Multi-receptor co-agonist compound as well as preparation method and application thereof

Abstract

Belongs to the technical field of therapeutic peptides, and relates to a compound shown in a general formula (I), or a racemate thereof, or an isomer thereof, or a pharmaceutically acceptable salt thereof, as a multi-receptor co-agonist compound, and application of the compound in treating diseases.

Description

Multiple receptor co-agonist compound and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of therapeutic peptides, and in particular relates to a multi-receptor co-agonist compound and a preparation method and application thereof. Background Art

[0002] Glucagon-like peptide (GLP-1) is a polypeptide hormone secreted by the intestine after food stimulation. GLP-1 stimulates insulin secretion and reduces glucagon secretion in a glucose-dependent manner. GLP-1 receptors are widely distributed in multiple organs and tissues throughout the body, including the central nervous system, gastrointestinal tract, cardiovascular system, liver, adipose tissue, and muscle in addition to the pancreas. GLP-1 receptor agonists exert their hypoglycemic effects through multiple mechanisms, including slowing gastric emptying, central appetite suppression, and reducing food intake. However, natural GLP-1 is easily degraded by dipeptidyl peptidases in the body, losing its activity. Its half-life in the body is only 1-2 minutes, which greatly limits its clinical application.

[0003] The glucose-dependent insulinotropic hormone GIP (gluocose-dependent insulinotropic hormone) is currently believed to be primarily secreted by enteroendocrine K cells in the duodenum and upper jejunum. Similar to GLP-1, GIP can stimulate insulin secretion. The GIP receptor, GIPR, is widely distributed throughout the body and is expressed in the pancreas, stomach, small intestine, adipose tissue, heart, and brain. Furthermore, activation of the GIP-GIPR pathway can also exert a weight-loss effect. However, the biological activity half-life of GIP in vivo is relatively short, less than 2 minutes in mice, 7 minutes in healthy subjects, and 5 minutes in patients with type 2 diabetes.

[0004] Glucagon (GCG) is a hormone produced in the alpha cells of the pancreas. It acts on the liver in response to stress, such as cold and hunger, breaking down glycogen in the liver and raising blood sugar. In addition to its blood sugar-raising effects, GCG also promotes lipolysis, fat oxidation, and fever (see Diabetologia, 2017, 60, 1851-1861). Long-term administration can increase energy metabolism and lead to weight loss. However, these beneficial effects on energy metabolism have limited widespread use due to its inherent blood sugar-raising effects.

[0005] Currently, although multiple receptor co-agonists and their potential medical uses are described in multiple patent applications, for example, CN201880081212.3, CN201680082352.3, CN202310659610.1, WO2010 / 011439, WO2013 / 164483, WO 2014 / 192284, WO 2015 / 067715, WO2015055801, WO2016209707, etc., there is still a need for multiple agonists with longer acting properties and suitable for oral administration.

[0006] Summary of the Invention

[0007] In view of the problems existing in the prior art, the present application provides a multi-receptor co-agonist compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt, and its preparation method and application, which can improve bioavailability.

[0008] In a first aspect, the present application provides a compound represented by formula (I), or an isomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof, including:

[0009] wherein A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide;

[0010] R 1 、R 2 solely selected from hydrogen, C 1-6 The alkyl group, R 3 Selected alone R 4 Selected from carboxylic acid, phosphoric acid,

[0011] R 5 solely selected from hydrogen, C 1-6 The alkyl group, X is selected from C 1-6 alkyl,

[0012] Y is selected from One of the following, or a combination of any two or more;

[0013] wherein m is selected from 0, 1, 2, 3 or 4; n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4; and Z is selected from an integer of 1-20, preferably an integer of 8-20.

[0014] In a second aspect, the present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above compounds, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0015] In a third aspect, the present invention also provides a use of a therapeutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a condition, wherein the disease is a chronic related disease selected from diabetes, obesity, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, cardiovascular disease, neurodegenerative disorder, chronic kidney disease, diabetic nephropathy, peripheral arterial disease, and / or heart failure.

[0016] Specifically, the present invention is achieved through the following technical solutions:

[0017] A GLP-GIP compound represented by formula (I), or an isomer, a racemate, or a pharmaceutically acceptable salt thereof, comprising:

[0018] wherein A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide;

[0019] R 1 、R 2 solely selected from hydrogen, C 1-6 The alkyl group,

[0020] R 3 Selected alone R 4 Selected from carboxylic acid, phosphoric acid,

[0021] R 5 solely selected from hydrogen, C 1-6 The alkyl group, X is selected from C 1-6 alkyl,

[0022] Y is selected from One of the following, or a combination of any two or more;

[0023] wherein m is selected from 0, 1, 2, 3 or 4; n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4; and Z is selected from an integer of 1-20, preferably an integer of 8-20.

[0024] As a preferred technical solution of the present invention, the compound selected from formula (II) is

[0025] wherein A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide;

[0026] X is selected from C 1-6 alkyl,

[0027] Y is selected from One of the following, or a combination of any two or more;

[0028] R 4 Selected from carboxylic acids, R 1 、R 5 solely selected from hydrogen, C 1-6 Alkyl;

[0029] wherein m is selected from 0, 1, 2, 3 or 4, n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4, and Z is selected from an integer of 1-20, preferably an integer of 8-20.

[0030] As a preferred technical solution of the present invention, the compound is selected from the compounds represented by formula (IIIa), (IIIb), (IIIc), and (IIId):

[0031] Wherein, Z1, Z2, Z3, and Z4 are independently selected from 12, 13, 14, and 15; and A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide.

[0032] As a preferred technical solution of the present invention, the A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide;

[0033] R 1 、R 2 independently selected from hydrogen, methyl, R 3 Selected alone

[0034] R 5 independently selected from hydrogen, methyl, R 4 、R 6 Independently selected from carboxylic acids; X is selected from ethyl, butyl, Y is selected from Among them, when the R 1 、R 2 One of the When the R 5independently selected from hydrogen or methyl; m is selected from 0 or 1, n and p are selected from 1, and Z is selected from an integer of 12-15.

[0035] As a preferred technical solution of the present invention, the C 1-6 The alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, 1-ethylpropyl, 2-methylbutyl, tert-pentyl, 1,2-dimethylpropyl, isopentyl, neopentyl, n-hexyl, isohexyl, sec-hexyl, tert-hexyl, neohexyl, 2-methylpentyl, 1,2-dimethylbutyl, and 1-ethylbutyl.

[0036] As a preferred technical solution of the present invention, at least one hydrogen atom of the compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt is substituted by deuterium isotope.

[0037] As a preferred technical solution of the present invention, the compound is selected from Table 1, Table 2, Table 3, Table 4 and the following structures:

[0038] As a preferred technical solution of the present invention, the pharmaceutically acceptable salt refers to the compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt prepared with a pharmaceutically acceptable acid or base, including acetate, hydrochloride, sulfate, trifluoroacetate, etc.

[0039] The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

[0040] The present invention further provides the medical use of the compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt, in particular, its use in the preparation of a medicament for treating a disease, wherein the disease is a chronic related disease selected from diabetes, obesity, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, cardiovascular disease, neurodegenerative disorder, chronic kidney disease, diabetic nephropathy, peripheral arterial disease, and / or heart failure.

[0041] For the sake of clarity, general terms used in the description of the compounds are defined herein.

[0042] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered as undefined or unclear in the absence of a specific definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0043] As used herein, the term "compound" refers to a molecular entity and, therefore, a "compound" may have different structural elements in addition to the minimum elements defined for each compound or group of compounds. The term compound may be used interchangeably with the term "construct". The term "compound" may be used to describe the prodrugs of the present invention. The compounds of the present invention may be referred to as "compounds", but the term "compound" is also intended to cover their pharmaceutically relevant forms, i.e., the present invention relates to compounds as defined herein or pharmaceutically acceptable salts, amides or esters thereof.

[0044] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is prepared by reacting a compound having a specific substituent discovered in the present invention with a pharmaceutically acceptable acid or base.

[0045] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrates. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0046] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, atropisomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0047] Optically active (R)- and (S)-isomers, as well as D and L isomers, atropisomers, etc., can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are resolved by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0048] The atoms of the molecules of the compounds of the present invention are isotopes, and isotope derivatization can generally extend half-life, reduce clearance, stabilize metabolism, and increase in vivo activity. In addition, an embodiment is included in which at least one atom is replaced by an atom having the same atomic number (number of protons) and a different mass number (protons and neutrons). Examples of isotopes included in the compounds of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, which respectively include 2 H. 3 H. 13 C. 14 C. 15 N. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36 In particular, radioactive isotopes that emit radiation as they decay, such as 3 H or 14 C can be used for local anatomy testing of pharmaceutical preparations or compounds in vivo. Stable isotopes neither decay nor change with their amount nor are they radioactive, so they can be used safely. When the atoms constituting the molecules of the compounds of the present invention are isotopes, the isotopes can be converted according to general methods by replacing the reagents used in the synthesis with reagents containing the corresponding isotopes.

[0049] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as deuterium ( 2 H), iodine-125( 125 I) or C-14( 14C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0050] Furthermore, the compounds of the present invention may have one or more hydrogen atoms replaced by deuterium isotopes ( 2 After deuteration, the compounds of the present invention have the effects of extending half-life, reducing clearance rate, stabilizing metabolism and improving in vivo activity.

[0051] The preparation method of the isotopic derivative generally includes a phase transfer catalytic method. For example, a preferred deuteration method uses a phase transfer catalyst (e.g., a tetraalkylammonium salt, NBu4HSO4). The use of a phase transfer catalyst to exchange the methylene protons of the diphenylmethane compound results in a higher deuterium incorporation than reduction with a deuterated silane (e.g., triethyldeuterated monosilane) in the presence of an acid (e.g., methanesulfonic acid) or with a Lewis acid such as aluminum trichloride using sodium deuterated borate.

[0052] As used herein, the term "polypeptide" or "polypeptide sequence" refers to a compound comprising a series of two or more amino acids interconnected by amide (or peptide) bonds. The term polypeptide is used interchangeably with the term "peptide" and the term "protein."

[0053] In one embodiment, the compound of the present invention comprises a GLP-GIP polypeptide. In one embodiment, the GLP-GIP polypeptide is the amino acid sequence of Tirzepatide, which has the structure:

[0054] In one embodiment, the compound of the present invention comprises a GLP-GCG polypeptide. In one embodiment, the GLP-GCG polypeptide is the amino acid sequence of mazdutide, whose structure is:

[0055] In one embodiment, the compound of the present invention comprises a GLP-GCG polypeptide. In one embodiment, the GLP-GCG polypeptide is an amino acid sequence of survodutide, the structure of which is:

[0056] The polypeptide sequence of survodutide is: HH-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2.

[0057] In one embodiment, the compound of the present invention comprises a GLP-GIP-GCG polypeptide. In one embodiment, the GLP-GIP-GCG polypeptide is the amino acid sequence of Retatrutide, whose structure is:

[0058] Among them, the polypeptide structure of Retatrutide is:

[0059] Among them, αMeL is an unnatural amino acid, and its structure is: The structure of Aib is:

[0060] In another embodiment, the GLP-GIP-GCG polypeptide is NN9423, whose polypeptide structure is:

[0061] As used herein, the term "GLP-GIP polypeptide" refers to a polypeptide capable of binding to a GLP receptor and / or activating a GIP receptor. In other words, a GLP polypeptide is a polypeptide having GLP-1 activity, and a GIP polypeptide is a polypeptide having GIP activity. In other words, a GLP-1 polypeptide is a GLP-1 receptor agonist, and a GIP polypeptide is a GIP receptor agonist.

[0062] As used herein, the term "GLP-GCG polypeptide" refers to a polypeptide capable of binding to a GLP receptor and / or activating a GCG receptor. In other words, a GLP polypeptide is a polypeptide having GLP-1 activity, and a GCG polypeptide is a polypeptide having GCG activity. In other words, a GLP-1 polypeptide is a GLP-1 receptor agonist, and a GCG polypeptide is a GCG receptor agonist.

[0063] As used herein, the term "GLP-GIP-GCG polypeptide" refers to a polypeptide that is capable of binding to a GLP receptor, a GIP receptor, and / or a GCG receptor. In other words, a GLP polypeptide is a polypeptide having GLP-1 activity, a GIP polypeptide is a polypeptide having GIP activity, and a GCG polypeptide is a polypeptide having GCG activity.

[0064] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity increasers, transdermal enhancers, etc. Their preparation is well known to those skilled in the field of cosmetics or topical medicine. For additional information on carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0065] With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a non-toxic amount of the drug or agent sufficient to achieve the intended effect. For the oral dosage forms of the present invention, an "effective amount" of an active substance in the composition means the amount required to achieve the intended effect when used in combination with another active substance in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

[0066] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0067] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG1 is a mass spectrum of the compound of Example 1 of the present invention. DETAILED DESCRIPTION

[0069] The present application is further described in detail below with reference to examples, but the implementation methods of the present application are not limited thereto.

[0070] Example 1

[0071] The synthesis route of Example 1 can refer to the synthesis route in WO2022096636A1, and the contents of WO2022096636A1 can also be fully introduced into this application by reference.

[0072] The specific synthetic route of the compound is as follows:

[0073] Step 1: Synthesis of F1:

[0074] Synthesis process description: Compound F1 was obtained by solid phase synthesis:

[0075] a. Using 2-CTC resin as a carrier, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it several times with DMF.

[0076] b. After washing, add methanol and DIEA respectively to cap the resin. After capping, wash the resin several times with DMF.

[0077] c. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0078] d. 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0079] e. Fmoc-aminoethanol, diisopropyl azodicarboxylate (DIAD), and triphenylphosphine were mixed in THF and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0080] f. Repeat step c. to remove the Fmoc protecting group.

[0081] g. Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction is complete, wash the resin with DMF.

[0082] h. Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out the condensation reaction.

[0083] i. Separately, mercaptoethanol, dicyclic amidine (DBU) and DMF were mixed and added to the resin to remove the Ns protecting group. After the reaction was completed, the resin was washed with DMF.

[0084] j. Weigh Boc-Gly-OH and repeat step g. to carry out condensation reaction.

[0085] k. The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0086] 1. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to obtain compound F1.

[0087] Step 2: Synthesis of F2 (Telpotide Fully Protected Peptide Resin)

[0088] The synthetic route of tilpotide refers to the synthetic route in patent application number CN201680005007.X.

[0089] H-Tyr(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr-Ser(tBu)-Ile(tBu)-Aib(tBu)-Leu-Asp(OtBu)-Lys-Ile(Trt)-Ala- Gln-Lys(AEEA-AEEA-γ-Glu(OtBu)-nonadecanedioic acid monotert-butyl ester)-Ala(OtBu)-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly(Boc)-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Rink aMide Resin

[0090] F2

[0091] Synthesis steps:

[0092] a. Use RINK amide resin as the carrier, first swell it with DMF, and after swelling, wash the resin with DMF.

[0093] b. Weigh Fmoc-Ser(tBu)-OH, HOBt, and 4-dimethylaminopyridine (DMAP) and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction, wash the resin with DMF.

[0094] c. After washing, add acetic anhydride and DIEA respectively for end-capping. After end-capping, wash the resin several times with DMF.

[0095] d. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After deprotection, the resin was washed with DMF.

[0096] e. Weigh Fmoc-Pro-OH and HOBt, add DMF, and dissolve them. Then, add DIC for activation for 3-5 minutes. After activation, add the mixture to the reactor and begin the coupling reaction. The amino acid coupling reaction should proceed for 1.0-3.0 hours. Ninhydrin should be used to monitor the reaction endpoint. After the coupling is complete, wash the resin with DMF.

[0097] f. Repeat step e. and sequentially couple amino acids according to the peptide sequence to obtain F2 (telport peptide fully protected peptide resin).

[0098] After the reaction, the peptide resin was washed with dichloromethane and methanol and dried at room temperature.

[0099] Step 3 Preparation of target compound (F1+F2)

[0100] a. F2 (Telpotide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0101] b. Weigh F1 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0102] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0103] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0104] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0105] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0106] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0107] (4) The freeze-dried samples were packaged and stored as required. Samples were taken for testing. The mass spectrum of compound 1 is shown in Figure 1.

[0108] Example 17

[0109] Step 1: Synthesis of F3

[0110] Synthesis process description: Compound F3 was obtained by solid phase synthesis:

[0111] (1) 2-CTC resin was used as a carrier, which was first swollen with N,N-dimethylformamide (DMF), and then Fmoc-Sar-OH and N,N-diisopropylethylamine (DIPEA) were added to react for several hours. After the reaction, it was washed with DMF several times.

[0112] (2) After washing, methanol and DIPEA were added for end-capping. After end-capping, the resin was washed several times with DMF.

[0113] (3) Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0114] (4) Weigh Fmoc-Lys(Boc)-OH and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N′-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin to react. After the reaction is complete, wash the resin with DMF.

[0115] (5) Weigh Fmoc-Glu(OtBu)-OH and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N′-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and react. After the reaction is complete, wash the resin with DMF.

[0116] (6) Weigh mono-tert-butyl hexadecanedioate and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N′-diisopropylcarbodiimide (DIC) and mix thoroughly. After mixing, add the mixture to the resin to react. After the reaction is complete, wash the resin with DMF.

[0117] (7) The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0118] (8) The resin was cleaved with 20% TFE / DCM, filtered and concentrated to obtain compound F3.

[0119] Step 2: Synthesis of F2 (Telpotide Fully Protected Peptide Resin)

[0120] The synthetic route of tilpotide refers to the synthetic route in patent application number CN201680005007.X.

[0121] Tyr(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr-Ser(tBu)-Ile(tBu)-Aib(tBu)-Leu-Asp(OtBu)-Lys-Ile(Trt)-Ala-G ln-Lys(AEEA-AEEA-γ-Glu(OtBu)-nonadecanedioic acid monotert-butyl ester)-Ala(OtBu)-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly(Boc)-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Set-Rink aMide Resin

[0122] F2

[0123] Synthesis steps:

[0124] (1) RINK amide resin was used as the carrier, which was first swollen with DMF. After swelling, the resin was washed with DMF.

[0125] (2) Weigh Fmoc-Ser(tBu)-OH, HOBt, and 4-dimethylaminopyridine (DMAP) and dissolve them in DMF. Add DIC and mix thoroughly. After mixing, add the mixture to the resin and react. After the reaction, wash the resin with DMF.

[0126] (3) After washing, acetic anhydride and DIEA were added for end-capping. After end-capping, the resin was washed several times with DMF.

[0127] (4) Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0128] (5) Weigh Fmoc-Pro-OH and HOBt, add DMF and dissolve them. Then add DIC to activate for 3-5 minutes. After activation, add them to the reactor to start the coupling reaction. The amino acid coupling reaction lasts for 1.0-3.0 hours. The reaction endpoint is monitored using ninhydrin during the coupling process. After the coupling is completed, wash the resin with DMF.

[0129] (6) Repeat step e. to couple amino acids sequentially according to the peptide sequence to obtain F2 (telportin fully protected peptide resin).

[0130] (7) After the reaction, the peptide resin was washed with dichloromethane and methanol and dried at room temperature.

[0131] Step 3 Preparation of target compound (F3+F2)

[0132] (1) F2 (Telpotide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0133] (2) Weigh F3 and HOBt, dissolve them in DMF, add DIC and mix thoroughly. After mixing, add the mixture to the resin for reaction. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and dry it.

[0134] (3) Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to ether with 8 times the volume of the lysis buffer to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0135] (4) The crude product was added to an acetonitrile / water solution and ultrasonically dissolved, and then filtered through a 0.45 μm filter membrane. The filtered solution was transferred to a purification HPLC crude fraction.

[0136] (5) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and qualified fractions were collected.

[0137] (6) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0138] (7) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0139] (8) The freeze-dried samples are packaged and stored as required. Samples are taken for testing to obtain the target structure.

[0140] Examples 2-16 and 18

[0141] The compounds of Examples 2-16 and 18 were prepared by referring to the synthetic routes of Examples 1 and 17. The structures of Examples 2-18 are shown in Table 1.

[0142] Table 1 shows the structures of Examples 2-18

[0143] Example 19

[0144] The synthetic route of Example 19 can be synthesized with reference to the synthetic route of Example 1, except that the telpotide in steps 2 and 3 of Example 1 is replaced with mashidu peptide, wherein the synthetic route of mashidu peptide refers to patent document CN201680036771.3, and the molecular weight of compound 19 is 5117.8.

[0145] Example 25

[0146] The synthesis steps of F3 can be prepared by referring to Step 1 in Example 17.

[0147] Step 2: Synthesis of F4 (Masdu peptide fully protected peptide resin)

[0148] His(Trt)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Glu(OtBu)-Lys(Boc)-Ly s(Boc)-Ala-Lys(diacid-C20-gamma-Glu-(AEEA)2)-Glu(OtBu)-Phe-Val-Glu(OtBu)-Trp(Boc)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Rink aMide Resin

[0149] F4

[0150] Synthesis process description: Compound F4 was obtained by solid phase synthesis:

[0151] (1) Using Rink MBHA resin as a support, first swell it with 10-15 times the volume of the resin in DMF for 30 minutes. After swelling, remove the solvent. Wash the resin four times with 10-15 times the volume of the resin in DMF.

[0152] (2) Deprotection was performed by adding 20% ​​piperidine / DMF in a volume 10-15 times the weight of the resin. The first deprotection was performed for 10 min and the second deprotection was performed for 15 min.

[0153] (3) After deprotection, the resin was washed with DMF six times, each washing lasting 1 to 3 minutes.

[0154] (4) Weigh and prepare the amino acid coupling solution. Weigh 2.0 eq. Fmoc-Gly-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2 eq. DIC to activate for 3-5 min. After activation, add to the reactor to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored using ninhydrin during the coupling process.

[0155] (5) The resin was washed four times with 10-15 volumes of DMF.

[0156] (6) Follow steps 2 to 5 to couple Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp (B oc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(diacid-C20-gamma-Glu-(AEEA)2)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH , Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fm oc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Aib-OH, Fmoc-His(Trt)-OH.

[0157] (7) After all amino acid couplings were completed, the resin was washed four times with DMF, four times with DCM, and four times with methanol.

[0158] (8) The above resin was placed in a vacuum drying oven at 15-30°C and dried to constant weight.

[0159] Step 3 Preparation of target compound (F3+F4)

[0160] (1) F4 (Masto peptide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0161] (2) Weigh F3 and HOBt, dissolve them in DMF, add DIC and mix thoroughly. After mixing, add the mixture to the resin for reaction. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and dry it.

[0162] (3) Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to ether with 8 times the volume of the lysis buffer to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0163] (4) The crude product was added to an acetonitrile / water solution and ultrasonically dissolved, and then filtered through a 0.45 μm filter membrane. The filtered solution was transferred to a purification HPLC crude fraction.

[0164] (5) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and qualified fractions were collected.

[0165] (6) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0166] (7) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0167] (8) The freeze-dried samples are packaged and stored as required. Samples are taken for testing to obtain the target compound.

[0168] Examples 20-24, 26

[0169] The synthetic routes of Examples 20-24 and 26 can refer to Examples 19 and 25, and the specific structures are shown in Table 2.

[0170] Table 2 shows the structures of Examples 20-26

[0171] Example 27

[0172] The synthetic route of Example 27 can be synthesized with reference to the synthetic route of Example 1, except that the telportide in steps 2 and 3 of Example 1 is replaced with sildopeptide, wherein the synthesis of sildopeptide is referenced to patent document CN201480056639.X. The molecular weight of compound 27 is 4786.4. The specific synthetic route is as follows:

[0173] Step 1: The synthesis process of F1 can refer to Step 1 in Example 1.

[0174] Step 2: Synthesis of F5 (Svidin fully protected peptide resin)

[0175] His(Trt)-{1-amino-1-cyclobutanecarboxylic acid}-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Thr(tBu)-Leu-Asp(OtBu)--Glu(OtBu)-Arg(Pbf)-Al a-Ala-Lys(Boc)-Asp(OtBu)-Phe-Ile-Lys{Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-γGlu(OtBu)-C18diacid}-Trp(Boc)-Leu-Glu(OtBu)-Ser(tBu)-Ala-Rink aMide Resin

[0176] F5

[0177] Synthesis process description: Compound F5 was obtained by solid phase synthesis:

[0178] (1) Using Rink MBHA resin as a support, first swell it with 10-15 times the volume of the resin in DMF for 30 minutes. After swelling, remove the solvent. Wash the resin four times with 10-15 times the volume of the resin in DMF.

[0179] (2) Deprotection was performed by adding 20% ​​piperidine / DMF in a volume 10-15 times the weight of the resin. The first deprotection was performed for 10 min and the second deprotection was performed for 15 min.

[0180] (3) After deprotection, the resin was washed with DMF six times, each washing lasting 1 to 3 minutes.

[0181] (4) Weigh and prepare the amino acid coupling solution. Weigh 2.0 eq. Fmoc-Ala-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2 eq. DIC to activate for 3-5 min. After activation, add to the reactor to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored using ninhydrin during the coupling process.

[0182] (5) The resin was washed four times with 10-15 volumes of DMF.

[0183] (6) Follow step 2N5 to couple Fmoc-Ser(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Ile-OH, Fmoc-P in sequence he-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu )-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-1-amino-1-cyclobutanecarboxylic acid, Fmoc-His(Trt)-OH.

[0184] (7) After all amino acid couplings were completed, the resin was washed four times with DMF.

[0185] (8) Add 2% hydrazine hydrate / DMF (10-15 times the resin weight) to remove the Dde protecting group. The first deprotection was performed for 15 min and the second deprotection was performed for 15 min.

[0186] (9) After deprotection, the resin was washed with DMF six times, each wash lasting 1 to 3 minutes.

[0187] (10) Weigh and prepare the amino acid coupling solution. Weigh 2.0 eq. Fmoc-Gly-OH and 2.2 eq. HOBt, respectively, add DMF to dissolve, then add 2.2 eq. DIC to activate for 3-5 min. After activation, add the solution to the reactor to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored using ninhydrin during the coupling process.

[0188] (11) The resin was washed four times with 10-15 volumes of DMF.

[0189] (12) According to steps 2 to 5, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-γmGlu-OtBu, and mono-tert-butyl octadecanediolate were coupled in sequence.

[0190] (13) After all amino acid couplings were completed, the resin was washed four times with DMF, four times with DCM, and four times with methanol.

[0191] (14) The resin was placed in a vacuum drying oven at 15-30°C and dried to a constant weight.

[0192] Step 3 Preparation of target compound (F1+F5)

[0193] (1) F5 (Svide peptide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0194] (2) Weigh F1 and HOBt, dissolve them in DMF, add DIC and mix thoroughly. After mixing, add the mixture to the resin for reaction. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and dry it.

[0195] (3) Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to ether with 8 times the volume of the lysis buffer to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0196] (4) The crude product was added to an acetonitrile / water solution and ultrasonically dissolved, and then filtered through a 0.45 μm filter membrane. The filtered solution was transferred to a purification HPLC crude fraction.

[0197] (5) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and qualified fractions were collected.

[0198] (6) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0199] (7) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0200] (8) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0201] Example 33

[0202] Step 1: The synthesis step of F3 was prepared by referring to Step 1 in Example 17.

[0203] Step 2: The synthesis of F5 (Svidin fully protected peptide resin) was carried out with reference to Step 2 in Example 27.

[0204] Step 3 Preparation of target compound (F3+F5)

[0205] (1) F5 (Svide peptide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0206] (2) Weigh F3 and HOBt, dissolve them in DMF, add DIC and mix thoroughly. After mixing, add the mixture to the resin for reaction. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and dry it.

[0207] (3) Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to ether with 8 times the volume of the lysis buffer to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0208] (4) The crude product was added to an acetonitrile / water solution and ultrasonically dissolved, and then filtered through a 0.45 μm filter membrane. The filtered solution was transferred to a purification HPLC crude fraction.

[0209] (5) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and qualified fractions were collected.

[0210] (6) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0211] (7) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0212] (8) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0213] Examples 28-32, 34

[0214] The synthetic routes of Examples 28-32 and 34 refer to Examples 27 and 33, and the specific structures are shown in Table 3.

[0215] Table 3 shows the structures of Examples 28-34

[0216] Example 35

[0217] The synthetic route of Example 1 can refer to the synthetic route in WO2022096636A1, and the contents of WO2022096636A1 can also be fully introduced into this application by reference. The specific synthetic route of the compound is as follows:

[0218] Step 1: The synthesis of F1 was prepared by referring to step 1 in Example 1.

[0219] Step 2: Synthesis of Frag.2 (Retaglutide fully protected peptide resin)

[0220] The synthetic route of retaglutide refers to the synthetic route in patent application number CN201880081212.3.

[0221] I-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeL-Leu-Asp(OtBu)-Lys(Boc)-Lys(A EEA-γ-Glu(OtBu)-Eicosanedioic acid monotert-butyl ester)-Ala-Gln(Trt)-Aib-Phe-Ile-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink aMide Resin, F6

[0222] Synthesis steps:

[0223] a. Use RINK amide resin as the carrier, first swell it with DMF, and after swelling, wash the resin with DMF.

[0224] b. Weigh Fmoc-Ser(tBu)-OH, HOBt, and 4-dimethylaminopyridine (DMAP) and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction, wash the resin with DMF.

[0225] c. After washing, add acetic anhydride and DIEA respectively for end-capping. After end-capping, wash the resin several times with DMF.

[0226] d. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After deprotection, the resin was washed with DMF.

[0227] e. Weigh Fmoc-Pro-OH and HOBt, add DMF, and dissolve them. Then, add DIC for activation for 3-5 minutes. After activation, add the mixture to the reactor and begin the coupling reaction. The amino acid coupling reaction should proceed for 1.0-3.0 hours. Ninhydrin should be used to monitor the reaction endpoint. After the coupling is complete, wash the resin with DMF.

[0228] f. Repeat step e. and sequentially couple amino acids according to the peptide sequence to obtain Frag.2 (retaglutide fully protected peptide resin).

[0229] After the reaction, the peptide resin was washed with dichloromethane and methanol and dried at room temperature.

[0230] Step 3: Preparation of target compound (F1+F6)

[0231] a. F6 (retaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0232] b. Weigh F1 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0233] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0234] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0235] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0236] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0237] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0238] The freeze-dried samples were packaged and stored as required. Samples were taken for testing, and the molecular weight of compound 35 was 5286.1.

[0239] Example 41

[0240] Step 1: The synthesis step of F3 was prepared by referring to Step 1 in Example 17.

[0241] Step 2: The synthesis steps of F6 (retaglutide fully protected peptide resin) refer to step 2 of Example 35 and the preparation of patent application number CN201880081212.3.

[0242] Step 3: Preparation of target compound (F3+F6)

[0243] a. F6 (retaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0244] b. Weigh F3 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0245] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0246] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0247] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0248] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0249] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0250] Examples 36-40, 42

[0251] The synthetic routes of Examples 36-40 and 42 can refer to Examples 35 and 41, and the specific structures are shown in Table 4.

[0252] Table 4 shows the compounds of Examples 36-42

[0253] Example 43 Transformation Half-Life Determination

[0254] Prepare peptide stock solutions by dissolving the test sample in PBS buffer to a target of 200 μM. PBS buffer is Ca-free. 2+ and Mg 2+ Dulbecco's phosphate-buffered saline (PBS), pH 7.4. Adjust the pH of the peptide stock solution to 7.4 with HCl or NaOH. Transfer the sample to an HPLC vial. Cap the vial tightly to prevent evaporation. Incubate the HPLC vial at 37°C, and remove samples at various time points over a 2-week period, flash-freeze at -80°C, and store at -20°C until analysis. Analyze the samples using LC / MS or liquid chromatography, and calculate the conversion half-life of the test sample based on the reduction in the test sample prototype.

[0255] The conversion half-life of the test sample of the present invention is not less than 24 hours, preferably not less than 50 hours, and the conversion half-life is relatively long. The specific results are shown in Table 5.

[0256] Table 5 shows the conversion half-life

[0257] Among them, A≥60h.

[0258] Example 44 Terminal Half-Life Determination

[0259] The terminal half-life of the test samples in vivo was evaluated in miniature pigs. Three miniature pigs were used for each sample and administered intravenously. The samples were prepared with a pH 7.4 solvent and the administration volume was 0.05 mL / kg. Approximately 0.8 mL of venous blood was collected at different time points after administration. Plasma was prepared by centrifugation at 4000 rpm for 10 minutes and stored at -20°C until analysis. The concentration of the active ingredient was detected by LC / MS or liquid chromatography. The plasma concentration (relative to time) curve of the test compound was evaluated by non-compartmental analysis (NCA) using WinNonlin software and the terminal half-life pharmacokinetic parameters were calculated.

[0260] The terminal half-life of the compound of the present invention after administration is not less than 30 hours.

[0261] Example 45 GLP-1 activity assay

[0262] The U2OS-GLP-1R stable cell line and HTRF detection method were used to determine the agonist effect of the test substance on GLP-1R

[0263] Experimental methods:

[0264] 1. Follow the cAMP-Gs Dynamic HTRF kit protocol and dilute the 5x Stimulation buffer (SB) provided with the kit with ddH2O to 1xSB. Add IBMX to a final concentration of 500 μM to prevent cAMP degradation. Prepare the test substance working solution in 1xSB and perform a series of serial dilutions.

[0265] 2. Digest and collect U2OS-GLP-R cells, resuspend and count, and dilute the cell density to 2x10^ using 1xSB solution. 6 The cells were plated at 5 μL per well in a 384-well plate to obtain 10,000 cells per well.

[0266] 3. Add 5 μL of the test substance at different concentrations to the corresponding cell wells and incubate in a 37°C incubator for 30 minutes.

[0267] 4. Prepare cAMP standard solutions of different concentrations according to the steps provided by the cAMP-Gs Dynamic HTRF kit and add them to the 384-well cell plate.

[0268] 5. Dilute the cAMP d2 reagent and Eu Cryptate antibody in the kit to 1x using Lysis & Detection Buffer. Add 5 μL of d2 and Eu, respectively, to a 384-well plate. Incubate at room temperature for 2 hours, then detect using a microplate reader HTRF module (665 / 620 nm) and collect experimental data.

[0269] 6. Signal values ​​were plotted against compound concentrations, and curve fitting and EC50 calculation were performed using GraphPad Prism software using nonlinear regression. The test results showed that the preferred compounds of the present invention exhibited excellent GLP-1 activity, with EC50 < 100 nM. The results are shown in Table 6.

[0270] Table 6 shows GLP-1 activity

[0271] Among them, B≤300nM, 300nM <C≤600nM,600nM<D≤1000nM,E>1000nM。

[0272] Example 46 Pharmacokinetic Experiment

[0273] 1. Reagents and instruments

[0274] Dulbecco's phosphate buffered saline (DPBS) (Lot No.: F2326001, Shanghai Aladdin Biochemical Technology Co., Ltd.) LC-MS instrument (Thermo TSQ Altis Plus).

[0275] 2. Experimental Animals

[0276] SD rats: male, 180-250 g, purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0277] 3. Preparation

[0278] Accurately weigh the test sample powder to make sure it does not contain Ca 2+ / Mg 2+ The drug was completely dissolved in Dulbecco's phosphate buffer, mixed to 0.1 mg / mL, and administered intravenously at a dose of 2 mL / kg.

[0279] 4. Blood sample collection

[0280] After intravenous administration to rats, 200 μL of venous blood was collected before administration and 0, 0.5, 2, 5, 24, 48, and 72 hours after administration in EDTA-K2 anticoagulant EP tubes, centrifuged at 12,000 rpm for 2 minutes, and the plasma was frozen at -20°C for testing.

[0281] 5. Biological analysis

[0282] Accurately weigh a certain amount of test sample and dissolve it in DMSO to 2 mg / mL as a stock solution. Accurately aspirate an appropriate amount of compound stock solution and dilute it with acetonitrile aqueous solution (ACN:H2O=1:1) to prepare a standard series of solutions. Accurately aspirate 4 μL of each of the above standard series solutions, add 36 μL of blank plasma, vortex mix, and prepare plasma samples equivalent to plasma concentrations of 0.3, 1, 3, 10, 30, 100, 300, 500, 1000, 3000, 5000 and 10000 ng / mL to establish a standard curve. Take 30 μL of plasma, add 150 μL of acetonitrile / methanol (1:1) solution of internal standard propranolol (5 ng / mL), vortex mix, centrifuge at 4000 rpm for 10 minutes, take 100 μL of the supernatant, add 100 μL of ultrapure water and perform LC-MS analysis. The LC-MS detection conditions are as follows:

[0283] Chromatographic column: YMC-Triart C18, 33*2.1mm, 5μm.

[0284] Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile, flow rate: 0.5 mL / min, gradient elution see Table 7 below:

[0285] Table 7

[0286] 6. Data processing

[0287] After LC-MS detection of blood drug concentration, WinNonlin 6.1 software was used to calculate the pharmacokinetic parameters of rats after administration using a non-compartmental model. The results showed that the compound of the present invention had a better exposure.

[0288] Example 47 In vivo efficacy

[0289] The experiment used 16-week-old DIO (diet-induced obesity) mice (purchased from Guangdong Weitong Lihua Laboratory Animal Technology Co., Ltd.) induced by high-fat diet. Wild-type mice of the same age served as normal controls. Before the experiment, the DIO mice were randomly divided into three groups of five mice each according to body weight. Five wild-type mice served as a blank control group. Both DIO and wild-type mice were administered subcutaneously every three days. Detailed dosing schedule is shown in Table 8. During the experiment, animal behavior, coat color, water intake, and urination were observed. Animals were weighed every other day, and abnormalities were recorded. The experimental period lasted 15 days.

[0290] Table 8: Grouping of mice for drug administration

[0291] The results showed that there were no abnormalities in the mice in each administration group during the administration process. After the last administration, the compound of the present invention had a better weight loss effect on DIO mice.

[0292] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A compound represented by formula (I), or an isomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, characterized in that: include: wherein A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide; R 1 , R 2 exclusively selected from hydrogen, C 1-6 The alkyl group R 3 Selected from R 5 exclusively selected from hydrogen, C 1-6 The alkyl group R 4 , R 6 Independently selected from carboxylic acid, phosphoric acid, X is selected from C 1-6 alkyl, Y is selected from One of the following, or a combination of any two or more; wherein m is selected from 0, 1, 2, 3 or 4, n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4, and Z is selected from an integer of 1-20, preferably an integer of 8-20.

2. The compound according to claim 1, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: Selected from compounds represented by formula (II), wherein A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide; X is selected from C 1-6 alkyl, Y is selected from One of the following, or a combination of any two or more; R 4 Selected from carboxylic acids, R 1 , R 5 exclusively selected from hydrogen, C 1-6 The alkyl group; wherein m is selected from 0, 1, 2, 3 or 4, n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4, and Z is selected from an integer of 1-20, preferably an integer of 8-20.

3. The compound according to claim 1 or 2, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: The C 1-6 The alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, 1-ethylpropyl, 2-methylbutyl, tert-pentyl, 1,2-dimethylpropyl, isopentyl, neopentyl, n-hexyl, isohexyl, sec-hexyl, tert-hexyl, neohexyl, 2-methylpentyl, 1,2-dimethylbutyl, and 1-ethylbutyl.

4. The compound according to claim 1 or 2, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: A compound selected from the group consisting of: Wherein, Z1, Z2, Z3, and Z4 are independently selected from 12, 13, 14, and 15; and A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide.

5. The compound according to claim 1 or 2, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: The A is selected from Tirzepatide, mazdutide, survodutide, and Retatrutide; R 1 , R 2 independently selected from hydrogen, methyl, R 3 Selected from R 5 independently selected from hydrogen, methyl, R 4 , R 6 independently selected from carboxylic acids; X is selected from ethyl, butyl, Y is selected from Among them, when the R 1 , R 2 One of the When the R 5 independently selected from hydrogen or methyl; m is selected from 0 or 1, n and p are selected from 1, and Z is selected from an integer of 12-15.

6. The compound according to claim 1 or 2, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from Table 1, Table 2, Table 3, Table 4 and the following structures:

7. A pharmaceutical composition, characterized in that The invention comprises a therapeutically effective amount of the compound according to any one of claims 1 to 6, or an isomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. The medical use of the compound according to any one of claims 1 to 6, or its isomer, or its racemate, or its pharmaceutically acceptable salt, in particular, its use in the preparation of a medicament for treating a disease, wherein the disease is a chronic related disease selected from diabetes, obesity, non-alcoholic fatty liver disease and non-alcoholic fatty hepatitis, cardiovascular disease, neurodegenerative disorders, chronic kidney disease, diabetic nephropathy, peripheral arterial disease, and / or heart failure.

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