

R ₃ represents (CH ₃ ) ₂ CH-, C ₆ H ₅ CH ₂ -, p-HO-C ₆ H ₄ CH ₂ -;

R ₄ , R ₅ , R ₆ and R ₇ each independently represent H, hydroxyl, R ₈ COO;

R ₈ represents unsubstituted or X-substituted linear or branched chain alkyl, alkenyl, alkyne, aryl and heteroaryl with 1 to 20 carbons;

X represents H, F, Cl, Br, I, CN, NO ₂ , NH ₂ , CF ₃ , SH, OH, OCH ₃ , OC ₂ H ₅ , COOH, straight or branched chain alkyl with 1 to 10 carbons , Alkenyl, Alkynyl, Aryl, Heteroaryl.

Preferred compounds among the above-mentioned compounds are:

X ₁ , X ₂ , X ₃ and X ₄ are all C or one of X ₁ , X ₂ , X ₃ and X ₄ is N and the others must be C;

R ₁ and R ₁ ' are each independently H, halogen, cyano;

R ₂ represents CH ₃ -, H ₂ NCH ₂ CH ₂ CH ₂ CH ₂ -;

R ₃ represents (CH ₃ ) ₂ CH-, C ₆ H ₅ CH ₂ -;

R ₄ , R ₅ , R ₆ and R ₇ are each independently H and hydroxyl;

X represents H, F, Cl, Br, I, CN, NO ₂ , NH ₂ , CF ₃ , SH, OH, OCH ₃ , OC ₂ H ₅ , COOH, straight or branched chain alkyl with 1 to 10 carbons , Alkenyl, Alkynyl, Aryl, Heteroaryl.

More preferred compounds are:

The compounds of the present invention can be prepared by the reported process, and can also be prepared by the following methods:

a) Dissolve the prepared glycogen phosphorylase inhibitor containing exposed hydroxyl and the dipeptide protected by tert-butoxycarbonyl (Boc) or fluorenylmethoxycarbonyl (Fmoc) at the amino end in an organic solvent, and add the coupling The reagent undergoes an ester-forming reaction for 1-72 hours at a temperature of 0°C to 45°C. Coupling reagents can use conventional condensation reagents, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N, N'-dicyclohexyl carbodiimide (DCC), O-benzotriazole-N, N, N', N'-tetramethyluronium tetrafluoroboric acid (TBTU), 2-(7-azobenzotriazole)-N,N , N', N'-tetramethyluronium hexafluorophosphate (HATU), 1-propylphosphoric tricyclic anhydride (T ₃ P). Depending on the progress of the reaction, the catalyst is appropriately selected, such as 4-dimethylaminopyridine;

b) Dissolving the product a in an organic solvent, adding a deprotection reagent to remove the protection of the amino terminal, and the temperature is from 0°C to reflux. Among them, the a product whose amino terminal is protected with tert-butoxycarbonyl (Boc) is deprotected with trifluoroacetic acid, and the a product whose amino terminal is protected with fluorenylmethoxycarbonyl (Fmoc) is deprotected with piperidine. The solvent used can be acetonitrile, methanol, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, chloroform, toluene, n-hexane, cyclohexane, tert-butyl methyl ether or a mixed solvent of the above solvents, preferably acetonitrile or dichloromethane as a solvent;

c) dissolving the cholic acid free at the carboxyl end and its derivatives and the product of b in an organic solvent, adding a coupling reagent and an organic amine or an inorganic base, and reacting for 1-72 hours at a temperature of 0°C to 45°C. The solvent is generally an inert solvent, especially an aprotic solvent, including acetonitrile, chloroform, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, toluene, n-hexane, cyclohexane, tetrahydrofuran , tert-butyl methyl ether or a mixed solvent of the above solvents, preferably dichloromethane, 1,2-dichloroethane or N,N-dimethylformamide. Coupling reagents can use conventional condensation reagents, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N, N'-dicyclohexyl carbodiimide (DCC), O-benzotriazole-N, N, N', N'-tetramethyluronium tetrafluoroboric acid (TBTU), 2-(7-azobenzotriazole)-N,N , N', N'-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazole (HATU), 1-propylphosphoric tricyclic anhydride (T ₃ P). The inorganic base used is sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate, and the organic base used is N, N-diisopropylethylamine or triethylamine.

The present invention also includes pharmaceutical preparations, which contain the compound of general formula (I) or its pharmaceutically acceptable salt, ester or pharmaceutically acceptable carrier as an active agent.

The above-mentioned pharmaceutically acceptable carrier refers to the conventional drug carrier in the field of pharmacy, and refers to one or more inert, non-toxic solid or liquid fillers, diluents, auxiliary agents, etc., which do not reversely interact with active compounds or patients. take effect.

The dosage forms of the composition of the present invention can be commonly used pharmaceutical dosage forms such as tablets, capsules, pills, suppositories, soft capsules, oral liquids, suspensions, and injections.

Tablets and capsules for oral use contain conventional excipients such as fillers, diluents, lubricants, dispersants and binders.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to well-known methods in the field of pharmacy.

Dosages of the above active agents will vary from formulation to formulation.

In general, amounts which have proven to be advantageous, are in the total range of about 0.01-800 mg per kilogram of compound of formula (1) administered per 24 hours, preferably in the total range of 0.1-100 mg/kg, to achieve the desired result. Administer in several single doses, if necessary. However, it is also possible, if necessary, to deviate from the above-mentioned dosages, i.e. it depends on the type and body weight of the subject to be treated, the individual's behavior towards the drug, the nature and severity of the disease, the type of formulation and administration, and the time of administration. and interval.

Description of drawings

Fig. 1 shows the preparation process of some derivatives of the present invention.

In Figure 1, X ₁ , X ₂ , X ₃ , X ₄ , R ₁ , R ₁ ', R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and X are as defined above As defined in formula (I), R ₉ represents N-terminal protecting groups of various amino acids, such as tert-butoxycarbonyl (Boc) or fluorenylmethoxycarbonyl (Fmoc).

detailed description:

The content of the present invention is specifically described below by way of examples. In the present invention, the examples described below are for better illustrating the present invention, and are not intended to limit the scope of the present invention.

Further illustrate the implementation of the present invention below by embodiment

Example 1

(S)-2-tert-butoxycarbonylamino-3-(4-fluorophenyl)-1-(4-hydroxypiperidin-1-yl)acetone

Dissolve BOC-4-fluoro-L-phenylalanine (15.6g, 55.1mmol) in anhydrous dichloromethane (160mL), add HATU (25g, 66.1mmol) and DIPEA (8.54g, 66.1 mmol), stirred at room temperature for 10 minutes, then added 4-hydroxypiperidine (6.7 g, 66.1 mmol), stirred at room temperature overnight. The reaction solution was washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated. The residue was separated by reverse-phase HPLC to obtain a white solid (50 mg, 29.8%). Flash column chromatography (petroleum ether/ethyl acetate l/l, V/V) gave a white solid (19.7 g, 98%).

ESI-MS m/z: 367.2[M+H] ⁺ .

¹ H NMR (CDCl ₃ , 400MHz): 7.14-7.18 (m, 2H), 6.95-7.00 (m, 2H), 5.47 (dd, J=8.8, 14.8Hz, 1H), 4.83 (dd, J=6.0, 13.6Hz, 1H), 3.81-4.01(m, 2H), 3.46-3.62(m, 1H), 3.15-3.33(m, 1H), 2.89-3.00(m, 2H), 1.73-1.83(m, 2H) , 1.42-1.52(m, 2H), 1.40(s, 9H).

(S)-2-Amino-3-(4-fluorophenyl)-1-(4-hydroxypiperidin-1-yl)acetone

Dissolve (S)-2-tert-butoxycarbonylamino-3-(4-fluorophenyl)-1-(4-hydroxypiperidin-1-yl)acetone (19 g, 52 mmol) in dichloromethane (50 mL) , under ice-cooling, slowly add freshly prepared hydrogen chloride in dichloromethane solution (2N, 50 mL) dropwise, and stir overnight at room temperature. The next day, the reaction solution was concentrated under reduced pressure to obtain a white solid (14 g, 89%).

ESI-MS m/z: 267.2[M+H] ⁺ .

¹ H NMR (MeOD, 400MHz): 8.52 (brs, 1H), 7.29 (dd, J=7.2, 13.6Hz, 2H), 7.09-7.14 (m, 2H), 4.63 (brs, 1H), 3.86-4.06 (m, 1H), 3.73-3.83(m, 1H), 3.35-3.62(m, 1H), 2.78-3.21(m, 4H), 0.92-1.85(m, 4H).

(S)-1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-4-hydroxypiper Pyridine

In a similar manner to the preparation of (S)-2-tert-butoxycarbonylamino-3-(4-fluorophenyl)-1-(4-hydroxypiperidin-1-yl)acetone, (S)-2-amino -3-(4-fluorophenyl)-1-(4-hydroxypiperidin-1-yl)acetone (14g, 46.4mmol) and 5-chloro-1-hydrogen-pyrrole[2,3-c]pyridine -2-Carboxylic acid (9.08g, 46.4mmol) was reacted to obtain a white solid (14.2g, 99%).

ESI-MS m/z: 444.9[M+H] ⁺ .

¹ H NMR (DMSO-d ₆ , 400MHz): 12.27(s, 1H), 9.22(t, J=8.8Hz, 1H), 8.57(s, 1H), 7.77(s, 1H), 7.36(d, J =9.6, 3H), 7.06(d, J=3.6, 1H), 5.16(d, J=6.4, 1H), 4.75(s, 1H), 3.67-4.04(m, 3H), 2.91-3.30(m, 4H), 1.57-1.67(m, 2H), 1.13-1.24(m, 2H).

(S)-Methyl 2-(2-tert-butoxycarbonylamino-3-methyl-n-butyramide)propionate

Dissolve N-tert-butoxycarbonyl-L-valine (4g, 18.4mmol) and alanine methyl ester hydrochloride (2.58g, 18.5mmol) in dry DMF (80mL), add EDCI under ice-cooling (5.3g, 27.7mmol), HOBt (3.73g, 27.65mmol) and DIPEA (5.93g, 46mmol), stirred overnight at room temperature under nitrogen protection. The reaction solution was washed successively with 1N hydrochloric acid solution, saturated sodium bicarbonate solution and saturated brine, dried over anhydrous Na ₂ SO ₄ , and evaporated to dryness under reduced pressure to obtain a white solid (4.5 g, 80%).

ESI-MS m/z: 325.2[M+Na] ⁺ .

¹ H-NMR (CDCl ₃ , 400MHz): 6.38 (brs, 1H), 5.07 (brs, 1H), 4.58-4.65 (m, 1H), 3.93-3.97 (m, 1H), 3.78 (s, 3H), 2.11-2.20(m, 1H), 1.47(s, 9H), 1.43(d, J=7.2Hz, 3H), 0.99(d, J=6.8Hz, 3H), 0.94(d, J=6.8Hz, 3H );

(S)-2-(2-tert-butoxycarbonylamino-3-methyl-n-butyramide)propionic acid

Dissolve (S)-2-(2-tert-butoxycarbonylamino-3-methyl n-butyramide) propionate methyl ester (2.5g, 8.3mmol) in methanol/water mixed solution (20mL, V/ V=l/l), sodium hydroxide (0.5 g, 12.5 mmol) was added, and stirred at room temperature for 3 h. Methanol was evaporated under reduced pressure, and the remaining reaction solution was neutralized to pH 2-3 with 1N hydrochloric acid aqueous solution, filtered, the filter cake was washed with dichloromethane, and dried in vacuo to obtain a white solid (1.6 g, 46%).

ESI-MS m/z: 287.0[M-H]-.

¹ H-NMR (CDCl ₃ , 400MHz): 6.86(brs, 1H), 5.26-5.34(m, 1H), 4.59-4.63(m, 1H), 3.99-4.02(m, 1H), 2.04-2.25(m , 1H), 1.47-1.49(m, 12H), 0.98(d, J=6.4, 3H), 0.95(d, J=6.8, 3H).

(S)-N-(2-tert-butoxycarbonylamino-3-methyl-n-butyryl)alanine-{1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine -2-Carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl}ester

(S)-2-(2-tert-butoxycarbonylamino-3-methyl-n-butyramide)propionic acid (1g, 3.5mmol) and (S)-1-[2-(5-chloro-1H- Pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-4-hydroxypiperidine (1.54 g, 3.5 mmol) was dissolved in DMF (30 mL), DCC (1.1 g, 5.2 mmol) was added under ice-cooling, and stirred at room temperature overnight. The reaction mixture was filtered, the filtrate was evaporated to remove the solvent, the residue was dissolved in ethyl acetate, placed in the refrigerator overnight, filtered, the filtrate was evaporated to remove the solvent, and the residue was separated by reverse phase HPLC to obtain a white solid (1.3 g, 57%).

ESI-MS m/z: 715.0[M+H] ⁺ .

¹ H-NMR (MeOD, 400MHz): 8.53(s, 1H), 7.64(s, 1H), 7.30-7.35(m, 2H), 7.14(s, 1H), 7.00-7.06(m, 2H), 5.31 -5.36(m, 1H), 4.89-4.96(m, 1H), 4.36-4.42(m, 1H), 3.40-3.95(m, 5H), 3.17-3.30(m, 1H), 3.08-3.13(m, 1H), 2.00-2.07(m, 1H), 1.78-1.89(m, 1H), 1.54-1.67(m, 1H), 1.29-1.45(m, 14H), 0.89-1.00(m, 6H).

(S)-N-(2-amino-3-methyl-n-butyryl)alanine {1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide )-3-(4-fluorophenyl)propionyl]-piperidin-4-yl}ester

(S)-N-(2-tert-butoxycarbonylamino-3-methyl n-butyryl)alanine {1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine -2-Carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl} ester (1g, 1.4mmol) was dissolved in dichloromethane (10mL), and trifluoro Acetic acid (1 mL, 14 mmol) was added and stirred at this temperature for 2 hours after dropping. The reaction solution was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate, washed with saturated sodium bicarbonate solution and saturated brine successively, and dried over anhydrous Na ₂ SO ₄ . The desiccant was removed by filtration and concentrated to obtain a white solid crude product, which was directly used in the next reaction without further purification.

(S)-N-{2-[N-(3α,7α,12α-trihydroxy-5β-cholaneamido)]-3-methyl-n-butyryl}alanine {1-[2-(5 -Chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl}ester

Dissolve cholic acid (330mg, 0.80mmol) in DMF (5mL), add HATU (456mg, 1.2mmol) and triethylamine (121mg, 1.2mmol) under ice-cooling, stir at room temperature for 10 minutes, then add (S)- N-(2-amino-3-methyl-n-butyryl)alanine {1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3- (4-Fluorophenyl)propionyl]-piperidin-4-yl} ester (450 mg, crude), stirred at room temperature overnight. The solvent was evaporated under reduced pressure, the residue was dissolved in ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated. The residue was separated by reverse-phase HPLC to obtain a light yellow solid (150 mg, 24%).

ESI-MS m/z: 1005.0[M+H] ⁺ .

¹ H-NMR (MeOD, 400MHz): 8.58(s, 1H), 7.71(s, 1H), 7.31-7.36(m, 2H), 7.17-7.20(m, 1H), 7.01-7.07(m, 2H) , 4.95-5.00(m, 1H), 4.35(td, J=7.2, 14.4Hz, 1H), 4.15-4.27(m, 1H), 3.90-3.96(m, 1H), 3.78-3.81(m, 2H) , 3.69(s, 1H), 3.55-3.62(m, 2H), 3.36-3.48(m, 2H), 3.08-3.24(m, 2H), 2.23-2.25(m, 4H), 1.13-1.84(envelope, 31H), 1.40(s, 3H), 1.38(s, 3H), 0.85-1.08(envelope, 16H), 0.69-0.71(m, 1H), 0.68(d, J=26.0Hz, 3H).

Example 2

(S)-2-[(S)-2-tert-butoxycarbonylamino-3-phenylpropanylamino]-6-(benzyloxyamido)hexanoic acid methyl ester

In a similar manner to the preparation of (S)-2-(2-tert-butoxycarbonylamino-3-methyl-n-butyramide)propionic acid methyl ester, lysine methyl ester hydrochloride (2.49 g, 7.6 mmlo) Reaction with N-tert-butoxycarbonyl-L-phenylalanine (2 g, 7.6 mmol) gave a white solid (3 g, 73%).

ESI-MS m/z: 542.3[M+H] ⁺ .

¹ H-NMR (CDCl ₃ , 400MHz): 7.38(d, J=4.4Hz, 4H), 7.29-7.35(m, 5H), 7.20-7.27(m, 3H), 6.49(d, J=7.2Hz, 1H), 5.11(dd, J=12.4, 16.4Hz, 2H), 4.54-4.59(m, 1H), 4.35-4.40(m, 1H), 3.72(s, 3H), 3.16(dd, J=6.0, 12.0Hz, 2H), 3.07(t, J=6.0Hz, 2H), 1.77-1.88(m, 1H), 1.64-1.71(m, 1H), 1.49-1.56(m, 2H), 1.40(s, 9H ), 1.27-1.36(m, 2H).

(S)-2-[(S)-2-tert-butoxycarbonylamino-3-phenylpropanylamino]-6-(benzyloxyamido)hexanoic acid

According to the similar method of preparing (S)-2-(2-tert-butoxycarbonylamino-3-methyl n-butyramide) propionic acid, (S)-2-[(S)-2-tert-butoxycarbonyl Amino-3-phenylpropanamido]-6-(benzyloxyamido)hexanoic acid methyl ester was hydrolyzed to give a white solid (2.5 g, 91%).

ESI-MS m/z: 526.0[MH] ^- .

¹ H-NMR (CDCl ₃ , 400MHz): 7.35 (brs, 4H), 7.17-7.29 (m, 6H), 704 (brs, 1H), 5.32 (d, J=44.4Hz, 1H), 5.09 (dd, J=12.4, 18.8Hz, 2H), 4.51(dd, J=14.8, 41.2Hz, 1H), 2.98-3.18(m, 4H), 1.88(brs, 1H), 1.73(brs, 1H), 1.43-1.51 (m, 2H), 1.38(s, 9H), 1.24-1.29(m, 2H).

(S)-2-[(S)-2-tert-butoxycarbonylamino-3-phenylpropanylamino]-6-(fluorenylmethoxycarbonylamino)hexanoic acid

Dissolve (S)-2-[(S)-2-tert-butoxycarbonylamino-3-phenylpropanamido]-6-(benzyloxyamido)hexanoic acid (1 g, 1.9 mmol) in methanol (10 mL) In , a catalytic amount of 10% Pd/C (20 mg) was added, and hydrogenation was carried out at room temperature and pressure for 24 hours. Pd/C was removed by normal pressure filtration, and the filtrate was evaporated to dryness. The residue was dissolved in dioxane (10 mL), NaHCO ₃ (404 mg, 4.75 mmol) and fluorenylmethoxycarbonyl chloride (518 mg, 2.0 mmol) were added slowly under ice cooling, and stirred for 5 hours under ice cooling. The solvent was evaporated under reduced pressure, the residue was diluted with ethyl acetate, washed successively with 1N aqueous hydrochloric acid solution, saturated aqueous NaHCO ₃ solution and saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to give a white solid (1 g, 86%).

ESI-MS m/z: 614.0[MH] ^- .

¹ H NMR (400 MHz, DMSO-d ₆ ): 7.89 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 7.6 Hz, 2H), 7.53 (d, J = 6.4 Hz, 1 H), 7.41 ( t, J=7.6Hz, 2H), 7.33(t, J=7.2Hz, 2H), 7.24-7.29(m, 4H), 7.15-7.19(m, 2H), 4.26(d, J=6.8Hz, 2H ), 4.19(t, J=6.4Hz, 1H), 4.05-4.11(m, 1H), 3.83(dd, J=5.6, 11.6Hz, 1H), 3.05-3.09(m, 1H), 2.93(dd, J=6.4, 13.2Hz, 2H), 2.70-2.76(m, 1H), 1.68-1.76(m, 1H), 1.52-1.61(m, 1H), 1.34-1.14(m, 2H), 1.29(s, 9H), 1.20-1.23(m, 2H).

(S)-N-[(S)-2-tert-butoxycarbonylamino-3-phenylpropionyl)]-N'-fluorenylmethoxycarbonyllysine-{1-[2-(5-chloro- 1H-Pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl}ester

(S)-2-[(S)-2-tert-butoxycarbonylamino-3-phenylpropanylamino]-6-(fluorenylmethoxycarbonylamino)hexanoic acid (380mg, 0.6mmol) and (S) -1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-4-hydroxypiperidine (550mg , 0.6 mmol) was dissolved in dry dichloromethane (30.0 mL), and a solution of 1-propylphosphoric tricyclic anhydride in ethyl acetate (T ₃ P, 50 wt.%, 0.95 mL, 3.1 mmol) was slowly added. After the addition was complete, the mixture was stirred overnight at room temperature under nitrogen protection. After the reaction, the reaction solution was washed with 1N hydrochloric acid aqueous solution, saturated NaHCO ₃ aqueous solution and saturated brine successively, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was separated by reverse phase HPLC (mobile phase: ACN---H ₂ O (0.05% TFA), gradient: 10%-30%) to give a white solid (380mg, 60%).

ESI-MS m/z: 1042.0[M+H] ⁺ .

¹ H-NMR (MeOD, 400MHz): 8.55(d, J=7.6Hz, 1H), 7.71-7.79(m, 2H), 7.60-7.66(m, 3H), 7.36(dd, J=7.2, 14.4Hz , 2H), 7.22-7.31(m, 9H), 7.13(d, J=10.0Hz, 1H), 7.00(dd, J=8.0, 16.0Hz, 2H), 5.26-5.33(m, 1H), 4.31- 4.38(m, 4H), 4.14-4.20(m, 1H), 3.39-3.77(m, 4H), 3.08-3.16(m, 5H), 2.67-2.87(m, 2H), 1.05-1.85(envelope, 20H) ), 1.32(s, 9H).

(S)-N-[(S)-2-amino-3-phenylpropionyl)]-N'-fluorenylmethoxycarbonyllysine-{1-[2-(5-chloro-1H-pyrrolo [2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl}ester

According to the preparation of (S)-N-(2-amino-3-methyl n-butyryl)alanine {1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine-2- (S)-N-[(S)-2-tert-butoxycarbonylamino-3 -Phenylpropionyl)]-N'-Fremoxycarbonyllysine-{1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3 -(4-fluorophenyl)propionyl]-piperidin-4-yl} ester was deprotected to obtain a white solid crude product, which was directly used in the next reaction without further purification.

(S)-N-{2-[N-(3α, 7α, 12α-trihydroxy-5β-cholaneamido)]-3-phenylpropanamide}-N'-fluorenylmethoxycarbonyllysine {1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl} ester

Prepare (S)-N-{2-[N-(3α,7α,12α-trihydroxy-5β-cholaneamido)]-3-methyl-n-butyryl}alanine {1-[2- In a similar manner to (5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl}ester, the Cholic acid (155 mg, 0.38 mmol) and (S)-N-[(S)-2-amino-3-phenylpropionyl)]-N'-fluorenylmethoxylysine-{1-[2- (5-Chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl) propionyl]-piperidin-4-yl} ester 350 mg crude product reaction, to obtain White solid (300 mg, 61%).

ESI-MS m/z: 666.6[M/2+H] ⁺ .

¹ H-NMR (MeOD, 400MHz): 8.70(d, J=7.6Hz, 1H), 7.73-7.81(m, 3H), 7.61-7.68(m, 2H), 7.39(t, J=7.2Hz, 2H ), 7.25-7.33(m, 6H), 7.14-7.20(m, 1H), 7.23(d, J=4.4Hz, 2H), 7.00-7.05(m, 2H), 5.26-5.37(m, 1H), 4.94-4.98(m, 1H), 4.61-4.72(m, 1H), 4.29-4.39(m, 2H), 4.17-4.24(m, 1H), 3.86-3.93(m, 1H), 3.71-3.76(m , 2H), 3.47-3.56(m, 2H), 3.04-3.22(m, 4H), 2.70-2.95(m, 3H), 2.14-2.33(m, 3H), 1.91-2.11(m, 3H), 1.30 -1.82(envelope, 23H), 1.30(s, 3H), 1.12-1.21(m, 3H), 0.89-1.03(envelope, 8H), 0.91(d, J=4.4Hz, 3H), 0.63-0.68(m , 1H), 0.62 (d, J=18.8Hz, 3H).

(S)-N-{2-[N-(3α, 7α, 12α-trihydroxy-5β-cholaneamido)]-3-phenylpropanamide}-lysine {1-[2-(5 -Chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl}ester

(S)-N-{2-[N-(3α, 7α, 12α-trihydroxy-5β-cholaneamido)]-3-phenylpropionamido}-N'-fluorenylmethoxycarbonyllysine Acid {1-[2-(5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide)-3-(4-fluorophenyl)propionyl]-piperidin-4-yl } ester (260mg, 0.20mmol) was dissolved in acetonitrile (3mL), piperidine solution (0.6mL) was slowly added dropwise under ice-cooling, and stirred at room temperature for 1h after dropping. The solvent was evaporated under reduced pressure, and the residue was separated by reverse-phase HPLC (mobile phase: ACN---H ₂ O (0.05% TFA), gradient: 10%-30%) to obtain a light brown solid (100 mg, 45%).

ESI-MS m/z: 1110.0[M+H] ⁺ .

¹ H-NMR (MeOD, 400MHz): 7.70 (d, J=3.6Hz, 1H), 7.33 (t, J=5.2Hz, 3H), 7.27 (dd, J=4.4, 24.4Hz, 4H), 7.18( d, J=2.8Hz, 1H), 7.02-7.06(m, 2H), 5.32(dd, J=7.2, 14.0Hz, 1H), 4.56-4.63(m, 1H), 4.37(ddd, J=5.2, 12.2, 23.0Hz, 1H), 3.92(d, J=23.6Hz, 1H), 3.71-3.82(m, 2H), 3.39-3.60(m, 3H), 3.08-3.23(m, 5H), 2.88-2.95 (m, 3H), 2.16-2.35(m, 4H), 1.31-1.90(m, 30H), 0.95-1.17(m, 3H), 0.93(d, J=5.2Hz, 3H), 0.67(d, J =21.2Hz, 3H).

Glycogen phosphorylase inhibitory activity test in vitro:

Preparation of reagents: 1) Preparation of chromogenic solution: Weigh 5 g of ammonium molybdate, dissolve it in 500 ml of 1M HCl, stir with a stirrer, add 190 mg of malachite green after it is completely dissolved, continue to stir until it is completely dissolved, and protect it with tinfoil paper. Light; 2) Preparation of buffer solution: ① Precisely weigh 0.5958g of Hepes, dissolve in 5ml H ₂ O, adjust the pH to 7.2 with 10M NaOH, and prepare Hepes with a final concentration of 0.5M; ② Precisely weigh 0.3728g of KCl , dissolved in 5ml H ₂ O to prepare KCl with a final concentration of 1M; ③accurately weigh 0.0255g of MgCl ₂ , dissolve it in 1ml H ₂ O, and prepare MgCl ₂ with a final concentration of 125mM; ④accurately weigh EGTA0 .0476g, dissolved in 5ml H ₂ O, adjusted the pH to 7.0 with 10M NaOH, and prepared EGTA with a final concentration of 25mM _; G-1-P with a final concentration of 5mM; ⑥ Precisely weigh 10mg of glycogen, dissolve it in 1ml of H ₂ O, and prepare glycogen with a final concentration of 10mg/ml; 3) Preparation of positive drug caffeine solution: dissolve caffeine in 10ml Prepare 0.5, 5, 50 and 500 μM solutions with H ₂ O; 4) Prepare GPa solution: take 1 μl of GPa and add it to a 100 μl reaction system, with a final concentration of 250 ng/100 μl; 5) Prepare the solution of the compound to be tested: The compound was dissolved in DMSO to prepare a solution with a concentration of 10 mM, and an appropriate amount of the compound solution was added to the reaction system to reach different final concentrations.

Measure the dose-effect curve of rabbit muscle glycogen phosphorylase activity: measure the dose-effect curve by reading the OD value at 655nm after different concentrations of GPa are added to the chromogenic solution. According to the dose-effect curve, the amount of GPa can be selected as 250ng.

Experimental steps: 1) Design PC (positive control), Blank (blank control), positive drug (caffeine); 2) Add reaction buffer 52 μl; 3) Add test compound to the final concentration; 4) Add enzyme 1 μl, the final concentration is 250ng /100 μl; 5) Add 150 μl of chromogenic solution; 6) React for 20 minutes at 30 degrees Celsius; 7) Colorimetric under the condition of wavelength 655nm; 8) Reading of data and calculation of inhibition rate: inhibition rate=[positive control- Test sample]/[positive control-blank control].

Test results: the table lists the inhibitory activity data of glycogen phosphorylase liver-targeted prodrug molecules on rabbit muscle glycogen phosphorylase, and the results show that this type of glycogen phosphorylase liver-targeted prodrug molecules have Prophosphorylases have varying degrees of inhibitory activity.

Inhibitory activity of glycogen phosphorylase liver-targeted prodrug molecules on rabbit muscle glycogen phosphorylase

^a _IC50 values are the average of three experiments;

^b NI means no activity at 100M concentration.

The above pharmacological data show that the compound of general formula (I) of the present invention has the inhibitory effect of glycogen phosphorylase, so it can be used for the prevention and treatment of diabetes and its complications, hyperlipidemia, obesity, hyperglucagonism, insulin Resistance, fasting hyperglycemia, hypertension and its complications, atherosclerosis, metabolic syndrome or tumors.

Claims (11)

1. the compound being shown below or its pharmaceutically acceptable salt:

Wherein:

X₁、X₂、X₃And X₄It is all C or X₁、X₂、X₃And X₄One of for N other be necessary for C；

R₁And R₁' each independently represent H, halogen, hydroxyl, cyano group, C₄Alkyl, C_1-4Alkoxyl, fluoromethyl, difluoromethyl, three Methyl fluoride, vinyl or acetenyl；

R₂Represent CH₃-、(CH₃)₂CHCH₂-、CH₃CH₂(CH₃) CH-or H₂NCH₂CH₂CH₂CH₂-；

R₃Represent (CH₃)₂CH-、C₆H₅CH₂-or p-HO-C₆H₄CH₂-；

R₄、R₅、R₆And R₇Each independently represent H, hydroxyl or R₈COO-；

R₈Represent the non-substituted of 1～20 carbon or X substituted straight or branched alkyl, alkylene, alkynes base, aryl or heteroaryl Base；

X represents H, F, Cl, Br, I, CN, NO₂、NH₂、CF₃、SH、OH、OCH₃、OC₂H₅, COOH, the straight or branched of 1～10 carbon Alkyl, alkylene, alkynes base, aryl or heteroaryl.

2. compound as claimed in claim 1 or its pharmaceutically acceptable salt, it is characterised in that:

X₁、X₂、X₃And X₄It is all C or X₁、X₂、X₃And X₄One of for N other be necessary for C；

R₁And R₁' each stand alone as H, halogen, hydroxyl or cyano group；

R₂Represent CH₃-or H₂NCH₂CH₂CH₂CH₂-；

R₃Represent (CH₃)₂CH-or C₆H₅CH₂-；

R₄、R₅、R₆And R₇Each stand alone as H or hydroxyl.

3. compound as claimed in claim 1 or its pharmaceutically acceptable salt, wherein, compound is following arbitrary compound Or its pharmaceutically acceptable salt:

4. the compound as according to any one of claim 1-3 or the preparation method of its pharmaceutically acceptable salt, including such as Lower step:

A) glycogen phosphorylase inhibitors containing exposed hydroxyl that will prepare, with aminoterminal tertbutyloxycarbonyl or fluorenes methoxy carbonyl The dipeptides of base protection dissolves in organic solvent, adds coupling reagent and carries out into ester reaction, reacts 1-72 hour, and temperature is 0 DEG C To 45 DEG C；Coupling reagent uses 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, N, N '-dicyclohexyl carbon two sub- Amine, O-BTA-N, N, N ', N '-tetramethylurea Tetrafluoroboric acid, 2-(7-azo BTA)-N, N, N ', N '-four MU hexafluorophosphoric acid ester or 1-propyl group tricresyl phosphate cyclic acid anhydride；

B) being dissolved in organic solvent by the product of step a), add deprotecting regent and slough N-terminal protection, temperature is 0 DEG C To backflow；Wherein, a) the product trifluoroacetic acid of aminoterminal tertbutyloxycarbonyl protection sloughs protection, aminoterminal fluorenes methoxy carbonyl A) the product piperidines of base protection sloughs protection；The solvent used be acetonitrile, methanol, oxolane, dichloromethane, 1,2-bis- Ethyl chloride, chloroform, toluene, normal hexane, hexamethylene, t-butyl methyl ether or the mixed solvent of above-mentioned solvent；

C) by the cholic acid or derivatives thereof that c-terminus is free, dissolve in organic solvent with the product of step b), add coupling examination Agent and organic amine or inorganic base, react 1-72 hour, and temperature is 0 DEG C to 45 DEG C；Solvent be acetonitrile, chloroform, dichloromethane, 1,2- Dichloroethanes, DMF, toluene, normal hexane, hexamethylene, oxolane, t-butyl methyl ether or above-mentioned solvent Mixed solvent；Coupling reagent uses 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, N, N '-dicyclohexyl carbon Diimine, O-BTA-N, N, N ', N '-tetramethylurea Tetrafluoroboric acid, 2-(7-azo BTA)-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester or 1-propyl group tricresyl phosphate cyclic acid anhydride；The inorganic base used is sodium carbonate, sodium bicarbonate, carbon Acid potassium or potassium bicarbonate, the organic amine used is DIPEA or triethylamine.

5. method as claimed in claim 4, wherein, adds catalyst DMAP in step a).

6. method as claimed in claim 4, wherein, the solvent employed in step b) is acetonitrile or dichloromethane.

7. method as claimed in claim 4, wherein, the solvent in step c) be dichloromethane, 1,2-dichloroethanes or N, N- Dimethylformamide.

8. a pharmaceutical composition, wherein contain therapeutically effective amount the compound as according to any one of claim 1-3 or Its pharmaceutically acceptable salt, and pharmaceutically acceptable carrier.

9. compound or its pharmaceutically acceptable salt as according to any one of claim 1-3 are used for preventing or controlling in preparation Treat the purposes in the medicine of hypertension or its complication, atherosclerosis, metabolism syndrome or tumor.

10. purposes as claimed in claim 9, it is characterised in that: described metabolism syndrome is diabetes or its complication, high blood Fat disease, obesity, high glucagon disease, insulin resistant or fasting hyperglycaemia.

11. purposes as claimed in claim 10, it is characterised in that: described diabetes are type 2 diabetes mellitus, and its complication includes: Diabetic nephropathy, diabetic foot, diabetic neuropathy or diabetes complicated cardiovascular and cerebrovascular disease.