Two-arm branched fatty acid chain, receptor agonist modified therewith, and use thereof

Abstract

The present application discloses a two-arm branched fatty acid side chain, a receptor agonist modified therewith, and a use thereof. By adjusting the number and structure of side chain arms of the fatty acid chain, optimizing the type of a core molecule, and using the fatty acid chain to modify a receptor agonist, the present application significantly improves the physical and chemical stability of the receptor agonist and the therapeutic effect of the receptor agonist on reducing blood glucose and body weight. The fatty acid chain and the receptor agonist modified therewith disclosed in the present application can reduce blood glucose levels and improve sugar tolerance and has the effects of controlling appetite, food intake, and caloric intake, increasing energy consumption, preventing weight gain, promoting weight loss, and reducing excess body weight. In addition, the fatty acid chain and the receptor agonist modified therewith disclosed in the present application have relatively long half-life and can effectively prolong in vivo drug actions.

Description

A two-arm branched fatty acid chain, receptor agonists modified with it, and their applications. Technical Field

[0001] This application relates to the pharmaceutical field, and in particular to a two-arm branched fatty acid chain, receptor agonists modified therefrom, and their applications. Background Technology

[0002] With social development and changing lifestyles, the number of overweight and obese individuals worldwide is constantly increasing. Obesity leads to metabolic abnormalities, causing numerous health problems such as cardiovascular disease and type 2 diabetes. Type 2 diabetes is the most common form of diabetes, stemming from impaired β-cell function, relative insulin deficiency, and insulin resistance; it is also a common cause of non-alcoholic fatty liver disease. Incretins secreted by the gastrointestinal tract play a crucial role in maintaining blood glucose homeostasis. Among them, glucose-dependent insulin-releasing peptide (GIP) and glucagon-like peptide-1 (GLP-1) are two major incretin hormones that increase glucose-dependent insulin secretion to exert a hypoglycemic effect.

[0003] US patent document US20230233650A1 discloses a fatty acid-modified urocortin 2 derivative, belonging to the corticotropin-releasing factor (CRF) family, which can be used to treat and / or prevent obesity and / or diabetes. It comprises a polypeptide and substituents, wherein the substituents are equivalent to the fatty acid chain of this application, and it has the structure shown in the following formula:

[0004] Patent document CN102918055A discloses a glucagon peptide containing substituents, wherein the substituents are equivalent to the fatty acid chain of this application, and it has the structure shown in the following formula:

[0005] Modifying glucagon peptides with substituents can improve the physical stability and solubility of glucagon agonists.

[0006] However, the peptide compounds in the prior art are not very effective in terms of physical and chemical stability, as well as in lowering blood sugar, reducing weight, improving glucose tolerance, and prolonging drug half-life. They have poor activity, and there are no reports of modifying peptide compounds with two-arm branched fatty acid side chains to improve the drug's effects in lowering blood sugar, reducing weight, improving glucose tolerance, and prolonging drug half-life. Summary of the Invention

[0007] To overcome the deficiencies in the prior art and improve the physical and chemical stability of receptor agonists as well as their properties in lowering blood sugar, reducing weight, improving glucose tolerance, and prolonging half-life, this application provides a fatty acid chain and receptor agonists modified therefrom, and their applications.

[0008] The first aspect disclosed in this application provides a fatty acid chain having the structure shown in formula (Ⅰ):

[0009] Where X1 and X2 are each independently C 1-25 Straight-chain / branched alkyl groups, in some embodiments, X1 and X2 are each independently C1. 10-20 Straight-chain / branched alkyl groups, in some embodiments, X1 and X2 are each independently C1. 13-20 Straight-chain / branched alkyl groups.

[0010] In some implementations, X1 and X2 are both C 14-18 Straight-chain / branched alkyl groups.

[0011] In some implementations, X1 and X2 are both C 16 Straight-chain / branched alkyl groups.

[0012] In some implementations, X1 and X2 are both C 18 Straight-chain / branched alkyl groups.

[0013] R1 and R2 are each independent of each other.

[0014] In some implementations, R1 and R2 are both

[0015] L1 and L2 are each independent of each other. Among them, R3 is X3 is C 1- 10 Straight-chain / branched alkylene, in some embodiments, X3 is C 1-8 Straight-chain / branched alkylene, in some embodiments, X3 is C 1-5 Straight-chain / branched alkylene compounds.

[0016] In some implementations, X3 is ethylene.

[0017] In some implementations, R3 is

[0018] L3 is C 1-10 In some embodiments, L3 is a straight-chain / branched alkylene group. 1-8 In some embodiments, L3 is a straight-chain / branched alkylene group. 1-5Straight-chain / branched alkylene compounds.

[0019] In some implementations, L3 is butylene.

[0020] a and b are each independently selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In some implementations, a and b are each independently selected from integers from 1 to 5.

[0021] In some implementations, a is 2.

[0022] In some implementations, b is 2.

[0023] In some embodiments, the fatty acid chain has the structure shown in formula (II):

[0024] In some embodiments, the fatty acid chain has the structure shown in formula (Ⅲ):

[0025] In some embodiments, the fatty acid chain has the structure shown in formula (Ⅲ-1):

[0026] In some embodiments, the fatty acid chain has the structure shown in formula (Ⅲ-2):

[0027] In other embodiments, the fatty acid chain has the structure shown in formula (Ⅳ):

[0028] In other embodiments, the fatty acid chain has the structure shown in formula (V):

[0029] A second aspect of this application discloses a receptor agonist modified by a fatty acid chain as described in the first aspect.

[0030] Furthermore, the amino acid sequence of the receptor agonist is shown in SEQ ID NO: 1:

[0031] Tyr-{Aib}-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{Aib}-Leu-Asp-Lys-Ile-Ala-Gln-Ly s-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2.

[0032] Furthermore, the lysine residue at position 20 of the receptor agonist is linked to a fatty acid chain as described in the first aspect.

[0033] A third aspect of this application discloses the use of a fatty acid chain as described in the first aspect in modifying receptor agonists.

[0034] Furthermore, the receptor agonist is selected from monoreceptor agonists, bireceptor agonists, or trireceptor agonists.

[0035] In some embodiments, the receptor agonist is a dual receptor agonist.

[0036] In some implementations, the receptors are GLP-1 and GIP.

[0037] The fourth aspect disclosed in this application provides the use of the receptor agonist as described in the second aspect.

[0038] Furthermore, the application includes at least one of the following:

[0039] (1) Application in the preparation of products for the prevention and treatment of diabetes;

[0040] (2) Application in the preparation of products that improve glucose tolerance;

[0041] (3) Application in the preparation of products that lower blood sugar;

[0042] (4) Application in the preparation of products for controlling appetite or food intake or calorie intake;

[0043] (5) Application in the preparation of products that increase the body's energy consumption;

[0044] (6) Application in the preparation of products that prevent weight gain;

[0045] (7) Application in the preparation of products that promote weight loss;

[0046] (8) Application in the preparation of products that reduce body weight;

[0047] (9) Application in the preparation of products for the prevention and treatment of obesity;

[0048] (10) Application in the preparation of products for the prevention and treatment of dyslipidemia;

[0049] (11) Application in the preparation of products for the prevention and treatment of metabolic syndrome.

[0050] Furthermore, the product also includes pharmaceutically acceptable carriers, diluents, or excipients.

[0051] The fifth aspect disclosed in this application provides the use of the receptor agonist as described in the second aspect.

[0052] Furthermore, the application includes at least one of the following:

[0053] (1) Its application in the prevention and treatment of diabetes;

[0054] (2) Application in improving glucose tolerance;

[0055] (3) Application in lowering blood sugar;

[0056] (4) Application in controlling appetite or food intake or calorie intake;

[0057] (5) Application in increasing the body's energy consumption;

[0058] (6) Application in preventing weight gain;

[0059] (7) Application in promoting weight loss;

[0060] (8) Application in weight loss;

[0061] (9) Application in the prevention and treatment of obesity;

[0062] (10) Application in the prevention and treatment of dyslipidemia;

[0063] (11) Application in the prevention and treatment of metabolic syndrome. Beneficial effects:

[0064] This application significantly improves the physical and chemical stability of receptor agonists and their therapeutic effects in lowering blood sugar and reducing weight by adjusting the number and structure of the side chain arms of fatty acid chains and improving the types of core molecules, and using them to modify receptor agonists. The fatty acid chains and their modified receptor agonists disclosed in this application can lower blood sugar levels and improve glucose tolerance; at the same time, they can control appetite, food intake and calorie intake, increase energy expenditure, prevent weight gain, promote weight loss and reduce overweight; in addition, the fatty acid chains and their modified receptor agonists disclosed in this application have a long half-life, which can effectively prolong the duration of drug action in the body.

[0065] The term C as used in this application 1-25 Straight-chain / branched alkyl groups, including methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, C5 straight-chain / branched alkyl groups, C6 straight-chain / branched alkyl groups, C7 straight-chain / branched alkyl groups, C8 straight-chain / branched alkyl groups, C9 straight-chain / branched alkyl groups, C 10 Straight-chain / branched alkyl, C 11 Straight-chain / branched alkyl, C 12 Straight-chain / branched alkyl, C 13 Straight-chain / branched alkyl, C 14Straight-chain / branched alkyl, C 15 Straight-chain / branched alkyl, C 16 Straight-chain / branched alkyl, C 17 Straight-chain / branched alkyl, C 18 Straight-chain / branched alkyl, C 19 Straight-chain / branched alkyl, C 20 Straight-chain / branched alkyl, C 21 Straight-chain / branched alkyl, C 22 Straight-chain / branched alkyl, C 23 Straight-chain / branched alkyl, C 24 Straight-chain / branched alkyl, C 25 Straight-chain / branched alkyl groups.

[0066] The term C as used in this application 10-20 Straight-chain / branched alkyl groups, including C 10 Straight-chain / branched alkyl, C 11 Straight-chain / branched alkyl, C 12 Straight-chain / branched alkyl, C 13 Straight-chain / branched alkyl, C 14 Straight-chain / branched alkyl, C 15 Straight-chain / branched alkyl, C 16 Straight-chain / branched alkyl, C 17 Straight-chain / branched alkyl, C 18 Straight-chain / branched alkyl, C 19 Straight-chain / branched alkyl, C 20 Straight-chain / branched alkyl groups.

[0067] The term C as used in this application 13-20 Straight-chain / branched alkyl groups, including C 13 Straight-chain / branched alkyl, C 14 Straight-chain / branched alkyl, C 15 Straight-chain / branched alkyl, C 16 Straight-chain / branched alkyl, C 17 Straight-chain / branched alkyl, C 18 Straight-chain / branched alkyl, C 19 Straight-chain / branched alkyl, C 20 Straight-chain / branched alkyl groups.

[0068] The term C as used in this application 14-18 Straight-chain / branched alkyl groups, including C 14 Straight-chain / branched alkyl, C 15 Straight-chain / branched alkyl, C 16 Straight-chain / branched alkyl, C 17 Straight-chain / branched alkyl, C 18 Straight-chain / branched alkyl groups.

[0069] The term C as used in this application1-10 Straight-chain / branched alkyl groups, including methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, C5 straight-chain / branched alkyl groups, C6 straight-chain / branched alkyl groups, C7 straight-chain / branched alkyl groups, C8 straight-chain / branched alkyl groups, C9 straight-chain / branched alkyl groups, C 10 Straight-chain / branched alkyl groups.

[0070] The term C as used in this application 1-8 Straight-chain / branched alkyl groups, including methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, C5 straight-chain / branched alkyl, C6 straight-chain / branched alkyl, C7 straight-chain / branched alkyl, and C8 straight-chain / branched alkyl.

[0071] The term C as used in this application 1-5 Straight-chain / branched alkyl groups, including methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, and C5 straight-chain / branched alkyl groups. Attached Figure Description

[0072] Figure 1 shows the HPLC chromatogram of JK-1246R-009.

[0073] Figure 2 shows the mass spectrum of JK-1246R-009.

[0074] Figure 3 shows the HPLC chromatogram of JK-1246R-010.

[0075] Figure 4 shows the mass spectrum of JK-1246R-010.

[0076] Figure 5 shows the HPLC chromatogram of JK-1246R-011.

[0077] Figure 6 shows the mass spectrum of JK-1246R-011.

[0078] Figure 7 shows the HPLC spectrum of JK-1246R-012.

[0079] Figure 8 shows the mass spectrum of JK-1246R-012.

[0080] Figure 9 shows the improved IPGTT results in wild-type mice of Example 7 after a single subcutaneous injection of 30 nmol / kg for 72 hours.

[0081] Figure 10 shows the improved IPGTT results in wild-type mice of Example 7 after a single subcutaneous injection of 30 nmol / kg for 72 hours.

[0082] Figure 11 shows the improved IPGTT results in wild-type mice of Example 7 after a single subcutaneous injection of 30 nmol / kg for 144 hours.

[0083] Figure 12 shows the improved IPGTT results in wild-type mice of Example 7 after a single subcutaneous injection of 30 nmol / kg for 144 hours.

[0084] Figure 13 shows the improved IPGTT results in wild-type mice of Example 7 after a single subcutaneous injection of 30 nmol / kg for 192 hours.

[0085] Figure 14 shows the improved IPGTT results in wild-type mice of Example 7 after a single subcutaneous injection of 30 nmol / kg for 192 hours.

[0086] Figure 15 shows the results of weight loss in diet-induced obese mice in Example 8 after repeated subcutaneous injection of a drug at a dose of 30 nmol / kg.

[0087] Figure 16 shows the results of weight loss in diet-induced obese mice in Example 8 after repeated subcutaneous injection of a drug at a dose of 30 nmol / kg.

[0088] Figure 17 shows the results of reducing serum triglycerides in diet-induced obese mice in Example 8 by repeated subcutaneous injection at a dose of 30 nmol / kg.

[0089] Figure 18 shows the results of reducing serum total cholesterol in diet-induced obese mice in Example 8 by repeated subcutaneous injection at a dose of 30 nmol / kg. Detailed Implementation

[0090] In order to better understand the technical content disclosed in this application, the following embodiments are provided in detail to clearly and completely describe the technical solution of this application. The purpose is only to better understand the content of this application and not to limit the scope of protection of this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0091] Example 1: Preparation and Characterization of JK-1246R-009

[0092] (1) Synthesis of the side chain tBuO-DOC-Glu(PEG3-PEG2-OH)-OtBu

[0093] Dissolve 0.5 g of tert-butyl docosanoate in 10 ml of dichloromethane, add 0.21 ml of DIEA, then add 0.39 g of TSTU, stir for 3 hours, add 0.2 ml of TEA, then add 0.26 g of 1-tert-butyl-L-glutamic acid, stir overnight. After the reaction is complete, wash twice with 10 ml of 0.2N HCl, dry the DCM phase and filter, add 0.21 ml of DIEA to the filtrate, then add 0.39 g of TSTU, stir for 3 hours, then add 0.2 ml of TEA and 0.5 g of NH2-PEG3-PEG2-CM, stir overnight, wash twice with 10 ml of 0.2N HCl after the reaction is complete, dry the dichloromethane phase, filter and concentrate to obtain the crude product. The crude product was purified by column chromatography (silica gel column chromatography, mobile phase: dichloromethane-methanol (100:1, v / v)) to give fatty acid side chain tBuO-DOC-Glu(PEG3-PEG2-OH)-OtBu (0.41 g, white solid), yield 37%. MS m / z (ESI): 946.7 [M+1] + .

[0094] (2) Amino acid coupled with fatty acid side chain (tBuO-DOC-Glu(PEG3-PEG2-OH)-OtBu)

[0095] 250 mg of tBuO-DOC-Glu(PEG3-PEG2-OH)-OtBu was dissolved in 3 mL of acetonitrile, and 82 mg of triethylamine was added. 77 mg of 3,5-dichloro-2-hydroxybenzenesulfonyl chloride was dissolved in tetrahydrofuran solution, and this tetrahydrofuran solution was added dropwise to the reaction system. After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, the solvent was removed by rotary evaporation of the reaction solution, MTBE was added, and the mixture was placed in an ice bath for half an hour, resulting in the precipitation of a white insoluble substance. The precipitate was removed by suction filtration, and the filtrate was collected and rotary evaporated to obtain a yellow oily substance. The oily substance from the previous step was dissolved in 10 times its volume of formic acid, and stirred at room temperature for about 20 hours. After the reaction was complete, the reaction solution was removed by rotary evaporation to obtain 200 mg of the oily active ester HO-DOC-Glu(PEG3-PEG2-HBSA)-OH, with a yield of 64%. MS m / z (ESI): 1058.44 [M+1]. + It is used directly in subsequent coupling reactions.

[0096] (3) Fatty acid side chain modification of peptides

[0097] Take 200 mg of peptide (YAibEGTFTSDYSIAibLDKIAQKAFVQWLIAGGPSSGAPPPS-NH2), add 3 mL of water, and adjust the pH to 10 with TEA. Dissolve 102 mg (2.0 eq, 0.098 mmol) of the active ester HO-DOC-Glu(PEG3-PEG2-HBSA)-OH in 0.5 mL of NMP, and add it dropwise to the alkaline aqueous solution of the peptide. During the reaction, adjust the pH of the system to maintain it between 11.0 and 12.0 with 1 M NaOH, and react for 2 h.

[0098] After the reaction was complete, acetic acid was added dropwise to quench the reaction, and 3 mL of acetonitrile was added to clarify the system. Purification was performed using reversed-phase high-performance liquid chromatography (HPLC), and the high-purity fraction was collected. The fraction was desalted by gel column chromatography and lyophilized to obtain JK-1246R-009 (28 mg, white solid powder), with a yield of 10%. The final structural formula of JK-1246R-009 is as follows:

[0099] (4) Characterization of JK-1246R-009

[0100] Characterization of JK-1246R-009 is shown in Figures 1 and 2. HPLC-UV: 84.439% (Column: Phenomenex Kinetex XB-C18 250*4.6mm 5um, 100A; Acquisition wavelength: 210nm; Mobile phase A: 0.1% TFA water; Mobile phase B: Acetonitrile-methanol (8:2, v / v)); Side chain substitution sites determined by enzymatic digestion: Lys20, Lys16; LC-MS: m / z: 1900.9945 (M+3H). 3+ .

[0101] Example 2: Preparation and Characterization of JK-1246R-010

[0102] (1) Fatty acid side chain modification of peptides

[0103] Take 200 mg of peptide (YAibEGTFTSDYSIAibLDKIAQKAFVQWLIAGGPSSGAPPPS-NH2), add 3 mL of water, and adjust the pH to 10 with TEA. Dissolve 102 mg (2.0 eq, 0.098 mmol) of the active ester HO-DOC-Glu(PEG3-PEG2-HBSA)-OH in 0.5 mL of NMP, and add it dropwise to the alkaline aqueous solution of the peptide. During the reaction, adjust the pH of the system to maintain it between 11.0 and 12.0 with 1 M NaOH, and react for 2 h.

[0104] After the reaction was complete, acetic acid was added dropwise to quench the reaction, and 3 mL of acetonitrile was added to clarify the system. The mixture was then purified using reversed-phase high-performance liquid chromatography (HPLC), and the high-purity fraction was collected. The fraction was desalted by gel column chromatography and lyophilized to obtain JK-1246R-010 (9 mg, white solid powder), with a yield of 3%.

[0105] (2) Characterization of JK-1246R-010

[0106] Characterization of JK-1246R-010 is shown in Figures 1 and 2. HPLC-UV: 90.217% (Column: Phenomenex Kinetex XB-C18 250*4.6mm 5um, 100A; Acquisition wavelength: 210nm; Mobile phase A: 10mM ammonium formate buffer-acetonitrile (9:1, v / v); Mobile phase B: Acetonitrile-isopropanol-water (3:1:1, v / v / v)); Side chain substitution site determined by enzymatic digestion: N-terminus, Lys20; LC-MS: m / z: 1900.9949 (M+3H). 3+ The final structural formula of JK-1246R-010 is as follows:

[0107] Example 3: Preparation and Characterization of JK-1246R-011

[0108] (1) Synthesis of side chain (tBuO-ICO-Glu)2-Lys(AEEA-AEEA-OH)

[0109] 40 g of eicosanoic acid monotert-butyl ester was dissolved in 800 ml of dichloromethane. 15.2 g of TEA and 33 g of TSTU were added. After stirring for 3 hours, 7.6 g of TEA and 24.4 g of 1-tert-butyl-L-glutamic acid were added, and the mixture was stirred overnight. After the reaction was complete, the mixture was washed twice with 500 ml of 0.2 N HCl, and then once with 500 ml of saturated brine. The dichloromethane phase was dried, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (silica gel column chromatography, mobile phase: dichloromethane-methanol (100:1, v / v)) to give compound 3 (42 g, white solid), yield 72%. MS m / z (ESI): 584.7 [M+1] + .

[0110] 6.3 g of compound 3 was dissolved in 130 mL of dichloromethane, 1.5 g of TEA was added, followed by 5.6 g of HATU and 1.52 g of L-lysine benzyl ester. The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the mixture was washed twice with 100 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography (silica gel column chromatography, mobile phase: dichloromethane-methanol (100:1, v / v)) to give compound 4 (6.0 g, white solid), in 68% yield. MS m / z (ESI): 1368.0 [M+1] + .

[0111] 6.0 g of compound 4 was dissolved in a mixed solvent of 30 mL methanol and 30 mL ethyl acetate. 0.6 g of Pd / C (10%) was added, and the mixture was stirred at room temperature under hydrogen atmosphere for 4 h. Solid Pd / C was removed by filtration, and the solution was concentrated to obtain compound 5 (5.3 g, a pale yellow oil), with a yield of 97%. This compound was used directly in the next reaction. MS m / z (ESI): 1278.1 [M+1] + .

[0112] 2.5 g of compound 5 was dissolved in 40 mL of dichloromethane. 484 mg of DCC and 270 mg of N-hydroxysuccinimide were added, and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the white solid was removed by filtration. 395 mg of TEA and 904 mg of AEEA-AEEA were added to the filtrate, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was washed twice with 30 mL of 0.2 N HCl, and then once with 30 mL of saturated brine. The dichloromethane phase was dried, filtered, and concentrated to obtain the crude product. The crude product was purified once by silica gel column chromatography, and then purified by high-performance liquid chromatography (acetonitrile / 0.1 TFA water) to obtain the product (tBuO-ICO-Glu)2-Lys(AEEA-AEEA-OH) (0.9 g, off-white semi-solid), with a yield of 30%. MS m / z (ESI): 1568.7 [M+1] + .

[0113] (2) Synthesis of active ester (tBuO-ICO-Glu)2-Lys(AEEA-AEEA-HBSA)

[0114] Dissolve 250 mg of (tBuO-ICO-Glu)2-Lys(AEEA-AEEA-OH) in 3 mL of acetonitrile, and add 82 mg of triethylamine. Dissolve 50 mg of 3,5-dichloro-2-hydroxybenzenesulfonyl chloride in tetrahydrofuran solution, and add the tetrahydrofuran solution dropwise to the reaction system. After the addition is complete, stir the reaction mixture overnight at room temperature. After the reaction is complete, remove the solvent by rotary evaporation, add MTBE, and incubate in an ice bath for half an hour. A white insoluble substance precipitates out. Filter to remove the precipitate, collect the filtrate, and rotary evaporate the filtrate to obtain a yellow oil. Dissolve the oil in 10 times its volume of formic acid, stir at room temperature for about 20 h, and after the reaction is complete, remove the oil by rotary evaporation to obtain 200 mg of the oily active ester (tBuO-ICO-Glu)2-Lys(AEEA-AEEA-HBSA), with a yield of 70%. MS m / z (ESI): 1567.78 [M+1]. + It is used directly in subsequent coupling reactions.

[0115] (3) Fatty acid side chain modification of peptides

[0116] Take 200 mg of peptide (YAibEGTFTSDYSIAibLDKIAQKAFVQWLIAGGPSSGAPPPS-NH2), add 3 mL of water, and adjust the pH to 10 with TEA. Dissolve 107 mg (1.2 eq, 0.06 mmol) of active ester (tBuO-ICO-Glu)2-Lys(AEEA-AEEA-HBSA) in 0.5 mL of NMP, and add it dropwise to the alkaline aqueous solution of the peptide. During the reaction, adjust the pH of the system with 1M NaOH to maintain it between 11.0 and 12.0, and react for 2 h.

[0117] After the reaction was complete, acetic acid was added dropwise to quench the reaction, and 3 mL of acetonitrile was added to clarify the system. Purification was performed using reversed-phase high-performance liquid chromatography (HPLC), and the high-purity fraction was collected. The fraction was desalted by gel column chromatography and lyophilized to obtain JK-1246R-011 (89 mg, white solid powder), with a yield of 33%. The final structural formula of JK-1246R-011 is as follows:

[0118] (4) Characterization of JK-1246R-011

[0119] Characterization of JK-1246R-011 is shown in Figures 5 and 6. HPLC-UV: 98.33% (Column: Phenomenex Kinetex XB-C18 250*4.6mm 5um, 100A; Acquisition wavelength: 210nm; Mobile phase A: 10mM ammonium formate buffer-acetonitrile (9:1, v / v); Mobile phase B: Acetonitrile-isopropanol-water (3:1:1, v / v / v)); Side chain substitution site determined by enzymatic digestion: Lys 20 (97.85%); LC-MS: m / z: 1349.8 (M+4H). 4+ .

[0120] Example 4: Preparation and Characterization of JK-1246R-012

[0121] Synthesis of (1))(tBuO-STE-Glu)2-Lys(AEEA-AEEA-OH)

[0122] 37 g of tert-butyl octadecanoate was dissolved in 800 ml of dichloromethane. 15.2 g of TEA and 33 g of TSTU were added. After stirring for 3 hours, 7.6 g of TEA and 24.4 g of 1-tert-butyl-L-glutamic acid were added, and the mixture was stirred overnight. After the reaction was complete, the mixture was washed twice with 500 ml of 0.2 N HCl, and then once with 500 ml of saturated brine. The dichloromethane phase was dried, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to give compound 8 (38 g, white solid), with a yield of 69%. MS m / z (ESI): 557.1 [M+1] + .

[0123] 7.0 g of compound 8 was dissolved in 150 mL of dichloromethane, 2.3 g of TEA was added, followed by 5.5 g of HATU and 1.77 g of L-lysine benzyl ester. The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the mixture was washed twice with 100 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography to give compound 9 (8.1 g, white solid), in 82% yield. MS m / z (ESI): 1312.3 [M+1] + .

[0124] 8.0 g of compound 9 was dissolved in a mixed solvent of 40 mL methanol and 40 mL ethyl acetate. 0.8 g of Pd / C (10%) was added, and the mixture was stirred at room temperature under hydrogen atmosphere for 4 h. Solid Pd / C was removed by filtration, and the solution was concentrated to obtain product compound 10 (6.2 g, a pale yellow oil), with a yield of 83%. This product was used directly in the next reaction. MS m / z (ESI): 1222.3 [M+1] + .

[0125] 1.2 g of compound 10 was dissolved in 20 mL of dichloromethane. 245 mg of DCC and 130 mg of N-hydroxysuccinimide were added, and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the white solid was removed by filtration. 210 mg of TEA and 454 mg of AEEA-AEEA were added to the filtrate, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was washed twice with 15 mL of 0.2 N HCl, and then once with 15 mL of saturated brine. The dichloromethane phase was dried, filtered, and concentrated to obtain the crude product. The crude product was purified once by silica gel column chromatography, and then purified by high-performance liquid chromatography (acetonitrile / 0.1 TFA water) to obtain product 11 (0.6 g, off-white semi-solid), yield 40%. MS m / z (ESI): 1512.7 [M+1] + .

[0126] (2) Synthesis of active ester (tBuO-STE-Glu)2-Lys(AEEA-AEEA-HBSA)

[0127] Dissolve 250 mg of (tBuO-STE-Glu)2-Lys(AEEA-AEEA-OH) in 3 mL of acetonitrile, and add 90 mg of triethylamine. Dissolve 53 mg of 3,5-dichloro-2-hydroxybenzenesulfonyl chloride in tetrahydrofuran solution, and add the tetrahydrofuran solution dropwise to the reaction system. After the addition is complete, stir the reaction mixture overnight at room temperature. After the reaction is complete, remove the solvent by rotary evaporation, add MTBE, and incubate in an ice bath for half an hour. A white insoluble precipitate forms. Filter to remove the precipitate, collect the filtrate, and rotary evaporate the filtrate to obtain a yellow oily substance. The oily substance from the previous step was dissolved in 10 times its volume of formic acid and stirred at room temperature for about 20 hours. After the reaction was complete, the reaction solution was removed by rotary evaporation to obtain 205 mg of the oily active ester (tBuO-STE-Glu)2-Lys(AEEA-AEEA-HBSA), with a yield of 71% and MS m / z (ESI): 1513.68 [M+1]. + It is used directly in subsequent coupling reactions.

[0128] (3) Fatty acid side chain modification of peptides

[0129] Take 200 mg of peptide (YAibEGTFTSDYSIAibLDKIAQKAFVQWLIAGGPSSGAPPPS-NH2), add 3 mL of water, and adjust the pH to 10 with TEA. Dissolve 105 mg (1.2 eq, 0.06 mmol) of active ester (tBuO-STE-Glu)2-Lys(AEEA-AEEA-HBSA) in 0.5 mL of NMP, and add it dropwise to the alkaline aqueous solution of the peptide. During the reaction, adjust the pH of the system with 1M NaOH to maintain it between 11.0 and 12.0, and react for 2 h.

[0130] After the reaction was complete, acetic acid was added dropwise to quench the reaction, and 3 mL of acetonitrile was added to clarify the system. The mixture was then purified using reversed-phase high-performance liquid chromatography (HPLC), and the high-purity fraction was collected. The fraction was desalted by gel column chromatography and lyophilized to obtain JK-1246R-012 (81 mg, white solid powder), with a yield of 29%. The final structural formula of JK-1246R-012 is as follows:

[0131] (4) Characterization of JK-1246R-012

[0132] Characterization of JK-1246R-012 is shown in Figures 9 and 10. HPLC-UV: 97.82% (Column: Phenomenex Kinetex XB-C18 250*4.6mm 5um, 100A; Acquisition wavelength: 210nm; Mobile phase A: 10mM ammonium formate buffer-acetonitrile (9:1, v / v); Mobile phase B: Acetonitrile-isopropanol-water (3:1:1, v / v / v)); Side chain substitution site determined by enzymatic digestion: Lys 20 (97.47%); LC-MS: m / z: 1335.7 (M+4H). 4+ .

[0133] Example 5, Cell Viability

[0134] I. Methods: Functional activity was determined using cAMP in HEK-293 clonal cell lines expressing hGIPR and hGLP-1R.

[0135] 1. Preparation of test culture medium and diluent

[0136] Prepare the plating medium and store at 2–8°C for up to 6 months. Preparation method: Mix 90% DMEM and 5% FBS thoroughly. (e.g., 450 ml DMEM and 50 ml FBS).

[0137] 2. Preparation of subculture medium

[0138] DMEM,10% FBS,300μg / ml G418,50μg / ml HygromycinB

[0139] 3. Preparation of test solution: Using the Bright-Lite Luciferase Assay System, add 100ml of staining solution to the substrate and mix well to obtain the test solution.

[0140] 4. Preparation of sample gradient solution

[0141] Weigh a certain amount of sample and control standard and dissolve them in sterile water to prepare a 1mM stock solution.

[0142] The stock solution was diluted to 120 nM (working concentration 60 nM) with stimulation buffer to obtain solution SD1. Starting from SD1, a total of 10 concentration gradients were obtained by 4-fold serial dilution.

[0143] 5. Cell inoculation

[0144] Cells were passaged and expanded beforehand. When the cell density reached 80%, the cells were washed 1-2 times with PBS, digested with 0.25% trypsin, and digestion was stopped after the cells became rounded under a microscope. Cells with a viability greater than 95% were collected by trypan blue staining and prepared into a density of 0.3 × 10⁻⁶ cells / year. 6 20 μl of cell suspension per well of a 384-well plate (6000 cells / well) was seeded.

[0145] 6. Drug treatment

[0146] The following day, 20 μl of serially diluted sample was added to each well and transferred to a 37°C cell culture incubator for further incubation for 3 hours.

[0147] 7. Detection solution treatment

[0148] After incubation, remove the cell plate and allow it to equilibrate to room temperature for 15 minutes. Then, add 40 μl of room temperature detection solution to each well. Place the culture plate on a microplate shaker and shake at 500 rpm for 3 minutes. Finally, collect the data using a microplate reader.

[0149] 8. Detection and Data Processing

[0150] Using a SpectraMax Paradigm microplate reader, signal values ​​were collected under emission conditions of 616 nm and 665 nm after excitation at 340 nm. The ratio of acceptor to donor emission signals for each individual well was calculated, and a graph showing the relationship between the HTRF ratio and compound concentration was plotted. The concentration of cAMP was determined by standard curve analysis, and a graph showing the relationship between compound concentration and cAMP concentration was plotted, with E0.05 values ​​provided. max and EC 50 value.

[0151] II. Sample Information

[0152] III. Results

[0153] Table 1. Measurement of functional hGIPR and hGLP-1R

[0154] The experimental results are shown in Table 1, and the specific analysis is as follows:

[0155] (1) GLP-1R activity: tirzepatide>011>012>>009.

[0156] (2) GIPR activity: tirzepatide>012>011>>009.

[0157] Based on the above results, JK-0246R-011 and JK-0246R-012, with Lys 20 as the side chain substitution site, exhibit better cell activity than JK-0246R-009, with Lys 20 and Lys 16 as the side chain substitution sites.

[0158] Example 6: Affinity of Human Serum Albumin

[0159] I. Methods: An amino-coupled method was employed, in which Human Serum Albuim (HSA) was directly immobilized onto a CM5 chip using a Biacore 8K microarray. The analyte was diluted to the desired concentration gradient using running buffer (10 mM HEPES, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% P2O). Multiple cycles of kinetic analysis were performed, with each cycle consisting of 180 seconds of injection followed by 180 seconds of dissociation before proceeding to the next cycle. Affinity kinetic data between the human HSA receptor protein and the analyte were obtained. The final data were then analyzed using Biacore Insight Evaluation Software (V 2.0.15.12933) with a 1:1 model for kinetic fitting.

[0160] 1) Prepare running buffer: 10mM HEPES, pH7.4, 137mM NaCl, 2.7mM KCl, 0.05% P20.

[0161] 2) CM5 chip activation: Activate with 400mM EDC and 100mM NHS at a flow rate of 10μL / min for 600s.

[0162] 3) Target protein coupling: HSA was diluted to 10 μg / mL with 10 mM sodium acetate (pH 5.0) (this has been used in other projects; HSA coupling on the chip is more effective at pH 5.0). Coupling was performed at a flow rate of 10 μL / min for 600 s. For different experiments, the coupling value was evaluated based on the molecular weight analysis of the analyte to determine if it met the requirements.

[0163] 4) CM5 chip sealing: seal with 1M ethanolamine at a flow rate of 10μL / min for 600s.

[0164] 6) Analyte concentration: Dilute the analyte to the desired concentration using running buffer. Note: In the preliminary experiments, the analyte concentrations (20 μM, 2 μM, 0.2 μM, 0 μM) were determined based on the preliminary experimental results, using a 2-fold dilution, resulting in 8 concentration points.

[0165] 7) Sample injection analysis: Each concentration of the analyte working solution is one cycle, with a flow rate of 30 μL / min for 180 s for binding and 180 s for dissociation.

[0166] 8) All results were subjected to kinetics fitting analysis using a 1:1 model.

[0167] II. Sample Information

[0168] III. Results

[0169] Table 2 Affinity of Human Albumin

[0170] The experimental results are shown in Table 2, and the specific analysis is as follows:

[0171] Compared to trizepatide, JK-1246R-009 and JK-1246R-010 bind to and dissociate from albumin very slowly, JK-1246R-0011 binds to and dissociates from albumin slightly slower, and JK-1246R-012 binds to and dissociates from albumin slightly faster.

[0172] Based on the above results, JK-0246R-011 and JK-0246R-012, with a side chain substitution site of Lys 20, have better affinity for human serum albumin than JK-0246R-009, with a side chain substitution site of Lys 20 and Lys 16, and JK-1246R-010, with a side chain substitution site at the N-terminus and Lys 20.

[0173] Example 7: Improving glucose tolerance in mice (IPGTT)

[0174] I. Methods

[0175] C57BL / 6J mice were acclimatized for 5 days. After measuring body weight and fasting blood glucose levels, they were randomly divided into four groups based on body weight: tirzepatide group, JK-1246R-009 group, JK-1246R-010 group, JK-1246R-011 group, and JK-1246R-012 group. Glucose tolerance was measured at 72h, 144h, and 192h after drug administration. Before glucose tolerance testing, mice were placed in metabolic cages and fasted for 12h. Blood was collected from the tail 1 hour before glucose administration (recorded as 0-minute blood glucose). Glucose (2g / kg) was injected intraperitoneally, and blood glucose levels were measured at 15, 30, 60, and 120 minutes after glucose administration. The animals were kept fasted during the experiment to prevent interference from food intake.

[0176] The experimental design is shown in Table 1:

[0177] Table 3

[0178] The solvent solution was prepared as follows: 40 mM Tris-HCl, pH 8.0, containing 0.02% PS-80.

[0179] II. Sample Information

[0180] III. Results

[0181] 72 hours post-drug administration: As shown in Figures 9 and 10, blood glucose levels in wild-type mice in the solvent group rose rapidly 15 minutes after intraperitoneal glucose injection 72 hours post-drug administration, and then decreased to near baseline levels by 120 minutes. Compared with the solvent group, the JK-1246R-009 group showed no activity in improving glucose tolerance (p>0.05). Compared with the solvent group, the tirzepatide group and the JK-1246R-010 group showed weaker activity in improving glucose tolerance at a dose of 30 nmol / kg (p<0.05), and glucose levels at 60 minutes post-glucose administration and thereafter were no difference from the solvent group (p>0.05). Compared with the solvent groups, the JK-1246R-011 and JK-1246R-012 groups showed significant glucose tolerance improvement activity 72 hours after a single subcutaneous injection in wild-type mice (p < 0.05); compared with the tirzepatide group, JK-1246R-009 group and JK-1246R-010 group, the JK-1246R-011 and JK-1246R-012 groups showed significant glucose tolerance improvement activity (p < 0.05); there was no difference in glucose tolerance improvement activity between the JK-1246R-011 and JK-1246R-012 groups (p > 0.05).

[0182] 144 h post-drug administration: As shown in Figures 11 and 12, blood glucose levels in wild-type mice in the solvent group rose rapidly 15 min after intraperitoneal injection of glucose 144 h post-drug administration, and then decreased to near baseline levels by 120 min. Compared with the solvent group, the tirzepatide group, JK-1246R-010 group, and JK-1246R-011 group showed weaker glucose tolerance-improving activity 144 h after a single subcutaneous injection of 30 nmol / kg into wild-type mice. (p<0.05); Compared with the solvent group, the JK-1246R-012 group showed significant glucose tolerance improvement activity 144h after a single subcutaneous injection of the drug at a dose of 30 nmol / kg into wild-type mice (p<0.05); Compared with the tirzepatide group, the JK-1246R-010 group and the JK-1246R-011 group, the JK-1246R-012 group showed significant glucose tolerance improvement activity (p<0.05).

[0183] 192 h post-drug administration: As shown in Figures 13 and 14, blood glucose levels in wild-type mice in the solvent group rose rapidly 15 min after intraperitoneal injection of glucose 192 h post-drug administration, and then decreased to near baseline levels by 120 min. Compared with the solvent group, the JK-1246R-012 group showed weaker glucose tolerance-improving activity 192 h after a single subcutaneous injection into wild-type mice (p < 0.05).

[0184] Example 8: Evaluation of the weight-reducing effects of JK1246R-011 and JK1246R-012

[0185] 1. Experimental Design

[0186] Each group consisted of 6 male C57BL / 6N mice, which were subsequently administered the test substance. The experimental design was as follows:

[0187] 2. Modeling Method

[0188] After a week of acclimatization feeding with the basic diet, all animals were given a high-fat diet to induce modeling.

[0189] Measures to ensure safety: To reduce fighting among mice, provide toys, observe the mice for injuries each time the water is changed, and promptly separate injured mice into individual cages; To reduce the impact on the mice's ability to eat due to overly soft high-fat feed making it difficult for them to wear down their teeth, provide chew sticks.

[0190] 3. Grouping method

[0191] Before the first administration, the animals were randomly divided into 4 groups of 6 each, based on their body weight.

[0192] 4. Administration method

[0193] On the day of administration, the dosage was calculated based on the animal's most recent weight and administered subcutaneously, 30-90 minutes before the start of the dark cycle. Dosing frequency was once every 3, 5, or 6 days, continuing until day 14. Animals in each group were randomly assigned to receive the medication in alternating order.

[0194] 5. Weigh yourself

[0195] All animals were weighed once a week during modeling. After drug administration began, they were weighed once a day. All animals were weighed upon discovery of death, upon unplanned euthanasia due to near death, and before planned euthanasia.

[0196] 6. Blood lipids

[0197] Approximately 0.8 mL of blood was collected from the heart of all animals on day 15, placed in tubes without anticoagulants, allowed to stand for 1–2 hours, centrifuged, and then used to detect triglycerides and total cholesterol (TC). A fully automated animal biochemical testing system was used for blood lipid detection; fasting was required for 6 hours prior to blood collection, but water intake was permitted.

[0198] 7. Data Analysis and Test Report

[0199] All data will be entered into an Excel document and expressed as mean ± standard error. Statistical analysis will be performed using GraphPad Prism 8 (one-way ANOVA, Dunnett's method for multiple comparisons), with a p-value less than 0.05 considered statistically significant*.

[0200] 8. Experimental Results

[0201] As shown in Figures 15 and 16, compared with the model control group at the same time point after drug administration, tirzepatide Q3D, JK-1246R-011Q5D, and JK-1246R-012Q6D, administered continuously for 14 days at a dose of 30 nmol / kg, resulted in a decrease in body weight and corresponding AUC (p < 0.05). Compared with the tirzepatide Q3D group at the same time point after drug administration, the decrease in AUC of body weight for JK-1246R-011Q5D and JK-1246R-012Q6D was stronger than that for tirzepatide (p < 0.05). Compared with the JK-1246R-011Q5D group at the same time point after drug administration, the decrease in AUC of body weight for JK-1246R-012Q6D was stronger than that for JK-1246R-011 (p < 0.05).

[0202] As shown in Figure 17, compared with the model control group, after 14 consecutive days of administration of tirzepatide Q3D, JK-1246R-011Q5D, and JK-1246R-012Q6D at a dose of 30 nmol / kg, serum triglycerides on day 15 decreased (p < 0.05); compared with the tirzepatide Q3D group, there was no difference in serum triglycerides between JK-1246R-011Q5D and JK-1246R-012Q6D (p > 0.05).

[0203] As shown in Figure 18, compared with the model control group, after 14 consecutive days of administration of tirzepatide Q3D, JK-1246R-011Q5D, and JK-1246R-012Q6D at a dose of 30 nmol / kg, serum total cholesterol on day 15 decreased (p < 0.05); compared with the tirzepatide Q3D group, there was no difference in serum total cholesterol between JK-1246R-011Q5D and JK-1246R-012Q6D (p > 0.05).

Claims

1. A fatty acid chain having the structure shown in formula (Ⅰ): in, X1 and X2 are each independently C 1-25 Straight-chain / branched alkyl groups; R1 and R2 are each independent of each other. L1 and L2 are each independent of each other. Among them, R3 is X3 is C 1-10 Straight-chain / branched alkylene chains; L3 is C 1-10 Straight-chain / branched alkylene chains; a and b are each independently selected from integers from 1 to 10.

2. The fatty acid chain as described in claim 1, characterized in that, X1 and X2 are each independently C 10-20 Straight-chain / branched alkyl groups.

3. The fatty acid chain as described in claim 2, characterized in that, X1 and X2 are each independently C 13-20 Straight-chain / branched alkyl groups.

4. The fatty acid chain as described in claim 1, characterized in that, Both R1 and R2 are 5. The fatty acid chain as described in claim 1, characterized in that, X3 is C 1-8 Straight-chain / branched alkylene compounds.

6. The fatty acid chain as described in claim 5, characterized in that, X3 is C 1-5 Straight-chain / branched alkylene compounds.

7. The fatty acid chain as described in claim 6, characterized in that, X3 is ethylene.

8. The fatty acid chain as described in claim 1, characterized in that, L3 is C 1-8 Straight-chain / branched alkylene compounds.

9. The fatty acid chain as described in claim 8, characterized in that, L3 is C 1-5 Straight-chain / branched alkylene compounds.

10. The fatty acid chain as described in claim 9, characterized in that, L3 is butylene.

11. The fatty acid chain as described in claim 1, characterized in that, a and b are each independently selected from integers from 1 to 5.

12. The fatty acid chain as described in claim 1, characterized in that, The fatty acid chain has the structure shown in formula (II):

13. The fatty acid chain as described in claim 12, characterized in that, The fatty acid chain has the structure shown in formula (Ⅲ):

14. The fatty acid chain as described in claim 1, characterized in that, The fatty acid chain has the structure shown in formula (Ⅲ-1) or (Ⅲ-2):

15. The fatty acid chain as described in claim 1, characterized in that, The fatty acid chain has the structure shown in formula (Ⅳ) or (Ⅴ):

16. A receptor agonist, characterized in that, The receptor agonist is modified with a fatty acid chain as described in any one of claims 1-15.

17. The receptor agonist of claim 16, characterized in that, The amino acid sequence of the receptor agonist is shown in SEQ ID NO: 1: Tyr-{Aib}-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{Aib}-Leu-Asp-Lys-Ile-Ala-Gln-Ly s-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2.

18. The receptor agonist of claim 17, characterized in that, The lysine residue at position 20 of the receptor agonist is linked to the fatty acid chain as described in any one of claims 1-15.

19. The use of the fatty acid chain according to any one of claims 1-15 in modifying receptor agonists, characterized in that, The receptor agonist is selected from monoreceptor agonists, bireceptor agonists, or trireceptor agonists.

20. The application as described in claim 19, characterized in that, The receptor agonist is a dual receptor agonist.

21. The use of the receptor agonist according to any one of claims 16-18, characterized in that, The applications include at least one of the following: (1) Application in the preparation of products for the prevention and treatment of diabetes; (2) Application in the preparation of products that improve glucose tolerance; (3) Application in the preparation of products that lower blood sugar; (4) Application in the preparation of products for controlling appetite or food intake or calorie intake; (5) Application in the preparation of products that increase the body's energy consumption; (6) Application in the preparation of products that prevent weight gain; (7) Application in the preparation of products that promote weight loss; (8) Application in the preparation of products that reduce body weight; (9) Application in the preparation of products for the prevention and treatment of obesity; (10) Application in the preparation of products for the prevention and treatment of dyslipidemia; (11) Application in the preparation of products for the prevention and treatment of metabolic syndrome.

Images