Long-acting GLP-1 compounds

Abstract

Compared with liraglutide, semaglutide and other commercially available GLP-1 derivatives, the novel GLP-1 derivative has equivalent or better efficacy, drug effect or efficacy, longer or equivalent in-vivo continuous acting time or in-vivo half-life period, better or equivalent GLP-1 receptor binding affinity and better or equivalent DPP-IV stability.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of December 29, 2020, application number 202080091240.0, and invention name “Long-acting GLP-1 Compounds”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to Chinese invention patent applications No. CN201911397405.2 filed on December 30, 2019 and No. CN202011053306.5 filed on September 29, 2020, which are incorporated herein by reference. Technical Field

[0004] The present invention relates to the field of therapeutic peptides, and in particular to a novel long-acting GLP-1 compound, a pharmaceutical preparation thereof, a pharmaceutical composition thereof and long-acting insulin, and medical uses of the compound, the pharmaceutical preparation and the pharmaceutical composition. Background Art

[0005] Glucagon-like peptide 1 (GLP-1) and its analogs and derivatives are very effective in treating type 1 and type 2 diabetes, however, the high clearance rate limits the effectiveness of these compounds. In order to provide GLP-1 compounds with longer duration of action in vivo, a series of different methods have been used to modify the structure of glucagon-like peptide 1 (GLP-1). For example, WO99 / 43708 discloses GLP-1 (7-35) and GLP-1 (7-36) derivatives with lipophilic substituents attached to the C-terminal amino acid residue. WO00 / 34331 discloses acylated GLP-1 analogs. WO00 / 69911 discloses activated insulinotropic peptides for injection into patients.

[0006] The GLP-1 drugs currently on the market include, for example: the natural GLP-1 analog Exenatide, which is administered twice a day; Liraglutide and Lixisenatide, which are administered once a day. The former is a hexadecanoic acid-modified GLP-1 compound, and the latter is a new molecule obtained by structural modification of Exenatide; and Semaglutide, Exenatide LAR, Abiglutide, Dulaglutide (also known as dulaglutide) and polyethylene glycol loxenatide, which are administered once a week. Among them, exenatide microspheres are prepared by encapsulating exenatide into a poly(lactic-co-glycolic acid) copolymer matrix through a microencapsulation method; albiglutide is prepared by fusing two modified GLP-1 peptide chains with human albumin in the form of a dimer to form a recombinant fusion protein; dulaglutide is obtained by fusing the modified GLP-1 chain to the Fc fragment of the recombinant G4 immunoglobulin through a disulfide bond; polyethylene glycol loxenatide is prepared by amino acid modification and polyethylene glycol modification based on the chemical structure of exenatide; semaglutide is mainly achieved by replacing Ala at the 8th position with the non-protein amino acid Aib on the GLP-1 (7-37) peptide to achieve once-a-week administration, but the presence of non-protein amino acids in semaglutide, compared with natural amino acids, may pose unknown and various potential side effects risks in the human body.

[0007] On the one hand, there is still a need to develop compounds that have better efficacy, pharmacodynamics or effectiveness, less risk of potential side effects, better weight loss and diet inhibition effects, longer or equivalent in vivo duration of action or in vivo half-life than similar drugs already on the market, such as liraglutide, dulaglutide and semaglutide, so as to provide better medication options for diabetic patients.

[0008] On the other hand, with the rapid increase in the number of people with type 2 diabetes worldwide, there is a greater demand for more effective drugs with simpler administration. For example, a compound preparation containing two active ingredients, insulin and GLP-1 peptide, may be a very effective therapeutic agent. Therefore, there is still a need for a compound preparation that can synergistically achieve better physical and chemical stability, longer duration of action, and better efficacy. Summary of the invention

[0009] In order to overcome or improve at least one disadvantage of the prior art, or to provide a useful alternative, the first aspect of the present invention provides a new GLP-1 compound (also called a GLP-1 derivative). Compared with the GLP-1 derivatives that have been marketed, such as liraglutide, dulaglutide, and semaglutide, the new GLP-1 compound has better efficacy, pharmacodynamics or efficacy, less risk of potential side effects, better weight loss effect, longer in vivo sustained action time or in vivo half-life, better or equivalent GLP-1 receptor binding affinity, and better or equivalent DPP-IV stability. And the long-acting GLP-1 compound of the present invention and the pharmaceutical composition or combined preparation of the long-acting insulin provided by the present invention will not only not weaken the physical stability of the GLP-1 compound and the insulin compound, but also the combined preparation has better physical stability than the single-drug preparation. Compared with the combined preparations of other long-acting GLP-1 compounds (such as the combined preparations of liraglutide and degludec insulin), the physical stability of the combined preparation of the present invention is unexpected. In addition, compared with the single-drug preparation, the combined preparation also increases the chemical stability of the GLP-1 compound and the acylated insulin. The GLP-1 compound of the present invention, and the combination preparation containing the GLP-1 compound and the pancreatic islet compound provided by the present invention can well achieve long-term pharmacokinetic (hereinafter also referred to as PK) characteristics, making it possible to perform subcutaneous treatment of diabetic patients twice a week, once a week, once every two weeks, or less frequently.

[0010] The GLP-1 compound provided in the first aspect of the present invention is a compound of formula B, or a pharmaceutically acceptable salt, amide or ester thereof:

[0011] [Acy-(L1) r -(L2) q ]-G1(B),

[0012] wherein G1 is a GLP-1 analog having Arg at position 34 and Ala or Gly at position 8 corresponding to GLP-1 (7-37) (SEQ ID NO: 1), [Acy-(L1) r -(L2) q ] is a substituent attached to the epsilon amino group of the Lys residue at position 26 of the GLP-1 analog, wherein

[0013] r is an integer from 1 to 10, and q is 0 or an integer from 1 to 10;

[0014] Acy is an aliphatic diacid comprising 20 to 24 carbon atoms, wherein a hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid;

[0015] L1 is an amino acid residue selected from the group consisting of γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp;

[0016] L2 is a neutral, alkylene glycol-containing amino acid residue;

[0017] Acy, L1, and L2 are connected by an amide bond; and

[0018] The order in which L1 and L2 appear in formula (B) can be independently interchanged.

[0019] In one embodiment, G1 is [Gly8, Arg34]GLP-1-(7-37) peptide or [Arg34]GLP-1-(7-37) peptide, preferably [Gly8, Arg34]GLP-1-(7-37) peptide.

[0020] In one embodiment, r is 1, 2, 3, 4, 5 or 6, preferably, r is 1, 2, 3 or 4, preferably, r is 1 or 2, preferably, r is 1.

[0021] In another embodiment, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably, q is 0, 1, 2, 3 or 4, more preferably, q is 0, 1, or 2.

[0022] In one embodiment, Acy is an aliphatic diacid containing 20-23 carbon atoms, preferably Acy is an aliphatic diacid containing 20, 21, or 22 carbon atoms, wherein a hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid.

[0023] In one embodiment, L2 is: -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2- H2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-, -HN-(CH2 )3-O-(CH2)4-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)2-O -(CH2)2-O-CH2-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)3-O -(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)2-CO-, -HN-(C H2)2-O-(CH2)2-O-(CH2)2-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-C O-, -HN-(CH2)3-O-(CH2)3-O-CH2-CO-, or -HN-(CH2)4-O-(CH2)4-O-CH2-CO-; preferably L2 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-.

[0024] In one embodiment, L1 is selected from γGlu or βAsp, preferably L1 is γGlu.

[0025] In one embodiment, Acy is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21-CO- or HOOC-(CH2) 22 -CO-, Acy is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

[0026] In one embodiment, Acy, L1, and L2 in formula (B) are sequentially linked by amide bonds, and the C-terminus of L2 is linked to the epsilon amino group of the Lys residue at position 26 of the GLP-1 analog.

[0027] In one embodiment, the compound of the first aspect of the present invention is selected from the following compounds:

[0028] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0029] N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0030] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0031] N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0032] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0033] N-ε 26-[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0034] N-ε 26 -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide,

[0035] N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide,

[0036] N-ε 26 -(21-carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide,

[0037] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0038] N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0039] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0040] N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0041] N-ε 26-[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0042] N-ε 26 -[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0043] N-ε 26 -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide,

[0044] N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, or

[0045] N-ε 26 -(21-Carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide.

[0046] In one embodiment, the compound of the first aspect of the present invention is selected from the following compounds:

[0047] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0048] N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0049] N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide,

[0050] N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide,

[0051] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0052] N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0053] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0054] N-ε 26 -[2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0055] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0056] N-ε 26 -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide,

[0057] N-ε 26 -(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide,

[0058] N-ε 26 -(22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide,

[0059] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0060] N-ε 26 -[2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0061] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0062] N-ε 26 -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide,

[0063] N-ε 26 -(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, or

[0064] N-ε 26 -(22-Carboxydocosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide.

[0065] In one embodiment, the compound of the first aspect of the present invention is selected from the following compounds:

[0066] N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyhenicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide.

[0067] The second aspect of the present invention provides a pharmaceutical preparation comprising the compound according to the first aspect of the present invention and a pharmaceutically acceptable excipient.

[0068] In one embodiment, the pharmaceutically acceptable excipient is selected from one or more of a buffer, a preservative, an isotonic agent, a stabilizer, and a chelating agent. In another embodiment, the pharmaceutically acceptable excipient is a buffer, a preservative, and an isotonic agent.

[0069] In one embodiment, the pharmaceutical preparation comprises: the compound described in the first aspect of the present invention, an isotonic agent, a preservative and a buffer. Preferably, in the pharmaceutical preparation, the compound described in the first aspect of the present invention is N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyhenicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide.

[0070] In one embodiment, the isotonic agent is selected from one or more of sodium chloride, propylene glycol, mannitol, sorbitol, glycerol, glucose and xylitol, preferably propylene glycol, mannitol or sodium chloride.

[0071] In another embodiment, the preservative is selected from one or more of phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, and benzyl alcohol, preferably phenol or m-cresol.

[0072] In another embodiment, the buffer is selected from one or more of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, preferably sodium acetate, citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate.

[0073] In one embodiment, the pH of the formulation is from about 6.0 to about 10.0, preferably from about 6.5 to about 10.0, preferably from about 6.5 to about 9.5, preferably from about 6.5 to about 8.5, more preferably from about 7.0 to about 8.5, more preferably from about 7.0 to about 8.1, and further preferably from about 7.3 to about 8.1.

[0074] In one embodiment, the pharmaceutical formulation contains the following ingredients:

[0075] About 0.1-1.2 mM, preferably about 0.2-1 mM, preferably about 0.3-0.7 mM, more preferably about 0.48-0.6 mM of the compound according to the first aspect of the present invention;

[0076] About 10-1500mM, preferably about 13-800mM, preferably about 65-400mM, preferably about 90-240mM, preferably about 150-250mM, preferably about 180-200mM, more preferably about 183-195mM isotonic agent, preferably, the isotonic agent is selected from one or more of propylene glycol, glycerol, mannitol or sodium chloride;

[0077] About 1-200mM, preferably about 5-150mM, preferably about 10-100mM, preferably about 20-85mM, preferably about 30-75mM, preferably about 45-60mM, more preferably about 50-60mM of a preservative, preferably, the preservative is selected from one or more of phenol or m-cresol;

[0078] About 3-35 mM, preferably about 5-20 mM, more preferably about 5-15 mM, more preferably about 7-10 mM of a buffer selected from one or more of sodium acetate, citrate, sodium dihydrogen phosphate or disodium hydrogen phosphate; and

[0079] The pH of the pharmaceutical preparation is about 6.0 to about 10.0, preferably about 6.5 to about 9.5, preferably about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, further preferably about 7.3 to about 8.1.

[0080] In another experimental embodiment, the pharmaceutical preparation comprises: about 0.3-0.7 mM, more preferably about 0.48-0.6 mM N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; about 180-200mM, more preferably about 183-195mM propylene glycol; about 45-60mM, more preferably about 50-60mM phenol; about 5-15mM buffer, preferably about 7-10mM disodium hydrogen phosphate; and the pH of the pharmaceutical preparation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, and further preferably about 7.3 to about 8.3.

[0081] In another experimental scheme, the pharmaceutical preparation comprises: about 0.5 mM N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; about 184 mM propylene glycol; about 58.5 mM phenol; about 10 mM disodium hydrogen phosphate; and the pH of the pharmaceutical formulation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and further preferably about 7.3 to about 8.1.

[0082] In another experimental embodiment, the pharmaceutical preparation comprises: about 2.0 mg / ml of N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; about 14 mg / ml propylene glycol; about 5.5 mg / ml phenol; about 1.42 mg / ml disodium hydrogen phosphate; and

[0083] The pH of the pharmaceutical preparation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.1.

[0084] The third aspect of the present invention provides a pharmaceutical composition comprising the GLP-1 compound according to the first aspect of the present invention and acylated insulin.

[0085] In one embodiment, the acylated insulin is B29K (N (ε) -docosandioyl-γGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-2xOEG), desB30 human insulin; or B29K (N (ε) -docosandioyl-γGlu-12xPEG), desB30 human insulin.

[0086] In one embodiment, the acylated insulin is the following insulin: the insulin parent of the acylated insulin is a naturally occurring insulin or an insulin analog, and comprises at least one lysine residue, the acyl moiety of the acylated insulin is linked to the lysine residue or the amino group of the N-terminal amino acid residue of the insulin parent, and the acyl moiety is as shown in formula (A):

[0087] III-(II) m -(I) n -(A), wherein m is 0 or an integer from 1 to 10, and n is an integer from 5 to 20; I is a neutral amino acid residue containing an alkylene glycol; II is an acidic amino acid residue; III is an aliphatic diacid containing 20 to 24 carbon atoms, wherein a hydroxyl group is formally removed from one of the carboxyl groups of the aliphatic diacid; III, II, and I are connected by an amide bond; and the order in which II and I appear in formula (A) can be independently interchanged.

[0088] In one embodiment, n is 5-15, preferably, n is 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, preferably, n is 5, 6, 7, 8, 9, 10, 11 or 12, preferably, n is 5, 6, 7, 8, 9, or 10, preferably, n is 5, 6, 7, 8 or 9, preferably, n is 5, 6, 7, or 8.

[0089] In another embodiment, m is 1-6, preferably, m is 1, 2, 3, or 4, preferably, m is 1 or 2, preferably, m is 1.

[0090] In yet another embodiment, III is an aliphatic diacid containing 20-23 carbon atoms, preferably III is an aliphatic diacid containing 20, 21, or 22 carbon atoms, wherein a hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid.

[0091] In another embodiment, the insulin precursor comprises one lysine residue.

[0092] In one embodiment, I is: -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2- 2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-, -HN-(CH2) 3-O-(CH2)4-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)2-O -(CH2)2-O-CH2-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)3-O -(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)2-CO-, -HN-(C H2)2-O-(CH2)2-O-(CH2)2-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-C O-, -HN-(CH2)3-O-(CH2)3-O-CH2-CO-, or -HN-(CH2)4-O-(CH2)4-O-CH2-CO-; preferably I is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-.

[0093] In another embodiment, II is an amino acid residue selected from the group consisting of γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; preferably, II is selected from γGlu or βAsp.

[0094] In another embodiment, III is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 19-CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO- or HOOC-(CH2) 22 -CO-, preferably III is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

[0095] In one embodiment, formula (A) is linked via the C-terminus of I to the amino group of a lysine residue or the N-terminal amino acid residue of the insulin precursor.

[0096] In one embodiment, the acyl moiety is linked to the epsilon amino group of a lysine residue of the insulin precursor.

[0097] In one embodiment the lysine residue of the insulin precursor is at position B29.

[0098] In one embodiment, the insulin parent is selected from the following insulins or insulin analogs: desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 5, representing the A chain and the B chain, respectively); A14E, B16H, B25H, desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 7, representing the A chain and the B chain, respectively); A14E, B16E, B25H, desB30 human insulin (SEQ ID NO: 8 and SEQ ID NO: 9, representing the A chain and the B chain, respectively); human insulin (SEQ ID NO: 10 and SEQ ID NO: 11, representing the A chain and the B chain, respectively); A21G human insulin (SEQ ID NO: 12 and SEQ ID NO: 13, representing the A chain and the B chain, respectively); A21G, desB30 human insulin (SEQ ID NO: 14 and SEQ ID NO: 15, representing the A chain and the B chain, respectively); or B28D human insulin (SEQ ID NO: 16 and SEQ ID NO: 17). NO:17, representing A chain and B chain respectively).

[0099] In one embodiment, the acylated insulin is selected from the following insulins: B29K (N (ε) -eicosanedioyl-γGlu-5xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-γGlu-5xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-5xOEG-γGlu), desB30 human insulin; eicosanedioyl-6xOEG-γGlu), desB30 human insulin; B29K(N(ε)-eicosanedioyl-6xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-eicosanedioyl-5xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-eicosanedioyl-βAsp-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-βAsp-6xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-5xOEG), desB30 human insulin; B29K( N(ε)-eicosanedioyl-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αAsp-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanedioyl-γGlu-5xOEG), desB30 human insulin EG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-5xOEG-γGlu), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16H, B 25H,B29K(N(ε)-eicosanedioyl-βAsp-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-αGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-αGlu-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-αGlu-αGlu-5xOEG),desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-6xOEG) ,desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-6xOEG-γGlu), desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K 9K (N (ε) -eicosanedioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-αAs p-5xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αAsp-6xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-γGlu-7xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-γGlu-8xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-γGlu-γGlu-7xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-γGlu-γGlu-8xOEG),desB30 human insulin;B2 9K(N(ε)-eicosanedioyl-7xOEG-γGlu),desB30 human insulin; B29K(N(ε)-eicosanedioyl-8xOEG-γGlu),desB30 human insulin; B29K(N(ε)-eicosanedioyl-8xOEG-γGlu-γGlu),desB30 human insulin; B29K(N(ε)-eicosanedioyl-7xOEG-γGlu-γGlu),desB30 human insulin; B29K(N(ε)-eicosanedioyl-βAsp-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-βAsp-8xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-αGlu-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-8xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-αGlu-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-αGlu-8xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αAsp-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αAsp-8xOEG),desB30 human insulin Insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl- βAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αGlu-αGlu-7xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αGlu-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-γGlu-7xOEG), desB30 human insulin; 9K (N (ε) -eicosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl- A14E, B16E, B25H, B29K (N (ε)-eicosanedioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-eicosanedioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-eicosanedioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-eicosanedioyl-βAsp-8xOEG ),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αGlu-αGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αGlu-αGlu-8xOEG),desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-αAsp-8xOEG), desB30 human insulin; B29K (N(ε)-docosanedioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosanedioyl-γGlu-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-βAsp-5xO EG), desB30 human insulin; B29K(N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αAsp-5xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; 5H,B29K(N(ε)-docosandioyl-5xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-βAsp-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-βAsp-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-βAsp-5xOEG),desB30 human insulin A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αAsp-6xOEG ),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-γGlu-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-γGlu-6xOEG),desB30 human insulin;A14E, B16E, B25H, B29K (N (ε) -docosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H ,B29K(N(ε)-docosandioyl-βAsp-5xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-βAsp-6xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-5xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-6xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-α Glu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-αAsp-6xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N (ε)-docosanedioyl-γGlu-γGlu-8xOEG), desB30 human insulin; B29K (N (ε)-docosanedioyl-γGlu-γGlu-7xOEG), desB30 human insulin; B29K (N (ε)-docosanedioyl-γGlu-γGlu-8xOEG), desB30 human insulin; B29K (N (ε)-docosanedioyl-7xOEG-γGlu), desB30 human insulin; B29K (N (ε)-docosanedioyl-8xOEG-γGlu), desB30 human insulin; B29K (N (ε)-docosanedioyl-8xOEG-γGlu-γGlu), desB30 human insulin;B29K (N (ε) -docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; B29K (N (ε) -docosandioyl-βAsp-7xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-βAsp-8xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αGlu-7xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αGlu-8xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αGlu-7xOEG), desB30 human insulin EG), desB30 human insulin; B29K (N (ε) -docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αAsp-7xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-8xOEG), desB30 human insulin G), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-8xOEG-γGlu), desB 30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-βAsp-8xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; 25H,B29K(N(ε)-docosandioyl-αAsp-7xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-αAsp-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl- γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-αGlu-7xOEG), desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-αGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-αGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αAsp-7xOEG),desB30 human insulin; 5H,B29K(N(ε)-docosanedioyl-αAsp-8xOEG),desB30 human insulin; B29K(N(ε)-henicosanedioyl-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-henicosanedioyl-γGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-henicosanedioyl-γGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-henicosanedioyl-γGlu-6xOEG),desB30 human insulin; E, B25H, B29K (N (ε) - heneicosandioyl - γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) - heneicosandioyl - γGlu-6xOEG), desB30 human insulin; B29K (N (ε) - heneicosandioyl - γGlu-7xOEG), desB30 human insulin; B29K (N (ε) - heneicosandioyl - γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - heneicosandioyl - γGlu-7xOEG), desB30 human insulin; A14E ,B16H,B25H,B29K(N(ε)-henicosanedioyl-γGlu-8xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-henicosanedioyl-γGlu-7xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-henicosanedioyl-γGlu-8xOEG),desB30 human insulin;B29K(N(ε)-tricosanedioyl-γGlu-5xOEG),desB30 human insulin;B29K(N(ε)-tricosanedioyl-γGlu-6xOEG),desB30 human insulin;A14E, B16H, B25H, B29K (N (ε) -tricosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -tricosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -tricosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -tricosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -tricosandioyl-γGlu- Glu-7xOEG), desB30 human insulin; B29K (N (ε)-tricosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε)-tricosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε)-tricosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-tricosanedioyl-γGlu-7xOEG), desB30 human insulin; H, B29K (N (ε) -tricosanodiaoyl -γGlu-8xOEG), desB30 human insulin; B29K (N (ε) -tetracosanodiaoyl -γGlu-5xOEG), desB30 human insulin; B29K (N (ε) -tetracosanodiaoyl -γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -tetracosanodiaoyl -γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -tetracosanodiaoyl -γGlu-6xOEG), desB30 human insulin; 16E, B25H, B29K (N (ε) -tetracosandiaoyl -γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -tetracosandiaoyl -γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -tetracosandiaoyl -γGlu-7xOEG), desB30 human insulin; B29K (N (ε) -tetracosandiaoyl -γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -tetracosandiaoyl -γGlu-7xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N (ε) - tetracosanedioyl - γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) - tetracosanedioyl - γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) - tetracosanedioyl - γGlu-8xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu-12xOEG), desB30 human insulin; B29K (N (ε) - docosanedioyl - γGlu-12xOEG), desB30 Human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 Human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 Human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 Human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 Human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 Human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu- 10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - docosandioyl - γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - heneicosandioyl - γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - tricosandioyl - γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - tetracosandioyl - γGlu-12xOEG), desB30 human insulin;A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-11xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-10xOEG), desB30 human insulin dodecandioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-γGlu-11xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-heneicosandioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-tricosandioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-tetracosandioyl-γGlu-12xOEG), desB30 human insulin esB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu -9xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu -10xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu -11xOEG), desB30 human insulin; B29K (N (ε) - docosanedioyl - γGlu -9xOEG), desB30 human insulin; B29K (N (ε) - docosanedioyl - γGlu -10xOEG), desB30 human insulin; B29K (N (ε) - docosanedioyl - γGlu -11xOEG), des B30 human insulin; B29K (N (ε) - heneicosandioyl -γGlu-12xOEG), desB30 human insulin; B29K (N (ε) - tricosandioyl -γGlu-12xOEG), desB30 human insulin; B29K (N (ε) - tetracosandioyl -γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - docosandioyl -γGlu-18xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N (ε) - docosandioyl -γGlu-24xOEG), desB30 human insulin. ;

[0100] The inventor unexpectedly found that the pharmaceutical composition of the compound and acylated insulin described in the first aspect of the present invention not only does not weaken the physical stability of the compound, but also the combined preparation has better physical stability than the single-drug preparation. Compared with the combined preparations of other long-acting insulin derivatives (such as degludec insulin and liraglutide), the physical stability of the combined preparation of the present invention is unexpected. In addition, the combined preparation also increases the chemical stability of the acylated insulin compared to the single-drug preparation.

[0101] The fourth aspect of the present invention provides use of the compound described in the first aspect of the present invention, the pharmaceutical preparation described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention as a medicine.

[0102] In one embodiment, the compound of the first aspect of the invention, the pharmaceutical formulation of the second aspect of the invention or the pharmaceutical composition of the third aspect of the invention is used to treat or prevent hyperglycemia, diabetes, and / or obesity.

[0103] The fifth aspect of the present invention provides use of the compound described in the first aspect of the present invention, the pharmaceutical preparation described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention in the preparation of a medicament for treating or preventing hyperglycemia, diabetes, and / or obesity.

[0104] The sixth aspect of the present invention provides a method for treating or preventing hyperglycemia, diabetes, and / or obesity, the method comprising administering an effective amount of the compound described in the first aspect of the present invention, the pharmaceutical preparation described in the second aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention, wherein the disease includes, but is not limited to, hyperglycemia, diabetes, and obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1a The hypoglycemic effects and duration of the title compounds of Examples 1-3 of the present invention, liraglutide, and vehicle on db / db mice are shown, wherein the percentage on the ordinate refers to the percentage of blood sugar at the corresponding time point obtained by comparing the blood sugar at each monitoring point after administration with the baseline blood sugar before administration (the same below).

[0106] Figure 1b (and Figure 1a Correspondingly) shows the AUC of the hypoglycemic effects of the title compounds of Examples 1-3 of the present invention, liraglutide, and vehicle on db / db mice.

[0107] Figure 2a The graph shows the hypoglycemic effect and duration of the title compound of Example 2 of the present invention, semaglutide, and a vehicle on db / db mice.

[0108] Figure 2b (and Figure 2a Correspondingly) shows the AUC of the hypoglycemic effect of the title compound of Example 2 of the present invention, semaglutide, and vehicle on db / db mice.

[0109] Figure 3a The graph shows the hypoglycemic effect and duration of the title compound of Example 3-4 of the present invention, liraglutide, and a vehicle on db / db mice.

[0110] Figure 3b (and Figure 3a Correspondingly, the AUC of the hypoglycemic effect of the title compound of Example 3-4 of the present invention, liraglutide, and vehicle on db / db mice is shown.

[0111] Figure 4a The hypoglycemic effects and action time of the title compounds of Examples 1-3 of the present invention, the title compounds of Comparative Examples 3-4, and the vehicle on db / db mice are shown.

[0112] Figure 4b (and Figure 4a Correspondingly, the AUC of the hypoglycemic effects of the title compounds of Examples 1-3 of the present invention, the title compounds of Comparative Examples 3-4, and the vehicle on db / db mice are shown.

[0113] Figure 5a The hypoglycemic effect and duration of the title compound of Example 11 of the present invention at doses of 100 μg / kg and 300 μg / kg, the title compound of Control Example 2, and the vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control) are shown.

[0114] Figure 5b (and Figure 5a Correspondingly, the AUC of the hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control) are shown.

[0115] Figure 5c The weight loss effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control) are shown.

[0116] Figure 6a The graph shows the hypoglycemic effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control) when ipGTT was performed 48 hours after the first administration.

[0117] Figure 6b (and Figure 6a Correspondingly, ΔAUC of the hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control) when ipGTT was performed 48 hours after the first administration is shown.

[0118] Figure 7a The graph shows the hypoglycemic effects of the title compound of Example 2 of the present invention, the title compound of Control Example 2 and the vehicle on db / db mice.

[0119] Figure 7b (and Figure 7a Correspondingly, ΔAUC of the hypoglycemic effect of the title compound of Example 2 of the present invention, the title compound of Control Example 2 and the vehicle on db / db mice is shown.

[0120] Figure 7c The results show the controlling effects of the title compound of Example 2 of the present invention, the title compound of Control Example 2 and the vehicle on the food intake of db / db mice.

[0121] Figure 7d The control effect of the title compound of Example 2 of the present invention, the title compound of Control Example 2 and the solvent on the drinking water amount of db / db mice is shown.

[0122] Figure 8a The long-term hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Control Example 2 and the vehicle on db / db mice is shown.

[0123] Figure 8b (and Figure 8a Correspondingly, the AUC of the long-term hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Control Example 2 and the vehicle on db / db mice is shown.

[0124] Figure 8c The long-term weight loss effect of the title compound of Example 11 of the present invention, the title compound of Control Example 2 and the vehicle on db / db mice is shown.

[0125] Figure 8d The results show the effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2 and the vehicle on the long-term food intake of db / db mice.

[0126] Figure 8e The results show the effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2 and the vehicle on the long-term water intake of db / db mice.

[0127] Figure 9a The hypoglycemic effects of the title compound of Example 11 of the present invention, the title compound of Example 2, dulaglutide and a solvent on Kkay mice are shown.

[0128] Figure 9b (and Figure 9a Correspondingly, the AUC of the hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Example 2, dulaglutide and the vehicle on Kkay mice is shown.

[0129] Fig.9c The results show the HbA1c lowering effects of the title compound of Example 11 of the present invention, the title compound of Example 2, dulaglutide and a solvent on Kkay mice.

[0130] Fig.10a The long-term hypoglycemic effects of the title compound of Example 11 of the present invention, dulaglutide, and vehicle (model control group) on db / db mice or normal mice (normal control) are shown.

[0131] Fig.10b (and Fig.10a Correspondingly, ΔAUC of the long-term hypoglycemic effect of the title compound of Example 11 of the present invention, dulaglutide, and vehicle (model control group) on db / db mice or normal mice (normal control) are shown.

[0132] Fig.10c The random blood glucose values ​​before, at the third, fifth and eleventh injections of the title compound of Example 11 of the present invention, dulaglutide and vehicle (model control group) in db / db mice or normal mice (normal control) are shown.

[0133] Fig.10d The graph shows the hypoglycemic effects of the title compound of Example 11 of the present invention, dulaglutide, and vehicle (model control group) on db / db mice or normal mice (normal control group) when ipGTT was performed 48 hours after the first administration.

[0134] Fig.10e (and Fig.10d Correspondingly, the AUC of the hypoglycemic effect of the title compound of Example 11 of the present invention, dulaglutide, and vehicle (model control group) on db / db mice or normal mice (normal control group) when ipGTT was performed 48 hours after the first administration is shown.

[0135] Fig.11a The long-term weight loss effects of the title compound of Example 11 of the present invention, dulaglutide, and vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control group) are shown.

[0136] Fig.11b The graph shows the controlling effects of the title compound of Example 11 of the present invention, dulaglutide and vehicle (model control group) on the long-term food intake of obese C57BL mice induced by a high-fat diet.

[0137] Fig.11c The graph shows the effect of the title compound of Example 11 of the present invention, dulaglutide and vehicle (model control group) on reducing peri-ovarian fat in obese C57BL female mice induced by a high-fat diet.

[0138] Fig.11d The graph shows the epididymal fat reducing effect of the title compound of Example 11 of the present invention, dulaglutide and vehicle (model control group) on high-fat diet-induced obese C57BL male mice. DETAILED DESCRIPTION

[0139] definition

[0140] GLP-1 analogs and GLP-1 derivatives

[0141] The term "GLP-1 analogue" or "analogue of GLP-1" as used herein refers to a peptide or compound that is a variant of human glucagon-like peptide-1 (GLP-1 (7-37)), wherein one or more amino acid residues of GLP-1 (7-37) are replaced, and / or one or more amino acid residues are deleted, and / or one or more amino acid residues are added. Specifically, the sequence of GLP-1 (7-37) is shown in SEQ ID NO: 1 in the sequence listing. The peptide having the sequence shown in SEQ ID NO: 1 may also be referred to as "native" GLP-1 or "native" GLP-1 (7-37).

[0142] In the sequence listing, the first amino acid residue (histidine) of SEQ ID NO: 1 is numbered 1. However, hereinafter, according to the established practice in the art, the histidine residue is numbered 7, and the amino acid residues thereafter are numbered accordingly, ending with glycine 37. Therefore, generally, the amino acid residue numbering or position numbering of the GLP-1 (7-37) sequence referred to herein is a sequence starting with His at position 7 and ending with Gly at position 37.

[0143] [Gly8, Arg34]GLP-1-(7-37) peptide is a GLP-1 analog having Gly and Arg at positions corresponding to position 8 and position 34 of GLP-1(7-37) (SEQ ID NO: 1), respectively, and [Arg34]GLP-1-(7-37) peptide is a GLP-1 analog having Arg at a position corresponding to position 34 of GLP-1(7-37) (SEQ ID NO: 1). Specifically, the amino acid sequences of [Gly8, Arg34]GLP-1-(7-37) peptide and [Arg34]GLP-1-(7-37) peptide are shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing, respectively.

[0144] In the context of a GLP-1 peptide or analog thereof, the term "derivative" as used herein refers to a chemically modified GLP-1 peptide or analog wherein one or more substituents have been covalently attached to the peptide. The substituents may also be referred to as side chains.

[0145] Unless otherwise stated, when reference is made to acylation with a lysine residue, this is understood to be with the epsilon-amino group thereof.

[0146] The GLP-1 derivatives of formula (B) of the present invention may exist in different stereoisomeric forms, which have the same molecular formula and linked atomic sequence, but differ only in the three-dimensional orientation of their atomic space. Unless otherwise specified, the present invention relates to all stereoisomeric forms of the claimed derivatives.

[0147] The term "peptide" when applied to, for example, the GLP-1 analogs of the present invention, refers to a compound comprising a series of amino acids interconnected by amide (or peptide) bonds.

[0148] In a specific embodiment, the peptide is largely or mainly composed of amino acids linked to each other by amide bonds (e.g., at least 50%, 60%, 70%, 80% or at least 90% of the molar mass). In another specific embodiment, the peptide is composed of amino acids linked to each other by peptide bonds.

[0149] Amino acids are molecules containing an amino group and a carboxylic acid group, optionally with one or more additional groups, often called side chains.

[0150] The term "amino acid" includes proteinogenic amino acids (encoded by the genetic code, including natural amino acids and standard amino acids), as well as non-proteinogenic (not found in proteins, and / or not encoded in the standard genetic code), and synthetic amino acids. Non-proteinogenic amino acids are parts that can be incorporated into peptides through peptide bonds, but are not proteinogenic amino acids. Synthetic non-proteinogenic amino acids include amino acids produced by chemical synthesis, i.e., D-isomers of amino acids encoded by the genetic code, such as D-alanine and D-leucine, Aib (α-aminoisobutyric acid), Abu (α-aminobutyric acid), 3-aminomethylbenzoic acid, anthranilic acid, deamino-histidine, β-analogs of amino acids such as β-alanine, etc., D-histidine, deamino-histidine, 2-amino-histidine, β-hydroxy-histidine, and homohistidine, etc.

[0151] Non-limiting examples of amino acids not encoded by the genetic code are γ-carboxyglutamate, ornithine, D-alanine, D-glutamine and phosphoserine. Non-limiting examples of synthetic amino acids are the D-isomers of amino acids, such as D-alanine and D-leucine, Aib (α-aminoisobutyric acid), β-alanine and des-amino-histidine (desH, alternative name imidazole propionic acid, abbreviated Imp).

[0152] In the following, all amino acids not indicating the optical isomer are understood to refer to the L-isomer (unless otherwise specified).

[0153] Pharmaceutically acceptable salts, amides or esters

[0154] The GLP-1 derivatives, analogs and intermediates of the present invention may be in the form of pharmaceutically acceptable salts, amides or esters. The salt may be a basic salt, an acidic salt, or a neutral salt. Basic salts produce hydroxide ions in water, and acidic salts produce hydronium ions. Salts of the derivatives of the present invention may be formed with added cations or anions that react with anionic groups or cationic groups, respectively. These groups may be located within the peptide portion and / or within the side chains of the derivatives of the present invention.

[0155] The non-limiting examples of the anionic groups of derivatives of the present invention include free carboxyl groups in side chains (if any) and peptide moieties. The peptide moiety typically includes the free carboxyl groups at the C-end, and it may also include free carboxyl groups on internal acidic amino acid residues such as Asp and Glu.

[0156] Non-limiting examples of cationic groups on the peptide portion include the free amino group at the N-terminus (if any) and any free amino groups on internal basic amino acid residues such as His, Arg and Lys.

[0157] The esters of the derivatives of the present invention can be formed, for example, by reacting a free carboxylic acid group with an alcohol or phenol, which results in at least one hydroxyl group being substituted with an alkoxy or aryloxy group. The formation of esters can involve the free carboxyl group at the C-terminus of the peptide, and / or any free carboxyl group of a side chain.

[0158] The amides of the derivatives of the present invention can be generated, for example, by reacting a free carboxylic acid group with an amine or substituted amine, or by reacting a free or substituted amino group with a carboxylic acid. The formation of amides can involve the free carboxyl group at the C-terminus of the peptide, any free carboxyl group of a side chain, the free amino group at the N-terminus of the peptide, and / or any free or substituted peptide amino group in the peptide and / or side chain.

[0159] In a specific embodiment, the GLP-1 compound or GLP-1 derivative of the present invention is in the form of a pharmaceutically acceptable salt. In another specific embodiment, it is in the form of a pharmaceutically acceptable amide, preferably with an amide group at the C-terminus of the peptide. In a further specific embodiment, the peptide or derivative is in the form of a pharmaceutically acceptable ester.

[0160] The preparation methods of the peptides of GLP-1 (7-37) and GLP-1 analogs of the present invention are well known in the art. For example, the GLP-1 peptide portion (or fragment thereof) of the derivative of the present invention and the GLP-1 analogs of the present invention can be produced by classical peptide synthesis, such as solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well-established techniques, see, for example, Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza "Organic Synthesis on solid Phase", Wiley-VCH Verlag GmbH, 2000, and "Fmoc Solid Phase Peptide Synthesis" edited by WC Chan and PD White, Oxford University Press, 2000.

[0161] In one embodiment, the complete GLP-1 analogs of the present invention, such as [Gly8, Arg34]GLP-1-(7-37) peptides, can be produced by recombinant methods, i.e., by culturing host cells that contain a DNA sequence encoding the analog and are capable of expressing the peptide in a suitable nutrient medium under conditions that allow expression of the peptide. Non-limiting examples of host cells suitable for expressing these peptides are: Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines. In some embodiments, this fully recombinant fermentation step of the production process is desirable, for example, for production economics considerations.

[0162] The fusion protein inclusion body containing the GPL-1 compound main chain is denatured and renatured to obtain a fusion protein containing the correct conformation, and a series of treatments such as enzyme digestion, precipitation, centrifugation, etc. are performed to obtain a GLP-1 compound main chain with a high content. After purification by ion exchange chromatography, a GLP-1 compound main chain with a high purity is obtained.

[0163] The term "excipient" broadly refers to any ingredient other than the active therapeutic ingredient. An excipient can be an inert substance, an inactive substance, and / or a non-pharmaceutically active substance.

[0164] Excipients may serve a variety of purposes, such as serving as carriers, solvents, diluents, tableting aids, and / or to improve administration and / or absorption of the active substance.

[0165] The formulation of pharmaceutically active ingredients with various excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th Edition (1995), and any later editions).

[0166] Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, isotonic agents, chelating agents, and stabilizers.

[0167] The GLP-1 derivatives and analogs of the present invention have GLP-1 activity, which refers to the ability to bind to the GLP-1 receptor and initiate a signal transduction pathway to produce an insulinotropic effect or other physiological effects.

[0168] In a specific embodiment, potency, efficacy and / or activity refers to in vitro efficacy, ie, performance in a functional GLP-1 receptor assay, more particularly the ability to stimulate cAMP formation in a cell line expressing a cloned human GLP-1 receptor.

[0169] In another specific embodiment, the derivatives of the invention are potent in vivo, which can be determined in any suitable animal model and in clinical trials in accordance with known methods in the art. For example, diabetic db / db mice are an example of a suitable animal model, and the blood glucose lowering effect can be determined in such mice, for example, as described in the Examples section of the present invention.

[0170] The term "insulin" includes naturally occurring insulin, such as human insulin, as well as insulin analogs and insulin derivatives thereof.

[0171] The term "insulin analogs" includes polypeptides having a molecular structure that is formally derived from the structure of naturally occurring insulin (e.g., human insulin) by deletion and / or substitution (replacement) of one or more amino acid residues present in natural insulin and / or addition of at least one amino acid residue. Preferably, the substituted amino acid residue is a codable amino acid residue.

[0172] Here, the term "insulin derivative" refers to naturally occurring insulin or insulin analogs that have been chemically modified, such as by introducing side chains at one or more positions of the insulin backbone or by oxidizing or reducing the groups of amino acid residues on the insulin or by converting free carboxyl groups into ester groups or acylating free amino or hydroxyl groups. The acylated insulin of the present invention belongs to insulin derivatives.

[0173] The term "insulin parent" refers to the insulin portion of an insulin derivative or acylated insulin (also referred to herein as parent insulin), for example, in the present invention, the portion of the acylated insulin without an additional acyl group. The insulin parent can be a naturally occurring insulin, such as human insulin or porcine insulin. On the other hand, the parent insulin can be an insulin analog.

[0174] Here, the term "amino acid residue" includes amino acids from which hydrogen atoms have been removed from amino groups and / or hydroxyl groups have been removed from carboxyl groups and / or hydrogen atoms have been removed from sulfhydryl groups. Inaccurately, the amino acid residue may be called an amino acid.

[0175] Unless otherwise specified, all amino acids mentioned herein are L-amino acids.

[0176] Here, the term alkylene glycol comprises oligo- and polyalkylene glycol moieties and monoalkylene glycol moieties.Monoalkylene glycol and polyalkylene glycol comprise for example based on mono- and polyethylene glycol, based on mono- and polypropylene glycol and based on mono- and polybutylene glycol chains, i.e. based on repeating units-CH2CH2O-,-CH2CH2CH2O- or-CH2CH2CH2CH2O- chains.Alkylene glycol moieties can be monodisperse (with well-defined length / molecular weight) and polydisperse (with less well-defined length / mean molecular weight).Monoalkylene glycol moieties include-OCH2CH2O-,-OCH2CH2CH2O- or-OCH2CH2CH2CH2O- comprising different groups at each end.

[0177] The term "fatty acid" includes straight or branched chain aliphatic carboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non-limiting examples of fatty acids are, for example, myristic acid, palmitic acid, stearic acid and eicosanoic acid.

[0178] Herein, the term "aliphatic diacid" includes straight or branched aliphatic dicarboxylic acids, which have at least two carbon atoms and are saturated or unsaturated. Non-limiting examples of aliphatic diacids are adipic acid, suberic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, eicosanedioic acid, docosanedioic acid, and tetracosanedioic acid.

[0179] In this article, the naming of insulin or GLP-1 compounds is carried out according to the following principles: the name is given according to the mutation and modification (such as acylation) relative to human insulin, or the mutation and modification (such as acylation) of native GLP-1 (7-37). For the naming of the acyl part, it is named according to the IUPAC nomenclature and in other cases according to the peptide nomenclature. For example, the following acyl part is named:

[0180]

[0181] For example, it can be named "eicosanedioyl-γGlu-OEG-OEG", "eicosanedioyl-γGlu-2xOEG" or "eicosanedioyl-gGlu-2xOEG", "19-carboxynonadecanoyl-γGlu-2xOEG" or "19-carboxynonadecanoyl-γGlu-OEG-OEG", where OEG represents the abbreviation of the group -NH(CH2)2O(CH2)2OCH2CO- (i.e., 2-[2-(2-aminoethoxy)ethoxy]acetyl), and γGlu (and gGlu) is a shorthand representation of the amino acid γglutamic acid in the L configuration. Alternatively, the acyl moiety can be named according to the IUPAC nomenclature (OpenEye, IUPAC format). According to this nomenclature, the above-mentioned acyl moieties of the present invention are referred to as the following names: “[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butanoyl]-amino]-ethoxy]-ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]” or “[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]”.

[0182] For example, the insulin of Example 6 of the present invention (having the sequence / structure given below) is referred to as "B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin", "B29K(Nε-eicosandioyl-γGlu-5xOEG), desB30 human insulin", or "B29K(Nε-eicosandioyl-gGlu-5xOEG), desB30 human insulin" to indicate that the amino acid K at position B29 in human insulin has been modified by acylation with the residue eicosandioyl-gGlu-2xOEG on the epsilon nitrogen of the lysine residue of B29 (referred to as Nε or (N(ε)), and the amino acid T at position B30 in human insulin has been deleted. For another example, the insulin of Control Example 5 (having the sequence / structure given below) is referred to as "B29K(Nε-eicosandioyl-γGlu-5xOEG), desB30 human insulin", or "B29K(Nε-eicosandioyl-gGlu-5xOEG), desB30 human insulin" to indicate that the amino acid K at position B29 in human insulin has been modified by acylation with the residue eicosandioyl-gGlu-2xOEG on the epsilon nitrogen of the lysine residue of B29 (referred to as Nε or (N(ε)), and the amino acid T at position B30 in human insulin has been deleted. structure) is referred to as "A14E,B16H,B25H,B29K (Nεeicosanedioyl-gGlu-2xOEG),desB30 human insulin" or "A14E,B16H,B25H,B29K (N(ε)-eicosanedioyl-γGlu-2xOEG),desB30 human insulin", indicating that the amino acid Y at position A14 in human insulin has been mutated to E, the amino acid Y at position B16 in human insulin has been mutated to H, the amino acid F at position B25 in human insulin has been mutated to H, the amino acid K at position B29 in human insulin has been modified by acylation on the epsilon nitrogen (referred to as Nε) of the lysine residue of B29 with the residue eicosanedioyl-gGlu-2xOEG, and the amino acid T at position B30 in human insulin has been deleted.

[0183]

[0184] In this article, "nxPEG" means -NH(CH2CH2O) n CH2CO-, where n is an integer. For example, "12xPEG" represents the group -NH(CH2CH2O) 12 CH2CO-.

[0185] Insulin is a polypeptide hormone secreted by β-cells in the pancreas, and is composed of two polypeptide chains, A and B, which are linked by two interchain disulfide bonds. In addition, the A chain is characterized by one intrachain disulfide bond.

[0186] There are three main methods for preparing human insulin in microorganisms. Two involve Escherichia coli, one is by expressing fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, III. 729-739 pages), and the other is to use a signal peptide to enable secretion to the periplasmic space (Chan et al. (1981) PNAS 78: 5401-5404). The third method is to use Saccharomyces cerevisiae to secrete insulin precursors into the culture medium (Thim et al. (1986) PNAS 83: 6766-6770). The prior art discloses many methods for expressing insulin precursors in E. coli or Saccharomyces cerevisiae, see for example US Pat. No. 5,962,267, WO95 / 16708, EP0055945, EP0163529, EP0347845 and EP0741188.

[0187] The construction, expression, processing and purification of the vector of the insulin analog can be carried out using techniques known to those skilled in the art. For example, the insulin analog can be prepared by expressing a DNA sequence encoding the target insulin analog in a suitable host cell using the well-known techniques disclosed in U.S. Pat. No. 6,500,645. For example, the insulin analog can also be prepared by the method reported in the following literature: Glendorf T, AR, Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding; Biochemistry 2008 474743-4751. In this document, overlap extension PCR was used to introduce mutations into the insulin encoding vector. Insulin analogs were expressed in the yeast MT663 as pre-insulin-like fusion proteins with Ala-Ala-Lys small C-peptide. The single-chain precursor was enzymatically converted into a double-chain desB30 analog using an A. lyticus endoprotease.

[0188] The isolated insulin analogue can be acylated at a desired position by an acylation method known in the art. Examples of such insulin analogues are described in, for example, Chinese patent applications with publication numbers CN1029977C, CN1043719A and CN1148984A.

[0189] Nucleic acid sequences encoding individual insulin analog polypeptides may be prepared synthetically by established standard procedures, such as those described in Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869 or Matthes et al. (1984) EMBO Journal 3:801-805.

[0190] The present invention is further described below by way of examples. It should be noted that these examples do not limit the scope of protection of the present invention.

[0191] Example

[0192] Abbreviations

[0193] cAMP is cyclic adenosine monophosphate;

[0194] BHK is baby hamster kidney cell;

[0195] DNA is deoxyribonucleic acid;

[0196] Na2HPO4 is disodium hydrogen phosphate;

[0197] NaOH is sodium hydroxide;

[0198] OEG is the amino acid residue -NH(CH2)2O(CH2)2OCH2CO-;

[0199] OSu is succinimidyl-1-yloxy-2,5-dioxo-pyrrolidin-1-yloxy;

[0200] OtBu is tert-butyloxy;

[0201] HCl is hydrogen chloride;

[0202] γGlu or gGlu is γL-glutamyl;

[0203] NHS is N-hydroxysuccinimide;

[0204] DCC is dicyclohexylcarbodiimide;

[0205] AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid;

[0206] OH is hydroxide;

[0207] Gly is glycine;

[0208] Arg is arginine;

[0209] TFA is trifluoroacetic acid;

[0210] HbA1c is glycosylated hemoglobin.

[0211] Example 1

[0212] Title compound: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (Compound 1)

[0213]

[0214] 1. N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyrylamino [amino]ethoxy]ethoxy]acetyl][Gly8,Arg34]GLP-1-(7-37) peptide preparation

[0215] [Gly8, Arg34]GLP-1-(7-37) peptide was prepared by a general protein recombinant expression method (for specific methods, see Molecular Cloning: A Laboratory Manual (Fourth Edition), Michael R. Green, Cold Spring Harbor Press, 2012). [Gly8, Arg34]GLP-1-(7-37) peptide (5 g, 1.48 mmol) was dissolved in 100 mM Na2HPO4 aqueous solution (150 mL), and acetonitrile (100 mL) was added, and the pH was adjusted to pH 10-12.5 with 1N NaOH. Tert-butyl eicosanedioyl-γGlu(2xOEG-OSu)-OtBu (1.59 g, 1.63 mmol) was dissolved in acetonitrile (50 mL) and slowly added to the [Gly8, Arg34]GLP-1-(7-37) peptide solution. The pH was maintained at 10-12.5. After 120 minutes, the reaction mixture was added to water (150 mL), and the pH was adjusted to 5.0 with 1N HCl aqueous solution. Precipitate by centrifugation and freeze-dry. The crude product was added to a mixed solution of trifluoroacetic acid (60 mL) and dichloromethane (60 ml), and stirred at room temperature for 30 minutes. The mixture was concentrated to about 30 ml, poured into ice-cold n-heptane (300 mL), the precipitated product was separated by filtration, and washed twice with n-heptane. After vacuum drying, the product was purified by ion exchange chromatography (Ressource Q, 0.25%-1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA), the purified fractions were combined, the pH was adjusted to 5.2 with 1N HCl, the precipitate was separated, and freeze-dried to obtain the title compound.

[0216] LC-MS (electrospray): m / z = 1028.79 [M + 4H] 4+

[0217] 2. Intermediate tert-butyl eicosanedioyl- γ Preparation of Glu-(2xOEG-OSu)-OtBu

[0218] 2.1 tert-Butyl eicosanedioyl-OSu

[0219] Under nitrogen protection, mono-tert-butyl eicosanedioate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane (400 mL), triethylamine (13.95 mL) was added, and the resulting turbid mixture was stirred at room temperature, and then DCC (11.39 g, 55.19 mmol) was added, and it was further stirred overnight. Filter, concentrate the obtained filtrate to almost dryness, mix the residue with cold water and ethyl acetate, stir for 20 minutes, separate, add saturated brine to wash the upper organic phase, dry the upper organic phase with anhydrous sodium sulfate after separation, and concentrate the filtrate under reduced pressure to almost dryness after filtration, and vacuum dry overnight to obtain 24.12 g (yield 97%) of tert-butyl eicosanedioyl-OSu.

[0220] LC-MS(Scie×100API): m / z=496.36(M+1) +

[0221] 2.2 tert-Butyl eicosanedioyl-γGlu-OtBu

[0222] Tert-butyl eicosanedioyl-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred, and H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water (25 mL) were added in sequence, and heated to obtain a clear solution, which was stirred at room temperature for 4 hours. Then 10% citric acid aqueous solution (200 mL) was added, and the liquid was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to almost dryness under reduced pressure, and vacuum dried overnight. 27.27 g (yield 96%) of tert-butyl eicosanedioyl-γGlu-OtBu was obtained.

[0223] LC-MS(Scie×100API): m / z=584.44(M+1) +

[0224] 2.3 tert-Butyleicosanedioyl-γGlu(OSu)-OtBu.

[0225] Under nitrogen protection, tert-butyleicosadianoyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added and stirred for 10 minutes, then NHS (5.38 g, 50.17 mmol) was added, followed by DCC (10.60 g, 51.38 mmol). The mixture was stirred overnight at room temperature. Filtered, the filtrate was concentrated to almost dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated brine, the upper organic phase was dried over anhydrous sodium sulfate after separation, the filtrate was concentrated to almost dryness under reduced pressure after filtration, methyl tert-butyl ether was added, stirred for 30 minutes, filtered, and the filter cake was vacuum dried overnight to obtain 25.76 g (yield 81%) of tert-butyleicosadianoyl-γGlu-(OSu)-OtBu.

[0226] LC-MS(Scie×100API): m / z=681.46(M+1) +

[0227] 2.4 tert-Butyl eicosanedioyl-γGlu-(2xOEG-OH)-OtBu

[0228] Tert-butyleicosanoyl-γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred, and 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added in sequence, and heated to obtain a clear solution, which was stirred at room temperature for 4 hours. Then, 10% citric acid aqueous solution (200 mL) was added for separation, and the lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated to almost dryness under reduced pressure after filtration, and vacuum dried overnight. 30.75 g (yield 93%) of tert-butyleicosanoyl-γGlu-(2xOEG-OH)-OtBu was obtained.

[0229] LC-MS(Scie×100API): m / z=874.59(M+1) +

[0230] 2.5 tert-Butyleicosanedioyl-γGlu-(2xOEG-OSu)-OtBu.

[0231] Under nitrogen protection, tert-butyleicosadianoyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added and stirred for 10 minutes, and NHS (4.05 g, 35.18 mmol) was added, followed by DCC (7.98 g, 38.70 mmol). The mixture was stirred overnight at room temperature. Filtered, the filtrate was concentrated to almost dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated brine, the upper organic phase was dried with anhydrous sodium sulfate after separation, filtered, the filtrate was concentrated to almost dryness under reduced pressure, and vacuum dried overnight to obtain 31.09 g (yield 91%) of tert-butyleicosadianoyl-γGlu-(2xOEG-OSu)-OtBu.

[0232] LC-MS(Scie×100API): m / z=971.61(M+1) +

[0233] Example 2

[0234] Title compound: N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (Compound 2)

[0235]

[0236] Prepare N-ε by a procedure similar to that of Example 1, Part 1 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide

[0237] LC-MS (electrospray): m / z = 992.52 [M + 4H] 4+

[0238] Intermediates Tert-Butyleicosanedioyl-γGlu-(OEG-OSu)-OtBu Prepared by a procedure similar to Example 1, part 2.

[0239] LC-MS(Scie×100API): m / z=826.54(M+1) +

[0240] Example 3

[0241] Title compound: N-ε 26-(19-Carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide (Compound 3)

[0242]

[0243] Prepared in a similar manner to Example 1, Part 1 N-ε 26 -(19-Carboxynonadecanoylamino)-4(S)-carboxy GLP-1-(7-37) peptide

[0244] LC-MS (electrospray): m / z = 956.25 [M + 4H] 4+

[0245] Intermediates Tert-Butyleicosanedioyl-γGlu-(OSu)-OtBu Prepared by a procedure similar to Example 1, part 2.

[0246] LC-MS(Scie×100API): m / z=681.46(M+1) +

[0247] Example 4

[0248] Title compound: N-ε 26 -(19-Carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide (Compound 4)

[0249]

[0250] Prepared in a similar manner to Example 1, Part 1 N-ε 26 -(19-Carboxynonadecanoylamino)-4(S)-carboxy Arg34-GLP-1-(7-37) peptide

[0251] LC-MS (electrospray): m / z = 959.75 [M + 4H] 4+

[0252] Intermediates Tert-Butyleicosanedioyl-γGlu-(OSu)-OtBu Prepared by a procedure similar to Example 1, part 2.

[0253] LC-MS(Scie×100API): m / z=681.46(M+1) +

[0254] Comparative Example 1

[0255] The reference compound liraglutide was prepared according to Example 37 of patent CN1232470A.

[0256] Comparative Example 2

[0257] The reference compound semaglutide was prepared according to Example 4 of patent CN101133082A.

[0258] Comparative Example 3

[0259] Title compound: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide

[0260]

[0261] Prepared in a similar manner to Example 1, Part 1 N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxy [(heptadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)

[0262] [ethoxy]ethoxy)acetyl] [Gly8, Arg34] GLP-1-(7-37) peptide

[0263] LC-MS (electrospray): m / z = 1021.78 [M+4H] 4+

[0264] Comparative Example 4

[0265] Title compound: N-ε 26 -(17-Carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide

[0266]

[0267] Prepared in a similar manner to Example 1, Part 1 N-ε 26 -(17-Carboxyheptadecanoylamino)-4(S)-carboxy GLP-1-(7-37) peptide

[0268] LC-MS (electrospray): m / z = 949.24 [M + 4H] 4+

[0269] Intermediates Tert-Butyl Octadecanedioyl-γGlu-(OSu)-OtBu Prepared by a procedure similar to Example 1, part 2.

[0270] LC-MS(Scie×100API): m / z=653.43(M+1) +

[0271] Example 5: Pharmacodynamic study in db / db mice

[0272] The purpose of this study is to confirm the regulatory effect of the GLP-1 derivatives of the present invention on hyperglycemia (BG) in the case of diabetes.

[0273] The title compounds of Examples 1-4 and Control Examples 1-4 (also known as GLP-1 derivatives) were tested in a single-dose study on an obese type 2 diabetes (T2DM) mouse model (db / db mice). The pharmacodynamic effects of the GLP-1 derivatives in lowering blood sugar were tested at different doses of 100 μg / kg.

[0274] Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in a suitable size feeding box in a barrier environment with free access to standard food and purified water. The environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22°C-24°C. After a 1-2 week adaptation period, they were used in the experiment.

[0275] Before the experiment started on the same day, basal blood glucose was assessed at about 9:30 am and the mice were weighed. Mice were matched to vehicle group or treatment group according to random blood glucose and body weight, and received the following treatments: subcutaneous injection of vehicle, or subcutaneous injection of GLP-1 derivative 100 μg / kg, wherein the vehicle contained: propylene glycol 14 mg / ml, phenol 5.5 mg / ml, disodium hydrogen phosphate 1.133 mg / ml, and the pH of the vehicle was 8.12.

[0276] The GLP-1 derivative was dissolved in a solvent to a dosing concentration of 20 μg / ml, and the dosing volume was 5 ml / kg (i.e., 50 μl / 10 g body weight). Subcutaneous administration was adopted, and the drug was administered once by subcutaneous injection at the back of the neck. The corresponding GLP-1 derivative was administered at approximately 10:30 am (time 0), and the animals were free to eat and drink during the administration. The blood glucose of mice was evaluated at 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours after administration. The tail of the mouse was cleaned with an alcohol cotton ball, and blood drops were collected from the tail using a disposable blood collection needle, and measured with a blood glucose meter and matching test strips (Roche). The food intake of the mice and the weight of each mouse were measured at 24, 48 and 72 hours after administration.

[0277] The blood glucose level before administration was taken as the benchmark, and the blood glucose level at each monitoring point after administration was compared with the baseline to obtain the blood glucose percentage at the corresponding time point. For each single dose of GLP-1 derivative, a dose-response curve of blood glucose percentage versus time was drawn. In order to quantitatively illustrate the effect of GLP-1 derivatives on blood glucose, for each individual dose-response curve, the area under the curve of blood glucose percentage-time from 0 to 72 hours (AUC 0-72h ). Among them, AUC is the area under the curve of the time-blood glucose percentage curve. The smaller the AUC value, the better the hypoglycemic effect and the better the efficacy.

[0278] Figure 1a-4bIt shows that the GLP-1 derivatives of the present invention have unexpectedly increased efficacy, for example, the title compounds of Examples 1-4 have significantly better hypoglycemic effects on db / db mice than liraglutide and the compounds of Control Examples 3-4. In particular, the hypoglycemic effect of the compound of Example 2 of the present invention is better than that of semaglutide. In addition, the effective duration of action of the GLP-1 derivatives of the present invention, such as the compounds of Examples 1-4, in db / db mice is significantly prolonged compared to liraglutide and the compounds of Control Examples 3-4, and in particular, the effective duration of action of the compound of Example 2 in db / db mice is longer than that of semaglutide.

[0279] Example 6

[0280] B29K (N(ε)-eicosanedioyl-γGlu-5xOEG), desB30 human insulin (Compound 5)

[0281]

[0282] 1. Synthesis of des(B30) human insulin

[0283] des(B30) human insulin was prepared according to the method described in Example 101 of Chinese patent CN1056618C.

[0284] 2. Preparation of target insulin

[0285] DesB30 human insulin (5g, 0.876mmol) is dissolved in 100mM NaHPO aqueous solution (150mL), and acetonitrile (100mL) is added, and pH is adjusted to pH10-12.5 with 1N NaOH. Tert-butyl eicosanedioyl-γGlu-(5xOEG-OSu)-OtBu (1.36g, 0.964mmol) is dissolved in acetonitrile (50mL), and slowly added in insulin solution. pH is maintained at 10-12.5. After 120 minutes, reaction mixture is added in water (150mL), and pH is adjusted to 5.0 with 1N HCl aqueous solution. Precipitate by centrifugation, and freeze-drying. Crude product is added in trifluoroacetic acid (60mL) and methylene chloride (60ml) mixed solution, and at room temperature stirred 30 minutes. The mixture was concentrated to about 30 ml, poured into ice-cold n-heptane (300 mL), the precipitated product was separated by filtration, and washed twice with n-heptane. After vacuum drying, it was purified by ion exchange chromatography (Ressource Q, 0.25%-1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA), and the purified fractions were combined, the pH was adjusted to 5.2 with 1N HCl, and the precipitate was separated and freeze-dried to obtain the title compound Compound 5.

[0286] LC-MS (electrospray): m / z = 1377.53 [M + 5H] 5+

[0287] 3. Preparation of the intermediate tert-butyleicosanedioyl-γGlu-(5xOEG-OSu)-OtBu

[0288] 3.1 tert-Butyl eicosanedioyl-OSu

[0289] Under nitrogen protection, mono-tert-butyl eicosandioate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane, triethylamine (13.95 mL) was added, and the resulting turbid mixture was stirred at room temperature, and then DCC (11.39 g, 55.19 mmol) was added, and further stirred overnight. Filter, concentrate the obtained filtrate to almost dryness, mix the residue with cold water and ethyl acetate, stir for 20 minutes, separate, add saturated brine to wash the upper organic phase, dry the upper organic phase with anhydrous sodium sulfate after separation, and concentrate the filtrate under reduced pressure to almost dryness after filtration, and vacuum dry overnight to obtain 24.12 g (yield 97%) of tert-butyl eicosandioyl-OSu.

[0290] LC-MS(Scie×100API): m / z=496.36(M+1) +

[0291] 3.2 tert-Butyl eicosanedioyl-γGlu-OtBu

[0292] Tert-butyleicosanoyl-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred, and H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water were added in sequence, and heated to obtain a clear solution, which was stirred at room temperature for 4 hours. Then, 10% citric acid aqueous solution (200 mL) was added, and the liquid was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to almost dryness under reduced pressure, and vacuum dried overnight. 27.27 g (yield 96%) of tert-butyleicosanoyl-γGlu-OtBu was obtained.

[0293] LC-MS(Scie×100API): m / z=584.44(M+1) +

[0294] 3.3 tert-Butyleicosanedioyl-γGlu-(OSu)-OtBu.

[0295] Under nitrogen protection, tert-butyleicosandioyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added in turn and stirred for 10 minutes, then NHS (5.38 g, 50.17 mmol) was added, followed by DCC (10.60 g, 51.38 mmol). The mixture was stirred overnight at room temperature. Filtered, the filtrate was concentrated to almost dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated brine, the upper organic phase was dried over anhydrous sodium sulfate after separation, the filtrate was concentrated to almost dryness under reduced pressure after filtration, methyl tert-butyl ether was added, stirred for 30 minutes, filtered, and the filter cake was vacuum dried overnight to obtain 25.76 g (yield 81%) of tert-butyleicosandioyl-γGlu-(OSu)-OtBu.

[0296] LC-MS(Scie×100API): m / z=681.46(M+1) +

[0297] 3.4 tert-Butyl eicosanedioyl-γGlu-(2xOEG-OH)-OtBu

[0298] Tert-butyleicosanoyl-γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred, and 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added in sequence, and heated to obtain a clear solution, which was stirred at room temperature for 4 hours. Then 10% citric acid aqueous solution (200 mL) was added, and the liquid was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to almost dryness under reduced pressure, and vacuum dried overnight. 30.75 g (yield 93%) of tert-butyleicosanoyl-γGlu-(2xOEG-OH)-OtBu was obtained.

[0299] LC-MS(Scie×100API): m / z=874.59(M+1) +

[0300] 3.5 tert-Butyleicosanedioyl-γGlu-(2xOEG-OSu)-OtBu.

[0301] Under nitrogen protection, tert-butyleicosadianoyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added and stirred for 10 minutes, and NHS (4.05 g, 35.18 mmol) was added, followed by DCC (7.98 g, 38.70 mmol). The mixture was stirred overnight at room temperature. Filtered, the obtained filtrate was concentrated to almost dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated brine, and the upper organic phase was dried over anhydrous sodium sulfate after separation. After filtering, the filtrate was concentrated to almost dryness under reduced pressure, and vacuum dried overnight to obtain 31.09 g (yield 91%) of tert-butyleicosadianoyl-γGlu-(2xOEG-OSu)-OtBu.

[0302] LC-MS(Scie×100API): m / z=971.61(M+1) +

[0303] 3.6 tert-Butyl eicosanedioyl-γGlu-(5xOEG-OH)-OtBu

[0304] Tert-butyl eicosanedioyl-γGlu-(2xOEG-OSu)-OtBu (31.09 g, 32.01 mmol) was dissolved in dichloromethane (350 mL) and stirred, and 3xAEEA (14.52 g, 32.01 mmol), triethylamine (8.90 mL), and water (25 mL) were added in sequence, and heated to obtain a clear solution, which was stirred at room temperature for 4 hours. Then, 10% citric acid aqueous solution (200 mL) was added, and the liquid was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to almost dryness under reduced pressure, and vacuum dried overnight. 38.99 g (yield 93%) of tert-butyl eicosanedioyl-γGlu-(5xOEG-OH)-OtBu was obtained.

[0305] LC-MS(Scie×100API): m / z=1309.81(M+1)+

[0306] 3.7 tert-Butyl eicosanedioyl-γGlu-(5xOEG-OSu)-OtBu

[0307] Under nitrogen protection, tert-butyleicosanoyl-γGlu-(5xOEG-OH)-OtBu (38.99 g, 29.77 mmol) was dissolved in dichloromethane (400 mL), triethylamine (8.28 mL) was added and stirred for 10 minutes, and NHS (3.43 g, 29.77 mmol) was added, followed by DCC (6.76 g, 32.75 mmol). The mixture was stirred overnight at room temperature. Filtered, the obtained filtrate was concentrated to almost dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated brine, the upper organic phase was dried over anhydrous sodium sulfate after separation, the filtrate was concentrated to almost dryness under reduced pressure after filtration, and vacuum dried overnight to obtain 38.11 g (yield 91%) of tert-butyleicosanoyl-γGlu-(5xOEG-OSu)-OtBu.

[0308] LC-MS(Scie×100API): m / z=1406.83(M+1) +

[0309] Embodiment 7:

[0310] B29K (N(ε)-eicosanedioyl-γGlu-6xOEG), desB30 human insulin (Compound 6)

[0311]

[0312] Compound 6 was prepared by a procedure similar to Example 6, part 2.

[0313] LC-MS (electrospray): m / z=1406.28 [M+5H] 5+

[0314] Intermediates Tert-Butyleicosanedioyl-γGlu-(6xOEG-OSu)-OtBu Follow the similar procedure as in Example 6, part 3.

[0315] LC-MS(Scie×100API): m / z=1551.90(M+1) +

[0316] Embodiment 8:

[0317] B29K (N(ε)-eicosanedioyl-γGlu-8xOEG), desB30 human insulin (Compound 7)

[0318]

[0319] Compound 7 was prepared by a procedure similar to Example 6, part 2.

[0320] LC-MS (electrospray): m / z=1464.30 [M+5H] 5+

[0321] Intermediates Tert-butyl eicosanedioyl γ Glu-(8xOEG-OSu)-OtBu Follow the similar procedure as in Example 6, part 3.

[0322] LC-MS(Scie×100API): m / z=1814.02(M+1) +

[0323] Embodiment 9:

[0324] B29K (N(ε)-docosanedioyl-γGlu-6xOEG), desB30 human insulin (Compound 8)

[0325]

[0326] Compound 8 was prepared by a procedure similar to Example 6, part 2.

[0327] LC-MS (electrospray): m / z=1411.88 [M+5H] 5+

[0328] Intermediates Tert-Butyldocosapentaenoyl-γGlu-(6xOEG-OSu)-OtBu The preparation of was carried out in a similar manner to that of Example 6, part 3.

[0329] LC-MS(Scie×100API): m / z=1579.94(M+1) +

[0330] Embodiment 10:

[0331] B29K (N(ε)-docosanedioyl-γGlu-8xOEG), desB30 human insulin (Compound 9)

[0332]

[0333] Compound 9 was prepared by a procedure similar to Example 6, part 2.

[0334] LC-MS (electrospray): m / z = 1469.91 [M+5H] 5+

[0335] Intermediates Tert-butyl docosanoyl- γ Glu-(8xOEG-OSu)-OtBu The preparation of was carried out in a similar manner to that of Example 6, part 3.

[0336] LC-MS(Scie×100API): m / z=1870.08(M+1) +

[0337] Embodiment 11:

[0338] Title compound: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (Compound 10)

[0339]

[0340] Prepare N-ε by a procedure similar to that of Example 1, Part 1 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide

[0341] LC-MS (electrospray): m / z=1035.80 [M+4H] 4+

[0342] Intermediates Tert-Butyldocosapentaenoyl-γGlu-(2xOEG-OSu)-OtBu Prepared by a procedure similar to Example 1, part 2.

[0343] LC-MS(Scie×100API): m / z=999.64(M+1) +

[0344] Example 12: In vitro potency or activity

[0345] The purpose of this example is to test the in vitro potency or activity of the GLP-1 derivatives of the present invention

[0346] The cells expressing GLP-1R were revived and inoculated into a 25 mL cell culture flask using ham's-F12 medium and cultured overnight at 37°C with 5% CO2. On the day of the experiment, the title compound of Example 11 of the present invention (Compound 10) and liraglutide were prepared to 150 μg / mL, and then the samples were graded diluted to 750 ng / ml, 150 ng / ml, 30 ng / ml, 6 ng / ml, 1.2 ng / ml, 0.24 ng / ml, 0.048 ng / ml, 0.0096 ng / ml and 0.00192 ng / ml. The cell concentration was adjusted to 1×10 5100 μl of diluted sample was added to each well of the 96-well plate, and the mixture was mixed. After 4 hours of culture, the luciferase reagent was added to each well of the 96-well plate. After oscillation and mixing, the luciferase reagent was transferred from the 96-well plate to a new 96-well white flat-bottom plate. The signal value was read by an ELISA reader. The data were processed with GraphPad Prism 6 to calculate the EC. 50 The in vitro potency experiments were repeated 4 times on different days.

[0347] Table 1: In vitro potency

[0348] Experimental subjects <![CDATA[EC 50 (nM)]]> Liraglutide 0.439 Compound 10 0.677

[0349] It can be seen from the experimental results that the GLP-1 derivative of the present invention has satisfactory in vitro efficacy, and its in vitro activity is close to that of liraglutide, which confirms that it has GLP-1 receptor agonist activity.

[0350] Example 13: Pharmacodynamics experiment in high-fat diet-induced obese C57BL mice

[0351] The purpose of this study is to confirm the blood sugar regulating effect and weight loss effect of the GLP-1 derivative of the present invention in high-fat diet-induced obese C57BL mice.

[0352] C57BL mice (half male and half female) aged 5 weeks and weighing 17-22 g were housed in feeding boxes of appropriate specifications (3-5 mice / box) in a barrier environment. The high-fat diet-induced group had free access to high-fat feed and purified water, and the normal control group had free access to standard food and purified water. The environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22°C-24°C. After 10 weeks of breeding, mice whose body weight exceeded that of the normal control group by 30%-50% were selected for drug efficacy evaluation.

[0353] Before the start of the experiment on the same day, the basal blood glucose was assessed at time -1 / 1h (9:30 am) and the mice were weighed. According to random blood glucose and body weight, the mice in the high-fat diet-induced group were matched and assigned to the vehicle group (i.e., the model control group) or the treatment group, and received the following treatments: subcutaneous injection of the vehicle, or subcutaneous injection of the control compound semaglutide 100 μg / kg, or subcutaneous injection of the title compound of Example 11 of the present invention 100 μg / kg and 300 μg / kg. The solvent contains: propylene glycol 14 mg / ml, phenol 5.5 mg / ml, disodium hydrogen phosphate 1.133 mg / ml, and the pH of the solvent is 7.4.

[0354] Subcutaneous administration was adopted, and the mice were subcutaneously administered once at the back of the neck (5 μl / g body weight). The GLP-1 derivative was administered at about 10:30 am (time 0), and the blood glucose of the mice was evaluated at 3, 6, 24, 48 and 72 hours after administration. At the same time, the body weight of the mice was monitored every day.

[0355] A Δ blood glucose-time curve is drawn for each single dose of the GLP-1 derivative. Wherein, Δ refers to the actual blood glucose at a given time minus the baseline, where the baseline is the blood glucose at time 0. Therefore, in these curves, y=0 represents the baseline. For each individual dose-response curve, the area under the blood glucose-time curve (ΔAUC) from 0 to the monitoring endpoint is calculated. The smaller the ΔAUC value, the better the hypoglycemic effect and the better the efficacy.

[0356] An intraperitoneal glucose tolerance test (ipGTT) was performed 48 hours after the first administration, with the following steps: blood was collected from the tip of the tail at the designated time point to measure fasting blood glucose (0 min), followed by intraperitoneal administration of glucose solution (200 mg / ml, 10 ml / kg), and then blood glucose was measured 30 min, 60 min and 120 min after glucose loading.

[0357] The mouse tail was cleaned with an alcohol cotton ball, and a blood drop was collected from the tail using a disposable blood collection needle, and the blood glucose was measured using a blood glucose meter (Roche) and matching test strips.

[0358] For each single dose of GLP-1 derivative, a dose response curve of blood glucose versus time and a dose response curve of daily weight change versus time are plotted. In order to more intuitively and quantitatively illustrate the effect of the GLP-1 derivative of the present invention on blood glucose, for each individual dose response curve, the relative blood glucose-time curve area difference (ΔAUC) from 0 to the monitoring endpoint is calculated. Among them, the smaller the ΔAUC value, the better the hypoglycemic effect and the better the efficacy.

[0359] Figure 5a-6b The results show that the GLP-1 derivatives of the present invention have unexpectedly increased efficacy. For example, the hypoglycemic effect of compound 10 in Example 11 on high-fat diet-induced obese C57BL mice was not significantly different from that of the marketed control compound semaglutide at the same dose, and even the quantitative Figure 5b and 6b It can be seen that the hypoglycemic effect of the GLP-1 derivatives of the present invention is slightly better than that of semaglutide. In particular, at 72 hours after administration, the average blood sugar level of the compound 10 group with the same dose is lower than the average blood sugar level of the semaglutide group with the same dose. In addition, the hypoglycemic effect of the GLP-1 derivatives of the present invention is dose-dependent, and as the dose of the GLP-1 of the present invention increases, its hypoglycemic effect is also significantly improved.

[0360] Figure 6a-6b It was shown that in the ipGTT experiment, compound 10 of Example 11, compared with the vehicle, had a significant inhibitory effect on blood glucose after the ipGTT experiment was performed 48 hours after the first administration to high-fat diet-induced obese C57BL mice, and was slightly better than the hypoglycemic effect of the same dose of semaglutide.

[0361] Figure 5c It shows that the GLP-1 derivatives of the present invention, such as compound 10 of Example 11, have excellent weight loss effect, and its weight loss effect is better than semaglutide.

[0362] Example 14: Pharmacodynamic study in type II diabetic db / db mice

[0363] The purpose of this study is to confirm the blood sugar regulating effect of the GLP-1 derivatives of the present invention in the case of diabetes.

[0364] The blood glucose lowering effect of the title compound of Example 11 and the control compound liraglutide were tested in db / db mice at different doses of 0.3, 1, 3, 10, 30 and 100 nmol / kg, and the ED 50 .

[0365] 8-9 week old male db / db (BKS / Lepr) mice were housed in a barrier environment in a suitable size feeding box with free access to standard food and purified water. The environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22°C-24°C. After a 1-2 week adaptation period, they were used in the experiment.

[0366] Before the experiment started on the same day, the basal blood glucose was assessed at 9:00 am and the mice were weighed. The diabetic mice were matched and assigned to the vehicle group or the treatment group according to the random blood glucose and body weight, and received the following treatments: subcutaneous injection of the vehicle, or subcutaneous injection of the compound of Example 11 or the control group compound liraglutide 0.3, 1, 3, 10, 30 and 100 nmol / kg, wherein the vehicle contained: propylene glycol 14 mg / ml, phenol 5.5 mg / ml, disodium hydrogen phosphate 1.133 mg / ml, and the pH of the vehicle was 7.4.

[0367] The compound of Example 11 was administered subcutaneously (50 μl / 10 g body weight) once at the back of the neck. The compound of Example 11 was administered at approximately 10:00 am (time 0), and the blood glucose of the mice was evaluated at 1, 2, 3, 6, 12, 24, 48 and 72 hours after administration.

[0368] The mouse tail was cleaned with an alcohol cotton ball, and a blood drop was collected from the tail using a disposable blood collection needle, and the blood glucose was measured using a blood glucose meter (Roche) and matching test strips.

[0369] A dose response curve of Δ blood glucose versus time was plotted for each single dose of the GLP-1 derivative. Δ refers to the actual blood glucose at a given time minus the baseline, where the baseline is the blood glucose at time 0. To illustrate the effect of the GLP-1 derivative on blood glucose, for each individual dose response curve, the area under the curve of Δ blood glucose from 0 to 72 hours, ΔAUC, was calculated, and the effective dose 50% (ED50) was calculated for ΔAUC.50 , the dose of the GLP-1 derivative that produces a response halfway between baseline and maximal effect). The ED values ​​obtained are shown in Table 2 below. 50 value.

[0370] Table 2: ED effects on blood glucose in db / db mice 50 value

[0371] Sample name <![CDATA[ED 50 (nmol / kg)]]> Liraglutide 9.68 Compound 10 8.42

[0372] The test results show that the in vivo glucose-lowering efficacy of compound 10 of the present invention is significantly better than that of liraglutide.

[0373] Example 15 Pharmacodynamic study in type II diabetic db / db mice

[0374] The purpose of this study is to confirm the control of blood sugar, food intake and water intake by the GLP-1 derivatives of the present invention.

[0375] The title compound of Example 2 and the control compound semaglutide were tested in a single dose study on type II diabetic db / db mice.

[0376] 8-9 week old male db / db (BKS / Lepr) mice were housed in a barrier environment in a suitable size feeding box with free access to standard food and purified water. The environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22°C-24°C. After a 1-2 week adaptation period, they were used in the experiment.

[0377] Before the experiment started on the same day, basal blood glucose was assessed at about 9:00 am and the mice were weighed. Diabetic mice were matched and assigned to a vehicle group or a treatment group according to random blood glucose and body weight, and received the following treatments: subcutaneous injection of a vehicle, or subcutaneous injection of the compound of Example 2 or a control compound, semaglutide 100 μg / kg, wherein the vehicle contained propylene glycol 14 mg / ml, phenol 5.5 mg / ml, and disodium hydrogen phosphate 1.133 mg / ml, pH 7.4.

[0378] The GLP-1 derivative was dissolved in a solvent to a dosing concentration of 20 μg / ml and administered subcutaneously (50 μl / 10 g body weight) once by subcutaneous injection at the back of the neck. The compound of Example 2 was administered at approximately 10:00 a.m. (time 0), and the blood glucose of mice was evaluated at 1, 2, 3, 6, 12, 24, 48 and 72 hours after administration. The tail of the mouse was cleaned with an alcohol cotton ball, and a blood drop was collected from the tail using a disposable blood collection needle, and measured using a blood glucose meter (Roche) and matching test strips. At the same time, the food intake and water intake of the mice were measured every day.

[0379] For each single dose of the GLP-1 derivative, a dose response curve of blood glucose versus time, a dose response curve of food intake versus time, and a dose response curve of water intake versus time are plotted. In order to illustrate the effect of the GLP-1 derivative of the present invention on blood glucose, for each individual dose response curve, the area under the curve (ΔAUC) of blood glucose-time from 0 to the monitoring endpoint is calculated. Among them, the smaller the ΔAUC value, the better the hypoglycemic effect and the better the efficacy.

[0380] Figure 7a-7d It is shown that the GLP-1 derivatives of the present invention have unexpectedly increased hypoglycemic efficacy and improved inhibitory effects on food intake and water intake after administration. This further proves that the title compound of Example 2 has a better hypoglycemic effect on db / db mice after administration than semaglutide at the same dose. In addition, the title compound of Example 2 can effectively control food intake and water intake, and its effect is better than semaglutide, indicating that the GLP-1 derivatives of the present invention have better weight loss effects.

[0381] Example 16 Long-term pharmacodynamic study in type II diabetic db / db mice

[0382] The purpose of this study is to confirm the long-term hypoglycemic effect, weight loss, and diet control effect of the GLP-1 derivative of the present invention on type II diabetic db / db mice.

[0383] The GLP-1 derivative of Example 11 and the control compound semaglutide were tested on type II diabetic db / db mice. The GLP-1 derivative was administered to mice at different doses of 100 and 300 μg / kg and semaglutide was administered at a dose of 100 μg / kg to measure the effects of the GLP-1 derivative and the control compound semaglutide on lowering blood sugar, body weight, food intake and water intake.

[0384] 8-9 week old male db / db (BKS / Lepr) mice were housed in a barrier environment in a suitable size feeding box with free access to standard food and purified water. The environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22°C-24°C. After a 1-2 week adaptation period, they were used in the experiment.

[0385] Before the experiment started on the same day, basal blood glucose was assessed at approximately 9:00 am and the mice were weighed. Diabetic mice were matched to the vehicle group or the treatment group according to random blood glucose and body weight, and received the following treatments: subcutaneous injection of the vehicle, or subcutaneous injection of 100 and 300 μg / kg of GLP-1 derivatives, or subcutaneous injection of the control compound semaglutide 100 μg / kg. The solvent contained: propylene glycol 14 mg / ml, phenol 5.5 mg / ml, disodium hydrogen phosphate 1.133 mg / ml, and the pH value of the solvent was 7.4.

[0386] The GLP-1 derivative was administered subcutaneously (50 μl / 10 g body weight) at about 10:00 am (time 0) on days 0, 3, 7, 10, 13, 16, 19, 22, 25, and 28, and the blood glucose of mice was evaluated before each administration and 72 hours after the last administration. The body weight, food intake, and water intake of mice were measured every day from day 0 to day 17, and every 3 days after day 17.

[0387] Figure 8a-8e It is shown that the GLP-1 derivatives of the present invention still have unexpectedly increased hypoglycemic efficacy, improved weight loss effect, and the effect of inhibiting food and water intake after long-term administration. Figure 8a and 8b It was shown that compared with the same dose of semaglutide, compound 10 of Example 11 had a better hypoglycemic effect on db / db mice after long-term administration. Figure 8c-8d It is shown that, compared with the same dose of semaglutide, the GLP-1 derivatives of the present invention, such as the title compound of Example 11, have better weight loss effect and effect of inhibiting food intake and water intake.

[0388] Example 17 Long-term pharmacodynamic study in type II diabetic Kkay mice

[0389] The purpose of this study is to confirm the hypoglycemic effect of the GLP-1 derivatives of the present invention on type II diabetic Kkay mice

[0390] Compound 10 of Example 11, Compound 2 of Example 2, and control compound dulaglutide (also known as dulaglutide) were tested on type II diabetic Kkay mice. Compound 10 and Compound 2 were administered to mice at different doses of 100 and 300 μg / kg, and dulaglutide was administered at a dose of 600 μg / kg to determine the blood sugar lowering effect and HbA1c effect of the GLP-1 derivative of the present invention and the control compound dulaglutide.

[0391] Male Kkay mice aged 12-14 weeks were housed in a suitable size feeding box in a barrier environment with free access to standard food and purified water. The environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22°C-24°C. After an adaptation period of 1-2 weeks, they were used in the experiment.

[0392] Before the experiment started on the same day, basal blood glucose was assessed at about 9:00 am and the mice were weighed. Diabetic mice were matched and assigned to the vehicle group or the treatment group according to random blood glucose and body weight, and received the following treatments: subcutaneous injection of the vehicle, or subcutaneous injection of 100 and 300 μg / kg of the GLP-1 derivative of the present invention, or subcutaneous injection of 600 μg / kg of the control compound dulaglutide. The vehicle contained: propylene glycol 14 mg / ml, phenol 5.5 mg / ml, disodium hydrogen phosphate 1.133 mg / ml, pH 7.4.

[0393] The GLP-1 derivative of the present invention, dulaglutide or vehicle was administered subcutaneously (50 μl / 10 g body weight) at approximately 10:00 a.m. (time 0) and once every 2 days for 16 consecutive times. The blood glucose of mice was evaluated at 3 h, 6 h, 1 day and 2 days after the first administration. EDTA anticoagulation was used to detect HbA1c 48 h after the last administration.

[0394] Figure 9a-9b It is shown that the GLP-1 derivatives of the present invention have unexpectedly increased hypoglycemic efficacy after administration, and the title compounds of Example 11 and Example 2 have significantly better hypoglycemic effects on Kkay mice than dulaglutide. Fig.9c It shows that the effect of the GLP-1 derivative of the present invention on reducing HbA1c in type II diabetic Kkay mice is significantly better than that of dulaglutide.

[0395] Example 18: Pharmacokinetics

[0396] The purpose of this example is to illustrate the in vivo pharmacokinetic properties of the compounds of the present invention.

[0397] Pharmacokinetics in SD rats

[0398] Thirty-two SD rats, 8 in each group (half male and half female), were divided into a low-dose group of compound 10, a medium-dose group of compound 10, and a high-dose group of compound 10, which were subcutaneously injected with 15, 90, and 540 μg / kg, respectively, and an intravenous injection group of compound 10, which was intravenously injected with 90 μg / kg. Blood was collected before (0 min) and 1, 3, 5, 8, 12, 16, 24, 36, 48, 72, 96, and 120 h after administration for the determination of blood drug concentration in the low-, medium-, and high-dose groups of compound 10, respectively, and blood was collected before (0 min) and 1 min, 10 min, 1, 3, 5, 8, 12, 24, 48, 72, 96, and 120 h after administration for the intravenous injection group of compound 10 for the determination of blood drug concentration. The non-compartmental model of WinNonLin v6.4 software was used to calculate the pharmacokinetic parameter C max , T max , T 1 / 2 , AUC 0-t, MRT, and the test results are shown in Table 3.

[0399] Table 3: Pharmacokinetic parameters of compound 10 after subcutaneous injection into SD rats

[0400]

[0401] C max = maximum measured plasma concentration, T max = the time corresponding to the maximum measured blood drug concentration, T 1 / 2 = terminal elimination half-life, AUC 0-t =0-t time-area under the plasma drug concentration time curve, MRT = mean residence time

[0402] Pharmacokinetics in cynomolgus monkeys

[0403] Twenty-four cynomolgus monkeys, 6 in each group (half male and half female), were divided into low-dose group, medium-dose group, and high-dose group of compound 10, which were subcutaneously injected with 10, 60, and 360 μg / kg, respectively, and intravenous injection group of compound 10, which was intravenously injected with 60 μg / kg of compound 10. Blood was collected before (0 min) and 1, 3, 6, 8, 10, 12, 16, 24, 48, 72, 120, 168, and 240 h after administration for the determination of blood drug concentration in the low-, medium-, and high-dose groups of compound 10, respectively, and blood was collected before (0 min) and 1 min, 10 min, 1, 3, 6, 8, 10, 12, 24, 48, 72, 120, 168, and 240 h after administration for the intravenous injection group of compound 10 for the determination of blood drug concentration. The non-compartmental model of WinNonLin v6.4 software was used to calculate the pharmacokinetic parameters, C max , T max , T 1 / 2 , AUC 0-t , MRT, and the test results are shown in Table 4.

[0404] Table 4: Pharmacokinetic parameters of compound 10 after subcutaneous injection in cynomolgus monkeys

[0405]

[0406]

[0407] The above experimental results show that the GLP-1 derivative compound 10 of the present invention exhibits a longer half-life and a larger AUC in rats and cynomolgus monkeys. 0-t , and a longer MRT. The GLP-1 derivatives of the present invention are all dose-dependent, and their efficacy increases with increasing doses.

[0408] Embodiment 19

[0409] The purpose of this experiment is to measure the chemical stability of the GLP-1 derivative formulations of the present invention.

[0410] GLP-1 derivative preparations

[0411] Compound 10 was dissolved in 5.68 mg / ml disodium hydrogen phosphate solution to a final concentration of 8 mg / ml. According to the amount of each component in the table below, auxiliary liquid containing propylene glycol and phenol was added in sequence, and the pH was adjusted to the value in the table below to produce a final GLP-1 compound concentration of 2 mg / ml.

[0412] The chemical stability of the preparation in this example can be shown by the change in high molecular weight protein (HMWP) relative to day 0 after storage at 37°C for 27 days, and can also be shown by the change in the amount of related substances measured after storage at 37°C for 28 days.

[0413] Determination of high molecular weight protein (HMWP)

[0414] Determined by high performance liquid chromatography (HPLC) High molecular weight protein (HMWP) content , tested on a Waters TSKgelG2000SWXL (7.8*300mm), 5μm column, at a column temperature of 30°C and a sample cell temperature of 5°C, with a mobile phase at a flow rate of 0.5ml / min, wherein the mobile phase contained 300ml isopropanol, 400ml glacial acetic acid and 300ml purified water. The detection wavelength was 276nm and the injection volume was 25μl. Table 5 shows the increase in HMWP at 37°C for 27 days relative to day 0.

[0415] Determination of the amount of relevant substances

[0416] The content of impurities in GLP-1 derivatives was determined by high performance liquid chromatography (HPLC) on a Waters Kromasil 100-3.5-C8 (4.6*250 mm) column at a column temperature of 35°C and a sample cell temperature of 5°C, using an elution phase at a flow rate of 1.0 ml / min. The elution was performed using a mobile phase consisting of:

[0417] Phase A contained 90 mM potassium dihydrogen phosphate and 10% acetonitrile (v / v), pH 2.4

[0418] Phase B was 75% (v / v) acetonitrile.

[0419] Gradient: linear change from 75% / 25% A / B to 55% / 45% A / B in 0-5 min, linear change from 5-12 min to 50% / 50% A / B, linear change from 12-42 min to 40% / 60% A / B, linear change from 42-60 min to 10% / 90% A / B, linear change from 60-61 min to 75% / 25% A / B, isocratic gradient from 61-70 min to 85% / 15% A / B.

[0420] The detection wavelength was 214 nm, the flow rate was 1.0 ml / min, and the injection volume was 15 μl. Table 5 shows the increase in the amount of related substances relative to day 0 after storage at 37°C for 28 days.

[0421] Table 5

[0422]

[0423] As can be seen from the above table, the preparations have good chemical stability when the pH value is between 6.5-8.4, and the chemical stability of the preparations is the best when the pH value is between 7.0-8.0.

[0424] Embodiment 20

[0425] The purpose of this experiment is to measure the chemical stability of the GLP-1 derivative formulations of the present invention.

[0426] According to the amounts of the components in Tables 6 and 7 below, the GLP-1 derivative preparations in Tables 6 and 7 were prepared according to procedures similar to Example 19. Changes in HMWP and related substances were measured according to procedures similar to Example 19. Tables 6 and 7 below show changes in HMWP and related substances of GLP-1 derivative preparations with different formulations.

[0427] Table 6

[0428]

[0429]

[0430] Table 7

[0431]

[0432] As can be seen from the above table, the amount of HMWP and the amount of related substances in the above GLP-1 derivative preparations of the present invention increase very slowly over time, indicating that the above GLP-1 derivative preparations have excellent chemical stability.

[0433] Comparative Example 5

[0434] A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-2xOEG), desB30 human insulin (reference compound 5)

[0435]

[0436] 1. Preparation of A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-2xOEG), desB30 human insulin

[0437] A14E, B16H, B25H, and desB30 human insulins were prepared by conventional methods for preparing insulin analogs (for specific methods, see Glendorf T, AR, Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding; Biochemistry 2008 474743-4751). A14E, B16H, B25H, desB30 human insulin (5 g, 0.888 mmol) was dissolved in 100 mM Na2HPO4 aqueous solution (150 mL), and acetonitrile (100 mL) was added, and the pH was adjusted to pH 10-12.5 with 1 N NaOH. Tert-butyl eicosanedioyl-γGlu-(2xOEG-OSu)-OtBu (0.948 g, 0.976 mmol) was dissolved in acetonitrile (50 mL) and slowly added to the insulin solution. The pH was maintained at 10-12.5. After 120 minutes, the reaction mixture was added to water (150 mL), and the pH was adjusted to 5.0 with a 1N HCl aqueous solution. Precipitate was separated by centrifugation and freeze-dried. The freeze-dried crude product was added to a mixed solution of trifluoroacetic acid (60 mL) and dichloromethane (60 ml), and stirred at room temperature for 30 minutes. The mixture was concentrated to about 30 ml, poured into ice-cold n-heptane (300 mL), and the precipitated product was separated by filtration and washed twice with n-heptane. After vacuum drying, the purified fractions were combined by ion exchange chromatography ((Ressource Q, 0.25%-1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA), and the pH was adjusted to 5.2 with 1N HCl, and the precipitate was separated and freeze-dried to obtain control compound 5.

[0438] LC-MS (electrospray): m / z = 1063.6852 [M+6H] 6+

[0439] 2. Preparation of the intermediate tert-butyleicosanedioyl-γGlu-(2xOEG-OSu)-OtBu: Some similar steps are carried out.

[0440] 2.1 tert-Butyl eicosanedioyl-OSu

[0441] Under nitrogen protection, mono-tert-butyl eicosandioate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane, triethylamine (13.95 mL) was added, and the resulting turbid mixture was stirred at room temperature, and then DCC (11.39 g, 55.19 mmol) was added, and further stirred overnight. Filter, concentrate the obtained filtrate to almost dryness, mix the residue with cold water and ethyl acetate, stir for 20 minutes, separate the liquids, wash the upper organic phase with saturated brine, dry the upper organic phase with anhydrous sodium sulfate after separation, and concentrate the filtrate under reduced pressure to almost dryness after filtration, and vacuum dry overnight to obtain 24.12 g (yield 97%) of tert-butyl eicosandioyl-OSu.

[0442] LC-MS(Scie×100API): m / z=496.36(M+1) +

[0443] 2.2 tert-Butyl eicosanedioyl-γGlu-OtBu

[0444] Tert-butyleicosanoyl-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred, and H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water were added in sequence, and heated to obtain a clear solution, which was stirred at room temperature for 4 hours. Then, 10% citric acid aqueous solution (200 mL) was added, and the liquid was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to almost dryness under reduced pressure, and vacuum dried overnight. 27.27 g (yield 96%) of tert-butyleicosanoyl-γGlu-OtBu was obtained.

[0445] LC-MS(Scie×100API): m / z=584.44(M+1) +

[0446] 2.3 tert-Butyleicosanedioyl-γGlu-(OSu)-OtBu.

[0447] Under nitrogen protection, tert-butyleicosadianoyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added and stirred for 10 min, then NHS (5.38 g, 50.17 mmol) was added, followed by DCC (10.60 g, 51.38 mmol). The mixture was stirred overnight at room temperature. Filtered, the filtrate was concentrated to almost dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated brine, the upper organic phase was dried over anhydrous sodium sulfate after separation, the filtrate was concentrated to almost dryness under reduced pressure after filtration, methyl tert-butyl ether was added, stirred for 30 min, filtered, and the filter cake was vacuum dried overnight to obtain 25.76 g (yield 81%) of tert-butyleicosadianoyl-γGlu-(OSu)-OtBu.

[0448] LC-MS(Scie×100API): m / z=681.46(M+1) +

[0449] 2.4 tert-Butyl eicosanedioyl-γGlu-(2xOEG-OH)-OtBu

[0450] Tert-butyl eicosanedioyl-γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred, and 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added in sequence, and heated to obtain a clear solution, which was stirred at room temperature for 4 hours. Then 10% citric acid aqueous solution (200 mL) was added, and the liquid was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried with anhydrous sodium sulfate, and the filtrate was concentrated to almost dryness under reduced pressure after filtration, and vacuum dried overnight. 30.75 g (yield 93%) of tert-butyl eicosanedioyl-γGlu-(2xOEG-OH)-OtBu was obtained.

[0451] LC-MS(Scie×100API): m / z=874.59(M+1) +

[0452] 2.5 tert-Butyleicosanedioyl-γGlu-(2xOEG-OSu)-OtBu.

[0453] Under nitrogen protection, tert-butyleicosadianoyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added and stirred for 10 minutes, and NHS (4.05 g, 35.18 mmol) was added, followed by DCC (7.98 g, 38.70 mmol). The mixture was stirred overnight at room temperature. Filtered, the filtrate was concentrated to almost dryness, the residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, separated, the upper organic phase was washed with saturated brine, the upper organic phase was dried over anhydrous sodium sulfate after separation, the filtrate was concentrated to almost dryness under reduced pressure after filtration, and vacuum dried overnight to obtain 31.09 g (yield 91%) of tert-butyleicosadianoyl-γGlu-(2xOEG-OSu)-OtBu.

[0454] LC-MS(Scie×100API): m / z=971.61(M+1) +

[0455] Embodiment 21

[0456] A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-6xOEG), desB30 human insulin (Compound 11)

[0457]

[0458] Compounds A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-6xOEG), desB30 human insulin were prepared by similar procedures as in Part 1 of Control Example 5

[0459] LC-MS (electrospray): m / z=1160.3997 [M+6H] 6+

[0460] The preparation of the intermediate tert-butyleicosanedioyl-γGlu-(6xOEG-OSu)-OtBu was carried out in a similar manner to that of Control Example 5, Part 2.

[0461] LC-MS(Scie×100API): m / z=1551.90(M+1) +

[0462] Embodiment 22

[0463] A14E, B16H, B25H, B29K (N(ε)-docosanedioyl-γGlu-6xOEG), desB30 human insulin (Compound 12)

[0464]

[0465] Compounds A14E, B16H, B25H, B29K (N(ε)-docosanedioyl-γGlu-6xOEG), desB30 human insulin were prepared by similar procedures as in Part 1 of Control Example 5

[0466] LC-MS (electrospray): m / z=1165.0674 [M+6H] 6+

[0467] The preparation of the intermediate tert-butyldocosanedioyl-γGlu-(6xOEG-OSu)-OtBu was carried out in a similar manner to that of Control Example 5, Part 2.

[0468] LC-MS(Scie×100API): m / z=1579.94(M+1) +

[0469] Embodiment 23

[0470] A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-12xOEG), desB30 human insulin (Compound 13)

[0471]

[0472] Compounds A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-12xOEG), desB30 human insulin were prepared by similar procedures as in Part 1 of Control Example 5

[0473] LC-MS (electrospray): m / z=1305.4716 [M+6H] 6+

[0474] The preparation of the intermediate tert-butyleicosanedioyl-γGlu-(12xOEG-OSu)-OtBu was carried out in a similar manner to that of Control Example 5, Part 2.

[0475] LC-MS(Scie×100API): m / z=2423.35(M+1) +

[0476] Embodiment 24

[0477] A14E, B16H, B25H, B29K (N(ε)-docosanedioyl-γGlu-12xOEG), desB30 human insulin (Compound 14)

[0478]

[0479] Compounds A14E, B16H, B25H, B29K (N(ε)-docosanedioyl-γGlu-12xOEG), desB30 human insulin were prepared by similar procedures as in Part 1 of Control Example 5

[0480] LC-MS (electrospray): m / z=1310.1425 [M+6H] 6+

[0481] The preparation of the intermediate tert-butyldocosanedioyl-γGlu-(12xOEG-OSu)-OtBu was carried out in a similar manner to that of Control Example 5, Part 2.

[0482] LC-MS(Scie×100API): m / z=2451.38(M+1) +

[0483] Example 25 GLP-1 receptor binding

[0484] The purpose of this example was to test the in vitro receptor binding affinity of the GLP-1 derivatives of the invention and how the presence of albumin potentially affects binding. Receptor binding is a measure of the affinity of a GLP-1 derivative for the human GLP-1 receptor.

[0485] The binding affinity of the GLP-1 derivatives and control compounds of the present invention to the human GLP-1 receptor was determined by measuring the amount of GLP-1 they displace from the receptor. 125 I-GLP-1 ability. In order to determine the binding of GLP-1 derivatives to albumin (HSA), low albumin concentration (0.005% (w / v)) and high albumin concentration (2% (w / v)) were used for determination. Binding affinity IC 50 The change in indicates that the GLP-1 derivatives bind to albumin, thereby predicting the potential prolonged pharmacokinetic characteristics of the GLP-1 derivatives in animal models.

[0486] For receptor binding assays in the presence of low HSA (0.005% (w / v)), 50 μl of assay buffer was added to each well of the assay plate. For receptor binding assays in the presence of high HSA (2% (w / v)), 50 μl of 8% (w / v) albumin stock solution was added to each well of the assay plate. Test compounds were prepared with 10 mM Na2HPO4, pH 7.3, and reference control GLP-1 (7-37) was prepared with ultrapure water to 1 mM stock solution. Under 0.005% HSA conditions, all test compounds and reference controls were diluted to 2 μM with assay buffer and then subjected to 4-fold serial gradient dilutions for a total of 10 concentration gradients. Under 2% HSA conditions, reference control GLP-1 (7-37) was diluted to 2 μM, liraglutide was diluted to 20 μM, compound 10 and semaglutide were diluted to 800 μM, and then all samples were subjected to 4-fold serial gradient dilutions for a total of 10 concentration gradients. Add 25 μl of different concentrations of test compound or reference control to the appropriate wells of the assay plate. Thaw and dilute the cell membrane protein aliquot to its working concentration (40 μg / mL), and add 50 μl of the cell membrane solution to each well of the assay plate. 125 I]-GLP-1 600pM solution to start incubation. Incubate the assay plate at room temperature for 1h. After incubation, collect the reaction solution onto the GF / C filter plate using a cell harvester, wash 6 times with plate washing buffer, and dry in a 50°C oven for 1h. Add 50μl scintillation fluid, block, and read using Microbeta2. Use nonlinear regression analysis in GraphPad Prism to calculate IC 50 The values ​​are reported in nM. Each test compound was repeated at least three times. The reported values ​​are the average of all the measured values ​​for each test compound.

[0487] Table 8: GLP-1 receptor binding affinity

[0488]

[0489] The ratio is [(IC 50 / nM)High HSA] / [(IC 50 / nM) low HSA]

[0490] In general, binding to the GLP-1 receptor should be as good as possible at low albumin concentrations, which corresponds to a low IC 50 At high albumin concentrations, IC 50 The IC value is a measure of the effect of albumin on the binding of GLP-1 derivatives to the GLP-1 receptor. As is known, GLP-1 derivatives also bind to albumin, which is generally a desired effect that will extend their plasma lifetime. Therefore, at high albumin, the IC50 Values ​​are usually higher than IC at low albumin 50 =1, which corresponds to a decrease in binding to the GLP-1 receptor due to albumin binding competing with GLP-1 receptor binding. 50 Value (high albumin) / IC 50 value (low albumin)), as the target derivative binds well to albumin (thereby determining that it has a long half-life), and also binds well to the GLP-1 receptor itself (IC 50 Value (high albumin) is high, IC 50 Value (low albumin) low) indication.

[0491] It can be seen from the above table that the ratio of the GLP-1 derivative of the present invention is higher than that of the reference compounds semaglutide, liraglutide and GLP-1 (7-37), indicating that the compound of the present invention has a longer half-life and binds well to the GLP-1 receptor.

[0492] Example 26 Long-term pharmacodynamic study in type II diabetic db / db mice

[0493] Referring to the similar experimental steps of Example 16, a long-term pharmacodynamic study was conducted in type II diabetic db / db mice, except that the control compound used was dulaglutide, and the dosage of dulaglutide was 300 μg / kg. The GLP-1 derivative was administered subcutaneously (50 μl / 10 g body weight) at the back of the neck at about 10:00 am (time 0), and was administered on days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30. The blood glucose of mice was evaluated at 3, 6, 9, 12, 24, 48, and 72 hours after the first administration, and the change in the area under the blood glucose-time curve (ΔAUC) was calculated. Fasting blood glucose was monitored for 6 hours after fasting for 48 hours before administration and after the 3rd, 5th, and 11th administrations. An intraperitoneal glucose tolerance test (ipGTT) was performed 48 hours after the first administration, with the following steps: blood was collected from the tip of the tail at a specified time point to measure fasting blood glucose (0 min), followed by intraperitoneal administration of glucose solution (200 mg / ml, 10 ml / kg), and then blood glucose was measured 30 min, 60 min, and 120 min after the glucose load. The tail of the mouse was cleaned with an alcohol cotton ball, and a blood drop was collected from the tail using a disposable blood collection needle. The blood glucose was measured using a blood glucose meter (Roche) and matching test strips, and the time-blood glucose curve was drawn, and the area under the time-blood glucose curve (AUC) was calculated.

[0494] Figures 10a-10e This shows that the GLP-1 derivatives of the present invention still have unexpectedly increased hypoglycemic efficacy after long-term administration. Fig.10a and 10bThe results showed that compared with dulaglutide, compound 10 of Example 11 had a better hypoglycemic effect on db / db mice after administration. Fig.10c The results showed that compared with dulaglutide, compound 10 had a better hypoglycemic effect on db / db mice after long-term administration. Figure 10d-10e It is shown that compared with dulaglutide, the GLP-1 derivative of the present invention has a more obvious inhibitory effect on blood sugar and is better than the blood sugar lowering effect of dulaglutide.

[0495] Example 27 Pharmacodynamics experiment in high-fat diet-induced obese C57BL mice

[0496] Referring to the similar experimental steps of Example 13, a pharmacodynamic experiment was carried out in high-fat diet-induced obese C57BL mice, except that the control compound used was dulaglutide, and the dosage of dulaglutide was 300 μg / kg.

[0497] Subcutaneous administration was adopted, and the drug was administered subcutaneously once at the back of the neck (5 μl / g body weight), once every 3 days, for a total of 11 times. The GLP-1 derivative was administered at about 10:30 am (time 0), and the blood glucose of mice was evaluated at 3, 6, 9, 12, 24, 48 and 72 hours after administration. At the same time, the body weight and food intake of mice were monitored every 3 days. Subcutaneous fat, perirenal fat, and perigenital fat were weighed at the end of the experiment.

[0498] Figures 11a-11d It was shown that the GLP-1 derivatives of the present invention have unexpectedly improved weight loss effects, dietary control effects, and lipid-lowering effects.

[0499] Embodiment 28

[0500] B29K (N(ε)-docosanedioyl-γGlu-12xOEG), desB30 human insulin (Compound 15)

[0501]

[0502] Compound B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin was prepared by a procedure similar to that of Example 6, part 2.

[0503] LC-MS (electrospray): m / z = 1585.98 [M+5H] 5+

[0504] Intermediates Tert-Butyldocosapentaenoyl-γGlu-(12xOEG-OSu)-OtBu Follow the similar procedure as in Example 6, part 3.

[0505] LC-MS(Scie×100API): m / z=2451.38(M+1) +

[0506] Embodiment 29

[0507] A14E, B16H, B25H, B29K (N(ε)-docosanedioyl-γGlu-18xOEG), desB30 human insulin (Compound 16)

[0508]

[0509] Compounds A14E, B16H, B25H, B29K (N(ε)-docosanedioyl-γGlu-18xOEG), desB30 human insulin were prepared by similar procedures as in Part 1 of Control Example 5

[0510] LC-MS (electrospray): m / z=1247.47 [M+7H] 7+

[0511] The preparation of the intermediate tert-butyldocosanedioyl-γGlu-(18xOEG-OSu)-OtBu was carried out in a similar manner to that of Control Example 5, Part 2.

[0512] LC-MS(Scie×100API): m / z=3320.83(M+1) +

[0513] Embodiment 30

[0514] A14E, B16H, B25H, B29K (N(ε)-docosanedioyl-γGlu-24xOEG), desB30 human insulin (Compound 17)

[0515]

[0516] Compounds A14E, B16H, B25H, B29K (N(ε)-docosanedioyl-γGlu-24xOEG), desB30 human insulin were prepared by similar procedures as in Part 1 of Control Example 5

[0517] LC-MS (electrospray): m / z=873.35 [M+11H] 11+

[0518] The preparation of the intermediate tert-butyldocosanedioyl-γGlu-(24xOEG-OSu)-OtBu was carried out in a similar manner to that of Control Example 5, Part 2.

[0519] LC-MS(Scie×100API): m / z=4192.27(M+1) +

[0520] Embodiment 31

[0521] B29K (N(ε)-docosanedioyl-γGlu-OEG), desB30 human insulin (Compound 18)

[0522]

[0523] Compound B29K (N(ε)-docosandioyl-γGlu-OEG), desB30 human insulin was prepared by a procedure similar to that of Example 6, part 2.

[0524] LC-MS (electrospray): m / z = 1266.8122 [M+5H] 5+

[0525] Intermediates Tert-Butyldocosapentaenoyl-γGlu-(OEG-OSu)-OtBu Follow the similar procedure as in Example 6, part 3.

[0526] LC-MS(Scie×100API): m / z=854.57(M+1) +

[0527] Embodiment 32

[0528] B29K (N(ε)-docosanedioyl-γGlu-12xPEG), desB30 human insulin (Compound 19)

[0529]

[0530] Compound B29K (N(ε)-docosandioyl-γGlu-12xPEG), desB30 human insulin was prepared by a procedure similar to that of Example 6, part 2.

[0531] LC-MS (electrospray): m / z=1354.8667 [M+5H] 5+

[0532] Intermediates Tert-Butyldocosanedioyl-γGlu-(12xPEG-OSu)-OtBu Follow the similar procedure as in Example 6, part 3.

[0533] LC-MS(Scie×100API): m / z=1294.83(M+1) +

[0534] The present invention has been described by the above embodiments, but it should be understood that the above embodiments are only for the purpose of example and description, and are not intended to limit the present invention to the scope of the described embodiments. In addition, it can be understood by those skilled in the art that the present invention is not limited to the above embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications all fall within the scope of the protection claimed by the present invention. The protection scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. A compound of formula B, or a pharmaceutically acceptable salt, amide or ester thereof: [Acy-(L1) r -(L2) q ]-G1(B), wherein G1 is a GLP-1 analog having Arg at position 34 and Ala or Gly at position 8 corresponding to GLP-1 (7-37) (SEQ ID NO: 1), [Acy-(L1) r -(L2) q ] is a substituent attached to the epsilon amino group of the Lys residue at position 26 of the GLP-1 analog, wherein r is an integer from 1 to 10, and q is 0 or an integer from 1 to 10; Acy is an aliphatic diacid comprising 20 to 24 carbon atoms, wherein a hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid; L1 is an amino acid residue selected from the group consisting of γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; L2 is a neutral, alkylene glycol-containing amino acid residue; Acy, L1, and L2 are connected by an amide bond; and The order in which L1 and L2 appear in formula (B) can be independently interchanged.

2. The compound according to claim 1, wherein G1 is [Gly8, Arg34]GLP-1-(7-37) peptide (SEQ ID NO: 2) or [Arg34]GLP-1-(7-37) peptide (SEQ ID NO: 3), preferably [Gly8, Arg34]GLP-1-(7-37) peptide; and / or r is 1, 2, 3, 4, 5 or 6, preferably, r is 1, 2, 3 or 4, preferably, r is 1 or 2, preferably, r is 1; and / or q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably, q is 0, 1, 2, 3 or 4, more preferably, q is 0, 1 or 2; and / or Acy is an aliphatic diacid containing 20-23 carbon atoms, preferably Acy is an aliphatic diacid containing 20, 21, or 22 carbon atoms.

3. The compound according to claim 1 or 2, wherein L2 is: -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O -(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-( CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-, -HN-(CH2)3-O-(C H2)4-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)2-O-(CH2) 2-O-CH2-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)3-O-(CH2 )2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)2-CO-, -HN-(CH2)2- O-(CH2)2-O-(CH2)2-NH-CO-CH2-O-CH2-CO-,-HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-,- HN-(CH2)3-O-(CH2)3-O-CH2-CO-, or -HN-(CH2)4-O-(CH2)4-O-CH2-CO-; preferably L2 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-; and / or L1 is selected from γGlu or βAsp, preferably L1 is γGlu; ​​and / or Acy is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO- or HOOC-(CH2) 22 -CO-, preferably, Acy is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

4. The compound according to any one of claims 1 to 3, wherein In formula (B), Acy, L1, and L2 are sequentially connected by amide bonds, and the C-terminus of L2 is connected to the epsilon amino group of the Lys residue at position 26 of the GLP-1 analog.

5. The compound according to claim 1, wherein the compound is selected from the following compounds: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(21-carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -(21-carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, or N-ε 26 -(22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide; Preferably, the compound is selected from the following compounds: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyhenicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; Preferably, the compound is selected from the following compounds:

6. A pharmaceutical preparation comprising a compound according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient; Preferably: the pharmaceutically acceptable excipient is selected from one or more of a buffer, a preservative, an isotonic agent, a stabilizer and a chelating agent; preferably, the pharmaceutically acceptable excipient is a buffer, a preservative and an isotonic agent.

7. The pharmaceutical preparation according to claim 6, wherein the isotonic agent is selected from one or more of sodium chloride, propylene glycol, mannitol, sorbitol, glycerol, glucose and xylitol, preferably propylene glycol, mannitol or sodium chloride; and / or The preservative is selected from one or more of phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, and benzyl alcohol, preferably phenol or m-cresol; and / or The buffer is selected from one or more of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, preferably sodium acetate, citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate.

8. The pharmaceutical preparation according to any one of claims 6 to 7, wherein The pH of the preparation is about 6.0 to about 10.0, preferably about 6.5 to about 10.0, preferably about 6.5 to about 9.5, preferably about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, further preferably about 7.3 to about 8.

1.

9. Pharmaceutical preparations containing the following ingredients: About 0.1-1.2 mM, preferably about 0.2-1 mM, preferably about 0.3-0.7 mM, more preferably about 0.48-0.6 mM of a compound according to any one of claims 1 to 5; About 10-1500mM, preferably about 13-800mM, preferably about 65-400mM, preferably about 90-240mM, preferably about 150-250mM, preferably about 180-200mM, more preferably about 183-195mM isotonic agent, preferably, the isotonic agent is selected from one or more of propylene glycol, glycerol, mannitol or sodium chloride; About 1-200mM, preferably about 5-150mM, preferably about 10-100mM, preferably about 20-85mM, preferably about 30-75mM, preferably about 45-60mM, more preferably about 50-60mM of a preservative, preferably, the preservative is selected from one or more of phenol or m-cresol; About 3-35 mM, preferably about 5-20 mM, more preferably about 5-15 mM, more preferably about 7-10 mM of a buffer selected from one or more of sodium acetate, citrate, sodium dihydrogen phosphate or disodium hydrogen phosphate; and The pH of the pharmaceutical preparation is about 6.0 to about 10.0, preferably about 6.5 to about 9.5, preferably about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, further preferably about 7.3 to about 8.1; Preferably: The pharmaceutical preparation comprises: About 0.3-0.7 mM, more preferably about 0.48-0.6 mM N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; About 180-200 mM, more preferably about 183-195 mM propylene glycol; About 45-60 mM, more preferably about 50-60 mM phenol; about 5-15 mM of a buffer, preferably about 7-10 mM of disodium hydrogen phosphate; and The pH of the pharmaceutical preparation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, and even more preferably about 7.3 to about 8.3; Preferably: The pharmaceutical preparation comprises: About 0.5mM N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; About 184 mM propylene glycol; about 58.5 mM phenol; about 10 mM sodium phosphate dibasic; and The pH of the pharmaceutical preparation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.1; Preferably: The pharmaceutical preparation comprises: About 2.0mg / ml N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; About 14 mg / ml of propylene glycol; About 5.5 mg / ml of phenol; about 1.42 mg / ml of disodium hydrogen phosphate; and The pH of the pharmaceutical preparation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.

1.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 and an acylated insulin, preferably, the acylated insulin is B29K (N (ε) -docosandioyl-γGlu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-2xOEG), desB30 human insulin; or B29K (N (ε) -docosandioyl-γGlu-12xPEG), desB30 human insulin; More preferably, the acylated insulin is the following acylated insulin, wherein the insulin parent of the acylated insulin is a naturally occurring insulin or an insulin analog and comprises at least one lysine residue, the acyl moiety of the acylated insulin is linked to the lysine residue or the amino group of the N-terminal amino acid residue of the insulin parent, and the acyl moiety is as shown in formula (A): III-(II) m -(I) n -(A), in, m is 0 or an integer of 1-10, and n is an integer of 5-30 (preferably 5-20); I is a neutral, alkylene glycol-containing amino acid residue; II is an acidic amino acid residue; III is an aliphatic diacid containing 20 to 24 carbon atoms, wherein a hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid; III, II, and I are connected by an amide bond; and The order in which II and I appear in formula (A) can independently be interchanged.

11. The pharmaceutical composition according to claim 10, wherein n is an integer of 5-15, preferably, n is 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, preferably, n is 5, 6, 7, 8, 9, 10, 11 or 12, preferably, n is 5, 6, 7, 8, 9, or 10, preferably, n is 5, 6, 7, 8 or 9, preferably, n is 5, 6, 7, or 8; and / or m is an integer of 1-6, preferably, m is 1, 2, 3, or 4, preferably, m is 1 or 2, preferably, m is 1; and / or III is an aliphatic diacid containing 20-23 carbon atoms, preferably III is an aliphatic diacid containing 20, 21, or 22 carbon atoms, wherein the hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid; and / or The insulin precursor contains one lysine residue.

12. The pharmaceutical composition according to claim 10 or 11, wherein I is: -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O- (CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-( CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-, -HN-(CH2)3-O-(C H2)4-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)2-O-(CH2) 2-O-CH2-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-(CH2)2-CO-, -HN-(CH2)3-O-(CH2 )2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)2-CO-, -HN-(CH2)2 -O-(CH2)2-O-(CH2)2-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)3-O-CH2-CO-, or -HN-(CH2)4-O-(CH2)4-O-CH2-CO-; preferably I is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-; and / or II is an amino acid residue selected from the group consisting of γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; and / or III is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO- or HOOC-(CH2) 22 -CO-.

13. The pharmaceutical composition according to any one of claims 10 to 12, wherein Formula (A) is connected to the amino group of the lysine residue or the N-terminal amino acid residue of the insulin parent through the C-terminus of I; Preferably, the acyl moiety is linked to the epsilon amino group of a lysine residue of the insulin precursor; Preferably, the lysine residue of the insulin parent is located at position B29; Preferably, the insulin precursor is selected from the following insulins or insulin analogs: desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; human insulin; A21G human insulin; A21G, desB30 human insulin; or B28D human insulin.

14. The pharmaceutical composition according to claim 10, wherein The acylated insulin is selected from the following insulins: B29K (N (ε) -eicosanedioyl-γGlu-5xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-γGlu-5xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-5xOEG-γGlu), desB30 human insulin; 6xOEG-γGlu),desB30 human insulin;B29K(N(ε)-eicosanedioyl-6xOEG-γGlu-γGlu),desB30 human insulin;B29K(N(ε)-eicosanedioyl-5xOEG-γGlu-γGlu),desB30 human insulin;B29K(N(ε)-eicosanedioyl-βAsp-5xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-βAsp-6xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-αGlu-5xOEG),desB30 human insulin;B29K(N(ε)- Eicosanedioyl-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αAsp-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanedioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanedioyl-γGlu-5xOEG ),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-γGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-γGlu-γGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-γGlu-γGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-5xOEG-γGlu),desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16H, B 25H,B29K(N(ε)-eicosanedioyl-βAsp-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-αGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-αGlu-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanedioyl-αGlu-αGlu-5xOEG),desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-6xOEG) ,desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-6xOEG-γGlu), desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-βAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-βAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K 9K (N (ε) -eicosanedioyl-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-αAs p-5xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αAsp-6xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-γGlu-7xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-γGlu-8xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-γGlu-γGlu-7xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-γGlu-γGlu-8xOEG),desB30 human insulin;B2 9K(N(ε)-eicosanedioyl-7xOEG-γGlu),desB30 human insulin; B29K(N(ε)-eicosanedioyl-8xOEG-γGlu),desB30 human insulin; B29K(N(ε)-eicosanedioyl-8xOEG-γGlu-γGlu),desB30 human insulin; B29K(N(ε)-eicosanedioyl-7xOEG-γGlu-γGlu),desB30 human insulin; B29K(N(ε)-eicosanedioyl-βAsp-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-βAsp-8xOEG),desB30 human insulin;B29K(N(ε)-eicosanedioyl-αGlu-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-8xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-αGlu-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αGlu-αGlu-8xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αAsp-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-αAsp-8xOEG),desB30 human insulin Insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl- βAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αGlu-αGlu-7xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αGlu-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-γGlu-7xOEG), desB30 human insulin; 9K (N (ε) -eicosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl- A14E, B16E, B25H, B29K (N (ε)-eicosanedioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-eicosanedioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-eicosanedioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-eicosanedioyl-βAsp-8xOEG ),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αGlu-αGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosanedioyl-αGlu-αGlu-8xOEG),desB30 human insulin;A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-αAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosanedioyl-αAsp-8xOEG), desB30 human insulin; B29K (N(ε)-docosanedioyl-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N(ε)-docosanedioyl-γGlu-γGlu-5xOEG), desB30 human insulin; B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosandioyl-βAsp-5xO EG), desB30 human insulin; B29K(N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αAsp-5xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; 5H,B29K(N(ε)-docosandioyl-5xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-βAsp-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-βAsp-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-βAsp-5xOEG),desB30 human insulin A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-αAsp-6xOEG ),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-γGlu-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-γGlu-6xOEG),desB30 human insulin;A14E, B16E, B25H, B29K (N (ε) -docosandioyl-5xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-6xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H ,B29K(N(ε)-docosandioyl-βAsp-5xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-βAsp-6xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-5xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-6xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-α Glu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-αAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-αAsp-6xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K (N (ε)-docosanedioyl-γGlu-γGlu-8xOEG), desB30 human insulin; B29K (N (ε)-docosanedioyl-γGlu-γGlu-7xOEG), desB30 human insulin; B29K (N (ε)-docosanedioyl-γGlu-γGlu-8xOEG), desB30 human insulin; B29K (N (ε)-docosanedioyl-7xOEG-γGlu), desB30 human insulin; B29K (N (ε)-docosanedioyl-8xOEG-γGlu), desB30 human insulin; B29K (N (ε)-docosanedioyl-8xOEG-γGlu-γGlu), desB30 human insulin;B29K (N (ε) -docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; B29K (N (ε) -docosandioyl-βAsp-7xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-βAsp-8xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αGlu-7xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αGlu-8xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αGlu-7xOEG), desB30 human insulin EG), desB30 human insulin; B29K (N (ε) -docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αAsp-7xOEG), desB30 human insulin; B29K (N (ε) -docosandioyl-αAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-8xOEG), desB30 human insulin G), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-8xOEG-γGlu), desB 30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-βAsp-8xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin; 25H,B29K(N(ε)-docosandioyl-αAsp-7xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosandioyl-αAsp-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-γGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl- γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin; γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-βAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-βAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -docosandioyl-αGlu-7xOEG), desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-αGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αGlu-αGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosandioyl-αAsp-7xOEG),desB30 human insulin; 5H,B29K(N(ε)-docosanedioyl-αAsp-8xOEG),desB30 human insulin; B29K(N(ε)-henicosanedioyl-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-henicosanedioyl-γGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-henicosanedioyl-γGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-henicosanedioyl-γGlu-6xOEG),desB30 human insulin; E, B25H, B29K (N (ε) - heneicosandioyl - γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) - heneicosandioyl - γGlu-6xOEG), desB30 human insulin; B29K (N (ε) - heneicosandioyl - γGlu-7xOEG), desB30 human insulin; B29K (N (ε) - heneicosandioyl - γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - heneicosandioyl - γGlu-7xOEG), desB30 human insulin; A14E ,B16H,B25H,B29K(N(ε)-henicosanedioyl-γGlu-8xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-henicosanedioyl-γGlu-7xOEG),desB30 human insulin;A14E,B16E,B25H,B29K(N(ε)-henicosanedioyl-γGlu-8xOEG),desB30 human insulin;B29K(N(ε)-tricosanedioyl-γGlu-5xOEG),desB30 human insulin;B29K(N(ε)-tricosanedioyl-γGlu-6xOEG),desB30 human insulin;A14E, B16H, B25H, B29K (N (ε) -tricosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -tricosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -tricosandioyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -tricosandioyl-γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -tricosandioyl-γGlu- Glu-7xOEG), desB30 human insulin; B29K (N (ε)-tricosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε)-tricosanedioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε)-tricosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-tricosanedioyl-γGlu-7xOEG), desB30 human insulin; H, B29K (N (ε) -tricosanodiaoyl -γGlu-8xOEG), desB30 human insulin; B29K (N (ε) -tetracosanodiaoyl -γGlu-5xOEG), desB30 human insulin; B29K (N (ε) -tetracosanodiaoyl -γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -tetracosanodiaoyl -γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -tetracosanodiaoyl -γGlu-6xOEG), desB30 human insulin; 16E, B25H, B29K (N (ε) -tetracosandiaoyl -γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -tetracosandiaoyl -γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -tetracosandiaoyl -γGlu-7xOEG), desB30 human insulin; B29K (N (ε) -tetracosandiaoyl -γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -tetracosandiaoyl -γGlu-7xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG), desB30 human insulin; or A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin; B29K (N(ε)-eicosanedioyl-γGlu-12xOEG), desB30 human insulin; B29K (N(ε)-eicosanedioyl-γGlu-12xOEG), desB3 0 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu- 10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - docosandioyl - γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - heneicosandioyl - γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - tricosandioyl - γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - tetracosandioyl - γGlu-12xOEG), desB30 human insulin;A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-11xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε) -eicosanedioyl-γGlu-10xOEG), desB30 human insulin dodecandioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-docosandioyl-γGlu-11xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-heneicosandioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-tricosandioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N (ε)-tetracosandioyl-γGlu-12xOEG), desB30 human insulin esB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu -9xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu -10xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu -11xOEG), desB30 human insulin; B29K (N (ε) - docosanedioyl - γGlu -9xOEG), desB30 human insulin; B29K (N (ε) - docosanedioyl - γGlu -10xOEG), desB30 human insulin; B29K (N (ε) - docosanedioyl - γGlu -11xOEG), des B30 human insulin; B29K (N (ε) - heneicosandioyl -γGlu-12xOEG), desB30 human insulin; B29K (N (ε) - tricosandioyl -γGlu-12xOEG), desB30 human insulin; B29K (N (ε) - tetracosandioyl -γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - docosandioyl -γGlu-18xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N (ε) - docosandioyl -γGlu-24xOEG), desB30 human insulin; Preferably, the acylated insulin is selected from the following insulins: B29K (N (ε) - eicosanedioyl - γGlu-5xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu-6xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu-5xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu-6xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu-7xOEG), desB30 human insulin; B29K (N (ε) - eicosanedioyl - γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin; B29K(N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin; B29K(N(ε)- B29K(N(ε)-eicosanedioyl-γGlu-11xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-γGlu-12xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-γGlu-11xOEG),desB30 human insulin; B29K(N(ε)-eicosanedioyl-γGlu-12xOEG),desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanedioyl-γGlu-5x OEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - eicosanedioyl - γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - eicosanedioyl - γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - eicosanedioyl - γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - eicosanedioyl - γGlu-7xOEG), desB30 human insulin;A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -docosandioyl-γGlu-8xOEG), desB30 human insulin; 14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-9xOEG), desB30 human insulin; E, B16H, B25H, B29K (N (ε) - eicosanedioyl - γGlu-10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - eicosanedioyl - γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) - eicosanedioyl - γGlu-12xOEG), desB30 human insulin; A14 E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-18xOEG), desB30 human insulin; Preferably, the acylated insulin is selected from the following insulins: B29K (N (ε) -eicosanedioyl-γGlu-5xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-5xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-6xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-8xOEG), desB30 human insulin; B29K (N (ε) -eicosanedioyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosanedioyl-γGlu-6 xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanedioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin; Preferably, the acylated insulin is selected from the following insulins: A14E, B16H, B25H, B29K (N (ε) -eicosandioyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosandioyl-γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N (ε) -eicosandioyl-γGlu-6xOEG), desB 30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin.

15. Use of the compound of any one of claims 1 to 5, the pharmaceutical preparation of any one of claims 6 to 9 or the pharmaceutical composition of any one of claims 10 to 14 in the preparation of a medicament for treating or preventing hyperglycemia, diabetes, and / or obesity; preferably, the diabetes is type II diabetes.

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