Polypeptide compound having GLP-1 / GIP receptor dual agonism, preparation method therefor, and use thereof

Abstract

The present application discloses a polypeptide compound having GLP-1 / GIP receptor dual agonism, or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof, and a use thereof in the preparation of GLP-1 / GIP receptor dual agonists.

Description

Polypeptide compounds with dual GLP-1 / GIP receptor agonism, methods of making and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to polypeptide compounds with dual GLP-1 / GIP receptor agonism, methods of making and uses thereof. BACKGROUND

[0002] According to a research paper published in 2023 in The Lancet (Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis for the Global Burden of Disease Study 2021), as of 2021, there were approximately 530 million people worldwide with diabetes, with a prevalence rate of 6%; by 2050, it is estimated that there will be approximately 1.3 billion people with diabetes, with a prevalence rate close to 10%. The reality of diabetes in China is even more severe, as Chinese people have both weaker beta cell function and higher obesity prevalence and accompanying insulin resistance, leading to a serious diabetes epidemic in China, with a continuously rising prevalence rate - the total diabetes prevalence rate among Chinese adults is 12.8%, which is much higher than the global prevalence rate. The continuously rising prevalence rate suggests a medical need for more effective treatment methods.

[0003] Incretins are a class of gut-derived hormones in human body, GIP and GLP-1 are two main incretins currently found. Human GLP-1 is a polypeptide containing 30 or 31 amino acids, mainly secreted by L cells in the terminal ileum and colon, and GIP is a polypeptide containing 42 amino acids, mainly secreted by K cells in the duodenum and jejunum. Under physiological conditions, the effect of incretins contributes 50% to 70% of insulin secretion, among which GIP accounts for about 44% of insulin secretion, and GLP-1 accounts for about 22%, both of which have the effect of promoting insulin secretion and glucose-dependent hypoglycemic effect. GLP-1 / GIP receptors can be expressed in islet cells and central nervous system, and adipocytes express GIP receptors. GLP-1 / GIP binds to the receptors to realize multi-dimensional regulation of energy metabolism balance in terms of intake, storage and metabolism through synergistic or complementary effects. Recent studies have shown that compared with the use of each incretin alone, the combined use of GLP-1 and GIP can significantly enhance insulin secretion, inhibit glucagon response, reduce energy intake, and improve insulin sensitivity. On the other hand, GIP receptor agonists can be used as antiemetics or as prophylaxis for diseases accompanied by vomiting or nausea, and thus can be used to inhibit or alleviate symptoms such as vomiting or nausea caused by GLP-1 receptor agonists. These research results have promoted the research and development of GLP-1 / GIP receptor dual agonists in the field of metabolic diseases such as diabetes and obesity.

[0004] Both natural GIP and GLP-1 can be degraded by the widely existing protease DPP-4 in the body to rapidly lose activity, although some non-natural amino acid substitutions can resist DPP-4 enzyme degradation, but the disadvantage of reducing the half-life by overcoming kidney clearance still needs to be overcome. In order to improve the convenience and compliance of such polypeptide drugs, therefore, the implementation of long-acting strategy is the focus and hotspot of the research in this field. It is particularly necessary to provide a new compound with long-acting GIP / GLP-1 receptor dual agonism, which supports the possibility of oral administration once a week or once a month in humans. SUMMARY

[0005] One of the purposes of the present application is to provide a polypeptide compound with long-acting GLP-1 / GIP receptor dual agonism, as described in formula (I), formula (I-1), formula (I-2), formula (I-3) and the like, and pharmaceutically acceptable salts, stereoisomers, solvates or hydrates thereof;

[0006] Another purpose of the present application is to provide a pharmaceutical composition comprising any of the above polypeptide compounds, or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof as an active ingredient, and a pharmaceutically acceptable excipient;

[0007] Another object of the present application is to provide use of the above-mentioned compound or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a hydrate thereof in the manufacture of a GLP-1 / GIP receptor dual agonist.

[0008] Another object of the present application is to provide a pharmaceutical composition for treating and / or preventing Type I diabetes, Type II diabetes, malnutrition-related diabetes, hyperglycemia, diabetic complications, obesity, obesity-related inflammation, non-alcoholic steatohepatitis (NASH), insulin resistance, glucose intolerance, hypertension, hyperlipidemia, arteriosclerosis, coronary heart disease, cerebral infarction, metabolic syndrome, obstructive sleep apnea, heart failure, Parkinson's disease, and / or dementia, which comprises the above-mentioned compound or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a hydrate thereof.

[0009] One or more embodiments of the present application provide a polypeptide compound of the present application or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a hydrate thereof for use as a medicament.

[0010] One or more embodiments of the present application provide a pharmaceutical composition of the present application for use as a medicament.

[0011] One or more embodiments of the present application provide a polypeptide compound of the present application or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a hydrate thereof or a pharmaceutical composition for use as a medicament for preventing and / or treating a disease mediated by GLP-1 receptor and / or GIP receptor.

[0012] One or more embodiments of the present application provide a polypeptide compound of the present application or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a hydrate thereof or a pharmaceutical composition for use as a GLP-1 / GIP receptor dual agonist.

[0013] One or more embodiments of the present application provide a method for treating and / or preventing a disease mediated by GLP-1 receptor and / or GIP receptor, which comprises administering a therapeutically and / or prophylactically effective amount of a polypeptide compound of the present application or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a hydrate thereof or a pharmaceutical composition to a subject in need thereof.

[0014] In one or more embodiments, the disease treated and / or prevented by the GLP-1 / GIP receptor dual agonist or the disease mediated by GLP-1 receptor and / or GIP receptor is type I diabetes, type II diabetes, malnutrition-related diabetes, hyperglycemia, diabetic complications, obesity, obesity-related inflammation, non-alcoholic steatohepatitis (NASH), insulin resistance, glucose intolerance, hypertension, hyperlipidemia, arteriosclerosis, coronary heart disease, cerebral infarction, metabolic syndrome, obstructive sleep apnea, heart failure, Parkinson's disease, and / or dementia.

[0015] Another object of the present application is to provide a preparation method of the above-mentioned compound.

[0016] To achieve the above object, the technical solution adopted by the present application is as follows:

[0017] One or more embodiments of the present application provide a GLP-1 / GIP dual agonist polypeptide compound having a structure of formula (I), or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof,

[0018] or a pharmaceutically acceptable salt thereof, wherein,

[0019] X0 is selected from Aib, or G;

[0020] X1 is selected from Aib or Y;

[0021] X2 is selected from A or Aib;

[0022] X3 is selected from Q, N or E;

[0023] Xa is selected from: or is absent;

[0024] Xb is selected from:

[0025] R1 is each independently selected from a hydrogen atom or a C1-C6 alkyl group;

[0026] R2 is each independently selected from:

[0027] R3 is each independently selected from: and when Xa is absent, R3 is

[0028] q is each independently selected from 2, 3, 4 or 5;

[0029] p is each independently selected from 13, 15, 17 or 19;

[0030] each n is independently selected from 13, 15, 17, or 19;

[0031] each L1is independently selected from the following formula:

[0032] Aib has the structure

[0033] In one or more embodiments of the present application, there is provided a GLP-1 / GIP dual agonistic polypeptide compound having the structure of Formula (I-1) or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof:

[0034] wherein:

[0035] X0is selected from Aib,

[0036] X1is selected from Aib or Y;

[0037] X2is selected from A or Aib;

[0038] X3is selected from Q, N, or E;

[0039] Xbis selected from:

[0040] each R1is independently selected from a hydrogen atom or a C1-C6alkyl group;

[0041] each R2is independently selected from:

[0042] each R3is independently selected from: and when Xais absent, R3is

[0043] each q is independently selected from 2, 3, 4, or 5;

[0044] each p is independently selected from 13, 15, 17, or 19;

[0045] each n is independently selected from 13, 15, 17, or 19;

[0046] each L1is independently selected from the following formula:

[0047] In one or more embodiments,

[0048] X1is Aib;

[0049] X3is selected from N or E;

[0050] Xbis

[0051] R1is a hydrogen atom;

[0052] R2is

[0053] q is selected from 2 or 3;

[0054] p is selected from 13 or 15;

[0055] n is selected from 15 or 17;

[0056] L1is selected from Formula 1 and Formula 2:

[0057] In one or more embodiments of the present application, there is provided a GLP-1 / GIP dual agonistic polypeptide compound having a structure of Formula (I-2) or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof:

[0058] wherein:

[0059] X0is selected from Aib,

[0060] X1is selected from Aib or Y;

[0061] X2is selected from A or Aib;

[0062] X3is selected from Q, N or E;

[0063] Xbis selected from:

[0064] R1is each independently selected from: a hydrogen atom, a methyl group or an ethyl group;

[0065] R2is each independently selected from:

[0066] R3is each independently selected from: and when Xais absent, R3is

[0067] q is each independently selected from 2, 3, 4 or 5;

[0068] p is each independently selected from 13, 15, 17 or 19;

[0069] n is each independently selected from 13, 15, 17 or 19;

[0070] L1is each independently selected from the following formulae:

[0071] In one or more embodiments,

[0072] X1is Aib;

[0073] X3is selected from N or E;

[0074] Xbis

[0075] R1is methyl;

[0076] R2is

[0077] q is 4;

[0078] p is selected from 13 or 15;

[0079] n is selected from 15 or 17;

[0080] L1is selected from Formula 1 and Formula 2:

[0081] In one or more embodiments of the present application, there is provided a GLP-1 / GIP dual agonistic polypeptide compound having the structure of Formula (I-3) or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof:

[0082] wherein:

[0083] X0is selected from Aib,

[0084] X1is selected from Aib or Y;

[0085] X2is selected from A or Aib;

[0086] X3is selected from Q, N or E;

[0087] Xbis selected from:

[0088] R3is each independently

[0089] n is each independently selected from 13, 15, 17 or 19.

[0090] In one or more embodiments,

[0091] X1is Aib;

[0092] X3is selected from N or E;

[0093] n is selected from 15 or 17.

[0094] In one or more embodiments, in the structure of Formula (I)

[0095] X0is selected from Aib or

[0096] X1is selected from Aib or Y;

[0097] X2is A;

[0098] X3is selected from Q or N;

[0099] Xais absent;

[0100] Xbis

[0101] R3is

[0102] N is 17;

[0103] Aib is each independently

[0104] In some embodiments, there is provided a polypeptide compound of the following formula:

[0105] In some embodiments, there is provided a pharmaceutical composition comprising at least one of the above polypeptide compounds or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof, and a pharmaceutically acceptable excipient.

[0106] In some embodiments, there is provided use of the above polypeptide compound or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof, or the above pharmaceutical composition in the manufacture of a GLP-1 / GIP receptor dual agonist.

[0107] In some embodiments, the above GLP-1 / GIP receptor dual agonist is a medicament for the treatment and / or prevention of Type I diabetes, Type II diabetes, malnutrition-related diabetes, hyperglycemia, diabetic complications, obesity, obesity-related inflammation, non-alcoholic steatohepatitis (NASH), insulin resistance, glucose intolerance, hypertension, hyperlipidemia, arteriosclerosis, coronary heart disease, cerebral infarction, metabolic syndrome, obstructive sleep apnea, heart failure, Parkinson's disease, and / or dementia.

[0108] Definitions and general terms:

[0109] The following terms as used in this application, including the specification and claims, have the definitions given below unless otherwise indicated.

[0110] As used herein, a bond represented by indicates the position at which the group is attached to the rest of the compound or molecule. In general, groups represented by a bond to a indicate attachment in the left direction to the general structure.

[0111] The term "compound" is meant to refer to a molecular entity, and thus, a "compound" can have different structural elements in addition to the minimum elements defined for each compound or each group of compounds. Thus, a compound can be a peptide or a derivative thereof, as long as the compound comprises the defined structural and / or functional elements.

[0112] The term "polypeptide" or "polypeptide sequence" refers to a compound comprising a series of two or more amino acids that are linked to one another by amide (or peptide) bonds. A "peptide" can also include amino acid elongations at the N-terminal and / or C-terminal position and / or truncations at the N-terminal and / or C-terminal position.

[0113] In the context of this document, "compound", "polypeptide" and "polypeptide compound" are synonymous and used interchangeably, unless clearly contradicted by the context.

[0114] The term "receptor agonist" refers to a substance that acts on a receptor, causing activation of the receptor, and thereby causing a biological effect, which can be an increase or decrease in a particular manifestation of cellular activity.

[0115] The terms "GLP-1 / GIP receptor dual agonist compound", "GLP-1 / GIP receptor dual agonist", "GLP-1 / GIP receptor agonist" or "GLP-1 / GIP receptor agonistic polypeptide compound" all refer to a polypeptide, protein or polypeptide compound that can activate both the GLP-1 receptor and the GIP receptor.

[0116] The term "amino acid" includes proteinogenic (or natural) amino acids (of which there are 20 standard amino acids) as well as non-proteinogenic (or unnatural) amino acids. Proteinogenic amino acids are amino acids that are naturally incorporated into proteins. Standard amino acids are those encoded by the genetic code, and amino acid residues can be represented by the standard one-letter code, three-letter code, or full name of the amino acid, all of which are fully equivalent and used interchangeably. Non-proteinogenic amino acids either do not occur in proteins or are not produced by standard cellular machinery (e.g., they can have undergone post-translational modification).

[0117] Amino acids in this application are represented as follows:

[0118] A or Ala represents alanine, with structure

[0119] R or Arg represents arginine, with structure

[0120] N or Asn represents asparagine, with structure

[0121] D or Asp represents aspartic acid, with structure

[0122] C or Cys represents cysteine, with structure

[0123] Q or Gin represents glutamine, with structure

[0124] E or Glu represents glutamic acid, with structure

[0125] G or Gly represents glycine, with structure

[0126] H or His represents histidine, with structure

[0127] I or lie represents isoleucine, with structure

[0128] L or Leu represents leucine, with structure

[0129] K or Lys represents lysine, with structure

[0130] M or Met represents methionine, with structure

[0131] F or Phe represents phenylalanine, with structure

[0132] P or Pro represents proline, with structure

[0133] S or Ser represents serine, with structure

[0134] T or Thr represents threonine, with structure

[0135] W or Trp represents tryptophan, with structure

[0136] Y or Tyr represents tyrosine, which has the structure When Xa is not present, the tyrosine represented by Y is present in the general structure with the complete structure including the amino terminus (NH2-).

[0137] V or Val represents valine, which has the structure

[0138] A non-limiting example of a non-proteinogenic amino acid is Aib, "Aib" is alpha amino isobutyric acid or 2-amino isobutyric acid, which has the structure 1 -aminocyclopropane carboxylic acid has the structure 1 -aminocyclobutane carboxylic acid has the structure Each amino acid of the peptides of the present application, unless otherwise specified, should be understood to mean the L-isomer, regardless of the optical isomer.

[0139] In glutamic acid residues, the gamma-carboxy group is the carboxy group attached to the 4-carbon. The atoms in glutamic acid are numbered according to standard IUPAC nomenclature, indicating the carbon atom number as well as the alpha to gamma positions:

[0140] Fmoc-AEEA-OH represents

[0141] The term "prodrug" refers to a compound that undergoes chemical conversion in vivo by enzymatic or non-enzymatic chemical processes to yield a parent drug. The term "parent drug" refers to the compound that is released from the prodrug upon conversion of the prodrug, which can occur enzymatically or non-enzymatically to release the parent drug. The rate at which conversion occurs can be quantified by the "conversion half-life," which is the length of time required for the concentration of the prodrug to be halved by conversion. The conversion half-life can also be referred to as the "prodrug-to-drug conversion half-life" or the "prodrug-to-parent drug conversion half-life." Conversion half-life can be measured in vitro, for example, at pH 7.4 and 37°C.

[0142] The term "C1-C6alkyl" refers to an alkyl group having from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, hexyl.

[0143] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present application, prepared from a compound of the present application having particular substituents discovered to be made with a pharmaceutically acceptable acid or base.

[0144] The term "solvate" is used to describe a molecular complex comprising a polypeptide compound of any of the above formulae or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules. When the solvent is water, the term "hydrate" is employed.

[0145] The term "pharmaceutically acceptable carrier" means any formulation carrier or medium which does not interfere with the biological activity of the active substance to be delivered and is nontoxic to the host or patient in which it is administered, representative carriers include water, oil, mineral, paste base, lotion base and ointment base, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc.

[0146] The polypeptide compounds of the present application can also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds can be radiolabeled with a radioactive isotope, such as deuterium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14 C). All isotopic variations of the compounds of the present application, whether radioactive or not, are encompassed within the scope of the present application.

[0147] One or more embodiments of the present application can achieve one or more of the following beneficial technical effects:

[0148] (1) The in vitro conversion half-life of the polypeptide compounds of the present application is greater than 120 h, and some of the compounds have an ultra-long conversion half-life, which has the potential to develop long-acting formulations;

[0149] (2) The polypeptide compounds of the present application have both agonistic activity on GLP-1 receptor and agonistic activity on GIP receptor. For GLP-1R activity: the activity of the compounds of the present application is at least 2 times that of the comparative compound, and the activity of compound 27 on GLP-1R is 24 times that of Tirzepatide and 18.3 times that of DB01. For GIPR activity: the activity of the compounds of the present application is at least 2 times that of Tirzepatide, and the activity of compound 25 on GIPR is 3.36 times that of Tirzepatide, which is comparable to or slightly better than the activity of DB01. It has a better weight loss and blood glucose lowering effect. When compared with Tirzepatide and DB01, some of the compounds of the present application have a larger HSA ratio, which reveals that the binding affinity to albumin is greater than that of Tirzepatide and DB01;

[0150] (3) After a single subcutaneous injection of the compounds of the present application (0.03 or 0.3 mg / kg), compound 27 shows a better blood glucose lowering effect than Tirzepatide on day 10, and the prodrug still exhibits an excellent blood glucose lowering effect compared with the vehicle group on day 14. It indicates that the compounds of the present application have a long half-life in vivo and a long-lasting blood glucose lowering effect;

[0151] (4) The compound 24 provided by the embodiment of the present application can be effectively converted into the original drug component in the rat body, and the half-life thereof is about 2 times of that of Tirzepatide, which has obvious advantages and is suitable for the development of long-acting preparations;

[0152] (5) In the active experiment of 7-day cumulative food intake, the effect of reducing food intake of the compound (for example, compounds 25 and 27) in the present application is better than that of Tirzepatide and DB01 at the same dose (0.1 mg / kg). BRIEF DESCRIPTION OF DRAWINGS

[0153] FIG. 1 is a mass spectrum of compound 24;

[0154] FIG. 2 is a mass spectrum of compound 25;

[0155] FIG. 3 is a mass spectrum of compound 27;

[0156] FIG. 4 is a graph of in-vitro GLP-1R agonistic activity of compounds 25, 26, 27, Tirzepatide and DB01;

[0157] FIG. 5 is a graph of in-vitro GIPR agonistic activity of compounds 25, 26, 27, Tirzepatide and DB01;

[0158] FIG. 6 is a graph of the rat in-vivo drug-time curve of compound 24;

[0159] FIG. 7 is a graph of cumulative food intake change of mice in the solvent control group, the Tirzepatide group, the DB01 group and the test compound group. DETAILED DESCRIPTION

[0160] The present application will be described in detail below by way of examples, but it does not mean any unfavorable limitation of the present application. The specific synthesis steps described in the present application also include new intermediates and methods that can be used to synthesize the polypeptide compounds or pharmaceutically acceptable salts thereof of the present application. In the specific synthesis steps of the following examples, those skilled in the art can combine in different ways to prepare the polypeptide compounds or salts thereof of the present application.

[0161] Unless otherwise specified, all reagents and raw materials used in the present disclosure can be purchased through commercial channels.

[0162] Preparation Example 1: Preparation of fatty acid side chain CL-1

[0163] Step 1: Preparation of side chain CL-1A

[0164] NaH (60%, 0.72 g, 17.90 mmol) was suspended in 20 mL of anhydrous DMF, and dibenzyl phosphite (3.91 g, 14.91 mmol) was added portionwise slowly. After stirring at room temperature for about 1 h, it was completely dissolved. Then methyl 15-bromopentadecanoate (5.00 g, 14.91 mmol) was added, and the mixture was stirred at room temperature overnight. TLC spot plate showed new spots generated. 60 mL of water was added, and the mixture was extracted with 60 mL of ethyl acetate twice. The organic phase was combined, washed with 60 mL of water twice and 60 mL of saturated NaCl solution twice, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc:PE = 40:60) to give the side chain CL-1A compound 5.78 g (yield 75.0%).

[0165] Step 2: Preparation of side chain CL-1B

[0166] Compound CL-1A (5.50 g, 10.65 mmol) was weighed into a 250 mL reaction flask, 66 mL of tetrahydrofuran was added and stirred uniformly, 1 N aqueous lithium hydroxide solution (21.3 mL, 21.3 mmol) was added dropwise slowly, and after the dropwise addition was completed, the mixture was stirred at 40-50 °C, and the reaction condition was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, 132 mL of water was added, and the mixture was extracted with 100 mL of ethyl acetate (to remove impurities). The aqueous phase was adjusted to pH = 3-4 with dilute hydrochloric acid (1 N), and then 100 mL of ethyl acetate was added for extraction three times. The combined organic phase was washed with 150 mL of saturated NaCl solution twice, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the side chain CL-1B compound 4.37 g (yield 81.6%), which was directly used in the next step reaction. LCMS M / Z = 503.2 [M+H] + .

[0167] Step 3: Preparation of side chain CL-1C

[0168] The CL-1B compound (4.30 g, 8.55 mmol) was dissolved in 86 mL of ethyl acetate at room temperature, and NHS (N-hydroxysuccinimide, 1.08 g, 9.41 mmol) was added. After stirring uniformly, DCC (2.12 g, 10.26 mmol) was added portionwise, and the mixture was stirred at room temperature overnight. The reaction condition was monitored by TLC spot plate. The solid was filtered, and the organic phase was washed with 45 mL of saturated NaCl solution three times, dried over anhydrous sodium sulfate, filtered, and concentrated to almost dryness to give the side chain CL-1C compound 4.74 g (yield 92.4%).

[0169] Step 4: Preparation of side chain CL-1D

[0170] Compound CL-1C (4.50 g, 7.50 mmol) was weighed into 45 mL DCM, H-Glu-OtBu (1.68 g, 8.25 mmol), triethylamine (2.1 mL, 15.00 mmol), water (4.5 mL) were added under stirring and the solution was heated slowly to dissolve and then cooled to room temperature and stirred. After completion of the reaction, 40 mL of 10% citric acid solution was added, stirred and allowed to separate. The organic layer was washed with 20 mL of saturated NaCl solution twice, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to get the side chain CL-1D compound 4.51 g (yield 87.5%)

[0171] Step 5: Preparation of side chain CL-1E

[0172] Compound CL-1D (4.50 g, 6.54 mmol) was dissolved in 90 mL of ethyl acetate at room temperature, NHS (N-hydroxysuccinimide, 0.83 g, 7.19 mmol) was added and stirred well, then DCC (1.62 g, 7.85 mmol) was added in portions and stirred at room temperature overnight. The reaction was monitored by TLC and the solid was filtered. The organic layer was washed with 45 mL of saturated NaCl solution three times and dried over anhydrous sodium sulfate, filtered and the organic layer was concentrated to almost dryness to get the side chain CL-1E compound 4.50 g (yield 87.6%).

[0173] Step 6: Preparation of side chain CL-1

[0174] Compound CL-1E (4.50 g, 5.73 mmol) was weighed into 67.5 mL DCM, 2 x AEEA (1.77 g, 5.73 mmol), triethylamine (1.2 g, 11.46 mmol), water (4.5 mL) were added under stirring and the solution was heated slowly to dissolve at 40°C and then cooled to room temperature and stirred. After completion of the reaction, 40 mL of 10% citric acid solution was added, stirred and allowed to separate. The organic layer was washed with 20 mL of saturated NaCl solution twice, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to dryness. The crude product was purified by column chromatography to get the side chain CL-1 compound 4.24 g (yield 75.8%) LCMS M / Z = 978.6 [M+H] + .

[0175] Preparation example 2: Preparation of fatty acid side chain CL-2

[0176] Prepared as per the procedure for the preparation of fatty acid side chain CL-1, only the starting material 15-bromopentadecanoic acid methyl ester was replaced with 17-bromohexadecanoic acid methyl ester LCMS M / Z = 1006.5 [M+H] + .

[0177] Preparation Example 3: Preparation of fatty acid side chain CL-3

[0178] Prepared according to the preparation method of fatty acid side chain CL-1, only replacing the starting material 15-bromopentadecanoic acid methyl ester with 19-bromononadecanoic acid methyl ester. LCMS M / Z = 1034.5 [M+H] + .

[0179] Preparation Example 4: Preparation of fatty acid side chain CL-4

[0180] Prepared according to the preparation method of fatty acid side chain CL-1, replacing the starting material 15-bromopentadecanoic acid methyl ester with hexadecandioic acid mono-tert-butyl ester. LCMS M / Z = 762.5 [M+H] + .

[0181] Preparation Example 5: Preparation of fatty acid side chain CL-5

[0182] The synthesis conditions of each step refer to the corresponding steps of the preparation of fatty acid side chain CL-1, and the same method can be used to synthesize tert-butyl octadecandioyl-γGlu-OtBu and tert-butyl eicosandioyl-γGlu-OtBu side chains. LCMS M / Z = 528.3 [M+H] + .

[0183] Preparation Example 6: Preparation of structural unit (Boc-Gly-Aeg(Fmoc)-OH)

[0184] Boc-Gly-OH (6.0 g, 34.3 mmol) was stirred and dissolved in DMF (120 mL) at room temperature, followed by the addition of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 10.3 g, 34.3 mmol) and DIPEA (10.4 g, 102.9 mmol). After stirring the mixture for 1 h, H-Aeg(Fmoc)-OH hydrochloride (11.7 g, 34.3 mmol) was added. The reaction was continued to stir at room temperature for 4 h. Then 240 mL of water was added and extracted twice with dichloromethane 300 mL, and the organic phase was washed with water (300 mL x 2), 10% aqueous citric acid solution (300 mL x 2), 5% aqueous sodium bicarbonate solution (300 mL x 2) and saturated NaCl solution (300 mL x 2) in turn. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (DCM / CH3OH = 4:1) to obtain the product 13.5 g (yield 78.9%).

[0185] Preparation Example 7: Preparation of compound 1

[0186] The peptide backbone was synthesized by solid phase synthesis using Fmoc / tBu strategy, mainly including steps of resin swelling / deprotection, coupling of amino acids and side chains, crude peptide cleavage and purification, etc. The specific implementation steps are as follows:

[0187] Step 1: Take 5g Fmoc Rink amide-MBHA resin (Substitution degree Sub = 0.45mmol / g), use DMF (75mL) to nitrogen bubble the reaction column for 2-3h, and then wash the waste to dryness. Add 20% piperidine / DMF (75mL) solution to nitrogen bubble for 30min, deprotect, and then wash the waste to dryness. Add DMF (75mL) to wash 4 times for 1.5min each time, and then wash the waste to dryness. Take a small amount of resin (about 10-15 resin particles) for ninhydrin detection, and the resin shows blue color.

[0188] Step 2: Coupling of amino acids

[0189] (1) Coupling of Fmoc-Ser(tBu)-OH

[0190] Take Fmoc-Ser(tBu)-OH (2.0eq) and add it to the above deprotected resin, add DIPEA (4.00eq) and 35mL MF to the reaction column and bubble nitrogen, and then add HBTU (2.0eq) after complete dissolution. React at 25°C for 1h, take a small amount of resin for ninhydrin detection, and the resin does not show blue color. Remove the reaction solution, wash with 75mL DMF for 4 times for 1.5min each time, and then wash the waste to dryness.

[0191] (2) Coupling of Fmoc-Pro-OH

[0192] Add 20% piperidine / DMF solution (50mL) to the reaction column, and then bubble nitrogen for 30min, and then wash the waste to dryness; again add 20% piperidine / DMF solution (50mL) to the reaction column, and then bubble nitrogen for 30min, and then wash the waste to dryness. Take a small amount of resin for ninhydrin detection, and the resin shows blue color. Wash with 75mL DMF for 4 times for 1.5min each time, and then wash the waste to dryness. Take Fmoc-Pro-OH (3.0eq) and add it to the above deprotected resin, add DIPEA (6.00eq) and 35mL DMF to the reaction column and bubble nitrogen, and then add HBTU (3.0eq) after complete dissolution. React at 25°C for 1.5h, take a small amount of resin for ninhydrin detection, and the resin does not show blue color. Remove the reaction solution, wash with 75mL DMF for 4 times for 1.5min each time, and then wash the waste to dryness.

[0193] (3) Repeat step (2) above to sequentially couple the following protected amino acids to the resin: 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-Aib-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-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, Fmoc-Tyr(tBu)-OH, Boc-Gly-Aeg(Fmoc)-OH;

[0194] Step 3: Fatty acid side chain CL-5 coupling

[0195] Add 20% piperidine in DMF (50 mL) to the resin and allow to stand for 30 min under a stream of nitrogen. Drain to waste and repeat once. Take a small sample of the resin and test by ninhydrin. The resin should be blue. Wash the resin with 75 mL of DMF for 1.5 min each time, draining to waste between washes. Weigh CL-5 (1.5 eq) and add to the resin. Add DIPEA (3.00 eq) and 35 mL of DMF to the reaction column and allow to stand until the reagents are evenly distributed. Add HBTU (1.5 eq) and allow the reaction to proceed for 3-4 h at 25 °C. Take a small sample of the resin and test by ninhydrin. The resin should not be blue. Drain the reaction mixture. Wash the resin with 75 mL of DMF for 1.5 min each time, draining to waste between washes.

[0196] Step 4: De-Alloc

[0197] Add scavenger PhSiH3(15.0 eq) and dichloromethane (50 mL) to the resin above, after purging with nitrogen, add Pd(PPh3)4(0.15 eq), purging with nitrogen, mix well for 60 min, repeat this step for 1 time, drain to waste.

[0198] Step 5: Coupling of Fmoc-Ida-OH

[0199] Weigh Fmoc-Ida-OH (3.0 eq) and add to the above deprotected resin, add DIPEA (6.00 eq) and 50 mL DMF to the reaction column, purging with nitrogen, after fully dissolved, add HBTU (3.0 eq). React at room temperature for 2 h, take a small amount of resin for ninhydrin test. After removing the reaction solution, wash with 75 mL DMF for 4 times, 1.5 min each time, drain to waste.

[0200] Step 6: Coupling of fatty acid side chain CL-1

[0201] Add 10% DBU / DMF (75 mL) to the above resin, purging with nitrogen, mix well for 30 min, drain to waste; repeat this step for 1 time. Wash with 75 mL DMF for 4 times, 1.5 min each time, drain to waste. Weigh fatty acid side chain CL-1 (1.50 eq) and add to the above resin, add DIPEA (3.00 eq) and 50 mL DMF to the reaction column, purging with nitrogen, after fully dissolved, add HBTU (1.5 eq). React at room temperature for 3-4 h, take a small amount of resin for ninhydrin test. After removing the reaction solution, wash with 75 mL DMF for 4 times, 1.5 min each time, drain to waste.

[0202] Step 7: Remove Dde

[0203] Add 2% hydrazine / DMF (70 mL) to the above resin, purging with nitrogen, react for 10 min, drain to waste, repeat this step for 2 times, then drain to waste. Wash with 75 mL DMF for 4 times, 1.5 min each time, drain to waste. Take a small amount of resin for ninhydrin test, the resin shows blue color.

[0204] Step 8: Cyclization

[0205] To the resin above, 55 mL of DMF was added, followed by the prepared HOBT (1.5 eq) solution (dissolved in 10 mL of DMF) and DIC (1.5 eq) solution (dissolved in 10 mL of DMF). The reaction was stirred at room temperature under a nitrogen atmosphere for 3-4 h. A small amount of resin was taken for ninhydrin test. The resin appeared colorless and transparent. The effluent was discarded. The resin was washed with 75 mL of DMF for 4 times, 2.0 min each, and the effluent was discarded. The resin was washed with MeOH for 3 times, and the effluent was discarded. The resin was poured out and dried.

[0206] Step 9: Cleavage and purification

[0207] The dried resin was added to the prepared cleavage solution (trifluoroacetic acid: triisopropylsilane: 1,2-ethanedithiol: water = 92:4:2:2) and stirred at room temperature for about 3.0 h to complete the cleavage of the resin and removal of the side chain protecting groups. The resin was washed with a small amount of trifluoroacetic acid. The combined filtrate was precipitated with 8-10 volumes of ice-cold methyl tert-butyl ether. The precipitate was filtered and washed. The crude peptide was dried under vacuum at reduced pressure for 2 h. The crude peptide was purified by reverse phase HPLC chromatography after confirmation by LCMS. Calculated [M+4H] 4+ The LCMS detected value was 1396.1.

[0208] Preparation Example 8: Preparation of compound 7

[0209] The peptide synthesis was performed based on Fmoc chemistry. The synthesis was performed using 0.5 mmol Rink Amide-AM resin (loading: 0.35 mmol / g) and standard Fmoc-amino acids. The coupling reagents were HBTU and HOBT, DIEA, and the deprotection was achieved using 20% piperidine in DMF. The resin was modified using 3-fold excess of Fmoc-Ser(tBu)-OH and 3-fold excess of coupling reagents.

[0210] Step 1: Synthesis of the main chain peptide

[0211] Fmoc-Pro-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-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, Fmoc-Tyr(tBu)-OH, Fmoc-Aeg(Alloc)-OH, Boc-Gly-OH, the coupling reaction is generally for 60 min, the deprotection time is 20 min x 2, the control monitoring (indanetrione coloration), the resin is washed with DMF for 6 times between each deprotection and reaction, and the main chain peptide is obtained.

[0212] Step 2: de-Alloc protection

[0213] The main chain peptide obtained in the above step is swelled in DMF (50 mL) for 30 min, then Pd(PPh3)4 (0.2 eq) and phenylsilane (10 eq) are added respectively, the reaction solution is reacted at room temperature for 2 h, then the reaction solution is filtered off, and the resin is washed with DMF for 8 times (after washing, indanetrione detection (blue color) is used to confirm that the de-Alloc protection product is obtained.

[0214] Step 3: prodrug side chain coupling

[0215] Fmoc-γ-Glu(OtBu)-OH, 15-(bis(benzyloxy)phosphoryl)pentadecanoic acid (coupling reagent is HBTU and HOBT, DIEA, and deprotection is achieved using 20% piperidine in DMF) were sequentially condensed using a 3-fold excess of Fmoc-amino acid, 3-fold excess of coupling reagent. The coupling reaction was typically 60 min, and deprotection was 20 min x 2, with monitoring (ninhydrin color) and washing the resin 6 times with DMF between each deprotection and reaction to give the prodrug side chain containing compound.

[0216] Step 4: Cyclization

[0217] The Dde protecting group was removed from the compound of the previous step using 2% N2H4 / DMF for 20 min x 2. The resin was then washed 6 times with DMF. Bis(2,5-dioxopyrrolidin-l-yl) 2,2'-((((9H-fluoren-9-yl)methoxy)carbonyl)azanediyl)diacetate (1.1 eq) in DMF (50 mL) and DIEA (4 eq) were added, and the reaction was allowed to proceed for 1 h at room temperature. The reaction was then drained, and the resin was washed 6 times with DMF (after washing, the resin was tested with ninhydrin (colorless) to give the cyclized compound.

[0218] Step 5: Main chain peptide side chain coupling

[0219] The Fmoc protecting group was removed from the compound of the previous step using 20% piperidine in DMF for 20 min x 2, and the resin was washed 6 times with DMF. Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid (coupling reagent is HBTU and HOBT, DIEA, and deprotection is achieved using 20% piperidine in DMF) were sequentially condensed using a 3-fold excess of Fmoc-amino acid, 3-fold excess of coupling reagent. The coupling reaction was typically 60 min, and deprotection was 20 min x 2, with monitoring (ninhydrin color) and washing the resin 6 times with DMF between each deprotection and reaction to give 3.5 g of crude product.

[0220] Step 6: Cleavage and purification

[0221] The crude product from the previous step was cleaved using 40 mL (V TFA :V Tis :V 水 = 90:5:5) for 3 hours. After cleavage, the used resin was removed by filtration and the filtrate was added to methyl tert-butyl ether which had been cooled at -20 °C, centrifuged, the supernatant was discarded and the solid was washed with cold methyl tert-butyl ether twice more. A small sample was taken to analyze the molecular weight and purity of the crude product by mass spectrometry and HPLC. The peptide was then dissolved in 10 mL of DMF and loaded on a C18 preparative HPLC column at a flow rate of 20 mL / min with water (0.1% TFA) / acetonitrile mobile phase, gradient 30-55% acetonitrile for 30 min. Compound 7 was obtained after lyophilization (white solid, 58 mg, purity: 96.3%, yield: 2.1%). LCMS of the polypeptide molecule: [M+4H] 4+ / 4: 1385.3; [M+5H] 5+ / 5: 1108.2.

[0222] Preparation Example 9: Preparation of compound 12

[0223] Peptide synthesis was performed based on Fmoc chemistry using solid phase synthesis method with 0.5 mmol Rink Amide-AM resin (loading: 0.35 mmol / g) and standard Fmoc-amino acids. The coupling reagents were HBTU and HOBT, DIEA and deprotection was achieved with 20% piperidine / DMF solution. Resin modification was performed using 3-fold excess of Fmoc-Ser(tBu)-OH and 3-fold excess of coupling reagents.

[0224] Step 1: Main chain peptide synthesis

[0225] Fmoc-Pro-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-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-1-amino-1-cyclobutanecarboxylic acid-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Sar-OH, Boc-Lys(Fmoc)-OH, Fmoc-γ-Glu(OtBu)-OH, hexadecanedioic acid mono-tert-butyl ester, using a 3-fold excess of Fmoc-amino acid, a 3-fold excess of coupling reagent, a coupling reaction time of typically 60 min, a deprotection time of 20 min*2, and monitoring by ninhydrin coloration between each deprotection and reaction, with 6 washes of the resin with DMF between each deprotection and reaction, to give the main chain peptide.

[0226] Step 2: cyclization

[0227] The compound from the previous step was deprotected using 2% N2H4 / DMF for 20 min x 2. The resin was then washed with DMF 6 times. Bis(2,5-dioxopyrrolidin-l-yl) 2,2'-((((9H-fluoren-9-yl)methoxy)carbonyl)azanediyl)diacetate (1.1 eq) in DMF (50 mL) and DIEA (4 eq) were added and the reaction was allowed to proceed for 1 h at room temperature. The reaction was then drained and the resin was washed with DMF 6 times (after washing, the resin was tested with ninhydrin (colorless)). The cyclized compound was obtained.

[0228] Step 3: Side chain coupling of the main chain peptide

[0229] The Fmoc protecting group was removed using 20% piperidine in DMF and the resin was washed with DMF 6 times. Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid (coupling reagents were HBTU and HOBT, DIEA, and deprotection was achieved using 20% piperidine in DMF) were sequentially coupled using a 3-fold excess of each reagent. The coupling reaction was allowed to proceed for 60 min and deprotection was achieved using 20 min x 2. The reaction was monitored by ninhydrin test. The resin was washed with DMF 6 times between each deprotection and reaction. The crude product was obtained as 3.6 g.

[0230] Step 4: Cleavage and purification

[0231] The crude product from the previous step was cleaved from the resin using 40 mL (V TFA :V Tis :V 水= 90:5:5) for 3 hours. After cleavage, the used resin was removed by filtration and the filtrate was added to methyl tert-butyl ether, which had been cooled at -20°C, centrifuged, the supernatant was discarded and the solid was washed with cold methyl tert-butyl ether twice more. A small sample was taken to analyze the molecular weight and purity of the crude product by mass spectrometry and HPLC, then the peptide was dissolved in 10 mL of DMF and loaded on a C18 preparative HPLC column at a flow rate of 20 mL / min with a water (0.1% TFA) / acetonitrile mobile phase gradient of 30-55% acetonitrile over 30 min. After lyophilization, compound 12 was obtained (white solid, 55 mg, purity: 95.08%, yield: 1.97%). LCMS of the polypeptide molecule: [M+4H] 4+ / 4: 1396.8; [M+5H] 5+ / 5: 1117.6.

[0232] Preparation Example 10: Preparation of compound 15

[0233] Peptide synthesis was performed based on Fmoc chemistry using solid phase synthesis with 0.5 mmol Rink Amide-AM resin (loading: 0.35 mmoL / g) and standard Fmoc-amino acids. The coupling agent was HBTU and HOBT, DIEA, and deprotection was achieved with 20% piperidine / DMF solution. Resin modification was performed using a 3-fold excess of Fmoc-Ser(tBu)-OH and a 3-fold excess of coupling reagent.

[0234] Step 1: Main chain peptide synthesis

[0235] Fmoc-Pro-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-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, Fmoc-Tyr(tBu)-OH, Fmoc-Sar-OH, Boc-Lys(Fmoc)-OH, Fmoc-γ-Glu(OtBu)-OH, 15-(bis(benzyloxy)phosphoryl)pentadecanoic acid, with a 3-fold excess of Fmoc-amino acid, a 3-fold excess of coupling reagent, a coupling reaction time of 60 min, a deprotection time of 20 min x 2, and monitoring by ninhydrin, with 6 washes of the resin with DMF between each deprotection and reaction, to give the main chain peptide.

[0236] Step 2: cyclization

[0237] The compound from the previous step was deprotected of the Dde protecting group using 2% N2H4 / DMF for 20 min x 2. The resin was then washed with DMF 6 times. Bis(2,5-dioxopyrrolidin-l-yl) 2,2'-((((9H-fluoren-9-yl)methoxy)carbonyl)azanediyl)diacetate (1.1 eq) in DMF (50 mL) and DIEA (4 eq) were added and the reaction was allowed to proceed for 1 h at room temperature. The reaction was then drained and the resin washed with DMF 6 times (after washing, the resin was tested with ninhydrin (colorless)). The cyclized compound was obtained.

[0238] Step 3: Side chain coupling of the main chain peptide

[0239] The product from the previous step was deprotected of the Fmoc protecting group using 20% piperidine in DMF for 20 min x 2. The resin was then washed with DMF 6 times. Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid (coupling reagents were HBTU and HOBT, DIEA, and deprotection was achieved using 20% piperidine in DMF) were sequentially coupled using a 3-fold excess of each Fmoc-amino acid. The coupling reactions were allowed to proceed for 60 min and the deprotection was achieved for 20 min x 2. The reactions were monitored by ninhydrin. The resin was washed with DMF 6 times between each deprotection and reaction. The crude product was obtained as 3.7 g.

[0240] Step 4: Cleavage and purification

[0241] The crude product from the previous step was cleaved from the resin using 40 mL (V TFA :V Tis :V 水= 90:5:5) for 3 hours. After cleavage, the used resin was removed by filtration and the filtrate was added to methyl tert-butyl ether, which had been cooled at -20°C, centrifuged, the supernatant was discarded and the solid was washed with cold methyl tert-butyl ether twice more. A small sample was taken to analyze the molecular weight and purity of the crude product by mass spectrometry and HPLC, then the peptide was dissolved in 10 mL of DMF and loaded on a C18 preparative HPLC column at a flow rate of 20 mL / min with a water (0.1% TFA) / acetonitrile mobile phase gradient of 30-55% acetonitrile over 30 min. After lyophilization, compound 15 was obtained (white solid, 37.5 mg, purity: 96.45%, yield: 1.3%). LCMS of the polypeptide molecule: [M+4H] 4+ / 4: 1395.8; [M+5H] 5+ / 5: 1116.6.

[0242] Preparation Example 11: Preparation of compound 23

[0243] Peptide synthesis was performed based on Fmoc chemistry using solid phase synthesis with 0.5 mmol Rink Amide-AM resin (loading: 0.35 mmol / g) and standard Fmoc-amino acids. The coupling reagents were HBTU and HOBT, DIEA, and deprotection was achieved with 20% piperidine / DMF. Resin modification was performed using a 3-fold excess of Fmoc-Ser(tBu)-OH and a 3-fold excess of coupling reagents.

[0244] Step 1: Main chain peptide synthesis

[0245] The main chain peptide was obtained by sequentially condensing Fmoc-Pro-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-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, Fmoc-Tyr(tBu)-OH, Fmoc-Aeg(Alloc)-OH, Boc-Gly-OH using a 3-fold excess of Fmoc-amino acid, 3-fold excess of coupling reagent, the coupling reaction was generally for 60 min, the deprotection time was 20 min x 2, the reaction was monitored by ninhydrin test, and the resin was washed with DMF 6 times between each deprotection and reaction.

[0246] Step 2: de-Alloc protection

[0247] The main chain peptide obtained in the previous step was swelled in DMF (50 mL) for 30 min, and then Pd(PPh3)4(0.2 eq), phenylsilane (10 eq) were added, respectively. The reaction was carried out at room temperature for 2 h, and then the reaction was filtered off, and the resin was washed with DMF 8 times (after washing, ninhydrin test was used (blue color was shown)) to obtain the de-Alloc protected product.

[0248] Step 3: prodrug side chain coupling

[0249] The Alloc-protected peptide from the previous step was condensed with 3x excess Fmoc-amino acid, 3x excess coupling reagent sequentially with Fmoc-γ-Glu(OtBu)-OH, hexadecanedioic acid mono-tert-butyl ester (coupling reagent was HBTU and HOBT, DIEA, and deprotection was achieved with 20% piperidine in DMF). The coupling reaction was typically 60 min, and deprotection was 20 min x 2, with monitoring (ninhydrin color), and the resin was washed 6 times with DMF between each deprotection and reaction to give the compound with the prodrug side chain.

[0250] Step 4: Cyclization

[0251] The Dde protecting group was removed from the compound from the previous step using 2% N2H4 / DMF for 20 min x 2. The resin was then washed 6 times with DMF. Bis(2,5-dioxopyrrolidin-l-yl) 2,2'-((((9H-fluoren-9-yl)methoxy)carbonyl)azanediyl)diacetate (1.1 eq) in DMF (50 mL) and DIEA (4 eq) were added, and the reaction was allowed to proceed for 1 h at room temperature. The reaction was then drained, and the resin was washed 6 times with DMF (after washing, the resin was tested with ninhydrin (colorless) to give the cyclized compound.

[0252] Step 5: Main chain peptide side chain coupling

[0253] The Fmoc protecting group was removed using 20% piperidine in DMF, and the resin was washed 6 times with DMF. The Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid (coupling reagent was HBTU and HOBT, DIEA, and deprotection was achieved with 20% piperidine in DMF) were condensed sequentially with 3x excess Fmoc-amino acid, 3x excess coupling reagent. The coupling reaction was typically 60 min, and deprotection was 20 min x 2, with monitoring (ninhydrin color), and the resin was washed 6 times with DMF between each deprotection and reaction to give 3.7 g of crude product.

[0254] Step 6: Cleavage and purification

[0255] The crude product from the previous step was cleaved from the resin using 40 mL (V TFA :V Tis :V 水= 90:5:5) for 3 hours. After cleavage, the used resin was removed by filtration and the filtrate was added to methyl tert-butyl ether, which had been cooled at -20°C, centrifuged, the supernatant was discarded and the solid was washed with cold methyl tert-butyl ether twice more. A small sample was taken to analyze the molecular weight and purity of the crude product by mass spectrometry and HPLC, then the peptide was dissolved in 10 mL of DMF and loaded on a C18 preparative HPLC column at a flow rate of 20 mL / min with a water (0.1% TFA) / acetonitrile mobile phase gradient of 30-55% acetonitrile over 30 min. After lyophilization, compound 23 was obtained (white solid, 72 mg, purity: 98.75%, yield: 2.62%). LCMS of the polypeptide molecule: [M+4H] 4+ / 4: 1376.3; [M+5H] 5+ / 5: 1101.1.

[0256] Preparation Example 12: Preparation of compound 24

[0257] Peptide synthesis was performed based on Fmoc chemistry, using solid phase synthesis with 0.5 mmol Rink Amide-AM resin (loading: 0.35 mmol / g) and standard Fmoc-amino acids. The coupling agent was HBTU and HOBT, DIEA, and deprotection was achieved with 20% piperidine / DMF. Resin modification was performed using a 3-fold excess of Fmoc-Ser(tBu)-OH and a 3-fold excess of coupling reagent.

[0258] Step 1: Main chain peptide synthesis

[0259] The main chain peptide was obtained by sequentially condensing Fmoc-Pro-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-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-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-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, Fmoc-Tyr(tBu)-OH, Fmoc-Aeg(Alloc)-OH, Boc-Gly-OH using a 3-fold excess of Fmoc-amino acid, 3-fold excess of coupling reagent, a coupling reaction time of 60 min, a deprotection time of 20 min x 2, and monitoring by ninhydrin (blue color) after each deprotection and reaction. The resin was washed with DMF 6 times between each deprotection and reaction.

[0260] Step 2: de-Alloc protection

[0261] The main chain peptide from the previous step was swelled in DMF (50 mL) for 30 min, Pd(PPh3)4 (0.2 eq) and phenylsilane (10 eq) were added, and the reaction was allowed to proceed at room temperature for 2 h. The reaction was then filtered off and the resin was washed with DMF 8 times (after washing, ninhydrin was used to check (blue color) to ensure that the Alloc protection was removed).

[0262] Step 3: prodrug side chain coupling

[0263] Fmoc-γ-Glu(OtBu)-OH, 15-(bis(benzyloxy)phosphoryl)pentadecanoic acid (coupling reagent is HBTU and HOBT, DIEA, and deprotection is achieved using 20% piperidine in DMF) were sequentially coupled using a 3-fold excess of Fmoc-amino acid, 3-fold excess of coupling reagent. The coupling reaction was typically 60 min, and deprotection was 20 min x 2, with monitoring (ninhydrin color), and the resin was washed 6 times with DMF between each deprotection and reaction to give the prodrug side chain containing compound.

[0264] Step 4: Cyclization

[0265] The Dde protecting group was removed from the compound of the previous step using 2% N2H4 / DMF for 20 min x 2. The resin was then washed 6 times with DMF. Bis(2,5-dioxopyrrolidin-l-yl) 2,2'-((((9H-fluoren-9-yl)methoxy)carbonyl)azanediyl)diacetate (1.1 eq) in DMF (50 mL) and DIEA (4 eq) were added, and the reaction was allowed to proceed for 1 h at room temperature. The reaction was then drained, and the resin was washed 6 times with DMF (after washing, the resin was tested with ninhydrin (colorless) to give the cyclized compound.

[0266] Step 5: Main chain peptide side chain coupling

[0267] Fmoc protecting groups were removed using 20% piperidine in DMF, and the resin was washed 6 times with DMF. Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid (coupling reagent is HBTU and HOBT, DIEA, and deprotection is achieved using 20% piperidine in DMF) were sequentially coupled using a 3-fold excess of Fmoc-amino acid, 3-fold excess of coupling reagent. The coupling reaction was typically 60 min, and deprotection was 20 min x 2, with monitoring (ninhydrin color), and the resin was washed 6 times with DMF between each deprotection and reaction to give 3.7 g of crude product.

[0268] Step 6: Cleavage and purification

[0269] The crude product from the previous step was cleaved from the resin using 40 mL (VTFA :V Tis :V 水 = 90:5:5) for 3 hours. After cleavage, the used resin was removed by filtration and the filtrate was added to methyl tert-butyl ether, which had been cooled at -20°C, centrifuged, the supernatant was discarded and the solid was washed with cold methyl tert-butyl ether twice more. A small sample was taken to analyze the molecular weight and purity of the crude product by mass spectrometry and HPLC, then the peptide was dissolved in 10 mL of DMF and loaded on a C18 preparative HPLC column at a flow rate of 20 mL / min with water (0.1% TFA) / acetonitrile as mobile phase with a gradient of 30-55% acetonitrile for 30 min. Compound 24 was obtained after lyophilization (white solid, 8 mg, purity: 97.51%, yield: 0.28%). LCMS of the polypeptide molecule: [M+4H] 4+ / 4: 1408.3; [M+5H] 5+ / 5: 1126.6, see Figure 1.

[0270] Preparation Example 13: Preparation of compound 25

[0271] Peptide synthesis was performed based on Fmoc chemistry using solid phase synthesis with 0.5 mmol Rink Amide-AM resin (loading: 0.35 mmol / g) and standard Fmoc-amino acids. The coupling reagents were HBTU and HOBT, DIEA and deprotection was achieved with 20% piperidine / DMF. Resin modification was performed using a 3-fold excess of Fmoc-Ser(tBu)-OH and a 3-fold excess of coupling reagents.

[0272] Step 1: Main chain peptide synthesis

[0273] The Fmoc-Pro-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-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 were sequentially condensed using a 3-fold excess of Fmoc-amino acid and a 3-fold excess of coupling reagent, the coupling reaction was generally for 60 min, the deprotection time was 20 min x 2, and the reaction was monitored by ninhydrin coloration. The resin was washed with DMF 6 times between each deprotection and reaction, to obtain the main chain peptide.

[0274] Step 2: cyclization

[0275] The Dde protecting group of the compound from the previous step was removed using 2% N2H4 / DMF, and the deprotection time was 20 min x 2. The resin was washed with DMF 6 times. Bis(2,5-dioxopyrrolidin-1-yl) 2,2'-((((9H-fluoren-9-yl)methoxy)carbonyl)azanediyl)diacetate (1.1 eq) and a DIEA (4 eq) solution in DMF (50 ml) were added, and the reaction was allowed to proceed at room temperature for 1 h. The reaction solution was then filtered off, and the resin was washed with DMF 6 times (after washing, ninhydrin detection was performed (colorless)), to obtain the cyclization compound.

[0276] Step 3: main chain peptide side chain coupling

[0277] Fmoc protecting group removal was achieved using 20% piperidine in DMF and the resin was washed 6 times with DMF. Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid (coupling reagent was HBTU and HOBT, DIEA, and deprotection was achieved using 20% piperidine in DMF) were sequentially condensed using 3-fold excess of Fmoc-amino acid and 3-fold excess of coupling reagent. The coupling reaction was typically 60 min, and the deprotection time was 20 min x 2, with monitoring (ninhydrin coloration) in between each deprotection and reaction by washing the resin 6 times with DMF. The crude product was obtained as 3.6 g.

[0278] Step 4: Cleavage and purification

[0279] The crude product from the previous step was cleaved using 40 mL (V TFA :V Tis :V 水 = 90:5:5) for 3 hours. After cleavage, the used resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether, which had been cooled at -20 °C, centrifuged, the supernatant was discarded, and the solid was washed with cold methyl tert-butyl ether twice more. A small sample was taken to analyze the molecular weight and purity of the crude product by mass spectrometry and HPLC, and then the peptide was dissolved in 10 mL of DMF and loaded on a C18 preparative HPLC column at a flow rate of 20 mL / min with a water (0.1% TFA) / acetonitrile mobile phase with a gradient of 30-55% acetonitrile over 30 min. After lyophilization, compound 25 was obtained as a white solid (80 mg, purity: 97.68%, yield: 3.23%). LCMS of the polypeptide molecule: [M+4H] 4+ / 4: 1237.6; [M+5H] 5+ / 5: 990.4, see Figure 2.

[0280] Preparation Example 14: Preparation of compound 26

[0281] Peptide synthesis was performed based on Fmoc chemistry using solid phase synthesis with 0.5 mmol Rink Amide-AM resin (loading: 0.35 mmol / g) and standard Fmoc-amino acids. The coupling reagent was HBTU and HOBT, DIEA, and deprotection was achieved using 20% piperidine in DMF. Resin modification was performed using 3-fold excess of Fmoc-Ser(tBu)-OH and 3-fold excess of coupling reagent.

[0282] Step 1: Backbone peptide synthesis

[0283] Fmoc-Pro-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-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-1-amino-1-cyclobutanecarboxylic acid, Boc-Tyr(tBu)-OH, with a coupling time of 60 min, deprotection time of 20 min x 2, and monitoring by ninhydrin, and washing the resin with DMF 6 times between each deprotection and reaction to give the backbone peptide.

[0284] Step 2: Cyclization

[0285] The compound from the previous step was deprotected using 2% N2H4 / DMF for 20 min x 2. The resin was then washed with DMF 6 times. Bis(2,5-dioxopyrrolidin-l-yl) 2,2'-((((9H-fluoren-9-yl)methoxy)carbonyl)azanediyl)diacetate (1.1 eq) in DMF (50 mL) and DIEA (4 eq) were added and the reaction was allowed to proceed for 1 h at room temperature. The reaction was then drained and the resin was washed with DMF 6 times (after washing, the resin was tested with ninhydrin (colorless)). The cyclized compound was obtained.

[0286] Step 3: Side chain coupling of the main chain peptide

[0287] The Fmoc protecting group was removed using 20% piperidine in DMF and the resin was washed with DMF 6 times. Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid (coupling reagents were HBTU and HOBT, DIEA, and deprotection was achieved using 20% piperidine in DMF) were sequentially coupled using a 3-fold excess of each reagent. The coupling reaction was allowed to proceed for 60 min and deprotection was achieved using 20 min x 2. The reaction was monitored by ninhydrin test. The resin was washed with DMF 6 times between each deprotection and reaction. The crude product was obtained as 3.4 g.

[0288] Step 4: Cleavage and purification

[0289] The crude product from the previous step was cleaved from the resin using 40 mL (V TFA :V Tis :V 水= 90:5:5) for 3 hours. After cleavage, the used resin was removed by filtration and the filtrate was added to methyl tert-butyl ether, which had been cooled at -20°C, centrifuged, the supernatant was discarded and the solid was washed with cold methyl tert-butyl ether twice more. A small sample was taken to analyze the molecular weight and purity of the crude product by mass spectrometry and HPLC, then the peptide was dissolved in 10 mL of DMF and loaded on a C18 preparative HPLC column at a flow rate of 20 mL / min with a water (0.1% TFA) / acetonitrile mobile phase gradient of 30-55% acetonitrile over 30 min. After lyophilization, compound 26 was obtained (white solid, 71 mg, purity: 97.31%, yield: 2.86%). LCMS of the polypeptide molecule: [M+4H] 4+ / 4: 1240.6; [M+5H] 5+ / 5: 992.6.

[0290] Preparation Example 15: Preparation of compound 27

[0291] Peptide synthesis was performed based on Fmoc chemistry using solid phase synthesis with 0.5 mmol Rink Amide-AM resin (loading: 0.35 mmol / g) and standard Fmoc-amino acids. The coupling reagents were HBTU and HOBT, DIEA, and deprotection was achieved with 20% piperidine / DMF. Resin modification was performed using a 3-fold excess of Fmoc-Ser(tBu)-OH and a 3-fold excess of coupling reagent.

[0292] Step 1: Main chain peptide synthesis

[0293] Fmoc-Pro-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-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-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-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, in general for 60 min, deprotection for 20 min x 2, monitored by ninhydrin, and the resin was washed with DMF 6 times between each deprotection and reaction to give the main chain peptide.

[0294] Step 2: cyclization

[0295] The compound from the previous step was deprotected of the Dde protecting group using 2% N2H4 / DMF for 20 min x 2. The resin was then washed with DMF 6 times. Bis(2,5-dioxopyrrolidin-l-yl) 2,2'-((((9H-fluoren-9-yl)methoxy)carbonyl)azanediyl)diacetate (1.1 eq) in DMF (50 ml) and DIEA (4 eq) were added and the reaction was allowed to proceed for 1 h at room temperature. The reaction was then drained and the resin washed with DMF 6 times (after washing, the resin was checked with ninhydrin (colorless) to give the cyclized compound.

[0296] Step 3: Side chain coupling of the main chain peptide

[0297] The product from the previous step was deprotected using 20% piperidine in DMF and the resin was washed 6 times with DMF. Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-γ-Glu(OtBu)-OH, 19-(bis(benzyloxy)phosphoryl)nonadecanoic acid (coupling reagents were HBTU and HOBT, DIEA, and deprotection was achieved using 20% piperidine in DMF) were sequentially condensed using a 3-fold excess of the Fmoc-amino acid and a 3-fold excess of the coupling reagent. The coupling reaction was typically 60 min, and deprotection was 20 min x 2, with monitoring (ninhydrin color) in between each deprotection and reaction by washing the resin 6 times with DMF. A 3.4 g crude product was obtained.

[0298] Step 4: Cleavage and purification

[0299] The crude product from the previous step was cleaved using 40 mL (V TFA :V Tis :V 水 = 90:5:5) for 3 hours. After cleavage, the used resin was removed by filtration, and the filtrate was added to methyl tert-butyl ether that had been cooled at -20 °C, centrifuged, the supernatant was discarded, and the solid was washed with cold methyl tert-butyl ether twice more. A small sample was taken to analyze the molecular weight and purity of the crude product by mass spectrometry and HPLC, and then the peptide was dissolved in 10 mL of DMF and loaded on a C18 preparative HPLC column at a flow rate of 20 mL / min with a water (0.1% TFA) / acetonitrile mobile phase with a gradient of 30-55% acetonitrile over 30 min. After lyophilization, compound 27 was obtained as a white solid (5.6 mg, purity: 95.1%, yield: 0.22%). LCMS of the polypeptide molecule: [M+4H] 4+ / 4: 1260.6; [M+5H] 5+ / 5: 1008.6, see Figure 3.

[0300] Preparation Example 16: Preparation of other compounds

[0301] Compounds 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22 can be obtained using the methods of the above preparation examples.

[0302] Preparation Example 17: Preparation of comparative compound DB01

[0303] Prepared according to the method of Example 5 in patent CN117015551A, LCMS of polypeptide molecule: [M+3H] 3+ / 3: 1642.2; [M+4H] 4+ / 4: 1231.8.

[0304] Biological test data

[0305] 1. Measurement of conversion half-life

[0306] A stock solution of a certain concentration was prepared by dissolving the synthesized polypeptide compound in PBS buffer without CaCl2 and MgCl2, and the pH of the peptide stock solution was adjusted to 7.4 with 0.02M HC1 or 0.02M NaOH. The sample was loaded into an HPLC vial with a fixed insert, and the vial was capped. The HPLC vial was incubated at 37°C (SHP-250 type biochemical incubator), and samples were taken at different time points within 2 weeks for analysis.

[0307] The prodrug and parent drug were confirmed by LCMS, and the peak area% was plotted as the natural logarithm against time, and the first-order half-life (T 1 / 2 ) = T 1 / 2 = Ln(2) / k.

[0308] Table 1. Determination of conversion half-life of compounds

[0309] The in vitro conversion half-life of the compounds of the present application is greater than 120h, and some compounds have an ultra-long conversion half-life.

[0310] 2. Detection experiment of GLP-1R / GIPR agonistic activity

[0311] Experimental method: GLP-1R / CRE Luc HEK293 cells (Nanjing Kebai, CBP71117) or GIPR / CRE Luc HEK293 cells (Nanjing Kebai, CBP71346) in logarithmic growth phase were digested, and the digested cells were resuspended in DMEM+10% FBS medium, and the cell density was adjusted to 3.5x10 5 / mL (3x10 5 / mL for GIPR cells). 90μL / well was inoculated in a 96-well cell culture plate, and the culture plate was incubated in an incubator overnight (37°C, 5% CO2).

[0312] DMSO was used to dissolve the compounds, and a stock solution with an initial concentration of 0.5 mM was prepared, and then DMSO was used for dilution (the initial concentration of GLP-1R cells was 1 mM, and the initial concentration of GIPR cells was 0.1 mM, diluted by 5 times, 10 concentration points, and the 11th point was DMSO), configured as a 1000x working solution. Then 2 μL of each concentration sample was added to 198 μL of test medium (DMEM), configured as a 10x working solution. Then 10 μL of different concentrations of 10x working solution was added to each well of the cell culture plate, and two duplicate wells were used for each sample. The culture plate was incubated in an incubator for 5 hours (37°C, 5% CO2). The 96-well cell culture plate was removed, and 100 μL of luciferase reagent was added to each well, gently shaken to mix, and then placed at room temperature for 10 minutes. The chemiluminescence signal value was measured using an enzyme-labeled instrument. Microsoft Excel was used to process and analyze the data using Graphpad Prism 8, and the EC values of the compounds were obtained. 50

[0313] Table 2. GLP-1R / GIPR agonistic activity of compounds

[0314] The compounds of the present application exhibit strong agonistic activity on both GLP-1R and GIPR, and the activity is better than Tirzepatide and DB01. For GLP-1R activity, the activity of the compounds of the present application is at least 2 times that of the comparative compound, and the activity of compound 27 on GLP-1R is 17.5 times that of Tirzepatide and 7.4 times that of DB01. For GIPR activity, the activity of the compounds of the present application is at least 2 times that of Tirzepatide, and the activity of compound 25 on GIPR is 3.36 times that of Tirzepatide, which is comparable to or slightly better than the activity of DB01, as shown in Figures 4 and 5.

[0315] Therefore, the compounds of the present application have more excellent agonistic activity on GLP-1R / GIP dual targets, and are expected to be developed into new drugs with better therapeutic effect and lower dosage.

[0316] 3. In vitro HSA-regulated GLP-1R and GIPR agonistic activity

[0317] Experimental method: GLP-1R / CRE Luc HEK293 cells (Nanjing Kebai, CBP71117) or GIPR / CRE Luc HEK293 cells (Nanjing Kebai, CBP71346) in logarithmic growth phase were digested, and the digested cells were resuspended in DMEM+10% FBS medium, and the cell density was adjusted to 3.5x10 5 / mL (3x10 5 ​ / mL). Seed 96-well cell culture plates at 90 μL / well, and incubate the plates overnight in the incubator (37 °C, 5% CO2).

[0318] Remove the 96-well plates and replace the cell culture medium with cell culture medium containing 2% HSA (human serum albumin, Aladdin, rp218441) or without HSA.

[0319] Dissolve the compounds with DMSO to prepare a stock solution with an initial concentration of 0.5 mM, and then use DMSO to prepare a 1000x working solution by diluting by a factor of 2 (GLP-1R cell initial concentration of 1 mM, GIPR cell initial concentration of 0.1 mM, 5-fold dilution, 10 concentration points, and the 11th point is DMSO). Then take 2 μL of each concentration sample and add it to 198 μL of test medium to prepare a 10x working solution. Then add 10 μL / well of the 10x working solution of different concentrations to the cell culture plates, with two replicate wells for each sample. Incubate the plates in the incubator for 5 hours (37 °C, 5% CO2).

[0320] Remove the 96-well cell culture plates and add 100 μL of luciferase reagent to each well, mix gently, and incubate at room temperature for 10 minutes. Measure the chemiluminescence signal value using a microplate reader. Use Microsoft Excel and Graphpad Prism 8 to process and analyze the data to obtain the EC 50 values of the compounds.

[0321] Table 3. HSA ratios of the compounds

[0322] For GLP-1R and GIPR, Tirzepatide has an HSA ratio of 37.2 and 1.3, respectively, DB01 has an HSA ratio of 21.7 and 1.6, respectively, compound 25 has an HSA ratio of 95.6 and 3.1, respectively, compound 26 has an HSA ratio of 91.5 and 7.5, respectively, and compound 27 has an HSA ratio of 92.9 and 13.1, respectively. Compared with Tirzepatide and DB01, the compounds of the present application have a larger HSA ratio, revealing that their binding affinity to albumin is greater than that of Tirzepatide and DB01.

[0323] 4. In vivo hypoglycemic experiment of the original drug compound in mice

[0324] Experimental method: C57BL / 6 mice, male, purchased from Beijing Huafukang Biotechnology Co., Ltd., were subcutaneously injected with a single dose of solvent or corresponding compound, and intraperitoneally injected with glucose solution (2 g / kg) after overnight fasting on day 6 and day 10. The blood glucose value was measured before the sugar was given (equivalent to 0 min blood glucose value) and 30 min after the glucose was given. The solvent was PBS, and the dose was 0.03 mg / kg (n = 3). The experimental results are shown in Table 4.

[0325] Table 4. Hypoglycemic effect of prodrugs in mice

[0326] The results of the hypoglycemic test of the compound of the present application in mice by single subcutaneous administration showed that on day 6, the compound of the present application exhibited a strong hypoglycemic effect compared with the solvent group, and exhibited a more optimal hypoglycemic effect compared with the Tirzepatide group; on day 10, the Tirzepatide group had a weaker hypoglycemic effect compared with the solvent group, while the compound of the present application still exhibited a strong hypoglycemic effect compared with the solvent group, and exhibited a more optimal hypoglycemic effect compared with the Tirzepatide group, for example, compound 27.

[0327] In addition, the compound of the present application also exhibited a more optimal hypoglycemic effect compared with DB01.

[0328] 5. Hypoglycemic experiment of prodrug compounds in mice

[0329] Experimental method: C57BL / 6 mice, male, purchased from Beijing Huafukang Biotechnology Co., Ltd., were subcutaneously injected with a single dose of solvent or corresponding compound, and intraperitoneally injected with glucose solution (2 g / kg) after overnight fasting on day 6 and day 10. The blood glucose value was measured before the sugar was given (equivalent to 0 min blood glucose value) and 30 min after the glucose was given. The solvent was PBS, and the dose was 0.03 mg / kg (n = 3). The experimental results are shown in Table 4.

[0330] Table 5. Hypoglycemic effect of prodrugs in mice

[0331] The results of the hypoglycemic test of the compound of the present application in mice by single subcutaneous administration showed that on day 6, the compound of the present application exhibited a strong hypoglycemic effect compared with the solvent group, and exhibited a more optimal hypoglycemic effect compared with the Tirzepatide group; on day 10, the Tirzepatide group had a weaker hypoglycemic effect compared with the solvent group, while the compound of the present application still exhibited a strong hypoglycemic effect compared with the solvent group, and exhibited a more optimal hypoglycemic effect compared with the Tirzepatide group, for example, compound 27.

[0332] 6. Pharmacokinetics in rats

[0333] Three healthy male SD rats (Beijing Huafukang Biosciences Co., Ltd.) were selected for each compound, and 0.5 mg / kg of the compound or the compound of the comparative example (the solvent was PBS) was subcutaneously injected, and the volume of the drug was 5 ml / kg. Before and after the administration, 0.5, 1, 2, 4, 8, 24, 48, 72 and 96 h, the whole blood of the rats was collected from the orbital venous plexus, about 200 μL, and placed in a centrifuge tube containing EDTA-K2 anticoagulant, then centrifuged at 3000-4000 rpm for 10 min, and the upper plasma was transferred to another clean centrifuge tube, the blood drug concentration in the plasma sample was detected by LC-MS / MS method, and the pharmacokinetic parameters were calculated by non-compartment model.

[0334] Conclusion: The compound 24 provided by the embodiment of the application can be effectively converted into the original drug component in the rat body, and the half-life is about 2 times of Tirzepatide, which has obvious advantages, is suitable for the development of long-acting preparations, and is shown in FIG. 6.

[0335] 7. Mouse food intake test

[0336] Experimental method: C57BL / 6 mice, male, purchased from Beijing Huafukang Biosciences Co., Ltd., were used for the experiment, and the animals were divided into a solvent control group, a positive control group (Tirzepatide-0.1 mg / kg, DB01-0.1 mg / kg), and a test product group (compound 0.1 mg / kg) according to the weight, and each group contained 6 mice. After grouping, the mice in the solvent group were subcutaneously injected with a single dose of solvent, and the mice in each test product group were given the corresponding test product, and the mice were subcutaneously administered once every 3 days, and the remaining amount of feed was weighed every day.

[0337] Experimental results: Under the same dose (0.1 mg / kg, subcutaneously administered once every 3 days), the compounds 25 and 27 of the application were lower than Tirzepatide and DB01 in the cumulative food intake in 7 days, as shown in FIG. 7.

Claims

1. A polypeptide compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof: wherein X0is selected from Aib, or G; X1is selected from Aib or Y; X2is selected from A or Aib; X3is selected from Q, N or E; Xa is selected from or is absent; Xb is selected from the group consisting of R1is each independently selected from a hydrogen atom or a C1-C6alkyl group; R2is each independently selected from the group consisting of R3is each independently selected from the group consisting of and when Xa is absent, R3 is q is each independently selected from 2, 3, 4 or 5; p is each independently selected from 13, 15, 17 or 19; n is each independently selected from 13, 15, 17 or 19; each L1is independently selected from the group consisting of: Aib is each independently 2. The polypeptide compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof, having a structure according to Formula (I-1): wherein X0is selected from Aib, X1is selected from Aib or Y; X2is selected from A or Aib; X3is selected from Q, N or E; Xb is selected from: R1is each independently selected from a hydrogen atom or a C1-C6alkyl group; R2is each independently selected from the group consisting of R3is each independently selected from the group consisting of and when Xa is absent, R3 is q is each independently selected from 2, 3, 4 or 5; p is each independently selected from 13, 15, 17 or 19; n is each independently selected from 13, 15, 17 or 19; each L1is independently selected from the group consisting of: Preferably, X1is Aib; X3is selected from N or E; Xb is R1is a hydrogen atom; R2 is q is selected from 2 or 3; p is selected from 13 or 15; n is selected from 15 or 17; L1is selected from 3. The polypeptide compound according to claim 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof, selected from the following:

4. The polypeptide compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof, having the structure shown in formula (I-2): wherein X0is selected from Aib, X1is selected from Aib or Y; X2is selected from A or Aib; X3is selected from Q, N or E; Xb is selected from: R1is each independently selected from a hydrogen atom, a methyl group or an ethyl group; R2is each independently selected from: R3is each independently selected from: and when Xa is absent, R3 is q is each independently selected from 2, 3, 4 or 5; p is each independently selected from 13, 15, 17 or 19; n is each independently selected from 13, 15, 17 or 19; each L1is independently selected from the group consisting of: Preferably, X1is Aib; X3is selected from N or E; Xbis R1is a methyl group; R2 is q is 4; p is selected from 13 or 15; n is selected from 15 or 17; L1is selected from 5. The polypeptide compound according to claim 4, or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof, selected from the following:

6. The polypeptide compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof, having the structure shown in formula (I-3): wherein X0is selected from Aib, X1is selected from Aib or Y; X2is selected from A or Aib; X3is selected from Q, N or E; Xb is selected from: R3are each independently n is each independently selected from 13, 15, 17 or 19; Preferably, X1is Aib; X3is selected from N or E; n is selected from 15 or 17.

7. The polypeptide compound or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof according to claim 1, wherein X0is selected from Aib or X1is selected from Aib or Y; X2is A; X3is selected from Q or N; Xais absent; Xbis R3 is N is 17; Aib is each independently 8. The polypeptide compound according to claim 6 or 7, or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof, selected from the following:

9. A pharmaceutical composition comprising a polypeptide compound or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof according to any one of claims 1 to 8, and a pharmaceutically acceptable excipient.

10. Use of a polypeptide compound or a pharmaceutically acceptable salt, stereoisomer, solvate or hydrate thereof according to any one of claims 1 to 8, or of a pharmaceutical composition according to claim 9, for the manufacture of a GLP-1 / GIP receptor dual agonist or for the manufacture of a medicament for the prevention and / or treatment of a disease mediated by GLP-1 receptor and / or GIP receptor.

11. The use according to claim 10, wherein the disease treated and / or prevented by the GLP-1 / GIP receptor dual agonist or the disease mediated by GLP-1 receptor and / or GIP receptor is type I diabetes, type II diabetes, malnutrition-related diabetes, hyperglycemia, diabetic complications, obesity, obesity-related inflammation, non-alcoholic steatohepatitis (NASH), insulin resistance, impaired glucose tolerance, hypertension, hyperlipidemia, arteriosclerosis, coronary heart disease, cerebral infarction, metabolic syndrome, obstructive sleep apnea, heart failure, Parkinson's disease and / or dementia.

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