WO2023207107A1

WO2023207107A1 - Glp-1/gcg receptor co-agonist, pharmaceutical composition comprising same and use thereof

Info

Publication number: WO2023207107A1
Application number: PCT/CN2022/137122
Authority: WO
Inventors: 吕佩; 田长麟; 郑勇; 吴文奎; 王辉
Original assignee: 苏州星洲生物科技有限公司
Priority date: 2022-04-29
Filing date: 2022-12-07
Publication date: 2023-11-02
Also published as: CN116970064A

Abstract

Provided is a co-agonist of a GLP-1 receptor and a GCG receptor. Also provided are a pharmaceutical composition comprising these compounds, use of these compounds, and a method for treating and/or preventing a metabolic disease or disorder.

Description

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

Technical field

The present disclosure relates to a GLP-1/GCG 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.

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.

Oxyntomodulin OXM (oxyntomodulin) can activate GLP-1 receptors and GCG receptors at the same time, which has the effect of slowing down weight gain and lowering blood sugar. However, endogenous OXM is rapidly degraded by dipeptidyl peptidase IV and other peptidases in the body, which greatly limits its clinical application.

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

However, there is still a need for alternative co-agonists with co-agonism at GLP-1 receptors and GCG receptors. 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 -Leu ₂₇ -Ala ₂₈ -X ₂₉ -X ₃₀ -Pro ₃₁ -Ser ₃₂ -Ser ₃₃ -Gly ₃₄ -NH _2Formula I,

in,

L ₁ is a peptide analog of the OXM (1-26) peptide, said L ₁ is a peptide consisting of 26 amino acids, and the amino acid sequence of said L ₁ is at least 69% identical to SEQ ID NO: 1,

X ₂₉ and X ₃₀ are each independently Gly, or X ₂₉ and X ₃₀ as a whole represent -NH-(CH ₂ ) _n -C(O)-,

n is any integer from 2 to 6, and

The compound has GLP-1 receptor agonist activity and GCG receptor agonist activity.

By modifying the peptide chain of the OXM(1-26) peptide, in particular by adopting the glycine residue Gly at both positions 29 and 30 of the peptide chain, or by combining positions 29 and 30 as a whole and at this The -NH-(CH ₂ ) _n -C(O)-unit (n is an integer from 2 to 6) is used at the site to replace the common -NH-(CH ₂ )-C(O)-unit in the peptide chain to obtain The compounds of formula I surprisingly retain high activity at GLP-1 receptors and GCG receptors, providing hypoglycemic and weight loss effects.

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

His ₁ -X ₂ -X ₃ -Gly ₄ -Thr ₅ -Phe ₆ -X ₇ -Ser ₈ -Asp ₉ -Tyr ₁₀ -Ser ₁₁ -X ₁₂ -Tyr ₁₃ -X ₁₄ -Asp ₁₅ -Ser ₁₆ -Lys ₁₇ -Lys ₁₈ -Ala ₁₉ -Lys ₂₀ -X ₂₁ -Phe ₂₂ -Val ₂₃ -Glu ₂₄ -Trp ₂₅ -Leu 26 -Leu ₂₇ -Ala ₂₈ _-X ₂₉ -X ₃₀ -Pro ₃₁ -Ser ₃₂ -Ser ₃₃ -Gly ₃₄ -NH ₂

Formula II,

Wherein, X ₂ , X ₃ , X ₇ , X ₁₂ , X ₁₄ and X ₂₁ are each independently selected from natural _amino acids or non-natural amino acid residues, and X ₂₉ _and ₃₀ as a whole represents -NH-(CH ₂ ) ₄ - C(O)-.

By modifying the peptide chain of the OXM(1-26) peptide, in particular by adopting the glycine residue Gly at both positions 29 and 30 of the peptide chain, or by combining positions 29 and 30 as a whole and at this The -NH-(CH ₂ ) _n -C(O)-unit (n is an integer from 2 to 6) is used at the site to replace the common -NH-(CH ₂ )-C(O)-unit in the peptide chain to obtain The compounds of Formula II of the present disclosure surprisingly possess co-agonism at GLP-1 receptors and GCG receptors, providing hypoglycemic and weight loss therapeutic effects. At the same time, the compound of formula II has high plasma stability, thereby having pharmacokinetic characteristics that support once-weekly subcutaneous injection administration in humans, thereby improving patient compliance.

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-C007 according to the present disclosure.

Figure 2 is a high performance liquid chromatography analysis chart of compound NBB-C007 according to the present disclosure.

Figure 3 is a mass spectrum of compound NBB-C002 according to the present disclosure.

Figure 4 is a high performance liquid chromatography analysis chart of compound NBB-C002 according to the present disclosure.

Figure 5 is a mass spectrum of compound NBB-C003 according to the present disclosure.

Figure 6 is a high performance liquid chromatography analysis chart of compound NBB-C003 according to the present disclosure.

Figure 7 is a mass spectrum of compound NBB-C004 according to the present disclosure.

Figure 8 is a high performance liquid chromatography analysis chart of compound NBB-C004 according to the present disclosure.

Figure 9 is a mass spectrum of compound NBB-C006 according to the present disclosure.

Figure 10 is a high performance liquid chromatography analysis chart of compound NBB-C006 according to the present disclosure.

Figure 11 is a mass spectrum of compound NBB-C008 according to the present disclosure.

Figure 12 is a high performance liquid chromatography analysis of compound NBB-C008 according to the present disclosure.

Figure 13 is a mass spectrum of compound NBB-C009 according to the present disclosure.

Figure 14 is a high performance liquid chromatography analysis of compound NBB-C009 according to the present disclosure.

Figure 15 is a potency-concentration curve of compound NBB-C002 acting on a target according to the present disclosure.

Figure 16 is a potency versus concentration curve of compound NBB-C003 acting on a target according to the present disclosure.

Figure 17 is a potency versus concentration curve of compound NBB-C004 acting on a target according to the present disclosure.

Figure 18 is a potency versus concentration curve of compound NBB-C006 acting on a target according to the present disclosure.

Figure 19 is a potency versus concentration curve of compound NBB-C007 acting on a target according to the present disclosure.

Figure 20 is a potency versus concentration curve of compound NBB-C008 acting on a target according to the present disclosure.

Figure 21 is a potency versus concentration curve of compound NBB-C009 acting on a target according to the present disclosure.

Figure 22 is the blood glucose-time change curve of the db/db mice tested.

Figure 23 is the blood glucose reduction percentage-time curve of the db/db mice tested.

Figure 24 is the body weight-time change curve of the db/db mice tested.

Figure 25 is the blood glucose-time change curve of normal mice tested.

Figure 26(a), Figure 26(b) and Figure 26(c) are respectively the concentration-time change curves of the compound NBB-C007 according to the present invention in the plasma of the subject SD rats injected subcutaneously.

Figure 27(a) is a concentration-time change curve of semaglutide in plasma as a control administered subcutaneously to SD rats in the test.

Figure 27(b) and Figure 27(c) are respectively the concentration-time change curves of Tirzepatide in the plasma of the subject SD rats injected subcutaneously as a control.

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

Figure 29 is the blood glucose-time change curve of the DIO mice tested.

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

Figure 31 is a bar graph showing changes in serum insulin levels in DIO mice.

Figure 32 is a histogram of serum biochemical index changes (UREA, TG, CHO, HDL, LDL, CREA) in DIO mice.

Figure 33 is a bar graph showing changes in serum biochemical indicators (ALT, AST, ALB, TBIL) in DIO mice.

Figure 34 Histogram of changes in body fat percentage of DIO mice tested.

Figure 35 is a bar graph showing changes in triglyceride content in DIO mice.

Figure 36 is a body weight-time change curve of DIO mice tested after long-term administration.

Figure 37 is a weight loss percentage-time change curve of DIO mice administered with different doses.

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. Lys represents lysine, and its structural formula is NH ₂ (CH ₂ ) ₄ CH(NH ₂ )COOH. In the polypeptide skeleton, Lys is connected to other amino acid residues with -NH-CH-C(O)-, NH ₂ ( CH ₂ ) ₄ - (i.e., ε-amino group) is attached as a side group to the α carbon atom, and the α-amino group is located in the polypeptide backbone. ε-Lys also represents lysine, and its structural formula is also NH ₂ (CH ₂ ) ₄ CH(NH ₂ )COOH. The difference is that ε-Lys is -NH-(CH ₂ ) ₄ -CH-C-(O )-connected to other amino acid residues, the ε-amino group is located in the polypeptide backbone, and the α-amino group is located in the side chain.

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 a 2-aminoisobutyric acid 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 histidine (His) at position 1, the C-terminal amino acid is glycine (Gly) at position 34, and lysine (Lys) is at position 20.

Compared with formula III to formula V and formula VII to formula IX, in the compound of formula VI, two adjacent glycines -NH-CH at positions 29 and 30 are replaced by -CH ₂ -CH ₂ - units. ₂ -C(O)-NH-CH ₂ -C(O)- contains the peptide bond. Although the -NH-(CH ₂ ) ₄ -C(O)- unit thus obtained is no longer the residue of the natural α-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 still formally numbered herein as amino acids 29 and 30 occupying 2 positions.

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 that, upon administration of a single or multiple doses to an individual in need thereof, provides the desired effect (i.e., can produce a clinically measurable effect of the individual's condition in the individual being diagnosed or treated). differences, such as, for example, a reduction in blood glucose and/or a reduction in weight or fat) in the 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 that is effective for an individual, many factors are considered, including, but not limited to, the species of the mammal, its size, age and 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 with the structure shown below.

[GLP-1 receptor and GCG receptor co-agonist]

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

in,

n is any integer from 2 to 6, and

SEQ ID NO:1 is the OXM (1-26) sequence: HSQGTFTSDYSKYLDSRRAQDFVQWL, namely His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg -Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu.

"The amino acid sequence of L ₁ is at least 69% identical to SEQ ID NO: 1" means that L ₁ has at least 18 amino acids in total at positions 1 to 26 among the 26 amino acids at positions 1 to 26 compared with SEQ ID NO: 1. Points have the same amino acid, for example at a total of 18, 19, 20, 21, 22, 23, 24, 25 or 26 positions.

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

In some examples, the amino acid sequence of L ₁ is at least 69% identical to SEQ ID NO: 1, for example, 69%, 73%, 77%, 81%, 85%, 88%, 92%, 96% Or 100% identical.

In some examples, the amino acid sequence of L ₁ is 69%, 73%, or 77% identical to SEQ ID NO:1.

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

According to the present disclosure, by modifying the peptide chain of wild-type OXM, in particular by adopting the glycine residue Gly at both positions 29 and 30 of the peptide chain, or by combining positions 29 and 30 as a whole and at the The -NH-(CH ₂ ) _n -C(O)-unit (n is an integer from 2 to 6) is used at the site to replace the common -NH-(CH ₂ )-C(O)-unit in the peptide chain to obtain The compounds of formula I can surprisingly maintain high activity on GLP-1 receptors and GCG receptors, providing blood sugar lowering and weight loss effects.

In some examples, L ₁ includes a substitution selected from S2(Aib) and S2(β-Ala) compared to SEQ ID NO:1.

In some examples, L ₁ contains a Q3E substitution compared to SEQ ID NO: 1.

In some examples, L ₁ contains a T7K substitution compared to SEQ ID NO: 1.

In some examples, L ₁ contains a K12 (epsilon-K) substitution compared to SEQ ID NO: 1.

In some examples, L ₁ contains an R17K substitution compared to SEQ ID NO: 1.

In some examples, L ₁ contains an R18K substitution compared to SEQ ID NO: 1.

In some examples, L ₁ contains a Q20K substitution compared to SEQ ID NO: 1.

In some examples, L ₁ includes a substitution selected from D21E and D20A compared to SEQ ID NO: 1.

In some examples, L ₁ contains the Q24E substitution compared to SEQ ID NO: 1.

In some examples, in Formula I, when the amino acid at position 20 is lysine (Lys), the C ₁₂ -C ₂₄ aliphatic diacid is conjugated to the lysine (Lys) via a direct bond or via a linker. ) side chain of the ε-amino group 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 to 2, thus giving the compound excellent in vivo and in vitro activity;

Preferably, the linker is (AEEA) ₂ -(γ-Glu), and the C ₁₂ -C ₂₄ aliphatic diacid is octadecanedioic acid or eicosanedioic acid.

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 acid at position 21 is alanine (Ala), the α-carbon atom of alanine at position 21 (Ala) is combined with alanine (Ala) at position 28 via an alkenyl group containing 16 carbon atoms. ) are connected to the α-carbon atoms.

In some examples, in formula I, when aspartic acid (Asp) or glutamic acid (Glu) containing side chain carboxyl groups, and lysine (Lys), arginine (Lys), arginine ( Arg) or histidine (His), the side chain carboxyl group of aspartic acid (Asp) or glutamic acid (Glu) is in contact with lysine (Lys), arginine (Arg) or histidine (His) The side chain amino group can form a ring by forming an amide bond;

Preferably, when the amino acid at position 3 is glutamic acid (Glu) and the amino acid at position 7 is lysine (Lys), the γ-carboxyl group of glutamic acid (Glu) at position 3 is in contact with the lysine at position 7 (Lys). The ε-amino group of Lys) forms a ring by forming an amide bond.

According to the present disclosure, there is also provided a compound of Formula II below or a pharmaceutically acceptable salt thereof:

His ₁ -X ₂ -X ₃ -Gly ₄ -Thr ₅ -Phe ₆ -X ₇ -Ser ₈ -Asp ₉ -Tyr ₁₀ -Ser ₁₁ -X ₁₂ -Tyr ₁₃ -X ₁₄ -Asp ₁₅ -Ser ₁₆ -Lys ₁₇ -Lys ₁₈ -Ala ₁₉ -Lys ₂₀ -X ₂₁ -Phe ₂₂ -Val ₂₃ -Glu ₂₄ -Trp 25 -Leu ₂₆ -Leu ₂₇ -Ala ₂₈ _-X ₂₉ -X ₃₀ -Pro ₃₁ -Ser ₃₂ -Ser ₃₃ -Gly ₃₄ -NH ₂

Formula II,

Wherein, X ₂ , X ₃ , X ₇ , X ₁₂ , X ₁₄ and X ₂₁ are each independently selected from natural _amino acids or non-natural amino acid residues, and X ₂₉ _and ₃₀ as a whole represents -NH-(CH ₂ ) ₄ -C(O)-.

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

_X3 is an amino acid residue selected from Gln and Glu,

X ₇ is an amino acid residue selected from Thr and Lys,

X ₁₂ is an amino acid residue selected from Lys and (ε-Lys),

X ₁₄ is an amino acid residue selected from Leu and (α-meL), and

X ₂₁ is an amino acid residue selected from Glu and Ala.

As previously mentioned, the compounds of Formula II of the present disclosure surprisingly possess co-agonism at GLP-1 receptors and GCG receptors, providing hypoglycemic and weight loss effects. At the same time, the compound of formula II has high plasma stability, thereby having pharmacokinetic characteristics that support once-weekly subcutaneous injection administration in humans, thereby improving patient compliance.

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

His-(Aib)-Gln-Gly-(D-Thr)-Phe-(D-Thr)-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys- Glu-Phe-Val-Glu-Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH ₂

Formula III,

His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-(α-meL)-Asp-Ser-Lys-Lys-Ala-Lys-Glu-Phe- Val-Glu-Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH ₂

Formula IV,

His-(β-Ala)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys-Glu-Phe-Val- Glu-Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH ₂

Formula V,

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

Formula VI,

His-(Aib)-Glu-Gly-Thr-Phe-Lys-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu- Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH ₂

Formula VII,

His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-(ε-Lys)-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys-Glu-Phe- Val-Glu-Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH ₂

Formula VIII, and

His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys-Ala-Phe-Val-Glu- Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH ₂

Formula IX.

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

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

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

In some examples, in Formula I or Formula II, the amino acids at positions 5 and 7 are both L-Thr or both are D-Thr.

In some examples, in any of Formulas I to IX, 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, decenyl or hexadecenyl; 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).

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 one embodiment, 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 one embodiment, conjugation is acylation.

In one embodiment, a compound of the invention includes an aliphatic diacid moiety conjugated to the epsilon-amino group of the K side chain of lysine at position 20 via a linker.

In one embodiment, compounds of the present invention include an aliphatic diacid moiety conjugated directly, without a linker, to a natural or non-natural amino acid having functional groups available for conjugation.

In some examples, wherein in any of Formula II to Formula IX, at lysine (Lys) at position 20, the C ₁₂ -C ₂₄ aliphatic diacid is conjugated via a direct bond or via a linker. The linker is chemically modified by being coupled to the ε-amino group of the lysine (Lys) side chain, 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 to 2, thereby imparting excellent in vivo and in vitro activity to the compound. 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 the context of the present invention, AEEA is an abbreviation for [2-(2-amino-ethoxy)-ethoxy]-acetyl, which means [2-(2-amino-ethoxy)-ethoxy]-acetyl. base. γ-Glu represents γ-glutamyl. Ahx is the abbreviation of amino hexanoyl, which means aminocaproyl. β-Ala represents β-alanyl group.

In some examples, the linker is (AEEA) ₂ -(γ-Glu), and the C ₁₂ -C ₂₄ aliphatic diacid is octadecanedioic acid or eicosanedioic acid.

In some examples, in any of Formula II to Formula IX, octadecanedioic acid or eicosanedioic acid is conjugated to lysine at position 20 via (AEEA) _2- (γ-Glu) (Lys) side chain ε-amino group is chemically modified.

Taking the compound NBB-C007 shown below (corresponding to formula VI) as an example, at the lysine at position 20, the first [2-(2-amino-ethoxy)-ethoxy]-acetyl The base (AEEA) unit is connected to the ε-amino group of the lysine side chain through an acyl group, and the second [2-(2-amino-ethoxy)-ethoxy]-acetyl (AEEA) unit is connected to the ε-amino group of the lysine side chain through an acyl group. The amino group of one [2-(2-amino-ethoxy)-ethoxy]-acetyl (AEEA) unit is connected, and the γ-glutamyl (γ-Glu) group is connected to the second [2 -(2-Amino-ethoxy)-ethoxy]-acetyl (AEEA) unit is connected to the amino group, and one terminal carboxyl group of eicosandioic acid (C ₂₀ diacid) is removed from the hydroxyl group to obtain the terminal acyl group, and via The terminal acyl group is connected to the amino group of γ-glutamyl (γ-Glu), 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 IX helps provide the compounds with GLP-1 receptors and GCP receptors, and offers the potential to generate long-acting compounds.

In some examples, in any one of Formula I to Formula IX, 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 16 carbon atoms. In some examples, in Formula IX, the alpha-carbon atom of alanine (Ala) at position 21 is connected to the alpha-carbon atom of alanine (Ala) at position 28 via an alkenyl group containing 16 carbon atoms. .

In particular, compounds of formula VI contain -NH-(CH ₂ ) ₄ -C(O)- units at positions 29 and 30, rather than two adjacent glycine residues as in other compounds herein. Based on -NH-CH ₂ -C(O)-NH-CH ₂ -C(O)-, it unexpectedly maintains co-agonism on GLP-1 receptors and GCG receptors, providing the effects of lowering blood sugar and reducing weight. . At the same time, the compound of Formula VI has a long half-life, which supports the pharmacokinetic characteristics 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, in any one of Formula II to Formula IX, 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. In some examples, in Formula VII, the γ-carboxyl group of glutamic acid (Glu) at position 3 and the ε-amino group of lysine (Lys) at position 7 form a ring by forming an amide bond.

In some examples, in any of Formulas I to IX, the histidine (His) at position 1 (i.e., the N-terminus) is amidated, for example, by -C _m H _2m+1 - C(O)- is attached to the amino group of histidine (His) to be amidated, 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.

[Preparation method of GLP-1/GCG 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) and the side chain amino group of lysine (Lys) are protected by Boc (tert-butoxycarbonyl group) group, tert-butoxycarbonyl) protection; the side chain hydroxyl group of serine (Ser), the side chain hydroxyl group of threonine (Thr) and the side chain phenol group of tyrosine (Tyr) are protected by tBu (tert-butyl, 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 amino acids from position 2 to position 4 protected by Fmoc (from the rightmost C end); 2 glycines protected by Fmoc (for compounds other than compound NBB-C007 Compounds other than the NBB-C series), or 5-aminovaleric acid (Fmoc-NH-(CH ₂ ) ₄ -C(O)OH) protected by Fmoc (for compound NBB-C007); and protected by Fmoc (from the rightmost C-terminal) to the 34th amino acid; repeat the previous coupling reaction to form an amide bond, deprotection to remove the Fmoc protecting group, and cleaning cycles in sequence.

iii) Deprotection and chemical modification of lysine at position 15 (from the rightmost C terminus) (corresponding to lysine at position 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 15 (from the rightmost C end) and wash.

Then, the Fmoc-protected linker Fmoc-AEEA was used to couple and deprotect the ε-amino group of lysine at position 15 (from the rightmost C end). In a similar manner, repeat this coupling and deprotection operation once to connect the second adapter AEEA.

Then, Fmoc and OtBu protected glutamic acid (Fmoc-Glu(OtBu)-OH) and condensation agent (HCTU) are used to couple with the amino group of the second linker AEEA 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:

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 to a subject 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), and 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, as follows: 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-C007]

(1) Resin swelling

a) Weigh 0.63g (0.2mmol) of Rink-Amide-AM resin 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 "(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, the polypeptide is formed.

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

a) Weigh 237 mg (0.8 mmol) of Fmoc-Gly-OH (glycine) and 314 mg (0.8 mmol) of the condensation agent HCTU into a 10 mL EP tube, add 6 mL of DMF to the EP tube to dissolve, shake well, and then Add 265 μL (1.6 mmol) of N,N-diisopropylethylamine (DIEA) to the EP tube to obtain a mixed solution.

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

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

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

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

Similar to connecting the first amino acid (from the rightmost C end) in step (3), connect the second to fourth amino acids in sequence, the only difference is that Fmoc-Ser(tBu)-OH (serine) is used respectively , Fmoc-Ser(tBu)-OH (serine), and Fmoc-Pro-OH (proline), as raw materials for coupling the second to fourth amino acids.

(5) Connect (from the rightmost C end) the "5th and 6th" 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".

(6) Connect (from the rightmost C end) the 7th to the 34th amino acid

Similar to connecting the first amino acid (from the rightmost C end) in step (3), connect the 7th to 34th amino acids in sequence, the only difference is that Fmoc-Ala-OH (alanine), Fmoc-Leu-OH(leucine), Fmoc-Leu-OH(leucine), Fmoc-Trp(Boc)-OH(tryptophan), Fmoc-Glu(OtBu)-OH(glutamic acid), Fmoc-Val-OH(valine), Fmoc-Phe-OH(phenylalanine), Fmoc-Glu(OtBu)-OH(glutamic acid), Fmoc-Lys(Boc)-OH(lysine) , Fmoc-Ala-OH(alanine), Fmoc-Lys(Boc)-OH(lysine), Fmoc-Lys(Boc)-OH(lysine), Fmoc-Ser(tBu)-OH(serine) ), Fmoc-Asp(OtBu)-OH(aspartic acid), Fmoc-Leu-OH(leucine), Fmoc-Tyr(tBu)-OH(tyrosine), Fmoc-Lys(Boc)-OH (lysine), Fmoc-Ser(tBu)-OH(serine), Fmoc-Tyr(tBu)-OH(tyrosine), Fmoc-Asp(OtBu)-OH(aspartic acid), Fmoc-Ser (tBu)-OH(serine), Fmoc-Thr(tBu)-OH(threonine), Fmoc-Phe-OH(phenylalanine), Fmoc-Thr(tBu)-OH(threonine), Fmoc -Gly-OH(glycine), Fmoc-Gln(Trt)-OH(glutamine), Fmoc-(Aib)-OH(2-aminoisobutyric acid) and Fmoc-His(Trt)-OH(histidine ), as the raw material for coupling the 7th to 34th amino acids (from the rightmost C-terminus).

(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 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/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) Add 40 mL of glacial ether to the 50 mL centrifuge tube. After shaking the centrifuge tube appropriately, put the centrifuge tube into the centrifuge at a speed of 3500 rpm and centrifuge for 3 minutes; after the centrifugation is completed, discard the supernatant.

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

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

The purified peptide was separated using Shimadzu semi-preparative liquid chromatography. Figure 1 shows the ESI-MS (electrospray mass spectrometry) characterization of the purified peptide NBB-C007. The peaks on the figure represent the molecular weights of different mass-to-charge ratios. Figure 2 is a high performance liquid chromatography analysis chart, showing that the synthesized NBB-C007 has a purity of 95%.

[Synthesis of other compounds in the NBB-C series]

Other compounds of the NBB-C series were prepared in a manner similar to the preparation of compound NBB-C007, except that the Fmoc protected amino acid raw materials, linker raw materials and/or aliphatic diacid sources, and other various modification groups were changed according to each structural formula ( if so).

The cyclization operation of NBB-C006 is as follows:

At the 7th and 14th positions (from the rightmost C end), Fmoc-S8-OH (Cas No.: 288617-75-4) and Fmoc-R8-OH (Cas No.: 945212-26- 0) As the amino acid raw material, weigh 64 mg of Grubbs catalyst (Cas No. 172222-30-9) into a 10 mL EP tube, and then add 4 mL 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 R8 unit and the S8 unit undergo olefin metathesis reaction, resulting in cyclization. After this step is completed, continue to extend the peptide chain, and sequentially access the 15th to 34th amino acids (from the rightmost C end).

The cyclization operation of NBB-C008 is as follows:

In the extension of the peptide chain: at the 14th, 28th and 32nd positions (from the rightmost C end), Fmoc-Lys(Dde)-OH, Fmoc-Lys(Alloc)-OH and Fmoc-Glu are used respectively. (OAll)-OH as amino acid raw material. After the peptide chain elongation is completed, the resin is washed.

(Counting from the leftmost N segment) Dehydration cyclization of the ε-amino group of lysine at position 7 and the γ-carboxyl group of glutamic acid at position 3: Weigh 232 mg (0.2 mmol) of tetrakis triphenylphosphine palladium and add 10 mL of In the EP tube, add 4 mL of DCM and 4 mL of DMF to dissolve, mix thoroughly, and then add 248 μL (0.2 mmol) of phenylsilane to the EP tube. Transfer the solution to a peptide synthesis tube. Transfer the peptide synthesis tube to a 33°C constant-temperature shaker and shake for 3 hours, then take it out and wash it. This step removes the side chain protecting groups at positions 7 and 3 (from the leftmost N segment), exposing the ε-amino group of lysine at position 7 and glutamine at position 3 (from the leftmost N segment). The γ-carboxyl group of the acid. Weigh 78 mg (1.52 mmol) of HOBt into a 10 mL EP tube, add 6 mL of DMF to dissolve, then add 250 μL (1.52 mmol) of DIC, and shake well. Mix the above mixture evenly and then add it to the peptide synthesis tube, transfer to a 33°C constant temperature shaker and shake for 12 hours, then take it out, thus (from the leftmost N segment) the ε-amino group of lysine at position 7 and the ε-amino group at position 3 The γ-carboxyl group of glutamic acid is dehydrated to form an amide group and cyclized.

Remove Dde from the 20th Lys (Dde) (from the leftmost N end): pipette 0.3mL of hydrazine hydrate into a 10mL EP tube, then add 8mL of DMF and shake thoroughly. Mix the above mixture evenly and transfer it to a peptide synthesis tube, then transfer it to a 33°C constant-temperature shaker for shaking for 10 minutes, then take it out and drain the solvent. Repeat the above steps once to completely remove the Dde protecting group.

Figures 1 to 14 are respectively ESI-MS (electrospray mass spectrometry) characterization and high performance liquid chromatography analysis of NBB-C series compounds. It can be seen that the purity of these compounds is ≥95%.

[In vitro activity assay: cAMP detection]

Experimental reagents

Laboratory equipment

名称name	公司company	型号model
酶标仪microplate reader	TECANTECAN		INFINITE 200 PROINFINITE 200 PRO
二氧化碳培养箱carbon dioxide incubator	赛默飞Thermo Fisher	31313131
生物洁净工作台Biological clean workbench	苏州安泰空气技术有限公司Suzhou Antai Air Technology Co., Ltd.	BLB-1300BLB-1300
显微镜microscope	NikonNikon	ECLIPSE Ts2-FLECLIPSE Ts2-FL
细胞计数仪cell counter	BIO-RADBIO-RAD	TC20 ^TM ^TC20TM
冰箱refrigerator	海尔Haier	BCD-252WXPSBCD-252WXPS
医用低温冰箱Medical low temperature refrigerator	赛默飞Thermo Fisher	902-ULTS902-ULTS

Human GLP-1R receptor, GIPR receptor and GCGR receptor were cloned into pcDNA3.1 vector respectively. Use Lipofectamin3000 Transfection 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, dilute it to 2×10 ⁶ cells/mL, and spread 5 μL of cells into a 384-well plate. Then add 5 μl DMSO dissolved and ten-fold gradient dilution (2×10 ^-6 , 2×10 ^-7 ,..., 2×10 ^-15 M) of the previously prepared NBB-C series compounds in DMEM containing 500 μM IBMX. Or as a comparison, any of the semaglutide (self-made), incubate at 37°C for 30 minutes, add 5 μL each of cAMP-d2 and anti-cAMP in the cAMP-Gs Dynamic kit, incubate at room temperature for 1 hour, and then use TECAN enzyme label The instrument reads, the excitation wavelength is 340nm, and the emission wavelengths are 620nm and 655nm. 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 15 to Figure 21 and the table below.

As can be seen from the above table, compounds NBB-C003, NBB-C004, NBB-C006 and NBB-C007 exhibit dual activity on human GLP-1 receptors and GCG receptors. In particular, for compound NBB-C007, compared with other NBB-C series compounds, NH-(CH ₂ ) ₄ -C(O)-units are included in the peptide chain instead of two adjacent glycine-NH-CH The ₂ -C(O)-NH-CH ₂ -C(O)-unit can still surprisingly maintain excellent dual activity on human GLP-1 receptor and GCG receptor.

[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 vehicle control ("solvent group", 0.05% NaHCO ₃ solution), the semaglutide control (dose 50 nmol/kg) dissolved in the vehicle (0.05% NaHCO ₃ solution), and the previous implementation were used. The NBB-C007 compound (dose 50 nmol/kg) prepared in the example 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 measured continuously for 72 hours. Blood glucose levels in mice. At the same time, the body weight of db/db mice was monitored at 0h, 24h, and 72h. The test results are shown in Figure 22 to Figure 24 and the table below.

It can be seen that the NBB-C007 compound according to the present disclosure has obvious blood sugar-lowering and weight-loss effects that are comparable to or better than semaglutide, and can maintain the pharmacodynamic effect for 72 hours, which shows that NBB- The C007 compound can achieve clinical treatment of type II diabetes and weight loss, and can be administered once a week.

[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 (dose 30 nmol/kg), Tirzepatide control (dose 30 nmol/kg), and NBB-C007 prepared in the previous examples were used respectively. The compound (dose 30 nmol/kg) was administered to db/db mice ad libitum by 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 the 4th, 6th, 8th, and 10th weeks 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 compound NBB-C007 according to the present disclosure 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-min 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 - C007 compound (dose 25 nmol/kg) was administered to C57BL/6 mice ad libitum by subcutaneous injection, and glucose solution (2 g/kg) was administered intraperitoneally at the same time, and blood glucose was measured at 15 min, 30 min, 60 min, and 120 min after administration. value. 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). See the table below and Figure 25 for test results.

It can be seen from the above glucose tolerance test that the NBB-C007 compound according to the present disclosure has a comparable hypoglycemic effect to that of semaglutide, and the glucose AUC was reduced in both the first and second days of testing, indicating that according to the present disclosure The disclosed NBB-C007 compound has good efficacy in postprandial blood sugar control.

[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 vehicle control (0.05% NaHCO ₃ solution), as well as the NBB-C007 compound prepared in the previous example (dose 1 mg/kg), semaglutide (dose 1 mg/kg) and alkaline dissolved in the vehicle were administered subcutaneously. Tirzepatide control (dose 0.5 mg/kg) (dose 1 mg/kg), respectively, at 0.25h, 0.5h, 1h, 2h, 4h, 8h, 24h, 48h, 72h, 96h, 108h, 120h, At 144h, 168h, 204h, 240h, 300h and/or 324h, collect 0.3mL of venous blood from the jugular vein and place it in an EDTA2K anticoagulant tube. Centrifuge at 8000rpm for 5 minutes to collect the plasma. Determine the plasma concentration of the test substance by LC-MS/MS method. , use methanol to extract compounds from plasma samples. 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 minutes, centrifuge for 10 minutes (3,900 rpm), and transfer the supernatant to another clean 96-well plate. LC-MS/MS analysis. The test results are shown in the table below and Figure 26(a) to Figure 27(c).

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

As can be seen, compound NBB-C007 reaches mean maximum plasma concentrations approximately 24 hours after subcutaneous administration. Among them, compound NBB-C007 has a half-life of 11.88 hours in SD rats and has good metabolic stability, supporting the possibility of 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-C007 (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 compound NBB-C007 has a half-life of 79.19 hours in SD rats and has good metabolic stability, 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 of 50 nmol/kg), and NBB-C007 group (administration dose of 50 nmol/kg). Mice were administered weekly The above test substances were administered by two subcutaneous injections, with a dosage volume of 5 mL/kg. The normal model group and the model control group ("solvent group") were administered vehicle control (0.05% NaHCO ₃ solution) for 4 consecutive weeks. Stop taking the drug, or continue taking it. 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 28.

As can be seen from Figure 28, 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 NBB prepared in the previous embodiment -The body weight of mice tested with C007 compound decreased by approximately 40%.

2. 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 29.

As can be seen from Figure 29, the test mice administered the NBB-C007 compound prepared in the previous example had better insulin sensitivity compared to the mice administered semaglutide.

3. 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 30.

As can be seen from Figure 30, the test mice administered the NBB-C007 compound prepared in the previous example had better postprandial blood sugar control compared to the mice administered semaglutide.

4. 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 31 to Figure 33.

As can be seen from Figures 31 to 33, the model control group showed high serum insulin content, a typical diabetes indicator. Compared with mice administered semaglutide, test mice administered the NBB-C007 compound prepared in the previous example had a comparable or better tendency to reduce serum insulin levels, and comparable or better protection of liver function. , protect kidney function and improve lipid metabolism.

5. 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. See Figure 34 and Figure 35 for test results.

As can be seen from Figure 34 and Figure 35, compared with mice administered semaglutide, the test mice administered the NBB-C007 compound prepared in the previous embodiment had comparable or better postprandial body fat reduction. and triglyceride reduction.

[Long-term weight loss effect of obese DIO mice]

C57BL/6 mice (Nanjing Jicui Yaokang), male, were selected and fed with high-fat feed (60% Kcal fat, D12492) for 10-12 weeks. The weight of the mice at the beginning of the experiment was between 38-45g. It was determined that the weight of the mice reached the ratio of The animals on a normal diet were more than 30% higher. After random blood sugar and body weight were detected, they were randomly divided into groups, with 6 animals in each group. Six 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 30nmol/kg), NBB-C007 group (administration dose 30nmol/kg), Tirzepatide group ( Dosage: 30 nmol/kg). DIO 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), continuously Administration is given for 50 days, during which body weight, food intake and other indicators are monitored. Among them, the results of body weight are shown in Figure 36.

As can be seen from Figure 36, during the 50-day long-term administration period, the weight loss of obese DIO mice after administration of the NBB-C007 compound prepared in the previous example was significantly better than that after administration of semaglutide, Tirzepatide and solvent, and surprisingly, the weight of obese DIO mice even dropped to that of normal mice.

[Screening of dosage in obese DIO mice]

C57BL/6 mice (Nanjing Jicui Yaokang), male, were selected and fed with high-fat feed (60% Kcal fat, D12492) for 10-12 weeks. The weight of the mice at the beginning of the experiment was between 38-45g. It was determined that the weight of the mice reached the ratio of The animals on a normal diet were more than 30% higher. After random blood sugar and body weight were detected, they were randomly divided into groups, with 5 animals in each group. Five 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") and NBB-C007 group (administration dosages of 5 nmol/kg, 10 nmol/kg, and 15 nmol/kg). DIO 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), continuously Administration was given for 28 days, during which body weight, food intake and other indicators were monitored. Among them, the results of body weight are shown in Figure 37 and the table below.

As can be seen from Figure 37 and the table above, as the dosage increases from 5 nmol/kg to 15 nmol/kg, the weight loss effect of obese DIO mice becomes more and more obvious.

Claims

A compound of formula I below or a pharmaceutically acceptable salt thereof:

L 1 -Leu 27 -Ala 28 -X 29 -X 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -NH 2Formula I,

in,

L 1 is a peptide analog of the OXM (1-26) peptide, said L 1 is a peptide consisting of 26 amino acids, and the amino acid sequence of said L 1 is at least 69% identical to SEQ ID NO: 1,

X 29 and X 30 are each independently Gly, or X 29 and X 30 as a whole represent -NH-(CH 2 ) n -C(O)-,

n is any integer from 2 to 6, and

The compound has GLP-1 receptor agonist activity and GCG receptor agonist activity.
The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein compared to SEQ ID NO: 1, L 1 contains a substitution selected from S2 (Aib) and S2 (β-Ala).
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 Q3E substitution.
The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein L 1 contains a T7K 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 contains a K12 (ε-K) substitution.
The compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein L 1 contains an R17K 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 an R18K substitution.
The compound according to any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein L 1 contains a Q20K substitution compared to SEQ ID NO: 1.
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 D21E and D20A.
The compound according to any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein L 1 contains a Q24E substitution compared to SEQ ID NO: 1.
The compound according to any one of claims 1-10 or a pharmaceutically acceptable salt thereof, wherein in formula I, when the amino acid at position 20 is lysine (Lys), via a direct bond or via a linker The C 12 -C 24 aliphatic diacid is conjugated to the ε-amino group of the lysine (Lys) side chain for chemical modification, 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 to 2;

Preferably, the linker is (AEEA) 2 -(γ-Glu), and the C 12 -C 24 aliphatic diacid is octadecanedioic acid or eicosanedioic acid.
The compound according to any one of claims 1 to 11 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 acid at position 21 is alanine (Ala), the α-carbon atom of alanine at position 21 (Ala) is combined with alanine (Ala) at position 28 via an alkenyl group containing 16 carbon atoms. ) are connected to the α-carbon atoms.
The compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein in formula I, when aspartic acid (Asp) or glutamic acid (Asp) containing a side chain carboxyl group is present at the same time Glu), and lysine (Lys), arginine (Arg) or histidine (His) containing side chain amino groups, the side chain carboxyl group of aspartic acid (Asp) or glutamic acid (Glu) and The side chain amino group of lysine (Lys), arginine (Arg) or histidine (His) can form a ring by forming an amide bond;

Preferably, when the amino acid at position 3 is glutamic acid (Glu) and the amino acid at position 7 is lysine (Lys), the γ-carboxyl group of glutamic acid (Glu) at position 3 is in contact with the lysine at position 7 (Lys). The ε-amino group of Lys) forms a ring by forming an amide bond.
A compound of formula II below or a pharmaceutically acceptable salt thereof:

His 1 -X 2 -X 3 -Gly 4 -Thr 5 -Phe 6 -X 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -X 12 -Tyr 13 -X 14 -Asp 15 -Ser 16 -Lys 17 -Lys 18 -Ala 19 -Lys 20 -X 21 -Phe 22 -Val 23 -Glu 24 -Trp 25 -Leu 26 -Leu 27 -Ala 28 -X 29 -X 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -NH 2

Formula II,

Wherein, X 2 , X 3 , X 7 , X 12 , X 14 and X 21 are each independently selected from natural amino acids or non-natural amino acid residues, and X 29 and 30 as a whole represents -NH-(CH 2 ) 4 -C(O)-.
The compound according to claim 14 or a pharmaceutically acceptable salt thereof, wherein,

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

X3 is an amino acid residue selected from Gln and Glu,

X 7 is an amino acid residue selected from Thr and Lys,

X 12 is an amino acid residue selected from Lys and (ε-Lys),

X 14 is an amino acid residue selected from Leu and (α-meL), and

X 21 is an amino acid residue selected from Glu and Ala.
The compound according to claim 14 or 15, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure selected from any one of the following formula III to the following formula IX:

His-(Aib)-Gln-Gly-(D-Thr)-Phe-(D-Thr)-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys- Glu-Phe-Val-Glu-Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH 2

Formula III,

His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-(α-meL)-Asp-Ser-Lys-Lys-Ala-Lys-Glu-Phe- Val-Glu-Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH 2

Formula IV,

His-(β-Ala)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys-Glu-Phe-Val- Glu-Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH 2

Formula V,

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

Formula VI,

His-(Aib)-Glu-Gly-Thr-Phe-Lys-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys-Glu-Phe-Val-Glu- Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH 2

Formula VII,

His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- (ε-Lys)-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys-Glu-Phe- Val-Glu-Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH 2

Formula VIII, and

His-(Aib)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys-Lys-Ala-Lys-Ala-Phe-Val-Glu- Trp-Leu-Leu-Ala-GLy-Gly-Pro-Ser-Ser-Gly-NH 2

Formula IX.
The compound according to any one of claims 14-16 or a pharmaceutically acceptable salt thereof, wherein in any one of formula II to formula IX, at the lysine (Lys) at position 20 , 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 to 2;

Preferably, the linker is (AEEA) 2 -(γ-Glu), and the C 12 -C 24 aliphatic diacid is octadecanedioic acid or eicosanedioic acid.
The compound according to any one of claims 14-17 or a pharmaceutically acceptable salt thereof, wherein in any one of formula II to formula IX, 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 IX, the α-carbon atom of alanine (Ala) at position 21 is connected to the α-carbon atom of alanine (Ala) at position 28 via an alkenyl group containing 16 carbon atoms.
The compound according to any one of claims 14-18 or a pharmaceutically acceptable salt thereof, wherein in any one of Formula II to Formula IX, aspartic acid (Asp) or glutamic acid ( The side chain carboxyl group of Glu) and the side chain amino group of lysine (Lys), arginine (Arg) or histidine (His) can form a ring by forming an amide bond;

Preferably, in formula VII, the γ-carboxyl group of glutamic acid (Glu) at position 3 and the ε-amino group of lysine (Lys) at position 7 form a ring by forming an amide bond.
Pharmaceutical compositions containing:

The compound according to any one of claims 1-19 or a pharmaceutically acceptable salt thereof, and

Pharmaceutically acceptable carrier, diluent or excipient.
Use of a pharmaceutical composition according to claim 20 or a compound according to any one of claims 1 to 19 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 thereof an effective amount of a pharmaceutical composition according to claim 20 or a compound according to any one of claims 1-19 or its Pharmaceutically acceptable salt.
The method of claim 22, wherein the pharmaceutical composition, the compound, or a pharmaceutically acceptable salt thereof is administered to the individual by subcutaneous injection.
The method of claim 22 or 23, wherein the pharmaceutical composition, the compound, or a pharmaceutically acceptable salt thereof is administered to the subject once a week.