Multi-receptor co-agonist, and preparation method therefor and use thereof

Abstract

The present application belongs to the technical field of therapeutic peptides, and relates to a compound as represented by general formula (I), or a racemate thereof, an isomer thereof or a pharmaceutically acceptable salt thereof, wherein same acts as a GLP-1 / amylin or GLP / GIP / amylin compound, and the use thereof for treating diseases. X-Z-Ya (I)

Description

Multiple receptor co-agonists, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of therapeutic peptide technology, specifically relating to a multiple receptor co-agonist and its preparation method and application, specifically relating to GLP-1 / amylin, GLP / GIP / amylin compounds, or their isomers, racemates, or pharmaceutically acceptable salts, and their preparation methods, as well as their use in treating diseases. Background Technology

[0002] Overweight and obesity are abnormal or excessive accumulations of body fat that pose a threat to an individual's overall health. A body mass index (BMI) over 25 is considered overweight, and a BMI over 30 is considered obese. Obesity is a major risk factor for many serious diseases, including type 2 diabetes and its associated comorbidities, as well as cardiovascular diseases such as heart disease and stroke, which are leading causes of death worldwide. Currently, diet and exercise alone are insufficient to reduce the body mass index (BMI) of obese individuals to an acceptable level.

[0003] Amylin is a long polypeptide hormone composed of 37 amino acids, produced in pancreatic beta (β) cells and co-secreted with insulin. The half-life of endogenous amylin is approximately 15–20 minutes. It acts in several different organ systems, primarily through amylin receptors 1–3 (AMYR1–3). Amylin is an important regulator of energy metabolism in both health and disease, inhibiting glucagon secretion, delaying gastric emptying, signaling satiety, and suppressing appetite. Other effects of amylin, such as those on the cardiovascular system and bones, have also been reported.

[0004] GLP-1 is a polypeptide hormone composed of 30 or 31 amino acids, synthesized and secreted by enteroendocrine L-cells. GLP-1 is an intestinal glucagon that lowers blood glucose levels in a glucose-dependent manner by enhancing insulin secretion. Endogenous GLP-1 is primarily rapidly degraded by dipeptidyl peptidase-4 (DPP-4), resulting in a half-life of approximately 2 minutes. Clinical studies have shown that both amylin receptor agonists and GLP-1 receptors can be used to treat overweight, obesity, type 1 diabetes, and / or type 2 diabetes.

[0005] Glucose-dependent insulinotropic hormone (GIP) 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, 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, GIP has a short biological activity half-life in vivo, less than 2 minutes in mice, 7 minutes in healthy individuals, and 5 minutes in patients with type 2 diabetes.

[0006] Currently, several patent reports have been published regarding amylin, which can be used to treat overweight and obesity. Examples include patent applications with patent numbers CN96196092.2, CN200580012043.0, CN201780055088.9, CN201280028554.1, and CN201580046850.8. Similarly, several patent reports have been published regarding GLP-1, which can be used to treat overweight, obesity, type 1 diabetes, and / or type 2 diabetes. Examples include patent applications with patent numbers CN201610420028.X, CN200880106356.6, and CN201480038380.6. There are also many patent reports related to GLP-GIP, which can be used to treat overweight, obesity, type 1 diabetes and / or type 2 diabetes, for example: patent application numbers: CN201680005007.X, CN202310851003.5, CN201980049095.7, etc.

[0007] Although patent document with application number CN202180085551.0 discloses a dual-target molecule of Amylin and GLP-1 for the treatment of obesity-related diseases, there are currently no related products on the market. There is still a need to provide dual-target molecules of Amylin and GLP-1 or triple-target molecules of Amylin, GLP-1, and GLP-1 that are more effective in treating obesity and suitable for oral administration. Summary of the Invention

[0008] In view of the problems existing in the prior art, this application provides a glucagon-like peptide-1 compound, or its isomer, racemate, or pharmaceutically usable salt thereof, and its preparation method and application, which can improve bioavailability.

[0009] In a first aspect, this application provides a compound of formula (I), or an isomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof, comprising: XZ-Ya (I)

[0010] Wherein, X is selected from peptides with GLP-1 activity and peptides with GLP-GIP activity;

[0011] Z is selected from connector;

[0012] Ya is selected from polypeptides that exhibit amylin activity.

[0013] As a preferred embodiment of the present invention, when X is selected from a polypeptide having GLP-1 activity, the compound, or its isomer, racemate, or pharmaceutically acceptable salt thereof, is selected from the structure shown in formula (II).

[0014] Among them, R 1 Selected from

[0015] m is selected from 0 or 1, and n is selected from 10, 11, 12, 13, 14 or 15.

[0016] As a preferred embodiment of the present invention, the polypeptide with GLP-1 activity is selected from:

[0017] As a preferred embodiment of the present invention, the GLP-GIP polypeptide is selected from Tirzepatide, SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, with Tirzepatide being preferred.

[0018] As a preferred embodiment of the present invention, the amylin polypeptide is selected from canagliflozin (SEQ ID NO:4), SEQ ID NO:5, and SEQ ID NO:6.

[0019] As a preferred embodiment of the present invention, the connector is selected from the connectors shown in Table 4, and the connector is connected to GLP-1 peptide and amylin peptide respectively via amide.

[0020] As a preferred embodiment of the present invention, the compound is selected from the structures shown in Tables 5, 6, 7, 8 and 9.

[0021] As a preferred embodiment of the present invention, the compound is selected from the structures shown in Tables 10 and 11.

[0022] In a second aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the compound as described above, or an isomer thereof, a racemic mixture thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0023] Thirdly, the present invention also provides a pharmaceutical use of the compound as described above, or its isomers, racemates, or pharmaceutically usable salts, or pharmaceutical compositions as described above, specifically, in the preparation of a medicament for treating a disease selected from diabetes, obesity, cardiovascular disease, nonsteroidal steatohepatitis, and / or cognitive impairment, wherein the cognitive impairment is cognitive impairment caused by Alzheimer's disease.

[0024] The GLP / GIP / amylin receptor agonist compounds of the present invention exhibit enhanced agonistic activity against GLP-1R, GIPR, and / or calcitonin (CT), and / or islet amyloid peptide (AMY3), and / or islet amyloid peptide (AMY1) receptors. Preferably, the in vitro agonistic activity against calcitonin (CT) is ECG. 50 <100 nM, preferably less than 10 nM, more preferably less than 1 nM, and / or in vitro agonistic activity against pancreatic amyloid peptide (AMY1) receptor EC 50 <100 nM, preferably less than 10 nM, more preferably less than 1 nM, and / or in vitro agonistic activity against islet amyloid peptide (AMY3) EC 50 <100 nM, and / or in vitro agonistic activity against GLP-1R EC 50 <100 nM, preferably less than 10 nM, more preferably less than 1 nM; and / or in vitro agonistic activity against GIPR EC 50 <100nM, preferably less than 10nM, more preferably less than 1nM.

[0025] The GLP-1 / amylin receptor agonist compounds of the present invention exhibit enhanced agonistic activity against GLP-1R and / or calcitonin (CT), and / or islet amyloid peptide (AMY3), and / or islet amyloid peptide (AMY1) receptors. Preferably, the in vitro agonistic activity against calcitonin (CT) is EC50. 50 <100 nM, preferably less than 10 nM, more preferably less than 1 nM, and / or in vitro agonistic activity against pancreatic amyloid peptide (AMY1) receptor EC 50 <100 nM, preferably less than 10 nM, more preferably less than 1 nM, and / or in vitro agonistic activity against islet amyloid peptide (AMY3) EC 50 <100 nM, and / or in vitro agonistic activity against GLP-1R EC 50 <100nM, preferably less than 10nM, more preferably less than 1nM.

[0026] For clarity, this article defines the general terminology used in the description of compounds.

[0027] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0028] As used herein, the term "compound" refers to a molecular entity; therefore, a "compound" may have different structural elements in addition to having the minimum elements defined for each compound or group of compounds. The term "compound" may be used interchangeably with the term "construction." 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," and the term "compound" is also intended to cover their pharmaceutically relevant forms, namely, compounds as defined herein or their pharmaceutically acceptable salts, amides, or esters.

[0029] The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention, prepared by combining a compound with specific substituents discovered in the present invention with a pharmaceutically acceptable acid or base, including hydrochloride, trifluoroacetate, acetate, etc.

[0030] Some compounds of this invention may exist in non-solventized or solvated forms, including hydrated forms. Generally, solvated and non-solventized forms are equivalent and both are included within the scope of this invention.

[0031] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, transisomers, racemic mixtures thereof, and other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0032] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, transisomers, etc., can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, 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 desired enantiomer in pure form. 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 salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0033] The atoms in the compounds of this invention are isotopes. Isotope derivatization can typically prolong half-life, reduce clearance rate, stabilize metabolism, and enhance in vivo activity. Furthermore, one embodiment is included, wherein at least one atom is replaced by an atom having the same number of atoms (protons) but different mass numbers (protons and neutrons). Examples of isotopes included in the compounds of this invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each comprising... 2 H, 3 H, 13 C 14 C 15 N、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 36 Cl. In particular, radioactive isotopes that emit radiation as they decay, such as 3 H or 14 C can be used for local anatomical examination of pharmaceutical preparations or compounds in vivo. Stable isotopes neither decay nor change with quantity and are not radioactive, therefore they can be used safely. When the atoms constituting the compounds of this invention are isotopes, the isotopes can be converted according to common methods by replacing the reagents used in the synthesis with reagents containing the corresponding isotopes.

[0034] The compounds of this invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting 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, regardless of radioactivity, are included within the scope of the present invention.

[0035] Furthermore, one or more hydrogen atoms in the compound of the present invention are coated with the isotope deuterium ( 2 The compounds of this invention, after being substituted with H), have the effects of prolonged half-life, reduced clearance rate, metabolic stabilization, and increased in vivo activity.

[0036] The preparation methods of the isotope derivatives typically include phase-transfer catalysis. For example, a preferred deuteration method employs a phase-transfer catalyst (e.g., tetraalkylammonium salt, NBu4HSO4). Using a phase-transfer catalyst to exchange the methylene protons of a diphenylmethane compound results in the introduction of higher levels of deuterium than reduction with deuterated silanes (e.g., triethyldeuterated silane) in the presence of an acid (e.g., methanesulfonic acid) or with Lewis acids such as aluminum trichloride using sodium deuterated borate.

[0037] As used herein, the term "polypeptide" or "polypeptide sequence" refers to a compound comprising a series of two or more amino acids linked together by amide (or peptide) bonds. The term polypeptide is used interchangeably with the terms "peptide" and "protein".

[0038] This invention relates to compounds comprising GLP-1 / amylin, GLP / GIP / amylin, or isomers thereof, racemates thereof, or pharmaceutically acceptable salts thereof, and methods for their preparation. The compounds disclosed herein are capable of activating or "activating" both the GLP-1 receptor and the amylin receptor system, or, alternatively, are capable of activating or "activating" both the GLP-GIP receptor and the amylin receptor system, and are "GLP-1 receptor-amylin receptor co-agonists" or "GLP-GIP receptor-amylin receptor co-agonists." The compound may further comprise one, two, or three elongation moieties. These elongation moieties may be able to non-covalently bind to albumin, thereby promoting the circulation of the GLP-1 receptor-amylin receptor co-agonist or the GLP-GIP receptor-amylin receptor co-agonist in the bloodstream and prolonging their half-life. Therefore, those skilled in the art may also refer to the elongation moieties as "albumin-binding moieties."

[0039] The compounds disclosed herein are “GLP-1 receptor-amylin receptor co-agonists” or “GLP-1-amylin receptor co-agonists” or “GLP-GIP receptor-amylin receptor co-agonists” or “GLP-GIP-amylin receptor co-agonists”. The GLP-1 receptor-amylin receptor co-agonist comprises a GLP-1 receptor agonist, an optional peptide linker, and an amylin receptor agonist. The GLP-1 receptor agonist component binds to and activates the GLP-1 receptor. The GLP-GIP receptor-amylin receptor co-agonist comprises a GLP-GIP receptor agonist, an optional peptide linker, and an amylin receptor agonist. The GLP-GIP receptor agonist component binds to and activates the GLP-GIP receptor, while the amylin receptor agonist component binds to and activates at least the human amylin 3 receptor (AMYR3), the human amylin 2 receptor (AMYR2), and the human amylin 1 receptor (AMYR1).

[0040] The compounds disclosed herein include GLP-1 receptor agonists. A “GLP-1 receptor agonist” can be defined as a ligand capable of binding to the GLP-1 receptor and producing a biological response similar to that of the natural ligand, glucagon-like peptide-1 (GLP-1). A “full” GLP-1 receptor agonist can be defined as a GLP-1 receptor agonist capable of eliciting a biological response of the same magnitude as that of GLP-1.

[0041] As used herein, the term "GLP-GIP peptide" refers to a peptide capable of binding to and / or activating the GLP receptor. In other words, a GLP peptide is a peptide with GLP-1 activity, and a GIP peptide is a peptide with GIP activity. In other words, a GLP-1 peptide is a GLP-1 receptor agonist, and a GIP peptide is a GIP receptor agonist.

[0042] In one embodiment, the compound of the present invention comprises a GLP-1 polypeptide. In one embodiment, the GLP-1 polypeptide is the amino acid sequence of semaglutide or a semaglutide analogue, the structure of which is as follows:

[0043] Specifically, GLP-1 receptor agonists must have a free N-terminus. Therefore, the compounds disclosed herein comprise GLP-1 receptor agonists whose C-terminus is linked to an optional peptide or amylin receptor agonist.

[0044] 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, with the following structure:

[0045] In one embodiment, the GLP-GIP polypeptide has the amino acid sequence of SEQ ID NO:1 and its structure is as follows:

[0046] (SEQ ID NO:1).

[0047] In one embodiment, the GLP-GIP polypeptide has the amino acid sequence of SEQ ID NO:2 and its structure is as follows:

[0048] (SEQ ID NO:2).

[0049] In one embodiment, the GLP-GIP polypeptide has the amino acid sequence of SEQ ID NO:3 and its structure is as follows:

[0050] The compounds disclosed herein include amylin receptor agonists. An "amylin receptor agonist" can be defined as a chemical entity capable of binding to and activating an amylin receptor. In the context of this invention, an "amylin receptor agonist" is at least capable of binding to and activating the AMYR3 complex. The amylin receptor agonist may also be capable of agonizing calcitonin receptors and AMYR1-2.

[0051] Examples of endogenous amylin receptor agonists are human amylin and human calcitonin. Examples of exogenous amylin receptor agonists are pramlintide and canagliptin (disclosed in WO2012 / 168432). The structure of canagliptin is as follows:

[0052] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium capable of delivering an effective amount of the active substance of this invention without interfering with the biological activity of the active substance and without toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, ointment bases, lotion bases, and ointment bases. These bases include suspending agents, thickeners, and transdermal penetration enhancers. Their formulations are well known to those skilled in the art of cosmetics or topical pharmaceuticals. For further information on carriers, see Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0053] For pharmaceuticals or pharmacologically active agents, the term "effective amount" or "therapeutic effective amount" refers to a sufficient quantity of a drug or agent that is non-toxic but achieves the desired effect. For the oral dosage forms of this invention, the "effective amount" of one active substance in the composition refers to the quantity required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the recipient's age and general condition, as well as the specific active substance. A suitable effective amount in any given case can be determined by a person skilled in the art through routine testing.

[0054] "Optional" or "optionally" means that the event or condition described below may occur but is not required to occur, and the description includes both the scenario in which said event or condition occurs and the scenario in which said event or condition does not occur.

[0055] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention. Attached Figure Description

[0056] Figure 1 shows the mass spectrum of compound 110 of the present invention.

[0057] Figure 2 shows the mass spectrum of compound A1 of the present invention. Detailed Implementation

[0058] The present application will be described in further detail below with reference to the embodiments, but the implementation of the present application is not limited thereto.

[0059] Example 1: Preparation of GLP-1 Compounds

[0060] The synthetic route for GLP-1 compounds can be prepared by referring to the synthetic route of WO2022096636A1, or the contents of WO2022096636A1 can be incorporated into this application by reference. In this invention, the GLP-1 receptor agonist is selected from the structures shown in Table 1.

[0061] Table 1 shows the structures of GLP-1 compounds.

[0062] The synthesis route for X1 is as follows:

[0063] Step 1: F1 Synthesis

[0064] Synthesis process description: Compound F1 was obtained by solid-state synthesis.

[0065] (1) Using 2-CTC resin as a carrier, it was first swollen with N,N-dimethylformamide (DMF), and then Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) were added and reacted for several hours. After the reaction was completed, it was washed several times with DMF.

[0066] (2) After washing, methanol and DIEA were added for end capping. After end capping, the resin was washed several times with DMF.

[0067] (3) Deprotection was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After the deprotection was completed, the resin was washed with DMF.

[0068] (4) Mix 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA with tetrahydrofuran (THF) and add them to the resin to carry out the reaction. After the reaction is completed, wash the resin with DMF.

[0069] (5) 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.

[0070] (6) Repeat step (3) to remove the Fmoc protecting group.

[0071] (7) Weigh out Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt), dissolve them in DMF, then add N,N'-diisopropylcarbodiimide (DIC) and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0072] (8) Weigh out tert-butyl hexadecanoate and repeat step (7) to carry out the condensation reaction.

[0073] (9) After mixing mercaptoethanol, DBU and DMF respectively, add them to the resin to remove Ns protecting groups. After the reaction is complete, wash the resin with DMF.

[0074] (10) Weigh out Boc-Pro-OH and repeat step (7) to carry out the condensation reaction.

[0075] (11) The resin was washed with dichloromethane and methanol respectively, and dried at room temperature to obtain peptide resin.

[0076] (12) The resin was cut with 20% TFE / DCM and the concentrated solution was filtered to obtain compound F1.

[0077] Step 2: Synthesis of F2 (Smigratide fully protected peptide resin)

[0078] The synthetic route of smegglutinin is referenced from the synthetic routes in patent applications CN200680006674.6 and WO2022096636A1.

[0079] His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtB u)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-octadecanedioic acid monotert-butyl ester)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-WangResin

[0080] F2

[0081] Synthesis steps:

[0082] (1) Wang resin was used as a carrier. It was first swollen with DMF. After the swelling was completed, the resin was washed with DMF.

[0083] (2) Weigh out Fmoc-Gly-OH, HOBt and 4-dimethylaminopyridine (DMAP), dissolve them in DMF, add DIC and mix well. Then add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the resin with DMF.

[0084] (3) After washing, acetic anhydride and DIEA were added for end capping. After end capping, the resin was washed several times with DMF.

[0085] (4) Deprotection was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After the deprotection was completed, the resin was washed with DMF.

[0086] (5) Weigh Fmoc-Arg(Pbf)-OH and HOBt, add DMF to mix and dissolve, then add DIC to activate for 3-5 min. After activation, add to the reactor to start the coupling reaction; the amino acid coupling reaction takes 1.0-3.0 h, and the reaction endpoint is monitored with ninhydrin throughout the coupling process. After coupling, wash the resin with DMF.

[0087] (6) Repeat step e. Couple amino acids sequentially according to the peptide sequence to obtain F2 (smegglutinin fully protected peptide resin).

[0088] (7) After the reaction is complete, wash the peptide resin with dichloromethane and methanol and air dry at room temperature.

[0089] Step 3: Preparation of the target compound (F2+F1)

[0090] (1) First, swell F2 (Smigratide fully protected peptide resin) with DMF. After swelling, wash the resin with DMF.

[0091] (2) Weigh out F1 and HOBt, dissolve them in DMF, then add DIC and mix well. After mixing, add the mixture to the resin to carry out the reaction. After the reaction is complete, wash the peptide resin with DMF, dichloromethane and methanol respectively and air dry.

[0092] (3) Using TFA / TIS / H2O = 95.0 / 2.5 / 2.5, the volume was prepared according to 10 ml of lysis buffer per gram of peptide resin. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the resin was filtered, and after concentration to remove part of the TFA, it was added to 8 times the volume of lysis buffer in diethyl ether to precipitate the precipitate. The crude product was collected by centrifugation and dried to constant weight.

[0093] (4) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0094] (5) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0095] (6) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0096] (7) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0097] The freeze-dried samples were aliquoted and stored according to requirements. Samples were taken for testing, and the target compound X1 was obtained.

[0098] Example 2 Preparation of GLP-GIP Compounds

[0099] The synthetic route for GLP-GIP compounds can be prepared by referring to the synthetic route of CN201680005007.X, or the contents of CN201680005007.X can be incorporated into this application by reference. In this invention, the GLP-GIP receptor agonist is selected from the structures shown in Table 2.

[0100] Table 2 shows the structures of GLP-GIP compounds.

[0101] Example 3: Preparation of amylin receptor agonists

[0102] In this invention, the amylin receptor agonist is selected from the structures shown in Table 3. The preparation routes of the structures shown in Table 3 are referred to patent application numbers CN201580046850.8 and CN201280028554.1.

[0103] Table 3 lists amylin receptor agonists.

[0104] The P structure is as follows:

[0105] Example 4: Preparation of GLP-1 and amylin receptor co-agonists

[0106] The connectors used in this invention are selected from those shown in Table 4.

[0107] Table 4 shows the connectors.

[0108] The preparation of the GLP-1 and amylin receptor co-agonists in this invention follows the synthetic route described in patent application CN202180085551.0, the entire contents of which are incorporated herein by reference. The structures of the GLP-1 and amylin receptor co-agonist compounds of this invention are shown in Tables 5, 6, 7, 8, and 9.

[0109] The specific synthetic route of compound 1 is as follows:

[0110] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0111] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0112] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0113] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0114] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0115] (6) Follow steps 2 to 5 to sequentially couple Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro- OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, FSmoc-Ser(tBu)-OH, Fmoc-His(Trt) -OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Al a-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-γ-Glu-OtBu, eicosanedioic acid monotert-butyl ester.

[0116] (7) After the above amino acid coupling is completed, the resin is washed with DMF four times in sequence.

[0117] (8) Add 2% hydrazine hydrate / DMF at 10-15 times the weight of the resin to remove the Dde protecting group. The first deprotection takes 15 minutes, and the second deprotection takes 15 minutes.

[0118] (9) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0119] (10) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Gly-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0120] (11) The resin was washed four times with 10-15 times its volume of DMF.

[0121] (12) Follow steps 2 to 5 to couple Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-T in sequence rp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln( Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Pro-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH

[0122] (13) After all amino acids were coupled, the resin was washed four times with DMF, four times with DCM, and four times with methanol.

[0123] (14) Place the above resin in a vacuum drying oven and dry it at 15-30°C until constant weight.

[0124] (15) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate and wash it with isopropyl ether more than 5 times. Dry the linear crude product after pyrolysis to constant weight.

[0125] (16) Oxidation: The linear crude product was dissolved in 20% acetic acid aqueous solution (1.0 mg / ml), and an appropriate amount of iodine ethanol solution was added for oxidation. The oxidation was carried out for 60 min. The linear crude product was detected by HPLC to be ≤3.0%. An appropriate amount of vitamin C was added to quench the reaction.

[0126] (17) The crude product oxidation solution was filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0127] (18) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0128] (19) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0129] (20) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0130] After lyophilization, the sample was aliquoted and stored as required. Sample analysis revealed that the compound had a measured molecular weight of 9327.6 and M / 5 = 1865.3.

[0131] The specific synthetic route for compound 3 is as follows:

[0132] The preparation steps are as follows:

[0133] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0134] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0135] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0136] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0137] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0138] (6) Follow steps 2 to 5 to sequentially couple Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro- OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt) -OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Al a-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-γ-Glu-OtBu, eicosanedioic acid monotert-butyl ester.

[0139] (7) After the above amino acid coupling is completed, the resin is washed with DMF four times in sequence.

[0140] (8) Add 2% hydrazine hydrate / DMF at 10-15 times the weight of the resin to remove the Dde protecting group. The first deprotection takes 15 minutes, and the second deprotection takes 15 minutes.

[0141] (9) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0142] (10) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Glu(OtBu)-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0143] (11) The resin was washed four times with 10-15 times its volume of DMF.

[0144] (12) Follow steps 2 to 5 to couple Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu- OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmo c-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Pro-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH

[0145] (13) After all amino acids were coupled, the resin was washed four times with DMF, four times with DCM, and four times with methanol.

[0146] (14) Place the above resin in a vacuum drying oven and dry it at 15-30°C until constant weight.

[0147] (15) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate and wash it with isopropyl ether more than 5 times. Dry the linear crude product after pyrolysis to constant weight.

[0148] (16) Oxidation: The linear crude product was dissolved in 20% acetic acid aqueous solution (1.0 mg / ml), and an appropriate amount of iodine ethanol solution was added for oxidation. The oxidation was carried out for 60 min. The linear crude product was detected by HPLC to be ≤3.0%. An appropriate amount of vitamin C was added to quench the reaction.

[0149] (17) The crude product oxidation solution was filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0150] (18) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0151] (19) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0152] (20) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0153] (21) The freeze-dried sample was dispensed and stored as required. The sample was taken for testing, and the measured molecular weight of the compound was 9456.8, M / 5 = 1891.6.

[0154] The synthetic route for compound 5 is as follows:

[0155] The preparation steps are as follows:

[0156] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0157] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0158] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0159] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0160] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0161] (6) Follow steps 2 to 5 to sequentially couple Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro- OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt) -OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Al a-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-γ-Glu-OtBu, eicosanedioic acid monotert-butyl ester.

[0162] (7) After the above amino acid coupling is completed, the resin is washed with DMF four times in sequence.

[0163] (8) Add 2% hydrazine hydrate / DMF at 10-15 times the weight of the resin to remove the Dde protecting group. The first deprotection takes 15 minutes, and the second deprotection takes 15 minutes.

[0164] (9) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0165] (10) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Gly-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0166] (11) The resin was washed four times with 10-15 times its volume of DMF.

[0167] (12) Follow steps 2 to 5 to couple Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Al in sequence a-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc -Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)- OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Pro-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH

[0168] (13) After all amino acids were coupled, the resin was washed four times with DMF, four times with DCM, and four times with methanol.

[0169] (14) Place the above resin in a vacuum drying oven and dry it at 15-30°C until constant weight.

[0170] (15) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate and wash it with isopropyl ether more than 5 times. Dry the linear crude product after pyrolysis to constant weight.

[0171] (16) Oxidation: The linear crude product was dissolved in 20% acetic acid aqueous solution (1.0 mg / ml), and an appropriate amount of iodine ethanol solution was added for oxidation. The oxidation was carried out for 60 min. The linear crude product was detected by HPLC to be ≤3.0%. An appropriate amount of vitamin C was added to quench the reaction.

[0172] (17) The crude product oxidation solution was filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0173] (18) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0174] (19) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0175] (20) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0176] (21) The freeze-dried sample was dispensed and stored as required. The sample was taken for testing, and the measured molecular weight of the compound was 9213.5, M / 5 = 1842.5.

[0177] Table 5

[0178] The structures of X1 and X2 are shown in Table 1, and the structures of Y1, Y2 and Y3 are shown in Table 3. The synthetic routes of compounds 2, 4 and 6-35 in Table 5 are prepared by referring to the synthetic routes of compounds 1, 3 and 5, the difference being the linker. The linker amino acids involved in steps 10 and 12 can be replaced with the linkers used in the corresponding compounds.

[0179] The synthetic route for compound 36 is as follows:

[0180] The preparation steps are as follows:

[0181] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0182] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0183] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0184] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0185] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0186] (6) Follow steps 2 to 5 to sequentially couple Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro- OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt) -OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Al a-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Dde)-OH, Fmoc-γ-Glu-OtBu, eicosanedioic acid monotert-butyl ester.

[0187] (7) After the above amino acid coupling is completed, the resin is washed with DMF four times in sequence.

[0188] (8) Add 2% hydrazine hydrate / DMF at 10-15 times the weight of the resin to remove the Dde protecting group. The first deprotection takes 15 minutes, and the second deprotection takes 15 minutes.

[0189] (9) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0190] (10) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Gly-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0191] (11) The resin was washed four times with 10-15 times its volume of DMF.

[0192] (12) Follow steps 2 to 5 to couple Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-T in sequence rp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln( Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Gly-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH

[0193] (13) After all amino acids were coupled, the resin was washed four times with DMF, four times with DCM, and four times with methanol.

[0194] (14) Place the above resin in a vacuum drying oven and dry it at 15-30°C until constant weight.

[0195] (15) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate and wash it with isopropyl ether more than 5 times. Dry the linear crude product after pyrolysis to constant weight.

[0196] (16) Oxidation: The linear crude product was dissolved in 20% acetic acid aqueous solution (1.0 mg / ml), and an appropriate amount of iodine ethanol solution was added for oxidation. The oxidation was carried out for 60 min. The linear crude product was detected by HPLC to be ≤3.0%. An appropriate amount of vitamin C was added to quench the reaction.

[0197] (17) Filter the crude product oxidation solution through a 0.45 μm filter membrane. Transfer the filtered solution to a purification system for crude HPLC separation.

[0198] (18) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0199] (19) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0200] (20) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0201] After lyophilization, the sample was aliquoted and stored as required. Sample analysis revealed that the compound had a measured molecular weight of 9287.6 and M / 5 = 1857.8.

[0202] Table 6

[0203] The structures of X1 and X2 are shown in Table 1, and the structures of Y1, Y2 and Y3 are shown in Table 3. The synthetic routes of compounds 37-70 are the same as those of compound 36, except that the linkers are different. The linker amino acids involved in steps 10 and 12 are replaced with the linkers used in the corresponding compounds.

[0204] The synthetic route for compound 71 is as follows:

[0205] The preparation steps are as follows:

[0206] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0207] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0208] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0209] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0210] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0211] (6) Couple Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH in sequence according to steps 2 - 5.Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Pro-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH,

[0212] (7) After all amino acids are coupled, the resin is washed four times with DMF, four times with DCM, and four times with methanol.

[0213] (8) Place the above resin in a vacuum drying oven and dry it at 15-30℃ until constant weight.

[0214] (9) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate and wash it with isopropyl ether more than 5 times. Dry the linear crude product after pyrolysis to constant weight.

[0215] (10) Oxidation: The linear crude product was dissolved in 20% acetic acid aqueous solution (1.0 mg / ml), and an appropriate amount of iodine ethanol solution was added for oxidation. The oxidation was carried out for 60 min. The linear crude product was detected by HPLC to be ≤3.0%. An appropriate amount of vitamin C was added to quench the reaction.

[0216] (11) The crude product oxidation solution was filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0217] (12) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0218] (13) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0219] (14) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0220] After lyophilization, the sample was aliquoted and stored as required. Sample analysis revealed that the compound had a measured molecular weight of 8874.0, with M / 5 = 1774.8.

[0221] Compound 73

[0222] The synthetic route of compound 73 can be referenced from that of compound 71, the difference being the linker. The measured molecular weight of compound 73 is 9003.2, M / 5 = 1801.2.

[0223] Table 7

[0224] The structures of X1 and X2 are shown in Table 1, and the structures of Y1, Y2 and Y3 are shown in Table 3. The synthetic routes of compounds 72-105 are the same as those of compound 71, except that the linkers are different. The linker amino acids involved in step 6 are replaced with the linkers used in the corresponding compounds.

[0225] The synthetic route for compound 106 is as follows:

[0226] The preparation steps are as follows:

[0227] (1) Using Rink MBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0228] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0229] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0230] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0231] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0232] (6) Couple Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH in sequence according to steps 2 - 5.Boc-Pro-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH、

[0233] (7) After all amino acids are coupled, the resin is washed four times with DMF, four times with DCM, and four times with methanol.

[0234] (8) Place the above resin in a vacuum drying oven and dry it at 15-30℃ until constant weight.

[0235] (9) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate, wash the precipitate with isopropyl ether more than 5 times, and dry the crude product to constant weight.

[0236] (10) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0237] (11) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0238] (12) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0239] (13) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0240] (14) The freeze-dried sample was dispensed and stored as required. The sample was taken for testing, and the measured molecular weight of the compound was 8270.3, M / 5 = 1653.9.

[0241] The synthetic route for compound 108 is as follows:

[0242] The preparation steps are as follows:

[0243] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0244] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0245] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0246] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0247] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0248] (6) Couple Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH in sequence according to steps 2 - 5.Boc-Pro-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH、

[0249] (7) After all amino acids are coupled, the resin is washed four times with DMF, four times with DCM, and four times with methanol.

[0250] (8) Place the above resin in a vacuum drying oven and dry it at 15-30℃ until constant weight.

[0251] (9) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate, wash the precipitate with isopropyl ether more than 5 times, and dry the crude product to constant weight.

[0252] (10) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0253] (11) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0254] (12) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0255] (13) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0256] After lyophilization, the sample was aliquoted and stored as required. Sample analysis revealed that the compound had a measured molecular weight of 8399.4 and M / 5 = 1679.8.

[0257] The synthetic route for compound 110 is as follows:

[0258] The preparation steps are as follows:

[0259] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0260] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0261] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0262] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0263] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0264] (6) Couple Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH in sequence according to steps 2 - 5.Boc-Pro-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH、

[0265] (7) After all amino acids are coupled, the resin is washed four times with DMF, four times with DCM, and four times with methanol.

[0266] (8) Place the above resin in a vacuum drying oven and dry it at 15-30℃ until constant weight.

[0267] (9) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate, wash the precipitate with isopropyl ether more than 5 times, and dry the crude product to constant weight.

[0268] (10) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0269] (11) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0270] (12) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0271] (13) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0272] After lyophilization, the sample was aliquoted and stored as required. Sample analysis revealed that the compound had a measured molecular weight of 8156.2, M / 5 = 1630.9, and the mass spectrum is shown in Figure 1.

[0273] The synthetic route for compound 112 is as follows:

[0274] The preparation steps are as follows:

[0275] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0276] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0277] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0278] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0279] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0280] (6) Couple Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH in sequence according to steps 2 - 5.Boc-Pro-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH、

[0281] (7) After all amino acids are coupled, the resin is washed four times with DMF, four times with DCM, and four times with methanol.

[0282] (8) Place the above resin in a vacuum drying oven and dry it at 15-30℃ until constant weight.

[0283] (9) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate, wash the precipitate with isopropyl ether more than 5 times, and dry the crude product to constant weight.

[0284] (10) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0285] (11) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0286] (12) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0287] (13) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0288] After lyophilization, the sample was aliquoted and stored as required. Sample analysis revealed that the compound had a measured molecular weight of 8228.2 and M / 5 = 1645.5.

[0289] Table 8

[0290] The structures of X1 and X2 are shown in Table 1, and the structures of Y1, Y2 and Y3 are shown in Table 3. The synthetic routes of compounds 107, 109, 111 and 113-140 are the same as those of compounds 106, 108, 110 and 112. The difference is that the linkers are different. The linker amino acids involved in step 6 are replaced with the linkers used in the corresponding compounds.

[0291] The synthetic route for compound 141 is as follows:

[0292] The preparation steps are as follows:

[0293] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0294] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0295] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0296] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0297] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0298] (6) Couple Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBuO-Ste-Glu(AEEA-AEEA)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH in sequence according to steps 2 - 5.Boc-Gly-(N-CH2CH2NH-γ-Glu(C16)-OtBu)Gly-His(Trt)-Aib-OH,

[0299] (7) After all amino acids are coupled, the resin is washed four times with DMF, four times with DCM, and four times with methanol.

[0300] (8) Place the above resin in a vacuum drying oven and dry it at 15-30℃ until constant weight.

[0301] (9) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate, wash the precipitate with isopropyl ether more than 5 times, and dry the crude product to constant weight.

[0302] (10) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0303] (11) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0304] (12) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0305] (13) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0306] After lyophilization, the sample was aliquoted and stored as required. Sample analysis revealed that the compound had a measured molecular weight of 8230.2 and M / 5 = 1646.0.

[0307] Table 9

[0308] The structures of X1 and X2 are shown in Table 1, and the structures of Y1, Y2 and Y3 are shown in Table 3. The synthetic routes of compounds 142-175 are the same as those of compound 141, except that the linkers are different. The linker amino acids involved in step 6 are replaced with the linkers used in the corresponding compounds.

[0309] Example 5: Preparation of GLP / GIP / Amylin receptor co-agonists

[0310] The connectors used in this invention are selected from those shown in Table 4.

[0311] The preparation of the GLP / GIP / Amylin receptor co-agonist in this invention follows the synthetic route described in patent application CN202180085551.0, the entire contents of which are incorporated herein by reference. The structures of the GLP / GIP / Amylin receptor co-agonist compounds of this invention are shown in Tables 10 and 11.

[0312] The specific synthetic route for compound A1 is as follows:

[0313] Step 1: Synthesize M1

[0314] GGGGEASELSTAALGRLSAELHELATLPRTETGSGSP-NH2

[0315] M1

[0316] The preparation steps are as follows:

[0317] (1) Using RinkMBHA resin with a substitution degree of 0.30 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0318] (2) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 minutes, and the second deprotection takes 15 minutes.

[0319] (3) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0320] (4) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Pro-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0321] (5) Wash the resin four times with 10-15 times its volume of DMF.

[0322] (6) Follow steps 2 to 5 to couple Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg( Pbf)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)- OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Se r(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH.

[0323] (7) After all amino acids are coupled, the resin is washed four times with DMF, four times with DCM, and four times with methanol.

[0324] (8) Place the above resin in a vacuum drying oven and dry it at 15-30℃ until constant weight.

[0325] (9) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate, wash the precipitate with isopropyl ether more than 5 times, and dry the crude product to constant weight.

[0326] (10) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0327] (11) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method, and the qualified fraction was collected.

[0328] (12) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0329] (13) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0330] After freeze-drying, the samples were aliquoted and stored according to requirements. Samples were then taken for testing.

[0331] Step 2: Prepare compound A1. The specific synthetic route is as follows:

[0332] (21) Using RinkMBHA resin with a substitution degree of 0.20 mmol / g as a carrier, it was first swollen with 10-15 times the volume of resin in DMF for 30 minutes. After swelling was completed, the solvent was removed. The resin was then washed 4 times with 10-15 times the volume of resin in DMF.

[0333] (22) Add 20% piperidine / DMF at 10-15 times the weight of the resin for deprotection. The first deprotection takes 10 min and the second deprotection takes 15 min.

[0334] (23) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0335] (24) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Ser(tBu)-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 reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0336] (25) The resin was washed four times with 10-15 times its volume of DMF.

[0337] (26) Follow steps 2 to 5 to couple Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc- Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-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-Glu(OtBu)-OH, Fmoc-Aib-OH, Boc-Tyr(tBu)-OH.

[0338] (27) After the above amino acid coupling is completed, the resin is washed with DMF four times in sequence.

[0339] (28) Add 2% hydrazine hydrate / DMF at 10-15 times the weight of the resin to remove the Dde protecting group. The first deprotection takes 15 minutes, and the second deprotection takes 15 minutes.

[0340] (29) After the deprotection is completed, the resin is washed with DMF six times, each time for 1 to 3 minutes.

[0341] (30) Weigh and prepare amino acid coupling solution. Weigh 2.0 eq. Fmoc-Glu(OtBu)-OH and 2.2 eq. HOBt respectively, add DMF to dissolve, then add 2.0 eq. DIC to activate for 3-5 min. After activation, add to the reaction vessel to start the coupling reaction. The amino acid coupling reaction lasts for 1-3 h. The reaction endpoint is monitored by ninhydrin during the coupling process.

[0342] (31) The resin was washed four times with 10-15 times its volume of DMF.

[0343] (32) Couple Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and eicosanoic acid sequentially according to steps 2 to 5.

[0344] (33) Weigh 2.0 eq. HBTU and 2.0 eq. HOBt, add DMF to dissolve, then add 2.0 eq. DIEA and stir evenly. Add to the reactor to activate the reaction and react for 1-3 hours. Wash the resin 4 times with 10-15 times its volume of DMF.

[0345] (34) Dissolve Y1 in DMF and add it to the reaction vessel and stir to react.

[0346] (35) After all amino acids were coupled, the resin was washed four times with DMF, four times with DCM, and four times with methanol.

[0347] (36) Place the above resin in a vacuum drying oven and dry it at 15-30°C until constant weight.

[0348] (37) Pyrolysis: Prepare a solution with a volume ratio of TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Add the resin to the prepared lysis buffer and stir at room temperature for 2 hours. After the reaction is complete, filter the resin and wash it twice with a small amount of TFA. Combine the filtrates, concentrate to remove some TFA, and add it to 8 times the volume of lysis buffer in isopropyl ether to precipitate the precipitate. Centrifuge to collect the precipitate, wash the precipitate with isopropyl ether more than 5 times, and dry the crude product to constant weight.

[0349] (38) The crude product was dissolved by sonication in acetonitrile / water solution and then filtered through a 0.45 μm filter membrane. The filtered solution was then transferred to a purification system for crude HPLC separation.

[0350] (39) HPLC crude fraction: The filtered sample solution is purified according to the crude fraction method, and qualified fractions are collected.

[0351] (40) HPLC purification: The qualified fraction collected by HPLC crude fraction is purified by HPLC purification method and the qualified fraction is collected.

[0352] (41) Concentration and freeze-drying: Concentrate the qualified fraction, and filter, divide and freeze-dry the concentrated sample according to the process specifications.

[0353] After lyophilization, the samples were aliquoted and stored according to requirements. Samples were taken for analysis; the measured molecular weight of compound A1 was (M+6H). 6+ =1396.1, (M+5H) 5+ =1675.0, and its mass spectrum is shown in Figure 2.

[0354] Table 10

[0355] The structure of X3 is shown in Table 2, and the structure of Y3 is shown in Table 3. The side chains of peptide Y3 in compounds A1-A35 are coupled to the side chains of X3 through a linker. The synthetic routes of compounds A2-A35 in Table 4 are prepared by referring to the synthetic route of compound A1, the difference being that the linkers are different. The linker amino acids in step 1 are replaced with the linkers used in the corresponding compounds.

[0356] The structure of compound B1 is as follows:

[0357] Table 11

[0358] The structures of X3 and Y3 are shown in Table 3. In compounds B1-B35, polypeptide Y3 is coupled to polypeptide sequence X3 via a linker.

[0359] The difference between compounds B2-B35 and compound B1 is the linker. Simply replace the linker in B1 with the linker used in the corresponding compound. The connection methods of X3 to the linker and Y3 in the structures of compounds B2-B35 in Table 11 are the same as those in compound B1. The synthetic routes for compounds B1-B35 in Table 11 can be found in patent document WO2025114501A1. Example 5: GLP-1 Activity Assay

[0360] The agonistic effect of the test substance on GLP-1R was determined using U2OS-GLP-1R stable cell lines and HTRF assay.

[0361] Experimental methods:

[0362] 1. Following the experimental procedures of the cAMP-Gs Dynamic HTRF kit, dilute the 5x Stimulation buffer (SB) provided with the kit to 1xSB using ddH2O. Add IBMX to the 1xSB solution to a final concentration of 500 μM to prevent cAMP degradation. Then, use the 1xSB to prepare the working solution of the test substance and perform a series of serial dilutions.

[0363] 2. U2OS-GLP-R cells were digested and collected, resuspended, counted, and then diluted to a density of 2 x 10^6 cells using 1xSB solution. 6 Cells / mL, and then seeded into a 384-well cell plate at a rate of 5 μL per well, so that the number of cells per well is 10,000.

[0364] 3. Add 5 μL of the test substance to each well and incubate at 37°C for 30 mins.

[0365] 4. Prepare cAMP standard solutions of different concentrations according to the steps provided in the cAMP-Gs Dynamic HTRF kit and add them to 384-well cell plates.

[0366] 5. Dilute the cAMP d2 reagent and Eu Cryptate antibody provided in the kit to 1x using lysis & Detection Buffer. Add 5 μL of d2 and Eu to each well of a 384-well plate. Incubate at room temperature for 2 hours and then detect the results using the HTRF module (665 / 620 nm) of a microplate reader. Collect the experimental data.

[0367] 6. By plotting the signal value against the compound concentration, curve fitting and EC50 calculation were performed using the nonlinear regression method in GraphPadPrism software. The results are shown in Table 12.

[0368] Table 12 shows the in vitro activity of the compounds of this invention against GLP-1.

[0369] Where B < 10nM.

[0370] As shown in Table 12, the GLP-1 and amylin receptor agonist compounds of the present invention have excellent agonistic activity against GLP-1.

[0371] Example 6: Determination of GLP-1R and GIPR agonist activity

[0372] In the stable cell lines HEK293-GLP-1R-CRE-LUC and HEK-293-GIPR-CRE-LUC, the HTRF assay was used to determine the effect of the test substance on cAMP formation in the stable cell lines and to determine the agonistic activity of the test substance on GLP-1R and GIPR.

[0373] The experimental method is as follows: HEK293-GLP-1R-CRE-LUC and HEK-293-GIPR-CRE-LUC stable cell lines were cultured at 37℃ and 5% CO2. The culture medium was high glucose DMEM + 10% FBS + 200μg / mL G418 + 200μg / mL hygromycin B. On the day of the experiment, HEK293-GLP-1R-CRE-LUC and HEK-293-GIPR-CRE-LUC cells in the logarithmic growth phase were collected by centrifugation and resuspended in 1×Stimulation buffer (cAMP-Gs Dynamic HTRF kit) containing 0.5 mM IBMX. The cell density was adjusted to 8×10^5 cells / mL, and 5 μL of each well (4000 cells / well) was transferred to a 384-well assay plate (PerkinElmer, Cat No. 6008280). 5 μL / well of 2× working solution of the test substance (diluted with 1×Stimulation buffer containing 0.5 mM IBMX, 0.4% DMSO) was added, and the plate was then incubated at 37°C and 5% CO2 for 30 minutes. An equal volume of 1×Stimulation buffer containing 0.5 mM IBMX was added to the control cells. Dilute cAMP d2 reagent (receptor fluorescent group) and cAMP Eu Cryptate antibody (donor fluorescent group) with Lysis & Detection Buffer I from the kit. Add 5 μL of cAMP-d2 reagent-reagent working solution to all wells of a 384-well plate except for the NC well. Add an equal volume of Lysis & Detection Buffer to the blank wells. Then add the cAMP Eu-Cryptate antibody-working solution to all wells. Incubate at room temperature in the dark for 1 hour. Then, use the HTRF module on a Pherastar FSX multi-plate reader to read the signals and calculate the donor-receptor emission ratio for each well: HTRF Ratio = 665nm signal / 620nm signal × 10. 4 Then, the HTRF Ratio is converted into Effect (%), where Effect (%) = (HTRF Ratio - positive control) / HTRF Ratio sample ) / (HTRF Ratio positive control-HTRF Rationegative control)×100%, with the concentration of the test substance as the x-axis and %Effect as the y-axis, the %Effect concentration curve was plotted using GraphpadPrism for nonlinear fitting, and the EC50 value (nM) of cAMP production induced by the test substance in HEK293-GLP-1R-CRE-LUC and HEK-293-GIPR-CRE-LUC cells was obtained.

[0374] Example 7: In vitro functional activity assay of CT / AMY3 / AMY1 agonists

[0375] Activation of calcitonin (CT), islet amyloid peptide (AMY3), and islet amyloid peptide (AMY1) receptors leads to an increase in intracellular cAMP levels. Changes in cellular cAMP levels were measured using CHHO-K1 cell lines stably expressing human CT, AMY3, or AMY1 receptors.

[0376] Cryopreserved CHO-K1 / Gα15 / AMY3 cell stock solution was rapidly thawed in a water bath and resuspended in Ham's F-12K (Gibco, 21127022) medium containing 10% fetal bovine serum (Gibco, 10099141C), 100 μg / mL hygromycin B (Invitrogen, 10687010), 200 μg / mL bleomycin (Invitrogen, R25001), and 200 μg / mL G418 (Gibco, 10131027). CHO-K1 / CT / Gα15 and CHO-K1 / AMY1 / Gα15 cells were cultured in the same medium, except that they lacked G418. The cultured cells were allowed to grow to 80% confluence and then incubated overnight in fresh medium.

[0377] On the day of assay, cells were isolated and cultured using trypsin (Gibco, 25200072). Following the instructions of the HTRF assay kit (Revity, 62AM4PEB), cells were resuspended in stimulation buffer (containing 0.5 mM IBMX), and 5 μL of a solution containing 3000 cells / well (CHO-K1 / AMY3 / Gα15 or CHO-K1 / CT / Gα15 or CHO-K1 / Gα15 / AMY1) was added to each well in a 384-well plate (Revity, 6008280). The test compound was prepared in DMSO (dimethyl sulfoxide) and diluted to the working concentration with stimulation buffer; 5 μL of the test compound was added to each well. The plate was incubated at 37°C for 30 minutes. 5 μL of cAMP-d2 assay solution and 5 μL of LEU Crytate antibody assay solution from the kit were then incubated with the treated cells at room temperature for 60 minutes. The HTRF signal was immediately detected using a microplate reader (BMG LABTEECH) to calculate the fluorescence ratio from 665 to 620 nm. The HTRF Ratio = (665 nm signal value / 620 nm signal value) × 10000. The calculated values ​​and the analyte concentration were then processed using GraphPadPrism to obtain the "S" curve and EC50.

[0378] The results are shown in Tables 13-16. The results indicate that the GLP-1 and amylin receptor agonist compounds of the present invention have better agonistic activity against GLP-1R, and / or calcitonin (CT), and / or islet amyloid peptide (AMY3), and / or islet amyloid peptide (AMY1) receptors.

[0379] Table 13 shows the in vitro agonistic activity data of the compounds of this invention against calcitonin (CT).

[0380] Where A≤1nM.

[0381] Table 14 shows the in vitro agonistic activity data of the compounds of this invention against the pancreatic amyloid peptide (AMY3) receptor.

[0382] Where A≤1nM.

[0383] Table 15 shows the in vitro agonistic activity data of the compounds of this invention against calcitonin (CT).

[0384] Where Z≤10nM.

[0385] Table 16 shows the in vitro agonistic activity data of the compounds of the present invention against the pancreatic amyloid peptide (AMY1) receptor.

[0386] Where Z≤10nM.

[0387] Example 8: In vivo efficacy

[0388] Experimental methods:

[0389] This experiment used approximately 6-week-old SD rats as the research model. Before the experiment, the SD rats were randomly divided into 7 groups of 4 rats each, with 2 rats per cage, for a total of 28 rats. The SD rats were administered the drug subcutaneously as a single dose at doses of 10 nmol / kg, 30 nmol / kg, 50 nmol / kg, or 100 nmol / kg. Detailed dosing designs are shown in Table 17 below. During the experiment, animal behavior, coat color, water intake, and urination were observed. Food intake was continuously recorded 24 hours after drug administration, and any abnormalities were noted. At the end of the experiment, the SD rats were euthanized. The experimental period was 2 days, 7 days, or 14 days. The results of the 2-day experimental period are shown in Table 18.

[0390] Table 17 SD rats were grouped according to drug administration

[0391] Table 18 Results of food intake inhibition in rats of each treatment group after 2 days

[0392] Among them, F > 10%.

[0393] The results showed that the preferred compounds of the present invention all showed significant food intake inhibition compared to the vehicle group after administration, with a preferred food intake inhibition rate of greater than 10%, and more preferably greater than 20%.

[0394] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound of formula (I), or an isomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, include: XZ-Ya (I) Wherein, X is selected from peptides with GLP-1 activity and peptides with GLP-GIP activity; Z is selected from connector; Ya is selected from peptides that exhibit amylin activity.

2. The compound according to claim 1, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that, When X is selected from a polypeptide with GLP-1 activity, it is selected from the structure shown in formula (II). Among them, R 1 Selected from m is selected from 0 or 1, and n is selected from 10, 11, 12, 13, 14 or 15.

3. The compound according to claim 1, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that, X is selected from polypeptides with GLP-GIP activity.

4. The compound according to claim 1 or 2, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that, The peptides with GLP-1 activity are selected from:

5. The compound according to claim 1 or 3, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that, The GLP-GIP peptide is selected from Tirzepatide, SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:

3.

6. The compound according to any one of claims 1-3, or an isomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The amylin polypeptide is selected from canagliflozin (SEQ ID NO:4), SEQ ID NO:5, and SEQ ID NO:

6.

7. The compound according to any one of claims 1-3, or an isomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The connectors are selected from those shown in Table 4.

8. The compound according to claim 1 or 2, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the structures shown in Tables 5, 6, 7, 8 and 9.

9. The compound according to claim 1 or 3, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the structures shown in Tables 10 and 11.

10. A pharmaceutical composition, characterized in that, The compound comprising a therapeutically effective amount of any one of claims 1-9, or an isomer thereof, a racemic mixture thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof.

11. The pharmaceutical use of the compound of any one of claims 1-9, or an isomer thereof, or a racemic mixture thereof, or a pharmaceutically usable salt thereof, or the pharmaceutical composition of claim 10, specifically, in the preparation of a medicament for treating a disease selected from diabetes, obesity, cardiovascular disease, nonsteroidal steatohepatitis, and / or cognitive impairment caused by Alzheimer's disease.

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