GLP-1 receptor compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of therapeutic peptides, and relates to a compound as shown in a general formula (I), or a racemate thereof, or an isomer thereof, or a pharmaceutically acceptable salt thereof, as a GLP-1 compound, and application of the GLP-1 compound in treating diseases.

Description

A GLP-1 receptor compound and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of therapeutic peptides, and specifically relates to a GLP-1 receptor compound and a preparation method and application thereof. Background Art

[0002] One of the major challenges in the oral delivery of proteins and peptides is that these compounds do not readily cross the gastrointestinal membrane. Human GLP-1 and its analogs have low oral bioavailability. Following oral administration, exposure and bioavailability of human GLP-1 and its analogs are extremely low. Human GLP-1 and its analogs can only achieve therapeutically relevant plasma concentrations after oral administration when formulated with specific amounts of certain absorption enhancers.

[0003] Currently available oral GLP-1 receptor agonist drugs must be administered once daily. This frequent dosing is detrimental to improved patient convenience and improved patient compliance, and therefore, the development of oral GLP-1 receptor agonists suitable for less frequent dosing than once daily would be a significant improvement in available treatment options. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present application provides a GLP-1 receptor compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt, and its preparation method and application, which has good GLP-1 activity and / or can improve bioavailability.

[0005] Specifically, the present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides a GLP-1 compound represented by formula (I), or an isomer, a racemate, or a pharmaceutically acceptable salt thereof, comprising:

[0007] wherein z is selected from 0, 1 or 2;

[0008] The R 1 、R 2 independently selected from hydrogen, C 1-6 alkyl, Alternatively, the R 1 、R 2 Cyclize together to form C 3-6 Cycloalkyl;

[0009] The R 3 、R 4 independently selected from hydrogen, C 1-6 alkyl, Alternatively, the R 3 、R 4Cyclize together to form C 3-6 Cycloalkyl;

[0010] X is selected from hydrogen, C 1-6 alkyl,

[0011] Y 1 Selected from NH2, OH or HS, the R 5 Selected from C 1-6 alkyl, -(CH2) y1 (CR 5a R 5b ) y2 (CH2) y3 (CR 5c R 5d ) y4 -or Among them, R 5a 、R 5b Cyclize together to form C 3-6 Cycloalkyl, R 5c 、R 5d Cyclize together to form C 3-6 wherein y1, y2, y3, y4 are selected from 0, 1, 2, 3, 4 or 5, and y2 and y4 are not 0 at the same time, y5, y6, y7, y8 are selected from 0, 1, 2, 3 or 4, and y6 and y7 are not 0 at the same time;

[0012] Among them, when X is selected from Y 1 When selected from NH2, R 1 、R 2 、R 3 、R 4 are not hydrogen at the same time, or, R 5 Not for C 1-6 alkyl;

[0013] R 6 Selected from One of the following, or a combination of any two or more;

[0014] R 7 Selected from C 10-20 of fatty acids;

[0015] m is selected from 0, 1, 2, 3 or 4, and n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4.

[0016] As a preferred technical solution of the present invention, when z is 0, Y 1 When NH2 is selected from the GLP-1 compound represented by formula (II),

[0017] Among them, the R 1 、R 2 independently selected from hydrogen, C 1-6 alkyl, Alternatively, the R 1 、R 2 Cyclize together to form C 3-6 Cycloalkyl;

[0018] The R 3 、R 4 independently selected from hydrogen, C 1-6 alkyl, Alternatively, the R 3 、R 4 Cyclize together to form C 3-6 Cycloalkyl;

[0019] X is selected from hydrogen, C 1-6 alkyl, Among them, the R 1 、R 2 、R 3 、R 4 , X, at least one of which is selected from

[0020] The R 5 Selected from C 1-6 alkyl, -(CH2) y1 (CR 5a R 5b ) y2 (CH2) y3 (CR 5c R 5d ) y4 -or Among them, R 5a 、R 5b Cyclize together to form C 3-6 Cycloalkyl, R 5c 、R 5d Cyclize together to form C 3-6 wherein y1, y2, y3, y4 are selected from 0, 1, 2, 3, 4 or 5, and y2 and y4 are not 0 at the same time, y5, y6, y7, y8 are selected from 0, 1, 2, 3 or 4, and y6 and y7 are not 0 at the same time;

[0021] Among them, when X is selected from When R 1 、R 2 、R 3 、R 4 are not hydrogen at the same time, or, R 5Not for C 1-6 alkyl;

[0022] R 6 Selected from One of the following, or a combination of any two or more;

[0023] R 7 Selected from C 10-20 of fatty acids;

[0024] m is selected from 0, 1, 2, 3 or 4, and n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4.

[0025] As a preferred technical solution of the present invention, when R 1 、R 3 When is hydrogen and z is 0, it is selected from the GLP-1 compound represented by formula (III),

[0026] Among them, the Y 1 Selected from OH, HS;

[0027] The X, R 2 、R 4 independently selected from hydrogen, C 1-6 alkyl, Wherein, the X, R 2 、R 4 At least one selected from The R 5 Selected from C 1-6 alkyl,

[0028] The R 6 Selected from One of the following, or a combination of any two or more;

[0029] R 7 Selected from C 10-20 of fatty acids;

[0030] m is selected from 0, 1, 2, 3 or 4, and n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4.

[0031] As a preferred technical solution of the present invention, the C 1-6The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, 1-ethylpropyl, 2-methylbutyl, tert-pentyl, 1,2-dimethylpropyl, isopentyl, neopentyl, n-hexyl, isohexyl, sec-hexyl, tert-hexyl, neohexyl, 2-methylpentyl, 1,2-dimethylbutyl, 1-ethylbutyl; the C 3-6 The cycloalkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0032] As a preferred technical solution of the present invention, C 10-20 The fatty acids are selected from C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 of fatty acids.

[0033] As a preferred technical solution of the present invention, the R 1 、R 2 Cyclize together to form C 3-6 The cycloalkyl group, or the R 3 、R 4 Cyclize together to form C 3-6 A cycloalkyl group, or R 5a 、R 5b Cyclize together to form C 3-6 A cycloalkyl group, or R 5c 、R 5d Cyclize together to form C 3-6 A cycloalkyl group, or R 5 Selected from y5, y6, y7, and y8 are as defined above.

[0034] As a preferred technical solution of the present invention, the n is 1; the R 1 、R 2 are independently selected from hydrogen, or, said R 1 、R 2 Cyclize together to form cyclopropyl and cyclobutyl; the R 3 、R 4 are independently selected from hydrogen, or, said R 3 、R 4 Cyclize together to form cyclopropyl and cyclobutyl groups;

[0035] X is selected from The R 5 Selected from ethyl, -(CH2)y1 (CR 5a R 5b ) y2 (CH2) y3 (CR 5c R 5d ) y4 -or When R 5 When selected from ethyl, R 1 、R 2 、R 3 、R 4 are not hydrogen at the same time, where R 5a 、R 5b Cyclize together to form cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, R 5c 、R 5d Cyclize together to form cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein y1, y2, y3, and y4 are selected from 0 or 1, and y2 and y4 are not 0 at the same time, y5 and y8 are 0, and y6 and y7 are selected from 0, 1, 2, 3, or 4, and y6 and y7 are not 0 at the same time;

[0036] R 6 Selected from

[0037] R 7 Selected from C 15-20 of fatty acids.

[0038] As a preferred technical solution of the present invention, the -(CH2) y1 (CR 5a R 5b ) y2 (CH2) y3 (CR 5c R 5d ) y4 -Selected Wherein, a1 and a2 are different connection points; Selected from

[0039] As a preferred technical solution of the present invention, n is 1, and the Y 1 Selected from OH;

[0040] The R 2 、R 4 Independently selected from hydrogen, X is selected from The R 5 Selected from ethyl and butyl;

[0041] The R 6 Selected from R7 Selected from C 15-20 of fatty acids.

[0042] As a preferred technical solution of the present invention, the GLP-1 compound is selected from:

[0043] As a preferred technical solution of the present invention, the GLP-1 compound is selected from:

[0044] As a preferred technical solution of the present invention, the GLP-1 compound is selected from:

[0045] As a preferred technical solution of the present invention, the GLP-1 compound is selected from the structures shown in Table 1.

[0046] As a preferred technical solution of the present invention, at least one hydrogen atom of the GLP-1 compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt is substituted with deuterium isotope.

[0047] As a preferred technical solution of the present invention, the pharmaceutically acceptable salt refers to the GLP-1 compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt prepared with a pharmaceutically acceptable acid or base, and the salt includes hydrochloride, trifluoroacetate, acetate, etc.

[0048] In a second aspect, the present invention further provides an intermediate compound comprising the following structure:

[0049] The present invention further provides a pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of the GLP-1 compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

[0050] The present invention further provides the medical use of the GLP-1 compound, or its isomer, or its racemate, or its pharmaceutically acceptable salt, or pharmaceutical composition, specifically, its use in the preparation of a medicament for treating a disease, wherein the disease is a chronic related disease selected from diabetes, obesity, non-alcoholic fatty liver disease and non-alcoholic fatty liver hepatitis, cardiovascular disease, neurodegenerative disorders, chronic kidney disease, diabetic nephropathy, peripheral arterial disease, and / or heart failure.

[0051] For the sake of clarity, general terms used in the description of the compounds are defined herein.

[0052] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered as undefined or unclear in the absence of a specific definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0053] As used herein, the term "compound" refers to a molecular entity and, therefore, a "compound" may have different structural elements in addition to the minimum elements defined for each compound or group of compounds. The term compound may be used interchangeably with the term "construct". The term "compound" may be used to describe the prodrugs of the present invention. The compounds of the present invention may be referred to as "compounds", but the term "compound" is also intended to cover their pharmaceutically relevant forms, i.e., the present invention relates to compounds as defined herein or pharmaceutically acceptable salts, amides or esters thereof.

[0054] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is prepared by reacting a compound having a specific substituent discovered in the present invention with a pharmaceutically acceptable acid or base.

[0055] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrates. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0056] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, atropisomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0057] Optically active (R)- and (S)-isomers, as well as D and L isomers, atropisomers, etc., can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are resolved by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0058] The atoms of the molecules of the compounds of the present invention are isotopes, and isotope derivatization can generally extend half-life, reduce clearance, stabilize metabolism, and increase in vivo activity. In addition, an embodiment is included in which at least one atom is replaced by an atom having the same atomic number (number of protons) and a different mass number (protons and neutrons). Examples of isotopes included in the compounds of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, which respectively include 2 H. 3 H. 13 C. 14 C. 15 N. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36 In particular, radioactive isotopes that emit radiation as they decay, such as 3 H or 14 C can be used for local anatomy testing of pharmaceutical preparations or compounds in vivo. Stable isotopes neither decay nor change with their amount nor are they radioactive, so they can be used safely. When the atoms constituting the molecules of the compounds of the present invention are isotopes, the isotopes can be converted according to general methods by replacing the reagents used in the synthesis with reagents containing the corresponding isotopes.

[0059] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as deuterium ( 2 H), iodine-125( 125 I) or C-14( 14C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0060] Furthermore, one or more hydrogen atoms of the compounds of the present invention are replaced by the isotope deuterium (2H). After deuteration, the compounds of the present invention have the effects of extending half-life, reducing clearance rate, metabolic stability and improving in vivo activity.

[0061] The preparation method of the isotopic derivative generally includes a phase transfer catalytic method. For example, a preferred deuteration method uses a phase transfer catalyst (e.g., a tetraalkylammonium salt, NBu4HSO4). The use of a phase transfer catalyst to exchange the methylene protons of the diphenylmethane compound results in a higher deuterium incorporation than reduction with a deuterated silane (e.g., triethyldeuterated monosilane) in the presence of an acid (e.g., methanesulfonic acid) or with a Lewis acid such as aluminum trichloride using sodium deuterated borate.

[0062] As used herein, the term "polypeptide" or "polypeptide sequence" refers to a compound comprising a series of two or more amino acids interconnected by amide (or peptide) bonds. The term polypeptide is used interchangeably with the term "peptide" and the term "protein."

[0063] In one embodiment, the compound of the invention comprises a GLP-1 polypeptide. In one embodiment, the GLP-1 polypeptide is the amino acid sequence of semaglutide. In one embodiment, the compound of the invention comprises a GLP-1 polypeptide, wherein the GLP-1 polypeptide is a GLP-1 analog; and wherein the GLP-1 analog has a maximum of 3 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1). In one embodiment, the compound of the invention comprises a GLP-1 polypeptide, wherein the GLP-1 polypeptide is a GLP-1 analog; and wherein the GLP-1 analog has a maximum of 2 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1).

[0064] Among them, the GLP-1 (7-37) polypeptide sequence is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1).

[0065] In one embodiment, the compound of the present invention comprises a GLP-1 derivative. In one embodiment, the GLP-1 polypeptide is semaglutide, and the structure of semaglutide is as follows:

[0066] As used herein, the term "GLP-1 polypeptide" refers to a polypeptide that is capable of binding to and / or activating a GLP-1 receptor. In other words, a GLP-1 polypeptide is a polypeptide that has GLP-1 activity. In other words, a GLP-1 polypeptide is a GLP-1 receptor agonist. A GLP-1 polypeptide can bind to and / or activate other types of receptors, i.e., as long as the polypeptide binds to and / or activates the GLP-1 receptor, it qualifies as a GLP-1 polypeptide, regardless of any other receptor interactions that may be involved. In addition to the amino acid residues responsible for GLP-1 receptor interaction, a GLP-1 polypeptide may also contain other amino acid residues that are not involved in GLP-1 receptor interaction.

[0067] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity increasers, transdermal enhancers, etc. Their preparation is well known to those skilled in the field of cosmetics or topical drugs. For additional information on carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0068] With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a non-toxic amount of the drug or agent sufficient to achieve the intended effect. For the oral dosage forms of the present invention, an "effective amount" of an active substance in the composition means the amount required to achieve the intended effect when used in combination with another active substance in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

[0069] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0070] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG1 is a mass spectrum (MS) of the compound of Example 7 of the present invention. DETAILED DESCRIPTION

[0072] The present application is further described in detail below with reference to examples, but the implementation methods of the present application are not limited thereto.

[0073] Example 1

[0074] The synthetic route of Example 1 can refer to the synthetic route in WO2022096636A1, and the contents of WO2022096636A1 can also be fully introduced into this application by reference. The specific synthetic route of the compound is as follows:

[0075] Step 1: Synthesis of F1:

[0076] Synthesis process description: Compound F1 was obtained by solid phase synthesis:

[0077] a. Use 2-CTC resin as a support, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Acc-OH (Fmoc-protected 1-aminocyclopropanecarboxylic acid) and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it several times with DMF.

[0078] b. After washing, add methanol and DIEA respectively to cap the resin. After capping, wash the resin several times with DMF.

[0079] c. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0080] d. 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0081] e. Fmoc-aminoethanol, diisopropyl azodicarboxylate (DIAD), and triphenylphosphine were mixed in THF and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0082] f. Repeat step c. to remove the Fmoc protecting group.

[0083] g. Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction is complete, wash the resin with DMF.

[0084] h. Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out the condensation reaction.

[0085] i. Separately, mercaptoethanol, bicyclic amidine (DBU) and DMF were mixed and added to the resin to remove the Ns protecting group. After the reaction was completed, the resin was washed with DMF.

[0086] j. Weigh Boc-Gly-OH and repeat step g. to carry out condensation reaction.

[0087] k. The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0088] 1. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to obtain compound F1.

[0089] Step 2: Synthesis of F2 (semaglutide fully protected peptide resin)

[0090] The synthetic route of semaglutide refers to the synthetic route in patent application number CN200680006674.6 and WO2022096636A1. H-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-octadecanediol mono-tert-butyl ester)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)f)-Gly-Arg(Pbf)-Gly-Wang Resin

[0091] F2

[0092] Synthesis steps:

[0093] a. Use Wang resin as the carrier, first swell it with DMF, and then wash the resin with DMF after swelling.

[0094] b. Weigh Fmoc-Gly-OH, HOBt, and 4-dimethylaminopyridine (DMAP) and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin and react. After the reaction, wash the resin with DMF.

[0095] c. After washing, add acetic anhydride and DIPEA respectively for end-capping. After end-capping, wash the resin several times with DMF.

[0096] d. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution for 10 minutes each time. After deprotection, the resin was washed with DMF.

[0097] e. Weigh Fmoc-Arg(Pbf)-OH and HOBt, add DMF, and dissolve them. Then, add DIC to activate for 3-5 minutes. After activation, add the mixture to the reactor and begin the coupling reaction. The amino acid coupling reaction should proceed for 1.0-3.0 hours. Ninhydrin should be used to monitor the reaction endpoint. After the coupling is complete, wash the resin with DMF.

[0098] f. Repeat step e. and sequentially couple amino acids according to the peptide sequence to obtain F2 (semaglutide fully protected peptide resin).

[0099] After the reaction, the peptide resin was washed with dichloromethane and methanol and dried at room temperature.

[0100] Step 3: Preparation of target compound (F1+F2)

[0101] a. F2 (semaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0102] b. Weigh F1 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0103] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0104] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0105] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0106] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0107] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0108] (4) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0109] The specific synthetic route of the compound in Example 7 is as follows:

[0110] Step 1: Synthesis of F4:

[0111] Synthesis process description: Compound F4 was obtained by solid phase synthesis:

[0112] a: Use 2-CTC resin as the carrier, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it with DMF several times.

[0113] b: After washing, methanol and DIEA were added for end-capping. After end-capping, the resin was washed several times with DMF.

[0114] c: Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0115] d: 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0116] e: Fmoc-aminocyclobutanemethanol, 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.

[0117] f: Repeat step c to remove the Fmoc protecting group.

[0118] g: Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction, wash the resin with DMF.

[0119] h: Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out condensation reaction.

[0120] i: After respectively mixing mercaptoethanol, bicyclic amidine (DBU) and DMF, add them to the resin to remove the Ns protecting group. After the reaction is completed, wash the resin with DMF.

[0121] j: Weigh Boc-Gly-OH and repeat step g. to carry out condensation reaction.

[0122] k: The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain a peptide resin. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to obtain compound F4.

[0123] Step 2: Synthesis of F2 (semaglutide fully protected peptide resin). The synthesis of F2 was prepared according to Step 2 in Example 1.

[0124] Step 3: Preparation of target compound (F4+F2)

[0125] a. F2 (semaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0126] b. Weigh F4 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0127] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0128] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0129] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0130] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0131] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0132] (4) The freeze-dried samples were packaged and stored as required. Samples were taken for testing. The mass spectrum of the compound of Example 7 is shown in FIG1 .

[0133] The specific synthetic route of the compound in Example 9B is as follows:

[0134] Step 1: Synthesis of F5:

[0135] Synthesis process description: Compound F5 was obtained by solid phase synthesis:

[0136] a. Using 2-CTC resin as a carrier, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it several times with DMF.

[0137] b. After washing, add methanol and DIEA respectively to cap the resin. After capping, wash the resin several times with DMF.

[0138] c. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0139] d. 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0140] e. Fmoc-(1S,2R)-2-aminocyclohexanol, 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.

[0141] f. Repeat step c. to remove the Fmoc protecting group.

[0142] g. Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction is complete, wash the resin with DMF.

[0143] h. Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out the condensation reaction.

[0144] i. Separately, mercaptoethanol, bicyclic amidine (DBU) and DMF were mixed and added to the resin to remove the Ns protecting group. After the reaction was completed, the resin was washed with DMF.

[0145] j. Weigh Boc-Gly-OH and repeat step g. to carry out condensation reaction.

[0146] k. The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0147] 1. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to give compound F5.

[0148] Step 2: Synthesis of F2 (semaglutide fully protected peptide resin). The synthesis steps of F2 refer to Step 2 in Example 1.

[0149] Step 3: Preparation of target compound (F5+F2)

[0150] a. F2 (semaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0151] b. Weigh F5 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0152] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0153] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0154] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0155] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0156] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0157] (4) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0158] The specific synthetic route of Example 10B is as follows:

[0159] Step 1: Synthesis of F6:

[0160] Synthesis process description: Compound F6 was obtained by solid phase synthesis:

[0161] a. Using 2-CTC resin as a carrier, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it several times with DMF.

[0162] b. After washing, add methanol and DIEA respectively to cap the resin. After capping, wash the resin several times with DMF.

[0163] c. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0164] d. 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0165] e. Fmoc-(1S,3R)-3-aminocyclopentanol, 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.

[0166] f. Repeat step c. to remove the Fmoc protecting group.

[0167] g. Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction is complete, wash the resin with DMF.

[0168] h. Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out the condensation reaction.

[0169] i. Separately, mercaptoethanol, bicyclic amidine (DBU) and DMF were mixed and added to the resin to remove the Ns protecting group. After the reaction was completed, the resin was washed with DMF.

[0170] j. Weigh Boc-Gly-OH and repeat step g. to carry out condensation reaction.

[0171] k. The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0172] 1. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to give compound F6.

[0173] Step 2: Preparation of target compound (F6+F2)

[0174] a. F2 (semaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0175] b. Weigh F6 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0176] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0177] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0178] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0179] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0180] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0181] (4) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0182] The specific synthetic route of the compound in Example 11A is as follows:

[0183] Step 1: Synthesis of F7:

[0184] Synthesis process description: Compound F7 was obtained by solid phase synthesis:

[0185] a. Using 2-CTC resin as a carrier, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it several times with DMF.

[0186] b. After washing, add methanol and DIEA respectively to cap the resin. After capping, wash the resin several times with DMF.

[0187] c. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0188] d. 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0189] e. Fmoc-(1S,3R)-3-aminocyclohexanol, 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.

[0190] f. Repeat step c. to remove the Fmoc protecting group.

[0191] g. Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction is complete, wash the resin with DMF.

[0192] h. Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out the condensation reaction.

[0193] i. Separately, mercaptoethanol, bicyclic amidine (DBU) and DMF were mixed and added to the resin to remove the Ns protecting group. After the reaction was completed, the resin was washed with DMF.

[0194] j. Weigh Boc-Gly-OH and repeat step g. to carry out condensation reaction.

[0195] k. The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0196] 1. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to give compound F7.

[0197] Step 2: Preparation of target compound (F7+F2)

[0198] a. F2 (semaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0199] b. Weigh F7 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0200] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0201] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0202] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0203] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0204] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0205] (4) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0206] The specific synthetic route of the compound in Example 11B is as follows:

[0207] Step 1: Synthesis of F8:

[0208] Synthesis process description: Compound F8 was obtained by solid phase synthesis:

[0209] a. Using 2-CTC resin as a carrier, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it several times with DMF.

[0210] b. After washing, add methanol and DIEA respectively to cap the resin. After capping, wash the resin several times with DMF.

[0211] c. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0212] d. 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0213] e. Fmoc-(1R,3R)-3-aminocyclohexanol, diisopropyl azodicarboxylate (DIAD), and triphenylphosphine were mixed in THF and added to the resin for reaction. After the reaction, the resin was washed with DMF.

[0214] f. Repeat step c. to remove the Fmoc protecting group.

[0215] g. Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction is complete, wash the resin with DMF.

[0216] h. Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out the condensation reaction.

[0217] i. Separately, mercaptoethanol, bicyclic amidine (DBU) and DMF were mixed and added to the resin to remove the Ns protecting group. After the reaction was completed, the resin was washed with DMF.

[0218] j. Weigh Boc-Gly-OH and repeat step g. to carry out condensation reaction.

[0219] k. The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0220] 1. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to obtain compound F8.

[0221] Step 2: Preparation of target compound (F8+F2)

[0222] a. F2 (semaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0223] b. Weigh F8 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0224] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0225] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0226] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0227] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0228] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0229] (4) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0230] The specific synthetic route of the compound in Example 12A is as follows:

[0231] Step 1: Synthesis of F9:

[0232] Synthesis process description: Compound F9 was obtained by solid phase synthesis:

[0233] a. Using 2-CTC resin as a carrier, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it several times with DMF.

[0234] b. After washing, add methanol and DIEA respectively to cap the resin. After capping, wash the resin several times with DMF.

[0235] c. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0236] d. 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0237] e. Fmoc-(1r,4r)-4-aminocyclohexanol, 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.

[0238] f. Repeat step c. to remove the Fmoc protecting group.

[0239] g. Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction is complete, wash the resin with DMF.

[0240] h. Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out the condensation reaction.

[0241] i. Separately, mercaptoethanol, bicyclic amidine (DBU) and DMF were mixed and added to the resin to remove the Ns protecting group. After the reaction was completed, the resin was washed with DMF.

[0242] j. Weigh Boc-Gly-OH and repeat step g. to carry out condensation reaction.

[0243] k. The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0244] 1. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to obtain compound F9.

[0245] Step 2: Preparation of target compound (F9+F2)

[0246] a. F2 (semaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0247] b. Weigh F9 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0248] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0249] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0250] (4) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0251] (5) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0252] (6) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0253] (4) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0254] Example 14

[0255] The synthetic route of Example 14 can refer to the synthetic route in WO2022096636A1, and the contents of WO2022096636A1 can also be fully introduced into this application by reference.

[0256] The specific synthetic route of the compound is as follows:

[0257] Step 1: Synthesis of F10:

[0258] F10 synthesis process description: Compound F10 was obtained by solid phase synthesis:

[0259] a. Using 2-CTC resin as a carrier, first swell it with N,N-dimethylformamide (DMF), then add Fmoc-Gly-OH and N,N-diisopropylethylamine (DIEA) and react for several hours. After the reaction, wash it several times with DMF.

[0260] b. After washing, add methanol and DIEA respectively to cap the resin. After capping, wash the resin several times with DMF.

[0261] c. Deprotection of Fmoc was performed twice with a 20% piperidine / DMF mixed solution, each time for 10 minutes. After deprotection, the resin was washed with DMF.

[0262] d. 2-nitrobenzenesulfonyl chloride (NsCl) and DIEA were mixed with tetrahydrofuran (THF) and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0263] e. Fmoc-aminoethanol, diisopropyl azodicarboxylate (DIAD), and triphenylphosphine were mixed in THF and added to the resin for reaction. After the reaction was completed, the resin was washed with DMF.

[0264] f. Repeat step c. to remove the Fmoc protecting group.

[0265] g. Weigh Fmoc-Glu-OtBu and hydroxybenzotriazole (HOBt) and dissolve them in DMF. Add N,N'-diisopropylcarbodiimide (DIC) and mix thoroughly. Add the mixture to the resin and allow to react. After the reaction is complete, wash the resin with DMF.

[0266] h. Weigh mono-tert-butyl hexadecanedioate and repeat step g. to carry out the condensation reaction.

[0267] i. Separately, mercaptoethanol, bicyclic amidine (DBU) and DMF were mixed and added to the resin to remove the Ns protecting group. After the reaction was completed, the resin was washed with DMF.

[0268] j. Weigh the glycolic acid with Boc protection on the hydroxyl group and repeat step g. to carry out the condensation reaction.

[0269] k. The resin was washed with dichloromethane and methanol, respectively, and dried at room temperature to obtain the peptide resin.

[0270] 1. The resin was cleaved with 20% TFE / DCM, filtered and concentrated to obtain compound F10.

[0271] Step 2: Preparation of target compound (F2+F10)

[0272] a. F2 (semaglutide fully protected peptide resin) was first swollen with DMF. After swelling, the resin was washed with DMF.

[0273] b. Weigh F10 and HOBt and dissolve them in DMF. Add DIC and mix thoroughly. Add the mixture to the resin to react. After the reaction, wash the peptide resin with DMF, dichloromethane, and methanol, respectively, and air-dry.

[0274] c. Use TFA / TIS / H2O = 95.0 / 2.5 / 2.5, and prepare 10 ml of lysis buffer per gram of peptide resin. Stir the reaction at room temperature for 2 hours. After the reaction is completed, filter the resin, concentrate to remove part of the TFA, and add it to 8 times the volume of lysis buffer with ether to precipitate. Collect the crude product by centrifugation and dry it to constant weight.

[0275] d. Add the crude product to an acetonitrile / water solution and dissolve it by ultrasonication, then filter it through a 0.45 μm filter membrane. Transfer the filtered solution to a purification HPLC crude fraction.

[0276] (1) HPLC crude fraction: The filtered sample solution was purified according to the crude fraction method and qualified fractions were collected.

[0277] (2) HPLC fine fractionation: The qualified fractions collected from the HPLC crude fractionation were purified according to the HPLC fine fractionation method and the qualified fractions were collected.

[0278] (3) Concentration and freeze-drying: Concentrate the qualified fractions, and filter, separate and freeze-dry the concentrated samples according to the process regulations.

[0279] (4) The freeze-dried samples are packaged and stored as required. Samples are collected for testing.

[0280] Examples 2-6, 8-10, 12-13, 15-24

[0281] The compounds of Examples 2-6, 8-10, 12-13, and 15-24 were prepared by referring to the synthetic routes of Examples 1, 7, 11A, 11B, and 14. The structures of the compounds of Examples 1-24 are shown in Table 1.

[0282] Table 1 shows the compound structure of GLP-1

[0283] Comparative Example 1

[0284] The synthetic route of comparative example 1 can be prepared by referring to the preparation method of Example 1 in the patent document with application number CN202180075068.4.

[0285] Example 25 Determination of conversion half-life

[0286] Prepare peptide stock solutions by dissolving the test sample in PBS buffer to a target of 200 μM. PBS buffer is Ca-free. 2+ and Mg 2+ Dulbecco's phosphate-buffered saline (PBS), pH 7.4. Adjust the pH of the peptide stock solution to 7.4 with HCl or NaOH. Transfer the sample to an HPLC vial. Cap the vial tightly to prevent evaporation. Incubate the HPLC vial at 37°C, and remove samples at various time points over a 2-week period, flash-freeze at -80°C, and store at -20°C until analysis. Analyze the samples using LC / MS or liquid chromatography, and calculate the conversion half-life of the test sample based on the reduction in the test sample prototype.

[0287] The conversion half-life of the test sample of the present invention is not less than 24h, and the conversion half-life is longer; the more preferred compounds of the present invention, such as compound numbers 4, 5, 7, 8A, 8B, 9A, 9B, 10A, 10B, 11A, 11B, 12A, 12B, and 14, have a conversion half-life of not less than 50h.

[0288] Example 26 Terminal Half-Life Determination

[0289] The terminal half-life of the test samples in vivo was evaluated in miniature pigs. Three miniature pigs were used for each sample and administered intravenously. The samples were prepared with a pH 7.4 solvent and the administration volume was 0.05 mL / kg. Approximately 0.8 mL of venous blood was collected at different time points after administration. Plasma was prepared by centrifugation at 4000 rpm for 10 minutes and stored at -20°C until analysis. The concentration of the active ingredient was detected by LC / MS or liquid chromatography. The plasma concentration (relative to time) curve of the test compound was evaluated by non-compartmental analysis (NCA) using WinNonlin software and the terminal half-life pharmacokinetic parameters were calculated.

[0290] The terminal half-life of the compound of the present invention after administration is not less than 30 hours.

[0291] Example 27 GLP-1 activity assay

[0292] The U2OS-GLP-1R stable cell line and HTRF detection method were used to determine the agonist effect of the test substance on GLP-1R

[0293] Experimental methods:

[0294] 1. Follow the cAMP-Gs Dynamic HTRF kit protocol and dilute the 5x Stimulation buffer (SB) provided with the kit with ddH2O to 1xSB. Add IBMX to a final concentration of 500 μM to prevent cAMP degradation. Prepare the test substance working solution in 1xSB and perform a series of serial dilutions.

[0295] 2. Digest and collect U2OS-GLP-R cells, resuspend and count, and dilute the cell density to 2x10^ using 1xSB solution. 6 The cells were plated at 5 μL per well in a 384-well plate to obtain 10,000 cells per well.

[0296] 3. Add 5 μL of the test substance at different concentrations to the corresponding cell wells and incubate in a 37°C incubator for 30 minutes.

[0297] 4. Prepare cAMP standard solutions of different concentrations according to the steps provided by the cAMP-Gs Dynamic HTRF kit and add them to the 384-well cell plate.

[0298] 5. Dilute the cAMP d2 reagent and Eu Cryptate antibody in the kit to 1x using Lysis & Detection Buffer. Add 5 μL of d2 and Eu, respectively, to a 384-well plate. Incubate at room temperature for 2 hours, then detect using a microplate reader HTRF module (665 / 620 nm) and collect experimental data.

[0299] 6. Signal values ​​were plotted against compound concentrations, and curve fitting and EC50 calculation were performed using GraphPad Prism software using nonlinear regression. The test results showed that the preferred compounds of the present invention exhibited excellent GLP-1 activity, with EC50 < 100 nm. The results are shown in Table 2.

[0300] Table 2 GLP-1 activity of compounds

[0301] Example 28 Pharmacokinetic Experiment

[0302] 1. Reagents and instruments

[0303] Dulbecco's phosphate buffered saline (DPBS) (Lot No.: F2326001, Shanghai Aladdin Biochemical Technology Co., Ltd.) LC-MS instrument (Thermo TSQ Altis Plus).

[0304] 2. Experimental Animals

[0305] SD rats: male, 180-250 g, purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0306] 3. Preparation

[0307] Accurately weigh the test sample powder to make sure it does not contain Ca 2+ / Mg 2+ The drug was completely dissolved in Dulbecco's phosphate buffer, mixed to 0.1 mg / mL, and administered intravenously at a dose of 2 mL / kg.

[0308] 4. Blood sample collection

[0309] After intravenous administration to rats, 200 μL of venous blood was collected before administration and 0, 0.5 h, 2 h, 5 h, 24 h, 48 h, and 72 h after administration in EDTA-K2 anticoagulant EP tubes, centrifuged at 12000 rpm for 2 min, and the plasma was frozen at -20°C for testing.

[0310] 5. Biological analysis

[0311] Accurately weigh a certain amount of test sample and dissolve it in DMSO to 2 mg / mL as a stock solution. Accurately aspirate an appropriate amount of compound stock solution and dilute it with acetonitrile aqueous solution (ACN:H2O=1:1) to prepare a standard series of solutions. Accurately aspirate 4 μL of each of the above standard series solutions, add 36 μL of blank plasma, vortex mix, and prepare plasma samples equivalent to plasma concentrations of 0.3, 1, 3, 10, 30, 100, 300, 500, 1000, 3000, 5000 and 10000 ng / mL to establish a standard curve. Take 30 μL of plasma, add 150 μL of acetonitrile / methanol (1:1) solution of internal standard propranolol (5 ng / mL), vortex mix, centrifuge at 4000 rpm for 10 minutes, take 100 μL of the supernatant, add 100 μL of ultrapure water and perform LC-MS analysis. The LC-MS detection conditions are as follows:

[0312] Chromatographic column: YMC-Triart C18, 33*2.1mm, 5μm.

[0313] Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile, flow rate: 0.5 mL / min, gradient elution see Table 3 below:

[0314] Table 3

[0315] 6. Data processing

[0316] After LC-MS detection of blood drug concentration, WinNonlin 6.1 software was used to calculate the pharmacokinetic parameters of rats after administration using a non-compartmental model. The results are shown in Table 4.

[0317] Table 4 pk data of the compounds of the present invention converted into semaglutide

[0318] As shown in Table 4, the compound of the present invention and the compound of the present invention converted into semaglutide both have higher exposure amounts and are superior to the compound of Comparative Example 1.

[0319] Example 29 In vivo efficacy

[0320] The experiment used 16-week-old DIO (diet-induced obesity) mice (purchased from Guangdong Weitong Lihua Laboratory Animal Technology Co., Ltd.) induced by high-fat diet. Wild-type mice of the same age served as normal controls. Before the experiment, the DIO mice were randomly divided into three groups of five mice each according to body weight. Five wild-type mice served as a blank control group. Both DIO and wild-type mice were administered subcutaneously every three days. Detailed dosing schedule is shown in Table 5. During the experiment, animal behavior, coat color, water intake, and urination were observed. Animals were weighed every other day, and abnormalities were recorded. The experimental period lasted 15 days.

[0321] Table 5: Mice dosing groups

[0322] The results showed that there were no abnormalities in the mice in each administration group during the administration process. After the last administration, the results showed that the compound of the present invention had a better weight loss effect on DIO mice than the control group.

[0323] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A GLP-1 compound represented by formula (I), or an isomer, a racemate, or a pharmaceutically acceptable salt thereof, characterized in that: include: wherein z is selected from 0, 1 or 2; The R 1 , R 2 independently selected from hydrogen, C 1-6 alkyl, Alternatively, the R 1 , R 2 Cyclize to form C 3-6 Cycloalkyl; The R 3 , R 4 independently selected from hydrogen, C 1-6 alkyl, Alternatively, the R 3 , R 4 Cyclize to form C 3-6 Cycloalkyl; X is selected from hydrogen, C 1-6 alkyl, Y 1 Selected from NH2, OH or HS; The R 5 Selected from C 1-6 alkyl, -(CH2) y1 (CR 5a R 5b ) y2 (CH2) y3 (CR 5c R 5d ) y4 -or Among them, R 5a , R 5b Cyclize to form C 3-6 Cycloalkyl, R 5c , R 5d Cyclize to form C 3-6 wherein y1, y2, y3, y4 are selected from 0, 1, 2, 3, 4 or 5, and y2 and y4 are not 0 at the same time, y5, y6, y7, y8 are selected from 0, 1, 2, 3 or 4, and y6 and y7 are not 0 at the same time; Among them, when X is selected from Y 1 When selected from NH2, R 1 , R 2 , R 3 , R 4 are not hydrogen at the same time, or, R 5 Not for C 1-6 alkyl; R 6 Selected from One of the following, or a combination of any two or more; R 7 Selected from C 10-20 of fatty acids; m is selected from 0, 1, 2, 3 or 4, and n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4.

2. The GLP-1 compound according to claim 1, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: When z is 0, Y 1 When NH2, it is selected from the GLP-1 compound represented by formula (II), Among them, the R 1 , R 2 independently selected from hydrogen, C 1-6 alkyl, Alternatively, the R 1 , R 2 Cyclize to form C 3-6 Cycloalkyl; The R 3 , R 4 independently selected from hydrogen, C 1-6 alkyl, Alternatively, the R 3 , R 4 Cyclize to form C 3-6 Cycloalkyl; X is selected from hydrogen, C 1-6 alkyl, Among them, the R 1 , R 2 , R 3 , R 4 , at least one of X is selected from The R 5 Selected from C 1-6 alkyl, -(CH2) y1 (CR 5a R 5b ) y2 (CH2) y3 (CR 5c R 5d ) y4 -or Among them, R 5a , R 5b Cyclize to form C 3-6 Cycloalkyl, R 5c , R 5d Cyclize to form C 3-6 wherein y1, y2, y3, y4 are selected from 0, 1, 2, 3, 4 or 5, and y2 and y4 are not 0 at the same time, y5, y6, y7, y8 are selected from 0, 1, 2, 3 or 4, and y6 and y7 are not 0 at the same time; Among them, when X is selected from When R 1 , R 2 , R 3 , R 4 are not hydrogen at the same time, or, R 5 Not for C 1-6 alkyl; R 6 Selected from One of the following, or a combination of any two or more; R 7 Selected from C 10-20 of fatty acids; m is selected from 0, 1, 2, 3 or 4, and n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4.

3. The GLP-1 compound according to claim 1, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: When R 1 , R 3 When is hydrogen and z is 0, it is selected from the GLP-1 compound represented by formula (III), Among them, the Y 1 Selected from OH, HS; X, R 2 , R 4 independently selected from hydrogen, C 1-6 alkyl, Among them, X, R 2 , R 4 At least one selected from R5 is selected from C 1-6 alkyl, The R 6 Selected from One of the following, or a combination of any two or more; R7 is selected from C1 0-20 of fatty acids; m is selected from 0, 1, 2, 3 or 4, and n, p, q, r, s, t, u, v are selected from 1, 2, 3 or 4.

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

5. The GLP-1 compound according to any one of claims 1 to 2, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: The n is 1; the R 1 , R 2 are independently selected from hydrogen, or, said R 1 , R 2 Cyclize together to form cyclopropyl and cyclobutyl groups; The R 3 , R 4 are independently selected from hydrogen, or, said R 3 , R 4 Cyclize together to form cyclopropyl and cyclobutyl groups; X is selected from The R 5 Selected from ethyl, -(CH2) y1 (CR 5a R 5b ) y2 (CH2) y3 (CR 5c R 5d ) y4 -or When R 5 When selected from ethyl, R 1 , R 2 , R 3 , R 4 are not hydrogen at the same time, where R 5a , R 5b Cyclize together to form cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, R 5c , R 5d Cyclize together to form cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, wherein y1, y2, y3, y4 are selected from 0 or 1, and y2 and y4 are not 0 at the same time, y5 and y8 are 0, y6 and y7 are selected from 0, 1, 2, 3 or 4, and y6 and y7 are not 0 at the same time; R 6 Selected from R 7 Selected from C 15-20 of fatty acids.

6. The GLP-1 compound according to claim 5, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: The -(CH2) y1 (CR 5a R 5b ) y2 (CH2) y3 (CR 5c R 5d ) y4 -Selected from Among them, a1 and a2 are different connection points; Said Selected from 7. The GLP-1 compound according to any one of claims 1 or 3, or its isomer, or its racemate, or its pharmaceutically acceptable salt, characterized in that: n is 1; The Y 1 Selected from OH; The R 2 , R 4 independently selected from hydrogen, X selected from The R 5 Selected from ethyl and butyl; The R 6 Selected from R 7 Selected from C 15-20 of fatty acids.

8. The GLP-1 compound according to any one of claims 1 to 7, or its isomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: The GLP-1 compound is selected from A1-A7 and the structures shown in Table 1.

9. An intermediate compound, characterized in that Includes the following structures:

10. A pharmaceutical composition, characterized in that The invention comprises a therapeutically effective amount of the GLP-1 compound according to any one of claims 1 to 8, or an isomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

11. The medical use of the GLP-1 compound according to any one of claims 1 to 8, or its isomer, or its racemate, or its pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 10, specifically, its use in the preparation of a drug for treating a disease.

12. The use according to claim 11, characterized in that The disease is selected from diabetes, obesity, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, cardiovascular disease, a neurodegenerative disorder, chronic kidney disease, diabetic nephropathy, peripheral arterial disease, and / or heart failure.

Images