Acylated human insulin analogue

Abstract

The present application relates to the field of biomedicine, and specifically provides an acylated human insulin analogue, a pharmaceutical composition thereof and a preparation method therefor, and a method for using the acylated human insulin analogue or the pharmaceutical composition thereof to treat metabolic diseases.

Description

Acylated human insulin analogues TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to acylated human insulin analogues, as well as a preparation method, a pharmaceutical composition and a use thereof. BACKGROUND

[0002] Diabetes mellitus is a metabolic disease, which is hyperglycemia caused by impaired insulin secretion and varying degrees of peripheral insulin resistance. Persistent hyperglycemia and chronic metabolic disorders can lead to serious complications, such as damage, dysfunction and failure of the eye, kidney, cardiovascular system and even nervous system. Basic insulin therapy is an important means to control high blood sugar, and basic insulin mainly includes medium-acting insulin neutral protamine zinc insulin, long-acting insulin protamine zinc insulin and long-acting / ultra-long-acting human insulin analogues (such as insulin glargine U100, insulin glargine U300, insulin detemir, insulin degludec, etc.). The most important obstacle of basic insulin application is that frequent injection leads to limited treatment compliance of some patients.

[0003] Icodec insulin (icodec insulin) is a super-long-acting basic insulin developed by Novo Nordisk, which only needs to be injected once a week. Icodec insulin is based on human insulin, with 4 amino acid mutations and acylation modification of fatty acid side chains, realizing the long-acting and solubility improvement of icodec insulin. The substitution of three amino acids (A14E, B16H and B25H) provides molecular stability, which helps to weaken the binding with insulin receptor (IR), reduces the clearance mediated by IR binding, and prolongs the half-life. The side chain containing C20 fatty diacid improves the binding affinity with albumin.

[0004] Basic insulin products are constantly iterated and upgraded, and there is still a demand for basic insulin with long action time, low drug frequency and good efficacy in clinical application. SUMMARY

[0005] In one aspect, the present application provides an acylated human insulin analogue, which comprises a human insulin analogue and an acyl moiety, the acyl moiety being represented by formula (I):

[0006] X-(Y) m -(Z) n -(I),

[0007] wherein,

[0008] X is HOOC-(CH2) p -CO-;

[0009] Y is an acidic amino acid residue;

[0010] Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-;

[0011] m is 1, 2, 3, 4, 5 or 6;

[0012] n is 3 or 4 when p is an integer less than 20; n is 1 or 2 when p is an integer greater than or equal to 20;

[0013] X, Y and Z are connected by an amide bond;

[0014] the acyl moiety is connected to the ε-amino group of the lysine residue of the human insulin analogue via Z by an amide bond.

[0015] In one aspect, the present application provides an acylated human insulin analogue comprising a human insulin analogue and an acyl moiety, the acyl moiety being represented by formula (I):

[0016] X-(Y) m -(Z) n -(I),

[0017] wherein,

[0018] X is HOOC-(CH2) p -CO-;

[0019] Y is an acidic amino acid residue;

[0020] Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-;

[0021] m is 1, 2, 3, 4, 5 or 6;

[0022] n is 3 or 4 when p is an integer less than 20;

[0023] X, Y and Z are connected by an amide bond;

[0024] the acyl moiety is connected to the ε-amino group of the lysine residue of the human insulin analogue via Z by an amide bond.

[0025] In one aspect, the present application provides an acylated human insulin analogue comprising a human insulin analogue and an acyl moiety, the acyl moiety being represented by formula (I):

[0026] X-(Y) m -(Z) n -(I),

[0027] wherein,

[0028] X is HOOC-(CH2) p-CO-;

[0029] Y is an acidic amino acid residue;

[0030] Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-;

[0031] m is 1, 2, 3, 4, 5 or 6;

[0032] p is an integer greater than or equal to 20 and n is 1 or 2;

[0033] X, Y and Z are connected by an amide bond;

[0034] said acyl moiety is connected to the epsilon-amino group of the lysine residue of the human insulin analogue via an amide bond.

[0035] In one aspect, the present application provides a compound of formula (II),

[0036] X-(Y) m -(Z) n -OH (II),

[0037] wherein,

[0038] X is HOOC-(CH2) p -CO-;

[0039] Y is an acidic amino acid residue;

[0040] Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-;

[0041] m is 1, 2, 3, 4, 5 or 6;

[0042] p is an integer less than 20 and n is 3 or 4; p is an integer greater than or equal to 20 and n is 1 or 2;

[0043] X, Y and Z are connected by an amide bond.

[0044] In some embodiments, Y is γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp. In some specific embodiments, Y is γGlu.

[0045] In some embodiments, the lysine residue is the lysine residue in position B29 of the human insulin analogue.

[0046] In some embodiments, the human insulin analogue is selected from the group consisting of: desB30 human insulin; A14E, A18L, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, B1E, B25H, B27E, B28E, desB30 human insulin; A14E, B1E, B25H, B28E, desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B25H, desB30 human insulin; A14E, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, B25H, B27E, desB30 human insulin; A14E, B25H, desB27, desB30 human insulin; A14E, B25H, B29R, desB30 human insulin; A14E, A21G, B16H, B25H, desB30 human insulin; A14E, A21G, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, A21G, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; A21G human insulin; A21G, desB30 human insulin; and B28D human insulin. In some specific embodiments, the human insulin analogue is A14E, B16H, B25H, desB30 human insulin.

[0047] In some embodiments, m is 1, 2, or 3. In some embodiments, m is 1, 2, or 3, and n is 1. In some embodiments, m is 1, 2, or 3, and n is 2. In some embodiments, m is 1, 2, or 3, and n is 3. In some embodiments, m is 1, 2, or 3, and n is 4.

[0048] In some embodiments, p is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, and n is 3. In some embodiments, p is 12, 13, 14, 15, 16, 17, 18, or 19, and n is 4. In some embodiments, p is 20, 21, 22, 23, 24, 25, or 26, and n is 1. In some embodiments, p is 20, 21, 22, 23, 24, 25, or 26, and n is 2.

[0049] In some embodiments, m is 1, 2, or 3, p is 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, m is 1, 2, or 3, p is 18. In some embodiments, m is 1, p is 18. In some embodiments, m is 3, p is 18. In some embodiments, m is 1, 2, or 3, p is 12, 13, 14, 15, 16, 17, 18, or 19, n is 3. In some embodiments, m is 1, p is 12, 13, 14, 15, 16, 17, 18, or 19, n is 3. In some embodiments, m is 3, p is 12, 13, 14, 15, 16, 17, 18, or 19, n is 3. In some embodiments, m is 1, 2, or 3, p is 12, 13, 14, 15, 16, 17, 18, or 19, n is 4. In some embodiments, m is 1, p is 12, 13, 14, 15, 16, 17, 18, or 19, n is 4. In some embodiments, m is 3, p is 12, 13, 14, 15, 16, 17, 18, or 19, n is 4. In some specific embodiments, m is 1, p is 18, n is 3. In some specific embodiments, m is 3, p is 18, n is 3. In some specific embodiments, m is 1, p is 18, n is 4. In some specific embodiments, m is 3, p is 18, n is 4.

[0050] In some embodiments, m is 1, 2, or 3, p is 20, 21, 22, 23, 24, 25, or 26. In some embodiments, m is 1, 2, or 3, p is 20. In some embodiments, m is 1, p is 20. In some embodiments, m is 2, p is 20. In some embodiments, m is 3, p is 20. In some embodiments, m is 1, 2, or 3, p is 20, 21, 22, 23, 24, 25, or 26, n is 1. In some embodiments, m is 1, p is 20, 21, 22, 23, 24, 25, or 26, n is 1. In some embodiments, m is 2, p is 20, 21, 22, 23, 24, 25, or 26, n is 1. In some embodiments, m is 3, p is 20, 21, 22, 23, 24, 25, or 26, n is 1. In some embodiments, m is 1, 2, or 3, p is 20, 21, 22, 23, 24, 25, or 26, n is 2. In some embodiments, m is 1, p is 20, 21, 22, 23, 24, 25, or 26, n is 2. In some embodiments, m is 2, p is 20, 21, 22, 23, 24, 25, or 26, n is 2. In some embodiments, m is 3, p is 20, 21, 22, 23, 24, 25, or 26, n is 2. In some embodiments, m is 1, 2, or 3, p is 20, n is 1. In some specific embodiments, m is 1, p is 20, n is 1. In some specific embodiments, m is 2, p is 20, n is 1. In some specific embodiments, m is 3, p is 20, n is 1. In some embodiments, m is 1, 2, or 3, p is 20, n is 2. In some specific embodiments, m is 1, p is 20, n is 2. In some specific embodiments, m is 2, p is 20, n is 2. In some specific embodiments, m is 3, p is 20, n is 2.

[0051] In some embodiments, the acylated human insulin analogue is selected from: A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(Nε -21-carboxy-heneicosanoyl-3xγGlu-2xOEG), desB30 human insulin. ε -21-carboxy-heneicosanoyl-2xγGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-3xγGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-γGlu-2xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-2xγGlu-2xOEG), desB30 human insulin and A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-3xγGlu-2xOEG), desB30 human insulin.

[0052] In a particular embodiment, the acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-γGlu-3xOEG), desB30 human insulin.

[0053] In a particular embodiment, the acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-γGlu-4xOEG), desB30 human insulin.

[0054] In a particular embodiment, the acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-γGlu-OEG), desB30 human insulin.

[0055] In a particular embodiment, the acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-2xγGlu-OEG), desB30 human insulin.

[0056] In a particular embodiment, the acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε-21-carboxy-heneicosanoyl-3xγGlu-OEG), desB30 human insulin.

[0057] In one specific embodiment, the acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-3xγGlu-OEG), desB30 human insulin.

[0058] In another aspect, the present application provides a method of preparing the acylated human insulin analogue, the method comprising acylating modification of a human insulin analogue described herein with an acyl moiety described herein.

[0059] In another aspect, the present application provides a pharmaceutical composition comprising an acylated human insulin analogue described herein.

[0060] In another aspect, the present application provides a method of treating a metabolic disease, the method comprising administering to a subject in need thereof an acylated human insulin analogue described herein or a pharmaceutical composition described herein. In some embodiments, the metabolic disease is diabetes. In some specific embodiments, the diabetes is type 1 diabetes or type 2 diabetes.

[0061] In yet another aspect, the present application provides a kit comprising an acylated human insulin analogue described herein or a pharmaceutical composition described herein.

[0062] The acylated human insulin analogue provided by the present application is safe, has a longer half-life in vivo, improved biological activity and in vivo efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 shows the effect of acylated human insulin analogue on blood glucose of db / db mice. DETAILED DESCRIPTION

[0064] Terminology

[0065] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present application have the meanings that are commonly understood by one of ordinary skill in the art to which this application belongs.

[0066] The term "human insulin analogue" has a molecular structure formally derivable from the structure of native human insulin by deletion and / or substitution (replacement) of one or more amino acid residues of native human insulin and / or addition of one or more amino acid residues. Native human insulin consists of two peptide chains, the A chain having 21 amino acid residues comprising one intra-chain disulfide bridge (A6C-A11C) and the B chain having 30 amino acid residues, wherein the thiol groups of the four cysteines A7C-B7C, A20C-B19C form two disulfide bridges connecting the A and B chains. The sequences of the A and B chains of native human insulin are as follows:

[0067] A chain: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1);

[0068] B chain: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2).

[0069] Herein, the terms such as A1, A2, A3 indicate positions 1, 2 and 3, respectively, in the A chain of insulin (counted from the N-terminus). Similarly, the terms such as B1, B2, B3 indicate positions 1, 2 and 3, respectively, in the B chain of insulin (counted from the N-terminus). The terms such as A14Y, A14E indicate that the amino acid at position A21 is Y (Tyr, tyrosine) and E (Glu, glutamic acid), respectively. The amino acids can be denoted by the three letter code or the one letter code. The term desB30 indicates an insulin analogue wherein the B30 amino acid residue is deleted.

[0070] The term "amino acid residue" includes an amino acid from which a hydrogen atom has been removed from the amino group and / or a hydroxyl group has been removed from the carboxyl group and / or a hydrogen atom has been removed from the thiol group. Herein, the term "amino acid" encompasses "amino acid residue".

[0071] The term "acidic amino acid residue" refers to an amino acid residue that is negatively charged at physiological pH. Acidic amino acids include, but are not limited to, aspartic acid (Asp, D) and glutamic acid (Glu, E).

[0072] The term "acylation" refers to the introduction of an acyl group in an organic molecule.

[0073] Herein, the nomenclature of the insulin is according to the following principles: the name is given according to the mutations and modifications (such as acylation) relative to native human insulin. The nomenclature of the acyl moiety is according to the IUPAC nomenclature or the nomenclature of peptides. For example, the following acyl moieties:

[0074] which can be named "eicosanedioyl-γGlu-OEG-OEG", "eicosanedioyl-γGlu-2xOEG", "19-carboxy-nonadecanoyl-γGlu-OEG-OEG", or "19-carboxy-nonadecanoyl-γGlu-2xOEG", wherein OEG represents the group -HN-(CH2)2-O-(CH2)2-O-CH2-CO- (i.e., 2-[2-(2-aminoethoxy)ethoxy]acetyl). According to IUPAC nomenclature, the acyl moiety above can also be named: [2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl].

[0075] The term "pharmaceutical composition" refers to a mixture of one or more of the active ingredients or agents of the application with a pharmaceutically acceptable carrier.

[0076] The term "treatment" generally refers to any action that results in a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. As used herein, "treatment" covers any treatment of a patient, including, but not limited to, preventing the onset of a disease or recurrence of a disease, alleviating the symptoms of a disease, reducing any direct or indirect pathological consequences of a disease, preventing metastasis of a disease, slowing the progression of a disease, ameliorating or lessening the state of a disease, increasing the frequency and duration of symptom-free periods, and remission or improvement of the prognosis of a disease.

[0077] As used herein, the terms "subject" and "patient" are used interchangeably. A "subject" or "patient" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and hamsters. In some embodiments, the term "subject" or "patient" is a mammal. In some embodiments, the subject or patient is a mouse. In some embodiments, the subject or patient is a human.

[0078] The terms "comprising" or "including" or "containing" are to be construed open-ended, i.e., to the effect that "including but not limited to".

[0079] Examples

[0080] While the foregoing application has been described in some detail for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims and that the foregoing application is not to be limited to the exact construction details as described. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the application. One of ordinary skill in the art will readily recognize a variety of noncritical parameters that can be changed or modified to yield essentially similar results.

[0081] All reagents employed in the examples can be purchased commercially unless otherwise stated.

[0082] The present application also provides the following specific embodiments, but is not limited thereto:

[0083] Embodiment 1. An acylated human insulin analogue, wherein the acylated human insulin analogue comprises a human insulin analogue and an acyl moiety, the acyl moiety being represented by formula (I):

[0084] X-(Y) m -(Z) n -(I),

[0085] wherein,

[0086] X is HOOC-(CH2) p -CO-;

[0087] Y is an acidic amino acid residue;

[0088] Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-;

[0089] m is 1, 2, 3, 4, 5 or 6;

[0090] n is 3 or 4 when p is an integer less than 20; n is 1 or 2 when p is an integer greater than or equal to 20;

[0091] X, Y and Z are connected via an amide bond;

[0092] the acyl moiety is connected to the epsilon-amino group of a lysine residue of the human insulin analogue via an amide bond through Z.

[0093] Embodiment 2. An acylated human insulin analogue, wherein the acylated human insulin analogue comprises a human insulin analogue and an acyl moiety, the acyl moiety being represented by formula (I):

[0094] X-(Y) m -(Z) n -(I),

[0095] wherein,

[0096] X is HOOC-(CH2) p -CO-;

[0097] Y is an acidic amino acid residue;

[0098] Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-;

[0099] m is 1, 2, 3, 4, 5 or 6;

[0100] p is an integer less than 20, and n is 3 or 4;

[0101] X, Y and Z are connected by an amide bond;

[0102] said acyl moiety is connected by Z to the ε-amino group of a lysine residue of the human insulin analogue.

[0103] Embodiment 3. An acylated human insulin analogue, characterized in that said acylated human insulin analogue comprises a human insulin analogue and an acyl moiety, said acyl moiety being represented by formula (I):

[0104] X-(Y) m -(Z) n -(I),

[0105] wherein,

[0106] X is HOOC-(CH2) p -CO-;

[0107] Y is an acidic amino acid residue;

[0108] Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-;

[0109] m is 1, 2, 3, 4, 5 or 6;

[0110] p is an integer greater than or equal to 20, and n is 1 or 2;

[0111] X, Y and Z are connected by an amide bond;

[0112] said acyl moiety is connected by Z to the ε-amino group of a lysine residue of the human insulin analogue.

[0113] Embodiment 4. The acylated human insulin analogue according to any of embodiments 1-3, characterized in that Y is γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; preferably Y is γGlu.

[0114] Embodiment 5. The acylated human insulin analogue according to any of embodiments 1-4, characterized in that the lysine residue is the lysine residue in position B29 of the human insulin analogue.

[0115] Embodiment 6. The acylated human insulin analogue according to any of embodiments 1-5, characterized in that the human insulin analogue is selected from the group consisting of: desB30 human insulin; A14E, A18L, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, B1E, B25H, B27E, B28E, desB30 human insulin; A14E, B1E, B25H, B28E, desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B25H, desB30 human insulin; A14E, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, B25H, B27E, desB30 human insulin; A14E, B25H, desB27, desB30 human insulin; A14E, B25H, B29R, desB30 human insulin; A14E, A21G, B16H, B25H, desB30 human insulin; A14E, A21G, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, A21G, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; A21G human insulin; A21G, desB30 human insulin and B28D human insulin; preferably the human insulin analogue is A14E, B16H, B25H, desB30 human insulin.

[0116] Embodiment 7. The acylated human insulin analogue according to any of embodiments 1-2 or embodiments 4-6, characterized in that m is 1, 2 or 3, p is 12, 13, 14, 15, 16, 17, 18 or 19; preferably m is 1, p is 18.

[0117] Embodiment 8. The acylated human insulin analogue according to any of embodiments 1-2 or embodiments 4-7, characterized in that m is 1, p is 18, n is 3.

[0118] Embodiment 9. The acylated human insulin analogue according to any of Embodiments 1-2 or Embodiments 4-7, characterized in that m is 1, p is 18, and n is 4.

[0119] Embodiment 10. The acylated human insulin analogue according to any of Embodiments 1 or Embodiments 3-6, characterized in that m is 1, 2 or 3, and p is 20, 21, 22, 23, 24, 25 or 26; preferably m is 1, 2 or 3, and p is 20.

[0120] Embodiment 11. The acylated human insulin analogue according to any of Embodiments 1, Embodiments 3-6 or Embodiment 10, characterized in that m is 1, 2 or 3, p is 20, and n is 1.

[0121] Embodiment 12. The acylated human insulin analogue according to any of Embodiments 1, Embodiments 3-6 or Embodiments 10-11, characterized in that m is 1, p is 20, and n is 1.

[0122] Embodiment 13. The acylated human insulin analogue according to any of Embodiments 1, Embodiments 3-6 or Embodiments 10-11, characterized in that m is 2, p is 20, and n is 1.

[0123] Embodiment 14. The acylated human insulin analogue according to any of Embodiments 1, Embodiments 3-6 or Embodiments 10-11, characterized in that m is 3, p is 20, and n is 1.

[0124] Embodiment 15. The acylated human insulin analogue according to any of Embodiments 1, Embodiments 3-6 or Embodiment 10, characterized in that m is 1, 2 or 3, p is 20, and n is 2.

[0125] Embodiment 16. The acylated human insulin analogue according to any of Embodiments 1, Embodiments 3-6, Embodiment 10 or Embodiment 15, characterized in that m is 1, p is 20, and n is 2.

[0126] Embodiment 17. The acylated human insulin analogue according to any of Embodiments 1, Embodiments 3-6, Embodiment 10 or Embodiment 15, characterized in that m is 2, p is 20, and n is 2.

[0127] Embodiment 18. The acylated human insulin analogue according to any of Embodiments 1, Embodiments 3-6, Embodiment 10 or Embodiment 15, characterized in that m is 3, p is 20, and n is 2.

[0128] Embodiment 19. The acylated human insulin analogue according to any of embodiments 1-18, characterized in that said acylated human insulin analogue is selected from the group consisting of: A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-γGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-2xγGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-3xγGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-γGlu-2xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-2xγGlu-2xOEG), desB30 human insulin and A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-3xγGlu-2xOEG), desB30 human insulin.

[0129] Embodiment 20. The acylated human insulin analogue according to any of embodiments 1-6, characterized in that said acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-3xOEG), desB30 human insulin.

[0130] Embodiment 21. The acylated human insulin analogue according to any of embodiments 1-6, characterized in that said acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 19-carboxy-nonadecanoyl-γGlu-4xOEG), desB30 human insulin.

[0131] Embodiment 22. The acylated human insulin analogue according to any of embodiments 1-6, characterized in that said acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 21-carboxy-heneicosanoyl-2xγGlu-OEG), desB30 human insulin.

[0132] Embodiment 23. The acylated human insulin analogue according to any of embodiments 1-6, characterized in that said acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 21-carboxy-heneicosanoyl-2xγGlu-OEG), desB30 human insulin.

[0133] Embodiment 24. The acylated human insulin analogue according to any of embodiments 1-6, characterized in that said acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 21-carboxy-heneicosanoyl-2xγGlu-OEG), desB30 human insulin.

[0134] Embodiment 25. The acylated human insulin analogue according to any of embodiments 1-6, characterized in that said acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 21-carboxy-heneicosanoyl-2xγGlu-OEG), desB30 human insulin.

[0135] Embodiment 26. A method of preparing an acylated human insulin analogue according to any of embodiments 1-25, characterized in that said method comprises acylating modification of said human insulin analogue with said acyl moiety.

[0136] Embodiment 27. A pharmaceutical composition, characterized in that said pharmaceutical composition comprises an acylated human insulin analogue according to any of embodiments 1-25.

[0137] Embodiment 28. A method of treating a metabolic disease, comprising administering to a subject in need thereof the acylated human insulin analogue of any one of embodiments 1-25 or the pharmaceutical composition of embodiment 27; preferably, the metabolic disease is diabetes.

[0138] Embodiment 29. The method of embodiment 28, wherein the diabetes is type 1 diabetes or type 2 diabetes.

[0139] Embodiment 30. A kit, comprising the acylated human insulin analogue of any one of embodiments 1-25 or the pharmaceutical composition of embodiment 27.

[0140] Embodiment 31. The kit of embodiment 31, further comprising a container loaded with the acylated human insulin analogue or the pharmaceutical composition and / or instructions for administering the acylated human insulin analogue or the pharmaceutical composition to a subject in need thereof.

[0141] Embodiment 32. A compound, comprising:

[0142] X-(Y) m -(Z) n -OH (II),

[0143] wherein,

[0144] X is HOOC-(CH2) p -CO-;

[0145] Y is an acidic amino acid residue;

[0146] Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-;

[0147] m is 1, 2, 3, 4, 5 or 6;

[0148] n is 3 or 4 when p is an integer less than 20; n is 1 or 2 when p is an integer greater than or equal to 20;

[0149] X, Y and Z are connected via amide bonds.

[0150] Embodiment 33. The compound of embodiment 32, wherein Y is γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; preferably, Y is γGlu.

[0151] Embodiment 34. The acylated human insulin analogue according to embodiment 32 or 33, characterized in that m is 1, 2 or 3, and p is 12, 13, 14, 15, 16, 17, 18 or 19; preferably m is 1 and p is 18.

[0152] Embodiment 35. The acylated human insulin analogue according to any one of embodiments 32-34, characterized in that m is 1, p is 18 and n is 3.

[0153] Embodiment 36. The acylated human insulin analogue according to any one of embodiments 32-35, characterized in that m is 1, p is 18 and n is 4.

[0154] Embodiment 37. The acylated human insulin analogue according to any one of embodiments 32 or 33, characterized in that m is 1, 2 or 3, and p is 20, 21, 22, 23, 24, 25 or 26; preferably m is 1, 2 or 3, and p is 20.

[0155] Embodiment 38. The acylated human insulin analogue according to any one of embodiments 32-33 or embodiment 37, characterized in that m is 1, 2 or 3, p is 20 and n is 1.

[0156] Embodiment 39. The acylated human insulin analogue according to any one of embodiments 32-33 or embodiments 37-38, characterized in that m is 1, p is 20 and n is 1.

[0157] Embodiment 40. The acylated human insulin analogue according to any one of embodiments 32-33 or embodiments 37-38, characterized in that m is 2, p is 20 and n is 1.

[0158] Embodiment 41. The acylated human insulin analogue according to any one of embodiments 32-33 or embodiments 37-38, characterized in that m is 3, p is 20 and n is 1.

[0159] Embodiment 42. The acylated human insulin analogue according to any one of embodiments 32-33 or embodiment 37, characterized in that m is 1, 2 or 3, p is 20 and n is 2.

[0160] Embodiment 43. The acylated human insulin analogue according to any one of embodiments 32-33, embodiment 37 or embodiment 42, characterized in that m is 1, p is 20 and n is 2.

[0161] Embodiment 44. The acylated human insulin analogue according to any one of embodiments 32-33, embodiment 37 or embodiment 42, characterized in that m is 2, p is 20 and n is 2.

[0162] Embodiment 45. The acylated human insulin analogue according to any of Embodiment 32-33, Embodiment 37 or Embodiment 42, characterized in that m is 3, p is 20, n is 2.

[0163] Example 1. Preparation of acylated human insulin analogue

[0164] The positive control used in the examples of the present application is insulin icodec, i.e. A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-γGlu-2xOEG), desB30 human insulin.

[0165] 1.1 Preparation of A14E, B16H, B25H, desB30 human insulin

[0166] The preparation of A14E, B16H, B25H, desB30 human insulin can be performed using techniques routine for the skilled person, e.g. Glendorf T. et al., 2008. Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding. Biochemistry, 47, p. 4743-4751. In the present example, a plasmid containing a sequence encoding a precursor of a human insulin analogue (SEQ ID NO: 3) is electrotransformed into a Pichia pastoris host strain GS115 (C18100, invitrogen), and the recombinant strain is fermented. The human insulin analogue precursor is purified from the fermentation broth and subjected to lysyl endopeptidase to obtain A14E, B16H, B25H, desB30 human insulin.

[0167] EEGEPKFVNQHLCGSHLVEALHLVCGERGFHYTPKAMKGIVEQCCTSICSLEQLENYCN (SEQ ID NO: 3).

[0168] 1.2 Preparation of side chain compound (21-carboxy-heneicosanoyl-2xγGlu-2xOEG)

[0169] 1) Resin swelling: 1.00 g of 2-CTC resin was weighed into a reactor, 15 mL DCM was added, and the reactor was shaken at 120 rpm for 2 h. Then the resin was washed twice with DCM;

[0170] 2) Coupling OEG: 1.09 g of Fmoc-OEG was weighed and added to 10 mL of DCM / NMP (volume ratio of DCM and NMP = 1:1), after dissolving, 0.47 g of DIEA was added, after mixing, it was added to the reactor containing the swollen resin, and the shaker was shaken at 120 rpm for 2 h. The resin was washed with DCM / NMP (volume ratio of DCM and NMP = 1:1) for 6 times;

[0171] 3) 188 μL of methanol and 0.36 g of DIEA were added to 8 mL of DCM / NMP (volume ratio of DCM and NMP = 1:1), after mixing, it was added to the resin, and the shaker was shaken at 120 rpm for 0.5 h. The resin was washed with DCM / NMP (volume ratio of DCM and NMP = 1:1) for 6 times;

[0172] 4) Deprotection group: 10 mL of PIP / DMF (volume ratio of PIP and MDF = 1:4) was added to the resin, the shaker was shaken at 120 rpm for 10 min, the resin was washed with DMF for 6 times, and then 10 mL of PIP / DMF (volume ratio of PIP and MDF = 1:4) was added, the shaker was shaken at 120 rpm for 20 min, and the resin was washed with DMF for 6 times;

[0173] 5) Coupling OEG: 1.09 g of Fmoc-OEG, 0.38 g of HOBt was weighed and added to 10 mL of DCM / NMP (volume ratio of DCM and NMP = 1:1) and mixed, then 0.43 mL of DIC was added, after mixing, it was added to the resin, and the shaker was shaken at 120 rpm for 2 h. The resin was washed with DCM / NMP (volume ratio of DCM and NMP = 1:1) for 6 times;

[0174] 6) Repeat step 4;

[0175] 7) Coupling γGlu: 1.19 g of Fmoc-γGlu, 0.38 g of HOBt was weighed and added to 10 mL of DCM / NMP (volume ratio of DCM and NMP = 1:1) and mixed, then 0.43 mL of DIC was added, after mixing, it was added to the resin, and the shaker was shaken at 120 rpm for 2 h. The resin was washed with 8 mL of DCM / NMP (volume ratio of DCM and NMP = 1:1) for 6 times;

[0176] 8) Repeat step 5;

[0177] 9) Repeat steps 7 and 8;

[0178] 10) Coupling C22: Weigh 1.19 g C22(acid)-Otbu, 0.38 g HOBt, add to 10 mL DCM / NMP (volume ratio of DCM and NMP = 1:1), mix, then add 0.43 mL DIC, mix, and then add to the resin, shake at 120 rpm for 2 h. Wash the resin with 8 mL DCM / NMP (volume ratio of DCM and NMP = 1:1) for 3 times, then wash the resin with DCM for 3 times;

[0179] 11) Dry the resin at 35 °C under vacuum for 3 h;

[0180] 12) Dissociate the sample: add 20 mL TFE / DCM (volume ratio of TFE and DCM = 1:4), shake at 120 rpm for 1 h;

[0181] 13) Suck-filter to obtain the supernatant, distill the supernatant at 40 °C under reduced pressure to remove the organic reagent, and collect the side chain solution;

[0182] 14) Side chain activation: take 1 mL of the side chain solution, add 1.5 eq HOSU and 1.2 eq DCC, stir with a rotor for 1-2 h, filter, rotary evaporate, add an appropriate amount of ethyl acetate to the filtrate, shake well, filter, rotary evaporate, and repeat this step for 2-3 times;

[0183] 15) Side chain deprotection: take 1 volume of the activated side chain, crack with 4 volumes of TFA for 2 h, then precipitate with 40 volumes of isopropyl ether, centrifuge at 10000 rpm for 10 min, add an appropriate amount of ethyl ether to the precipitate, stir and centrifuge, repeat this step for 2-3 times, dry to obtain a solid side chain, and then dissolve in 16 mL DMF.

[0184] 1.3 Preparation of A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-2xγGlu-2xOEG), desB30 human insulin (compound S033)

[0185] Add triethylamine to a final concentration of 1.5% to a 60 mg A14E, B16H, B25H, desB30 human insulin solution, then add 1 mL of the side chain solution, react at room temperature for 30 min, detect the peak area of the target compound by liquid chromatography, continue to add 1 mL of the side chain to the reaction system, react at room temperature for 30 min, until the peak area of the target compound no longer increases, stop the reaction. Harvest the product by reverse phase chromatography.

[0186] Liquid chromatography instrument and conditions:

[0187] Instrument: high performance liquid chromatograph (1260-Bio, Agilent)

[0188] Mobile phase A: Sulfate and phosphate buffer: acetonitrile (90.4:9.6) Mobile phase B: acetonitrile - water (50:50) solution

[0189] Chromatographic column: Waters Symmetry Shield RP8 (4.6 mm x 150 mm, 3.5 μm)

[0190] Wavelength: 214 nm Flow rate: 1.0 mL / min Column temperature: 55 °C Sample chamber temperature: 5 °C Injection volume: 10 μL

[0191] Gradient:

[0192] All compounds in Table 1 were prepared following similar procedures as described in Sections 1.1-1.3 above. Table 2 shows the structures of some acylated human insulin analogs.

[0193] Table 1 Acylated human insulin analogs

[0194] Table 2 Structures of some acylated human insulin analogs

[0195] 1.4 Characterization of acylated human insulin analogs

[0196] Instrument: Liquid chromatography-mass spectrometry (1290-6420A, Agilent)

[0197] Mobile phase A: 0.1% formic acid - water solution

[0198] Mobile phase B: 0.1% formic acid - acetonitrile solution

[0199] Chromatographic column: Waters ACQUITY UPLC BEH C8 (2.1 mm x 100 mm, 1.7 μm)

[0200] Wavelength: 214 nm Flow rate: 0.4 mL / min Column temperature: 40 °C Sample chamber temperature: 5 °C Injection volume: 5 μL

[0201] Gradient:

[0202] Table 3 Mass spectrometry molecular weights of acylated human insulin analogs

[0203] Example 2 Affinity analysis of acylated human insulin analogs to human insulin receptor, human serum albumin

[0204] Amino coupling of human insulin receptor protein or human serum albumin to CM5 sensor chip, using running buffer (100 mL 10x PBS-P added to 900 mL ultrapure water, then 1.115 g EDTA-2Na, 8.069 g NaCl) dilute each sample to 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, 3.125 μmol / L, 1.5625 μmol / L, each concentration sample dilution at a flow rate of 50 μL / min through the experimental channel, binding time 150 s, dissociation time 180 s, Biacore Control Software 2.0 real-time data signal acquisition, Biacore Evaluation Software 2.0 data analysis, calculate the association rate constant ka, dissociation rate constant kd, equilibrium dissociation constant KD. The results are shown in Tables 4 and 5, the acylated human insulin analogs of the present application have high affinity for human insulin receptor, human serum albumin.

[0205] Table 4 Binding affinity of acylated human insulin analogs to human insulin receptor

[0206] Table 5 Binding affinity of acylated human insulin analogs to human serum albumin

[0207] Example 3 Biological activity analysis of acylated human insulin analogs

[0208] After acylated human insulin analogs bind to human insulin receptor (hIR), the level of intracellular tyrosine phosphorylation changes and shows a dose-dependent relationship, which is used to determine the biological activity of acylated human insulin analogs.

[0209] CHO-hIR cells were cultured in DMEM complete medium at 37°C in a 5% CO2 incubator to the logarithmic growth phase, trypsinized to detach the cells, and then seeded in 96-well plates at a density of 1.8 x 10 5The cells were inoculated into 96-well cell culture plates at a cell density of 1.5 x 105cells / mL, and after being cultured at 37°C in a 5% CO2incubator for 16-20 h, 50 μL of acylated human insulin analogues at different concentrations were added, and after being incubated at 37°C for 30 min, cell lysis solution was added. The lysis mixture was added to a 96-well high-adsorption plate coated with anti-insulin receptor antibody, and after being incubated for 1 h, the plate was washed 5 times per well with DELFIA washing solution, 200 μL of 0.3 μg / mL anti-phosphotyrosine antibody (AD0040, PerkinElmer) was added, and after being incubated for 1 h, the plate was washed 5 times per well with DELFIA washing solution, 200 μL per well of DELFIA enhancement solution was added, and after being incubated for 2 h, the plate was detected by a multifunctional microplate reader HTRF module.

[0210] The experimental data were analyzed by Prism 5 software, the logarithm of the concentration of acylated human insulin analogue was taken as the X axis, and the corresponding fluorescence value was taken as the Y axis, a four-parameter equation was selected for fitting, a dose-response curve was drawn, and the EC 50 value was obtained. The results are shown in Table 6, and the acylated human insulin analogues of the application have good binding activity with human insulin receptors.

[0211] Table 6 EC 50

[0212] Example 4 Effect of acylated human insulin analogues on blood glucose of ICR mice

[0213] SPF level 18-20 g normal male ICR mice were raised in an animal laboratory with a temperature of 20-26°C and a light-dark cycle of 12 hours each, and were allowed to drink water and eat freely. Before the experiment, the basal blood glucose and body weight of the mice were detected, and the mice were grouped according to the body weight and basal blood glucose value. The acylated human insulin analogues were diluted with PBS, and the dosage was 1.44 μmol / kg. The control group was injected with the same volume of PBS. The animals were not fasted during the administration period, and the blood glucose was measured at 24 h, 48 h and 72 h after administration. The results are shown in Tables 7 and 8, and the hypoglycemic effect of S014, S015, S033, S028, S029 and S030 is better than that of insulin icodec.

[0214] Table 7 Effect of acylated human insulin analogues on blood glucose of ICR mice Note: - indicates that the observation was terminated because the blood glucose did not decrease or the blood glucose rebounded significantly.

[0215] Table 8 Effect of acylated human insulin analogues on blood glucose of ICR mice

[0216] Effect of acylated human insulin analog on blood glucose of db / db mice

[0217] SPF level 5-6 weeks old male db / db mice, weighing 30-40 g, were adaptively fed at temperature 23±2℃, humidity 55±10%, light and dark alternation each 12 hours for 7 days, free drinking water and food. The body weight and basal blood glucose of mice were detected before the experiment, and according to the body weight and basal blood glucose value, they were randomly divided into control group, 3mg / kg Insulin icodec group, 6mg / kg Insulin icodec group, 3mg / kg S033 group and 6mg / kg S033 group, 2 days once abdominal subcutaneous injection of drug, continuous administration for 52 days, the control group was injected with PBS. The tail tip blood was taken to detect the blood glucose during the experiment, and the results are shown in Figure 1, S033 has a significantly better hypoglycemic effect than insulin icodec.

Claims

1. An acylated human insulin analogue, characterized in that, The acylated human insulin analogue comprises a human insulin analogue and an acyl moiety, which is of formula (I): X-(Y) m -(Z) n - (Ⅰ), wherein X is HOOC-(CH2) p -CO-; Y is an acidic amino acid residue; Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-; m is 1, 2, 3, 4, 5 or 6; n is 3 or 4 when p is an integer less than 20; n is 1 or 2 when p is an integer greater than or equal to 20; X, Y and Z are linked by amide bonds; said acyl moiety is linked by an amide bond to the epsilon-amino group of a lysine residue of a human insulin analogue.

2. The acylated human insulin analogue according to claim 1, characterized in that p is an integer less than 20, n is 3 or 4.

3. The acylated human insulin analogue of claim 1, wherein p is an integer greater than or equal to 20, n is 1 or 2.

4. The acylated human insulin analogue according to any one of claims 1-3, characterized in that, Y is γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; preferably Y is γGlu.

5. The acylated human insulin analogue according to any of claims 1-4, characterised in that, said lysine residue is the lysine residue in position B29 of a human insulin analogue.

6. The acylated human insulin analogue of any of claims 1-5, characterized in that, said human insulin analogue is selected from the group consisting of desB30 human insulin; A14E, A18L, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, B1E, B25H, B27E, B28E, desB30 human insulin; A14E, B1E, B25H, B28E, desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B25H, desB30 human insulin; A14E, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, B25H, B27E, desB30 human insulin; A14E, B25H, desB27, desB30 human insulin; A14E, B25H, B29R, desB30 human insulin; A14E, A21G, B16H, B25H, desB30 human insulin; A14E, A21G, B25H, B26G, B27G, B28G, desB30 human insulin; A14E, A21G, B25H, desB30 human insulin; A14E, A21G, B25H, desB27, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; A21G human insulin; A21G, desB30 human insulin and B28D human insulin; preferably said human insulin analogue is A14E, B16H, B25H, desB30 human insulin.

7. The acylated human insulin analogue of any of claims 1-2 or claims 4-6, characterised in that, m is 1, 2 or 3, p is 12, 13, 14, 15, 16, 17, 18 or 19; preferably m is 1, p is 18.

8. The acylated human insulin analogue of any of claims 1-2 or claims 4-7, characterized in that, m is 1, p is 18, n is 3.

9. The acylated human insulin analogue of any of claims 1-2 or claims 4-7, characterised in that, m is 1, p is 18, n is 4.

10. The acylated human insulin analogue of any one of claims 1 or 3-6, characterised in that, m is 1, 2 or 3, p is 20, 21, 22, 23, 24, 25 or 26; preferably m is 1, 2 or 3, p is 20.

11. The acylated human insulin analogue of any of claims 1, 3-6 or 10, characterized in that, m is 1, p is 20, n is 1.

12. The acylated human insulin analogue of any of claims 1, 3-6 or 10-11, characterized in that, m is 1, p is 20, n is 1.

13. The acylated human insulin analogue of any of claims 1, 3-6 or 10-11, characterized in that, m is 2, p is 20, n is 1.

14. The acylated human insulin analogue of any of claims 1, 3-6 or 10-11, characterized in that, m is 3, p is 20, n is 1.

15. The acylated human insulin analogue of any of claims 1, 3-6 or 10, characterized in that, m is 1, 2 or 3, p is 20, and n is 2.

16. The acylated human insulin analogue of any of claims 1, 3-6, 10 or 15, characterized in that, m is 1, p is 20, and n is 2.

17. The acylated human insulin analogue of any of claims 1, 3-6, 10 or 15, characterized in that, m is 2, p is 20, and n is 2.

18. The acylated human insulin analogue of any of claims 1, 3-6, 10 or 15, characterized in that, m is 3, p is 20, and n is 2.

19. The acylated human insulin analogue of any of claims 1-18, characterized in that, said acylated human insulin analogue is selected from: A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-4xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-4xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-4xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -19-carboxy-nonadecanoyl-3xγGlu-4xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-γGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-2xγGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-3xγGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-γGlu-2xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-2xγGlu-2xOEG), desB30 human insulin and A14E, B16H, B25H, B29K(N ε -21-carboxy-heneicosanoyl-3xγGlu-2xOEG), desB30 human insulin.

20. The acylated human insulin analogue of any of claims 1-6, characterized in that, The acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 19-carboxy-nonadecanoyl-γGlu-3xOEG), desB30 human insulin.

21. The acylated human insulin analogue of any of claims 1-6, characterized in that, The acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 19-carboxy-nonadecanoyl-γGlu-4xOEG), desB30 human insulin.

22. The acylated human insulin analogue of any of claims 1-6, characterized in that said acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 21-carboxy-heneicosanoyl-YGIu-OEG), desB30 human insulin.

23. The acylated human insulin analogue of any of claims 1-6, characterized in that, The acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 21-carboxy-heneicosanoyl-2xGlu-OEG), desB30 human insulin.

24. The acylated human insulin analogue of any of claims 1-6, characterized in that, The acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 21-carboxy-heneicosanoyl-3xGlu-OEG), desB30 human insulin.

25. The acylated human insulin analogue of any of claims 1-6, characterized in that, The acylated human insulin analogue is A14E, B16H, B25H, B29K(N ε - 21-carboxy-heneicosanoyl-2xγGlu-2xOEG), desB30 human insulin.

26. A method of preparing the acylated human insulin analogue of any of claims 1-25, characterized in that, The method comprises acylating modification of the human insulin analogue with the acyl moiety.

27. A pharmaceutical composition comprising, The pharmaceutical composition comprises the acylated human insulin analogue of any one of claims 1-25.

28. A method for treating metabolic diseases, characterized in that, The method comprises administering to a subject in need thereof the acylated human insulin analogue of any one of claims 1-25 or the pharmaceutical composition of claim 27; preferably, the metabolic disease is diabetes.

29. The method of claim 28, wherein, The diabetes is type 1 diabetes or type 2 diabetes.

30. A kit comprising, The kit comprises the acylated human insulin analogue of any one of claims 1-25 or the pharmaceutical composition of claim 27.

31. The kit of claim 31, wherein, The kit further comprises a container loaded with the acylated human insulin analogue or the pharmaceutical composition and / or instructions for administering the acylated human insulin analogue or the pharmaceutical composition to a subject in need thereof.

32. A compound characterized by, The compound is shown as formula (II): X-(Y) m -(Z) n -OH (II), wherein, X is HOOC-(CH2) p -CO-; Y is an acidic amino acid residue; Z is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-; m is 1, 2, 3, 4, 5 or 6; n is 3 or 4 when p is an integer less than 20; n is 1 or 2 when p is an integer greater than or equal to 20; X, Y and Z are connected via amide bonds.

33. The compound of claim 32, wherein Y is γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; preferably, Y is γGlu.

Images