WO2023207106A1 - Glp-1/gip receptor co-agonist, pharmaceutical composition comprising same, and use thereof - Google Patents

Abstract

Provided are a GLP-1/GIP receptor co-agonist, a pharmaceutical composition comprising the compound, use thereof, and a method for treating and/or preventing a metabolic disease or disorder.

Description

GLP-1/GIP receptor co-agonists, pharmaceutical compositions containing the same and uses thereof Technical field

The present disclosure relates to a GLP-1/GIP receptor co-agonist, pharmaceutical compositions containing the same, and uses and methods for treating and/or preventing metabolic diseases or disorders.

Background technique

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

Glucose-dependent insulinotropic hormone GIP (glucose-dependent insulinotropic) is currently believed to be mainly secreted by enteroendocrine K cells in the duodenum and upper jejunum. Similar to GLP-1, GIP stimulates insulin secretion. The GIP receptor GIPR is widely distributed in the body and is expressed in the pancreas, stomach, small intestine, adipose tissue, heart, and brain tissue. In addition, activating the GIP-GIPR pathway can also exert a weight loss effect. However, the biological activity half-life of GIP in the body is short, less than 2 minutes in mice, 7 minutes in normal people and 5 minutes in patients with type II diabetes.

Glucagon (GCG) is a hormone produced in the α-cells of the pancreas. It acts on the liver under stress conditions such as cold and hunger to decompose glycogen in the liver and increase blood sugar. In addition to its blood sugar-raising effect, GCG also has the effects of promoting lipolysis, fat oxidation, and heating in the body (see Diabetologia, 2017, 60, 1851–1861). Long-term administration can show weight loss effects by increasing energy metabolism. , but these beneficial effects of GCG on energy metabolism have not been widely used due to its inherent blood glucose effect.

Drug developers have developed a series of GLP-1 receptor agonists and GIP receptor agonists. GLP-1 receptor agonists and GIP receptor agonists can exert the same biological effects as natural GLP-1 and GIP, and can also avoid degradation and lose activity, thus extending the duration of action.

However, there is still a need for alternative GLP-1 receptor agonists, particularly co-agonists with co-agonism at GLP-1 receptors and GIP receptors. A co-agonist with co-agonistic properties. In particular, it is desired that the agonist has a good blood glucose lowering effect, especially a simultaneous blood glucose lowering and weight loss effect. It is also desirable that the agonist has high plasma stability and thus pharmacokinetic characteristics that support once-weekly subcutaneous administration in humans.

Contents of the invention

In order to solve the above technical problems, the inventor conducted in-depth research and proposed the technical solution of the present disclosure.

In one aspect, the present disclosure provides a compound of Formula I, or a pharmaceutically acceptable salt thereof:

L ₁ -NH-(CH ₂ ) _n -C(O)-L ₂ -NH ₂ Formula I

in,

L ₁ is a peptide analog of the GIP (1-28) peptide, said L ₁ is a peptide consisting of 28 amino acids, and the amino acid sequence of said L ₁ is at least 39% identical to SEQ ID NO: 1,

L ₂ is a peptide having the amino acid sequence consisting of SEQ ID NO:2,

n is any integer from 2 to 6, and

The compound has GLP-1 receptor agonist activity, GIP receptor agonist activity, or both.

By modifying the peptide chain of GIP(1-28) peptide, especially by replacing the common -NH in the peptide chain with -NH-(CH ₂ ) _n -C(O)-unit (n is an integer from 2 to 6) -(CH ₂ )-C(O)- unit, the obtained compound of formula I unexpectedly maintains high activity on GLP-1 receptors and even has co-agonism on GLP-1 receptors and GIP receptors, providing Effective in lowering blood sugar and reducing weight.

In another aspect, the present disclosure provides a compound of Formula II, or a pharmaceutically acceptable salt thereof:

Tyr ₁ -X ₂ -Glu ₃ -Gly ₄ -X ₅ -X ₆ -Thr ₇ -Ser ₈ -Asp 9 -X ₁₀ -Ser _{11 -X 12} -X ₁₃ -Leu ₁₄ -Asp ₁₅ -X ₁₆ -Ile ₁₇ -X ₁₈ -Gln ₁₉ -Lys ₂₀ -X ₂₁ -Phe ₂₂ -X ₂₃ -X ₂₄ -X ₂₅ -Leu ₂₆ -Ile 27 -Ala _{28 -NH-} (CH ₂ ) ₄ -C(O)-Pro ₃₁ - Ser ₃₂ -Ser ₃₃ -Gly ₃₄ -Ala ₃₅ -Pro ₃₆ -Pro ₃₇ -Pro ₃₈ -Ser ₃₉ -NH ₂

Formula II,

Among them, X ₂ , X _{5 ,} X ₆ , X ₁₀ , X ₁₂ , X ₁₃ , X ₁₆ , X ₁₈ , X ₂₁ , _{X 23} , .

By modifying the peptide chain of the GIP(1-28) peptide, especially by replacing the common -NH-CH ₂ -C(O)- in the peptide chain with -NH-(CH ₂ ) ₄ -C(O)- units. unit, the resulting compound of Formula II unexpectedly retains co-agonism at GLP-1 receptors and GIP receptors, providing hypoglycemic and weight loss effects. At the same time, the compound of formula II has a long half-life, supporting the pharmacokinetic profile of once-weekly subcutaneous injection in humans, which is superior to known drugs administered once-daily subcutaneous injection, thereby improving patient compliance.

In addition, the adverse gastrointestinal irritation of the compounds according to the present disclosure is mild and controllable, so the therapeutic effect can be improved by increasing the dosage.

In another aspect, the present disclosure provides a pharmaceutical composition comprising:

A compound according to the present disclosure or a pharmaceutically acceptable salt thereof, and

Pharmaceutically acceptable carrier, diluent or excipient.

In another aspect, the present disclosure provides the use of a pharmaceutical composition according to the present disclosure or a compound according to the present disclosure or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment and/or prevention of metabolic diseases or disorders .

In yet another aspect, the present disclosure provides a method of treating and/or preventing a metabolic disease or disorder, comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition according to the present disclosure or a compound according to the present disclosure or a pharmaceutical composition thereof. Acceptable salt.

Description of the drawings

Figure 1 is a mass spectrum of compound NBB-T007 according to the present disclosure.

Figure 2 is a high performance liquid chromatogram of compound NBB-T007 according to the present disclosure.

Figure 3 is a potency-concentration curve of semaglutide acting on the target as a control.

Figure 4 is a potency-concentration curve of compound NBB-T007-1 acting on a target according to the present disclosure.

Figure 5 is a potency-concentration curve of compound NBB-T007-2 acting on a target according to the present disclosure.

Figure 6 is a potency-concentration curve of compound NBB-T007-3 acting on a target according to the present disclosure.

Figure 7 is a potency-concentration curve of compound NBB-T007-4 acting on a target according to the present disclosure.

Figure 8 is a potency-concentration curve of compound NBB-T007-5 acting on a target according to the present disclosure.

Figure 9 is a potency versus concentration curve of compound NBB-T007-6 acting on a target according to the present disclosure.

Figure 10 is a potency versus concentration curve of compound NBB-T007-7 acting on a target according to the present disclosure.

Figure 11 is a potency-concentration curve of compound NBB-T007-8 acting on a target according to the present disclosure.

Figure 12 is a potency versus concentration curve of compound NBB-T007-9 acting on a target according to the present disclosure.

Figure 13 is a potency versus concentration curve of compound NBB-T007-10 acting on a target according to the present disclosure.

Figure 14 is a potency versus concentration curve of compound NBB-T007-11 acting on a target according to the present disclosure.

Figure 15 is a potency versus concentration curve of compound NBB-T007-12 acting on a target according to the present disclosure.

Figure 16 is a potency versus concentration curve of compound NBB-T007-14 acting on a target according to the present disclosure.

Figure 17 is a potency versus concentration curve of compound NBB-T007-15 acting on a target according to the present disclosure.

Figure 18 is a potency versus concentration curve of compound NBB-T007 acting on a target according to the present disclosure.

Figure 19 is a potency versus concentration curve of compound NBB-T007-17 acting on a target according to the present disclosure.

Figure 20 is a potency versus concentration curve of compound NBB-T007-18 acting on a target according to the present disclosure.

Figure 21 is a potency versus concentration curve of compound NBB-T007-19 acting on a target according to the present disclosure.

Figure 22 is a potency versus concentration curve of compound NBB-T007-21 acting on a target according to the present disclosure.

Figure 23 is a potency versus concentration curve of compound NBB-T007-22 acting on a target according to the present disclosure.

Figure 24 is a potency versus concentration curve of compound NBB-T007-23 acting on a target according to the present disclosure.

Figure 25 is a potency versus concentration curve of compound NBB-T007-24 on a target according to the present disclosure.

Figure 26 is a potency versus concentration curve of compound NBB-EX4 on a target according to the present disclosure.

Figure 27 is the blood glucose-time change curve of the db/db mice tested.

Figure 28 is the body weight-time change curve of the db/db mice tested.

Figure 29 is the blood glucose reduction percentage-time curve of the db/db mice tested.

Figure 30 is the blood glucose-time change curve of normal mice tested.

Figure 31 is a body weight-time change curve of normal mice tested.

Figure 32 is a concentration-time change curve in plasma of the compound NBB-T007 according to the present disclosure injected subcutaneously in the test SD rats.

Figure 33 is a concentration-time change curve of semaglutide in plasma injected subcutaneously as a control in SD rats.

Figure 34 is a concentration-time change curve in plasma of NBB-T007-10 according to the present disclosure injected subcutaneously into SD rats.

Figure 35 is a concentration-time change curve in plasma of NBB-T007-12 according to the present disclosure injected subcutaneously in SD rats.

Figure 36 is the body weight-time change curve of the DIO mice tested.

Figure 37 is the blood glucose-time change curve of the DIO mice tested.

Figure 38 is the blood glucose-time change curve of the DIO mice tested.

Figure 39(a) is a bar graph showing changes in serum insulin content of DIO mice.

Figure 39(b) is a histogram of changes in serum biochemical indicators (UREA, TG, CHO, HDL, LDL and CREA) of the DIO mice tested.

Figure 39(c) is a histogram of changes in serum biochemical indicators (ALT, AST, ALB and TBIL) of the DIO mice tested.

Figure 40(a) is a bar graph showing changes in body fat percentage of DIO mice tested.

Figure 40(b) is a bar graph showing changes in triglyceride content in DIO mice.

Detailed ways

[definition]

As used herein, "analog" means a compound, such as a natural or synthetic peptide or polypeptide, that activates a target receptor and elicits at least one in vivo or in vitro effect of an agonist on that receptor.

As used herein, the sequence or structural formula of a compound contains the standard one-letter or three-letter codes for the 20 natural amino acids that make up proteins (also known as proteinogenic amino acids or encoded amino acids). Except for proline (Pro), the amino and carboxyl groups of the remaining 19 protein amino acids are connected to the α carbon atom, also known as α-amino acids. For example, α-Ala represents (α-)alanine, with the structure CH ₃ CH(NH ₂ )COOH, in which the amino group is attached to the α carbon atom. β-Ala represents β-alanine, and its structural formula is NH ₂ CH ₂ CH ₂ COOH, in which the amino group is connected to the β carbon atom.

As used herein, each amino acid residue can be in the L-configuration or the D-configuration independently of each other, and can also have pendant substituents on the carbon atoms independently of each other.

As used herein, a compound may also contain unnatural amino acid residues in its sequence or structural formula. For example, Aib represents 2-aminoisobutyric acid residue; homo-Phe represents homophenylalanine residue; Cpa-Ala represents p-chlorophenylalanine residue; Fpa5-Ala represents pentafluorophenylalanine Residue; Na1 represents 1-naphthylalanine residue.

In this context, a compound of formula I or formula II and "polypeptide" are synonymous and may be used interchangeably unless clearly contradicted.

In this context, unless otherwise stated or clearly contradicted, the position numbering of amino acids is calculated from the leftmost N-terminus of the peptide chain or structural formula of the compound. For example, taking the compound of formula II as an example, the N-terminal amino acid is tyrosine (Tyr) at position 1, and the C-terminal amino acid is serine (Ser) at position 39. In the compounds of formula II, the -NH-( _CH2 ) ₄ -C(O)- unit is not a residue of the natural alpha-amino acid, nor is it 2 amino acid residues, but only 1 amino acid residue. However, for convenience, the -NH-( _CH2 ) ₄ -C(O)- units are formally numbered herein as occupying 2 positions at amino acid positions 29 and 30.

As used herein, an "individual in need thereof" means a mammal, preferably a human, but also a non-human animal, including non-human primates (e.g., monkeys), having a condition, disease, disorder or symptom in need of treatment or prevention. , cynomolgus monkeys, etc.), pets (such as cats, dogs, etc.), livestock (such as cattle, sheep, pigs, horses, etc.) and rodents (such as rats, mice, guinea pigs, etc.).

As used herein, an "effective amount" means one that provides the desired effect (i.e., can produce a clinically measurable difference in the condition of the individual) in the individual being diagnosed or treated upon administration of single or multiple doses to an individual in need thereof. , such as, for example, a reduction in blood glucose, and/or a reduction in weight or fat), an amount, concentration, or dose of one or more compounds of the present disclosure or a pharmaceutically acceptable salt thereof. Effective amounts are readily determined by those skilled in the art using known techniques and by observation of results obtained under similar circumstances. In determining the amount effective for an individual, many factors are considered, including, but not limited to, the species of the mammal, size, age, general health, the specific disease or condition involved, the severity of the disease or condition, the individual's response, The specific compound administered, the mode of administration, the bioavailability characteristics of the formulation administered, the dosage regimen selected, the use of concomitant drug therapy, and other relevant circumstances.

As used herein, the term "treating" means attenuating, inhibiting, reversing, slowing or stopping the progression or severity of an existing condition, disease, disorder or symptom.

As used herein, the term "C ₁₂ -C ₂₄ aliphatic diacid" means a linear or branched dicarboxylic acid having 12 to 24 carbon atoms. In one embodiment, C ₁₂ -C ₂₄ aliphatic diacids suitable for the present disclosure may be saturated or unsaturated diacids, with saturated diacids being preferred. _C12 - _C24 fatty acids suitable for compounds of the present disclosure include, but are not limited to, dodecanedioic acid ( _C12dioic acid), tridecanedioic acid ( _C13dioic acid), tetradecanedioic acid ( _C14dioic acid). acid), pentadecanedioic acid (C ₁₅ diacid), hexadecanedioic acid (C ₁₆ diacid), heptadecanedioic acid (C ₁₇ diacid), octadecanedioic acid (C ₁₈ diacid) , nonadecanedioic acid (C ₁₉ diacid), eicosanedioic acid (C ₂₀ diacid), undecanedioic acid (C ₂₁ diacid), behenedioic acid (C ₂₂ diacid) , tricosanedioic acid (C ₂₃ diacid), tetracosanedioic acid (C ₂₄ diacid), and their branched and/or substituted derivatives.

As used herein, the term "plasma half-life" or "half-life" refers to the time required for half of the compound of interest to be cleared from the plasma.

As used herein, "in vitro activity" refers to an indicator of a peptide's ability to activate GLP-1 receptors, GIP receptors, and/or GCG receptors in cell-based assays. In vitro activity is expressed as the "half maximal effective concentration ( _EC50 )", which is the effective concentration of compound that results in 50% activity in a single dose response experiment. As used herein, " _EC50 " means the effective concentration of a compound that results in 50% activation/stimulation of an assay endpoint, such as a dose-response curve (eg, cAMP).

In the context of this article, semaglutide refers to a chemically synthesized GLP-1 analog having the structure shown below:

In the context of the present invention, Tirzepatide (TZP) is a GLP-1/GIP receptor co-agonist.

[GLP-1/GIP receptor co-agonist]

The present disclosure provides compounds of Formula I, or a pharmaceutically acceptable salt thereof:

L ₁ -NH-(CH ₂ ) _n -C(O)-L ₂ -NH ₂ Formula I

in,

L ₁ is a peptide analog of the GIP (1-28) peptide, said L ₁ is a peptide consisting of 28 amino acids, and the amino acid sequence of said L ₁ is at least 39% identical to SEQ ID NO: 1,

L ₂ is a peptide having the amino acid sequence consisting of SEQ ID NO:2,

n is any integer from 2 to 6, and

The compound has GLP-1 receptor agonist activity, GIP receptor agonist activity, or both.

SEQ ID NO:1 is the GIP (1-28) sequence: YAEGTFISDYSIAMDKIHQQDFVNWLLA, namely Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile -His-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala.

SEQ ID NO:2 is PSSGAPPPS, which is Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser.

"The amino acid sequence of L ₁ has at least 39% identity with SEQ ID NO: 1" means that compared with SEQ ID NO: 1, L ₁ has a total of at least 28 amino acids from positions 1 to 28. 11 positions have the same amino acid, for example at a total of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 positions have the same amino acids.

In some examples, the amino acid sequence of L ₁ has identical amino acids at a total of 11, 18, 19, or 20 positions compared to SEQ ID NO: 1.

In some examples, the amino acid sequence of L ₁ is at least 39% identical to SEQ ID NO: 1, for example, has 39%, 43%, 46%, 50%, 54%, 57%, 61%, 64% , 68%, 71%, 75%, 79%, 82%, 86%, 89%, 93%, 96% or 100% identical.

In some examples, the amino acid sequence of L ₁ is 39%, 64%, 68%, or 71% identical to SEQ ID NO:1.

In some examples, n is 2, 3, 4, 5 or 6, with 4 being preferred.

By modifying the peptide chain of GIP(1-28) peptide, especially by replacing the common -NH in the peptide chain with -NH-(CH ₂ ) _n -C(O)-unit (n is an integer from 2 to 6) -(CH ₂ )-C(O)- unit, the resulting compound of formula I can unexpectedly maintain agonism on GIP receptors, and even have co-agonism on GLP-1 receptors and GIP receptors, providing reduced Efficacy in blood sugar and weight loss.

In some examples, L ₁ contains a Y1H substitution compared to SEQ ID NO: 1.

In some examples, compared to SEQ ID NO: 1, L ₁ includes a substitution selected from A2G, A2(Aib), and A2(β-Ala).

In some examples, L ₁ contains a T5S substitution compared to SEQ ID NO: 1.

In some examples, L ₁ includes a substitution selected from F6 (homo-Phe) and F6 (Cpa-Ala) compared to SEQ ID NO: 1.

In some examples, L ₁ contains an I7T substitution compared to SEQ ID NO: 1.

In some examples, compared to SEQ ID NO: 1, L ₁ includes a substitution selected from Y10L and Y10(Fpa5-Ala).

In some examples, L ₁ contains a I12K substitution compared to SEQ ID NO: 1.

In some examples, compared to SEQ ID NO: 1, L ₁ includes a substitution selected from A13Q, A13(Aib), and A13Y.

In some examples, L ₁ includes a substitution selected from M14L and M14(α-meL) compared to SEQ ID NO: 1.

In some examples, L ₁ contains a D15E substitution compared to SEQ ID NO: 1.

In some examples, L ₁ includes a substitution selected from K16E and K16(Ala) compared to SEQ ID NO: 1.

In some examples, L ₁ contains an I17E substitution compared to SEQ ID NO: 1.

In some examples, L ₁ includes a substitution selected from H18A and H18(Aib) compared to SEQ ID NO:1.

In some examples, L ₁ contains the Q19V substitution compared to SEQ ID NO: 1.

In some examples, L ₁ includes a substitution selected from Q20R and Q20K when compared to SEQ ID NO: 1.

In some examples, L ₁ includes a substitution selected from D21L, D21A, D21E, and D21(Abu) compared to SEQ ID NO:1.

In some examples, L ₁ includes a substitution selected from V23I and V23L compared to SEQ ID NO: 1.

In some examples, L ₁ includes a substitution selected from N24E and N24Q compared to SEQ ID NO: 1.

In some examples, L ₁ includes a substitution selected from W25(Na1) and W25(2-me-Trp) compared to SEQ ID NO:1.

In some examples, L ₁ includes a substitution selected from L27K and L27I compared to SEQ ID NO: 1.

In some examples, L ₁ contains an A28N substitution compared to SEQ ID NO: 1.

The present disclosure also provides compounds of the following formula XXI or pharmaceutically acceptable salts thereof:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula XXI.

By modifying the peptide chain of the GIP(1-28) peptide, especially by replacing the common -NH-CH ₂ -C(O)- in the peptide chain with -NH-(CH ₂ ) ₄ -C(O)- units. unit, the resulting compound of formula XXI unexpectedly retains agonism at the GLP-1 receptor, providing hypoglycemic and weight loss effects.

In some examples, in Formula I, when the amino acid at position 16 or 20 is lysine (Lys), the C ₁₂ -C ₂₄ aliphatic diacid is conjugated to the acid via a direct bond or via a linker. The ε-amino group of the lysine (Lys) side chain is chemically modified, and the linker is selected from (AEEA) ₂ -(γ-Glu) _a , AEEA-Ahx-(γ-Glu) _a , (Ahx) ₂ - (γ-Glu) _a and (β-Ala) ₂ -(γ-Glu) _a , where a is 1 or 2;

Preferably, the chemical modification is carried out by conjugating eicosanedioic acid to the ε-amino group of the lysine (Lys) side chain at position 16 or 20 via (AEEA) ₂ -(γ-Glu); or

_{Eicosanedioic} acid is conjugated to _lysine 20 ( Lys) side chain of the ε-amino group is chemically modified.

In some examples, in Formula I, histidine (His) or tyrosine (Tyr) at position 1 is amidated, e.g., linked to by -C _m H _2m+1 -C(O)- On the amino group of the histidine (His) or tyrosine (Tyr), and m is an integer from 1 to 20;

Preferably, the amino group of tyrosine (Tyr) at position 1 is amidated by being connected to CH ₃ -C(O) or to C ₁₉ H ₃₉ -C(O)-.

In some examples, in Formula I, the α-carbon atoms of any two amino acids can be connected to form a ring via a direct bond or via a linker selected from an alkyl or alkenyl group containing 2 to 20 carbon atoms. base;

Preferably, when the amino acids at positions 13 and 16 are both alanine, the α-carbon atom of alanine (Ala) at position 13 and alanine at position 16 are connected via an alkenyl group containing 10 carbon atoms. The α-carbon atoms of (Ala) are attached.

The present disclosure also provides compounds of Formula II below, or pharmaceutically acceptable salts thereof:

Tyr ₁ -X ₂ -Glu ₃ -Gly ₄ -X ₅ -X ₆ -Thr ₇ -Ser ₈ -Asp 9 -X ₁₀ -Ser _{11 -X 12} -X ₁₃ -Leu ₁₄ -Asp ₁₅ -X ₁₆ -Ile ₁₇ -X ₁₈ -Gln ₁₉ -Lys ₂₀ -X ₂₁ -Phe ₂₂ -X ₂₃ -X ₂₄ -X ₂₅ -Leu ₂₆ -Ile 27 -Ala _{28 -NH-} (CH ₂ ) ₄ -C(O)-Pro ₃₁ - Ser ₃₂ -Ser ₃₃ -Gly ₃₄ -Ala ₃₅ -Pro ₃₆ -Pro ₃₇ -Pro ₃₈ -Ser ₃₉ -NH ₂

Formula II,

Among them, X ₂ , X _{5 ,} X ₆ , X ₁₀ , X ₁₂ , X ₁₃ , X ₁₆ , X ₁₈ , X ₂₁ , _{X 23} , .

By modifying the peptide chain of the GIP(1-28) peptide, especially by replacing the common -NH-CH ₂ -C(O)- in the peptide chain with -NH-(CH ₂ ) ₄ -C(O)- units. unit, the resulting compound of Formula II unexpectedly retains co-agonism at GLP-1 receptors and GIP receptors, providing hypoglycemic and weight loss effects. At the same time, the compound of formula II has a long half-life, supporting the pharmacokinetic profile of once-weekly subcutaneous injection in humans, which is superior to known drugs administered once-daily subcutaneous injection, thereby improving patient compliance.

In some examples, X is an amino acid residue selected from ₂ -aminoisobutyric acid (Aib) and (β-Ala),

X ₅ is an amino acid residue selected from Thr and Ser,

X ₆ is an amino acid residue selected from Phe, homophenylalanine (homo-Phe) and p-chlorophenylalanine (Cpa-Ala),

X ₁₀ is an amino acid residue selected from Tyr and pentafluorophenylalanine (Fpa5-Ala),

X ₁₂ is an amino acid residue selected from Ile and Lys,

X ₁₃ is an amino acid residue selected from 2-aminoisobutyric acid (Aib), Tyr and Ala,

X ₁₆ is an amino acid residue selected from Lys and Ala,

X ₁₈ is an amino acid residue selected from Ala and Aib,

X ₂₁ is an amino acid residue selected from Ala, Glu and 2-aminobutyric acid (Abu),

X ₂₃ is an amino acid residue selected from Val and Leu,

X ₂₄ is an amino acid residue selected from Gln and Asn, and

X ₂₅ is an amino acid residue selected from Trp, 2-methyltryptophan (2-me-Trp) and 1-naphthylalanine (Nal).

In some examples, the compound has a structure selected from any one of Formula III below to Formula XX below:

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Ala-Leu-Asp-Ala-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln- Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula III,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula IV,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Tyr-(α-meL)-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe- Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula V,

Tyr-(β-Ala)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe- Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula VI,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Glu-Phe-Val- Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula VII,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Glu-Phe-Val- Asn-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula VIII,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Asn-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula IX,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Asn-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula X,

Tyr-(Aib)-Glu-Gly-(D-Thr)-(D-Phe)-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln- Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser -NH ₂

Formula XI,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-(D-Ala)-Pro-Pro-Pro-Ser-NH ₂

Formula XII,

Tyr-(Aib)-(D-Glu)-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala- Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula XIII,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Tyr-(α-meL)-Asp-Lys-Ile-(Aib)-Gln-Lys-Ala- Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula XIV,

Tyr-(Aib)-Glu-Gly-Thr-(homo-Phe)-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala- Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula XV,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-(Abu)-Phe- Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula XVI,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Gln-(2-me-Trp)-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula XVII,

Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Gln-(Nal)-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula XVIII,

Tyr-(Aib)-Glu-Gly-Ser-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Leu- Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Formula XIX, and

Tyr-(Aib)-Glu-Gly-Thr-(Cpa-Ala)-Thr-Ser-Asp-(Fpa5-Ala)-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln- Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser -NH ₂

Formula XX.

In some examples, in any of Formulas I to XXI, each amino acid residue is in the L-configuration or the D-configuration independently of one another.

In some examples, in any one of Formulas I to XXI, each amino acid residue is in the L-configuration independently of one another.

In some examples, in any one of Formulas I to XXI, there is at least 1 D-configured amino acid residue, such as 1-5, such as 1 or 2 D-configured amino acid residues. base, such as D-Glu, D-Thr, D-Phe and/or D-Ala, etc.; the remaining amino acid residues are in L-configuration.

In some examples, in Formula I or Formula II, the amino acid at position 3 is L-Glu or D-Glu.

In some examples, in Formula I or Formula II, the amino acid at position 5 is L-Thr or D-Thr.

In some examples, in Formula I or Formula II, the amino acid at position 6 is L-Phe or D-Phe.

In some examples, in Formula I or Formula II, the amino acid at position 35 is L-Ala or D-Ala.

In some examples, in any of Formulas I to XXI, the carbon atoms of each amino acid residue can have pendant substituents independently of each other. There are no particular limitations on the substituents as long as they do not affect the desired properties of the compounds according to the present disclosure. In some examples, the substituents are each independently selected from linear, branched or cyclic, saturated or unsaturated aliphatic groups, or aromatic groups. Optionally, the substituents may be further substituted. In some examples, the substituent is, for example, C ₁ -C ₂₀ alkyl, such as methyl; C ₂ -C ₂₀ alkenyl, such as pentenyl or deenyl; substituted or unsubstituted phenyl, such as p- Chlorophenyl, pentafluorophenyl; and/or substituted or unsubstituted condensed ring aryl, such as 1-naphthyl. Substituted amino acid residues include leucine residues in which the α carbon atom is replaced by methyl group (α-meL), tryptophan residues in which the 2-position carbon atom is replaced by methyl group (2-me-Trp), and p-chlorophenyl-substituted alanine residue (Cpa-Ala), and pentafluorophenyl-substituted alanine residue (Fpa5-Ala).

In some examples, in Formula I or Formula II, the amino acid at position 14 is a leucine residue in which the alpha carbon atom is replaced with a methyl group (α-meL).

In some examples, in Formula I or Formula II, the amino acid at position 25 is a tryptophan residue in which the 2-carbon atom is replaced by a methyl group (2-me-Trp).

When used herein, the term "C ₁₂ -C ₂₄ aliphatic diacid conjugated to" an amino acid refers to any natural or unnatural amino acid that has the ability to bind by covalent bond, or preferably by linker. A functional group conjugated to the aliphatic diacid. Examples of conjugated functional groups of amino acids include amino, carboxyl, chlorine, bromine, iodine, azide, alkynyl, alkenyl and thiol, with amino being preferred. Examples of natural amino acids including such functional groups include lysine K (having an amino group), cysteine C (having a thiol group), glutamic acid E (having a carboxyl group), and aspartic acid D (having a carboxyl group).

In some examples, the conjugated amino acid is lysine K. In such embodiments, conjugation refers to conjugation to the epsilon-amino group of the K side chain of lysine.

In some examples, the conjugation is acylation.

In some examples, compounds of the invention include an aliphatic diacid moiety conjugated to the epsilon-amino group of the K side chain of lysine at position 16 or 20 via a linker.

In some examples, compounds of the invention include an aliphatic diacid moiety that is conjugated directly, without a linker, to a natural or non-natural amino acid with functional groups available for conjugation.

In some examples, in any of Formula II to Formula XX, at the lysine (Lys) at position 16 or 20, a C ₁₂ -C ₂₄ aliphatic diacid is added via a direct bond or via a linker Conjugated to the ε-amino group of the lysine (Lys) side chain for chemical modification, the linker is selected from (AEEA) ₂ -(γ-Glu) _a , AEEA-Ahx-(γ-Glu) _a , (Ahx) ₂ -(γ-Glu) _a and (β-Ala) ₂ -(γ-Glu) _a , where a is 1 or 2, endow the compound with excellent in vivo and in vitro activity. In some examples, the linker is selected from (AEEA) ₂ -(γ-Glu), AEEA-Ahx-(γ-Glu), (Ahx) ₂ -(γ-Glu), and (β-Ala) ₂ -(γ- Glu).

When referring to linkers in this context, AEEA is an abbreviation for [2-(2-amino-ethoxy)-ethoxy]-acetyl, which means [2-(2-amino-ethoxy)-ethoxy]-acetyl. . γ-Glu represents γ-glutamyl. Ahx is the abbreviation of amino hexanoyl, which means aminocaproyl. β-Ala represents β-alanyl group.

In some examples, in any one of Formula II to Formula XX, via the linker (AEEA) ₂ -(γ-Glu), AEEA-Ahx-(γ-Glu), (Ahx) ₂ -(γ-Glu ) or (β-Ala) ₂ -(γ-Glu), by conjugating octadecanedioic acid or eicosanedioic acid to the ε-amino group of the lysine (Lys) side chain at position 16 or 20 Perform chemical modifications.

In some examples, in any of Formula III and Formula V to Formula XX, eicosanedioic acid is conjugated to lysine (Lys) at position 20 via (AEEA) ₂ -(γ-Glu) Chemical modification of the ε-amino group of the side chain.

In some examples, in Formula IV, chemical modification is performed by conjugating eicosanedioic acid to the epsilon-amino group of the lysine (Lys) side chain at position 16 via (AEEA) _2- (γ-Glu).

In some examples, in Formula IV, chemical modification occurs by conjugating eicosanedioic acid to the epsilon-amino group of the lysine (Lys) side chain at position 20 via (Ahx) _2- (γ-Glu).

In some examples, in Formula IV, eicosanedioic acid is chemically modified by conjugating eicosanedioic acid to the epsilon-amino group of the lysine (Lys) side chain at position 20 via AEEA-Ahx-(γ-Glu).

In some examples, in Formula IV, the chemistry is performed by conjugating eicosanedioic acid to the epsilon-amino group of the lysine (Lys) side chain at position 20 via (β-Ala) _2- (γ-Glu) Grooming.

Taking compound NBB-T007-12 (corresponding to formula XVI) shown below as an example, at the lysine at position 20, the first [2-(2-amino-ethoxy)-ethoxy group The ]-acetyl (AEEA) unit is linked to the ε-amino group of the lysine side chain via the acyl group, and the second [2-(2-amino-ethoxy)-ethoxy]-acetyl (AEEA) unit is via The acyl group is connected to the amino group of the first [2-(2-amino-ethoxy)-ethoxy]-acetyl (AEEA) unit, and the γ-glutamyl (γ-Glu) group is connected to the second through the γ-acyl group. The amino group of [2-(2-amino-ethoxy)-ethoxy]-acetyl (AEEA) unit is connected, and one terminal carboxyl group of eicosanedioic acid (C ₂₀ diacid) removes the hydroxyl group to obtain the terminal acyl group , and is connected to the amino group of γ-glutamyl (γ-Glu) via the terminal acyl group, thereby chemically modifying the ε-amino group of the lysine (Lys) side chain at position 20.

According to the present disclosure, the use of a linker to conjugate a C ₁₂ -C ₂₄ aliphatic diacid to the epsilon-amino group of the lysine (Lys) side chain of compounds of Formulas I to XXI helps provide the compounds with GLP-1 receptors and GIP receptors, and offers the potential to generate long-acting compounds.

In some examples, in any of Formula II to Formula XX, the histidine (His) or tyrosine (Tyr) at position 1 (i.e., the N-terminus) is amidated, for example, by- C _m H _2m+1 -C(O)- is linked to the amino group of the histidine (His) or tyrosine (Tyr) for amidation, and m is an integer from 1 to 30. In some examples, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29 or 30.

In some instances, m is 1 or 19. Preferably, in formula IV, the amino group of tyrosine (Tyr) at position 1 is amidated by being connected to CH ₃ -C(O)- or to C ₁₉ H ₃₉ -C(O)-, respectively, as in the following compounds T007-19 (acetylation) and T007-20 (eicosanoylation) are shown.

In some examples, in any one of Formula I to Formula XXI, the α-carbon atoms of any two amino acids can be connected to form a ring via a direct bond or via a linker selected from the group consisting of 2 to 20 Alkyl or alkenyl group of carbon atoms. This side chain modification is also called "staple peptide" modification, which is used to enhance the structural rigidity of the polypeptide and stabilize the activity of the polypeptide compound. When the linking group is an alkenyl group containing 2 to 20 carbon atoms, the linking group can be introduced by using a Grubbs catalyst. The ring-forming α-carbon atoms may come from the side chains of two adjacent amino acids or from the side chains of two amino acids separated by at least 1 amino acid. The linking group is an alkyl group containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms or alkenyl. In some examples, the linker is an alkenyl group containing 10 carbon atoms. In some examples, in Formula III, the alpha-carbon atom of alanine (Ala) at position 13 is connected to the alpha-carbon atom of alanine (Ala) at position 16 via an alkenyl group containing 10 carbon atoms. .

In some examples, in any one of Formula II to Formula XX, the side chain carboxyl group of aspartic acid (Asp) or glutamic acid (Glu) is in contact with lysine (Lys), arginine (Arg) Or the side chain amino group of histidine (His) can form a ring by forming an amide bond.

[Preparation method of GLP-1/GIP receptor co-agonist]

Compounds according to the present disclosure were prepared via a solid-phase synthesis method using Rink-amide-AM resin (Tianjin Nankai Hecheng Technology Co., Ltd.) as a synthetic carrier. The amino groups of each amino acid raw material used in the synthesis process are protected by the Fmoc group (9-fluorenyl-methyloxycarbonyl, Fmoc). For neutral polar amino acids, acidic amino acids and basic amino acids, according to the different side chain functional groups, select appropriate protective groups to protect the polar side chains of these amino acid raw materials. For example, but not limited to, the side chain thiol group of cysteine (Cys), the side chain amino group of glutamine (Gln), the side chain imidazolyl group of histidine (His), and the side chain amino group of asparagine (Asn). It is protected by Trt (trityl); the side chain guanidine group of arginine (Arg) is protected by Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) protection; the side chain indole group of tryptophan (Trp), the side chain hydroxyl group of serine (Ser) and the side chain amino group of lysine (Lys) It is protected by Boc (tert-butoxycarbonyl protecting group, tert-butoxycarbonyl); the side chain hydroxyl group of threonine (Thr) and the side chain phenol group of tyrosine (Tyr) are protected by tBu (tert-butyl group, tert-butyl) Protection; the side chain carboxyl groups of aspartic acid (Asp) and glutamic acid (Glu) are protected by OtBu (tert-butoxy).

In some examples, compounds according to the present disclosure are prepared by a method comprising:

i) Swelling and deprotection of resin carrier

The Rink-Amide-AM resin resin previously protected by Fmoc is swollen, and then the Fmoc protecting group of the Rink-Amide-AM resin resin is removed with a solution of N,N-dimethylformamide (DMF) containing 20% piperidine.

ii) Formation of polypeptides

Starting from the rightmost C terminus of the compound structural formula and moving toward the leftmost N terminus, connect the amino acids one by one in the following manner to form a polypeptide, in which the amide bond is formed through a condensation reaction with the carboxyl group of the amino acid whose amino group is protected by Fmoc:

First, the condensation agent 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3 , 3-tetramethyluronium hexafluorophosphate, HCTU), the carboxyl group of the first amino acid with Fmoc protection (from the rightmost C end) is condensed in the form of an amide bond onto the swollen and deprotected Rink-amide-AM resin. , then use N,N-dimethylformamide (DMF) solution containing 20% piperidine to remove the Fmoc protecting group on the amino group, and wash.

Then, in a similar manner, the condensing agent HCTU is used, and the 2nd to 9th amino acids (from the rightmost C end) protected by Fmoc, 5-aminovaleric acid (Fmoc-NH-() protected by Fmoc are respectively used CH ₂ ) ₄ -C(O)OH), and the Fmoc-protected amino acid from position 12 to position 39 (from the rightmost C end), repeat the previous coupling reaction to form an amide bond and remove Fmoc protection in sequence. cycle of base deprotection and cleaning.

iii) Deprotection and chemical modification of lysine at position 16 or 20 (from the leftmost N-terminus)

Use tetrakis triphenylphosphine palladium Pd(PPh ₃ ) ₄ to remove the Boc protecting group on the ε-amino group of lysine at position 16 or 20 (from the leftmost N end) and wash.

Then, use an Fmoc-protected linker, such as Fmoc-AEEA, Fmoc-Ahx, or Fmoc-β-Ala; and a condensing agent (HCTU), for lysine 16 or 20 (from the leftmost N-terminus) The ε-amino group is coupled and deprotected. In a similar manner, repeat this coupling and deprotection operation once to connect the second linker AEEA, Ahx, or β-Ala.

Then, Fmoc and OtBu protected glutamic acid (Fmoc-Glu(OtBu)-OH) and condensing agent (HCTU) are used to couple with the amino group of the second linker and deprotect, thereby connecting the third linker γ- Glu.

Then, C ₁₂ -C ₂₄ aliphatic diacid or its derivative (for example, monotert-butyl ester of the diacid) and a condensing agent (HCTU) are used to couple with the amino group of γ-Glu, thereby connecting C ₁₂ -C ₂₄ aliphatic diacid.

iv) Cleavage and characterization of polypeptides

A mixture of trifluoroacetic acid (TFA, trifluoroacetic acid)/water/phenol/triisopropylsilane (Tips, Triisopropyl silane) is used as the cleavage reagent. The cleavage reagent reacts with Rink-Amide-AM resin to remove the polypeptide from the resin. cleaved from the resin carrier. Precipitate from frozen ice ether to obtain the crude polypeptide solid product. The solid crude polypeptide is centrifuged, dried and crushed to obtain the purified polypeptide.

The purified peptides were subjected to electrospray mass spectrometry to determine the molecular structure, and high performance liquid chromatography was used to analyze the purity of the purified peptides.

[Pharmaceutical composition]

The present disclosure provides a pharmaceutical composition comprising:

A compound according to the present disclosure or a pharmaceutically acceptable salt thereof, and

Pharmaceutically acceptable carrier, diluent or excipient.

In addition to one or more compounds according to the present disclosure, or pharmaceutically acceptable salts thereof, pharmaceutical compositions may also contain other components, such as physiologically/pharmaceutically acceptable carriers, diluents and excipients, to facilitate administration to an individual The administration of the drug facilitates the absorption of the compound as the active ingredient or its pharmaceutically acceptable salt and thereby exerts biological activity.

"Pharmaceutically acceptable salts" refer to salts of compounds according to the present disclosure that are safe and effective when administered in vivo and are biologically active.

Compounds according to the present disclosure can react with a variety of inorganic and organic acids to form pharmaceutically acceptable salts. Pharmaceutically acceptable salts and common methods for preparing them are well known in the art. See, for example, P. Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd revised edition (Wiley-VCH, 2011); S. M. Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, Vol. 66, No.1, January 1977.

In some examples, a compound according to the present disclosure, or a pharmaceutically acceptable salt thereof, may be formulated as a pharmaceutical composition for administration by parenteral routes (eg, subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). Such pharmaceutical compositions and methods for their preparation are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy (ed. D.B. Troy, 21st ed., Lippincott, Williams & Wilkins, 2006).

Pharmaceutically acceptable salts according to the present disclosure include, but are not limited to, trifluoroacetate, hydrochloride, and acetate.

[use]

The present disclosure provides the use of a pharmaceutical composition according to the present disclosure or a compound according to the present disclosure or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment and/or prevention of metabolic diseases or disorders;

In particular, said metabolic diseases or disorders include diabetes and diabetes-related conditions, and obesity and obesity-related conditions;

In particular, the diabetes and diabetes-related conditions include insulin resistance, glucose intolerance, elevated fasting glucose, prediabetes, type I diabetes, type II diabetes mellitus (T2DM), gestational diabetes hypertension, dyslipidemia, atherosclerosis sclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease, and atherogenic dyslipidemia, dyslipidemia, elevated blood pressure, hypertension, prothrombotic and proinflammatory states, and combinations thereof;

In particular, obesity and obesity-related conditions include obesity-associated inflammation, obesity-associated gallbladder disease, obesity-induced sleep apnea, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and combinations thereof.

In some examples, drugs of the present disclosure are used to treat type II diabetes.

[Methods to treat and/or prevent metabolic diseases or disorders]

The present disclosure provides a method of treating and/or preventing a metabolic disease or disorder, comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition according to the present disclosure or a compound according to the present disclosure or a pharmaceutically acceptable salt thereof .

In some examples, the pharmaceutical composition, the compound, or a pharmaceutically acceptable salt thereof is administered to the subject by subcutaneous injection.

In some examples, the pharmaceutical composition, the compound, or a pharmaceutically acceptable salt thereof is administered to the subject once weekly.

In some examples, the pharmaceutical composition, the compound, or a pharmaceutically acceptable salt thereof is administered to the subject once weekly by subcutaneous injection.

[Example]

[Preparation Example]

Experimental reagents:

Unless otherwise stated, N,N-dimethylformamide (DMF) and dichloromethane (DCM) used in this article are common reagents with a purity of 99.7%, and the excipients are 0.05% NaHCO ₃ solution. The 20% piperidine solution refers to the volume percentage, which can be obtained, for example, by the following method: measure 100 mL of piperidine with a graduated cylinder, and add DMF to the graduated cylinder scale of 500 mL.

Unless otherwise stated, in the embodiments of the present disclosure, draining the solvent means, for example, using an air pump to pump the solvent in the polypeptide synthesis tube into a filter bottle, and draining the solvent until the resin becomes dry powder.

[Preparation of compound NBB-T007]

(1) Resin swelling

a) Weigh 0.63g (0.2mmol) of Fmoc-protected Rink-Amide-AM resin, weigh it and put it into the peptide synthesis tube.

b) Add 10 mL of DMF (N,N-dimethylformamide) and 10 mL of DCM (dichloromethane) to the peptide synthesis tube and leave it at room temperature for 30 minutes.

c) Use an air pump to drain the solvent.

d) Rinse with 10mL DMF and drain the solvent.

(2) Resin deprotection

a) Add 10 mL of 20% piperidine solution to the peptide synthesis tube to submerge the swollen resin obtained in step (1), and transfer to a 33°C constant-temperature shaker for 5 minutes.

b) Take out the peptide synthesis tube from the shaker.

c) Cleaning: First rinse the resin three times with DMF (10 mL each time) and drain the solvent; then rinse the resin three times with DCM (10 mL each time) and drain the solvent; finally rinse the resin three times with DMF (10 mL each time) and drain it. Solvent.

d) Deprotection: Add 10 mL of 20% piperidine solution to the peptide synthesis tube, shake in a constant temperature shaker at 33°C for 10 minutes, and take out the peptide synthesis tube.

e) Repeat cleaning step c) in step (2) "Resin Deprotection".

Then, the polypeptide is formed in the following way: starting from the first amino acid at the rightmost C-terminal of the polypeptide chain, and connecting the amino acids one by one toward the leftmost N-terminal.

(3) Connect (from the rightmost C end) to the first amino acid

a) Weigh 310mg (0.8mmol) Fmoc-Ser(Boc)-OH (serine) and 314mg (0.8mmol) condensation agent (HCTU) into a 10mL EP tube, add 6mL DMF to the EP tube to dissolve, Shake well, then add 265 μL (1.6 mmol) of N,N-diisopropylethylamine (DIEA) into the EP tube to obtain a mixed solution.

b) Transfer the above mixed solution to the polypeptide synthesis tube, then transfer the polypeptide synthesis tube to a 33°C constant temperature shaker and shake for 1 hour, then take out the polypeptide synthesis tube.

c) Cleaning: Repeat cleaning step c) in step (2) "Resin Deprotection".

d) Deprotection: Add 10 mL of 20% piperidine solution to the peptide synthesis tube, shake in a constant temperature shaker at 33°C for 10 minutes, and take out the peptide synthesis tube.

(4) Connect (from the rightmost C end) the 2nd to 9th amino acid

Similar to connecting the first amino acid (from the rightmost C end) in step (3), the second to ninth amino acids (from the rightmost C end) are connected in sequence. The only difference is that Fmoc-Pro is used respectively. -OH (proline), Fmoc-Pro-OH (proline), Fmoc-Pro-OH (proline), Fmoc-Ala-OH (alanine), Fmoc-Gly-OH (glycine), Fmoc-Ser(Boc)-OH(serine), Fmoc-Ser(Boc)-OH(serine) and Fmoc-Pro-OH(proline), as the second to coupling (from the rightmost C-terminus) Raw material for the 9th amino acid.

(5) Connect (from the rightmost C end) the "10th and 11th" amino acids

a) Weigh 205 mg (0.6 mmol) of Fmoc-5-aminovaleric acid (molecular formula: Fmoc-NH-(CH ₂ ) ₄ -C(O)-OH) and 235 mg (0.6 mmol) of the condensing agent (HCTU) and place into a 10 mL EP tube, add 6 mL DMF to the EP tube to dissolve, shake well, and then add 265 μL (1.6 mmol) of DIEA to the EP tube to obtain a mixed solution.

b) Shake the above mixed solution well and transfer it to a polypeptide synthesis tube; transfer the polypeptide synthesis tube to a 33°C constant temperature shaker and shake for 1 hour, then take out the polypeptide synthesis tube.

c) Cleaning: Repeat cleaning step c) in step (2) "Resin Deprotection".

d) Deprotection: Add 10 mL of 20% piperidine solution to the peptide synthesis tube, shake in a constant temperature shaker at 33°C for 10 minutes, and take out the peptide synthesis tube.

(6) Connect (from the rightmost C end) the 12th to the 39th amino acid

Similar to connecting the first amino acid (from the rightmost C end) in step (3), the 12th to 39th amino acids (from the rightmost C end) are sequentially connected, the only difference is that Fmoc-Ala is used respectively. -OH(Alanine), Fmoc-Ile-OH(Isoleucine), Fmoc-Leu-OH(Leucine), Fmoc-Trp(Boc)-OH(Tryptophan), Fmoc-Gln(Trt )-OH(Glutamine), Fmoc-Val-OH(Valine), Fmoc-Phe-OH(Phenylalanine), Fmoc-Ala-OH(Alanine), Fmoc-Lys(Boc)- OH(Lysine), Fmoc-Gln(Trt)-OH(Glutamine), Fmoc-Ala-OH(Alanine), Fmoc-Ile-OH(Isoleucine), Fmoc-Lys(Boc) - OH(lysine), Fmoc-Asp(OtBu)-OH(aspartic acid), Fmoc-Leu-OH(leucine), Fmoc-(Aib)-OH(2-aminoisobutyric acid), Fmoc-Ile-OH(isoleucine), Fmoc-Ser(Boc)-OH(serine), Fmoc-Tyr(tBu)-OH(tyrosine), Fmoc-Asp(OtBu)-OH(aspartic acid acid), Fmoc-Ser(Boc)-OH(serine), Fmoc-Thr(tBu)-OH(threonine), Fmoc-D-Phe-OH(phenylalanine), Fmoc-D-Thr(tBu )-OH(threonine), Fmoc-Gly-OH(glycine), Fmoc-Glu(OtBu)-OH(glutamic acid), Fmoc-(Aib)-OH(2-aminoisobutyric acid) and Fmoc- Tyr(tBu)-OH (tyrosine) is used as the raw material for coupling the 12th to 39th amino acids (from the rightmost C end).

(7) Remove the Boc on the 20th Lys (Boc) (from the leftmost N end)

After the peptide chain extension is completed, wash the resin with 10mL DMF, 10mL DCM and 10mL DMF respectively.

Then, weigh 116 mg (0.1 mmol) of tetraphenylphosphine palladium into a 10 mL EP tube, then add 3 mL of DCM and 3 mL of DMF to the EP tube to dissolve, and mix thoroughly. Add 124 μL (1 mmol) of phenylsilane to the EP tube, shake well, and transfer the solution to the peptide synthesis tube. Transfer the peptide synthesis tube to a 33°C constant-temperature shaker and shake for 2 hours, then take it out and wash it.

From the above addition of palladium tetrakis triphenylphosphine to the cleaning step, repeat the entire step of removing Boc once.

(8) Connection of side chain modifications

a) After washing, couple the first AEEA on the side chain of lysine at position 20 (from the leftmost N-terminus): combine 231mg (0.6mmol) of Fmoc-AEEA and 235mg (0.6mmol) of HCTU Dissolve in DMF, then add 200 μL (1.2 mmol) of DIEA, mix evenly, and transfer to a peptide synthesis tube. Transfer the peptide synthesis tube to a constant temperature shaker and shake at room temperature for 1 hour. Repeat step (2) "Resin Deprotection".

b) Coupling the second AEEA: Dissolve 231mg (0.6mmol) of Fmoc-AEEA and 235mg (0.6mmol) of HCTU in DMF, then add 200μL (1.2mmol) of DIEA, mix evenly and transfer to the peptide synthesis tube . Transfer the peptide synthesis tube to a constant temperature shaker and shake at room temperature for 1 hour. Repeat step (2) "Resin Deprotection".

c) Coupling γ-Glu: Dissolve 255mg (0.6mmol) Fmoc-Glu-(OtBu)-OH and 235mg (0.6mmol) HCTU in DMF, then add 200μL (1.2mmol) DIEA, mix evenly and transfer to the peptide synthesis tube. Transfer the peptide synthesis tube to a constant temperature shaker and shake at room temperature for 1 hour. Repeat step (2) "Resin Deprotection".

d) Coupling eicosanedioic acid: Dissolve 240 mg (0.6 mmol) of eicosanedioic acid mono-tert-butyl ester and 235 mg (0.6 mmol) of HCTU in DMF, then add 200 μL (1.2 mmol) of DIEA, and mix evenly Then transfer it to the peptide synthesis tube. Transfer the peptide synthesis tube to a constant temperature shaker and shake at room temperature for 1 hour.

(9) Cleavage of crude peptide

Take out the peptide synthesis tube, rinse the resin three times with DMF (10 mL each time), and drain the solvent after each rinse. Rinse the resin three times with DCM (10 mL each time), and drain the solvent after each rinse (drain until the resin is dry powder). After draining, prepare TFA (trifluoroacetic acid)/H ₂ O/phenol/Tips (volume ratio: 10mL/500μL/500mg/500μL) cutting reagent in a 10mL EP tube. Transfer the cleavage reagent to the above-mentioned peptide synthesis tube, place it in a 26°C constant-temperature shaker and shake for 2.5 hours. Take out the peptide synthesis tube. The solution in the tube is the peptide chain cleavage solution.

(10) Post-processing of polypeptides

a) Use an ear cleaning ball to transfer 10 mL of peptide chain lysis solution into a 50 mL centrifuge tube, and blow the lysate as dry as possible with nitrogen at room temperature to less than 5 mL.

b) Ice ether precipitation and centrifugation operation: Add 40 mL ice ether to the 50 mL centrifuge tube, shake the centrifuge tube appropriately, put the centrifuge tube into the centrifuge, set the speed to 3500 rpm, and centrifuge for 3 minutes; after centrifugation is completed, discard the supernatant liquid.

c) Repeat the above ice ether precipitation and centrifugation operations. After discarding the supernatant, the precipitate obtained is the crude peptide.

d) Dry at room temperature and mash to obtain purified polypeptide.

Purified peptides were separated using Shimadzu semi-preparative liquid chromatography. Figure 1 is an ESI-MS (electrospray mass spectrometry) characterization of the purified peptide compound NBB-T007. The peaks on the figure represent the molecular weights of different mass-to-charge ratios. Figure 2 is a high performance liquid chromatogram showing that the synthesized NBB-T007 has a purity of 95%.

[Preparation of other compounds of the NBB-T007 series and compound NBB-EX4]

Other compounds of the NBB-T007 series and compound NBB-EX4 were prepared in a manner similar to the preparation of compound NBB-T007, except that the Fmoc protected amino acid raw material, linker raw material and/or aliphatic diacid source, were changed according to the structural formula of each compound. and various other modifying groups (if any). After high-performance liquid chromatography analysis, the purity of these compounds was ≥95%.

Among them, when preparing NBB-T007-15, Fmoc-S5-OH (Cas No.: 288617-73-2) and Fmoc-R5 were used at the 24th and 27th positions (from the rightmost C end) respectively. -OH (Cas No.: 288617-77-6) is used as the amino acid raw material. After inserting the 27th Fmoc-R5-OH (from the rightmost C end), weigh 64 mg of Grubbs catalyst (Cas No. 172222- 30-9) into a 10mL EP tube, and then add 4mL of DCM to dissolve. After mixing evenly, transfer the mixed solution to the peptide synthesis tube. Then transfer the peptide synthesis tube to a 33°C constant temperature shaker, shake for 4 hours and then take out the peptide synthesis tube. Under the action of Grubbs catalyst, the side chains of the R5 unit and the S5 unit undergo olefin metathesis reaction, leading to cyclization. After this step is completed, continue to insert the 28th to 39th amino acids in sequence (from the rightmost C end).

[In vitro activity assay: cAMP detection]

Experimental reagents

Laboratory equipment

name company model microplate reader TECAN INFINITE 200 PRO carbon dioxide incubator Thermo Fisher 3131 Biological clean workbench Suzhou Antai Air Technology Co., Ltd. BLB-1300 microscope Nikon ECLIPSE Ts2-FL cell counter BIO-RAD ^TC20TM refrigerator Haier BCD-252WXPS Medical low temperature refrigerator Thermo Fisher 902-ULTS

Human GLP-1R receptor, GIPR receptor and GCGR receptor were cloned into pcDNA3.1 vector respectively. Use Lipofectamin3000Transfection kit to transfect pcDNA3.1-GLP1R, pcDNA3.1-GIPR and pcDNA3.1-GCGR into HKE293T cells cultured in 35mm culture dishes, and culture them in a carbon dioxide incubator for 24 hours. Resuspend the cells in DMEM containing 10% FBS, 1% P/S and 500 μM IBMX. Take 20 μL for cell counting and dilute it to 2×10 ⁶ cells/mL. Take 5 μL of the diluted DMEM to resuspend the cells and plate them on in 384-well plates. Then add 5 μL DMSO to each well of the well plate and use the ten-fold gradient dilution (2×10 ^-6 , 2×10 ^-7 ,..., 2×10 ^-15 M) of DMEM containing 500 μM IBMX to perform the previous implementation. Any one of the following polypeptides prepared in the example: NBB-T007 series compounds, compound NBB-EX4, or semaglutide (homemade) as a comparison, was incubated at 37°C for 30 minutes. Add 5 μL each of cAMP-d2 and anti-cAMP in the cAMP-Gs Dynamic kit and incubate at room temperature for 1 hour, then read with a TECAN microplate reader. The excitation wavelength is 340 nm, and the emission wavelengths are 620 nm and 655 nm. Calculate the signal ratio (655nm/620nm*10,000), and perform nonlinear fitting of the signal ratio and sample concentration using a four-parameter equation in GraphPad Prism 8 to obtain the EC ₅₀ value, see Figure 3 to Figure 26 and the table below.

As can be seen from the above table, semaglutide only has activity against human GLP-1R, while the NBB-T007 series compounds according to the present disclosure (except compound NBB-T007-7) unexpectedly show activity against human GIP receptors and Dual activity of GLP-1 receptor. In particular, compounds NBB-T007-1, NBB-T007-5, NBB-T007-8, NBB-T007-10, NBB-T007-12, NBB-T007-18, NBB-T007-19, NBB-T007- 21. NBB-T007-22, NBB-T007-23 and NBB-T007-24 have triple activity on human GIP receptor, GLP-1 receptor and GCG receptor.

Compound NBB-EX4 has high activity against human GLP-1R, indicating that by replacing two adjacent glycines at positions 29 and 30 with -CH ₂ -CH ₂ - units -NH-CH ₂ -C(O )-NH-CH ₂ -C(O)-contains peptide bonds to modify the peptide chain, which is widely applicable to peptide drugs and can maintain high activity against human GLP-1R.

[Db/db mice (type II diabetic mice) hypoglycemic test]

Male db/db mice (Changzhou Cavins), 7-8 weeks old, each weighing 33-40 g were used. The mice were housed individually in a temperature-controlled (22-25°C) facility with a 12-hour light/dark cycle (lighting started at 08:00) and with free access to food and water. After 1 week of acclimation to the facility, subjects were randomly assigned to treatment groups (n=6/group) based on body weight and blood glucose so that each group had similar starting mean body weight and blood glucose concentration. The excipient control ("solvent group", 0.05% NaHCO ₃ solution), the semaglutide control (dose 50 nmol/kg) dissolved in the excipient, and the NBB-T007 series compounds prepared in the previous examples ( A dose of 50 nmol/kg) was administered to db/db mice ad libitum by subcutaneous injection. Blood was collected from the tail vein using a Wenhao fast blood glucose meter (OneTouch UltraEasy, Johnson & Johnson), and the blood glucose levels of the mice were measured continuously for 48 hours. At the same time, the body weight of db/db mice was monitored at 0h, 24h and 72h. The test results are shown in the table below and Figure 27 to Figure 29.

Formula IV*: Use a linker (AEEA-Ahx-(γ-Glu)) at the 20th lysine (Lys) of formula IV to conjugate eicosanedioic acid;

Formula IV**: The linker (β-Ala)2-(γ-Glu) is used to conjugate eicosanedioic acid at lysine (Lys) position 20 of formula IV.

It can be seen that the NBB-T007 series of compounds according to the present disclosure have an obvious hypoglycemic effect that is equivalent to or better than that of semaglutide, and can maintain the pharmacodynamic effect for 48 hours; among them, compounds NBB-T007, NBB-T007-10 and NBB-T007-12 have particularly better blood sugar-lowering and weight-lowering effects.

[Long-term hypoglycemic effect of db/db mice (type II diabetic mice)]

Male db/db mice (Changzhou Cavins), 7-8 weeks old, each weighing 33-40 g were used. The mice were housed individually in a temperature-controlled (22-25°C) facility with a 12-hour light/dark cycle (lighting started at 08:00) and with free access to food and water. After 1 week of acclimation to the facility, subjects were randomly assigned to treatment groups (n=6/group) based on body weight and blood glucose, so that each group had similar starting mean body weight and blood glucose concentration. Semaglutide control (dose 30 nmol/kg), Tirzepatide control (dose 30 nmol/kg) dissolved in excipient (0.05% NaHCO ₃ solution), and NBB-prepared in the previous examples were used respectively. T007-12 compound (dose 30 nmol/kg) was administered to db/db mice ad libitum via subcutaneous injection twice a week for 12 weeks. During the administration period, the mice's weight, food intake, and blood sugar were monitored, and blood was collected at weeks 2, 4, 6, 8, and 10 for detection of glycated hemoglobin HbA1C. The test results for glycated hemoglobin inhibition (%) are shown in the table below.

It can be seen that the tested compound has a significant reducing effect on glycated hemoglobin in type II diabetic mice after long-term administration, suggesting that it can effectively improve blood sugar in db/db mice.

[Glucose tolerance IPGTT test in normal mice]

C57BL/6 mice (Beijing Vitong Lever), male, 7-10 weeks old, weighing 18-20g. The mice were housed individually in a temperature-controlled (22-25°C) facility with a 12-hour light/dark cycle (lighting started at 08:00) and with free access to food and water. After acclimating to the facility, the mice were randomly divided into groups of 6, according to blood glucose and body weight, so that each group had a similar starting average body weight and blood glucose concentration. The mice were fasted overnight for 12-16 hours. The mice were weighed the next day, and 0-minute blood glucose was measured using a Wenhao rapid blood glucose meter (OneTouch UltraEasy, Johnson & Johnson).

The following test substances were used respectively: excipient control ("solvent group", 0.05% NaHCO ₃ solution), and semaglutide control (25 nmol/kg) dissolved in the excipient and NBB prepared in the previous example -T007 series compounds (dose 25nmol/kg) were administered to C57BL/6 mice ad libitum by subcutaneous injection, and glucose solution (2g/kg) was administered intraperitoneally at the same time, and the concentrations at 15min, 30min, 60min, and 120min after administration were measured. Blood sugar level. At the same time on the second and third days, no test substance was administered, but glucose solution (2g/kg) was administered intraperitoneally, and blood glucose levels were measured at 15 min, 30 min, 60 min, and 120 min after administration. Calculate the area under the blood glucose-time curve AUC (area under the curve). Furthermore, the body weight of the mice was monitored at 24h, 48h and 72h after administration. The test results are shown in the table below and Figure 30 to Figure 31.

It can be seen from the above glucose tolerance test that the NBB-T007 series compounds according to the present disclosure have an obvious hypoglycemic effect better than semaglutide, and the glucose AUC in the first, second and third day tests All decreased.

[In vivo activity test: pharmacokinetic analysis in SD rats]

SD rats (Beijing Vitong Lever) were selected, male, 8-10 weeks old, weighing 180-200g. Rats were housed individually in temperature-controlled (22-25°C) facilities with a 12-h light/dark cycle (lighting started at 08:00) and had free access to food and water. After the rats adapted to the facility, they were randomly divided into groups, with 3 rats in each group. The excipient control (0.05% NaHCO ₃ solution), the NBB-T007 series compounds prepared in the previous examples (dose 1 mg/kg) and semaglutide (dose 1 mg/kg) dissolved in the excipient were administered subcutaneously, At 0.25h, 0.5h, 1h, 2h, 4h, 8h, 24h, 48h, and 72h after administration, 0.3 mL of venous blood was collected from the jugular vein and placed in EDTA2K anticoagulant tubes. The plasma was collected by centrifugation at 8000 rpm for 5 minutes and passed through LC- The MS/MS method is used to determine the plasma concentration of the test substance, and methanol is used to extract the compounds in the plasma sample. The sample processing steps are as follows:

Take 30.0 μL of sample, 50.0 μL of internal standard solution (semaglutide, 20,000ng/mL) and 200 μL of methanol, vortex for 10 min, centrifuge for 10 min (3900 rpm), take the supernatant into another clean 96-well plate, and perform LC -MS/MS analysis. See the table below and Figure 32 to Figure 35 for test results.

T _1/2 = half-life, T _max = time to reach maximum concentration, C _max = maximum plasma concentration, AUC _last AUC from the beginning of administration to the last point, AUC _INF _obs from the beginning of administration to theory Extrapolating the AUC to time at infinity, MRT _{INF_obs} is the average residence time from time zero to time at infinity.

It can be seen that compounds NBB-T007, NBB-T007-10 and NBB-T007-12 reach mean maximum plasma concentrations approximately 8 hours after subcutaneous administration. Among them, the half-lives of compounds NBB-T007 and NBB-T007-12 in rats are 11.82 and 9.29 hours respectively, supporting the possibility of once-weekly administration.

[In Vivo Activity Test: Pharmacokinetic Analysis in Cynomolgus Monkeys]

Male cynomolgus monkeys weighing 3-6kg were selected for pharmacokinetic analysis in non-rodent animals. A single intravenous administration of NBB-T007-12 (dose 0.5 mg/kg) dissolved in vehicle (0.05% _NaHCO solution) and Tirzepatide control (dose 0.5 mg/kg) was administered. Before administration (0h), and at 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 204h, 240h and 312h after administration, 0.5mL of blood was taken from the forelimb vein, and EDTA- In the K2 test tube, the whole blood was collected and temporarily stored in an ice water bath, centrifuged at 11,000 rpm for 5 minutes within 30 minutes, the plasma was separated, and placed in a plasma separation refrigerator to be frozen for testing. LC-MS/MS method was used to detect the concentration of prototype drug in plasma. Use WinNonlin software to calculate relevant pharmacokinetic parameters T _max , C _max , AUC _last , AUC _0-t (AUC from the beginning of administration to time t), AUC _{INF_obs} (the time from the beginning of administration to theoretical extrapolation to infinity AUC), T _1/2 , CL, etc. See the table below for test results.

T _1/2 = half-life, C _max = maximum plasma concentration, AUC _last AUC from the start of administration to the last point.

It can be seen that the half-life of compound NBB-T007-12 after a single intravenous administration in cynomolgus monkeys is 46.85 hours, further supporting the possibility of weekly administration.

[Weight loss and hypoglycemic effects in obese DIO mice]

70 male C57BL/6 mice (Nanjing Jicui Yaokang) were selected. The animal room environment was maintained at a temperature of 23±2°C, a humidity of 40-70%, and 12 hours of light and dark alternating (lighting started at 08:00). 4-5 mice were kept in each cage, and the bedding was changed twice a week. The mice were fed high-fat feed (60% Kcal fat, D12492) for 10-12 weeks. The body weight at the beginning of the experiment was between 38-45g. It was determined that the body weight of the mice was more than 30% higher than that of animals on a normal diet. Random blood sugar and body weight were measured after random testing. Group into groups, 8-10 in each group. Eight mice fed with normal chow served as the normal model group. Mice fed with high-fat diet were randomly divided into model control group ("solvent group"), semaglutide group (administration dose 50nmol/kg), NBB-T007 group (administration dose 50nmol/kg), NBB-T007 -10 group (administration dose: 50 nmol/kg), NBB-T007-12 group (administration dose: 50 nmol/kg). Mice were given the above test substances by subcutaneous injection twice a week, with a dosage volume of 5 mL/kg. The normal model group and the model control group ("solvent group") were given vehicle control (0.05% NaHCO ₃ solution), and were continuously given Medication for 4 weeks. Discontinue the medication, or continue the medication. Test the following indicators:

1. During the experiment, the body weight and food consumption of the mice were measured twice a week. The test results are shown in Figure 36.

As can be seen from Figure 36, after 40 days, the weight of mice in both the normal model group and the model control group ("solvent group") increased; the weight of mice administered with semaglutide remained almost unchanged, and the body weight of mice administered with the compound prepared in the previous example The body weight of the test mice of NBB-T007, NBB-T007-10 and NBB-T007-12 all decreased significantly, and the body weight of the test mice administered with the compounds NBB-T007-10 and NBB-T007-12 prepared in the previous examples was down about 20%.

3. Insulin tolerance ITT test: 72 hours after the last dose study, animals were enrolled for ITT study. After the animals were fasted for 1 hour, the 0h blood glucose level was measured. After intraperitoneal injection of 1U/kg insulin injection, the 15min, 30min, and 1h blood glucose were tested. The test results are shown in Figure 37.

As can be seen from Figure 37, compared with mice administered semaglutide, the test mice administered the compounds NBB-T007, NBB-T007-10 and NBB-T007-12 prepared in the previous examples had better Insulin sensitivity.

4. Glucose tolerance IPGTT test: 2 hours after the end of the ITT study, the animals were given the test substance once, and 72 hours later, the animals were enrolled for the IPGTT study. After the animals were fasted overnight for 16 hours, blood was collected from the tail tip to test 0-h fasting blood glucose. Inject a glucose solution with a concentration of 2g/kg intraperitoneally, and at the same time give the test solution by subcutaneous injection, and test the animal's blood sugar at 15min, 30min, 1h, and 2h (the above blood sugar tests all use the second drop of blood). The test results are shown in Figure 38.

As can be seen from Figure 38, compared with mice administered semaglutide, the test mice administered the compounds NBB-T007, NBB-T007-10 and NBB-T007-12 prepared in the previous examples had comparable or Better post-meal blood sugar control.

5. Serum biochemistry/serum insulin, C-peptide/blood HbA1C: After the ITT study, the animals were euthanized by carbon dioxide, blood was collected from the heart, and heparin sodium anticoagulated whole blood was collected. The blood was divided into two parts, one part was about 120 μL for testing the HbA1C level, and the other part was used to test the HbA1C level. A portion of 500 μL whole blood was separated into serum and tested for serum insulin and routine biochemical index tests (total cholesterol CHO, triglyceride TG, high-density lipoprotein HDL, low-density lipoprotein LDL, free fatty acid NEFA, urea UREA, inosine CREA (creatine ), albumin ALB (albumin), total bilirubin TBIL (total bilirubin), alanine aminotransferase ALT, aspartate aminotransferase AST), the test results are shown in Figure 39(a), Figure 39(b) and Figure 39(c).

As can be seen from Figure 39(a), Figure 39(b) and Figure 39(c), the model control group showed high serum insulin content, a typical diabetes indicator. Compared with mice administered semaglutide, test mice administered the compounds NBB-T007, NBB-T007-10 and NBB-T007-12 prepared in the previous examples had comparable or better reductions in serum insulin levels trend, and comparable or better protection of liver function, protection of kidney function, and improvement of lipid metabolism.

6. Gross anatomy/fat weight weighing: After the animal is euthanized by carbon dioxide, changes in the organs are observed, the abdominal or epididymal fat of the animal is collected, the weight is calculated, the body weight coefficient is calculated, part of the liver tissue of the animal is collected, and triglyceride TG is detected. The test results are shown in Figures 40(a) and 40(b).

As can be seen from Figures 40(a) and 40(b), compared with mice administered semaglutide, the mice administered the compounds NBB-T007, NBB-T007-10 and NBB-T007-12 prepared in the previous examples The test mice had comparable or better postprandial body fat reduction and triglyceride reduction.

Claims (37)

A compound of formula I below or a pharmaceutically acceptable salt thereof: L ₁ -NH-(CH ₂ ) _n -C(O)-L ₂ -NH ₂ Formula I in, L ₁ is a peptide analog of the GIP (1-28) peptide, said L ₁ is a peptide consisting of 28 amino acids, and the amino acid sequence of said L ₁ is at least 39% identical to SEQ ID NO: 1, L ₂ is a peptide having the amino acid sequence consisting of SEQ ID NO:2, n is any integer from 2 to 6, and The compound has GLP-1 receptor agonist activity, GIP receptor agonist activity, or both. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein _L1 contains a Y1H substitution compared to SEQ ID NO:1. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains a substitution selected from A2G, A2(Aib) and A2(β-Ala). The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein _L1 contains a T5S substitution compared to SEQ ID NO: 1. The compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ comprises F6 (homo-Phe) and F6 (Cpa-Ala ) replacement. The compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein L ₁ contains an I7T substitution compared to SEQ ID NO: 1. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains a substitution selected from Y10L and Y10(Fpa5-Ala). The compound according to any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains an I12K substitution. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains a substitution selected from A13Q, A13(Aib) and A13Y. The compound according to any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ comprises a substitution selected from M14L and M14(α-meL). The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein L ₁ contains a D15E substitution compared to SEQ ID NO: 1. The compound according to any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains a substitution selected from K16E and K16(Ala). The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein _L1 contains an I17E substitution compared to SEQ ID NO: 1. The compound according to any one of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains a substitution selected from H18A and H18(Aib). The compound according to any one of claims 1-14, or a pharmaceutically acceptable salt thereof, wherein L ₁ contains a Q19V substitution compared to SEQ ID NO: 1. The compound according to any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains a substitution selected from Q20R and Q20K. The compound according to any one of claims 1-16 or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ comprises a substitution selected from the group consisting of D21L, D21A, D21E and D21 (Abu) . The compound according to any one of claims 1-17, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains a substitution selected from V23I and V23L. The compound according to any one of claims 1-18, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ contains a substitution selected from N24E and N24Q. The compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof, wherein compared with SEQ ID NO: 1, L ₁ comprises selected from the group consisting of W25(Na1) and W25(2-me-Trp ) replacement. The compound according to any one of claims 1-20, or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L ₁ comprises a substitution selected from L27K and L27I. The compound of any one of claims 1-21, or a pharmaceutically acceptable salt thereof, wherein L ₁ contains an A28N substitution compared to SEQ ID NO: 1. Compounds of the following formula XXI or pharmaceutically acceptable salts thereof: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula XXI. The compound according to any one of claims 1-23 or a pharmaceutically acceptable salt thereof, wherein in formula I, when the amino acid at position 16 or 20 is lysine (Lys), via Chemical modification by conjugating a C ₁₂ -C ₂₄ aliphatic diacid to the ε-amino group of the lysine (Lys) side chain, either directly or via a linker selected from (AEEA) ₂ -(γ- Glu) _a , AEEA-Ahx-(γ-Glu) _a , (Ahx) ₂ -(γ-Glu) _a and (β-Ala) ₂ -(γ-Glu) _a , where a is 1 or 2; Preferably, the chemical modification is carried out by conjugating eicosanedioic acid to the ε-amino group of the lysine (Lys) side chain at position 16 or 20 via (AEEA) ₂ -(γ-Glu); or _{Eicosanedioic} acid is conjugated to _lysine 20 ( Lys) side chain of the ε-amino group is chemically modified. The compound according to any one of claims 1-24 or a pharmaceutically acceptable salt thereof, wherein in formula I, the histidine (His) or tyrosine (Tyr) at position 1 is amidated , for example, is connected to the amino group of the histidine (His) or tyrosine (Tyr) through -C _m H _2m+1 -C(O)-, and m is an integer from 1 to 20; Preferably, the amino group of tyrosine (Tyr) at position 1 is amidated by being connected to CH ₃ -C(O) or to C ₁₉ H ₃₉ -C(O)-. The compound according to any one of claims 1 to 25 or a pharmaceutically acceptable salt thereof, wherein in formula I, the α-carbon atoms of any two amino acids can be connected to form a ring via a direct bond or via a linker. , the linking group is selected from an alkyl or alkenyl group containing 2 to 20 carbon atoms; Preferably, when the amino acids at positions 13 and 16 are both alanine, the α-carbon atom of alanine (Ala) at position 13 and alanine at position 16 are connected via an alkenyl group containing 10 carbon atoms. The α-carbon atoms of (Ala) are attached. A compound of formula II below or a pharmaceutically acceptable salt thereof: Tyr ₁ -X ₂ -Glu ₃ -Gly ₄ -X ₅ -X ₆ -Thr ₇ -Ser ₈ -Asp 9 -X ₁₀ -Ser _{11 -X 12} -X ₁₃ -Leu ₁₄ -Asp ₁₅ -X ₁₆ -Ile ₁₇ -X ₁₈ -Gln ₁₉ -Lys ₂₀ -X ₂₁ -Phe ₂₂ -X ₂₃ -X ₂₄ -X ₂₅ -Leu ₂₆ -Ile 27 -Ala _{28 -NH-} (CH ₂ ) ₄ -C(O)-Pro ₃₁ - Ser ₃₂ -Ser ₃₃ -Gly ₃₄ -Ala ₃₅ -Pro ₃₆ -Pro ₃₇ -Pro ₃₈ -Ser ₃₉ -NH ₂ Formula II, Among them, X ₂ , X _{5 ,} X ₆ , X ₁₀ , X ₁₂ , X ₁₃ , X ₁₆ , X ₁₈ , X ₂₁ , _{X 23} , . The compound according to claim 27 or a pharmaceutically acceptable salt thereof, wherein, X ₂ is an amino acid residue selected from 2-aminoisobutyric acid (Aib) and (β-Ala), X ₅ is an amino acid residue selected from Thr and Ser, X ₆ is an amino acid residue selected from Phe, homophenylalanine (homo-Phe) and p-chlorophenylalanine (Cpa-Ala), X ₁₀ is an amino acid residue selected from Tyr and pentafluorophenylalanine (Fpa5-Ala), X ₁₂ is an amino acid residue selected from Ile and Lys, X ₁₃ is an amino acid residue selected from 2-aminoisobutyric acid (Aib), Tyr and Ala, X ₁₆ is an amino acid residue selected from Lys and Ala, X ₁₈ is an amino acid residue selected from Ala and Aib, X ₂₁ is an amino acid residue selected from Ala, Glu and 2-aminobutyric acid (Abu), X ₂₃ is an amino acid residue selected from Val and Leu, X ₂₄ is an amino acid residue selected from Gln and Asn, and X ₂₅ is an amino acid residue selected from Trp, 2-methyltryptophan (2-me-Trp) and 1-naphthylalanine (Nal). The compound according to claim 27 or 28, or a pharmaceutically acceptable salt thereof, having a structure selected from any one of the following formula III to the following formula XX: Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Ala-Leu-Asp-Ala-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln- Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula III, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula IV, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Tyr-(α-meL)-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe- Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula V, Tyr-(β-Ala)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe- Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula VI, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Glu-Phe-Val- Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula VII, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Glu-Phe-Val- Asn-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula VIII, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Asn-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula IX, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Asn-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula X, Tyr-(Aib)-Glu-Gly-(D-Thr)-(D-Phe)-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln- Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser -NH ₂ Formula XI, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-(D-Ala)-Pro-Pro-Pro-Ser-NH ₂ Formula XII, Tyr-(Aib)-(D-Glu)-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala- Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula XIII, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Tyr-(α-meL)-Asp-Lys-Ile-(Aib)-Gln-Lys-Ala- Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula XIV, Tyr-(Aib)-Glu-Gly-Thr-(homo-Phe)-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala- Phe-Val-Gln-Trp-Leu-Ile-Ala -[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula XV, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-(Abu)-Phe- Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula XVI, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Gln-(2-me-Trp)-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula XVII, Tyr-(Aib)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Val- Gln-(Nal)-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula XVIII, Tyr-(Aib)-Glu-Gly-Ser-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Ala-Phe-Leu- Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ Formula XIX, and Tyr-(Aib)-Glu-Gly-Thr-(Cpa-Ala)-Thr-Ser-Asp-(Fpa5-Ala)-Ser-Ile-(Aib)-Leu-Asp-Lys-Ile-Ala-Gln- Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-[NH-(CH ₂ ) ₄ -C(O)]-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser -NH ₂ Formula XX. The compound according to any one of claims 27-29 or a pharmaceutically acceptable salt thereof, wherein in any one of formula II to formula XX, the lysine at position 16 or 20 (Lys) is chemically modified by conjugating a C ₁₂ -C ₂₄ aliphatic diacid to the ε-amino group of the lysine (Lys) side chain via a direct bond or via a linker selected from (AEEA ) ₂ -(γ-Glu) _a , AEEA-Ahx-(γ-Glu) _a , (Ahx) ₂ -(γ-Glu) _a and (β-Ala) ₂ -(γ-Glu) _a , where a is 1 or 2; Preferably, in any of Formula III and Formula V to Formula XX, the eicosanedioic acid is conjugated to the lysine (Lys) side chain at position 20 via (AEEA) _2- (γ-Glu) chemical modification of the ε-amino group; or In Formula IV, the chemical modification is performed by conjugating eicosanedioic acid to the epsilon-amino group of the side chain of lysine (Lys) at position 16 via (AEEA) _2- (γ-Glu); or In formula IV, eicosanedioic acid is conjugated to the 20th via (Ahx) ₂ -(γ-Glu), AEEA-Ahx-(γ-Glu) or (β-Ala) ₂ -(γ-Glu). It is chemically modified at the ε-amino group of the lysine (Lys) side chain. The compound according to any one of claims 27-30 or a pharmaceutically acceptable salt thereof, wherein in any one of Formula II to Formula XX, the histidine (His) or tyrosine at position 1 The amino acid (Tyr) is amidated, for example, by -C _m H _2m+1 -C(O)- being attached to the amino group of the histidine (His) or tyrosine (Tyr), And m is an integer from 1 to 30; Preferably, in formula IV, the amino group of tyrosine (Tyr) at position 1 is amidated by being connected to CH ₃ -C(O)- or to C ₁₉ H ₃₉ -C(O)-. The compound according to any one of claims 27-31 or a pharmaceutically acceptable salt thereof, wherein in any one of Formula II to Formula XX, the α-carbon atoms of any two amino acids can be directly bond or connected to form a ring via a linking group selected from an alkyl or alkenyl group containing 2 to 20 carbon atoms; Preferably, in formula III, the α-carbon atom of alanine (Ala) at position 13 is connected to the α-carbon atom of alanine (Ala) at position 16 via an alkenyl group containing 10 carbon atoms. Pharmaceutical compositions comprising: The compound according to any one of claims 1-32 or a pharmaceutically acceptable salt thereof, and Pharmaceutically acceptable carrier, diluent or excipient. Use of a pharmaceutical composition according to claim 33 or a compound according to any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treatment and/or prevention metabolic disease or disorder; In particular, said metabolic diseases or disorders include diabetes and diabetes-related conditions, and obesity and obesity-related conditions; In particular, the diabetes and diabetes-related conditions include insulin resistance, glucose intolerance, elevated fasting glucose, prediabetes, type I diabetes, type II diabetes mellitus (T2DM), gestational diabetes hypertension, dyslipidemia, atherosclerosis sclerosis, arteriosclerosis, coronary heart disease, peripheral arterial disease, and atherogenic dyslipidemia, dyslipidemia, elevated blood pressure, hypertension, prothrombotic and proinflammatory states, and combinations thereof; In particular, obesity and obesity-related conditions include obesity-associated inflammation, obesity-associated gallbladder disease, obesity-induced sleep apnea, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and combinations thereof. A method of treating and/or preventing metabolic diseases or disorders, comprising administering to an individual in need an effective amount of a pharmaceutical composition according to claim 33 or a compound according to any one of claims 1-32 or a compound thereof. Pharmaceutically acceptable salt. The method of claim 35, wherein the pharmaceutical composition, the compound, or a pharmaceutically acceptable salt thereof is administered to the individual by subcutaneous injection. The method of claim 35 or 36, wherein the pharmaceutical composition, the compound, or a pharmaceutically acceptable salt thereof is administered to the subject once a week.

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