Long-acting glp-1 compounds

Abstract

A new GLP-1 derivative, which has comparable or better potency, efficacy or efficacy, longer or comparable in vivo duration of action or in vivo half-life, has better or comparable GLP-1 receptor binding affinity, has better or comparable DPP-IV stability, compared to the marketed GLP-1 derivatives such as liraglutide, semaglutide, etc.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on December 29, 2020, with application number 202080091240.0 and invention title "Long-acting GLP-1 Compound".

[0002] Cross-references 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] This invention relates to the field of therapeutic peptides, and more particularly to novel long-acting GLP-1 compounds, pharmaceutical formulations thereof, pharmaceutical compositions thereof with long-acting insulin, and the pharmaceutical uses of said compounds, pharmaceutical formulations and pharmaceutical compositions. Background Technology

[0005] Glucagon-like peptide-1 (GLP-1) and its analogues and derivatives are highly effective in treating type 1 and type 2 diabetes; however, high clearance limits the efficacy of these compounds. To provide GLP-1 compounds with longer durations of action in vivo, a range of different approaches have been used to modify the structure of GLP-1. For example, WO99 / 43708 discloses GLP-1 (7-35) and GLP-1 (7-36) derivatives having lipophilic substitutions linked to C-terminal amino acid residues. WO00 / 34331 discloses acylated GLP-1 analogues. WO00 / 69911 discloses activated insulinotropic peptides for injection into patients.

[0006] Currently marketed GLP-1 drugs include: exenatide, a natural GLP-1 analog administered twice daily; liraglutide and lixisenatide, administered once daily (the former being a hexadecanoic acid-modified GLP-1 compound, and the latter a new molecule obtained by structurally modifying exenaglutide); and semaglutide, exenatide LAR, abiglutide, dulaglutide (also known as dulaglutide), and polyethylene glycol loxenatide, administered once weekly. Among them, exenatide microspheres are prepared by encapsulating exenatide into a polylactic acid-glycolic acid copolymer matrix through microencapsulation; abiglutide is prepared by fusing two modified GLP-1 peptide chains in dimer form with human albumin to form a recombinant fusion protein; dulaglutide is prepared by fusing modified GLP-1 chains into the Fc fragment of recombinant G4 immunoglobulin through disulfide bonds; polyethylene glycol loxenatide is prepared by modifying the amino acid structure of exenatide and modifying it with polyethylene glycol; semaglutide is mainly achieved by replacing the 8th position of Ala with the non-protein amino acid Aib on the GLP-1 (7-37) peptide to achieve once-weekly administration. However, the presence of non-protein amino acids in semaglutide may pose unknown and various potential side effects risks in the human body compared to natural amino acids.

[0007] On the one hand, there is still a need to develop compounds that, compared to marketed drugs of the same class such as liraglutide, dulaglutide, and semaglutide, have better efficacy, pharmacodynamics, or therapeutic effects, lower risk of potential side effects, better weight loss and appetite suppression effects, and longer or comparable duration of action or half-life in vivo, in order to provide better medication options for diabetic patients.

[0008] On the other hand, with the rapid increase in the global population with type 2 diabetes, there is a greater demand for drugs that are easier to administer and more effective. For example, combination formulations containing both insulin and GLP-1 peptide as active ingredients may be very effective therapeutic agents. Therefore, there is still a need for combination formulations that can synergistically achieve better physical and chemical stability, longer duration of action, and better efficacy. Summary of the Invention

[0009] To overcome or improve at least one drawback of the prior art, or to provide a useful alternative, the first aspect of this invention provides novel GLP-1 compounds (also referred to as GLP-1 derivatives). Compared to marketed GLP-1 derivatives such as liraglutide, dulaglutide, and semaglutide, these novel GLP-1 compounds exhibit better potency, efficacy, or therapeutic effect; a lower risk of potential side effects; better weight loss; longer duration of action or half-life in vivo; better or comparable GLP-1 receptor binding affinity; and better or comparable DPP-IV stability. Furthermore, the pharmaceutical composition or combination formulation of the long-acting GLP-1 compound and the long-acting insulin provided by this invention not only does not impair the physical stability of the GLP-1 compound and the insulin compound, but the combination formulation also exhibits better physical stability than the monotherapy formulation. The physical stability of the combination formulation of this invention is unexpected compared to combination formulations of other long-acting GLP-1 compounds (e.g., a combination of liraglutide and degludec insulin). In addition, the combination formulation increases the chemical stability of the GLP-1 compound and acylated insulin compared to the monotherapy formulation. The GLP-1 compound of the present invention, and the combination formulations containing the GLP-1 compound and the islet compound provided by the present invention, can achieve long pharmacokinetic (PK) characteristics, making it possible to subcutaneously treat diabetic patients twice a week, once a week, once every two weeks, or at a lower frequency.

[0010] The GLP-1 compound provided in the first aspect of this 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 analogue having Arg at position 34 corresponding to GLP-1(7-37)(SEQ ID NO: 1) and having Ala or Gly at position 8, [Acy-(L1)] r -(L2) q ] is a substituent attached to the ε-amino group of the Lys residue at position 26 of the GLP-1 analogue, 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 containing 20-24 carbon atoms, wherein the hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid;

[0015] L1 is selected from the following amino acid residues: γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp;

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

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

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

[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 implementation, 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 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.

[0023] In one implementation, 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-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(C 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 implementation, 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 attached to the ε-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-carboxynonadecanylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0043] N-ε 26 -(23-Carboxy-tetradecanoylamino)-4(S)-Carboxybutyryl-[Arg34]GLP-1-(7-37)peptide

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

[0045] N-ε 26 -(21-Carboxy-2-O ...

[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-carboxynonadecanylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide,

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

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

[0050] N-ε 26 -(19-Carboxynonadecanylamino)-4(S)-Carboxybutyryl-[Arg34]GLP-1-(7-37)peptide

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

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

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

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

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

[0057] N-ε 26 -(20-Carboxyeicosicosylamino)-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-carboxyeicosicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37)peptide,

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

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

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

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

[0064] N-ε 26 -(22-Carboxydodecanoylamino)-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-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide, or N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide.

[0067] A second aspect of the present invention provides a pharmaceutical formulation comprising the compound described in 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 buffers, preservatives, isotonic agents, stabilizers, and chelating agents. In another embodiment, the pharmaceutically acceptable excipient is a buffer, preservative, or isotonic agent.

[0069] In one embodiment, the pharmaceutical formulation comprises: the compound described in the first aspect of the present invention, an isotonic agent, a preservative, and a buffer. Preferably, in the pharmaceutical formulation, the compound described in the first aspect of the present invention is N-ε. 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide, or N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]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 para-hydroxybenzoate, propyl para-hydroxybenzoate, 2-phenoxyethanol, butyl para-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, even 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 preparation contains the following components:

[0075] The compound described in the first aspect of the present invention, at a concentration of about 0.1-1.2 mM, preferably about 0.2-1 mM, more preferably about 0.3-0.7 mM, and more preferably about 0.48-0.6 mM;

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

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

[0078] A buffer of about 3-35 mM, preferably about 5-20 mM, more preferably about 5-15 mM, and even more preferably about 7-10 mM, wherein the buffer is 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 from about 6.0 to about 10.0, preferably from about 6.5 to about 9.5, more preferably from about 6.5 to about 8.5, more preferably from about 7.0 to about 8.5, even more preferably from about 7.0 to about 8.1, and even more preferably from about 7.3 to about 8.1.

[0080] In another experimental protocol, the pharmaceutical preparation comprises: about 0.3-0.7 mM, more preferably about 0.48-0.6 mM of N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)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 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.

[0081] In another experimental protocol, the pharmaceutical preparation comprises: approximately 0.5 mM of N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)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 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.

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

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

[0084] A third aspect of the present invention provides a pharmaceutical composition comprising the GLP-1 compound described in the first aspect of the present invention and acylated insulin.

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

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

[0087] III-(II) m -(I) n -(A), where 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 20-24 carbon atoms, wherein the hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid; III, II, and I are linked 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, and 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 the hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid.

[0091] In another embodiment, the insulin precursor contains a lysine residue.

[0092] In one embodiment, I is: -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O ... 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 following: 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 implementation, 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 to the amino group of the lysine residue or N-terminal amino acid residue of the insulin precursor via the C-terminus of I.

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

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

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

[0099] In one embodiment, the acylated insulin is selected from the following insulins: B29K(N(ε)-eicosanodicyl-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-eicosanodicyl-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-eicosanodicyl-γGlu-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-eicosanodicyl-γGlu-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-eicosanodicyl-5xOEG-γGlu),desB30 human insulin; B29K(N(ε)- Eicosyl-6xOEG-γGlu), desB30 human insulin; B29K(N(ε)-eicosyl-6xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-eicosyl-5xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-eicosyl-βAsp-5xOEG), desB30 human insulin; B29K(N(ε)-eicosyl-βAsp-6xOEG), desB30 human insulin; B29K(N(ε)-eicosyl-αGlu-5xOEG), desB30 human insulin; B29K( N(ε)-eicosanodicyl-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αAsp-5xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-γGlu-5xO EG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-γGlu-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-γGlu-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-5xOEG-γGlu), desB30 human insulin;A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-6xOEG-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-6xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-5xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-βAsp-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-βAsp-5xOEG),desB30 human insulin; A14E,B16H,B 25H,B29K(N(ε)-eicosanodicyl-βAsp-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-αGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-αGlu-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-αGlu-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-αGlu-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-βAsp-6xOEG),desB30 human insulin; Eicosyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosyl-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosyl-αAsp-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosyl-γGlu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N(ε)-eicosyl-γGlu-6xOEG) Human insulin desB30; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-γGlu-γGlu-5xOEG), desB30; Human insulin ...A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-6xOEG-γGlu-γGlu),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-5xOEG-γGlu-γGlu),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-βAsp-5xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-βAsp-6xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-βAsp-6xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-βAsp-6xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-βAsp-6xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-βAsp-6xOEG),desB30 human insulin; 9K(N(ε)-eicosicodiacyl-αGlu-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-αGlu-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-αGlu-αGlu-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-αGlu-αGlu-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-αGlu-αGlu-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-αAs p-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-αAsp-6xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-γGlu-γGlu-7xOEG), desB30 human insulin; B2 9K(N(ε)-eicosanodicyl-7xOEG-γGlu),desB30 human insulin; B29K(N(ε)-eicosanodicyl-8xOEG-γGlu),desB30 human insulin; B29K(N(ε)-eicosanodicyl-8xOEG-γGlu-γGlu),desB30 human insulin; B29K(N(ε)-eicosanodicyl-7xOEG-γGlu-γGlu),desB30 human insulin; B29K(N(ε)-eicosanodicyl-βAsp-7xOEG),desB30 human insulin; B29K(N(ε)-eicosanodicyl-βAsp-8xOEG),desB30 human insulin;B29K(N(ε)-eicosanodicyl-αGlu-7xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αGlu-αGlu-7xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αGlu-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αAsp-7xOEG), desB30 human insulin; B29K(N(ε)-eicosanodicyl-αAsp-8xOEG), desB30 human insulin Insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanodicyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanodicyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanodicyl-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-eicosanodicyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-8xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-8xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl- βAsp-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-βAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-αGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-αGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosanodicyl-αGlu-αGlu-7xOEG), desB30 human insulin;A14E,B16H,B25H,B29K(N(ε)-eicosicodiacyl-αGlu-αGlu-8xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosicodiacyl-αAsp-7xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosicodiacyl-αAsp-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-7xOEG),desB30 human insulin; 9K(N(ε)-eicosicodiacyl-γGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-γGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-7xOEG-γGlu),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-7xOEG-γGlu),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-eicosicodiacyl-γGlu-8xOEG),desB30 human insulin; Human insulin (N(ε)-eicosanoyl-8xOEG-γGlu-γGlu), desB30; human insulin (N(ε)-eicosanoyl-7xOEG-γGlu-γGlu), desB30; human insulin (N(ε)-eicosanoyl-7xOEG-γGlu-γGlu), desB30; human insulin (N(ε)-eicosanoyl-βAsp-8xOEG ... Human insulin, desB30; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-αGlu-7xOEG), desB30; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-αGlu-8xOEG), desB30; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-αGlu-αGlu-7xOEG), desB30; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-αGlu-αGlu-8xOEG), desB30;A14E, B16E, B25H, B29K(N(ε)-eicosicodiacyl-αAsp-7xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosicodiacyl-αAsp-8xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-γGlu-5xOEG),desB30 human insulin; B29K (N(ε)-docosadicyloyl-γGlu-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-docosadicyloyl-5xOEG-γGlu),desB30 human insulin; B29K(N(ε)-docosadicyloyl-6xOEG-γGlu),desB30 human insulin; B29K(N(ε)-docosadicyloyl-6xOEG-γGlu-γGlu),desB30 human insulin; B29K(N(ε)-docosadicyloyl-5xOEG-γGlu-γGlu),desB30 human insulin; B29K(N(ε)-docosadicyloyl-βAsp-5xO B29K(N(ε)-docosadicyloyl-βAsp-6xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αGlu-αGlu-5xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αGlu-αGlu-6xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αGlu-αGlu-6xOEG), desB30 human insulin; Didecanoic acid-αAsp-5xOEG), desB30 human insulin; B29K(N(ε)-dococanoic acid-αAsp-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-dococanoic acid-γGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-dococanoic acid-γGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-dococanoic acid-γGlu-γGlu-5xOEG), desB30 human insulin;A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-γGlu-γGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-5xOEG-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-6xOEG-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-6xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-6xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B29K(N(ε)-docosadicyloyl-6xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B29K(N(ε)-docosadicyloyl-6xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B29K(N(ε)-docosadicyloyl-6xOEG-γGlu-γGlu),desB30 human insulin; 5H,B29K(N(ε)-dococadecanidyl-5xOEG-γGlu-γGlu),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-dococadecanidyl-βAsp-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-dococadecanidyl-βAsp-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-dococadecanidyl-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-dococadecanidyl-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-dococadecanidyl-αGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-dococadecanidyl-αGlu-5xOEG),desB30 human insulin; Acyl-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosadicylate-αGlu-αGlu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosadicylate-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosadicylate-αAsp-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosadicylate-αAsp-6xOEG) Human insulin, desB30; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-γGlu-5xOEG), desB30; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-γGlu-6xOEG), desB30; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-γGlu-γGlu-5xOEG), desB30; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-γGlu-γGlu-6xOEG), desB30;A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-5xOEG-γGlu),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-6xOEG-γGlu),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-6xOEG-γGlu-γGlu),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-5xOEG-γGlu-γGlu),desB30 human insulin; A14E, B16E, B25H B29K(N(ε)-docosadicyloyl-βAsp-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosadicyloyl-βAsp-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosadicyloyl-αGlu-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosadicyloyl-αGlu-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-docosadicyloyl-αGlu-α Glu-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyl-αGlu-αGlu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyl-αAsp-5xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyl-αAsp-6xOEG), desB30 human insulin; B29K(N(ε)-docosadicyl-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-docosadicyl-γGlu-7xOEG), desB30 human insulin; Didecanoic acid-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-dococanoic acid-γGlu-γGlu-7xOEG), desB30 human insulin; B29K(N(ε)-dococanoic acid-γGlu-γGlu-8xOEG), desB30 human insulin; B29K(N(ε)-dococanoic acid-7xOEG-γGlu), desB30 human insulin; B29K(N(ε)-dococanoic acid-8xOEG-γGlu), desB30 human insulin; B29K(N(ε)-dococanoic acid-8xOEG-γGlu-γGlu), desB30 human insulin;B29K(N(ε)-docosadicyl-7xOEG-γGlu-γGlu), desB30 human insulin; B29K(N(ε)-docosadicyl-βAsp-7xOEG), desB30 human insulin; B29K(N(ε)-docosadicyl-βAsp-8xOEG), desB30 human insulin; B29K(N(ε)-docosadicyl-αGlu-7xOEG), desB30 human insulin; B29K(N(ε)-docosadicyl-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosadicyl-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosadicyl-αGlu-αGlu-7xO EG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αGlu-αGlu-8xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αAsp-7xOEG), desB30 human insulin; B29K(N(ε)-docosadicyloyl-αAsp-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicyloyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicyloyl-γGlu-8xOEG), desB30 human insulin; G), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicylo-γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicylo-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicylo-7xOEG-γGlu), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicylo-8xOEG-γGlu), desB 30 human insulins; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-8xOEG-γGlu-γGlu),desB 30 human insulins; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-7xOEG-γGlu-γGlu),desB 30 human insulins; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-βAsp-7xOEG),desB 30 human insulins; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-βAsp-8xOEG),desB 30 human insulins;A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-αGlu-7xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-αGlu-8xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-αGlu-αGlu-7xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-αGlu-αGlu-8xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-docosadicyloyl-αGlu-αGlu-8xOEG),desB30 human insulin; A14E,B16H,B 25H,B29K(N(ε)-dococadecanyl-αAsp-7xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-dococadecanyl-αAsp-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-dococadecanyl-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-dococadecanyl-γGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-dococadecanyl-γGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-dococadecanyl- γGlu-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-γGlu-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-7xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-8xOEG-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-8xOEG-γGlu- γGlu), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococadecanoyl-7xOEG-γGlu-γGlu), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococadecanoyl-βAsp-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococadecanoyl-βAsp-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococadecanoyl-αGlu-7xOEG), desB30 human insulin;A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-αGlu-8xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-αGlu-αGlu-7xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-αGlu-αGlu-8xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-αGlu-αGlu-8xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyloyl-αAsp-7xOEG),desB30 human insulin; A14E, B16E, B2 5H,B29K(N(ε)-Cocicosidic-αAsp-8xOEG),desB30 human insulin; B29K(N(ε)-Cocicosidic-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-Cocicosidic-γGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-Cocicosidic-γGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-Cocicosidic-γGlu-6xOEG),desB30 human insulin; A14E,B16 E, B25H, B29K(N(ε)-eicos ... B16H, B25H, B29K(N(ε)-Cocicosidic-γGlu-8xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-Cocicosidic-γGlu-7xOEG),desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-Cocicosidic-γGlu-8xOEG),desB30 human insulin; B29K(N(ε)-Cocicosidic-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-Cocicosidic-γGlu-6xOEG),desB30 human insulin;A14E,B16H,B25H,B29K(N(ε)-Coctidecyldiacyl-γGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-Coctidecyldiacyl-γGlu-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-Coctidecyldiacyl-γGlu-6xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-Coctidecyldiacyl-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-Coctidecyldiacyl-γGlu-6xOEG),desB30 human insulin; Glu-7xOEG), desB30 human insulin; B29K(N(ε)-docosadicyl-γGlu-8xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicyl-γGlu-7xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicyl-γGlu-8xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-docosadicyl-γGlu-7xOEG), desB30 human insulin; A14E, B16E, B25 H,B29K(N(ε)-tetradecanoic acid-γGlu-8xOEG),desB30 human insulin; B29K(N(ε)-tetradecanoic acid-γGlu-5xOEG),desB30 human insulin; B29K(N(ε)-tetradecanoic acid-γGlu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-tetradecanoic acid-γGlu-5xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-tetradecanoic acid-γGlu-6xOEG),desB30 human insulin; A14E,B 16E,B25H,B29K(N(ε)-tetracosyl-γGlu-5xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-tetracosyl-γGlu-6xOEG),desB30 human insulin; B29K(N(ε)-tetracosyl-γGlu-7xOEG),desB30 human insulin; B29K(N(ε)-tetracosyl-γGlu-8xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-tetracosyl-γGlu-7xOEG),desB30 human insulin;A14E,B16H,B25H,B29K(N(ε)-teicosicodiacyl-γGlu-8xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-teicosicodiacyl-γGlu-7xOEG),desB30 human insulin; A14E,B16E,B25H,B29K(N(ε)-teicosicodiacyl-γGlu-8xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-12xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-12xOEG),desB30 Human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosicodiacyl-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosicodiacyl-γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-γGlu-10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-γGlu-9xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-eicosicodiacyl-γGlu- 10xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicylo-γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicylo-γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicylo-γGlu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(ε)-docosadicylo-γGlu-12xOEG), desB30 human insulin;A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-γGlu-10xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-γGlu-11xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-γGlu-11xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-γGlu-9xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-γGlu-11xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-γGlu-9xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-eicosanodicyl-γGlu-11xOEG), desB30 human insulin; Dodecanoic acid-γGlu-10xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococanoic acid-γGlu-11xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococanoic acid-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococanoic acid-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococanoic acid-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococanoic acid-γGlu-12xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N(ε)-dococanoic acid-γGlu-12xOEG), desB30 human insulin; esB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-9xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-10xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-11xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-9xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-10xOEG),desB30 human insulin; B29K(N(ε)-eicosicodiacyl-γGlu-11xOEG),des B30 human insulin; B29K(N(ε)-eicosodecanediayl-γGlu-12xOEG),desB30 human insulin; B29K(N(ε)-teicosodecanediayl-γGlu-12xOEG),desB30 human insulin; B29K(N(ε)-teicosodecanediayl-γGlu-12xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N(ε)-eicosodecanediayl-γGlu-18xOEG),desB30 human insulin; or A14E,B16H,B25H,B29K(N(ε)-eicosodecanediayl-γGlu-24xOEG),desB30 human insulin.

[0100] The inventors unexpectedly discovered that the pharmaceutical composition of the compound and acylated insulin described in the first aspect of the present invention not only does not impair the physical stability of the compound, but also that the combination formulation exhibits better physical stability than the monotherapy formulation. The physical stability of the combination formulation of the present invention is unexpected compared to combination formulations of other long-acting insulin derivatives (e.g., insulin degludec and liraglutide). Furthermore, the combination formulation also increases the chemical stability of the acylated insulin compared to the monotherapy formulation.

[0101] The fourth aspect of the present invention provides the use of the compounds described in the first aspect of the present invention, the pharmaceutical preparations described in the second aspect of the present invention, or the pharmaceutical compositions described in the third aspect of the present invention as pharmaceuticals.

[0102] In one embodiment, 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 is used to treat or prevent hyperglycemia, diabetes, and / or obesity.

[0103] The fifth aspect of the present invention provides the use of the compounds described in the first aspect of the present invention, the pharmaceutical preparations described in the second aspect of the present invention, or the pharmaceutical compositions described in the third aspect of the present invention in the preparation of medicaments for the treatment or prevention of 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 a compound of the first aspect of the present invention, a pharmaceutical preparation of the second aspect of the present invention, or a pharmaceutical composition of the third aspect of the present invention, the diseases including but not limited to, for example, hyperglycemia, diabetes, and obesity. Attached Figure Description

[0105] Figure 1a The hypoglycemic effects and duration of action of the title compound, liraglutide, and vehicle of Examples 1-3 of this invention on db / db mice are shown. The percentages on the vertical axis refer to the percentage of blood glucose at each monitoring point after administration compared with the baseline blood glucose before administration (the same applies below).

[0106] Figure 1b and Figure 1a Correspondingly, the AUCs of the hypoglycemic effects of the title compounds, liraglutide, and solvents of Examples 1-3 of the present invention on db / db mice are shown.

[0107] Figure 2a The hypoglycemic effects and duration of action of the title compound, semaglutide, and solvent of Example 2 of this invention on db / db mice are shown.

[0108] Figure 2b and Figure 2a Correspondingly, the AUC of the hypoglycemic effects of the title compound, semaglutide, and solvent of Example 2 of the present invention on db / db mice is shown.

[0109] Figure 3a The hypoglycemic effects and duration of action of the title compound, liraglutide, and solvent of Examples 3-4 of this invention on db / db mice are shown.

[0110] Figure 3b and Figure 3a Correspondingly, the AUCs of the hypoglycemic effects of the title compounds, liraglutide, and solvents of Examples 3-4 of the present invention on db / db mice are shown.

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

[0112] Figure 4b and Figure 4a The AUCs of the hypoglycemic effects of the title compounds of Examples 1-3, the title compounds of Control Examples 3-4, and the solvents on db / db mice are shown accordingly.

[0113] Figure 5a The hypoglycemic effects and duration of action 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 solvent (model control group) on obese C57BL mice or normal mice (normal control) induced by a high-fat diet are shown.

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

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

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

[0117] Figure 6b and Figure 6a Correspondingly, the 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 solvent (model control group) on the ipGTT performed 48 hours after the first administration of the high-fat diet-induced obese C57BL mice or normal mice (normal control) is shown.

[0118] Figure 7a The hypoglycemic effects of the title compound of Example 2, the title compound of Control Example 2, and the solvent on db / db mice are shown.

[0119] Figure 7b and Figure 7a Correspondingly, the ΔAUC of the hypoglycemic effects of the title compound of Example 2, the title compound of Control Example 2, and the solvent on db / db mice is shown.

[0120] Figure 7c The effects of the title compound of Example 2, the title compound of Control Example 2, and the solvent on the control of food intake in db / db mice are shown.

[0121] Figure 7d The effects of the title compound of Example 2, the title compound of Control Example 2, and the solvent on water intake control in db / db mice are shown.

[0122] Figure 8a The long-term hypoglycemic effects of the title compound of Example 11, the title compound of Control Example 2, and the solvent on db / db mice are shown.

[0123] Figure 8b and Figure 8a Correspondingly, the AUC of the long-term hypoglycemic effects of the title compound of Example 11, the title compound of Control Example 2, and the solvent on db / db mice are shown.

[0124] Figure 8c The effects of the title compound of Example 11, the title compound of Control Example 2, and the solvent on long-term weight loss in db / db mice are shown.

[0125] Figure 8d The effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the solvent pair on the long-term control of food intake in db / db mice are shown.

[0126] Figure 8e The effects of the title compound of Example 11, the title compound of Control Example 2, and the solvent on long-term water intake control in db / db mice are shown.

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

[0128] Figure 9b and Figure 9a Correspondingly, the AUC of the hypoglycemic effects of the title compound of Example 11, the title compound of Example 2, dulaglutide, and solvent on Kkay mice are shown.

[0129] Figure 9c The effects of the title compound of Example 11, the title compound of Example 2, dulaglutide, and solvent on HbA1c reduction in Kkay mice are shown.

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

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

[0132] Figure 10c Random blood glucose levels are shown before, after the third, fifth, and eleventh injections of the title compound of Example 11 of the present invention, dulaglutide, and solvent (model control group) in db / db mice or normal mice (normal control).

[0133] Figure 10d The hypoglycemic effect of the title compound, dulaglutide, and solvent (model control group) of Example 11 of the present invention on ipGTT performed 48 hours after the first administration to db / db mice or normal mice (normal control group).

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

[0135] Figure 11a The results of Example 11 of this invention demonstrate the long-term weight loss effects of the title compound, dulaglutide, and solvent (model control group) on obese C57BL mice or normal mice (normal control group) induced by a high-fat diet.

[0136] Figure 11bThe effects of the title compound, dulaglutide, and solvent (model control group) of Example 11 of this invention on the long-term food intake of obese C57BL mice induced by a high-fat diet are shown.

[0137] Figure 11c The effects of the title compound, dulaglutide, and solvent (model control group) of Example 11 of this invention on reducing periovarian fat in obese C57BL female mice induced by a high-fat diet are shown.

[0138] Figure 11d The effects of the title compound, dulaglutide, and solvent (model control group) of Example 11 of this invention on reducing epididymal fat in obese C57BL male mice induced by a high-fat diet are shown. Detailed Implementation

[0139] definition

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

[0141] As used herein, the terms “GLP-1 analog” or “GLP-1 analogue” refer to peptides or compounds that are variants of human glucagon-like peptide-1 (GLP-1(7-37)) wherein one or more amino acid residues of GLP-1(7-37) are substituted, or one or more amino acid residues are deleted, 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. Peptides having the sequence shown in SEQ ID NO: 1 may also be referred to as “natural” GLP-1 or “natural” GLP-1(7-37).

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

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

[0144] In the case of GLP-1 peptides or their analogues, the term "derivative" as used herein refers to a chemically modified GLP-1 peptide or its analogue in which one or more substituents are covalently linked to the peptide. Substituents may also be referred to as side chains.

[0145] Unless otherwise stated, when referring to acylation with lysine residues, it is understood to refer to acylation with its ε-amino group.

[0146] The GLP-1 derivative of formula (B) of the present invention may exist in different stereoisomers having the same molecular formula and linked atomic sequences, but differing only in their three-dimensional orientation in atomic space. Unless otherwise stated, the present invention relates to all stereoisomers of the claimed derivatives.

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

[0148] In one specific embodiment, the peptide is largely or primarily composed of amino acids interconnected by amide bonds (e.g., at least 50%, 60%, 70%, 80%, or at least 90% by molar mass). In another specific embodiment, the peptide is composed of amino acids interconnected by peptide bonds.

[0149] Amino acids are molecules containing amino and carboxylic acid groups, and optionally one or more additional groups, commonly referred to as side chains.

[0150] The term "amino acid" encompasses protein-derived amino acids (encoded by the genetic code, including natural and standard amino acids), as well as non-protein-derived amino acids (not found in proteins and / or not encoded in the standard genetic code) and synthetic amino acids. Non-protein-derived amino acids are portions of peptides that can be integrated into the peptide via peptide bonds, but are not protein-derived amino acids. Synthetic non-protein-derived amino acids include amino acids produced through 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, deaminated histidine, β-analytes of amino acids such as β-alanine, D-histidine, deaminated histidine, 2-aminohistidine, β-hydroxyhistidine, and homohistidine, etc.

[0151] Non-limiting examples of amino acids not encoded by the genetic code are γ-carboxyglutamic acid, ornithine, D-alanine, D-glutamine, and phosphoserine. Non-limiting examples of synthetic amino acids are 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 propionate, abbreviated Imp).

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

[0153] Pharmaceutically acceptable salts, amides or esters

[0154] The GLP-1 derivatives, analogs, and intermediates of this invention can be in the form of pharmaceutically acceptable salts, amides, or esters. The salts can be basic, acidic, or neutral salts. In water, the basic salts produce hydroxide ions, and the acidic salts produce hydrated hydrogen ions. Salts of the derivatives of this invention can be formed by adding cations or anions that react with anionic or cationic groups, respectively. These groups can be located within the peptide moiety and / or within the side chain of the derivatives of this invention.

[0155] Non-limiting examples of anionic groups in the derivatives of this invention include side chains (if present) and free carboxyl groups in the peptide moiety. The peptide moiety typically comprises a C-terminal free carboxylic acid, and may also include free carboxyl groups on internal acidic amino acid residues such as Asp and Glu.

[0156] Non-limiting examples of cationic groups in peptide moieties include N-terminal free amino groups (if present) and any free amino groups on internal basic amino acid residues such as His, Arg, and Lys.

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

[0158] The amides of the derivatives of this invention can be generated, for example, by reacting a free carboxylic acid group with an amine or a substituted amine, or by reacting a free or substituted amino group with a carboxylic acid. The formation of the amide may involve a free carboxyl group at the C-terminus of the peptide, any free carboxyl group in the side chain, a 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 one 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 having an amide group at the C-terminus of the peptide. In a still further specific embodiment, the peptide or derivative is in the form of a pharmaceutically acceptable ester.

[0160] The methods for preparing the GLP-1 (7-37) peptide and GLP-1 analogs of the present invention are well known in the art. For example, the GLP-1 peptide moiety (or fragment thereof) of the derivatives 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 improved 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 W.C. Chan and PD. White, Oxford University Press, 2000.

[0161] In one embodiment, the complete GLP-1 analogue of the present invention, such as the [Gly8, Arg34]GLP-1-(7-37) peptide, can be produced by a recombinant method, i.e., by culturing host cells containing the DNA sequence encoding the analogue and capable of expressing the peptide in a suitable nutrient medium under conditions that allow for peptide expression. 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 economic reasons.

[0162] The fusion protein inclusion bodies containing the GLP-1 compound backbone were denatured and renatured to obtain a fusion protein with the correct conformation. After a series of treatments including enzyme digestion, sedimentation, and centrifugation, a high-content GLP-1 compound backbone was obtained. This backbone was then purified by ion exchange chromatography to obtain a high-purity GLP-1 compound backbone.

[0163] The term "excipient" broadly refers to any ingredient other than the active therapeutic ingredient. Excipients can be inert, inactive, and / or non-pharmaceutical active substances.

[0164] Excipients can be used for a variety of purposes, such as as carriers, solvents, diluents, tablet additives, and / or to improve administration and / or absorption of active substances.

[0165] The formulation of pharmaceutical active ingredients with different 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 include solvents, diluents, buffers, preservatives, isotonic agents, chelating agents, and stabilizers.

[0167] The GLP-1 derivatives and analogs of the present invention possess GLP-1 activity. GLP-1 activity refers to the ability to bind to the GLP-1 receptor and trigger signal transduction pathways to produce insulin-promoting effects or other physiological effects.

[0168] In one specific implementation, potency, efficacy, and / or activity refer to in vitro efficacy, i.e., performance in a functional GLP-1 receptor assay, and more particularly the ability to stimulate cAMP formation in cell lines expressing a cloned human GLP-1 receptor.

[0169] In another specific embodiment, the derivatives of the present invention are potent in vivo and can be measured in any suitable animal model and in clinical trials in a manner known in the art. For example, diabetic db / db mice are an example of a suitable animal model in which a glycemic-lowering effect can be measured, for example, as described in the Embodiments section of the present invention.

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

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

[0172] Here, the term "insulin derivative" refers to naturally occurring insulin or insulin analogues that have been chemically modified, such modification being, for example, the introduction of side chains at one or more positions on the insulin backbone, the oxidation or reduction of groups on amino acid residues of insulin, the conversion of free carboxyl groups into ester groups, or the acylation of free amino or hydroxyl groups. The acylated insulin of this invention belongs to the category of insulin derivatives.

[0173] The term "parent insulin" refers to the insulin portion of an insulin derivative or acylated insulin (also referred to herein as parent insulin), for example, in this invention, it refers to the portion of acylated insulin without the additional acyl group. Parent insulin can be naturally occurring insulin, such as human insulin or porcine insulin. Alternatively, parent insulin can be an insulin analogue.

[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 thiol groups. More precisely, an amino acid residue may be called an amino acid.

[0175] Unless otherwise stated, all amino acids mentioned in this article are L-amino acids.

[0176] Here, the term alkylene glycol includes both oligoalkylene glycol moieties and monoalkylene glycol moieties. Monoalkylene glycols and polyalkylene glycols include chains based on, for example, monoalkylene glycol, monoalkylene glycol, and monoalkylene glycol, i.e., chains based on repeating units -CH2CH2O-, -CH2CH2CH2O-, or -CH2CH2CH2CH2O-. Alkylene glycol moieties can be monodisperse (with a well-defined length / molecular weight) and polydisperse (with a less well-defined length / average molecular weight). Monoalkylene glycol moieties include -OCH2CH2O-, -OCH2CH2CH2O-, or -OCH2CH2CH2CH2O- containing different groups at each end.

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

[0178] Here, the term "aliphatic diacid" includes straight-chain or branched aliphatic dicarboxylic acids that have at least two carbon atoms and are saturated or unsaturated. Non-limiting examples of aliphatic diacids include adipic acid, octanoic acid, sebacic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, and tetradecanoic acid.

[0179] In this document, insulin or GLP-1 compounds are named according to the following principles: they are named according to mutations and modifications relative to human insulin (e.g., acylation), or mutations and modifications of native GLP-1 (7-37) (e.g., acylation). For the acyl moiety, nomenclature follows IUPAC nomenclature and, in other cases, peptide nomenclature. For example, the following acyl moieties are named:

[0180]

[0181] It can be named, for example, "eicosanodiacyl-γGlu-OEG-OEG", "eicosanodiacyl-γGlu-2xOEG", or "eicosanodiacyl-gGlu-2xOEG", "19-carboxynonadecanyl-γGlu-2xOEG", or "19-carboxynonadecanyl-γGlu-OEG-OEG", where OEG represents the abbreviation for the group -NH(CH2)2O(CH2)2OCH2CO- (i.e., 2-[2-(2-aminoethoxy)ethoxy]acetyl), and γGlu (and gGlu) are abbreviations for the L-configured amino acid γ-glutamic acid. Alternatively, the acyl moiety can be named according to the IUPAC nomenclature (OpenEye, IUPAC format). According to this nomenclature, the above-mentioned acyl group of the present invention is referred to by the following name: "[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(19-carboxy-nonadecanylamino)butyryl]-amino]-ethoxy]-ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]" or "[2-(2-[2-(2-[2-(2-[4-(19-carboxy-nonadecanylamino)-4(S)-carboxy-butyrylamino]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)-eicosanodicyl-γGlu-5xOEG),desB30 human insulin”, “B29K(Nε-eicosanodicyl-γGlu-5xOEG),desB30 human insulin”, or “B29K(Nε-eicosanodicyl-gGlu-5xOEG),desB30 human insulin” to indicate that the amino acid K at position B29 in human insulin has been modified by acylation of the ε nitrogen (referred to as Nε or (N(ε)) of the lysine residue at B29 with the residue eicosanodicyl-gGlu-2xOEG, and the amino acid T at position B30 in human insulin has been deleted. As another example, the insulin of Control Example 5 (having the sequence / structure given below) The structure is referred to as "A14E,B16H,B25H,B29K(Nε-eicosanodicyl-gGlu-2xOEG),desB30 human insulin" or "A14E,B16H,B25H,B29K(N(ε)-eicosanodicyl-γGlu-2xOEG),desB30 human insulin", indicating that amino acid Y at position A14 in human insulin has been mutated to E, amino acid Y at position B16 in human insulin has been mutated to H, amino acid F at position B25 in human insulin has been mutated to H, amino acid K at position B29 in human insulin has been modified by acylation of the ε nitrogen (called Nε) of the lysine residue at B29 with the residue eicosanodicyl-gGlu-2xOEG, and amino acid T at position B30 in human insulin has been deleted.

[0183]

[0184] In this article, "nxPEG" represents -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. It consists of two polypeptide chains, A and B, linked by two interchain disulfide bonds. Furthermore, chain A is characterized by having one intrachain disulfide bond.

[0186] There are three main methods for preparing human insulin in microorganisms. Two involve *Escherichia coli*, one by expressing a fusion protein in the cytoplasm (Frank et al. (1981) *Peptides: Proceedings of the 7th American Peptide Chemistry Symposium* (Rich & Gross, eds.), Pierce Chemical Co., Rockford, III. pp. 729-739), and the other by using a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78: 5401-5404). The third method uses *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 Escherichia coli or Saccharomyces cerevisiae, see, for example, U.S. Patent Nos. 5,962,267, WO95 / 16708, EP0055945, EP0163529, EP0347845 and EP0741188.

[0187] The construction, expression, processing, and purification of insulin analog vectors can be performed using techniques well-known to those skilled in the art. For example, insulin analogs can be prepared by expressing a DNA sequence encoding the target insulin analog in a suitable host cell using well-known techniques disclosed in U.S. Patent No. 6,500,645. Insulin analogs can also be prepared, for example, by methods reported in the following literature: Glendorf T, AR, Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chainα-Helix for Receptor Binding; Biochemistry 2008 474743-4751. This paper uses overlap-extension PCR to introduce mutations into an insulin-encoding vector. Insulin analogs are expressed as pre-insulin-like fusion proteins with a small Ala-Ala-Lys C-peptide in *Saccharomyces cerevisiae* strain MT663. The single-chain precursor is enzymatically converted to a double-chain desB30 analog using hydrolytic *Achromobacterium lyticus* endonuclease.

[0188] The isolated insulin analogue can be acylated at the desired location using acylation methods known in the art, examples of which have been described in, for example, Chinese patent applications with publication numbers CN1029977C, CN1043719A and CN1148984A.

[0189] The nucleic acid sequences encoding the various insulin analog polypeptides can be synthesized by established standard methods, such as those described by Beaucage et al. (1981) Tetrahedron Letters 22: 1859-1869, or by Mattes et al. (1984) EMBO Journal 3: 801-805.

[0190] The present invention will be further illustrated by the following embodiments. It should be noted that these embodiments do not constitute a limitation on the scope of protection of the present invention.

[0191] Example

[0192] Abbreviations

[0193] cAMP is cyclic adenosine monophosphate;

[0194] BHK is a kidney cell from a young hamster;

[0195] DNA is deoxyribonucleic acid;

[0196] Na2HPO4 is disodium hydrogen phosphate;

[0197] NaOH is sodium hydroxide;

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

[0199] OSu is succinimide-1-yloxy-2,5-dioxo-pyrrolidine-1-yloxy;

[0200] OtBu is tert-butyl oxygen;

[0201] HCl is hydrogen chloride;

[0202] γGlu or gGlu is γL-glutamyl;

[0203] NHS stands for N-hydroxysuccinimide;

[0204] DCC stands for dicyclohexylcarbodiimide;

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

[0206] OH is the hydroxide ion;

[0207] Gly is glycine;

[0208] Arg is arginine;

[0209] TFA stands for trifluoroacetic acid;

[0210] HbA1c is glycated hemoglobin.

[0211] Example 1

[0212] Title compound: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanylamino)-4(S)-carboxybutyrylamino]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-Carboxynonadecanylamino)-4(S)-Carboxybutyrylamino] [Gly8,Arg34]GLP-1-(7-37) peptide preparation

[0215] The [Gly8,Arg34]GLP-1-(7-37) peptide was prepared using a standard recombinant protein expression method (see *Molecular Cloning: A Laboratory Manual (Fourth Edition)*, Michael R. Green, ColdSpring Harbor Press, 2012 for details). The [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. The pH was adjusted to 10–12.5 with 1 N NaOH. Tert-butyleicosanoyl-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. The precipitate was separated by centrifugation and lyophilized. The crude product was added to a mixture of trifluoroacetic acid (60 mL) and dichloromethane (60 mL), and stirred at room temperature for 30 minutes. The mixture was concentrated to approximately 30 mL and poured into ice-cold n-heptane (300 mL). The precipitate was separated by filtration and washed twice with n-heptane. After vacuum drying, the product was purified by ion exchange chromatography (RessourceQ, 0.25%–1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5) and reversed-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 lyophilized to give the title compound.

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

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

[0218] 2.1 tert-butyleicosanoyl-OSu

[0219] Under nitrogen protection, tert-butyl eicosanoate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane (400 mL), and triethylamine (13.95 mL) was added. The resulting turbid mixture was stirred at room temperature, and then DCC (11.39 g, 55.19 mmol) was added, followed by further stirring overnight. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and separated. The upper organic phase was washed with saturated brine, separated again, and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure to almost dryness, then dried under vacuum overnight to give 24.12 g (97% yield) of tert-butyleicosanoyl-OSu.

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

[0221] 2,2-tert-butyleicosicodiayl-γGlu-OtBu

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

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

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

[0225] Under nitrogen protection, tert-butyleicosicodiacyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added, and the mixture was 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. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and separated. The upper organic phase was washed with saturated brine, separated again, and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure to almost dryness. Methyl tert-butyl ether was added, and the mixture was stirred for 30 minutes. The mixture was then filtered, and the filter cake was dried under vacuum overnight to give 25.76 g (81% yield) of tert-butyleicosicodiacyl-γGlu-(OSu)-OtBu.

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

[0227] 2,4-tert-butyleicosicodiacyl-γGlu-(2xOEG-OH)-OtBu

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

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

[0230] 2,5-tert-butyleicosicodiacyl-γGlu-(2xOEG-OSu)-OtBu.

[0231] Under nitrogen protection, tert-butyleicosicodiacyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added, and the mixture was stirred for 10 minutes. Then, 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. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and separated. The upper organic phase was washed with saturated brine, separated again, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to almost dryness under reduced pressure and dried under vacuum overnight to give 31.09 g (91% yield) of tert-butyleicosicodiacyl-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)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide (compound 2)

[0235]

[0236] N-ε was prepared using steps similar to those in Part 1 of Example 1. 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide

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

[0238] intermediate tert-butyleicosicodiacyl-γGlu-(OEG-OSu)-OtBu The preparation was carried out using steps similar to those in Part 2 of Example 1.

[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 using steps similar to those in Part 1 of Example 1 N-ε 26 -(19-Carboxynonadecanylamino)-4(S)-carboxyl Butyryl-[Gly8,Arg34]GLP-1-(7-37) peptide

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

[0245] intermediate tert-butyleicosicodiacyl-γGlu-(OSu)-OtBu The preparation was carried out using steps similar to those in Part 2 of Example 1.

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

[0247] Example 4

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

[0249]

[0250] Prepared using steps similar to those in Part 1 of Example 1 N-ε 26 -(19-Carboxynonadecanylamino)-4(S)-carboxyl Butyryl-[Arg34]GLP-1-(7-37) peptide

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

[0252] intermediate tert-butyleicosicodiacyl-γGlu-(OSu)-OtBu The preparation was carried out using steps similar to those in Part 2 of Example 1.

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

[0254] Compare with Example 1

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

[0256] Compare with Example 2

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

[0258] Compare with Example 3

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

[0260]

[0261] Prepared using steps similar to those in Part 1 of Example 1 N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyl decadecyl)] [Heptaneacylamino]-4(S)-Carboxybutyrylamino]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] Compare with Example 4

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

[0266]

[0267] Prepared using steps similar to those in Part 1 of Example 1 N-ε 26 -(17-Carboxyheptadecanoylamino)-4(S)-Carboxyl Butyryl-[Gly8,Arg34]GLP-1-(7-37) peptide

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

[0269] intermediate tert-Butyloctadecanoyl-γGlu-(OSu)-OtBu The preparation was carried out using steps similar to those in Part 2 of Example 1.

[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 demonstrate the regulatory effect of the GLP-1 derivative of this invention on hyperglycemia (BG) in diabetic patients.

[0273] In a single-dose study, the title compounds (also referred to as GLP-1 derivatives) of Examples 1-4 and Controls 1-4 were tested in an obese type 2 diabetes mellitus (T2DM) mouse model (db / db mice). The glycemic efficacy of the GLP-1 derivatives was tested at different doses of 100 μg / kg.

[0274] Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in appropriately sized enclosures within a barrier environment, with free access to standard food and purified water. Environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22℃-24℃. After an acclimatization period of 1-2 weeks, they were used in experiments.

[0275] Before the start of the experiment, basal blood glucose was assessed at approximately 9:30 AM, and mice were weighed. Mice were randomly assigned to either the solvent group or the treatment group based on their blood glucose and body weight, and received either a subcutaneous injection of the solvent or a subcutaneous injection of a GLP-1 derivative at a pH of 100 μg / kg, wherein the solvent contained 14 mg / ml propylene glycol, 5.5 mg / ml phenol, and 1.133 mg / ml disodium hydrogen phosphate, and the solvent pH was 8.12.

[0276] The GLP-1 derivative was dissolved in a solvent to a dosage concentration of 20 μg / ml, and the administration volume was 5 ml / kg (i.e., 50 μl / 10 g body weight). Subcutaneous administration was administered via a single subcutaneous injection into the neck and back. The corresponding GLP-1 derivative was administered at approximately 10:30 AM (time 0). Animals had free access to food and water during the administration period. Blood glucose levels in mice were assessed at 2, 4, 6, 8, 10, 12, 24, 48, and 72 hours post-administration. The tail of the mice was cleaned with an alcohol swab, and blood drops were collected from the tail using a disposable lancet. Blood glucose was measured using a blood glucose meter and accompanying test strips (Roche). Food intake and body weight were measured at 24, 48, and 72 hours post-administration.

[0277] Using baseline blood glucose levels before administration as a benchmark, the percentage of blood glucose at each monitoring point after administration was compared to this baseline to obtain the corresponding time point. A dose-response curve of blood glucose percentage versus time was plotted for each single dose of the GLP-1 derivative. To quantitatively illustrate the effect of the GLP-1 derivative on blood glucose, the area under the curve (AUC) of blood glucose percentage versus time was calculated for each individual dose-response curve from 0 to 72 hours. 0-72h AUC is the area under the time-glucose percentage curve. The smaller the AUC value, the better the blood sugar lowering effect and the better the drug efficacy.

[0278] Figures 1a-4bThe GLP-1 derivatives of the present invention have been shown to have unexpectedly increased efficacy. For example, the title compounds of Examples 1-4 showed significantly better hypoglycemic effects in db / db mice than liraglutide and the compounds of Control Examples 3-4. In particular, the compound of Example 2 of the present invention showed better hypoglycemic effects than semaglutide. Furthermore, 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 was significantly prolonged compared to liraglutide and the compounds of Control Examples 3-4, especially the compound of Example 2, which showed a longer effective duration of hypoglycemic action in db / db mice than semaglutide.

[0279] Example 6

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

[0281]

[0282] 1. Des (B30) Synthesis of 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 (5 g, 0.876 mmol) was dissolved in 100 mM Na2HPO4 aqueous solution (150 mL), and acetonitrile (100 mL) was added. The pH was adjusted to 10–12.5 with 1 N NaOH. Tert-butyleicosicodiayl-γGlu-(5xOEG-OSu)-OtBu (1.36 g, 0.964 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 1 N HCl aqueous solution. The precipitate was separated by centrifugation and lyophilized. The crude product was added to a mixture of trifluoroacetic acid (60 mL) and dichloromethane (60 mL) and stirred at room temperature for 30 minutes. The mixture was concentrated to approximately 30 mL and poured into 300 mL of ice-cold n-heptane. The precipitate 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) and reversed-phase chromatography (acetonitrile, water, TFA). The purified fractions were combined, the pH was adjusted to 5.2 with 1 N HCl, and the precipitate was separated, lyophilized, and the title compound, compound 5, was obtained.

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

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

[0288] 3.1 tert-butyleicosanoyl-OSu

[0289] Under nitrogen protection, tert-butyl eicosanoate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane, and triethylamine (13.95 mL) was added. The resulting turbid mixture was stirred at room temperature, and then DCC (11.39 g, 55.19 mmol) was added, followed by further stirring overnight. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and separated. The upper organic phase was washed with saturated brine, separated again, and dried over anhydrous sodium sulfate. The filtrate was filtered, concentrated under reduced pressure to almost dryness, and dried under vacuum overnight to give 24.12 g (97% yield) of tert-butyleicosanoyl-OSu.

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

[0291] 3.2-tert-butyleicosicodiacyl-γGlu-OtBu

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

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

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

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

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

[0297] 3,4-tert-butyleicosicodiacyl-γGlu-(2xOEG-OH)-OtBu

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

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

[0300] 3,5-tert-butyleicosicodiacyl-γGlu-(2xOEG-OSu)-OtBu.

[0301] Under nitrogen protection, tert-butyleicosicodiacyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added, and the mixture was stirred for 10 minutes. Then, 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. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and separated. The upper organic phase was washed with saturated brine, separated again, and dried over anhydrous sodium sulfate. The filtrate was filtered, concentrated under reduced pressure to almost dryness, and dried under vacuum overnight to give 31.09 g (91% yield) of tert-butyleicosicodiacyl-γGlu-(2xOEG-OSu)-OtBu.

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

[0303] 3,6-tert-butyleicosicodiayl-γGlu-(5xOEG-OH)-OtBu

[0304] 31.09 g (32.01 mmol) of tert-butyleicosicodiayl-γGlu-(2xOEG-OSu)-OtBu was dissolved in 350 mL of dichloromethane and stirred. Then, 3xAEEA (14.52 g (32.01 mmol)), triethylamine (8.90 mL), and water (25 mL) were added sequentially. The mixture was heated to obtain a clear solution, which was then stirred at room temperature for 4 hours. A 10% citric acid aqueous solution (200 mL) was then added. The mixture was separated, and 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 under reduced pressure to almost dryness and then dried under vacuum overnight. 38.99 g (93% yield) of tert-butyleicosicodiayl-γGlu-(5xOEG-OH)-OtBu was obtained.

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

[0306] 3,7-tert-butyleicosicodiacyl-γGlu-(5xOEG-OSu)-OtBu

[0307] Under nitrogen protection, tert-butyleicosicodiacyl-γGlu-(5xOEG-OH)-OtBu (38.99 g, 29.77 mmol) was dissolved in dichloromethane (400 mL), followed by the addition of triethylamine (8.28 mL) and stirring for 10 minutes. Then, 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. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and separated. The upper organic phase was washed with saturated brine, separated again, and dried over anhydrous sodium sulfate. The filtrate was filtered, concentrated under reduced pressure to almost dryness, and dried under vacuum overnight to give 38.11 g (91% yield) of tert-butyleicosicodiacyl-γGlu-(5xOEG-OSu)-OtBu.

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

[0309] Example 7:

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

[0311]

[0312] Compound 6 was prepared using steps similar to those in Part 2 of Example 6.

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

[0314] intermediate tert-butyleicosicodiacyl-γGlu-(6xOEG-OSu)-OtBu The steps are similar to those in Part 3 of Example 6.

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

[0316] Example 8:

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

[0318]

[0319] Compound 7 was prepared using steps similar to those in Part 2 of Example 6.

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

[0321] intermediate tert-butyleicosanoyl- γ Glu-(8xOEG-OSu)-OtBu The steps are similar to those in Part 3 of Example 6.

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

[0323] Example 9:

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

[0325]

[0326] Compound 8 was prepared using steps similar to those in Part 2 of Example 6.

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

[0328] intermediate tert-Butyl docosanodiacyl-γGlu-(6xOEG-OSu)-OtBu The preparation was carried out using steps similar to those in Part 3 of Example 6.

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

[0330] Example 10:

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

[0332]

[0333] Compound 9 was prepared using steps similar to those in Part 2 of Example 6.

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

[0335] intermediate tert-butyldocodiyl- γ Glu-(8xOEG-OSu)-OtBu The preparation was carried out using steps similar to those in Part 3 of Example 6.

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

[0337] Example 11:

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

[0339]

[0340] N-ε was prepared using steps similar to those in Part 1 of Example 1. 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]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] intermediate tert-Butyl docosanodiacyl-γGlu-(2xOEG-OSu)-OtBu The preparation was carried out using steps similar to those in Part 2 of Example 1.

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

[0344] Example 12: In vitro potency or activity

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

[0346] GLP-1R-expressing cells were revived and seeded into 25 mL cell culture flasks using Ham's-F12 medium. The cells were incubated overnight at 37°C with 5% CO2. On the day of the experiment, the title compound (compound 10) of Example 11 of this invention and liraglutide were prepared to a concentration of 150 μg / mL, and then serially 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 × 10⁻⁶ cells / mL. 5Cells / ml: Add 200 μl of cells and 200 μl of diluted sample to each well, mix well, and then transfer 100 μl to a new 96-well plate (3 replicates). Incubate for 4 hours in a cell culture incubator. Add luciferase reagent, vortex to mix, and transfer from the 96-well plate to a new 96-well white plate. Read the signal values ​​using a microplate reader. Process the data using GraphPad Prism 6 and calculate EC50. 50 The in vitro potency test was repeated four times on different dates.

[0347] Table 1: In Vitro Efficacy

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

[0349] The experimental results show 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: Pharmacodynamic experiments in high-fat diet-induced obese C57BL mice

[0351] The purpose of this study is to verify the effects of the GLP-1 derivative of the present invention on blood glucose regulation and weight loss in high-fat diet-induced obese C57BL mice.

[0352] Five-week-old C57BL mice (half male and half female), weighing 17-22g, were housed in a barrier environment in appropriately sized feeding boxes (3-5 mice / box). The high-fat diet induced group had free access to high-fat feed and purified water, while 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℃-24℃. After 10 weeks of housing, mice whose weight exceeded that of normal control mice by 30%-50% were selected for drug efficacy evaluation.

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

[0354] The GLP-1 derivative was administered subcutaneously once via the back of the neck (5 μl / g body weight). The mice were given the GLP-1 derivative at approximately 10:30 AM (time 0), and blood glucose levels were assessed at 3, 6, 24, 48, and 72 hours post-administration. Mouse body weight was also monitored daily.

[0355] For each single dose of the GLP-1 derivative, a Δglucose-time curve was plotted. Here, Δ represents the actual blood glucose level at a given time minus the baseline, where the baseline is the blood glucose level 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 difference (ΔAUC) from 0 to the monitoring endpoint was calculated. A smaller ΔAUC value indicates a better blood glucose-lowering effect and greater efficacy.

[0356] An intraperitoneal glucose tolerance test (ipGTT) was performed 48 hours after the first administration. The procedure was as follows: fasting blood glucose was measured at the tail tip at a specified time point (0 min), followed by intraperitoneal administration of glucose solution (200 mg / ml, 10 ml / kg), and blood glucose was measured at 30 min, 60 min and 120 min after the glucose load.

[0357] Clean the rat's tail with an alcohol swab, collect a blood drop from the tail using a disposable lancet, and measure the blood using a Roche blood glucose meter and the accompanying test strips.

[0358] For each single dose of the GLP-1 derivative, dose-response curves of blood glucose versus time and daily weight change versus time were plotted. To more intuitively and quantitatively illustrate the effect of the GLP-1 derivative of this invention on blood glucose, for each individual dose-response curve, the relative area under the blood glucose-time curve difference (ΔAUC) from 0 to the monitoring endpoint was calculated. A smaller ΔAUC value indicates a better hypoglycemic effect and better efficacy.

[0359] Figures 5a-6b The invention demonstrates that the GLP-1 derivatives exhibit unexpectedly enhanced pharmacological effects. For example, compound 10 of Example 11 showed no significant difference in hypoglycemic effect against high-fat diet-induced obese C57BL mice at the same dose compared to the marketed control compound semaglutide, and even showed a difference in quantified efficacy. Figure 5b and 6b It can be seen that the hypoglycemic effect of the GLP-1 derivative of the present invention is slightly better than that of semaglutide. In particular, at 72 hours after administration, the average blood glucose level of the 10 groups of compounds at the same dose was lower than that of the semaglutide group at the same dose. In addition, the hypoglycemic effect of the GLP-1 derivative of the present invention is dose-dependent; as the dose of GLP-1 of the present invention increases, its hypoglycemic effect also increases significantly.

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

[0361] Figure 5c The GLP-1 derivatives of the present invention, such as compound 10 of Example 11, have shown to have excellent weight loss effects, which are superior to those of semaglutide.

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

[0363] The purpose of this study is to demonstrate the blood glucose regulation effect of the GLP-1 derivative of the present invention in the context of diabetes.

[0364] In db / db mice, the glycemic effects of the title compound of Example 11 and the control compound liraglutide were tested at different doses of 0.3, 1, 3, 10, 30, and 100 nmol / kg, and the effect-lowering effect (ED) was calculated. 50 .

[0365] Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in appropriately sized enclosures within a barrier environment, with free access to standard food and purified water. Environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22℃-24℃. After an acclimatization period of 1-2 weeks, they were used in experiments.

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

[0367] The compound of Example 11 was administered subcutaneously (50 μl / 10 g body weight) via a single subcutaneous injection into the back of the neck. Mice were given the compound at approximately 10:00 AM (time 0), and blood glucose levels were assessed at 1, 2, 3, 6, 12, 24, 48, and 72 hours post-administration.

[0368] Clean the rat's tail with an alcohol swab, collect a blood drop from the tail using a disposable lancet, and measure the blood using a Roche blood glucose meter and the accompanying test strips.

[0369] For each single dose of the GLP-1 derivative, a dose-response curve Δglucose versus time was plotted. Δ is 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 (ΔAUC) of Δglucose from 0 to 72 hours was calculated, and the effective dose 50% (ED) was calculated against ΔAUC.50 (The GLP-1 derivative dose that produces half the response between baseline and maximum effect). The obtained EDs are shown in Table 2 below. 50 value.

[0370] Table 2: Effects of ED 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 experimental results show that the in vivo hypoglycemic effect 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 verify the effect of the GLP-1 derivative of this invention on the control of blood glucose, food intake and water intake.

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

[0376] Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in appropriately sized enclosures within a barrier environment, with free access to standard food and purified water. Environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22℃-24℃. After an acclimatization period of 1-2 weeks, they were used in experiments.

[0377] Before the start of the experiment that day, at approximately 9:00 AM, basal blood glucose was assessed and the mice were weighed. Based on random blood glucose and weight, diabetic mice were matched to either the solvent group or the treatment group and received the following treatment: subcutaneous injection of the solvent, or subcutaneous injection of the compound of Example 2 or the control compound semaglutide 100 μg / kg, wherein the solvent 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 concentration of 20 μg / ml and administered subcutaneously (50 μl / 10 g body weight) via a single subcutaneous injection into the neck and back. The compound of Example 2 was administered at approximately 10:00 AM (time 0), and mouse blood glucose levels were assessed at 1, 2, 3, 6, 12, 24, 48, and 72 hours post-administration. Blood was collected from the tail of the mice using a disposable lancet after cleaning the tail with an alcohol swab and measured using a Roche blood glucose meter and accompanying test strips. Food and water intake were measured daily.

[0379] For each single dose of the GLP-1 derivative, dose-response curves for blood glucose versus time, food intake versus time, and water intake versus time were plotted. To illustrate the effect of the GLP-1 derivative of this invention on blood glucose, for each individual dose-response curve, the area under the curve (ΔAUC) of blood glucose versus time from 0 to the monitoring endpoint was calculated. A smaller ΔAUC value indicates a better hypoglycemic effect and greater efficacy.

[0380] Figures 7a-7d The results showed that the GLP-1 derivative of the present invention exhibited an unexpectedly increased hypoglycemic effect and an inhibitory effect on increases in food intake and water consumption after administration. This further demonstrates that the title compound of Example 2 has a superior hypoglycemic effect on db / db mice compared to semaglutide at the same dose after administration. Furthermore, the title compound of Example 2 effectively controlled food intake and water consumption, with better results than semaglutide, suggesting that the GLP-1 derivative of the present invention has a better weight-loss effect.

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

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

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

[0384] Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in appropriately sized enclosures within a barrier environment, with free access to standard food and purified water. Environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22℃-24℃. After an acclimatization period of 1-2 weeks, they were used in experiments.

[0385] Before the start of the experiment, basal blood glucose was assessed at approximately 9:00 AM, and mice were weighed. Based on random blood glucose and body weight, diabetic mice were matched to either the solvent group or the treatment group and received the following treatments: subcutaneous injection of the solvent, or subcutaneous injection of GLP-1 derivatives 100 and 300 μg / kg, 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 of the solvent was 7.4.

[0386] GLP-1 derivatives were administered subcutaneously (50 μl / 10 g body weight) via subcutaneous injection into the neck and back at approximately 10:00 AM (time 0). Dosing was performed on days 0, 3, 7, 10, 13, 16, 19, 22, 25, and 28. Blood glucose levels were assessed before each administration and 72 hours after the last administration. Body weight, food intake, and water intake were measured daily from day 0 to 17. From day 17 onwards, these parameters were monitored every 3 days.

[0387] Figures 8a-8e The invention demonstrates that the GLP-1 derivatives, even after long-term administration, exhibit unexpectedly increased hypoglycemic efficacy, enhanced weight loss, and inhibition of food and water intake. Figure 8a and 8b The results showed that, compared with the same dose of semaglutide, compound 10 of Example 11 had a better hypoglycemic effect in db / db mice after long-term administration. Figures 8c-8d The results show that, compared to 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 effects and effects on inhibiting food intake and water consumption.

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

[0389] The purpose of this study is to verify the hypoglycemic effect of the GLP-1 derivative of this invention on type II diabetic Kkay mice.

[0390] Compound 10 of Example 11, compound 2 of Example 2, and the control compound dulaglutide (also known as dulaglutide) were tested in type II diabetic Kay mice. Mice were administered compounds 10 and 2 at different doses of 100 and 300 μg / kg, and dulaglutide at a dose of 600 μg / kg. The blood glucose-lowering and HbA1c-lowering effects of the GLP-1 derivative of the present invention and the control compound dulaglutide were determined.

[0391] Male Kkay mice aged 12-14 weeks were housed in appropriately sized enclosures within a barrier environment, with free access to standard food and purified water. Environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22℃-24℃. After an acclimatization period of 1-2 weeks, they were used in experiments.

[0392] Before the start of the experiment that day, at approximately 9:00 AM, basal blood glucose was assessed and the mice were weighed. Based on random blood glucose and body weight, diabetic mice were matched to either the solvent group or the treatment group and received the following treatments: subcutaneous injection of the solvent, or subcutaneous injection of 100 or 300 μg / kg of the GLP-1 derivative of this invention, or subcutaneous injection of the control compound dulaglutide 600 μg / kg. The solvent contained: propylene glycol 14 mg / ml, phenol 5.5 mg / ml, disodium hydrogen phosphate 1.133 mg / ml, pH 7.4.

[0393] The mice were administered the drug via subcutaneous injection (50 μl / 10 g body weight) into the back of the neck at approximately 10:00 AM (time 0). The GLP-1 derivative, dulaglutide, or solvent of the present invention was administered once every 2 days for 16 consecutive times. Blood glucose levels were assessed at 3 h, 6 h, 1 day, and 2 days after the first administration. HbA1c was measured by EDTA anticoagulation 48 h after the last administration.

[0394] Figures 9a-9b The invention demonstrates that the GLP-1 derivatives exhibit an unexpectedly increased hypoglycemic effect after administration, with the title compounds of Examples 11 and 2 showing significantly better hypoglycemic effects in Kkay mice than dulaglutide. Figure 9c The results show that the GLP-1 derivative of the present invention is significantly more effective than dulaglutide in reducing HbA1c in type II diabetic Kay mice.

[0395] Example 18: Pharmacokinetics

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

[0397] Pharmacokinetics of SD rats

[0398] Thirty-two SD rats, divided into eight groups (half male and half female), were randomly assigned to three groups: a low-dose group, a medium-dose group, and a high-dose group, receiving subcutaneous injections of 15, 90, and 540 μg / kg, respectively. A high-dose group received intravenous injection of 90 μg / kg of compound 10. Blood concentrations in the low-, medium-, and high-dose groups were measured before administration (0 min) and at 1, 3, 5, 8, 12, 16, 24, 36, 48, 72, 96, and 120 h post-administration. Blood concentrations in the intravenous group were measured before administration (0 min) and at 1 min, 10 min, 1, 3, 5, 8, 12, 24, 48, 72, 96, and 120 h post-administration. The pharmacokinetic parameter C was calculated using a non-compartmental model in WinNonLin v6.4 software. max T max T 1 / 2 AUC 0-tThe results of the MRT test are shown in Table 3.

[0399] Table 3: Pharmacokinetic parameters of compound 10 after subcutaneous injection in 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 concentration-time curve, MRT = mean residence time

[0402] Pharmacokinetics in cynomolgus monkeys

[0403] Twenty-four cynomolgus macaques, divided into groups of six (half male and half female), were randomly assigned to three groups: a low-dose group, a medium-dose group, and a high-dose group, receiving subcutaneous injections of 10, 60, and 360 μg / kg, respectively. A high-dose group received intravenous injection of 60 μg / kg of compound 10. Blood concentrations in the low-, medium-, and high-dose groups were measured before administration (0 min) and at 1, 3, 6, 8, 10, 12, 16, 24, 48, 72, 120, 168, and 240 h after administration. Blood concentrations in the intravenous group were measured before administration (0 min) and at 1 min, 10 min, 1, 3, 6, 8, 10, 12, 24, 48, 72, 120, 168, and 240 h after administration. Pharmacokinetic parameters were calculated using a non-compartmental model in WinNonLin v6.4 software. max T max T 1 / 2 AUC 0-t The results of the MRT test are shown in Table 4.

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

[0405]

[0406]

[0407] The experimental results above show that the GLP-1 derivative compound 10 of the present invention exhibits a long half-life and a large AUC in both rats and cynomolgus monkeys. 0-t The GLP-1 derivatives of this invention exhibit a relatively long MRT (metabolism response time). Furthermore, all GLP-1 derivatives of this invention are dose-dependent, with their efficacy increasing with increasing dosage.

[0408] Example 19

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

[0410] GLP-1 derivative formulations

[0411] Compound 10 was dissolved in a 5.68 mg / ml disodium hydrogen phosphate solution to a final concentration of 8 mg / ml. Based on the amounts of each component in the table below, auxiliary solutions containing propylene glycol and phenol were added sequentially, and the pH was adjusted to the values ​​in the table below to produce a final GLP-1 compound concentration of 2 mg / ml.

[0412] In this embodiment, the chemical stability of the formulation can be shown by the change in high molecular weight protein (HMWP) after storage at 37°C for 27 days relative to day 0, and can also be expressed 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 The experiment was conducted on a Waters TSKgel G2000SWXL (7.8*300mm), 5μm column at a column temperature of 30℃ and a sample cell temperature of 5℃, using a mobile phase at a flow rate of 0.5 ml / min. The mobile phase consisted of 300 ml isopropanol, 400 ml glacial acetic acid, and 300 ml purified water. The detection wavelength was 276 nm, and the injection volume was 25 μl. Table 5 shows the increase in HMWP after 27 days of storage at 37℃ relative to day 0.

[0415] Determination of the amount of substance

[0416] The content of impurities related to 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 eluent at a flow rate of 1.0 ml / min. Elution was performed using a mobile phase consisting of the following:

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

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

[0419] Gradients: Linear change from 75% / 25% A / B to 55% / 45% A / B from 0-5 min, linear change from 5-12 min to 50% / 50% A / B from 12-42 min to 40% / 60% A / B from 12-42 min, linear change from 42-60 min to 10% / 90% A / B from 42-60 min, linear change from 60-61 min to 75% / 25% A / B from 61-70 min, and isocratic gradient of 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 related substances after 28 days of storage at 37 °C relative to day 0.

[0421] Table 5

[0422]

[0423] As shown in the table above, the formulations exhibit good chemical stability at pH values ​​between 6.5 and 8.4, with the best chemical stability observed at pH values ​​between 7.0 and 8.0.

[0424] Example 20

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

[0426] Based on the amounts of each component in Tables 6 and 7 below, the GLP-1 derivative formulations shown in Tables 6 and 7 were prepared following procedures similar to those in Example 19. Changes in HMWP and related substances were then determined following procedures similar to those in Example 19. Tables 6 and 7 below show the changes in HMWP and related substances for different formulations of the GLP-1 derivative formulations.

[0427] Table 6

[0428]

[0429]

[0430] Table 7

[0431]

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

[0433] Compare with Example 5

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

[0435]

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

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

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

[0439] 2. Preparation of the intermediate tert-butyleicosicodiacyl-γGlu-(2xOEG-OSu)-OtBu: As in Example 1, section 3... Some similar steps are performed.

[0440] 2.1 tert-butyleicosanoyl-OSu

[0441] Under nitrogen protection, tert-butyl eicosanoate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane, and triethylamine (13.95 mL) was added. The resulting turbid mixture was stirred at room temperature, and then DCC (11.39 g, 55.19 mmol) was added, followed by further stirring overnight. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and separated. The upper organic phase was washed with saturated brine, separated again, and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure to almost dryness, then dried under vacuum overnight to give 24.12 g (97% yield) of tert-butyleicosanoyl-OSu.

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

[0443] 2,2-tert-butyleicosicodiayl-γGlu-OtBu

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

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

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

[0447] Under nitrogen protection, tert-butyleicosicodiacyl-γGlu-OtBu (27.27 g, 46.71 mmol) was dissolved in dichloromethane (300 mL), triethylamine (11.99 mL) was added, and the mixture was 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. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 min, and separated. The upper organic phase was washed with saturated brine, separated again, and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure to almost dryness. Methyl tert-butyl ether was added, and the mixture was stirred for 30 min. The mixture was then filtered, and the filter cake was dried under vacuum overnight to give 25.76 g (81% yield) of tert-butyleicosicodiacyl-γGlu-(OSu)-OtBu.

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

[0449] 2,4-tert-butyleicosicodiacyl-γGlu-(2xOEG-OH)-OtBu

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

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

[0452] 2,5-tert-butyleicosicodiacyl-γGlu-(2xOEG-OSu)-OtBu.

[0453] Under nitrogen protection, tert-butyleicosicodiacyl-γGlu-(2xOEG-OH)-OtBu (30.75 g, 35.18 mmol) was dissolved in dichloromethane (300 mL), triethylamine (9.03 mL) was added, and the mixture was stirred for 10 minutes. Then, 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. The mixture was filtered, and the filtrate was concentrated to almost dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and separated. The upper organic phase was washed with saturated brine, separated again, and dried over anhydrous sodium sulfate. The filtrate was filtered, concentrated under reduced pressure to almost dryness, and dried under vacuum overnight to give 31.09 g (91% yield) of tert-butyleicosicodiacyl-γGlu-(2xOEG-OSu)-OtBu.

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

[0455] Example 21

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

[0457]

[0458] Compounds A14E,B16H,B25H,B29K(N(ε)-eicosanodiacyl-γGlu-6xOEG),desB30 human insulin were prepared using procedures similar to those in Part 1 of Comparative Example 5.

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

[0460] The intermediate tert-butyleicosicodiacyl-γGlu-(6xOEG-OSu)-OtBu was prepared using steps similar to those in Part 2 of Comparative Example 5.

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

[0462] Example 22

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

[0464]

[0465] Compounds A14E,B16H,B25H,B29K(N(ε)-docosadicylo-γGlu-6xOEG),desB30 human insulin were prepared using steps similar to those in Part 1 of Comparative Example 5.

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

[0467] The intermediate tert-butyldocodiacyl-γGlu-(6xOEG-OSu)-OtBu was prepared using steps similar to those in Part 2 of Comparative Example 5.

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

[0469] Example 23

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

[0471]

[0472] Compounds A14E,B16H,B25H,B29K(N(ε)-eicosanodiacyl-γGlu-12xOEG),desB30 human insulin were prepared using procedures similar to those in Part 1 of Comparative Example 5.

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

[0474] The intermediate tert-butyleicosicodiacyl-γGlu-(12xOEG-OSu)-OtBu was prepared using steps similar to those in Part 2 of Comparative Example 5.

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

[0476] Example 24

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

[0478]

[0479] Compounds A14E, B16H, B25H, B29K (N(ε)-eicosadecanoyl-γGlu-12xOEG), desB30 human insulin were prepared using procedures similar to those in Part 1 of Comparative Example 5.

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

[0481] The intermediate tert-butyldocodiacyl-γGlu-(12xOEG-OSu)-OtBu was prepared using steps similar to those in Part 2 of Comparative Example 5.

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

[0483] Example 25 GLP-1 receptor binding

[0484] The purpose of this embodiment is to test the in vitro receptor-binding affinity of the GLP-1 derivative of the present invention, and how the presence of albumin may potentially affect binding. Receptor binding is a measure of the affinity of the 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 as follows: that is, by measuring their substitution from the receptor... 125 The binding affinity of I-GLP-1 was determined. To ascertain the binding of the GLP-1 derivative to albumin (HSA), low-concentration albumin (0.005% (w / v)) and high-concentration albumin (2% (w / v)) were used. The binding affinity IC50 was measured. 50 The changes indicate that the GLP-1 derivative binds to albumin, thereby predicting the potential for prolonged pharmacokinetic characteristics of the GLP-1 derivative in animal models.

[0486] For receptor binding assays with low HSA (0.005% (w / v)), add 50 μl of assay buffer to each well of the assay plate. For receptor binding assays with high HSA (2% (w / v)), add 50 μl of 8% (w / v) albumin stock solution to each well of the assay plate. Prepare test compounds with 10 mM Na₂HPO₄ at pH 7.3, and prepare 1 mM stock solutions of reference standard GLP-1 (7-37) with ultrapure water. Under 0.005% HSA conditions, dilute all test compounds and reference standards to 2 μM with assay buffer, then perform 4-fold serial dilutions for a total of 10 concentration gradients. Under 2% HSA conditions, dilute reference standard GLP-1 (7-37) to 2 μM, liraglutide to 20 μM, and compounds 10 and semaglutide to 800 μM, then perform 4-fold serial dilutions for a total of 10 concentration gradients. Add 25 μl of different concentrations of test compounds or reference standards to the appropriate wells of the assay plate. Thaw and dilute cell membrane proteins aliquots to their working concentration (40 μg / mL), and add 50 μl of the cell membrane-containing solution to each well of the assay plate. [The assay plate is then analyzed by adding 25 μl of […] to each well of the assay plate.] 125 Incubation was initiated with a 600 pM solution of 1-GLP-1. The assay plate was incubated at room temperature for 1 h. After incubation, the reaction solution was collected onto a GF / C filter plate using a cell collector, washed 6 times with wash buffer, and dried in a 50°C oven for 1 h. 50 μl of scintillation buffer was added, and the plate was blocked. Readings were performed using Microbeta2. The IC50 was calculated using nonlinear regression analysis in GraphPad Prism. 50 Values ​​are reported in nM. Each test compound is repeated at least three times. The reported value is the average of all measurements for each test compound.

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

[0488]

[0489] "Ratio" refers to [(IC 50 / nM)High HSA] / [(IC 50 [ / nM) Low HSA]

[0490] Typically, at low albumin concentrations, binding to the GLP-1 receptor should be as good as possible, which corresponds to low IC50. 50 Value. At high albumin concentrations, IC50... 50 The 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 often the desired effect, as this effect prolongs their plasma lifespan. Therefore, in high albumin levels, IC50...50 The value is usually higher than the IC50 value at low albumin levels. 50 The value corresponds to a decrease in binding to the GLP-1 receptor, which is caused by albumin binding, which competes with GLP-1 receptor binding. Therefore, a high ratio (IC50) can be used. 50 Value (high albumin) / IC 50 The target derivative (low albumin) binds well to albumin (thus confirming a long half-life) and also binds well to the GLP-1 receptor (IC50). 50 High albumin level, IC50 50 The value (low albumin) is an indication.

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

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

[0493] Following similar experimental procedures as in Example 16, long-term pharmacodynamic studies were 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. GLP-1 derivatives were administered subcutaneously (50 μl / 10 g body weight) via subcutaneous injection into the back of the neck at approximately 10:00 AM (time 0), on days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30. Blood glucose levels were assessed 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 before administration and after 48 hours of fasting followed by the 3rd, 5th, and 11th administrations. The intraperitoneal glucose tolerance test (ipGTT) was performed 48 hours after the initial drug administration. The procedure was as follows: fasting blood glucose was measured at the tail tip at a specified time point (0 min). Subsequently, glucose solution (200 mg / ml, 10 ml / kg) was administered intraperitoneally. Blood glucose was then measured at 30 min, 60 min, and 120 min after the glucose load. The rat tail was cleaned with an alcohol swab, and blood drops were collected from the tail using a disposable lancet. Blood glucose was measured using a Roche glucometer and accompanying test strips. A time-glucose curve was plotted, and the area under the time-glucose curve (AUC) was calculated.

[0494] Figures 10a-10e This demonstrates that the GLP-1 derivative of the present invention retains an unexpectedly increased hypoglycemic effect even after long-term administration. For example... Figure 10a and 10bThe results showed that, compared to dulaglutide, compound 10 of Example 11 had a superior hypoglycemic effect in db / db mice after administration. Figure 10c The results showed that, compared to dulaglutide, compound 10 had a superior hypoglycemic effect in db / db mice after long-term administration. Figures 10d-10e The results show that, compared to dulaglutide, the GLP-1 derivative of the present invention has a more significant inhibitory effect on blood glucose and is superior to the hypoglycemic effect of dulaglutide.

[0495] Example 27 Pharmacodynamic Experiment in High-Fat Diet-Induced Obese C57BL Mice

[0496] Following similar experimental procedures as in Example 13, pharmacodynamic experiments were conducted in C57BL mice that were induced to be obese by a high-fat diet, except that the control compound used was dulaglutide, and the dosage of dulaglutide was 300 μg / kg.

[0497] GLP-1 derivatives were administered subcutaneously via the back of the neck once (5 μl / g body weight), repeated every 3 days for a total of 11 doses. The GLP-1 derivative was administered at approximately 10:30 AM (time 0). Blood glucose levels were assessed at 3, 6, 9, 12, 24, 48, and 72 hours post-administration. Body weight and food intake were monitored every 3 days. Subcutaneous fat, perirenal fat, and perigenital fat were measured at the end of the experiment.

[0498] Figures 11a-11d The invention demonstrates that the GLP-1 derivative has unexpectedly enhanced weight loss, diet control, and lipid-lowering effects.

[0499] Example 28

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

[0501]

[0502] Compound B29K(N(ε)-docosadicyloyl-γGlu-12xOEG),desB30 human insulin was prepared using steps similar to those in Part 2 of Example 6.

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

[0504] intermediate tert-Butyl docosanodiacyl-γGlu-(12xOEG-OSu)-OtBu The steps are similar to those in Part 3 of Example 6.

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

[0506] Example 29

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

[0508]

[0509] Compounds A14E, B16H, B25H, B29K (N(ε)-docosadicyloyl-γGlu-18xOEG), desB30 human insulin were prepared using procedures similar to those in Part 1 of Comparative Example 5.

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

[0511] The intermediate tert-butyldocodiacyl-γGlu-(18xOEG-OSu)-OtBu was prepared using steps similar to those in Part 2 of Comparative Example 5.

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

[0513] Example 30

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

[0515]

[0516] Compounds A14E, B16H, B25H, B29K (N(ε)-eicosanodiacyl-γGlu-24xOEG), desB30 human insulin were prepared using steps similar to those in Part 1 of Comparative Example 5.

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

[0518] The intermediate tert-butyldocodiacyl-γGlu-(24xOEG-OSu)-OtBu was prepared using a similar procedure to that in Part 2 of Comparative Example 5.

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

[0520] Example 31

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

[0522]

[0523] Compound B29K(N(ε)-docosadicyloyl-γGlu-OEG),desB30 human insulin was prepared using steps similar to those in Part 2 of Example 6.

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

[0525] intermediate tert-Butyl docosanodiacyl-γGlu-(OEG-OSu)-OtBu The steps are similar to those in Part 3 of Example 6.

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

[0527] Example 32

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

[0529]

[0530] Compound B29K(N(ε)-docosadicyloyl-γGlu-12xPEG),desB30 human insulin was prepared using steps similar to those in Part 2 of Example 6.

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

[0532] intermediate tert-Butyl docosanodiacyl-γGlu-(12xPEG-OSu)-OtBu The steps are similar to those in Part 3 of Example 6.

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

[0534] The present invention has been described through the above embodiments. However, it should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and many more variations and modifications can be made based on the teachings of the present invention, all of which fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended 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 [Gly8, Arg34]GLP-1-(7-37) peptide (SEQ ID NO: 2), [Acy-(L1)] r -(L2) q ] is a substituent on the ε-amino group of the Lys residue at position 26 of the [Gly8,Arg34]GLP-1-(7-37) peptide, wherein, r is 1, q is 1 or 2; Acy is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 19 -CO-, or HOOC-(CH2) 20 -CO; L1 is the amino acid residue γGlu; L2 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-; Acy, L1, and L2 are connected by amide bonds; and In formula (B), Acy, L1, and L2 are connected sequentially by amide bonds, and the C-terminus of L2 is connected to the ε-amino group of the Lys residue at position 26 of the [Gly8,Arg34]GLP-1-(7-37) peptide.

2. The compound of claim 1, wherein, q is 2.

3. The compound of claim 1, wherein, Acy is HOOC-(CH2) 18 -CO- or HOOC-(CH2) 20 -CO-.

4. The compound of claim 1, wherein, In formula B, Acy is HOOC-(CH2). 18 -CO-.

5. The compound of claim 1, wherein, In formula B, Acy is HOOC-(CH2). 20 -CO-.

6. Use of the compound according to any one of claims 1-5 in the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

7. The use as described in claim 6, wherein the compound is selected from the following compounds: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide, and N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide.

8. The use as described in claim 6, wherein the compound is selected from the following compounds: ;and 。 9. The use as described in claim 6, wherein the compound is one of the following compounds: 。 10.N-ε 26 Use of -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide in the preparation of medicaments for the treatment of type 2 diabetes and / or obesity; in, The amino acid sequence of the [Gly8, Arg34]GLP-1-(7-37) peptide is shown in SEQ ID NO:

2.

11. The use of the following compounds in the preparation of medicaments for the treatment of type 2 diabetes, 。 12. The use of the following compounds in the preparation of medicaments for the treatment of obesity, 。 13. A pharmaceutical formulation comprising the compound of any one of claims 1-5 and a pharmaceutically acceptable excipient.

14. The pharmaceutical preparation of claim 13, wherein, The pharmaceutically acceptable excipient is selected from one or more of buffers, preservatives, isotonic agents, stabilizers, and chelating agents.

15. The pharmaceutical preparation of claim 14, wherein, The pharmaceutically acceptable excipients are selected from buffers, preservatives, and isotonic agents.

16. The pharmaceutical formulation of claim 14, wherein the isotonic agent is selected from one or more of sodium chloride, propylene glycol, mannitol, sorbitol, glycerol, glucose, and xylitol; and / or The preservative is selected from one or more of phenol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, butylparaben, 2-phenylethanol, and benzyl alcohol; 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.

17. The pharmaceutical preparation of claim 16, wherein, The isotonic agent is propylene glycol, mannitol, or sodium chloride; and / or The preservative is phenol or m-cresol; and / or The buffer is sodium acetate, citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate.

18. The pharmaceutical preparation according to any one of claims 14-17, wherein, The pH of the formulation is 6.0 to 10.

0.

19. The pharmaceutical preparation of claim 18, wherein, The pH of the formulation is 6.5 to 10.

0.

20. The pharmaceutical preparation of claim 18, wherein, The pH of the formulation is between 6.5 and 9.

5.

21. The pharmaceutical preparation of claim 18, wherein, The pH of the formulation is between 6.5 and 8.

5.

22. The pharmaceutical preparation of claim 18, wherein, The pH of the formulation is 7.0 to 8.

5.

23. The pharmaceutical preparation of claim 18, wherein, The pH of the formulation is 7.0 to 8.

1.

24. The pharmaceutical preparation of claim 18, wherein, The pH of the formulation is 7.3 to 8.

1.

25. Pharmaceutical preparations containing the following ingredients: 0.1-1.2 mM of the compound according to any one of claims 1-5; 10-1500mM isotonic agent; 1-200 mM preservatives; A 3-35 mM buffer, wherein the buffer is selected from one or more of sodium acetate, citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate; and The pH of the pharmaceutical preparation is between 6.0 and 10.

0.

26. The pharmaceutical preparation of claim 25, comprising 0.2-1 mM of the compound of any one of claims 1-5.

27. The pharmaceutical preparation of claim 26, comprising 0.3-0.7 mM of the compound of any one of claims 1-5.

28. The pharmaceutical preparation of claim 26, comprising 0.48-0.6 mM of the compound of any one of claims 1-5.

29. The pharmaceutical preparation of claim 25, wherein it contains 13-800 mM of an isotonic agent.

30. The pharmaceutical preparation of claim 29, wherein it contains 65-400 mM of an isotonic agent.

31. The pharmaceutical preparation of claim 29, wherein it contains 90-240 mM of an isotonic agent.

32. The pharmaceutical preparation of claim 29, wherein it contains 150-250 mM of an isotonic agent.

33. The pharmaceutical preparation of claim 29, wherein it contains 180-200 mM of an isotonic agent.

34. The pharmaceutical preparation of claim 29, wherein it contains 183-195 mM of an isotonic agent.

35. The pharmaceutical preparation according to any one of claims 29-34, wherein, The isotonic agent is selected from one or more of propylene glycol, glycerin, mannitol, or sodium chloride.

36. The pharmaceutical preparation of claim 25, wherein it contains 5-150 mM of preservative.

37. The pharmaceutical preparation of claim 36, wherein it contains 10-100 mM of preservative.

38. The pharmaceutical preparation of claim 36, wherein it contains 20-85 mM of preservative.

39. The pharmaceutical preparation of claim 36, wherein it contains 30-75 mM of preservative.

40. The pharmaceutical preparation of claim 36, wherein it contains 45-60 mM of preservative.

41. The pharmaceutical preparation of claim 36, wherein it contains 50-60 mM of preservative.

42. The pharmaceutical preparation according to any one of claims 36-41, wherein, The preservative is selected from one or more of phenol or m-cresol.

43. The pharmaceutical preparation of claim 25, wherein it contains 5-20 mM of buffer.

44. The pharmaceutical preparation of claim 43, wherein it contains 5-15 mM of buffer.

45. The pharmaceutical preparation of claim 43, wherein it contains 7-10 mM of buffer.

46. ​​The pharmaceutical preparation according to any one of claims 43-45, wherein, The buffer is selected from one or more of sodium acetate, citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate.

47. The pharmaceutical preparation of claim 25, wherein, The pH of the pharmaceutical preparation is between 6.5 and 9.

5.

48. The pharmaceutical preparation of claim 47, wherein, The pH of the pharmaceutical preparation is between 6.5 and 8.

5.

49. The pharmaceutical preparation of claim 47, wherein, The pH of the pharmaceutical preparation is 7.0 to 8.

5.

50. The pharmaceutical preparation of claim 47, wherein, The pH of the pharmaceutical preparation is 7.0 to 8.

1.

51. The pharmaceutical preparation of claim 47, wherein, The pH of the pharmaceutical preparation is 7.3 to 8.

1.

52. Pharmaceutical preparations, comprising: 0.3-0.7mM N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide; 180-200mM propylene glycol; 45-60mM phenol; 5-15mM buffer; and The pH of the pharmaceutical preparation is 6.5 to 8.5; in, The amino acid sequence of the [Gly8, Arg34]GLP-1-(7-37) peptide is shown in SEQ ID NO:

2.

53. The pharmaceutical formulation of claim 52, comprising 0.48-0.6 mM of N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide.

54. The pharmaceutical formulation of claim 52, comprising 183-195 mM of propylene glycol.

55. The pharmaceutical preparation of claim 52, comprising 50-60 mM phenol.

56. The pharmaceutical preparation of claim 52, wherein, The buffer is disodium hydrogen phosphate, and the content of disodium hydrogen phosphate is 7-10 mM.

57. The pharmaceutical preparation of claim 52, wherein, The pH of the pharmaceutical preparation is 7.0 to 8.

5.

58. The pharmaceutical preparation of claim 57, wherein, The pH of the pharmaceutical preparation is 7.3 to 8.

3.

59. Pharmaceutical preparations, comprising: 0.5mM N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide; 184 mM propylene glycol; 58.5 mM phenol; 10mM disodium hydrogen phosphate; and The pH of the pharmaceutical preparation is 6.5 to 8.5; in, The amino acid sequence of the [Gly8, Arg34]GLP-1-(7-37) peptide is shown in SEQ ID NO:

2.

60. The pharmaceutical preparation of claim 59, wherein, The pH of the pharmaceutical preparation is 7.0 to 8.

5.

61. The pharmaceutical preparation of claim 59, wherein, The pH of the pharmaceutical preparation is 7.0 to 8.

1.

62. The pharmaceutical preparation of claim 59, wherein, The pH of the pharmaceutical preparation is 7.3 to 8.

1.

63. Pharmaceutical preparations, comprising: 2.0 mg / ml N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxycoteicoacylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37)peptide; 14 mg / ml of propylene glycol; 5.5 mg / ml phenol; 1.42 mg / ml of disodium hydrogen phosphate; and The pH of the pharmaceutical preparation is 6.5 to 8.5; in, The amino acid sequence of the [Gly8, Arg34]GLP-1-(7-37) peptide is shown in SEQ ID NO:

2.

64. The pharmaceutical preparation of claim 63, wherein, The pH of the pharmaceutical preparation is 7.0 to 8.

5.

65. The pharmaceutical preparation of claim 64, wherein, The pH of the pharmaceutical preparation is 7.0 to 8.

1.

66. The pharmaceutical preparation of claim 64, wherein, The pH of the pharmaceutical preparation is 7.3 to 8.

1.

67. The compound according to any one of claims 1-5, used as a medicine.

68. The compound according to any one of claims 1-5, for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

69. The pharmaceutical preparation of claim 13, used for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

70. The pharmaceutical preparation of claim 18, used for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

71. The pharmaceutical preparation of claim 25, used for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

72. The pharmaceutical preparation of claim 35, used for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

73. The pharmaceutical preparation of claim 42, used for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

74. The pharmaceutical preparation of claim 46, used for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

75. The pharmaceutical preparation of any one of claims 14-17, 19-24, 26-34, 36-41 and 43-45, for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

76. Use of the pharmaceutical preparation of any one of claims 13-66 and 69-74 in the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, and / or obesity.

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