Polypeptide compound, pharmaceutical composition thereof and use thereof

Abstract

Disclosed in the present invention are a polypeptide compound, a pharmaceutical composition thereof and the use thereof. Specifically, disclosed in the present invention is a compound as shown in formula I or a pharmaceutically acceptable salt thereof. The polypeptide compound of the present invention has excellent solubility, or has a good binding activity toward an MC4R receptor, or has a relatively long conversion half-life, or has a good body weight reduction effect. A-Z (I)

Description

Polypeptide compound, pharmaceutical composition and use thereof

[0001] This application claims the benefit of Chinese patent application No. 2023117840602, filed on December 22, 2023. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to a polypeptide compound, a pharmaceutical composition and use thereof. Background Art

[0003] The melanocortin 4 receptor (MC4R) is a member of the melanocortin receptor family (MCRs). MCRs belong to class A of the G protein-coupled receptor (GPCR) superfamily and are composed of five subtypes (MC1R-MC5R). They are N-coupled receptors that increase intracellular cyclic adenosine monophosphate (cAMP) accumulation and mediate a variety of receptor responses in humans. MC4Rs are abundant in the mammalian central nervous system (CNS), particularly in the paraventricular nucleus of the hypothalamus, a region critical for energy homeostasis. In animal models and clinical pharmacological and genetic studies, MC4Rs have been shown to play a key role in regulating satiety. MC4R agonists have also been shown to be effective in suppressing appetite, reducing food intake, and reducing weight in patients with high body mass index (BMI) due to leptin, pro-opiomelanocortin (POMC), or MC4R deficiency. Therefore, MC4R is one of the most promising drug targets for the treatment of obesity.

[0004] Successful peptide agonists targeting the MC4R can be divided into two categories: linear peptide agonists and cyclic peptide agonists. Linear peptide agonists range in size from 4 to 16 amino acid residues, with 5 to 7 residues being the most common type. However, while linear peptide agonists activate the MC4R, they also activate other MC receptors, resulting in a lack of selectivity and a short half-life. Cyclic peptide agonists are based on linear peptides through cyclization via lactam bonds, lactonization, disulfide bonds, and triazole rings. Multiple studies have shown that cyclization can enhance agonist selectivity. However, for both linear and cyclic peptides, to exert MC4R agonistic activity, they must contain a conserved region called the "HFRW" in their amino acid sequence. In addition to peptide agonists, there are also small molecule agonists. These small molecules can incorporate various substituents within a rigid ring structure, resulting in higher MC receptor selectivity and potency, and are immune to protein degradation. In summary, all MC4R agonists typically contain at least one positively charged group and three aromatic rings, following the established HFRW motif. Early MC4R agonists were ineffective and associated with significant adverse reactions, including elevated blood pressure and heart rate. Setmelanotide, a second-generation MC4R agonist, has a favorable therapeutic index and good tolerability profile, with no reports of serious adverse cardiovascular events in early clinical studies. However, due to its short in vivo half-life of approximately 11 hours, setmelanotide requires daily subcutaneous administration, leading to poor patient compliance. Furthermore, its solubility is poor, requiring the addition of PEG to achieve a dosage form of only 10 mg / mL, and the PEG excipient also presents safety concerns.

[0005] Current strategies for extending the half-life of peptide drugs include the introduction of non-natural amino acids, cyclization, fusion with long-acting proteins (HAS (Human Serum Albumin) fusion, Fc fusion, XETN fusion, PAS fusion, etc.) or chemical coupling (fatty acid side chain modification, etc.), and conjugation with polymers (PEG modification, etc.).

[0006] Prodrug technology

[0007] In the present invention patent, unlike the currently marketed MC4R agonists, the strategy for extending the half-life of MC4R agonist analogs is to apply prodrug technology, which can be used to generate compounds with characteristics suitable for specific dosing frequencies. Prodrugs based on diketopiperazine (DKP) have been previously reported. Patents WO2010071807, WO2010080605, WO2011163012, and WO2011162968 all disclose various peptide-based prodrugs that are connected to, for example, glucagon superfamily peptides or other known drugs with amide bonds. Patents WO2014152460 and WO2016049174 disclose that peptide-based prodrugs of glucagon superfamily peptides and insulin have the effect of extending half-life. This technology chemically converts the parent drug in vivo through enzymatic or non-enzymatic processes through intramolecular cyclization of terminal dipeptidyl amide extension. Under physiological conditions, this intramolecular cyclization can occur as an enzyme-independent process. This prodrug technology can be used to optimize the properties of a drug to make it suitable for a specific dosing regimen, such as once-weekly dosing. Summary of the Invention

[0008] The present invention addresses the shortcomings of the prior art MC4R agonist setmelanotide, such as its short half-life, poor solubility, and safety of the co-solvent PEG. The present invention provides a polypeptide compound, pharmaceutical composition, and use thereof. The polypeptide compound of the present invention exhibits excellent solubility, strong binding activity to MC4R, a long conversion half-life, or a significant weight-reducing effect.

[0009] The present invention solves the above technical problems through the following technical solutions.

[0010] The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0011] AZ

[0012] I

[0013] Wherein, Z is a polypeptide analogue, and its amino acid sequence is Arg 1 -c[Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 ]-X1-X2-X3-X4-X5-X6-X7, where Arg 1 is the N-terminus of the amino acid sequence, and Z is connected to A via its N-terminus;

[0014] Aaa and Bbb independently represent a cysteine ​​residue, a homocysteine ​​(hCys) residue, or a penicillamine (Pen) residue;

[0015] Xxx is an alanine residue, a D-alanine residue, or does not exist;

[0016] Yyy is a threonine residue, a D-threonine residue, a serine residue, a D-serine residue, a histidine residue, a D-histidine residue, an asparagine residue, a D-asparagine residue, a glutamine residue or a D-glutamine residue;

[0017] c[Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 The "c" in ] means Aaa 2 The thiol group in Bbb 8 The sulfhydryl groups in the sulfhydryl group form a ring through a disulfide bond;

[0018] X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue, a D-lysine residue, a proline residue, a D-proline residue, a glutamic acid residue, a D-glutamic acid residue, an aspartic acid residue, a D-aspartic acid residue, a threonine residue, a D-threonine residue, a glycine residue, a D-glycine residue, an alanine residue, a D-alanine residue, an arginine residue, a D-arginine residue, a serine residue, a D-serine residue,

[0019] Cys 8 It is connected to X1 via an amide bond;

[0020] X1, X2, X3, X4, X5 and X6 are connected by amide bonds or ester bonds;

[0021] X7 is NH2 or OH;

[0022] X1, X2, X3, X4, X5 or X6 is connected to X7 through a carbonyl group;

[0023] When X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or D-lysine residue is unsubstituted or connected to L' in the form of an amide bond, and L' is -L1-L2-L3-L4-L5-L6-L7-L8;

[0024] L1, L2, L3, L4, L5 and L6 are each independently absent,

[0025] L7 is

[0026] L8 is n is 10, 11, 12, 13, 14, 15, 16, 17 or 18;

[0027] L1, L2, L3, L4, L5, L6, L7 and L8 are connected by amide bonds;

[0028] A does not exist, or B;

[0029] B is

[0030] X is H or -(CH2) m -NH-L;

[0031] m is 1, 2, 3 or 4;

[0032] L is H or L';

[0033] Y is H, C1-C4 alkyl or -(CH2) p -NH-L, p is 1, 2, 3 or 4;

[0034] The compound as shown in Formula I satisfies at least one of the following conditions:

[0035] Condition I: At least one of X1, X2, X3, X4, X5 and X6 is a lysine residue, a D-lysine residue, a proline residue, a D-proline residue, a glutamic acid residue, a D-glutamic acid residue, an aspartic acid residue, a D-aspartic acid residue, a threonine residue, a D-threonine residue, a glycine residue, a D-glycine residue, an alanine residue, a D-alanine residue, an arginine residue, a D-arginine residue, a serine residue, a D-serine residue, When X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond, and L' is -L1-L2-L3-L4-L5-L6-L7-L8;

[0036] Condition II: A is B.

[0037] The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0038] AZ

[0039] I

[0040] Wherein, Z is a polypeptide analogue, and its amino acid sequence is Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]-X1-X2-X3-X4-X5-X6-X7, where Arg 1 is the N-terminus of the amino acid sequence, and Z is connected to A via its N-terminus;

[0041] c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 The "c" in ] represents Cys 2 The thiol group and Cys 8 The sulfhydryl groups in the sulfhydryl group form a ring through a disulfide bond;

[0042] X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue, a D-lysine residue, a proline residue, a D-proline residue, a glutamic acid residue, a D-glutamic acid residue, an aspartic acid residue, a D-aspartic acid residue, a threonine residue, a D-threonine residue, a glycine residue, a D-glycine residue, an alanine residue, a D-alanine residue, an arginine residue, a D-arginine residue, a serine residue, a D-serine residue,

[0043] Cys 8 It is connected to X1 via an amide bond;

[0044] X1, X2, X3, X4, X5 and X6 are connected by amide bonds or ester bonds;

[0045] X7 is NH2 or OH;

[0046] X1, X2, X3, X4, X5 or X6 is connected to X7 through a carbonyl group;

[0047] When X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or D-lysine residue is unsubstituted or connected to L' in the form of an amide bond, and L' is -L1-L2-L3-L4-L5-L6-L7-L8;

[0048] L1, L2, L3, L4, L5 and L6 are each independently absent,

[0049] L7 is

[0050] L8 is n is 10, 11, 12, 13, 14, 15, 16, 17 or 18;

[0051] L1, L2, L3, L4, L5, L6, L7 and L8 are connected by amide bonds;

[0052] A does not exist, or B;

[0053] B is

[0054] X is H or -(CH2) m -NH-L;

[0055] m is 1, 2, 3 or 4;

[0056] L is H or L';

[0057] Y is H, C1-C4 alkyl or -(CH2) p -NH-L, p is 1, 2, 3 or 4;

[0058] When A is When at least one of X1, X2, X3, X4, X5 and X6 in Z exists.

[0059] In certain preferred embodiments of the present invention, the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, certain groups are defined as follows, and the unmentioned groups are the same as those described in any embodiment of the present invention (referred to as "in a certain embodiment of the present invention").

[0060] In one embodiment of the present invention, Aaa is a cysteine ​​residue or a homocysteine ​​residue.

[0061] In one embodiment of the present invention, Bbb is a cysteine ​​residue or a penicillamine residue.

[0062] In one embodiment of the present invention, Xxx is absent or a D-alanine residue.

[0063] In one embodiment of the present invention, Yyy is a threonine residue or a histidine residue.

[0064] In one embodiment of the present invention, Aaa 2 -Xxx 3-Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 is Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 .

[0065] In a certain embodiment of the present invention, when the compound as shown in Formula I satisfies at least Condition I, at least one of X1, X2, X3, X4, X5 and X6 is a lysine residue, a D-lysine residue, a glutamic acid residue, a D-glutamic acid residue, an arginine residue or a D-arginine residue, and when X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond.

[0066] In a certain embodiment of the present invention, when the compound of Formula I satisfies at least Condition I, at least one of X1, X2, X3, X4, X5 and X6 is a lysine residue, a glutamic acid residue or an arginine residue, and when X1, X2, X3, X4, X5 or X6 is a lysine residue, the ε-amino group of the side chain of the lysine residue is unsubstituted or connected to L' in the form of an amide bond.

[0067] In a certain embodiment of the present invention, when the compound of Formula I satisfies at least Condition I, at least one of X1, X2, X3, X4, X5 and X6 is a lysine residue.

[0068] In a certain embodiment of the present invention, when the compound of Formula I satisfies at least Condition I, -X1-X2-X3-X4-X5-X6- is -Lys-, -Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Glu-, -Glu-Glu-, -Glu -Glu-Glu-, -Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-Glu-, -Lys-Gl u-, -Lys-Glu-Lys-, -Lys-Glu-Glu-, -Glu-Lys-, -Lys-Glu-Glu-Lys-, -Arg-, -Arg-Arg, or any of the following structures:

[0069] In a certain embodiment of the present invention, when the compound of formula I satisfies at least condition 1, -X1-X2-X3-X4-X5-X6- is -Lys-, -Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Glu-, -Glu-Glu-, -Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-Glu-, -Lys-Glu-, -Lys-Glu-Lys-, -Lys-Glu-Glu-, -Glu-Lys-, -Lys-Glu-Glu-Lys-, -Arg-, -Arg-Arg, excellent It is selected from -Lys-, -Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys- or -Lys-Lys-Lys-Lys-Lys-Lys-, and -Lys-Lys-Lys-Lys- is more preferred.

[0070] In a certain embodiment of the present invention, when the compound as shown in Formula I satisfies Condition II, X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue, a D-lysine residue, a glutamic acid residue, a D-glutamic acid residue, an arginine residue or a D-arginine residue; when X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond.

[0071] In a certain embodiment of the present invention, when the compound as shown in Formula I satisfies Condition II, X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue, a glutamic acid residue or an arginine residue, preferably absent or a lysine residue; when X1, X2, X3, X4, X5 or X6 is a lysine residue, the ε-amino group of the side chain of the lysine residue is unsubstituted or connected to L' in the form of an amide bond.

[0072] In a certain embodiment of the present invention, when the compound of formula I satisfies condition II, -X1-X2-X3-X4-X5-X6- is absent, -Lys-, -Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Glu-, -Glu-Glu-, -G lu-Glu-Glu-, -Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-Glu-, -Lys-G lu-, -Lys-Glu-Lys-, -Lys-Glu-Glu-, -Glu-Lys-, -Lys-Glu-Glu-Lys-, -Arg-, -Arg-Arg, or any of the following structures:

[0073] In a certain embodiment of the present invention, when the compound of formula I satisfies condition II, -X1-X2-X3-X4-X5-X6- is absent, -Lys-, -Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Glu-, -Glu-Glu-, -Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu- -, -Glu-Glu-Glu-Glu-Glu-Glu-, -Lys-Glu-, -Lys-Glu-Lys-, -Lys-Glu-Glu-, -Glu-Lys-, -Lys-Glu-Glu-Lys-, -Arg-, -Arg-Arg, Preferred are -Lys-, -Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys- or -Lys-Lys-Lys-Lys-Lys-Lys-, and -Lys-Lys-Lys-Lys- is more preferred.

[0074] In one embodiment of the present invention, X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue or a D-lysine residue, and the ε-amino group of the side chain of the lysine residue or D-lysine residue is unsubstituted or connected to L' in the form of an amide bond.

[0075] In one embodiment of the present invention, X1, X2, X3, X4, X5 and X6 are each independently absent or a lysine residue, and the ε-amino group of the lysine residue side chain is unsubstituted or connected to L' in the form of an amide bond.

[0076] In one embodiment of the present invention, X1, X2, X3, X4, X5 and X6 are each independently absent or a lysine residue.

[0077] In one embodiment of the present invention, L1, L2, L3, L4, L5 and L6 are each independently absent,

[0078] In one embodiment of the present invention, in L', -L1-L2-L3-L4-L5-L6- is

[0079] Among them, the end marked with “#” represents the L1 end, and correspondingly, the end not marked with “#” represents the L6 end.

[0080] In a certain embodiment of the present invention, L8 is n is 10, 12, 16 or 18.

[0081] In one embodiment of the present invention, L' is:

[0082] Preferably

[0083] In one embodiment of the present invention, -X1-X2-X3-X4-X5-X6-X7 is -NH2, -Lys-NH2, -Lys-Lys-NH2, -Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-Lys-NH2, or any one of the following structures:

[0084] In one embodiment of the present invention, -X1-X2-X3-X4-X5-X6-X7 is -NH2, -Lys-NH2, -Lys-Lys-NH2, -Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-NH2 or -Lys-Lys-Lys-Lys-Lys-Lys-NH2.

[0085] In one embodiment of the present invention, -X1-X2-X3-X4-X5-X6-X7 is -NH2, -Lys-NH2, -Lys-Lys-NH2, -Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-NH2 or -Lys-Lys-Lys-Lys-Lys-Lys-NH2.

[0086] In one embodiment of the present invention, X is H, -(CH2)-NH-L, -(CH2)2-NH-L or -(CH2)4-NH-L.

[0087] In one embodiment of the present invention, X is H, Preferably H,

[0088] In one embodiment of the present invention, Y is H, C1-C4 alkyl or -(CH2)2-NH-L.

[0089] In one embodiment of the present invention, Y is H, CH3, -(CH2)2-NH2, Preferably it is CH3 or -(CH2)2-NH2, more preferably CH3.

[0090] In one embodiment of the present invention, A is absent or For example, if it does not exist,

[0091] In a certain embodiment of the present invention, when the compound shown in Formula I satisfies Condition I, A is absent,

[0092] In a certain embodiment of the present invention, when the compound shown in formula I satisfies condition II, A is

[0093] In one embodiment of the present invention, A is absent, and the compound shown in Formula I is Z, wherein Z is Arg 1 -c[Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8]-X1-X2-X3-X4-X5-X6-X7, -X1-X2-X3-X4-X5-X6- is -Lys-, -Lys-Lys-, -Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys- or -Lys-Lys-Lys-Lys-Lys-Lys-;

[0094] X7 is NH2;

[0095] Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 、hCys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 or hCys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 .

[0096] In one embodiment of the present invention, A is

[0097] In one embodiment of the present invention, A is

[0098] X is

[0099] Y is H or C1-C4 alkyl,

[0100] Z is Arg 1 -c[Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb8 ]-X1-X2-X3-X4-X5-X6-X7, X1-X2-X3-X4-X5-X6-X7 is -NH2 or -Lys-Lys-Lys-Lys-NH2;

[0101] Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 、hCys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 or hCys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 .

[0102] In one embodiment of the present invention, A is absent, and the compound shown in Formula I is Z, wherein Z is Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]-X1-X2-X3-X4-X5-X6-X7, X1, X2, X3, X4, X5 and X6 are each independently absent or a lysine residue, and X7 is NH2.

[0103] In one embodiment of the present invention, A is

[0104] X is H or -(CH2)4-NH2;

[0105] Y is C1-C4 alkyl or -(CH2) p -NH2, p is 1, 2, 3 or 4;

[0106] Z is Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]-X1-X2-X3-X4-X5-X6-X7, -X1-X2-X3-X4-X5-X6-X7 is -NH2 or -Lys-Lys-Lys-Lys-NH2.

[0107] In one embodiment of the present invention, A is

[0108] X is

[0109] Y is H or C1-C4 alkyl,

[0110] Z is Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]-X1-X2-X3-X4-X5-X6-X7, -X1-X2-X3-X4-X5-X6-X7 is -Lys-Lys-Lys-Lys-NH2.

[0111] In one embodiment of the present invention, the compound as shown in Formula I is any one of the following compounds:

[0112] The present invention also provides a pharmaceutical composition comprising a compound as shown in Formula I as described in any of the above schemes, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0113] The present invention also provides a use of the compound as shown in Formula I, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in any of the above schemes in the preparation of an MC4R agonist.

[0114] The present invention also provides a use of a compound as shown in Formula I, a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition according to any of the above schemes in the preparation of a drug for preventing and / or treating diseases mediated by MC4R excitation.

[0115] The present invention also provides a use of a compound as shown in Formula I, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in any of the above schemes in the preparation of a drug for preventing and / or treating obesity, polydactyly, retinal atrophy, gonadal dysgenesis, renal malformation, or cognitive impairment.

[0116] The present invention also provides a use of a compound as shown in Formula I according to any of the above schemes, a pharmaceutically acceptable salt thereof, or the above pharmaceutical composition in the preparation of a medicament for preventing and / or treating chronic weight management deficiency in adults and children aged 6 years and above with monogenic or syndromic obesity caused by proopiomelanocortin (POMC) deficiency, proprotein convertase subtilisin / kexin type 1 (PCSK1), leptin receptor (LEPR) deficiency, or Bardet-Biedl syndrome (BBS).

[0117] The present invention also provides a use of a compound as shown in Formula I or a pharmaceutically acceptable salt thereof according to any of the above schemes in the preparation of a health product for alleviating obesity.

[0118] It will be understood by those skilled in the art that the structural formulas used in the present invention to describe groups are based on the conventions used in the art. It means that the corresponding group is connected to other fragments and groups in the compound through this site.

[0119] As used herein, a substituent may be preceded by a single dash "-" to indicate that the named substituent is bonded to the parent moiety through a single bond.

[0120] When any variable (such as L1) appears multiple times in the definition of a compound, the definition of the variable at each position is independent of the definition at the remaining positions, and their meanings are independent of each other and do not affect each other.

[0121] In this article, "Arg 1 -c[Cys 2 -DAla 3-His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]”, the number in the upper right corner of the amino acid residue only indicates the position of the marked amino acid residue in the polypeptide sequence and has no other meaning, such as Arg 1 The "1" in the string indicates that Arg is located at the "Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]" is the first position in the polypeptide sequence.

[0122] As used herein, the term "polypeptide" refers to a compound comprising a series of amino acids interconnected by amide (or peptide) bonds.

[0123] As used herein, the term "amino acid" residue refers to the residue after an amino acid has reacted via its amino or carboxyl group to form a polypeptide.

[0124] As used herein, amino acid residues may be identified by their full names, single-letter abbreviations, or three-letter abbreviations. These three methods are fully equivalent and can be used interchangeably. Hereinafter, amino acid residues for which the optical configuration is not indicated are L-amino acids (unless otherwise indicated).

[0125] The term "alkyl" refers to a straight or branched chain alkyl group having a specified number of carbon atoms (e.g., C1-C4). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0126] The term "ε-amino group of the side chain of a lysine residue" refers to The amino group is connected to the carbon atom at the "ε" position.

[0127] As used herein, "prodrug" refers to a compound that undergoes chemical conversion within an organism via an enzymatic or non-enzymatic chemical process, resulting in the release of a parent drug. The term "parent drug" as used herein refers to the pharmaceutically active compound released by the prodrug when the prodrug is converted. The term "conversion" refers to the process by which a prodrug is converted via an enzymatic or non-enzymatic chemical process, resulting in the release of the parent drug. The rate at which conversion occurs can be quantified as the "conversion half-life." The "conversion half-life" is the time required for the prodrug concentration to be reduced to half. The "conversion half-life" may also be referred to as the "prodrug to drug conversion half-life" or the "prodrug to parent drug conversion half-life."

[0128] The term "pharmaceutically acceptable salt" refers to salts prepared from compounds of the present invention with relatively nontoxic, pharmaceutically acceptable acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in neat solution or in a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent.

[0129] The term "pharmaceutically acceptable excipients" refers to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These excipients are all substances, other than the active ingredient, contained in a pharmaceutical preparation. For more information, see Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition) or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009 Sixth Edition).

[0130] In the present invention, the "agonist" can be used in mammals; it can also be used in vitro, mainly for experimental purposes, for example, as a standard sample or control sample for comparison, or prepared into a kit according to conventional methods in the art.

[0131] The term "treat" refers to therapeutic treatment. When referring to a specific condition, treatment means: (1) alleviating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that leads to or causes the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0132] The term "prevent" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0133] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0134] The reagents and raw materials used in the present invention are commercially available.

[0135] The positive progress of the present invention is that the polypeptide compound of the present invention has one or more of the following advantages:

[0136] (1) Excellent solubility;

[0137] (2) have good in vitro MC4R agonist activity;

[0138] (3) having a long conversion half-life;

[0139] (4) It has a good effect in reducing weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] Figure 1 is a curve showing the body weight growth rate of normal rats after administration of prodrug-linked MC4R peptide agonists 10, 11, and 12.

[0141] Figure 2 shows the weight growth rate curve of DIO mice administered with the prodrug-linked MC4R peptide agonist 12. DETAILED DESCRIPTION

[0142] The present invention is further illustrated by way of examples, which are not intended to limit the invention to the scope of the examples. Experimental procedures in the following examples, where specific conditions are not specified, were performed using conventional techniques such as organic synthesis, biochemistry, and protein purification within the skill of the art, or according to product specifications, and are fully explained in the literature. In the following examples, efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account.

[0143] Those skilled in the art should understand that equivalent replacements or corresponding improvements made to the technical features of the present disclosure are still within the scope of protection of the present disclosure.

[0144] The detection, purification and characterization methods used in the examples are described below.

[0145] The list of abbreviations is as follows:

[0146] 1. RP-HPLC analysis method A:

[0147] The chromatographic column is Acclaim TM The elution was performed using a 120 C18 column (4.6 × 250 mm, 5 μm, 12 nm) at a flow rate of 1 ml / min and a detection wavelength of 215 nm. Mobile phase A consisted of 0.1 mol / L aqueous Na2SO4, pH 2.0, and mobile phase B consisted of acetonitrile. The gradient elution parameters are shown in Table 1.

[0148] Table 1 Gradient elution parameters

[0149] 2. RP-HPLC analysis method B:

[0150] The chromatographic column was a YMC-Pack Pro C4 (250 x 4.6 mm IDS-5 μm, 12 nm), with a flow rate of 1 ml / min and a detection wavelength of 215 nm. Mobile phase A consisted of 0.05% TFA / water, and mobile phase B consisted of 0.05% TFA / ACN. The gradient elution parameters are shown in Table 2.

[0151] Table 2 Gradient elution parameters

[0152] 3. RP-HPLC purification method A:

[0153] The chromatographic column was Sepax-GP-C18 (10.0 × 250 mm), the flow rate was 5 ml / min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 0.05% TFA / water, and the mobile phase B was 0.05% TFA / ACN. The gradient elution parameters are shown in Table 3.

[0154] Table 3 Gradient elution parameters

[0155] 4. RP-HPLC purification method B:

[0156] The chromatographic column was Kromasil 300-10-C4 (10.0 × 250 mm), the flow rate was 5 ml / min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 0.5% HAC / water solution, and the mobile phase B was 0.5% HAC / ACN solution. The gradient elution parameters are shown in Table 4.

[0157] Table 4 Gradient elution parameters

[0158] Example 1: Synthesis, Purification and Solubility Determination of Setmelanotide Analogs

[0159] Setmelanotide analog 1, Setmelanotide analog 2, Setmelanotide analog 3, Setmelanotide analog 4, Setmelanotide analog 5, Setmelanotide analog 6, Setmelanotide analog 7, Setmelanotide analog 8, Setmelanotide analog 9, Setmelanotide analog 10, Setmelanotide analog 11, Setmelanotide analog 12, Setmelanotide analog 13, Setmelanotide analog 14, Setmelanotide analog 15, Setmelanotide analog 16, Setmelanotide analog 17, Setmelanotide analog 18, Setmelanotide analog 19, Setmelanotide analog 20, Setmelanotide analog 21, Setmelanotide analog 22, Setmelanotide analog 23, Setmelanotide analog 24, Setmelanotide analog 25, Setmelanotide analog 26, Setmelanotide analog 27, Setmelanotide analog 28, Setmelanotide analog 29, Setmelanotide analog 30, Setmelanotide analog 31, Setmelanotide analog 32, Setmelanotide analog 33, Setmelanotide analog 34, Setmelanotide analog 35 Setmelanotide analogue 25, Setmelanotide analogue 26, Setmelanotide analogue 27, Setmelanotide analogue 28, Setmelanotide analogue 29, Setmelanotide analogue 30, Setmelanotide analogue 31, Setmelanotide analogue 32, Setmelanotide analogue 33, Setmelanotide analogue 34, Setmelanotide analogue 35, Setmelanotide analogue 36, Setmelanotide analogue Analog 37, Setmelanotide analog 38, Setmelanotide analog 39, Setmelanotide analog 40, Setmelanotide analog 41, Setmelanotide analog 42, Setmelanotide analog 43, Setmelanotide analog 44, Setmelanotide analog 45, Setmelanotide analog 46, Setmelanotide analog 47, Setmelanotide analog 48, Setmelanotide analog 49.

[0160] Materials and reagents

[0161] MBHA resin (Xi'an Lanxiao Biotechnology Co., Ltd., product number 09-160720) with a substitution value of 0.47 mmol / g.

[0162] Protected amino acids: Fmoc-Cys(Trt)-OH (CAS: 103213-32-7), Fmoc-Trp(Boc)-OH (CAS: 143824-78-6), Fmoc-Arg(Pbf)-OH (CAS: 154445-77-9), Fmoc-D-Phe-OH (CAS: 86123-10-6), Fmoc-D-Ala-OH (CAS: 79990-15-1), Fmoc-His(Trt) -OH(CAS:109425-51-6), Fmoc-Lys(Boc)-OH(CAS:71989-26-9), Fmoc-Glu(OtBu)-OH(CAS:71989-18-9), Fmo c-hCys(Trt)-OH(CAS:167015-23), Fmoc-Pen(Trt)-OH(CAS:201531-88-6), Fmoc-Thr-OH(CAS:73731-37-0).

[0163] Acetic anhydride (Sinopharm Chemical Reagent Co., Ltd., product number 20170822)

[0164] Synthesis reagents: HOBt, DIC, DMF, DCM, piperidine.

[0165] 1.1 Instrument

[0166] CS-BIO peptide synthesizer, Waters 600 semi-preparative high performance liquid chromatograph, Beckman centrifuge, Buchi rotary evaporator

[0167] 1.2 Operation steps

[0168] a. Solid-phase peptide synthesis

[0169] Weigh 0.5g of MBHA resin and place it in a CS-BIO peptide synthesizer reactor. Add 10mL of DCM and soak for 2 hours to fully swell the resin. Add 15mL of deprotection reagent to the swollen resin, mix for 20 minutes, and wash with DMF (10mL x 6). Weigh three times the amount of Fmoc-Cys(Trt)-OH, DIC, and HOBt, equivalent to the amount of resin, dissolve in 10mL of DMF, and then add to the reactor for reaction at room temperature. Monitor the reaction progress with ninhydrin. A blue-purple color indicates incomplete condensation, while a colorless color indicates complete reaction. After the reaction is complete, wash the resin six times with DMF. Then, add 15mL of deprotection reagent again, mix for 20 minutes to remove the amino protecting group, and wash with DMF (10mL x 6). At this point, the ninhydrin color will be purple.

[0170] The coupling reaction of the next amino acid was continued according to the above method, in the order of C-terminus to N-terminus, and this cycle was repeated until all amino acids were coupled. The last amino acid was deprotected and washed, and the resin was washed with DCM 6 times to obtain the resin peptide.

[0171] b. Lysis and precipitation

[0172] The resin peptide was vacuum dried and weighed. The dried resin peptide was placed in a 50 mL centrifuge tube and the corresponding amount of cleavage reagent (TFA:H2O:TIS = 95:2.5:2.5) was added according to the ratio of 1 mL of cleavage reagent per 100 mg of resin peptide. The reaction was stirred at room temperature for 2 hours. After the cleavage was completed, the filtrate was collected by filtration using a quartz sand funnel, and 10 times the volume of the filtrate was added. Anhydrous ether was added to precipitate the peptide, and the mixture was centrifuged (10000 × g) for 5 minutes. The supernatant was discarded and the operation was repeated 6 times. The precipitate was dried under reduced pressure for 24 hours to obtain a crude peptide sample, which was weighed.

[0173] c. Purify the peptide samples according to RP-HPLC purification method A, combine samples with purity above 90%, and verify by LC-MS mass spectrometry.

[0174] 1) Setmelanotide analogue 1

[0175] Sequence (amino acid sequence corresponding to SEQ ID NO: 1): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -NH2 (cycl 2→8) chemical structure:

[0176] LC-MS molecular weight verification: m / z = 538.65 [M+2H] 2+ consistent with the theoretical molecular weight.

[0177] 2) Setmelanotide analogue 2

[0178] Sequence (amino acid sequence corresponding to SEQ ID NO: 2): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -NH2(cycl2→8)

[0179] Chemical structure:

[0180] LC-MS molecular weight verification: m / z = 602.75 [M+2H] 2+ consistent with the theoretical molecular weight.

[0181] 3) Setmelanotide analogue 3

[0182] Sequence (amino acid sequence corresponding to SEQ ID NO: 2): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -NH2(cycl 2→8)

[0183] Chemical structure:

[0184] LC-MS molecular weight verification: m / z = 623.75 [M+2H] 2+ consistent with the theoretical molecular weight.

[0185] 4) Setmelanotide analogue 4

[0186] Sequence (amino acid sequence corresponding to SEQ ID NO: 3): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -NH2(cycl 2→8)

[0187] Chemical structure:

[0188] LC-MS molecular weight verification: m / z = 666.83 [M+2H] 2+ consistent with the theoretical molecular weight.

[0189] 5) Setmelanotide analogue 5

[0190] Sequence (amino acid sequence corresponding to SEQ ID NO: 3): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -NH2(cycl 2→8)

[0191] Chemical structure:

[0192] LC-MS molecular weight verification: m / z = 686.83 [M+2H] 2+ consistent with the theoretical molecular weight.

[0193] 6) Setmelanotide analogue 6

[0194] Sequence (amino acid sequence corresponding to SEQ ID NO: 4): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→8)

[0195] Chemical structure:

[0196] LC-MS molecular weight verification: m / z = 730.92 [M+2H] 2+ consistent with the theoretical molecular weight.

[0197] 7) Setmelanotide analogue 7

[0198] Sequence (amino acid sequence corresponding to SEQ ID NO: 4): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→8)

[0199] Chemical structure:

[0200] LC-MS molecular weight verification: m / z = 751.92 [M+2H] 2+ consistent with the theoretical molecular weight.

[0201] 8) Setmelanotide analogue 8

[0202] Sequence (amino acid sequence corresponding to SEQ ID NO: 5): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -Lys 11 -Lys 12 -NH2(cycl 2→8)

[0203] Chemical structure:

[0204] LC-MS molecular weight verification: m / z = 795.01 [M+2H] 2+ consistent with the theoretical molecular weight.

[0205] 9) Setmelanotide analogue 9

[0206] Sequence (amino acid sequence corresponding to SEQ ID NO: 5): Ac-Arg1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -Lys 11 -Lys 12 -NH2(cycl 2→8)

[0207] Chemical structure:

[0208] LC-MS molecular weight verification: m / z = 816.01 [M+2H] 2+ consistent with the theoretical molecular weight.

[0209] 10) Setmelanotide analogue 10

[0210] Sequence (amino acid sequence corresponding to SEQ ID NO: 6): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -Lys 11 -Lys 12 -Lys 13 -NH2(cycl 2→8)

[0211] Chemical structure:

[0212] LC-MS molecular weight verification: m / z = 859.09 [M+2H] 2+ consistent with the theoretical molecular weight.

[0213] 11) Setmelanotide analogue 11

[0214] Sequence (amino acid sequence corresponding to SEQ ID NO: 6): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp7 -Cys 8 -Lys 9 -Lys 10 -Lys 11 -Lys 12 -Lys 13 -NH2(cycl 2→8)

[0215] Chemical structure:

[0216] LC-MS molecular weight verification: m / z = 880.09 [M+2H] 2+ consistent with the theoretical molecular weight.

[0217] 12) Setmelanotide analogue 12

[0218] Sequence (amino acid sequence corresponding to SEQ ID NO: 7): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -Lys 11 -Lys 12 -Lys 13 -Lys 14 -NH2(cycl 2→8)

[0219] Chemical structure:

[0220] LC-MS molecular weight verification: m / z = 923.1 [M+2H] 2+ consistent with the theoretical molecular weight.

[0221] 13) Setmelanotide analogue 13

[0222] Sequence (amino acid sequence corresponding to SEQ ID NO: 7): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Lys 10 -Lys 11-Lys 12 -Lys 13 -Lys 14 -NH2(cycl 2→8)

[0223] Chemical structure:

[0224] LC-MS molecular weight verification: m / z = 944.1 [M+2H] 2+ consistent with the theoretical molecular weight.

[0225] 14) Setmelanotide analogue 14

[0226] Sequence (amino acid sequence corresponding to SEQ ID NO: 16): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -NH2(cycl2→8)

[0227] Chemical structure:

[0228] LC-MS molecular weight verification: m / z = 603.2 [M+2H] 2+ consistent with the theoretical molecular weight.

[0229] 15) Setmelanotide analogue 15

[0230] Sequence (amino acid sequence corresponding to SEQ ID NO: 16): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -NH2(cycl 2→8)

[0231] Chemical structure:

[0232] LC-MS molecular weight verification: m / z = 624.2 [M+2H] 2+ consistent with the theoretical molecular weight.

[0233] 16) Setmelanotide analogue 16

[0234] Sequence (amino acid sequence corresponding to SEQ ID NO: 17): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -NH2(cycl 2→8)

[0235] Chemical structure:

[0236] LC-MS molecular weight verification: m / z = 667.7 [M+2H] 2+ consistent with the theoretical molecular weight.

[0237] 17) Setmelanotide analogue 17

[0238] Sequence (amino acid sequence corresponding to SEQ ID NO: 17): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -NH2(cycl 2→8)

[0239] Chemical structure:

[0240] LC-MS molecular weight verification: m / z = 688.7 [M+2H] 2+ consistent with the theoretical molecular weight.

[0241] 18) Setmelanotide analogue 18

[0242] Sequence (amino acid sequence corresponding to SEQ ID NO: 18): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu9 -Glu 10 -Glu 11 -NH2(cycl 2→8)

[0243] Chemical structure:

[0244] LC-MS molecular weight verification: m / z = 732.3 [M+2H] 2+ consistent with the theoretical molecular weight.

[0245] 19) Setmelanotide analogue 19

[0246] Sequence (amino acid sequence corresponding to SEQ ID NO: 18): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -Glu 11 -NH2(cycl 2→8)

[0247] Chemical structure:

[0248] LC-MS molecular weight verification: m / z = 753.3 [M+2H] 2+ consistent with the theoretical molecular weight.

[0249] 20) Setmelanotide analogue 20

[0250] Sequence (amino acid sequence corresponding to SEQ ID NO: 19): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -Glu 11 -Glu 12 -NH2(cycl 2→8)

[0251] Chemical structure:

[0252] LC-MS molecular weight verification: m / z = 796.8 [M+2H] 2+ consistent with the theoretical molecular weight.

[0253] 21) Setmelanotide analogue 21

[0254] Sequence (amino acid sequence corresponding to SEQ ID NO: 19): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -Glu 11 -Glu 12 -NH2(cycl 2→8)

[0255] Chemical structure:

[0256] LC-MS molecular weight verification: m / z = 817.9 [M+2H] 2+ consistent with the theoretical molecular weight.

[0257] 22) Setmelanotide analogue 22

[0258] Sequence (amino acid sequence corresponding to SEQ ID NO: 20): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -Glu 11 -Glu 12 -Glu 13 -NH2(cycl 2→8)

[0259] Chemical structure:

[0260] LC-MS molecular weight verification: m / z = 861.4 [M+2H] 2+ consistent with the theoretical molecular weight.

[0261] 23) Setmelanotide analogue 23

[0262] Sequence (amino acid sequence corresponding to SEQ ID NO: 20): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -Glu 11 -Glu 12 -Glu 13 -NH2(cycl 2→8)

[0263] Chemical structure:

[0264] LC-MS molecular weight verification: m / z = 882.5 [M+2H] 2+ consistent with the theoretical molecular weight.

[0265] 24) Setmelanotide analogue 24

[0266] Sequence (amino acid sequence corresponding to SEQ ID NO: 21): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -Glu 11 -Glu 12 -Glu 13 -Glu 14 -NH2(cycl 2→8)

[0267] Chemical structure:

[0268] LC-MS molecular weight verification: m / z = 926 [M+2H] 2+ consistent with the theoretical molecular weight.

[0269] 25) Setmelanotide analogue 25

[0270] Sequence (amino acid sequence corresponding to SEQ ID NO: 21): Ac-Arg 1 -Cys 2 -DAla3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Glu 10 -Glu 11 -Glu 12 -Glu 13 -Glu 14 -NH2(cycl 2→8)

[0271] Chemical structure:

[0272] LC-MS molecular weight verification: m / z = 947 [M+2H] 2+ consistent with the theoretical molecular weight.

[0273] 26) Setmelanotide analogue 26

[0274] Sequence (amino acid sequence corresponding to SEQ ID NO: 22): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Glu 10 -NH2(cycl 2→8)

[0275] Chemical structure:

[0276] LC-MS molecular weight verification: m / z = 667.3 [M+2H] 2+ consistent with the theoretical molecular weight.

[0277] 27) Setmelanotide analogue 27

[0278] Sequence (amino acid sequence corresponding to SEQ ID NO: 22): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Glu 10-NH2(cycl 2→8)

[0279] Chemical structure:

[0280] LC-MS molecular weight verification: m / z = 688.3 [M+2H] 2+ consistent with the theoretical molecular weight.

[0281] 28) Setmelanotide analogue 28

[0282] Sequence (amino acid sequence corresponding to SEQ ID NO: 23): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Glu 10 -Lys 11 -NH2(cycl 2→8)

[0283] Chemical structure:

[0284] LC-MS molecular weight verification: m / z = 731.4 [M+2H] 2+ consistent with the theoretical molecular weight.

[0285] 29) Setmelanotide analogue 29

[0286] Sequence (amino acid sequence corresponding to SEQ ID NO: 23): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Glu 10 -Lys 11 -NH2(cycl 2→8)

[0287] Chemical structure:

[0288] LC-MS molecular weight verification: m / z = 752.4 [M+2H] 2+ consistent with the theoretical molecular weight.

[0289] 30) Setmelanotide analogue 30

[0290] Sequence (amino acid sequence corresponding to SEQ ID NO: 24): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Glu 10 -Glu 11 -NH2(cycl 2→8)

[0291] Chemical structure:

[0292] LC-MS molecular weight verification: m / z = 731.9 [M+2H] 2+ consistent with the theoretical molecular weight.

[0293] 31) Setmelanotide analogue 31

[0294] Sequence (amino acid sequence corresponding to SEQ ID NO: 24): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Glu 10 -Glu 11 -NH2(cycl 2→8)

[0295] Chemical structure:

[0296] LC-MS molecular weight verification: m / z = 752.8 [M+2H] 2+ consistent with the theoretical molecular weight.

[0297] 32) Setmelanotide analogue 32

[0298] Sequence (amino acid sequence corresponding to SEQ ID NO: 25): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg6 -Trp 7 -Cys 8 -Glu 9 -Lys 10 -NH2(cycl 2→8)

[0299] Chemical structure:

[0300] LC-MS molecular weight verification: m / z = 667.3 [M+2H] 2+ consistent with the theoretical molecular weight.

[0301] 33) Setmelanotide analogue 33

[0302] Sequence (amino acid sequence corresponding to SEQ ID NO: 25): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Glu 9 -Lys 10 -NH2(cycl 2→8)

[0303] Chemical structure:

[0304] LC-MS molecular weight verification: m / z = 688.3 [M+2H] 2+ consistent with the theoretical molecular weight.

[0305] 34) Setmelanotide analogue 34

[0306] Sequence (amino acid sequence corresponding to SEQ ID NO: 26): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Glu 10 -Glu 11 -Lys 12 -NH2(cycl 2→8)

[0307] Chemical structure:

[0308] LC-MS molecular weight verification: m / z = 795.9 [M+2H] 2+ consistent with the theoretical molecular weight.

[0309] 35) Setmelanotide analogue 35

[0310] Sequence (amino acid sequence corresponding to SEQ ID NO: 26): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Lys 9 -Glu 10 -Glu 11 -Lys 12 -NH2(cycl 2→8)

[0311] Chemical structure:

[0312] LC-MS molecular weight verification: m / z = 817 [M+2H] 2+ consistent with the theoretical molecular weight.

[0313] 36) Setmelanotide analogue 36

[0314] Sequence (amino acid sequence corresponding to SEQ ID NO: 27): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Arg 9 -NH2(cycl2→8)

[0315] Chemical structure:

[0316] LC-MS molecular weight verification: m / z = 616.7 [M+2H] 2+ consistent with the theoretical molecular weight.

[0317] 37) Setmelanotide analogue 37

[0318] Sequence (amino acid sequence corresponding to SEQ ID NO: 27): Ac-Arg 1 -Cys 2-DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Arg 9 -NH2(cycl 2→8)

[0319] Chemical structure:

[0320] LC-MS molecular weight verification: m / z = 637.7 [M+2H] 2+ consistent with the theoretical molecular weight.

[0321] 38) Setmelanotide analogue 38

[0322] Sequence (amino acid sequence corresponding to SEQ ID NO: 28): Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Arg 9 -Arg 10 -NH2(cycl 2→8)

[0323] Chemical structure:

[0324] LC-MS molecular weight verification: m / z = 694.8 [M+2H] 2+ consistent with the theoretical molecular weight.

[0325] 39) Setmelanotide analogue 39

[0326] Sequence (amino acid sequence corresponding to SEQ ID NO: 28): Ac-Arg 1 -Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -Arg 9 -Arg 10 -NH2(cycl 2→8)

[0327] Chemical structure:

[0328] LC-MS molecular weight verification: m / z = 715.8 [M+2H] 2+ consistent with the theoretical molecular weight.

[0329] 40) Setmelanotide analogue 40

[0330] Sequence (amino acid sequence corresponding to SEQ ID NO: 29): Arg 1 -hCys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 -Lys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→7)

[0331] Chemical structure:

[0332] LC-MS molecular weight verification: m / z = 762.5 [M+2H] 2+ consistent with the theoretical molecular weight.

[0333] 41) Setmelanotide analogue 41

[0334] Sequence (amino acid sequence corresponding to SEQ ID NO: 29): Ac-Arg 1 -hCys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 -Lys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→7)

[0335] Chemical structure:

[0336] LC-MS molecular weight verification: m / z = 783.5 [M+2H] 2+ consistent with the theoretical molecular weight.

[0337] 42) Setmelanotide analogues 42

[0338] Sequence (amino acid sequence corresponding to SEQ ID NO: 30): Arg1 -hCys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Pen 8 -Lys 9 -Lys 10 -Lys 11 -Lys 12 -NH2(cycl 2→8)

[0339] Chemical structure:

[0340] LC-MS molecular weight verification: m / z = 816 [M+2H] 2+ consistent with the theoretical molecular weight.

[0341] 43) Setmelanotide analogue 43

[0342] Sequence (amino acid sequence corresponding to SEQ ID NO: 30): Ac-Arg 1 -hCys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Pen 8 -Lys 9 -Lys 10 -Lys 11 -Lys 12 -NH2(cycl 2→8)

[0343] Chemical structure:

[0344] LC-MS molecular weight verification: m / z = 837.1 [M+2H] 2+ consistent with the theoretical molecular weight.

[0345] 44) Setmelanotide analogue 44

[0346] Sequence (amino acid sequence corresponding to SEQ ID NO: 31): Arg 1 -Cys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Cys 7 -Lys8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→7)

[0347] Chemical structure:

[0348] LC-MS molecular weight verification: m / z = 741.4 [M+2H] 2+ consistent with the theoretical molecular weight.

[0349] 45) Setmelanotide analogue 45

[0350] Sequence (amino acid sequence corresponding to SEQ ID NO: 31): Ac-Arg 1 -Cys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Cys 7 -Lys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→7)

[0351] Chemical structure:

[0352] LC-MS molecular weight verification: m / z = 762.5 [M+2H] 2+ consistent with the theoretical molecular weight.

[0353] 46) Setmelanotide analogue 46

[0354] Sequence (amino acid sequence corresponding to SEQ ID NO: 32): Arg 1 -hCys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 -Lys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→7)

[0355] Chemical structure:

[0356] LC-MS molecular weight verification: m / z = 780.5 [M+2H] 2+ consistent with the theoretical molecular weight.

[0357] 47) Setmelanotide analogue 47

[0358] Sequence (amino acid sequence corresponding to SEQ ID NO: 32): Ac-Arg 1 -hCys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 -Lys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→7)

[0359] Chemical structure:

[0360] LC-MS molecular weight verification: m / z = 801.5 [M+2H] 2+ consistent with the theoretical molecular weight.

[0361] 48) Setmelanotide analogue 48

[0362] Sequence (amino acid sequence corresponding to SEQ ID NO: 33): Arg 1 -Cys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Cys 7 -Lys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→7)

[0363] Chemical structure:

[0364] LC-MS molecular weight verification: m / z = 759.5 [M+2H] 2+ consistent with the theoretical molecular weight.

[0365] 49) Setmelanotide analogue 49

[0366] Sequence (amino acid sequence corresponding to SEQ ID NO: 33): Ac-Arg 1-Cys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Cys 7 -Lys 8 -Lys 9 -Lys 10 -Lys 11 -NH2(cycl 2→7)

[0367] Chemical structure:

[0368] LC-MS molecular weight verification: m / z = 780.5 [M+2H] 2+ consistent with the theoretical molecular weight.

[0369] 1.3 Determination of the solubility of setmelanotide analogs

[0370] 50 mg of each of the 21 setmelanotide analogs was weighed into separate transparent centrifuge tubes, and 200 μL of purified water was added to each tube. After sonication, the tubes were centrifuged (12,000 rpm) for 2 minutes. The supernatant was diluted 25-fold and analyzed by RP-HPLC. The solubility of each analog was calculated by comparing the peak area with that of a control substance of known concentration (i.e., each setmelanotide analog of known concentration).

[0371] The formula is as follows:

[0372] (C2 represents the concentration of the analyte, C1 represents the concentration of the known reference substance; A2 represents the peak area of ​​the analyte, A1 represents the peak area of ​​the known reference substance; D represents the dilution factor; V1 represents the injection volume of the reference substance; V2 represents the injection volume of the analyte)

[0373] The results are shown in Table 5 below (V1=10 μl, V2=2 μl, D=25):

[0374] Table 5 Solubility of Setmelanotide analogs

[0375] From the results in the above table, it can be seen that adding Lys to the C-terminus of semaphoride can significantly increase the solubility of semaphoride, and the solubility does not change regularly with the number of Lys. When the number of Lys is 4, the solubility is the best, that is, Setmelanotide analogue 8.

[0376] Example 2: Site-directed synthesis and purification of modified setmelanotide analogs

[0377] 2.1 Materials and Reagents

[0378] In addition to the materials and reagents used in 1.1, the following reagents were used: Fmoc-Glu-OtBu (CAS: 84793-07-7), Fmoc-AEEA-OH (CAS: 166108-71-0), Fmoc-Ser(tBu)-OH (CAS: 71989-33-8), octadecanediolatoic acid mono-tert-butyl ester (CAS: 843666-40-0), tetradecanediolatoic acid mono-tert-butyl ester (CAS: 234082-00-9), Fmoc-Lys(Mtt)-OH (CAS: 167393-62-6), Boc-Arg(pbf)-OH (CAS: 200124-22-7)

[0379] 2.2 Instruments

[0380] Same as 1.2

[0381] 2.3 Operation steps

[0382] 2.3.1 Solid-phase fixed-site synthesis

[0383] Weigh 0.5g of MBHA resin and place it in a CS-BIO peptide synthesizer reactor. Add 10mL of DCM and soak for 2 hours to fully swell the resin. Add 15mL of deprotection reagent to the swollen resin, mix for 20 minutes, and wash with DMF (10mL x 6). Weigh three times the amount of Fmoc-Lys(Boc)-OH, DIC, and HOBt, equivalent to the amount of resin, dissolve in 10mL of DMF, and then add to the reactor for reaction at room temperature. Monitor the reaction progress with ninhydrin. A blue-purple color indicates incomplete condensation, while a colorless color indicates complete reaction. After the reaction is complete, wash the resin six times with DMF. Then, add 15mL of deprotection reagent again, mix for 20 minutes to remove the amino protecting group, and wash with DMF (10mL x 6). At this point, the ninhydrin color will appear purple. Repeat the condensation and deprotection process until the main peptide chain is elongated. (Note: The lysine site-specific modification site was condensed using Fmoc-Lys(Mtt)-OH, and the lysine Mtt protecting group was removed with 1% TFA / DMF. The amino acid condensation and deprotection process was repeated on the lysine side chain amino group until the fatty acid side chain extension was completed. The resin was washed with DCM 6 times to obtain the resin peptide. For the site-specific modification of lysine, please refer to the following literature: Jiang He, Lu Jianghua, Lu Rongjian. Research progress on orthogonal protection of lysine in peptide synthesis by Fmoc method [J]. Journal of Chongqing University of Technology (Natural Science), 2011, 25(04): 23-27+32.)

[0384] 2.3.2 Lysis and precipitation

[0385] Same as 1.3.b

[0386] 2.3.3 Purification

[0387] The peptide samples were purified according to RP-HPLC purification method B, and samples with purity above 90% were combined and verified by LC-MS mass spectrometry.

[0388] ① Modified Setmelanotide analogue 1:

[0389] Sequence (amino acid sequence corresponds to SEQ ID NO: 3):

[0390] Chemical structure:

[0391] LC-MS determined m / z = 996.71 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0392] ②Modified Setmelanotide analogue 2:

[0393] Sequence (amino acid sequence corresponds to SEQ ID NO: 3):

[0394] Chemical structure:

[0395] LC-MS determined m / z = 996.71 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0396] ③Modified Setmelanotide analogue 3:

[0397] Sequence (amino acid sequence corresponds to SEQ ID NO: 5):

[0398] Chemical structure:

[0399] LC-MS determined m / z = 902.43 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0400] ④Modified Setmelanotide analogue 4:

[0401] Sequence (amino acid sequence corresponds to SEQ ID NO: 5):

[0402] Chemical structure:

[0403] LC-MS determined m / z = 902.43 [M+3H]3+ , which is consistent with the theoretical molecular weight.

[0404] ⑤Modified Setmelanotide analogue 5:

[0405] Sequence (amino acid sequence corresponds to SEQ ID NO: 5):

[0406] Chemical structure:

[0407] LC-MS determined m / z = 956.09 [M+4H] 4+ , which is consistent with the theoretical molecular weight.

[0408] ⑥ Modified Setmelanotide analogue 6:

[0409] Sequence (amino acid sequence corresponds to SEQ ID NO: 7):

[0410] Chemical structure:

[0411] LC-MS determined m / z = 987.84 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0412] ⑦Modified Setmelanotide analogue 7:

[0413] Sequence (amino acid sequence corresponds to SEQ ID NO: 7):

[0414] Chemical structure:

[0415] LC-MS determined m / z = 987.84 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0416] Example 3 In vitro activity determination of setmelanotide analogs and modified setmelanotide analogs

[0417] MC4R-CRE-bla CHO-K1 cells were used, and Setmelanotide was used as a positive control to measure the in vitro cell activity of the test samples (Setmelanotide analogs and modified Setmelanotide analogs). Setmelanotide can specifically bind to MC4R on the cell surface, leading to the activation of membrane adenylate cyclase and the formation of cyclic adenosine monophosphate (cAMP) in the cell. The amount of cAMP generated is positively correlated with the concentration of Setmelanotide. The amount of intracellular cAMP generated can be accurately measured by time-resolved fluorescence resonance energy transfer immunoassay (TR-FRET). (The detection kit is the LANCE Ultra cAMP Kit from PerkinElmer). The experimental data were analyzed using Prism5 software, with the sample concentration as the X-axis and the corresponding fluorescence ratio as the Y-axis. A four-parameter equation was used for fitting, and the dose-response curves of the positive control and the test sample were drawn, and the EC 50 EC values ​​of samples and positive controls 50 The values ​​are shown in Table 6.

[0418] Table 6 In vitro activities of Setmelanotide analogs and modified Setmelanotide analogs

[0419] Example 4 Synthesis and Preparation of Prodrug-Linked MC4R Polypeptide Agonists

[0420] The prodrug-linked MC4R polypeptide agonist was prepared according to the synthesis and purification method of Example 1 and confirmed by mass spectrometry.

[0421] Supplementary Example 1: The raw materials for protecting amino acids are: Fmoc-Aeg-OH (CAS: 172405-45-7), Fmoc-Dab(Boc)-OH (CAS: 125238-99-5), Fmoc-Dap-OH (CAS: 181954-34-7), and Fmoc-Dap(MTT)-OH (CAS: 851392-68-2).

[0422] The specific results are as follows:

[0423] ① Linked prodrug MC4R peptide agonist 1

[0424] Sequence (amino acid sequence corresponding to SEQ ID NO: 8): Gly 1 -Aeg 2 -Arg 3 -Cys 4 -DAla 5 -His 6-DPhe 7 -Arg 8 -Trp 9 -Cys 10 -NH2(cycl 4→10)

[0425] Chemical structure:

[0426] LC-MS determined m / z = 617.23 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0427] ② Linked prodrug MC4R peptide agonist 2

[0428] Sequence (amino acid sequence corresponding to SEQ ID NO: 9): DLys 1 -Sar 2 -Arg 3 -Cys 4 -DAla 5 -His 6 -DPhe 7 -Arg 8 -Trp 9 -Cys 10 -NH2(cycl 4→10)

[0429] Chemical structure:

[0430] LC-MS determined m / z = 638.27 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0431] ③ Linked prodrug MC4R peptide agonist 3

[0432] Sequence (amino acid sequence corresponding to SEQ ID NO: 10): Dab 1 -Gly 2 -Arg 3 -Cys 4 -DAla 5 -His 6 -DPhe 7 -Arg 8 -Trp 9 -Cys 10 -NH2(cycl 4→10)

[0433] Chemical structure:

[0434] LC-MS determined m / z = 617.23 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0435] ④ Connecting prodrug MC4R polypeptide agonist 4

[0436] Sequence (amino acid sequence corresponding to SEQ ID NO: 11): Dap 1 -Gly 2 -Arg 3 -Cys 4 -DAla 5 -His 6 -DPhe 7 -Arg 8 -Trp 9 -Cys 10 -NH2(cycl 4→10)

[0437] Chemical structure:

[0438] LC-MS determined m / z = 610.22 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0439] ⑤ Connecting prodrug MC4R peptide agonist 5

[0440] Sequence (amino acid sequence corresponding to SEQ ID NO: 12): Dap 1 -Gly 2 -Arg 3 -Cys 4 -DAla 5 -His 6 -DPhe 7 -Arg 8 -Trp 9 -Cys 10 -Lys 11 -Lys 12 -NH2(cycl 4→10)

[0441] Chemical structure:

[0442] LC-MS determined m / z = 738.39 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0443] ⑥ Connected prodrug MC4R polypeptide agonist 6

[0444] Sequence (amino acid sequence corresponding to SEQ ID NO: 13): Dap 1 -Gly 2 -Arg 3 -Cys 4 -DAla 5-His 6 -DPhe 7 -Arg 8 -Trp 9 -Cys 10 -Lys 11 -Lys 12 -Lys 13 -Lys 14 -NH2(cycl 4→10)

[0445] Chemical structure:

[0446] LC-MS determined m / z = 866.57 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0447] ⑦ Linked prodrug MC4R peptide agonist 7

[0448] Sequence (amino acid sequence corresponding to SEQ ID NO: 14): Dap 1 -Gly 2 -Arg 3 -Cys 4 -DAla 5 -His 6 -DPhe 7 -Arg 8 -Trp 9 -Cys 10 -Lys 11 -Lys 12 -Lys 13 -Lys 14 -Lys 15 -Lys 16 -NH2(cycl 4→10)

[0449] Chemical structure:

[0450] LC-MS determined m / z = 994.74 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0451] ⑧ Linked prodrug MC4R polypeptide agonist 8

[0452] Sequence (amino acid sequence corresponding to SEQ ID NO: 15): DLys 1 -Sar 2 -Arg 3 -Cys 4 -DAla 5 -His 6 -DPhe 7-Arg 8 -Trp 9 -Cys 10 -Lys 11 -Lys 12 -Lys 13 -Lys 14 -NH2(cycl 4→10)

[0453] Chemical structure:

[0454] LC-MS determined m / z = 894.62 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0455] ⑨ Linked prodrug MC4R peptide agonist 9

[0456] Sequence (amino acid sequence corresponding to SEQ ID NO: 15): C12 diacid 1 -Glu(γ) 2 -AEEA 3 -AEEA 4 -DLys 5 -Sar 6 -Arg 7 -Cys 8 -DAla 9 -His 10 -DPhe 11 -Arg 12 -Trp 13 -Cys 14 -Lys 15 -Lys 16 -Lys 17 -Lys 18 -NH2(cycl 8→14)

[0457] Chemical structure:

[0458] LC-MS determined m / z = 807.32 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0459] ⑩ Linked prodrug MC4R polypeptide agonist 10

[0460] Sequence (amino acid sequence corresponding to SEQ ID NO: 15): C14 diacid 1 -Glu(γ) 2 -AEEA 3 -AEEA 4 -DLys 5 -Sar6 -Arg 7 -Cys 8 -DAla 9 -His 10 -DPhe 11 -Arg 12 -Trp 13 -Cys 14 -Lys 15 -Lys 16 -Lys 17 -Lys 18 -NH2(cycl 8→14)

[0461] Chemical structure:

[0462] LC-MS determined m / z = 816.67 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0463] Linked prodrug MC4R peptide agonist 11

[0464] Sequence (amino acid sequence corresponding to SEQ ID NO: 15): C20 diacid 1 -Glu(γ) 2 -AEEA 3 -AEEA 4 -DLys 5 -Sar 6 -Arg 7 -Cys 8 -DAla 9 -His 10 -DPhe 11 -Arg 12 -Trp 13 -Cys 14 -Lys 15 -Lys 16 -Lys 17 -Lys 18 -NH2(cycl 8→14)

[0465] Chemical structure:

[0466] LC-MS determined m / z = 844.72 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0467] Linked prodrug MC4R peptide agonist 12

[0468] Sequence (amino acid sequence corresponding to SEQ ID NO: 15): C18 diacid 1 -Glu(γ) 2 -AEEA 3 -AEEA 4 -DLys 5 -Sar 6 -Arg 7 -Cys 8 -DAla 9 -His 10 -DPhe 11 -Arg 12 -Trp 13 -Cys 14 -Lys 15 -Lys 16 -Lys 17 -Lys 18 -NH2(cycl 8→14)

[0469] Chemical structure:

[0470] LC-MS determined m / z = 835.37 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0471] Linked prodrug MC4R peptide agonist 13

[0472] Sequence (amino acid sequence corresponding to SEQ ID NO: 34): C18 diacid 1 -Glu(γ) 2 -AEEA 3 -AEEA 4 -DLys 5 -Sar 6 -Arg 7 -hCys 8 -Thr 9 -DPhe 10 -Arg 11 -Trp 12 -Pen 13 -Lys 14 -Lys 15 -Lys 16 -Lys 17 -NH2(cycl 8→13)

[0473] LC-MS determined m / z = 813.6 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0474] Linked prodrug MC4R peptide agonist 14

[0475] Sequence (amino acid sequence corresponding to SEQ ID NO: 35): C18 diacid 1 -Glu(γ) 2 -AEEA 3 -AEEA 4 -DLys 5 -Sar 6 -Arg 7 -hCys 8 -His 9 -DPhe 10 -Arg 11 -Trp 12 -Pen 13 -Lys 14 -Lys 15 -Lys 16 -Lys 17 -NH2(cycl 8→13)

[0476] LC-MS determined m / z = 825.7 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0477] Example 5: Synthesis of Modifier

[0478] Example 5.1: Preparation of C18 diacid-Glu(γ)-Glu(γ)-Ser-Glu(γ)-Ser-Glu(γ)-Glu(γ)-COSU

[0479] The chemical structure of C18 diacid-Glu(γ)-Glu(γ)-Ser-Glu(γ)-Ser-Glu(γ)-Glu(γ)-COSU is shown below:

[0480] 5.1.1 Materials and reagents

[0481] 2-CTC resin (Xi'an Lanxiao Biotechnology Co., Ltd., product number: 03-210310), with a substitution value of 1.15 mmol / g.

[0482] The synthetic materials are: Fmoc-Glu-OtBu (CAS: 84793-07-7), Fmoc-AEEA-OH (CAS: 166108-71-0), Fmoc-Ser(tBu)-OH (CAS: 71989-33-8), and mono-tert-butyl octadecanediolate (CAS: 843666-40-0).

[0483] Synthesis reagents: HOBt, DIC, DMF, DCM, PIP, DIEA

[0484] 5.1.2 Instruments

[0485] CS-BIO peptide synthesizer, Waters 600 semi-preparative high performance liquid chromatograph, Beckman centrifuge, BUCHI rotary evaporator.

[0486] 5.1.3 Synthetic modifiers

[0487] a. Solid Phase Synthesis

[0488] Weigh 1.0 g of 2-CTC resin and place it in a CS-BIO peptide synthesizer reactor. Add 10 mL of DCM and soak for 1 hour to fully swell the resin. Weigh three times the amount of Fmoc-Glu-OtBu, six times the amount of DIEA, and dissolve them in 10 mL of DMF. Add these to the reactor and react at room temperature. Monitor the reaction progress with ninhydrin. A blue-purple color indicates incomplete condensation, while a colorless color indicates completion. After completion, wash the resin six times with DMF. Add 15 mL of deprotection reagent and mix for 20 minutes to remove the amino protecting groups. Wash with 6 additional volumes of DMF (10 mL x 10 mL). At this point, the ninhydrin color will be purple. Next, couple the second amino acid. Weigh three times the amount of Fmoc-Glu-OtBu, DIC, and HOBt, dissolve them in 10 mL of DMF, and add these to the reactor and react at room temperature. The reaction progress was monitored using ninhydrin. A blue-purple color indicated incomplete condensation, while a colorless color indicated complete reaction. After the reaction was complete, the resin was washed six times with DMF. Subsequent amino acids and octadecanedioic acid were coupled in the same manner as the second amino acid until all amino acids and octadecanedioic acid were coupled. Finally, the resin was washed six times with DCM to obtain the peptide on resin.

[0489] b. Lysis and precipitation

[0490] The resin peptide was vacuum dried and weighed. The dried resin peptide was placed in a 50 mL centrifuge tube and the corresponding amount of cleavage reagent (trifluoroethanol: DCM = 1:4) was added according to the ratio of 1 mL cleavage reagent per 100 mg of resin peptide. The reaction was stirred at room temperature for 1 hour. After the cleavage was completed, the filtrate was collected by filtration using a quartz sand funnel. The filtrate was evaporated under reduced pressure at 40°C to remove the solvent. 10 ml of DCM was added to the vacuum distillation flask and the solvent was evaporated under reduced pressure again. This process was repeated 2-3 times to obtain the modifier intermediate.

[0491] c. Activation and cleavage

[0492] 10 ml of tetrahydrofuran (THF) was added to the above modifier intermediate for dissolution, and 1.2 equivalents of HOSU and DIC were added, and the reaction was carried out at room temperature for 1-2 hours. The THF was removed by distillation under reduced pressure to obtain a yellow oil. A mixed solvent of TFA:H2O = 90:10 was added to remove the tert-butyl ester protecting group. After reacting at room temperature for 1 hour, glacial ether was slowly added for precipitation. The solid was obtained by centrifugation, washed with ether 5 times, and dried under reduced pressure to obtain a solid powder, which was weighed to obtain the target modifier.

[0493] LC-MS revealed m / z = 1232.3 [M+H] + , which is consistent with the theoretical molecular weight.

[0494] Example 5.2: Preparation of C14 diacid-Glu(γ)-AEEA-AEEA-COSU

[0495] C14 diacid-Glu(γ)-AEEA-AEEA-COSU was synthesized according to the method of Example 5.1. Its chemical structure is shown below:

[0496] LC-MS determined m / z = 775.86 [M+H] + , which is consistent with the theoretical molecular weight.

[0497] Example 5.3: Preparation of C12 diacid-Glu(γ)-AEEA-AEEA-COSU

[0498] C12 diacid-Glu(γ)-AEEA-AEEA-COSU was synthesized according to the method of Example 5.1. Its chemical structure is shown below:

[0499] LC-MS determined m / z = 747.81 [M+H] + , which is consistent with the theoretical molecular weight.

[0500] Example 5.4: Preparation of C18 diacid-Glu(γ)-AEEA-AEEA-COSU

[0501] C18diacid-Glu(γ)-AEEA-AEEA-COSU was synthesized according to the method of Example 5.1. Its chemical structure is shown below:

[0502] LC-MS determined m / z = 831.97 [M+H] + , which is consistent with the theoretical molecular weight.

[0503] Example 5.5: Preparation of C20 diacid-Glu(γ)-AEEA-AEEA-COSU

[0504] C20diacid-Glu(γ)-AEEA-AEEA-COSU was synthesized according to the method of Example 5.1. Its chemical structure is shown below:

[0505] LC-MS determined m / z = 860.02 [M+H] + , which is consistent with the theoretical molecular weight.

[0506] Example 5.6: Preparation of C18 diacid-Glu(γ)-Glu(γ)-AEEA-AEEA-Glu(γ)-Glu(γ)-COSU

[0507] C18 diacid-Glu(γ)-Glu(γ)-AEEA-AEEA-Glu(γ)-Glu(γ)-COSU was synthesized according to the method of Example 5.1. Its chemical structure is shown below:

[0508] LC-MS determined m / z = 610.16 [M+2H] 2+ , which is consistent with the theoretical molecular weight.

[0509] Example 6 Modification of Prodrug-Linked MC4R Polypeptide Agonists

[0510] (1) Modification of prodrug-linked MC4R peptide agonists

[0511] A prodrug-linked MC4R polypeptide agonist was dissolved in 1% triethylamine (the concentration of the prodrug-linked MC4R polypeptide agonist was 2 mg / ml). An amount of the modifier sample of Example 5.1 equivalent to 1.2 times the amount of the prodrug-linked MC4R polypeptide agonist was weighed and dissolved in a small amount of ethanol. The modifier was added dropwise to the polypeptide solution, and the reaction was carried out at room temperature for 0.5-1 hour. The reaction was monitored according to RP-HPLC analytical method B to obtain a reaction solution. The addition of 1% triethylamine adjusted the pH of the reaction solution to between 10 and 11. Generally, when the pH is above 10, the side chain amino group of the prodrug participates in the reaction, thereby completing the modification of the side chain. The N-terminal amino group can only participate in the reaction at a pH of 7-8.

[0512] (2) Purification and characterization of modified prodrug-linked MC4R peptide agonists

[0513] The reaction solution was purified according to RP-HPLC purification method B, and the products with a purity of more than 90% were combined and lyophilized to obtain a modified prodrug-type MC4R polypeptide agonist, which was confirmed by mass spectrometry.

[0514] The specific results are as follows:

[0515] ① Compound 1 (amino acid sequence corresponding to SEQ ID NO: 8) after modification by linking a prodrug-like MC4R polypeptide agonist

[0516] The prodrug-linked MC4R polypeptide agonist 1 was modified with the substituents of Example 5.1. The structure of the modified compound is as follows:

[0517] LC-MS determined m / z = 783.88 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0518] ② Compound 2 (amino acid sequence corresponding to SEQ ID NO: 8) after modification by linking a prodrug-like MC4R polypeptide agonist

[0519] The prodrug-linked MC4R polypeptide agonist 1 was modified with the substituents of Example 5.6. The structure of the modified compound is as follows:

[0520] LC-MS determined m / z = 779.56 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0521] ③ Compound 3 (amino acid sequence corresponding to SEQ ID NO: 8) after modification by linking a prodrug-like MC4R polypeptide agonist

[0522] The prodrug-linked MC4R polypeptide agonist 1 was modified with the substituents of Example 5.4. The structure of the modified compound is as follows:

[0523] LC-MS determined m / z = 650.45 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0524] ④ Compound 4 (amino acid sequence corresponding to SEQ ID NO: 8) after modification by linking a prodrug-like MC4R polypeptide agonist

[0525] The prodrug-linked MC4R polypeptide agonist 1 was modified with the substituents of Example 5.5. The structure of the modified compound is as follows:

[0526] LC-MS determined m / z = 659.79 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0527] ⑤ Compound 5 (amino acid sequence corresponding to SEQ ID NO: 8) after modification by linking a prodrug-like MC4R polypeptide agonist

[0528] The prodrug-linked MC4R polypeptide agonist 1 was modified with the substituents of Example 5.3. The structure of the modified compound is as follows:

[0529] LC-MS determined m / z = 622.39 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0530] ⑥ Compound 6 (amino acid sequence corresponding to SEQ ID NO: 8) after modification with a prodrug-like MC4R polypeptide agonist

[0531] The prodrug-linked MC4R polypeptide agonist 1 was modified with the substituents of Example 5.2. The structure of the modified compound is as follows:

[0532] LC-MS determined m / z = 631.74 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0533] ⑦ Compound 7 (amino acid sequence corresponding to SEQ ID NO: 15) after modification with a prodrug-like MC4R polypeptide agonist

[0534] The prodrug-linked MC4R polypeptide agonist 8 was modified with the substituents of Example 5.1. The structure of the modified compound is as follows:

[0535] LC-MS determined m / z = 797.91 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0536] ⑧ Compound 8 (amino acid sequence corresponding to SEQ ID NO: 15) after modification by linking a prodrug-like MC4R polypeptide agonist

[0537] The prodrug-linked MC4R polypeptide agonist 8 was modified with the substituents of Example 5.6. The structure of the modified compound is as follows:

[0538] LC-MS determined m / z = 793.59 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0539] 9. Compound 9 (amino acid sequence corresponding to SEQ ID NO: 15) after modification by linking a prodrug-like MC4R polypeptide agonist

[0540] The prodrug-linked MC4R polypeptide agonist 8 was modified with the substituents of Example 5.2. The structure of the modified compound is as follows:

[0541] LC-MS determined m / z = 645.77 [M+3H] 3+, which is consistent with the theoretical molecular weight.

[0542] ⑩ Compound 10 (amino acid sequence corresponding to SEQ ID NO: 10) after modification by linking a prodrug-like MC4R polypeptide agonist

[0543] The prodrug-linked MC4R polypeptide agonist 3 was modified with the substituents of Example 5.1. The structure of the modified compound is as follows:

[0544] LC-MS determined m / z = 783.88 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0545] Compound 11 (amino acid sequence corresponding to SEQ ID NO: 11) after modification of the prodrug-type MC4R polypeptide agonist

[0546] The prodrug-linked MC4R polypeptide agonist 4 was modified with the substituents of Example 5.1. The structure of the modified compound is as follows:

[0547] LC-MS determined m / z = 779.2 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0548] Example 7 Synthesis and Purification of Modified Compounds Linked to Prodrug-like MC4R Peptide Agonists

[0549] The modified compound of the prodrug-linked MC4R polypeptide agonist was prepared according to the synthesis and purification method of Example 2 and confirmed by mass spectrometry.

[0550] Supplementary Example 2: The raw materials for protecting amino acids are: Boc-Dap(Fmoc)-OH (CAS: 122235-70-5), mono-tert-butyl dodecanedioate (CAS: 234081-98-2), and Fmoc-Gly-OH (CAS: 29022-11-5).

[0551] The specific results are as follows:

[0552] ① Compound 12 modified by linking prodrug-like MC4R peptide agonist

[0553] Sequence (amino acid sequence corresponds to SEQ ID NO: 12):

[0554] LC-MS determined m / z = 703.17 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0555] ② Compound 13 after modification with prodrug-like MC4R peptide agonist

[0556] Sequence (amino acid sequence corresponds to SEQ ID NO: 12):

[0557] LC-MS determined m / z = 703.17 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0558] ③ Compound 14 after modification with prodrug-like MC4R peptide agonist

[0559] Sequence (amino acid sequence corresponds to SEQ ID NO: 12):

[0560] LC-MS determined m / z = 913.74 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0561] ④ Compound 15 modified by linking prodrug-like MC4R peptide agonist

[0562] Sequence (amino acid sequence corresponds to SEQ ID NO: 12):

[0563] Chemical structure:

[0564] LC-MS determined m / z = 712.52 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0565] ⑤ Compound 16 modified by linking prodrug-like MC4R peptide agonist

[0566] Sequence (amino acid sequence corresponds to SEQ ID NO: 13):

[0567] Chemical structure:

[0568] LC-MS determined m / z = 950.10 [M+3H] 3+ , which is consistent with the theoretical molecular weight.

[0569] ⑥ Compound 17 after modification with prodrug-like MC4R polypeptide agonist

[0570] Sequence (amino acid sequence corresponds to SEQ ID NO: 13):

[0571] Chemical structure:

[0572] LC-MS determined m / z = 950.10 [M+3H]3+ , which is consistent with the theoretical molecular weight.

[0573] ⑦ Compound 18 after modification with prodrug-like MC4R peptide agonist

[0574] Sequence (amino acid sequence corresponds to SEQ ID NO: 13):

[0575] Chemical structure:

[0576] LC-MS determined m / z = 991.87 [M+4H] 4+ , which is consistent with the theoretical molecular weight.

[0577] Example 8 Determination of the Conversion Half-Life of Prodrug-Linked MC4R Polypeptide Agonists

[0578] Prodrug-linked MC4R peptide agonists and modified compounds of prodrug-linked MC4R peptide agonists were dissolved in DPBS (without CaCl2 and MgCl2) to a final concentration of 200 μM. The pH of the sample solution was adjusted to 7.4 using 0.02 M HCl or 0.02 M NaOH. The solution was then aliquoted into small tubes and stored in a sealed cell culture incubator at 37°C. Samples were collected at different time points over two weeks to test the hydrolysis efficiency, i.e., the conversion half-life. The results are shown in Table 7 below:

[0579] Table 7 Conversion half-life of linked prodrug compounds

[0580] “-” means no hydrolysis and no conversion occurs.

[0581] Example 9 In vitro activity assay of prodrug-linked MC4R polypeptide agonists

[0582] The in vitro activity assay method of the prodrug-linked MC4R polypeptide agonist was consistent with that in Example 3. The results are shown in Table 8:

[0583] Table 8 In vitro activities of Setmelanotide analogs and modified Setmelanotide analogs

[0584] “-” indicates inactivity.

[0585] Example 9 Pharmacodynamics Evaluation in Rats

[0586] Twenty-five SPF-grade normal female Sprague-Dawley rats (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were housed in a normal animal laboratory with free access to water and food. Before the experiment, they were randomly divided into five groups of five rats each. The specific groupings and dosing schedules are shown in Table 9. (All drugs were prepared in normal saline at a 5 mg / ml solution. The dose was then calculated based on the animal's body weight and diluted with normal saline to the target dose.)

[0587] Table 9 Grouped Dosage Scheme

[0588] Groups A and B were dosed once daily, Group C was dosed every two days, and Groups D and E were dosed every four days. The body weights of the rats in each treatment group and the control group were then measured and recorded daily. The body weight growth rates of the rats were calculated.

[0589] The formula for calculating weight gain is as follows:

[0590] Body weight increase rate (%) = (Dt body weight - D1 body weight) / D1 body weight x 100%, where Dt body weight is the body weight on Dt day and D1 body weight is the initial body weight. The body weight change rate of rats is shown in Figure 1.

[0591] As shown in Figure 1, the prodrug-linked MC4R peptide agonist 12, administered once every four days, achieved similar weight gain to that observed in the positive drug setmelanotide group, demonstrating that the drug achieved long-term efficacy, meeting our goal of slow-release prodrugs to extend drug half-life. To this end, the dosage and dosing schedule of the prodrug-linked MC4R agonist 12 were adjusted, and further in vivo efficacy was validated in obese DIO mice.

[0592] Example 10 In vivo pharmacodynamic evaluation in DIO mice

[0593] Twenty-five SPF-grade diabetic inducible (DIO) mice and five normal mice of the same age (all from Changzhou Cavens Laboratory Animal Co., Ltd.) were housed in an SPF animal facility. The DIO mice were fed a 60% high-fat diet, while the normal mice of the same age were fed a maintenance diet. The DIO mice were randomly divided into five groups of five before the experiment. The specific groupings and dosing schedule are shown in Table 10. (All drugs were prepared in saline at a 5 mg / ml solution, then the dose was calculated based on animal weight and diluted with saline to the target dose.)

[0594] Table 10 Grouped Dosage Scheme

[0595] Groups A, B, and C were dosed once daily, while groups D, E, and F were dosed every three days. The body weights of DIO mice in each treatment group and the control group were then measured and recorded daily. The body weight growth rates of the DIO mice were calculated.

[0596] The formula for calculating weight gain is as follows:

[0597] Body weight increase rate (%) = (Dt body weight - D0 body weight) / D0 body weight × 100%, where Dt body weight is the body weight on Dt and D0 body weight is the initial body weight. The body weight change rates of DIO mice are shown in Figure 2, and the specific body weight change rates are shown in Table 11.

[0598] Table 11 Weight growth rate of animals in each group

[0599] As can be seen from the results in Figure 2, the prodrug-linked MC4R polypeptide agonist 12 has a good weight loss effect. The dosage of the low-dose administration group within three days is equivalent to that of the setmelanotide administration group, but it has a significant weight loss advantage compared with the setmelanotide administration group. The prodrug-linked MC4R agonist 12 is administered once every three days, which proves that it not only achieves the long-term purpose, but also has a significant advantage in weight loss compared with setmelanotide.

[0600] Example 11 Pharmacokinetic evaluation in SD rats

[0601] SD rats (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) (weight 240-260g) were randomly divided into groups, 3 in each group, and subcutaneously injected with a prodrug-linked MC4R peptide agonist 12 at a dose of 0.9nmol / kg. Blood was collected subcutaneously at 0.5, 1, 3, 6, 8, 10, 24, 30, 48, 72, and 96h after administration, and plasma samples were prepared at each time point. 500μL of the plasma sample to be tested was acidified (at a ratio of plasma: 10% perchloric acid = 95:5 (v / v)); 5μL of the standard working solution was added to the acidified mixture and mixed for 30s; 500μL of 4% H3PO4 and 0.2M ZnSO4 were added and mixed for 30s, and centrifuged at 13000rpm / min at low temperature (4°C) for 10min; 1mL was quantitatively taken and added to the activated Solid-phase extraction (SPE) was performed using a MAX column (96-Well Plate, 30 mg plate), including ammonia treatment, methanol elution, and elution with a mixture of acetonitrile:methanol (6:4, v / v) containing 2% formic acid. The eluate was dried with nitrogen at 40°C and then reconstituted with 150 μL of 50% methanol solution containing 1% formic acid. The mixture was vortexed for 1 minute and centrifuged at 13,000 rpm / min for 10 minutes at 4°C. The supernatant was then analyzed by LC-MS. A non-compartmental model was used to fit the pharmacokinetic parameters of the drug in each group of SD rats, including the elimination half-life (t 1 / 2 ), peak time (T max ), drug peak concentration (C max), area under the drug-time curve (AUC (0-t) ), the area under the infinite drug-time curve (AUC(0-inf)_obs), the observed value of apparent distribution volume (V_obs), the observed value of clearance (CL_obs), the mean residence time (MRT(0-t)), and the infinite mean residence time (MRT(0-inf)_obs) are shown in Table 12.

[0602] Table 12. Pharmacokinetic parameters in SD rats

[0603] Although the present disclosure has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made based on the present disclosure. Therefore, such modifications or improvements, which do not depart from the spirit of the present disclosure, are intended to be within the scope of protection claimed herein.

Claims

1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof, AZ I in, Z is a polypeptide analogue, whose amino acid sequence is Arg 1 -c[Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 ]-X1-X2-X3-X4-X5-X6-X7, where Arg 1 is the N-terminus of the amino acid sequence, and Z is connected to A via its N-terminus; Aaa and Bbb are independently a cysteine ​​residue, a homocysteine ​​(hCys) residue, or a penicillamine (Pen) residue; Xxx is an alanine residue, a D-alanine residue or does not exist; Yyy is a threonine residue, a D-threonine residue, a serine residue, a D-serine residue, a histidine residue, a D-histidine residue, an asparagine residue, a D-asparagine residue, a glutamine residue or a D-glutamine residue; c[Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 The "c" in ] means Aaa 2 The thiol group in Bbb 8 The sulfhydryl groups in the sulfhydryl group form a ring through disulfide bonds; X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue, a D-lysine residue, a proline residue, a D-proline residue, a glutamic acid residue, a D-glutamic acid residue, an aspartic acid residue, a D-aspartic acid residue, a threonine residue, a D-threonine residue, a glycine residue, a D-glycine residue, an alanine residue, a D-alanine residue, an arginine residue, a D-arginine residue, a serine residue, a D-serine residue, Cys 8 It is connected to X1 through an amide bond; X1, X2, X3, X4, X5 and X6 are connected by an amide bond or an ester bond; X7 is NH2 or OH; X1, X2, X3, X4, X5 or X6 is connected to X7 via a carbonyl group; When X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond, and L' is -L1-L2-L3-L4-L5-L6-L7-L8; L1, L2, L3, L4, L5 and L6 are each independently absent, L7 L8 n is 10, 11, 12, 13, 14, 15, 16, 17 or 18; L1, L2, L3, L4, L5, L6, L7 and L8 are connected by amide bonds; A does not exist, or B; B is X is H or -(CH2) m -NH-L; m is 1, 2, 3 or 4; L is H or L'; Y is H, C1-C4 alkyl or -(CH2) p -NH-L, p is 1, 2, 3 or 4; The compound as shown in Formula I satisfies at least one of the following conditions: Condition I: at least one of X1, X2, X3, X4, X5 and X6 is a lysine residue, a D-lysine residue, a proline residue, a D-proline residue, a glutamic acid residue, a D-glutamic acid residue, an aspartic acid residue, a D-aspartic acid residue, a threonine residue, a D-threonine residue, a glycine residue, a D-glycine residue, an alanine residue, a D-alanine residue, an arginine residue, a D-arginine residue, a serine residue, a D-serine residue, When X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond, and L' is -L1-L2-L3-L4-L5-L6-L7-L8; Condition II: A is B.

2. A compound as shown in formula I or a pharmaceutically acceptable salt thereof; AZ I in, Z is a polypeptide analogue, whose amino acid sequence is Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]-X1-X2-X3-X4-X5-X6-X7, where Arg 1 is the N-terminus of the amino acid sequence, and Z is connected to A via its N-terminus; c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 The "c" in ] stands for Cys 2 The thiol and Cys 8 The sulfhydryl groups in the sulfhydryl group form a ring through disulfide bonds; X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue, a D-lysine residue, a proline residue, a D-proline residue, a glutamic acid residue, a D-glutamic acid residue, an aspartic acid residue, a D-aspartic acid residue, a threonine residue, a D-threonine residue, a glycine residue, a D-glycine residue, an alanine residue, a D-alanine residue, an arginine residue, a D-arginine residue, a serine residue, a D-serine residue, Cys 8 It is connected to X1 through an amide bond; X1, X2, X3, X4, X5 and X6 are connected by an amide bond or an ester bond; X7 is NH2 or OH; X1, X2, X3, X4, X5 or X6 is connected to X7 via a carbonyl group; When X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond, and L' is -L1-L2-L3-L4-L5-L6-L7-L8; L1, L2, L3, L4, L5 and L6 are each independently absent, L7 L8 n is 10, 11, 12, 13, 14, 15, 16, 17 or 18; L1, L2, L3, L4, L5, L6, L7 and L8 are connected by amide bonds; A does not exist, or B; B is X is H or -(CH2) m -NH-L; m is 1, 2, 3 or 4; L is H or L'; Y is H, C1-C4 alkyl or -(CH2) p -NH-L, p is 1, 2, 3 or 4; When A is When at least one of X1, X2, X3, X4, X5 and X6 in Z exists.

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1) Aaa is a cysteine ​​residue or a homocysteine ​​residue; (2) Bbb is a cysteine ​​residue or a penicillamine residue; (3) Xxx is absent or a D-alanine residue; (4) Yyy is a threonine residue or a histidine residue; (5) When the compound of Formula I satisfies at least Condition I, at least one of X1, X2, X3, X4, X5 and X6 is a lysine residue, a D-lysine residue, a glutamic acid residue, a D-glutamic acid residue, an arginine residue or a D-arginine residue; when X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond. Preferably, when the compound as shown in Formula I satisfies at least Condition I, at least one of X1, X2, X3, X4, X5 and X6 is a lysine residue, a glutamic acid residue or an arginine residue, and when X1, X2, X3, X4, X5 or X6 is a lysine residue, the ε-amino group of the side chain of the lysine residue is unsubstituted or connected to L' in the form of an amide bond; More preferably, when the compound as shown in Formula I satisfies at least Condition I, at least one of X1, X2, X3, X4, X5 and X6 is a lysine residue; (6) When the compound of formula I satisfies condition II, X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue, a D-lysine residue, a glutamic acid residue, a D-glutamic acid residue, an arginine residue or a D-arginine residue; when X1, X2, X3, X4, X5 or X6 is a lysine residue or a D-lysine residue, the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond; Preferably, when the compound as shown in Formula I satisfies Condition II, X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue, a glutamic acid residue or an arginine residue, preferably absent or a lysine residue, and when X1, X2, X3, X4, X5 or X6 is a lysine residue, the ε-amino group of the side chain of the lysine residue is unsubstituted or connected to L' in the form of an amide bond; (7) When the compound as shown in formula I satisfies condition I, A is absent, (8) When the compound as shown in formula I satisfies condition II, A is 4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1)Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 is Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 、hCys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 、hCys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Pen 8 、Cys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Cys 7 、hCys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 or Cys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Cys 7 ,preferably Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 、hCys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 or hCys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 ; (2) L1, L2, L3, L4, L5 and L6 are each independently absent, Preferably, -L1-L2-L3-L4-L5-L6- is Among them, the end marked with "#" indicates the L1 end, and correspondingly, the end without "#" indicates the L6 end; (3) L8 n is 10, 12, 16 or 18; (4) X is H, -(CH2)-NH-L, -(CH2)2-NH-L or -(CH2)4-NH-L; (5) Y is H, C1-C4 alkyl or -(CH2)2-NH-L.

5. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: L' is: Preferably 6. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1) X is H, Preferably H, (2) Y is H, CH3, -(CH2)2-NH2, Preferably CH3 or -(CH2)2-NH2, more preferably CH3; (3) When the compound of Formula I satisfies at least Condition I, -X1-X2-X3-X4-X5-X6- is -Lys-, -Lys-Lys-, -Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Glu-, -Glu-Glu-, -Glu-Glu -Glu-, -Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-Glu-, -Lys-Glu- , -Lys-Glu-Lys-, -Lys-Glu-Glu-, -Glu-Lys-, -Lys-Glu-Glu-Lys-, -Arg-, -Arg-Arg, or any of the following structures: Preferably, when the compound as shown in Formula I satisfies at least Condition 1, -X1-X2-X3-X4-X5-X6- is -Lys-, -Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Glu-, -Glu-Glu-, -Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-, -Gl u-Glu-Glu-Glu-Glu-Glu-, -Lys-Glu-, -Lys-Glu-Lys-, -Lys-Glu-Glu-, -Glu-Lys-, -Lys-Glu-Glu-Lys-, -Arg-, -Arg-Arg, preferably is -Lys-, -Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys- or -Lys-Lys-Lys-Lys-Lys-Lys-, further preferably -Lys-Lys-Lys-Lys-; (4) When the compound of Formula I satisfies Condition II, -X1-X2-X3-X4-X5-X6- is absent, -Lys-, -Lys-Lys-, -Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu-Glu-, -Glu- lu-Glu-, -Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-Glu-, -Lys-Glu -, -Lys-Glu-Lys-, -Lys-Glu-Glu-, -Glu-Lys-, -Lys-Glu-Glu-Lys-, -Arg-, -Arg-Arg, or any of the following structures: Preferably, when the compound of formula I satisfies condition II, -X1-X2-X3-X4-X5-X6- is absent, -Lys-, -Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys-, -Glu-, -Glu-Glu-, -Glu-Glu-Glu-Glu-, -Glu-Glu-Glu-Glu-Glu-, - Glu-Glu-Glu-Glu-Glu-Glu-, -Lys-Glu-, -Lys-Glu-Lys-, -Lys-Glu-Glu-, -Glu-Lys-, -Lys-Glu-Glu-Lys-, -Arg-, -Arg-Arg, excellent It is selected from -Lys-, -Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys- or -Lys-Lys-Lys-Lys-Lys-Lys-, and -Lys-Lys-Lys-Lys- is more preferred.

7. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: It meets one or more of the following conditions: (1) X1, X2, X3, X4, X5 and X6 are each independently absent, a lysine residue or a D-lysine residue, wherein the ε-amino group of the side chain of the lysine residue or the D-lysine residue is unsubstituted or connected to L' in the form of an amide bond; (2) L1, L2, L3, L4, L5 and L6 are each independently absent, Preferably, in L', -L1-L2-L3-L4-L5-L6- is Among them, the end marked with "#" indicates the L1 end, and correspondingly, the end without "#" indicates the L6 end; (3) L8 n is 10, 12, 16 or 18; (4) X is H, -(CH2)-NH-L, -(CH2)2-NH-L or -(CH2)4-NH-L; (5) Y is H, C1-C4 alkyl or -(CH2)2-NH-L; (6) A does not exist or For example, if it does not exist, 8. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: It meets one or more of the following conditions: (1) X1, X2, X3, X4, X5 and X6 are each independently absent or a lysine residue, wherein the ε-amino group of the side chain of the lysine residue is unsubstituted or connected to L' in the form of an amide bond; (2) L' is: Preferably (3) X is H, Preferably H, (4) Y is H, CH3, -(CH2)2-NH2, Preferred is CH3 or -(CH2)2-NH2, and more preferred is CH3.

9. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: X1, X2, X3, X4, X5 and X6 are each independently absent or a lysine residue.

10. The compound of formula I as claimed in claim 2, or a pharmaceutically acceptable salt, amide or ester thereof, characterized in that: -X1-X2-X3-X4-X5-X6-X7 is any of the following: (1) -X1-X2-X3-X4-X5-X6-X7 is NH2, -Lys-NH2, -Lys-Lys-NH2, -Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-NH2, or any one of the following structures: (2) -X1-X2-X3-X4-X5-X6-X7 is -NH2, -Lys-NH2, -Lys-Lys-NH2, -Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-NH2 or -Lys-Lys-Lys-Lys-Lys-Lys-NH2; (3) -X1-X2-X3-X4-X5-X6-X7 is -NH2, -Lys-NH2, -Lys-Lys-NH2, -Lys-Lys-Lys-Lys-NH2, -Lys-Lys-Lys-Lys-Lys-NH2 or -Lys-Lys-Lys-Lys-Lys-Lys-NH2.

11. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The compound as shown in formula I is any one of the following: (1) A is absent, and the compound of formula I is Z, wherein Z is Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]-X1-X2-X3-X4-X5-X6-X7, X1, X2, X3, X4, X5 and X6 are each independently absent or a lysine residue, and X7 is NH2; (2) A is X is H or -(CH2)4-NH2; Y is C1-C4 alkyl or -(CH2) p -NH2, p is 1, 2, 3 or 4; Z is Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 ]-X1-X2-X3-X4-X5-X6-X7, -X1-X2-X3-X4-X5-X6-X7 is -NH2 or -Lys-Lys-Lys-Lys-NH2; (3) A is X is Y is H or C1-C4 alkyl, Z is Arg 1 -c[Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 -X1-X2-X3-X4-X5-X6-X7, -X1-X2-X3-X4-X5-X6-X7 is -Lys-Lys-Lys-Lys-NH2.

12. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound as shown in formula I is any one of the following: (1) A is absent, and the compound of formula I is Z, wherein Z is Arg 1 -c[Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 ]-X1-X2-X3-X4-X5-X6-X7, -X1-X2-X3-X4-X5-X6- is -Lys-, -Lys-Lys-, -Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-, -Lys-Lys-Lys-Lys-Lys- or -Lys-Lys-Lys-Lys-Lys-Lys-; X7 is NH2; Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 is Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 、hCys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 or hCys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 ; (2) A is X is Y is H or C1-C4 alkyl, Z is Arg 1 -c[Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 ]-X1-X2-X3-X4-X5-X6-X7, X1-X2-X3-X4-X5-X6-X7 is -NH2 or -Lys-Lys-Lys-Lys-NH2; Aaa 2 -Xxx 3 -Yyy 4 -DPhe 5 -Arg 6 -Trp 7 -Bbb 8 is Cys 2 -DAla 3 -His 4 -DPhe 5 -Arg 6 -Trp 7 -Cys 8 、hCys 2 -Thr 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 or hCys 2 -His 3 -DPhe 4 -Arg 5 -Trp 6 -Pen 7 。 13. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound as shown in formula I is any one of the following:

14. A pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 13, and a pharmaceutically acceptable excipient.

15. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, or the pharmaceutical composition according to claim 14 in the preparation of an MC4R agonist.

16. Use of a compound of formula I as described in any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described in claim 14 in the preparation of a medicament for preventing and / or treating chronic weight management deficiency in adults and children aged 6 years and above with monogenic or syndromic obesity caused by proprotein convertase subtilisin / kexin type 1, leptin receptor deficiency or Bardet-Biedl syndrome.

17. Use of a compound of formula I as described in any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described in claim 14 in the preparation of a medicament for preventing and / or treating obesity, polydactyly, retinal atrophy, gonadal dysgenesis, renal malformation or cognitive impairment.

18. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 in the preparation of a health product for alleviating obesity.

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