Long-acting GLP-1 compounds

Abstract

The present invention relates to novel long-acting GLP-1 compounds, i.e., novel GLP-1 derivatives, which have comparable or better efficacy, potency, efficacy, longer or comparable duration of action or half-life in vivo, better or comparable GLP-1 receptor binding affinity, and better or comparable DPP-IV stability compared to commercially available GLP-1 derivatives such as liraglutide and semaglutide.

Description

[Technical Field]

[0001] The present invention relates to the field of therapeutic peptides, and in particular to novel long-acting GLP-1 compounds, their pharmaceutical formulations, pharmaceutical compositions thereof with long-acting insulin, and the pharmaceutical uses of said compounds, pharmaceutical formulations and pharmaceutical compositions. [Background technology]

[0002] Glucagon-like peptide 1 (GLP-1) and its analogs and derivatives are highly effective in treating type 1 and type 2 diabetes, but high clearance limits the effectiveness of these compounds. To provide GLP-1 compounds with longer duration of action in the body, a series of different methods have been applied to modify the structure of glucagon-like peptide 1 (GLP-1). For example, WO 99 / 43708 discloses GLP-1(7-35) and GLP-1(7-36) derivatives with lipophilic substituents linked to the C-terminal amino acid residue. Patent document 1 discloses acylated GLP-1 analogs. Patent document 2 discloses activated insulinotropic peptides for injection into patients. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 00 / 34331 [Patent Document 2] International Publication No. 00 / 69911 Summary of the Invention [Problem to be solved by the invention]

[0004] Currently available GLP-1 drugs include exenatide, a natural GLP-1 analogue administered twice daily; liraglutide, a once-daily GLP-1 compound modified with hexadecanoic acid; lixisenatide, a new molecule obtained by modifying and altering the structure of exenatide; semaglutide, exenatide microspheres (Exenatide LAR), abiglutide, dulaglutide (also known as Trulicity), and polyethylene glycol loxenatide, all administered once weekly. Among them, exenatide microspheres are prepared by microencapsulating exenatide in a polylactic-co-glycolic acid matrix; aviglutide is a recombinant fusion protein formed by fusing two modified GLP-1 peptide chains with human albumin in a dimeric form; dulaglutide is obtained by fusing a modified GLP-1 chain with the Fc fragment of recombinant G4 immunoglobulin via a disulfide bond; and polyethylene glycol loxenatide is a compound based on the chemical structure of exenatide, modified with polyethylene glycol through amino acid modification; and semaglutide has been administered once a week mainly by substituting the non-protein amino acid Aib for Ala at position 8 in the GLP-1 (7-37) peptide. However, because semaglutide contains non-protein amino acids, there may be various unknown potential side effects in the human body compared to natural amino acids.

[0005] On the other hand, there is still a need to develop compounds that can have better efficacy, potency or potency, a lower risk of potential side effects, better weight loss and food suppression effects, and a longer or comparable duration of action or half-life in the body compared to similar drugs on the market, such as liraglutide, dulaglutide and semaglutide, in order to provide better drug options for diabetic patients.

[0006] On the other hand, with the rapid increase in the number of type 2 diabetes patients worldwide, there is a growing demand for drugs that are easier to administer and more effective. For example, a combination formulation containing two active ingredients, insulin and GLP-1 peptide, may be a highly effective therapeutic agent. 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. [Means for solving the problem]

[0007] To overcome or ameliorate at least one deficiency in the prior art or to provide a useful alternative, a first aspect of the present invention provides a novel GLP-1 compound (also referred to as a GLP-1 derivative). Compared to commercially available GLP-1 derivatives such as liraglutide, dulaglutide, and semaglutide, the novel GLP-1 compound has better efficacy, pharmacological activity, and efficacy, a lower risk of potential side effects, a better weight loss effect, a longer duration of action or half-life in the body, better or comparable GLP-1 receptor binding affinity, and better or comparable DPP-IV stability. Furthermore, the pharmaceutical composition or combined formulation of the long-acting GLP-1 compound and long-acting insulin provided by the present invention not only does not reduce the physical stability of the GLP-1 compound and the insulin compound, but also has better physical stability than the single formulations. Compared to other combined formulations of long-acting GLP-1 compounds (e.g., combined formulations of liraglutide and insulin degludec), the physical stability of the combined formulation of the present invention is greater than expected. Furthermore, the combined preparation also improves the chemical stability of the GLP-1 compound and acylated insulin compared to the single preparations. Both the GLP-1 compound of the present invention and the combined preparation comprising the GLP-1 compound and insulin compound provided by the present invention can favorably achieve long pharmacokinetic (hereinafter also referred to as PK) characteristics, enabling subcutaneous treatment of diabetic patients twice a week, once a week, once every two weeks, or less frequently.

[0008] The GLP-1 compound provided according to the first aspect of the present invention is a compound of formula B, or a pharmaceutically acceptable salt, amide or ester thereof: [Acy-(L1) r -(L2) q ]-G1 (B), wherein G1 is a GLP-1 analog having Arg at position corresponding to position 34 of GLP-1(7-37) (SEQ ID NO: 1) and Ala or Gly at position corresponding to position 8, and is [Acy-(L1) r -(L2) q is a substituent linked to the epsilon amino group of the Lys residue at position 26 of the GLP-1 analogue, r is an integer of 1 to 10, q is 0 or an integer of 1 to 10, Acy is an aliphatic diacid containing 20 to 24 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; L1 is an amino acid residue selected from γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp, or α-D-Asp; L2 is a neutral alkylene glycol-containing amino acid residue, Acy, L1 and L2 are connected by an amide bond, and The order of appearance of L1 and L2 in formula (B) is independently interchangeable.

[0009] In one embodiment, G1 is a [Gly8, Arg34]GLP-1-(7-37) peptide or a [Arg34]GLP-1-(7-37) peptide, preferably a [Gly8, Arg34]GLP-1-(7-37) peptide. In one embodiment, r is 1, 2, 3, 4, 5 or 6, preferably r is 1, 2, 3 or 4, preferably r is 1 or 2, preferably r is 1.

[0010] 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.

[0011] In one embodiment, Acy is an aliphatic diacid containing 20 to 23 carbon atoms, preferably Acy is an aliphatic diacid containing 20, 21, or 22 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid.

[0012] In one embodiment, L2 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN- (CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2 )2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-, -HN-(CH2)3-O -(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-(CH2)2-O-( L2 is preferably -HN-(CH2)2-O-(CH2)2-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)3-NH-CO-CH2-O-CH2-CO-, -HN-(CH2)3-O-(CH2)3-O-CH2-CO-, or -HN-(CH2)4-O-(CH2)4-O-CH2-CO-, and preferably L2 is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-.

[0013] In one embodiment, L1 is selected from γGlu or βAsp, preferably L1 is γGlu. In one embodiment, Acy is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO- or HOOC-(CH2) 22 -CO-, and preferably Acy is HOOC-(CH2) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

[0014] In one embodiment, in formula (B), Acy, L1 and L2 are sequentially linked by an amide bond, and the C-terminus of L2 is linked to the ε-amino group of the Lys residue at position 26 of the GLP-1 analog.

[0015] In one embodiment, the compound according to the first aspect of the invention is N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoiamino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(21-carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyric acid Tanoi (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26-[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(23-carboxytricosanoylamino)-4(S)-carboxybutyric acid Tanoi (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[23-carboxytricosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -(23-carboxytricosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, or N-ε 26 -(21-carboxyheneicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide.

[0016] In one embodiment, the compound according to the first aspect of the invention is N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[21-carboxyheneicosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -(22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(20-carboxyeicosanoylamino)-4(S)-carboxybutyric acid Tanoi (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[20-carboxyeicosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(22-carboxydocosanoylamino)-4(S)-carboxybutyric acid Tanoi(amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -[2-(2-[2-(4-[22-carboxydocosanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Arg34]GLP-1-(7-37) peptide, N-ε 26 -(20-carboxyeicosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, or N-ε 26 -(22-carboxydocosanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide.

[0017] In one embodiment, the compound according to the first aspect of the invention is N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8, Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi and the compound is selected from the group consisting of: (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]Gly8, Arg34]GLP-1-(7-37) peptide.

[0018] A second aspect of the invention provides a pharmaceutical formulation comprising a compound according to the first aspect of the invention and a pharmaceutically acceptable excipient.

[0019] In one embodiment, the pharmaceutically acceptable excipient is selected from one or more of a buffering agent, a preservative, a tonicity agent, a stabilizer, and a chelating agent, hi another embodiment, the pharmaceutically acceptable excipient is a buffering agent, a preservative, and a tonicity agent.

[0020] In one embodiment, the pharmaceutical formulation comprises a compound according to the first aspect of the present invention, an isotonicity agent, a preservative, and a buffer. Preferably, in the pharmaceutical formulation, the compound according to the first aspect of the present invention is N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8, Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi and GLP-1-(7-37) peptide.

[0021] In one embodiment, the tonicity agent is selected from one or more of sodium chloride, propylene glycol, mannitol, sorbitol, glycerin, glucose and xylitol, preferably propylene glycol, mannitol or sodium chloride.

[0022] In another embodiment, the preservative is selected from one or more of phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, and benzyl alcohol, preferably phenol or m-cresol.

[0023] In another embodiment, the buffering agent 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.

[0024] In one embodiment, the pH of the formulation is about 6.0 to about 10.0, preferably about 6.5 to about 10.0, preferably about 6.5 to about 9.5, preferably about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.1.

[0025] In one embodiment, the drug formulation comprises: about 0.1 to 1.2 mM, preferably about 0.2 to 1 mM, preferably about 0.3 to 0.7 mM, more preferably about 0.48 to 0.6 mM of the compound according to the first aspect of the present invention; an isotonicity agent at about 10 to 1500 mM, preferably about 13 to 800 mM, preferably about 65 to 400 mM, preferably about 90 to 240 mM, preferably about 150 to 250 mM, preferably about 180 to 200 mM, more preferably about 183 to 195 mM, preferably selected from one or more of propylene glycol, glycerin, mannitol, and sodium chloride; a preservative selected from one or more of about 1 to 200 mM, preferably about 5 to 150 mM, preferably about 10 to 100 mM, preferably about 20 to 85 mM, preferably about 30 to 75 mM, preferably about 45 to 60 mM, more preferably about 50 to 60 mM, and preferably phenol or m-cresol; a buffering agent selected from one or more of sodium acetate, citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate, at about 3 to 35 mM, preferably about 5 to 20 mM, more preferably about 5 to 15 mM, more preferably about 7 to 10 mM; and The pH of the above drug formulation is about 6.0 to about 10.0, preferably about 6.5 to about 9.5, preferably about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.1.

[0026] In another embodiment, the drug formulation has an N-ε concentration of about 0.3 to 0.7 mM, more preferably about 0.48 to 0.6 mM. 26-[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]Gly8, Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi and a buffering agent which is about 5 to 15 mM, preferably about 7 to 10 mM disodium hydrogen phosphate; and the pH of the pharmaceutical formulation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, and even more preferably about 7.3 to about 8.3.

[0027] In another embodiment, the drug formulation has about 0.5 mM N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]Gly8, Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi [(amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, about 184 mM propylene glycol, about 58.5 mM phenol, and about 10 mM disodium hydrogen phosphate, and the pH of the drug formulation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.1.

[0028] In another embodiment, the drug formulation has an N-ε of about 2.0 mg / mL26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]Gly8, Arg34]GLP-1-(7-37) peptide, or N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi

[0023] GLP-1-(7-37) peptide, about 14 mg / mL propylene glycol, about 5.5 mg / mL phenol, and about 1.42 mg / mL disodium hydrogen phosphate; and The pH of the above drug formulation is about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.1.

[0029] A third aspect of the invention provides a pharmaceutical composition comprising a GLP-1 compound according to the first aspect of the invention and an acylated insulin.

[0030] In one embodiment, the acylated insulin is B29K(N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, or B29K (N(ε)-docosand Hey Glu-γGlu-12xPEG), desB30 human insulin.

[0031] In one embodiment, the acylated insulin is an acylated insulin whose parent insulin is a naturally occurring insulin or insulin analogue and which contains at least one lysine residue, the acyl moiety being linked to the amino group of a lysine residue or the N-terminal amino acid residue of the parent insulin, said acyl moiety having the formula (A), i.e., III-(II)m -(I) n - (A), wherein m is 0 or an integer of 1 to 10, n is an integer of 5 to 20, I is a neutral amino acid residue containing alkylene glycol, II is an acidic amino acid residue, and III is an aliphatic diacid containing 20 to 24 carbon atoms, provided that in the form, a hydroxy group has been removed from one of the carboxy groups of the aliphatic diacid, III, II, and I are connected by an amide bond, and the order of appearance of II and I in formula (A) is independently interchangeable.

[0032] In one embodiment, n is 5 to 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. In another embodiment, m is 1 to 6, preferably m is 1, 2, 3 or 4, preferably m is 1 or 2, preferably m is 1.

[0033] 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, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of the aliphatic diacid.

[0034] In another embodiment, the insulin parent contains one lysine residue. In one embodiment, I is -HN-(CH2)2-O-(CH2)2-O-CH2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-( CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2) 2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-CO-, -HN-(CH2)3-O-(CH2)4-O-(CH2)3-NH-CO-, -HN-(CH2)3-O -(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-(CH2)2-O-( and preferably I is -HN-(CH)-O-(CH)-O-CH-CO-, -HN-(CH)-O-(CH)-O-(CH)-NH-CO-CH-O-CH-CO-, -HN-(CH)-O-(CH)-O-(CH)-O-(CH)-NH-CO-CH-O-CH-CO-, -HN-(CH)-O-(CH)-O-CH-CO-, or -HN-(CH)-O-(CH)-O-CH-CO-, and preferably I is -HN-(CH)-O-(CH)-O-CH-CO-.

[0035] In another embodiment, II is an amino acid residue selected from 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. In one embodiment, III is HOOC-(CH) 18-CO-, HOOC-(CH2) 19 -CO-, HOOC-(CH2) 20 -CO-, HOOC-(CH2) 21 -CO- or HOOC-(CH2) 22 -CO-, and preferably III is HOOC-(CH) 18 -CO-, HOOC-(CH2) 20 -CO- or HOOC-(CH2) 22 -CO-.

[0036] In one embodiment, Formula (A) is linked by the C-terminus of I to the amino group of the lysine residue or the N-terminal amino acid residue of the parent insulin. In one embodiment, the acyl moiety is linked to the epsilon amino group of a lysine residue of the insulin parent. In one embodiment, the parent insulin lysine residue is located at position B29. In one embodiment, the insulin parent is selected from the following insulin or insulin analogues: desB30 human insulin (SEQ ID NO:4 and SEQ ID NO:5, representing the A-chain and the B-chain, respectively), A14E, B16H, B25H, desB30 human insulin (SEQ ID NO:6 and SEQ ID NO:7, representing the A-chain and the B-chain, respectively), A14E, B16E, B25H, desB30 human insulin (SEQ ID NO:8 and SEQ ID NO:9, representing the A-chain and the B-chain, respectively), human insulin (SEQ ID NO:10 and SEQ ID NO:11, representing the A-chain and the B-chain, respectively), A21G human insulin (SEQ ID NO:12 and SEQ ID NO:13, representing the A-chain and the B-chain, respectively), A21G, desB30 human insulin (SEQ ID NO:14 and SEQ ID NO:15, representing the A-chain and the B-chain, respectively), or B28D human insulin (SEQ ID NO:16 and SEQ ID NO:17, representing the A-chain and the B-chain, respectively).

[0037] In one embodiment, the acylated insulin is B29K(N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand HeydesB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosanoid Hey desB30 human insulin, B29K (N(ε)-eicosanoid Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid HeydesB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid), desB30, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid), Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid), desB30, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid), Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand HeydesB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid Hey Leu-5xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosin Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand HeydesB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosanoid Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosanoid Hey desB30 human insulin, B29K (N(ε)-eicosanoid Hey desB30 human insulin, B29K (N(ε)-eicosand HeydesB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid), desB30, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid), Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid), desB30, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid), Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoid HeydesB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey Le-αGlu-αGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid HeydesB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey Human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid), desB30, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid), Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoid Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey Le-βAsp-6xOEG), desB30 human insulin, B29K(N(ε)-docosmid Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey le-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosmid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand HeydesB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandine), desB30, A14E, B16E, B25H, B29K (N(ε)-docosandine), Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand HeydesB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey Leu-γGlu-8xOEG), desB30 human insulin, B29K(N(ε)-docosmid Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand HeydesB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey Rudokosanji Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey Leu-γGlu-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosmid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey Human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandine), desB30 Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand HeydesB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-heteroglycoside) Ne Ikosanji Hey desB30 human insulin, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey Leu-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-he Ne Ikosanji Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji HeydesB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, B29K (N(ε)-trichosan Hey desB30 human insulin, B29K (N(ε)-trichosan Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-trichosan Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-trichosan Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosan Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosan Hey desB30 human insulin, B29K (N(ε)-trichosan Hey desB30 human insulin, B29K (N(ε)-trichosan Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-trichosan Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-trichosan HeydesB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosan Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosan Hey desB30 human insulin, B29K (N(ε)-tetracosand) Hey desB30 human insulin, B29K (N(ε)-tetracosand) Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosane Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosane Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tetracosane Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tetracosane Hey desB30 human insulin, B29K (N(ε)-tetracosand) Hey desB30 human insulin, B29K (N(ε)-tetracosand) Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosane Hey Leu-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosanol) Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tetracosane Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tetracosane HeydesB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosanoic acid Hey human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-trichosan Heyhuman insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosane Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosand Hey Leu-γGlu-9xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosin Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosanoic acid Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosan Hey desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tetracosane Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-eicosand Hey desB30 human insulin, B29K (N(ε)-docosand HeydesB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-docosand Hey desB30 human insulin, B29K (N(ε)-heteroglycoprotein), Ne Ikosanji Hey desB30 human insulin, B29K (N(ε)-trichosan Hey desB30 human insulin, B29K (N(ε)-tetracosand) Hey desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosand Hey The insulin is selected from the group consisting of insulins such as desB30 human insulin, insulin-γGlu-24xOEG, and desB30 human insulin.

[0038] The inventors have unexpectedly discovered that not only do pharmaceutical compositions of the compounds described in the first aspect of the present invention and acylated insulins not reduce the physical stability of the compounds, but that the combined formulations have superior physical stability to the single formulations. Compared with combined formulations of other long-acting insulin derivatives (e.g., insulin degludec and liraglutide), the physical stability of the combined formulations of the present invention is greater than expected. Furthermore, the combined formulations further improve the chemical stability of the acylated insulins compared to the single formulations.

[0039] A fourth aspect of the present invention provides the use of a compound according to the first aspect of the present invention, a pharmaceutical formulation according to the second aspect of the present invention or a pharmaceutical composition according to the third aspect of the present invention for use as a medicament.

[0040] In one embodiment, the compound according to the first aspect of the invention, the pharmaceutical formulation according to the second aspect of the invention or the pharmaceutical composition according to the third aspect of the invention is used for the treatment or prevention of hyperglycemia, diabetes, and / or obesity.

[0041] A fifth aspect of the invention provides the use of a compound according to the first aspect of the invention, a pharmaceutical formulation according to the second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention in the preparation of a medicament for the treatment or prevention of hyperglycemia, diabetes and / or obesity.

[0042] A sixth aspect of the present invention provides a method for treating or preventing hyperglycemia, diabetes, and / or obesity, said method comprising administering a therapeutically effective amount of a compound according to the first aspect of the invention, a pharmaceutical formulation according to the second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention, said diseases including, but not limited to, hyperglycemia, diabetes, and obesity. [Brief explanation of the drawings]

[0043] [Figure 1a] The blood glucose lowering effect and duration of action of the title compounds of Examples 1 to 3 of the present invention, liraglutide, and the vehicle in db / db mice are shown, in which the percentage on the ordinate represents the blood glucose percentage at the corresponding time point obtained by comparing the blood glucose at each monitoring point after administration with the baseline blood glucose before administration (the same applies below). [Figure 1b] FIG. 1A corresponds to FIG. 1A and shows the AUC of the hypoglycemic effect of the title compounds of Examples 1 to 3 of the present invention, liraglutide, and the solvent on db / db mice. [Figure 2a] 1 shows the hypoglycemic effect and duration of action of the title compound of Example 2 of the present invention, semaglutide, and a solvent in db / db mice. [Figure 2b] Corresponding to Figure 2a, the AUC of the hypoglycemic effect of the title compound of Example 2 of the present invention, semaglutide, and the solvent on db / db mice is shown. [Figure 3a] 1 shows the hypoglycemic effect and duration of action of the title compounds of Examples 3 and 4 of the present invention, liraglutide, and a solvent in db / db mice. [Figure 3b]FIG. 3 corresponds to FIG. 3a and shows the AUC of the hypoglycemic effect of the title compounds of Examples 3 and 4 of the present invention, liraglutide, and the solvent on db / db mice. [Figure 4a] 1 shows the hypoglycemic effect and duration of action of the title compounds of Examples 1 to 3 of the present invention, the title compounds of Control Examples 3 and 4, and the solvent in db / db mice. [Figure 4b] 4a, the AUC of the hypoglycemic effect of the title compounds of Examples 1 to 3 of the present invention, the title compounds of Control Examples 3 and 4, and the solvent in db / db mice is shown. [Figure 5a]

[0046] Figure 1 shows the hypoglycemic effect 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 high-fat diet-induced obese C57BL mice or normal mice (normal control). [Figure 5b] Corresponding to Figure 5a, this shows the AUC of the hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the solvent (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control). [Figure 5c] 1 shows the weight-reducing effects 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 high-fat diet-induced obese C57BL mice or normal mice (normal control). [Figure 6a]

[0049] Figure 1 shows the hypoglycemic effects 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 high-fat diet-induced obese C57BL mice or normal mice (normal control) when ipGTT is performed 48 hours after the first administration. [Figure 6b] FIG. 6a corresponds to FIG. 6b, and shows 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 high-fat diet-induced obese C57BL mice or normal mice (normal control) when ipGTT was performed 48 hours after the first administration. [Figure 7a] 1 shows the hypoglycemic effect of the title compound of Example 2 of the present invention, the title compound of Control Example 2, and the solvent on db / db mice. [Figure 7b] FIG. 7A corresponds to FIG. 7A and shows ΔAUC of the hypoglycemic effect of the title compound of Example 2 of the present invention, the title compound of Control Example 2, and the solvent on db / db mice. [Figure 7c] 1 shows the food intake controlling effects of the title compound of Example 2 of the present invention, the title compound of Control Example 2, and the solvent on db / db mice. [Figure 7d] 1 shows the effects of the title compound of Example 2 of the present invention, the title compound of Control Example 2, and the solvent on controlling water intake in db / db mice. [Figure 8a] 1 shows the long-term hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the solvent on db / db mice. [Figure 8b] 8a, which shows the AUC of the long-term hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the solvent in db / db mice. [Figure 8c] 1 shows the long-term weight-reducing effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the solvent on db / db mice. [Figure 8d] 1 shows the long-term food intake controlling effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the solvent on db / db mice. [Figure 8e] 1 shows the effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the solvent on controlling long-term water intake in db / db mice. [Figure 9a] 1 shows the hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Example 2, dulaglutide, and a solvent on Kkay mice. [Figure 9b] Corresponding to Figure 9a, the AUC of the hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Example 2, dulaglutide, and the solvent on Kkay mice is shown. [Figure 9c] 1 shows the HbA1c-lowering effects of the title compound of Example 11 of the present invention, the title compound of Example 2, dulaglutide, and a solvent in Kkay mice. [Figure 10a]1 shows the long-term hypoglycemic effect of the title compound of Example 11 of the present invention, dulaglutide, and the solvent (model control group) on db / db mice or normal mice (normal control). [Figure 10b] Corresponding to Figure 10a, the ΔAUC of the long-term hypoglycemic effect of the title compound of Example 11 of the present invention, dulaglutide, and the solvent (model control group) on db / db mice or normal mice (normal control) is shown. [Figure 10c] This shows random blood glucose levels before injection and after the third, fifth, and eleventh injections when the title compound of Example 11 of the present invention, dulaglutide, or solvent (model control group) was administered to db / db mice or normal mice (normal control). [Figure 10d] This shows the hypoglycemic effect of the title compound of Example 11 of the present invention, dulaglutide, and solvent (model control group) on db / db mice or normal mice (normal control group) when ipGTT is performed 48 hours after the first administration. [Figure 10e] Corresponding to Figure 10d, this shows the AUC of the hypoglycemic effect of the title compound of Example 11 of the present invention, dulaglutide, and solvent (model control group) on db / db mice or normal mice (normal control group) when ipGTT was performed 48 hours after the first administration. [Figure 11a] 1 shows the long-term weight-reducing effects of the title compound of Example 11 of the present invention, dulaglutide, and solvent (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control group). [Figure 11b] 1 shows the long-term food intake controlling effects of the title compound of Example 11 of the present invention, dulaglutide, and the solvent (model control group) on high-fat diet-induced obese C57BL mice. [Figure 11c] 1 shows the effect of the title compound of Example 11 of the present invention, dulaglutide, and the solvent (model control group) on reducing the periovarian fat in high-fat diet-induced obese C57BL female mice. [Figure 11d] 1 shows the epididymal fat-reducing effect of the title compound of Example 11 of the present invention, dulaglutide, and the solvent (model control group) on high-fat diet-induced obese C57BL male mice. DETAILED DESCRIPTION OF THE INVENTION

[0044] definition GLP-1 analogues and derivatives As used herein, the term "GLP-1 analog" or "analog of GLP-1" refers to a peptide or compound that is a variant of human glucagon-like peptide-1 (GLP-1(7-37)), in which one or more amino acid residues of GLP-1(7-37) have been substituted, and / or one or more amino acid residues have been deleted, and / or one or more amino acid residues have been added. Specifically, the sequence of GLP-1(7-37) is set forth in SEQ ID NO: 1 in the Sequence Listing. A peptide having the sequence set forth in SEQ ID NO: 1 may be referred to as "native" GLP-1 or "native" GLP-1(7-37).

[0045] In the sequence listing, the first amino acid residue (histidine) of SEQ ID NO: 1 is numbered 1. However, hereinafter, in accordance with established convention in the art, the histidine residue will be numbered 7, and subsequent amino acid residues will be numbered accordingly, culminating in glycine numbered 37. Therefore, in general, the amino acid residue numbering or position numbering of the GLP-1(7-37) sequence referred to in this application is the sequence starting with His at position 7 and ending with Gly at position 37.

[0046] The [Gly8, Arg34]GLP-1-(7-37) peptide is a GLP-1 analog having Gly and Arg at positions corresponding to positions 8 and 34 of GLP-1(7-37) (SEQ ID NO: 1), respectively, and the [Arg34]GLP-1-(7-37) peptide is a GLP-1 analog having Arg at position corresponding to position 34 of 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 set forth in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, in the Sequence Listing.

[0047] In the context of a GLP-1 peptide or analog thereof, the term "derivative" as used herein refers to a chemically modified GLP-1 peptide or analog in which one or more substituents, which may also be referred to as side chains, have been covalently attached to said peptide.

[0048] Unless otherwise specified, references to acylation with lysine residues are understood to be with the ε-amino group thereof.

[0049] The GLP-1 derivatives of formula (B) according to the invention may exist in different stereoisomeric forms, which have the same molecular formula and connected atomic arrangement but differ only in the three-dimensional orientation of the atoms in space. Unless otherwise specified, the invention relates to all stereoisomeric forms of the claimed derivatives.

[0050] The term "peptide", when used, for example, to describe a GLP-1 analogue according to the present invention, refers to a compound comprising a series of amino acids linked together by amide (or peptide) bonds.

[0051] In one specific embodiment, the peptide consists largely or primarily of amino acids linked to each other by amide bonds (e.g., at least 50%, 60%, 70%, 80%, or at least 90% of the molar mass). In another specific embodiment, the peptide consists of amino acids linked to each other by peptide bonds.

[0052] An amino acid is a molecule containing an amino group and a carboxylic acid group, and optionally one or more additional groups, commonly referred to as side chains.

[0053] The term "amino acid" includes proteinogenic amino acids (encoded by the genetic code, including natural and standard amino acids), as well as nonproteinogenic (not found in proteins and / or not encoded in the standard genetic code) and synthetic amino acids. Nonproteinogenic amino acids are moieties that can be incorporated into peptides via peptide bonds, but are not proteinogenic. Synthetic nonproteinogenic amino acids include amino acids produced by chemical synthesis, i.e., D-isomers of the genetically encoded amino acids, such as D-alanine and D-leucine, Aib (α-aminoisobutyric acid), Abu (α-aminobutyric acid), 3-aminomethylbenzoic acid, o-aminobenzoic acid, desaminohistidine, β-analogs of amino acids, such as β-alanine, D-histidine, desaminohistidine, 2-aminohistidine, β-hydroxyhistidine, and homohistidine.

[0054] 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: imidazolylpropionic acid, abbreviation: Imp).

[0055] Hereinafter, all amino acids not specified as optical isomers will be understood to be L-isomers (unless otherwise specified).

[0056] pharmaceutically acceptable salts, amides or esters The GLP-1 derivatives, analogs, and intermediate products of the present invention may be in the form of pharmaceutically acceptable salts, amides, or esters. The salts may be basic, acidic, or neutral salts. In water, basic salts yield hydroxide ions, and acidic salts yield hydronium ions. Salts of the derivatives of the present invention may be formed by an additional cation or anion reacting with an anionic or cationic group, respectively. These groups may be located within the peptide moiety and / or in the side chains of the derivatives of the present invention.

[0057] Non-limiting examples of anionic groups of the derivatives of the present invention include free carboxy groups in the side chains (if any) and in the peptide moiety, which typically includes a C-terminal free carboxylic acid and may include free carboxy groups in internal acidic amino acid residues, such as Asp and Glu.

[0058] Non-limiting examples of cationic groups of a peptide moiety include the free amino group at the N-terminus (if any) and any free amino groups on internal basic amino acid residues such as His, Arg and Lys.

[0059] Esters of the derivatives according to the invention may be formed, for example, by reaction of free carboxylic acid groups with alcohols or phenols, resulting in the replacement of at least one hydroxy group with an alkoxy or aryloxy group. Ester formation may involve the free carboxy group at the C-terminus of the peptide and / or any free carboxy group in the side chain.

[0060] Amide derivatives according to the invention may be produced, for example, by reaction of a free carboxylic acid group with an amine or substituted amine, or by reaction of a free or substituted amino group with a carboxylic acid. Amide formation may involve the free carboxy group at the C-terminus of the peptide, any free carboxy group in the side chain, the free amino group at the N-terminus of the peptide, and / or any free or substituted peptide amino group in the peptide and / or in the side chain.

[0061] In one specific embodiment, the GLP-1 compound or GLP-1 derivative according to the present invention is in the form of a pharmaceutically acceptable salt. In another specific embodiment, the pharmaceutically acceptable amide form is preferably an amide group at the C-terminus of the peptide. In a further specific embodiment, the peptide or derivative is in the form of a pharmaceutically acceptable ester.

[0062] Methods for preparing the GLP-1(7-37) peptide and GLP-1 analogs of the present invention are known in the art. The GLP-1 peptide moiety (or fragments thereof) of the derivatives of the present invention and the GLP-1 analogs of the present invention can be prepared by traditional peptide synthesis, such as solid-phase peptide synthesis using t-Boc or Fmoc chemistry, or other complete 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 W.C. Chan and P.D. White, eds., "Fmoc Solid Phase Peptide Synthesis," Oxford University Press, 2000.

[0063] In one embodiment, a complete GLP-1 analog of the present invention, e.g., [Gly8, Arg34]GLP-1-(7-37) peptide, can be produced by recombinant methods, i.e., by culturing host cells that contain a DNA sequence encoding the analog and are capable of expressing the peptide in an appropriate nutrient medium under conditions that allow for expression of the peptide. Non-limiting examples of host cells suitable for expression of these peptides include Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines. In some embodiments, this fully recombinant fermentation step in the manufacturing process is feasible, for example, from the perspective of production economics.

[0064] Fusion protein inclusion bodies containing the GLP-1 compound backbone are denatured and renatured to obtain a fusion protein with the correct conformation. After a series of processes such as enzymatic cleavage, precipitation, and centrifugation, a high content of the GLP-1 compound backbone is obtained. After purification and processing by ion exchange chromatography, a relatively high purity of the GLP-1 compound backbone is obtained.

[0065] The term "excipient" broadly refers to any ingredient other than the active therapeutic ingredient. An excipient may be an inactive, non-active, and / or non-pharmaceutically active substance.

[0066] Excipients can serve various purposes, eg, to serve as carriers, solvents, diluents, tableting aids, and / or to improve the administration and / or absorption of active substances.

[0067] The formulation of active pharmaceutical 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 updated editions).

[0068] Non-limiting examples of excipients are solvents, diluents, buffers, preservatives, tonicity agents, chelating agents and stabilizers.

[0069] The GLP-1 derivatives and analogs of the present invention have GLP-1 activity, which refers to the ability to bind to the GLP-1 receptor and initiate a signal transduction pathway to produce insulin secretion-promoting effects or other physiological effects.

[0070] In one specific embodiment, potency, efficacy and / or activity is expressed in vitro in a functional GLP-1 receptor assay, more particularly the ability to stimulate cAMP formation in a cell line expressing a clonal human GLP-1 receptor.

[0071] In another specific embodiment, the derivatives of the present invention are potent in vivo, as can be measured in any suitable animal model and clinical trial by methods known in the art, for example, diabetic db / db mice are an example of a suitable animal model, and in such mice the blood glucose lowering effect can be measured, for example, as described in the Examples section of the present invention.

[0072] The term "insulin" includes naturally occurring insulin, such as human insulin, as well as insulin analogues and insulin derivatives thereof.

[0073] The term "insulin analogue" includes polypeptides having a molecular structure that is derivable from the structure of naturally occurring insulin (e.g., human insulin) by deficiency and / or substitution (replacement) of one or more amino acid residues present in natural insulin and / or by the addition of at least one amino acid residue. Preferably, the substituted amino acid residue is a codable amino acid residue.

[0074] Here, the term "insulin derivative" refers to a naturally occurring insulin or insulin analogue that has been chemically modified, which modification may be, for example, the introduction of a side chain at one or more positions of the insulin skeleton, or the oxidation or reduction of a group of an amino acid residue on the insulin, or the conversion of a free carboxy group to an ester group, or the acylation of a free amino or hydroxy group. The acylated insulin of the present invention belongs to the insulin derivatives.

[0075] The term "parent insulin" refers to the insulin portion of an insulin derivative or acylated insulin (also referred to herein as parent insulin), e.g., in the present invention, the unacylated portion of an acylated insulin. The parent insulin may be a naturally occurring insulin, such as human insulin or porcine insulin. Alternatively, the parent insulin may be an insulin analogue.

[0076] Here, the term "amino acid residue" includes amino acids in which a hydrogen atom has been removed from an amino group, and / or a hydroxy group has been removed from a carboxy group, and / or a hydrogen atom has been removed from a sulfhydryl group. Amino acid residues may be referred to imprecisely as amino acids.

[0077] Unless otherwise specified, all amino acids referred to herein are L-amino acids.

[0078] Here, the term alkylene glycol includes oligo / polyalkylene glycol moieties and monoalkylene glycol moieties. Monoalkylene glycols and polyalkylene glycols include, for example, chains based on monoethylene glycol and polyethylene glycol, chains based on monopropylene glycol and polypropylene glycol, and chains based on monobutylene glycol and polybutylene glycol, i.e., chains based on the repeating units -CH2CHO-, -CH2CH2CHO-, or -CH2CH2CH2CHO-. The alkylene glycol moiety may be monodisperse (having a clearly defined length / molecular weight) or polydisperse (having an ill-defined length / average molecular weight). The monoalkylene glycol moiety includes -OCH2CHO-, -OCH2CH2CHO-, or -OCH2CH2CH2CHO-, each containing a different group at its end.

[0079] The term "fatty acid" includes straight- or branched-chain aliphatic carboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non-limiting examples of fatty acids are, for example, myristic acid, palmitic acid, stearic acid, and eicosanoic acid.

[0080] Here, the term "aliphatic diacid" includes straight or branched chain aliphatic dicarboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non-limiting examples of aliphatic diacids are adipic acid, suberic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, eicosane diacid, docosane diacid, and tetracosane diacid.

[0081] In this application, insulin or GLP-1 compounds are named according to the following principles: they are named according to mutations and modifications (e.g., acylation) of human insulin or natural GLP-1(7-37). The acyl moiety is named according to the IUPAC nomenclature and, in other cases, according to the peptide nomenclature. For example, the following acyl moieties are named:

[0082] [ka] For example, "Eiko Sanji Hey "Glu-γGlu-OEG-OEG", "Eicosanji Hey "Glu-γGlu-2xOEG" or "Eicosanoid Hey The acyl moiety can be named as "19-carboxynonadecanoyl-γGlu-2xOEG," "19-carboxynonadecanoyl-γGlu-2xOEG," or "19-carboxynonadecanoyl-γGlu-OEG-OEG," where OEG is an abbreviation for the group -NH(CH2)2O(CH2)2OCH2CO- (i.e., 2-[2-(2-aminoethoxy)ethoxy]acetyl), and γGlu (and gGlu) is an abbreviation for the amino acid γ-glutamic acid in the L-configuration. Alternatively, the acyl moiety can be named according to IUPAC nomenclature (OpenEye, IUPAC format). In accordance with this nomenclature, the acyl group moiety of the present invention is "[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butyryl]-amino]-ethoxy]-ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]" or "2-(2-(2-(2-(2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl)amino]-ethoxy]-ethoxy]acetyl" Tanoi It is called "amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl".

[0083] For example, insulin of Example 6 of the present invention (having the sequence / structure shown below) is "B29K(N(ε)-eicosand) Hey desB30 human insulin, B29K(N ε -Eiko Sanji Hey desB30 human insulin" or "B29K(N ε -Eiko Sanji Hey The amino acid K at position B29 in human insulin is already attached to the ε nitrogen (N) of the lysine residue at B29. εor (referred to as N(ε)) is a residue of eicosanoid Hey It also shows that the insulin of Control Example 5 (having the sequence / structure shown below) is modified by acylation with Glu-2xOEG and lacks the amino acid T at position B30 in human insulin. ε Eiko Sanji Hey human insulin" or "A14E, B16H, B25H, B29K (N(ε)-eicosane Hey The amino acid Y at position A14 in human insulin has been mutated to E, the amino acid Y at position B16 in human insulin has been mutated to H, the amino acid F at position B25 in human insulin has been mutated to H, and the amino acid K at position B29 in human insulin has been mutated to the ε-nitrogen (N) of the lysine residue at B29. ε (called eicosanoid residue) Hey Glu-2xOEG and is modified by acylation with Glu-2xOEG, and is missing the amino acid T at position B30 in human insulin.

[0084] [ka] As used herein, "nxPEG" refers to -NH(CH2CH2O) n For example, "12xPEG" represents -NH(CHCHO) 12 This represents the group CH2CO-.

[0085] Insulin is a polypeptide hormone secreted by β-cells in the pancreas and consists of two polypeptide chains, A and B, which are linked by two interchain disulfide bonds. The A chain is also characterized by having one intrachain disulfide bond.

[0086] There are three main methods for preparing human insulin in microorganisms. Two involve Escherichia coli. One involves expressing a fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, IL, pp. 729-739). Another uses a signal peptide to allow secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). The third method uses Saccharomyces cerevisiae, where the insulin precursor is secreted into the culture medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art is replete with methods for expressing insulin precursors in E. coli or Saccharomyces cerevisiae, see for example US Pat. No. 5,962,267, WO95 / 16708, EP0055945, EP0163529, EP0347845 and EP0741188.

[0087] The construction of vectors, expression, processing, and purification of insulin analogs can be carried out by techniques known to those skilled in the art. For example, the insulin analogs can be prepared by expressing a DNA sequence encoding the target insulin analog in a suitable host cell by known techniques disclosed in U.S. Pat. No. 6,500,645. For example, insulin analogs may be prepared by the methods reported in the following documents: Glendorf T, et al., (outside 1) TIFF2021136303000003.tif10170 Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding, Biochemistry 2008 47 4743-4751. In this paper, mutations are introduced into an insulin-encoding vector by overlap extension PCR. The insulin analog is expressed in the Saccharomyces cerevisiae strain MT663 as a pre-insulin-like fusion protein with an Ala-Ala-Lys small C-peptide. The single-chain precursor is enzymatically converted to the double-chain desB30 analog by hydrolytic A. lyticus endoprotease.

[0088] The isolated insulin analogs can be acylated at the desired position by acylation methods known in the art, and examples of such insulin analogs are described, for example, in Chinese patent applications with publication numbers CN1029977C, CN1043719A, and CN1148984A.

[0089] The nucleic acid sequence encoding each insulin analog polypeptide can be prepared synthetically by established standard methods, such as those described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869 or Matthes et al. (1984) EMBO Journal 3:801-805.

[0090] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0091] Example Abbreviation cAMP is cyclic adenosine monophosphate. BHK is baby hamster kidney cells. DNA is deoxyribonucleic acid. Na2HPO4 is disodium hydrogen phosphate. NaOH is sodium hydroxide. OEG is the amino acid residue -NH(CH2)2O(CH2)2OCH2CO-. OSu is succinimid-1-yloxy-2,5-dioxo-pyrrolidin-1-yloxy. OtBu is oxy-tert-butyl. HCl is hydrogen chloride. γGlu or gGlu is a γL-glutamyl group. NHS is N-hydroxysuccinimide. DCC is dicyclohexylcarbodiimide. AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid. OH is the hydroxyl radical. Gly is glycine. ARG is arginine. TFA is trifluoroacetic acid. HbA1c is glycosylated hemoglobin. [Example]

[0092] Title compound: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutyric acid Tanoi (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl[Gly8, Arg34]GLP-1-(7-37) peptide (compound 1)

[0093] [ka] 1, N-ε 26 Preparation of -[2-(2-[2-(2-[2-(2-[4-(19-carboxynonadecanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide [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, Cold Spring Harbor Press, 2012, for specific methods). [Gly8, Arg34]GLP-1-(7-37) peptide (5 g, 1.48 mmol) was dissolved in 100 mM NaHPO aqueous solution (150 mL), acetonitrile (100 mL) was added, and the pH was adjusted to 10-12.5 with 1 N NaOH. tert-Butyl eicosandine was added. Hey Leu-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 between 10 and 12.5. After 120 min, the reaction mixture was added to water (150 mL) and the pH was adjusted to 5.0 with 1N aqueous HCl. 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 min. The mixture was concentrated to approximately 30 mL and poured into ice-cold n-heptane (300 mL). The precipitated product was separated by filtration and washed twice with n-heptane. After drying in vacuo, the product was purified by ion exchange chromatography (Resource Q, 0.25% to 1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA), the purified fractions were combined, the pH was adjusted to 5.2 with 1 N HCl, the precipitate was isolated, and lyophilized to give the title compound. LC-MS (electrospray): m / z = 1028.79 [M+4H] 4+ 2. Intermediate ter t-Butyl eicosanoid oil-γ Preparation of Glu-(2xOEG-OSu)-OtBu 2.1 tert-Butyl eicosanoid Hey Lu-OSu Under nitrogen gas protection, mono-tert-butyl eicosanedioate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane (400 mL), triethylamine (13.95 mL) was added, and the resulting cloudy mixture was stirred at room temperature. DCC (11.39 g, 55.19 mmol) was added, and the mixture was further stirred overnight. After filtration, the resulting filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried in vacuo overnight to obtain 24.12 g (97% yield) of tert-butyl eicosanedioate. Hey Le-OSu was obtained. LC-MS(Scie×100API): m / z=496.36(M+1) + 2.2 tert-Butyl eicosanoid Hey Le-γGlu-OtBu tert-Butyl eicosanoid Hey 1-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred. H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water (25 mL) were added in this order and heated to obtain a clear solution. The solution was stirred at room temperature for 4 hours. 10% aqueous citric acid (200 mL) was then added, and the layers were separated. The lower organic layer was washed with saturated brine. After separation, the lower organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 27.27 g (96% yield) of tert-butyl eicosane dihydrate was obtained. Hey Glu-γGlu-OtBu was obtained. LC-MS(Scie×100API): m / z=584.44(M+1) + 2.3 tert-Butyl eicosanoid Hey Leu-γGlu(OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosanoid Heyγ-Glu-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. 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. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness. Methyl tert-butyl ether was added, stirred for 30 minutes, and suction filtered. The filter cake was dried overnight in vacuo to give 25.76 g (81% yield) of tert-butyl eicosane. Hey Glu-γGlu-(OSu)-OtBu was obtained. LC-MS(Scie×100API): m / z=681.46(M+1) + 2.4 tert-Butyl eicosanoid Hey Leu-γGlu-(2xOEG-OH)-OtBu tert-Butyl eicosanoid Hey γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred. 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added in this order and heated to obtain a clear solution. The solution was stirred at room temperature for 4 hours. Next, 10% aqueous citric acid (200 mL) was added, and the layers were separated. The lower organic layer was washed with saturated brine. After separation, the lower organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 30.75 g (93% yield) of tert-butyl eicosane dihydrate was obtained. Hey The resulting product was γ-Glu-(2xOEG-OH)-OtBu. LC-MS(Scie×100API): m / z=874.59(M+1) + 2.5 tert-butyl eicosanoid Hey Leu-γGlu-(2xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosanoid Hey γ-Glu-(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 min. NHS (4.05 g, 35.18 mmol) and DCC (7.98 g, 38.70 mmol) were added. The mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to near dryness and dried in vacuo overnight to give 31.09 g (91% yield) of tert-butyl eicosane. Hey The resulting product was γ-Glu-(2xOEG-OSu)-OtBu. LC-MS(Scie×100API): m / z=971.61(M+1) + [Example]

[0094] Title compound: N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi (amino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide (compound 2)

[0095] [ka] Following steps similar to those in Part 1 of Example 1, N-ε 26 -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutyric acid Tanoi [(amino)ethoxy]ethoxy)acetyl][Gly8, Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z = 992.52 [M+4H] 4+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(OEG-OSu)-OtBu was prepared following steps similar to those in Example 1, Part 2. LC-MS(Scie×100API): m / z=826.54(M+1) + [Example]

[0096] Title compound: N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide (compound 3)

[0097] [ka] Following steps similar to those in Part 1 of Example 1, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutylyl-[Gly8, Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z = 956.25 [M+4H] 4+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(OSu)-OtBu was prepared following steps similar to those in Example 1, Part 2. LC-MS(Scie×100API): m / z=681.46(M+1) + [Example]

[0098] Title compound: N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide (compound 4)

[0099] [ka] Following steps similar to those in Part 1 of Example 1, N-ε 26 -(19-carboxynonadecanoylamino)-4(S)-carboxybutyl-[Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z = 959.75 [M+4H] 4+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(OSu)-OtBu was prepared following steps similar to those in Example 1, Part 2. LC-MS(Scie×100API): m / z=681.46(M+1) + Control Example 1 The control compound, liraglutide, was prepared according to Example 37 of patent CN1232470A.

[0100] Control Example 2 The control compound, semaglutide, was prepared according to Example 4 of patent CN101133082A.

[0101] Control Example 3 Title compound: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)-carboxybutanol] Tanoi (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]Gly8, Arg34]GLP-1-(7-37) peptide

[0102] [ka] Following steps similar to those in Part 1 of Example 1, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoyl amino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino) Ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z = 1021.78 [M+4H] 4+ Control Example 4 Title compound: N-ε 26 -(17-carboxyheptadecanoylamino)-4(S)-carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide

[0103] [ka] Following steps similar to those in Part 1 of Example 1, N-ε 26 -(17-carboxyheptadecanoylamino)-4(S)-carboxybutylyl-[Gly8, Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z = 949.24 [M+4H] 4+ Intermediates tert-Butyloctadecanediol-γGlu-(OSu)-OtBu was prepared following steps similar to those in Example 1, Part 2. LC-MS(Scie×100API): m / z=653.43(M+1) + [Example]

[0104] Pharmacodynamic studies in db / db mice The purpose of this study is to confirm the effect of the GLP-1 derivative of the present invention on regulating hyperglycemia (BG) in cases of diabetes.

[0105] The title compounds (also referred to as GLP-1 derivatives) of Examples 1-4 and Control Examples 1-4 were tested in a single-dose study in an obese type 2 diabetes mellitus (T2DM) mouse model (db / db mice). The hypoglycemic efficacy of the GLP-1 derivatives was tested at a dose of 100 μg / kg.

[0106] Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in a barrier environment in an appropriate breeding box with free access to standard chow and purified water. The environmental conditions were controlled at a relative humidity of 40-60% and a temperature of 22-24°C. After an acclimation period of 1-2 weeks, the mice were used in the experiments.

[0107] Before the start of the experiment, at approximately 9:30 AM, basal blood glucose levels were assessed and mice were weighed. Mice were randomly assigned to vehicle or treatment groups based on blood glucose and body weight, and treated with either vehicle or 100 μg / kg of GLP-1 derivative subcutaneously. The vehicle contained 14 mg / mL propylene glycol, 5.5 mg / mL phenol, and 1.133 mg / mL disodium hydrogen phosphate, with a pH of 8.12.

[0108] The GLP-1 derivatives were dissolved in solvent to a concentration of 20 μg / mL, with a dosing volume of 5 mL / kg (i.e., 50 μL / 10 g body weight). A single subcutaneous injection was administered into the dorsal neck. The corresponding GLP-1 derivative was administered at approximately 10:30 AM (time 0). Animals were allowed free access to food and water during the dosing period. Blood glucose levels were assessed in mice at 2, 4, 6, 8, 10, 12, 24, 48, and 72 hours after dosing. The rats' tails were cleaned with an alcohol swab, and blood samples were collected from the tails using a disposable blood collection needle. Blood glucose levels were measured using a blood glucose meter and attached test strips (Roche). Food intake and body weight of each mouse were measured at 24, 48, and 72 hours after dosing.

[0109] The blood glucose at each monitoring point after administration was compared with the baseline blood glucose before administration to obtain the blood glucose percentage at the corresponding time point. A dose-response curve of blood glucose percentage versus time was then constructed for each single dose of GLP-1 derivative. To quantitatively describe the effect of the GLP-1 derivative on blood glucose, the area under the curve of blood glucose percentage versus time (AUC) was calculated for each individual dose-response curve. 0~72h ) was calculated. AUC is the area under the time-blood glucose percentage curve, and a smaller AUC value indicates a better blood glucose lowering effect and better drug efficacy.

[0110] 1a to 4b show that the GLP-1 derivatives according to the present invention have unexpectedly improved efficacy; for example, the blood glucose lowering effects of the title compounds of Examples 1 to 4 in db / db mice are clearly superior to those of liraglutide and the compounds of Control Examples 3 and 4. In particular, the blood glucose lowering effect of the compound of Example 2 of the present invention is superior to that of semaglutide. Furthermore, the GLP-1 derivatives according to the present invention, for example, the compounds of Examples 1 to 4, have a clearly longer effective duration of action in db / db mice than those of liraglutide and the compounds of Control Examples 3 and 4, and in particular, the compound of Example 2 has a longer effective duration of action in db / db mice than semaglutide. [Example]

[0111] B29K(N(ε)-Eikosanji Hey desB30 human insulin (compound 5)

[0112] [ka] 1. Synthesis of des(B30) human insulin Des(B30) human insulin was prepared according to the method described in Example 101 of Chinese patent CN1056618C.

[0113] 2. Preparation of target insulin DesB30 human insulin (5 g, 0.876 mmol) was dissolved in 100 mM NaHPO aqueous solution (150 mL), acetonitrile (100 mL) was added, and the pH was adjusted to 10-12.5 with 1 N NaOH. Hey Leu-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 between 10 and 12.5. After 120 min, the reaction mixture was added to water (150 mL) and the pH was adjusted to 5.0 with 1N aqueous HCl. 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 min. The mixture was concentrated to approximately 30 mL and poured into ice-cold n-heptane (300 mL). The precipitated product was separated by filtration and washed twice with n-heptane. After drying in vacuo, the product was purified by ion exchange chromatography (Resource Q, 0.25% to 1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5) and reverse-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 isolated and lyophilized to give the title compound, Compound 5. LC-MS (electrospray): m / z = 1377.53 [M+5H] 5+ 3. Preparation of intermediate tert-butyleicosandioyl-γGlu-(5xOEG-OSu)-OtBu 3.1 tert-Butyl eicosanoid Hey Lu-OSu Under nitrogen gas protection, mono-tert-butyl eicosanedioate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane, triethylamine (13.95 mL) was added, and the resulting cloudy mixture was stirred at room temperature. DCC (11.39 g, 55.19 mmol) was added and further stirred overnight. After filtration, the resulting filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried in vacuo overnight to obtain 24.12 g (97% yield) of tert-butyl eicosanedioate. Hey Le-OSu was obtained. LC-MS(Scie×100API): m / z=496.36(M+1) + 3.2 tert-Butyl eicosanoid Hey Le-γGlu-OtBu tert-Butyl eicosanoid Hey 1-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred. H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water were added in this order. The mixture was heated to obtain a clear solution, which was then stirred at room temperature for 4 hours. 10% aqueous citric acid (200 mL) was then added, and the layers were separated. The lower organic layer was washed with saturated brine and then separated. The lower organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 27.27 g (96% yield) of tert-butyl eicosane dihydrate was obtained. Hey Glu-γGlu-OtBu was obtained. LC-MS(Scie×100API): m / z=584.44(M+1) + 3.3 tert-Butyl eicosanoid Hey Leu-γGlu-(OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosanoid Heyγ-Glu-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. NHS (5.38 g, 50.17 mmol) and DCC (10.60 g, 51.38 mmol) were then added. The mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure. Methyl tert-butyl ether was added, stirred for 30 minutes, and suction filtered. The filter cake was dried overnight in vacuo to give 25.76 g (81% yield) of tert-butyl eicosane. Hey Glu-γGlu-(OSu)-OtBu was obtained. LC-MS(Scie×100API): m / z=681.46(M+1) + 3.4 tert-Butyl eicosanoid Hey Leu-γGlu-(2xOEG-OH)-OtBu tert-Butyl eicosanoid Hey γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred. 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added in this order and heated to obtain a clear solution. The solution was stirred at room temperature for 4 hours. Next, 10% aqueous citric acid (200 mL) was added, and the layers were separated. The lower organic layer was washed with saturated brine. After separation, the lower organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 30.75 g (93% yield) of tert-butyl eicosane dihydrate was obtained. Hey The resulting product was γ-Glu-(2xOEG-OH)-OtBu. LC-MS(Scie×100API): m / z=874.59(M+1) + 3.5 tert-butyl eicosanoid Hey Leu-γGlu-(2xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosanoid Hey γ-Glu-(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 min. NHS (4.05 g, 35.18 mmol) and DCC (7.98 g, 38.70 mmol) were added. The mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried in vacuo overnight to give 31.09 g (91% yield) of tert-butyl eicosane. Hey The resulting product was γ-Glu-(2xOEG-OSu)-OtBu. LC-MS(Scie×100API): m / z=971.61(M+1) + 3.6 tert-Butyl eicosanoid Hey Leu-γGlu-(5xOEG-OH)-OtBu tert-Butyl eicosanoid Hey γGlu-(2xOEG-OSu)-OtBu (31.09 g, 32.01 mmol) was dissolved in dichloromethane (350 mL) and stirred. 3xAEEA (14.52 g, 32.01 mmol), triethylamine (8.90 mL), and water (25 mL) were added in that order and heated to obtain a clear solution. The solution was stirred at room temperature for 4 h. Next, 10% aqueous citric acid (200 mL) was added, and the layers were separated. The lower organic layer was washed with saturated brine. After separation, the lower organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 38.99 g (93% yield) of tert-butyl eicosane dihydrate was obtained. Hey Glu-γGlu-(5xOEG-OH)-OtBu was obtained. LC-MS(Scie×100API): m / z=1309.81(M+1) + 3.7 tert-Butyl eicosanoid Hey Leu-γGlu-(5xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosanoid Hey γ-Glu-(5xOEG-OH)-OtBu (38.99 g, 29.77 mmol) was dissolved in dichloromethane (400 mL), triethylamine (8.28 mL) was added, and the mixture was stirred for 10 min. NHS (3.43 g, 29.77 mmol) and DCC (6.76 g, 32.75 mmol) were added. The mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 38.11 g (91% yield) of tert-butyl eicosane. Hey The resulting product was 5xOEG-γGlu-(5xOEG-OSu)-OtBu. LC-MS(Scie×100API): m / z=1406.83(M+1) + [Example]

[0114] B29K(N(ε)-Eikosanji Hey desB30 human insulin (compound 6)

[0115] [ka] Compound 6 was prepared following a procedure similar to that of Example 6, Part 2. LC-MS (electrospray): m / z=1406.28 [M+5H] 5+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(6xOEG-OSu)-OtBu was prepared following steps similar to those in Example 6, Part 3. LC-MS(Scie×100API): m / z=1551.90(M+1) + [Example]

[0116] B29K(N(ε)-Eikosanji Hey desB30 human insulin (compound 7)

[0117] [ka] Compound 7 was prepared following a procedure similar to that of Example 6, Part 2. LC-MS (electrospray): m / z = 1464.30 [M+5H] 5+ Intermediates tert-Butyl eicosanoidioyl-γGlu-(8xOEG-OSu)-OtBu was prepared following steps similar to those in Example 6, Part 3. LC-MS(Scie×100API): m / z=1814.02(M+1) + [Example]

[0118] B29K(N(ε)-Docosand Hey desB30 human insulin (compound 8)

[0119] [ka] Compound 8 was prepared following a procedure similar to that of Example 6, Part 2. LC-MS (electrospray): m / z = 1411.88 [M+5H] 5+ Intermediates tert-spotted Rudokosanji Oil-γ Glu-(6xOEG-OSu)-OtBu was prepared following steps similar to those in Example 6, Part 3. LC-MS(Scie×100API): m / z=1579.94(M+1) + [Example]

[0120] B29K(N(ε)-Docosand Hey desB30 human insulin (compound 9)

[0121] [ka] Compound 9 was prepared following a procedure similar to that of Example 6, Part 2. LC-MS (electrospray): m / z = 1469.91 [M+5H] 5+ Intermediates tert- Butyl docosandi oil-γG lu-(8xOEG-OSu)-OtBu was prepared following steps similar to those in Example 6, Part 3. LC-MS(Scie×100API): m / z=1870.08(M+1) + [Example]

[0122] Title compound: N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8, Arg34]GLP-1-(7-37) peptide (compound 10)

[0123] [ka] Following steps similar to those in Part 1 of Example 1, N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutyric acid Tanoi (amino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl[Gly8, Arg34]GLP-1-(7-37) peptide was prepared. LC-MS (electrospray): m / z = 1035.80 [M+4H] 4+ Intermediates tert- Butyl docosandi oil-γG lu-(2xOEG-OSu)-OtBu was prepared following steps similar to those in Example 1, Part 2. LC-MS(Scie×100API): m / z=999.64(M+1) + [Example]

[0124] In vitro potency or activity This example is aimed at testing the in vitro potency or activity of the GLP-1 derivatives according to the invention. The GLP-1R-expressing cells were regenerated, and the cells were inoculated into a 25 mL cell culture flask in Ham's-F12 medium and cultured overnight at 37°C and 5% CO2. On the day of the experiment, the title compound of Example 11 of the present invention (compound 10) and liraglutide were added to a concentration of 150 μg / mL, and then the sample was 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 1 x 10 5 The cells were adjusted to 100 μL / mL, and 200 μL of diluted sample was added to each well. After mixing, 100 μL of each sample was transferred to a new 96-well plate in three parallel wells. The cells were cultured in a cell incubator for 4 hours, after which luciferase reagent was added and the mixture was mixed by shaking. The mixture was then transferred from the 96-well plate to a new 96-well white flat-bottom plate. The signal was read using a microplate reader, and the data was processed using GraphPad Prism 6 to calculate EC. 50 The in vitro potency experiment was repeated four times on different days.

[0125] [Table 1] As can be seen from the experimental results, the GLP-1 derivative of the present invention has sufficient in vitro efficacy, its in vitro activity is close to that of liraglutide, and it has been confirmed that it has GLP-1 receptor agonist activity. [Example]

[0126] Pharmacodynamic studies in high-fat diet-induced obese C57BL mice The purpose of this study is to confirm the blood glucose regulating effect and weight loss effect of the GLP-1 derivative according to the present invention in high-fat diet-induced obese C57BL mice. Five-week-old C57BL mice (half male and half female) weighing 17-22g were housed in appropriate breeding boxes (3-5 mice / box) in a barrier environment. The high-fat diet-induced group was allowed free access to high-fat feed and purified water, while the normal control group was allowed free access to standard feed and purified water. The environmental conditions were controlled at a relative humidity of 40%-60% and a temperature of 22°C-24°C. The mice were housed for 10 weeks, and mice whose body weight exceeded that of the normal control group by 30%-50% were selected for evaluation of the drug efficacy.

[0127] Before the start of the experiment on the day, basal blood glucose was evaluated at time -1 / 1 h (9:30 AM) and the mice's weights were measured. According to random blood glucose and body weight, the mice in the high-fat diet-induced group were matched and assigned to a vehicle group (i.e., model control group) or a treatment group, and treated by subcutaneous injection of the vehicle, 100 μg / kg of the control compound semaglutide, or 100 μg / kg and 300 μg / kg of the title compound of Example 11 of the present invention. The vehicle contained 14 mg / mL propylene glycol, 5.5 mg / mL phenol, and 1.133 mg / mL disodium hydrogen phosphate, and the pH of the vehicle was 7.4. The mice were administered subcutaneously once in the dorsal neck (5 μl / g body weight). The GLP-1 derivative was administered at approximately 10:30 AM (time 0), and blood glucose levels were assessed 3, 6, 24, 48, and 72 hours after administration. At the same time, the mice's body weights were monitored daily.

[0128] For each single dose of GLP-1 derivative, a Δ blood glucose-time curve was constructed, where Δ is the actual blood glucose at a given time minus the baseline, where the baseline is the blood glucose at time 0. Therefore, in these curves, y=0 indicates the baseline. For each single dose-response curve, the difference in the area under the blood glucose-time curve (ΔAUC) from 0 to the monitoring endpoint was calculated, and a smaller ΔAUC value indicates a better blood glucose lowering effect and better drug efficacy.

[0129] An intraperitoneal glucose tolerance test (ipGTT) experiment was performed 48 hours after the initial administration. The procedure involved blood sampling at the tip of the tail at predetermined time points to measure fasting blood glucose (0 min), and then intraperitoneally administering a glucose solution (200 mg / mL, 10 mL / kg). Blood glucose was measured 30, 60, and 120 min after the glucose load.

[0130] The rat's tail was cleaned with an alcohol swab, and a drop of blood was collected from the tail with a disposable blood collection needle, and the blood glucose level was measured using a blood glucose meter (Roche) and the attached test strip.

[0131] For each single dose of the GLP-1 derivative, a dose-response curve for blood glucose versus time and a dose-response curve for daily weight change versus time were created. To more intuitively and quantitatively explain the effect of the GLP-1 derivatives of the present invention on blood glucose, the relative area under the blood glucose-time curve (ΔAUC) from 0 to the monitoring endpoint was calculated for each single dose-response curve. A smaller ΔAUC value indicates a better blood glucose-lowering effect and better efficacy.

[0132] Figures 5a to 6b show that the GLP-1 derivatives of the present invention have improved efficacy beyond expectations. For example, the blood glucose lowering effect of compound 10 of Example 11 in high-fat diet-induced obese C57BL mice was not significantly different from that of the commercially available control compound semaglutide at the same dose. Furthermore, quantitative results in Figures 5b and 6b show that the blood glucose lowering effect of the GLP-1 derivatives of the present invention is slightly better than that of semaglutide. In particular, 72 hours after administration, the mean blood glucose value in the compound 10 group at the same dose was lower than that in the semaglutide group at the same dose. Furthermore, the blood glucose lowering effect of the GLP-1 derivatives of the present invention is dose-dependent, and the blood glucose lowering effect improves significantly as the dose of GLP-1 of the present invention increases.

[0133] As shown in Figures 6a and 6b, in the ipGTT experiment, compound 10 of Example 11 had a significant inhibitory effect on blood glucose compared with the vehicle after the ipGTT experiment was performed 48 hours after the initial administration to high-fat diet-induced obese C57BL mice, and the blood glucose lowering effect was slightly better than that of semaglutide at the same dose.

[0134] FIG. 5c shows that the GLP-1 derivatives according to the present invention, such as compound 10 of Example 11, have excellent weight-reducing effects, and their weight-reducing effects are superior to those of semaglutide. [Example]

[0135] Pharmacodynamic study in type 2 diabetic db / db mice The purpose of this study is to confirm the blood glucose regulating effect of the GLP-1 derivative of the present invention in cases of diabetes. In db / db mice, the hypoglycemic 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 ED 50 asked for. Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in a barrier environment in an appropriate breeding box with free access to standard chow and purified water. The environmental conditions were controlled at a relative humidity of 40-60% and a temperature of 22-24°C. After an acclimation period of 1-2 weeks, the mice were used in the experiments.

[0136] Before the start of the experiment, at 9:00 am, basal blood glucose was evaluated and the mice were weighed. According to random blood glucose and body weight, the diabetic mice were assigned to a vehicle group or a treatment group, and treated by subcutaneous injection of the vehicle, or the compound of Example 11 or the control compound liraglutide at 0.3, 1, 3, 10, 30 and 100 nmol / kg, where the vehicle contained 14 mg / mL propylene glycol, 5.5 mg / mL phenol, and 1.133 mg / mL disodium hydrogen phosphate, and the pH of the vehicle was 7.4.

[0137] A single subcutaneous injection (50 μl / 10 g body weight) was administered in the dorsal neck. The compound of Example 11 was administered at approximately 10:00 AM (time 0), and the mice were assessed for blood glucose at 1, 2, 3, 6, 12, 24, 48, and 72 hours after administration.

[0138] The rat's tail was cleaned with an alcohol swab, and a drop of blood was collected from the tail with a disposable blood collection needle, and the blood glucose level was measured using a blood glucose meter (Roche) and the attached test strip. For each single dose of GLP-1 derivative, a dose-response curve of Δ blood glucose versus time was constructed. Δ was calculated by subtracting the baseline from the actual blood glucose at a given time, where the baseline is the blood glucose at time 0. To explain the effect of the GLP-1 derivative on blood glucose, the area under the curve of Δ blood glucose from 0 to 72 hours, ΔAUC, was calculated for each individual dose-response curve, and the 50% effective dose (ED) was calculated for ΔAUC. 50 The ED (the dose of GLP-1 derivative that produced a response halfway between the baseline and maximum effect) was calculated. Table 2 below shows the resulting ED 50 The values ​​are shown.

[0139] [Table 2] The test results show that the in vivo hypoglycemic effect of Compound 10 of the present invention is clearly superior to that of liraglutide. [Example]

[0140] Pharmacodynamic study in type 2 diabetic db / db mice The purpose of this study is to confirm the control of blood glucose, food intake and water intake by the GLP-1 derivative of the present invention. The title compound of Example 2 and the control compound semaglutide were tested in type 2 diabetic db / db mice in a single dose study. Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in a barrier environment in an appropriate breeding box with free access to standard chow and purified water. The environmental conditions were controlled at a relative humidity of 40-60% and a temperature of 22-24°C. After an acclimation period of 1-2 weeks, the mice were used in the experiments.

[0141] Before the start of the experiment on the day, basal blood glucose was assessed and the mice were weighed at approximately 9:00 a.m. Diabetic mice were randomly assigned to a vehicle group or a treatment group according to blood glucose and body weight, and treated by subcutaneous injection of the vehicle, or 100 μg / kg of the compound of Example 2 or the control compound semaglutide, where the vehicle contained 14 mg / mL propylene glycol, 5.5 mg / mL phenol, and 1.133 mg / mL disodium hydrogen phosphate, pH 7.4.

[0142] 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) once in the dorsal neck. The compound of Example 2 was administered at approximately 10:00 AM (time 0), and blood glucose levels were assessed in the mice 1, 2, 3, 6, 12, 24, 48, and 72 hours after administration. The rats' tails were cleaned with an alcohol swab, and blood samples were collected from the tails using a disposable blood collection needle. Blood glucose levels were measured using a blood glucose meter (Roche) and the accompanying test strips. Food and water intake was also measured daily.

[0143] For each single dose of the GLP-1 derivative, a dose-response curve for blood glucose versus time, a dose-response curve for food intake versus time, and a dose-response curve for water intake versus time were prepared. To explain the effect of the GLP-1 derivatives of the present invention on blood glucose, the difference in the area under the blood glucose-time curve (ΔAUC) from 0 to the monitoring endpoint was calculated for each single dose-response curve. A smaller ΔAUC value indicates a better blood glucose lowering effect and better efficacy.

[0144] Figures 7a to 7d show that the GLP-1 derivative according to the present invention has an unexpectedly improved hypoglycemic effect and an improved inhibitory effect on food and water intake after administration. This further proves that the hypoglycemic effect of the title compound of Example 2 on db / db mice after administration is superior to that of semaglutide at the same dose. Furthermore, the title compound of Example 2 can effectively control food and water intake, and this effect is superior to that of semaglutide, suggesting that the GLP-1 derivative according to the present invention has a better weight loss effect. [Example]

[0145] Long-term drug efficacy study in type 2 diabetic db / db mice The purpose of this study is to confirm the long-term blood glucose lowering effect, weight loss, and diet control effect of the GLP-1 derivative according to the present invention in type 2 diabetic db / db mice.

[0146] The GLP-1 derivative of Example 11 and the control compound semaglutide were tested in type 2 diabetic db / db mice. The GLP-1 derivative was administered to the mice at different doses of 100 and 300 μg / kg, and semaglutide at a dose of 100 μg / kg, and the effects of the GLP-1 derivative and the control compound semaglutide on lowering blood glucose, reducing body weight, and reducing food and water intake were measured.

[0147] Male db / db (BKS / Lepr) mice aged 8-9 weeks were housed in a barrier environment in an appropriate breeding box with free access to standard chow and purified water. The environmental conditions were controlled at a relative humidity of 40-60% and a temperature of 22-24°C. After an acclimation period of 1-2 weeks, the mice were used in the experiments.

[0148] Before the start of the experiment, basal blood glucose levels were assessed at approximately 9:00 AM, and the mice were weighed. Based on random blood glucose and body weight, diabetic mice were assigned to either a vehicle or treatment group, and treated with either a subcutaneous injection of the vehicle, 100 or 300 μg / kg of a GLP-1 derivative, or 100 μg / kg of the control compound semaglutide. The vehicle contained 14 mg / mL propylene glycol, 5.5 mg / mL phenol, and 1.133 mg / mL disodium hydrogen phosphate, with a pH of 7.4.

[0149] The GLP-1 derivatives were administered subcutaneously (50 μl / 10 g body weight) in the dorsal neck at approximately 10:00 AM (time 0) 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 final administration. Body weight, food intake, and water intake were measured daily from days 0 to 17. After day 17, body weight, food intake, and water intake were monitored every 3 days.

[0150] Figures 8a-8 e These results demonstrate that the GLP-1 derivatives of the present invention have unexpectedly improved hypoglycemic efficacy, improved weight loss, and inhibitory effects on food and water intake, even after prolonged administration. As shown in Figures 8a and 8b, compound 10 of Example 11 has a superior hypoglycemic effect in db / db mice after prolonged administration, compared with semaglutide at the same dose. As shown in Figures 8c and 8d, the GLP-1 derivatives of the present invention, such as the title compound of Example 11, have better weight loss and inhibitory effects on food and water intake, compared with semaglutide at the same dose. [Example]

[0151] Long-term drug efficacy study in type 2 diabetic Kkay mice The purpose of this study is to confirm the blood glucose lowering effect of the GLP-1 derivative according to the present invention on type 2 diabetic Kkay mice. Compound 10 of Example 11, compound 2 of Example 2, and the control compound dulaglutide (also known as Trulicity) were tested in type 2 diabetic Kkay mice. Compound 10 and compound 2 were administered to the mice at different doses of 100 and 300 μg / kg, and dulaglutide was administered at a dose of 600 μg / kg, and the blood glucose lowering effects and HbA1c effects of the GLP-1 derivative of the present invention and the control compound dulaglutide were measured. Male Kkay mice aged 12 to 14 weeks were housed in a barrier environment in an appropriate breeding box, with free access to standard chow and purified water. The environmental conditions were controlled at a relative humidity of 40% to 60% and a temperature of 22 to 24°C. After an acclimation period of 1 to 2 weeks, the mice were used in the experiments.

[0152] Before the start of the experiment, basal blood glucose levels were assessed and mice were weighed at approximately 9:00 AM. Diabetic mice were randomly assigned to vehicle or treatment groups based on blood glucose and body weight, and treated with subcutaneous injections of vehicle, 100 or 300 μg / kg of the GLP-1 derivatives of the present invention, or 600 μg / kg of the control compound dulaglutide. The vehicle contained 14 mg / mL propylene glycol, 5.5 mg / mL phenol, and 1.133 mg / mL disodium hydrogen phosphate, pH 7.4.

[0153] The mice were administered subcutaneously (50 μl / 10 g body weight) in the dorsal neck at approximately 10:00 a.m. (time 0) with the GLP-1 derivative of the present invention, dulaglutide, or solvent, once every two days for a total of 16 consecutive doses. The mice's blood glucose was evaluated 3 hours, 6 hours, 1 day, and 2 days after the first dose, and 48 hours after the final dose, EDTA anticoagulation was performed to detect HbA1c.

[0154] Figures 9a and 9b show that the GLP-1 derivatives of the present invention have an unexpectedly improved hypoglycemic effect after administration, and the hypoglycemic effect of the title compounds of Examples 11 and 2 in Kkay mice is clearly superior to that of dulaglutide. Figure 9c shows that the HbA1c-reducing effect of the GLP-1 derivatives of the present invention in Kkay mice with type 2 diabetes is clearly superior to that of dulaglutide. [Example]

[0155] Pharmacokinetics This example is intended to illustrate the in vivo pharmacokinetic properties of the compounds of the present invention.

[0156] Pharmacokinetics in SD rats Thirty-two SD rats (8 rats per group, half male and half female) were divided into three groups: a low-dose group (15, 90, 540 μg / kg) of Compound 10, a medium-dose group (12, 16, 24, 36, 48, 72, 96, and 120 h after administration) and a high-dose group (90 μg / kg) of Compound 10 administered subcutaneously. Blood samples were collected from the low-, medium-, and high-dose groups before administration (0 min) and 1, 3, 5, 8, 12, 16, 24, 36, 48, 72, 96, and 120 h after administration to measure blood drug concentrations. Blood samples were collected from the intravenous group before administration (0 min) and 1, 10, 1, 3, 5, 8, 12, 24, 48, 72, 96, and 120 h after administration to measure blood drug concentrations. The pharmacokinetic parameter C was calculated using a non-compartmental model in WinNonLin v6.4 software. max , T max , T 1 / 2 , AUC 0~t The MRT was calculated and the test results are shown in Table 3.

[0157] [Table 3] C max = maximum observed plasma concentration, T max = the corresponding time of maximum observed blood drug concentration, T 1 / 2 = terminal elimination half-life, AUC 0~t=0~t time-area under the blood glucose concentration time curve, MRT=mean residence time Pharmacokinetics in cynomolgus monkeys Twenty-four cynomolgus monkeys (6 monkeys per group, half male and half female) were divided into three groups: a low-dose group (10, 60, 360 μg / kg) of Compound 10, a medium-dose group (10), and a high-dose group (10, 60, 360 μg / kg) of Compound 10 administered subcutaneously, and an intravenous group (60 μg / kg) of Compound 10 administered intravenously. Blood samples were taken from the low-, medium-, and high-dose groups before administration (0 min) and 1, 3, 6, 8, 10, 12, 16, 24, 48, 72, 120, 168, and 240 h after administration to measure blood drug concentrations. Blood samples were taken from the intravenous group before administration (0 min) and 1, 10, 1, 3, 6, 8, 10, 12, 24, 48, 72, 120, 168, and 240 h after administration to measure blood drug concentrations. The pharmacokinetic parameter C was calculated using a non-compartmental model in the software WinNonLin v6.4. max , T max , T 1 / 2 , AUC 0~t , MRT was calculated, and the test results are shown in Table 4.

[0158] [Table 4] From the results of the above experiments, it was found that the GLP-1 derivative compound 10 according to the present invention has a relatively long half-life and a relatively large AUC 0~t It can be seen that each of the GLP-1 derivatives according to the present invention exhibits a relatively long MRT. Furthermore, all of the GLP-1 derivatives according to the present invention are dose-dependent, and their efficacy improves as the dose increases. [Example]

[0159] The purpose of this experiment is to measure the chemical stability of the GLP-1 derivative formulation according to the present invention.

[0160] GLP-1 derivatives Compound 10 was dissolved in 5.68 mg / mL disodium hydrogen phosphate solution to a final concentration of 8 mg / mL, and an auxiliary solution containing propylene glycol and phenol was added in order according to the amounts of each component in the table below, and the pH was adjusted to the value in the table below, resulting in a final concentration of the GLP-1 compound of 2 mg / mL. In this example, the chemical stability of the formulation may be represented by the change in high molecular weight protein (HMWP) relative to day 0 after 27 days of storage at 37°C, and may also be represented by the change in the amount of related substances measured after 28 days of storage at 37°C.

[0161] Measurement of high molecular weight proteins (HMWPs) By high performance liquid chromatography (HPLC) High molecular weight protein (HMWP) content The measurements were performed on a Waters TSKgel G2000SWXL (7.8 x 300 mm), 5 μm column at a flow rate of 0.5 s / min, with the column temperature at 30°C and the sample cell temperature at 5°C, using a mobile phase containing 300 mL of isopropanol, 400 mL of glacial acetic acid, and 300 mL of purified water. The detection wavelength was 276 nm, and the sample injection volume was 25 μl. Table 5 shows the increase in HMWP after 27 days of storage at 37°C relative to day 0.

[0162] Measurement of the amount of related substances The content of GLP-1 derivative-related impurities was determined by high-performance liquid chromatography (HPLC) and tested on a Waters Kromasil 100-3.5-C8 (4.6*250mm) column at a flow rate of 1.0mL / min with an elution phase at a column temperature of 35°C and a sample cell temperature of 5°C. Elution was performed with the following mobile phase: Phase A contained 90 mM potassium dihydrogen phosphate and 10% acetonitrile (v / v) at pH 2.4. Phase B was 75% (v / v) acetonitrile. Gradient: Linear change from 75% / 25% A / B to 55% / 45% A / B from 0 to 5 min, linear change to 50% / 50% A / B from 5 to 12 min, linear change to 40% / 60% A / B from 12 to 42 min, linear change to 10% / 90% A / B from 42 to 60 min, linear change to 75% / 25% A / B from 60 to 61 min, isocratic gradient to 85% / 15% A / B from 61 to 70 min. The detection wavelength was 214 nm, the flow rate was 1.0 mL / min, and the sample injection volume was 15 μL. Table 5 shows the increase in the amount of related substances after 28 days of storage at 37°C compared to day 0.

[0163] [Table 5] From the above table, it can be seen that all the formulations have good chemical stability when the pH value is between 6.5 and 8.4, and the chemical stability of the formulations is best when the pH value is between 7.0 and 8.0. [Example]

[0164] The purpose of this experiment is to measure the chemical stability of the GLP-1 derivative formulation according to the present invention. The GLP-1 derivative formulations in Tables 6 and 7 were formulated according to the amounts of each ingredient in Tables 6 and 7 below, using a process similar to that of Example 19. The changes in HMWP and related substances were also measured using a process similar to that of Example 19. Tables 6 and 7 below show the changes in HMWP and related substances in the GLP-1 derivative formulations using different formulation methods.

[0165] [Table 6]

[0166] [Table 7] As can be seen from the table above, the amount of HMWP and the amount of related substances in the above GLP-1 derivative preparations of the present invention increase very slowly over time, demonstrating that all of the above GLP-1 derivative preparations have excellent chemical stability.

[0167] Control Example 5 A14E, B16H, B25H, B29K(N(ε)-eicosanji Hey le-γGlu-2xOEG), desB30 human insulin (control compound 5)

[0168] [ka] 1, A14E, B16H, B25H, B29K(N(ε)-Eicosanji Hey Preparation of desB30 human insulin (γGlu-2xOEG) A14E, B16H, B25H, and desB30 human insulin were prepared by conventional methods for preparing insulin analogs (see Glendorf T, (outside 2) (See TIFF2021136303000024.tif10170 Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding, Biochemistry 2008 47 4743-4751.) A14E, B16H, B25H, and desB30 human insulin (5 g, 0.888 mmol) was dissolved in 100 mM aqueous NaHPO, 100 mL of acetonitrile was added, and the pH was adjusted to 10-12.5 with 1 N NaOH. tert-Butyl eicosand was added. HeyLeu-γGlu-(2xOEG-OSu)-OtBu (0.948 g, 0.976 mmol) was dissolved in acetonitrile (50 mL) and slowly added to the insulin solution. The pH was maintained between 10 and 12.5. After 120 min, the reaction mixture was added to water (150 mL) and the pH was adjusted to 5.0 with 1N aqueous HCl. 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 min. The mixture was concentrated to approximately 30 mL and poured into ice-cold n-heptane (300 mL). The precipitated product was separated by filtration and washed twice with n-heptane. After drying under vacuum, the product was purified by ion exchange chromatography (Resource Q, 0.25% to 1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5) and reverse phase chromatography (acetonitrile, water, TFA). The purified fractions were combined, the pH was adjusted to 5.2 with 1N HCl, and the precipitate was isolated and lyophilized to obtain reference compound 5. LC-MS (electrospray): m / z = 1063.6852 [M+6H] 6+ 2. Preparation of the intermediate tert-butyleicosandioyl-γGlu-(2xOEG-OSu)-OtBu was carried out following steps similar to those in Part 3 of Example 1.

[0169] 2.1 tert-Butyl eicosanoid Hey Lu-OSu Under nitrogen gas protection, mono-tert-butyl eicosanedioate (20 g, 50.17 mmol) and NHS (5.77 g, 50.17 mmol) were mixed in dichloromethane, triethylamine (13.95 mL) was added, and the resulting cloudy mixture was stirred at room temperature. DCC (11.39 g, 55.19 mmol) was added and further stirred overnight. After filtration, the resulting filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 minutes, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried in vacuo overnight to obtain 24.12 g (97% yield) of tert-butyl eicosanedioate. Hey Le-OSu was obtained. LC-MS(Scie×100API): m / z=496.36(M+1) + 2.2 tert-Butyl eicosanoid Hey Le-γGlu-OtBu tert-Butyl eicosanoid Hey 1-OSu (24.12 g, 48.66 mmol) was dissolved in dichloromethane (250 mL) and stirred. H-Glu-OtBu (10.88 g, 53.53 mmol), triethylamine (12.49 mL), and water were added in this order. The mixture was heated to obtain a clear solution, which was then stirred at room temperature for 4 hours. 10% aqueous citric acid (200 mL) was then added, and the mixture was separated. The lower organic phase was washed with saturated brine. After separation, the lower organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 27.27 g (96% yield) of tert-butyl eicosane dihydrate was obtained. Hey Glu-γGlu-OtBu was obtained. LC-MS(Scie×100API): m / z=584.44(M+1) + 2.3 tert-Butyl eicosanoid Hey Leu-γGlu-(OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosanoid Hey γ-Glu-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. 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. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure. Methyl tert-butyl ether was added, stirred for 30 min, and suction filtered. The filter cake was dried overnight in vacuo to give 25.76 g (81% yield) of tert-butyl eicosane. Hey Glu-γGlu-(OSu)-OtBu was obtained. LC-MS(Scie×100API): m / z=681.46(M+1) + 2.4 tert-Butyl eicosanoid Hey Leu-γGlu-(2xOEG-OH)-OtBu tert-Butyl eicosanoid Hey γGlu-(OSu)-OtBu (25.76 g, 37.83 mmol) was dissolved in dichloromethane (250 mL) and stirred. 2xAEEA (11.66 g, 37.83 mmol), triethylamine (9.71 mL), and water (25 mL) were added in that order and heated to obtain a clear solution. The solution was stirred at room temperature for 4 hours. Next, 10% aqueous citric acid (200 mL) was added, and the layers were separated. The lower organic layer was washed with saturated brine. After separation, the lower organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to near dryness and dried overnight in vacuo. 30.75 g (93% yield) of tert-butyl eicosane dihydrate was obtained. Hey The resulting product was γ-Glu-(2xOEG-OH)-OtBu. LC-MS(Scie×100API): m / z=874.59(M+1) + 2.5 tert-butyl eicosanoid Hey Leu-γGlu-(2xOEG-OSu)-OtBu Under nitrogen gas protection, tert-butyl eicosanoid Hey γ-Glu-(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 min. NHS (4.05 g, 35.18 mmol) and DCC (7.98 g, 38.70 mmol) were added. The mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated to near dryness. The residue was mixed with cold water and ethyl acetate, stirred for 20 min, and the layers were separated. The upper organic layer was washed with saturated brine. After separation, the upper organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to near dryness under reduced pressure and dried overnight in vacuo to give 31.09 g (91% yield) of tert-butyl eicosane. HeyThe resulting product was γ-Glu-(2xOEG-OSu)-OtBu. LC-MS(Scie×100API): m / z=971.61(M+1) + [Example]

[0170] A14E, B16H, B25H, B29K(N(ε)-eicosanji Hey desB30 human insulin (compound 11)

[0171] [ka] Following a procedure similar to that of Part 1 of Control Example 5, compounds A14E, B16H, B25H, B29K (N(ε)-eicosanoids) were synthesized. Hey Human insulin (de-γGlu-6xOEG) and desB30 were prepared. LC-MS (electrospray): m / z = 1160.3997 [M+6H] 6+ Intermediate tert-butyl eicosanoid Hey The preparation of 6xOEG-γGlu-(6xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 5, Part 2. LC-MS(Scie×100API): m / z=1551.90(M+1) + [Example]

[0172] A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin (compound 12)

[0173] [ka] Compounds A14E, B16H, B25H, B29K (N(ε)-docosandine) were synthesized according to a procedure similar to that of Part 1 of Control Example 5. Hey DesB30 human insulin was prepared using the γ-γGlu-6xOEG enzyme. LC-MS (electrospray): m / z = 1165.0674 [M+6H] 6+ Intermediate tert-butyl docosanol Hey The preparation of 6xOEG-γGlu-(6xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 5, Part 2. LC-MS(Scie×100API): m / z=1579.94(M+1) + [Example]

[0174] A14E, B16H, B25H, B29K(N(ε)-eicosanji Hey desB30 human insulin (compound 13)

[0175] [ka] Following a procedure similar to that of Part 1 of Control Example 5, compounds A14E, B16H, B25H, B29K (N(ε)-eicosanoids) were synthesized. Hey Human insulin (de-γGlu-12xOEG) and desB30 were prepared. LC-MS (electrospray): m / z = 1305.4716 [M+6H] 6+ Intermediate tert-butyl eicosanoid Hey The preparation of 12xOEG-γGlu-(12xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 5, Part 2. LC-MS(Scie×100API): m / z=2423.35(M+1) + [Example]

[0176] A14E, B16H, B25H, B29K (N(ε)-docosand Hey Glu-γGlu-12xOEG), desB30 human insulin (compound 14)

[0177] [ka] Compounds A14E, B16H, B25H, B29K (N(ε)-docosandine) were synthesized according to a procedure similar to that of Part 1 of Control Example 5. Hey Human insulin (de-γGlu-12xOEG) and desB30 were prepared. LC-MS (electrospray): m / z = 1310.1425 [M+6H] 6+ Intermediate tert-butyl docosanol Hey The preparation of 12xOEG-γGlu-(12xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 5, Part 2. LC-MS(Scie×100API): m / z=2451.38(M+1) + [Example]

[0178] GLP-1 receptor binding This example aims to examine the in vitro receptor binding affinity of the GLP-1 derivatives of the present invention and how the presence of albumin potentially affects binding. Receptor binding is a measure of the affinity of a GLP-1 derivative for the human GLP-1 receptor. The binding affinity of the GLP-1 derivatives and control compounds of the present invention to the human GLP-1 receptor was measured by measuring the binding affinity of the GLP-1 derivatives and control compounds to the human GLP-1 receptor. 125 The binding affinity (IC) was measured by measuring the ability of the derivatives to displace I-GLP-1. The binding of the GLP-1 derivatives to albumin (HSA) was measured using low-concentration albumin (0.005% (w / v)) and high-concentration albumin (2% (w / v)). 50 The change in β-glucose (β) represents the binding of GLP-1 derivatives to albumin, thereby predicting the potentially prolonged pharmacokinetic profile of GLP-1 derivatives in animal models. For receptor binding assays with low HSA (0.005% (w / v)), 50 μl of assay buffer was added to each well of the assay plate. For receptor binding assays with high HSA (2% (w / v)), 50 μl of 8% (w / v) albumin stock solution was added to each well of the assay plate. Test compounds and the reference control, GLP-1(7-37), were formulated in 10 mM NaHPO, pH 7.3, and then formulated to a 1 mM stock solution in ultrapure water. Under the 0.005% HSA condition, all test compounds and reference controls were diluted to 2 μM in assay buffer and then serially diluted in a 4-fold gradient for a total of 10 concentrations. Under 2% HSA conditions, the reference control GLP-1(7-37) was diluted to 2 μM, liraglutide to 20 μM, and compound 10 and semaglutide to 800 μM. All samples were then diluted with a 4-fold serial gradient for a total of 10 concentration gradients. 25 μL of test compound or reference control at different concentrations was added to the appropriate wells of the assay plate. Cell membrane protein was thawed and diluted to its working concentration (40 μg / mL), and 50 μL of the solution containing the cell membrane was added to each well of the assay plate. 25 μL of [ 125 Incubation was initiated by adding a 600 pM solution of [I]-GLP-1. The measurement plate was incubated at room temperature for 1 h. After incubation was completed, the reaction mixture was collected onto a GF / C filtration plate using a cell harvester, washed six times with plate washing buffer, and dried in a dry box at 50 °C for 1 h. 50 μl of scintillation fluid was added, the plate was sealed, and the readings were read using Microbeta2. Analysis was performed using nonlinear regression in GraphPad Prism, and the IC was calculated using the software. 50 Values ​​were calculated and reported in nM. At least three replicates were performed for each test compound. Reported values ​​are the average of all measurements for each test compound.

[0179] [Table 8] "Ratio" is [(IC 50 / nM) high HSA] / [(IC 50 / nM) low HSA Generally, at low albumin concentrations, binding to the GLP-1 receptor should be as good as possible, which leads to a low IC 50 At high albumin concentrations, the IC 50 The IC value is a measure of the effect of albumin on the binding of GLP-1 derivatives to the GLP-1 receptor. As is known, GLP-1 derivatives also bind to albumin, which is usually a desirable effect that prolongs their plasma lifetime. Therefore, at high albumin levels, the IC 50 IC values ​​are usually low for albumin 50 values, corresponding to a decrease in binding to the GLP-1 receptor, which is due to albumin binding competing with GLP-1 receptor binding.

[0180] Therefore, a high ratio (IC 50 Value (high albumin) / IC 50 value (low albumin)) to determine whether the target derivative has good binding to albumin (which can be determined to have a long half-life) and good binding to the GLP-1 receptor (IC 50 High values ​​(hyperalbuminemia) and IC 50 A low albumin level can be taken as an indication of low serum albumin levels.

[0181] From the above table, it can be seen that the ratio of the GLP-1 derivative according to the present invention is higher than that of the control compounds semaglutide, liraglutide and GLP-1(7-37), suggesting that the compounds according to the present invention have a longer half-life and also better binding to the GLP-1 receptor. [Example]

[0182] Long-term drug efficacy study in type 2 diabetic db / db mice A long-term pharmacodynamic study was conducted in type 2 diabetic db / db mice using experimental procedures similar to those in Example 16, except that the control compound used was dulaglutide, and the dose of dulaglutide was 300 μg / kg. The GLP-1 derivative was administered subcutaneously (50 μl / 10 g body weight) in the dorsal neck at approximately 10:00 AM (time 0) on days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30, respectively. Blood glucose was assessed in the mice 3, 6, 9, 12, 24, 48, and 72 hours after the first administration, and the change in area under the blood glucose-time curve (ΔAUC) was calculated. Fasting blood glucose was monitored 48 hours after the 3rd, 5th, and 11th administrations, with a 6-hour fast. An intraperitoneal glucose tolerance test (ipGTT) was performed 48 h after the initial administration. Blood samples were collected from the tip of the tail at designated time points to measure fasting blood glucose (0 min). A glucose solution (200 mg / mL, 10 mL / kg) was then intraperitoneally administered, and blood glucose was measured 30, 60, and 120 min after the glucose load. The mouse tails were cleaned with an alcohol swab, and a drop of blood was collected from the tail with a disposable blood collection needle. Blood glucose levels were measured using a blood glucose meter (Roche) and the attached test strip. A time-glucose curve was plotted, and the area under the time-glucose curve (AUC) was calculated.

[0183] Figures 10a to 10e show that the GLP-1 derivatives of the present invention have unexpectedly improved hypoglycemic efficacy, even after prolonged administration. As shown in Figures 10a and 10b, compound 10 of Example 11 has a superior hypoglycemic effect in db / db mice after administration compared to dulaglutide. As shown in Figure 10c, compound 10 has a superior hypoglycemic effect in db / db mice after prolonged administration compared to dulaglutide. As shown in Figures 10d to 10e, the GLP-1 derivatives of the present invention have a more significant inhibitory effect on blood glucose compared to dulaglutide, and the hypoglycemic effect is superior to that of dulaglutide. [Example]

[0184] Pharmacodynamic studies in high-fat diet-induced obese C57BL mice Pharmacological experiments were conducted in high-fat diet-induced obese C57BL mice using experimental procedures similar to those in Example 13, except that the control compound used was dulaglutide and the dose of dulaglutide was 300 μg / kg.

[0185] The mice were administered subcutaneously in the dorsal neck (5 μl / g body weight) once every three days for a total of 11 doses. The GLP-1 derivative was administered at approximately 10:30 AM (time 0), and blood glucose levels were assessed 3, 6, 9, 12, 24, 48, and 72 hours after administration. At the same time, body weight and food intake were monitored every three days. At the end of the study, subcutaneous fat, perirenal fat, and perigonal fat were weighed. 11a to 11d show that the GLP-1 derivative according to the present invention has unexpectedly improved weight loss, food control and fat-lowering effects. [Example]

[0186] B29K(N(ε)-Docosand Hey Glu-γGlu-12xOEG), desB30 human insulin (compound 15)

[0187] [ka] Compound B29K (N(ε)-docosandy) was prepared according to a procedure similar to that of Part 2 of Example 6. Hey Human insulin (de-γGlu-12xOEG) and desB30 were prepared. LC-MS (electrospray): m / z = 1585.98 [M+5H] 5+ Intermediates tert- Butyl docosandi oil γGlu-(12xOEG-OSu)-OtBu was prepared following steps similar to those in Example 6, Part 3. LC-MS(Scie×100API): m / z=2451.38(M+1) + [Example]

[0188] A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin (compound 16)

[0189] [ka] Compounds A14E, B16H, B25H, B29K (N(ε)-docosandine) were synthesized according to a procedure similar to that of Part 1 of Control Example 5. Hey (L-γGlu-18xOEG), desB30 human insulin was prepared. LC-MS (electrospray): m / z = 1247.47 [M+7H] 7+ Intermediate tert-butyl docosanol Hey The preparation of 18xOEG-γGlu-(18xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 5, Part 2. LC-MS(Scie×100API): m / z=3320.83(M+1) + [Example]

[0190] A14E, B16H, B25H, B29K (N(ε)-docosand Hey desB30 human insulin (compound 17)

[0191] [ka] Compounds A14E, B16H, B25H, B29K (N(ε)-docosandine) were synthesized according to a procedure similar to that of Part 1 of Control Example 5. Hey Human insulin (de-γGlu-24xOEG) and desB30 were prepared. LC-MS (electrospray): m / z = 873.35 [M+11H] 11+ Intermediate tert-butyl docosanol Hey The preparation of ru-γGlu-(24xOEG-OSu)-OtBu was carried out following steps similar to those in Control Example 5, Part 2. LC-MS(Scie×100API): m / z=4192.27(M+1) + [Example]

[0192] B29K(N(ε)-Docosand Hey desB30 human insulin (compound 18)

[0193] [ka] Compound B29K (N(ε)-docosandy) was prepared according to a procedure similar to that of Part 2 of Example 6. Hey DesB30 human insulin was prepared using the γ-γGlu-OEG (de-γGlu-OEG) enzyme. LC-MS (electrospray): m / z = 1266.8122 [M+5H] 5+ Intermediates tert- Butyl docosandi oil-γ Glu-(OEG-OSu)-OtBu was prepared following steps similar to those in Example 6, Part 3. LC-MS(Scie×100API): m / z=854.57(M+1) + [Example]

[0194] B29K(N(ε)-Docosand Hey desB30 human insulin (compound 19)

[0195] [ka] Compound B29K (N(ε)-docosandy) was prepared according to a procedure similar to that of Part 2 of Example 6. Hey Human insulin (de-γGlu-12xPEG) and desB30 were prepared. LC-MS (electrospray): m / z = 1354.8667 [M+5H] 5+ Intermediates tert-Butyldocosandioyl-γGlu-(12xPEG-OSu)-OtBu was prepared following steps similar to those in Example 6, Part 3. LC-MS(Scie×100API): m / z=1294.83(M+1) + Although the present invention has been described with reference to the above embodiments, it should be understood that the above embodiments are merely for illustrative and explanatory purposes and are not intended to limit the present 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 that many further variations and modifications can be made based on the teachings of the present invention, and that all of these variations and modifications are included within the scope of the claims of the present invention. The scope of the claims of the present invention is limited by the scope of 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 a GLP-1 analog having Arg at a position corresponding to position 34 of GLP-1(7-37) (SEQ ID NO: 1) and Ala or Gly at a position corresponding to position 8, [Acy-(L1) ｒ -(L2) ｑ is a substituent linked to the epsilon amino group of the Lys residue at position 26 of said GLP-1 analog, with the proviso that r is an integer from 1 to 10, q is 0 or an integer from 1 to 10, Acy is an aliphatic diacid containing 20 to 24 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; L1 is an amino acid residue selected from γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp, and α-D-Asp; L2 is a neutral, alkylene glycol-containing amino acid residue; Acy, L1 and L2 are linked by an amide bond, and The order of appearance of L1 and L2 in formula (B) is independently interchangeable. or a pharmaceutically acceptable salt, amide or ester thereof.

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

3. L2は、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-CH ２ -O-CH ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-CO-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-CO-CH ２ -O-CH ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ —NH—CO—CH ２ -O-CH ２ -CO-, -HN-(CH ２ ) ３ -O-(CH ２ ) ３ -O-CH ２ -CO-, or -HN-(CH ２ ) ４ -O-(CH ２ ) ４ -O-CH ２ -CO-, and preferably L2 is -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-, and / or L1 is selected from γGlu or βAsp, preferably L1 is γGlu, and / or Acy is HOOC-(CH ２ ) １８ -CO-, HOOC-(CH ２ ) １９ -CO-, HOOC-(CH ２ ) ２０ -CO-, HOOC-(CH ２ ) ２１ -CO- or HOOC-(CH ２ ) ２２ -CO-, and preferably Acy is HOOC-(CH ２ ) １８ -CO-, HOOC-(CH ２ ) ２０ -CO- or HOOC-(CH ２ ) ２２ -CO-, 3. The compound of claim 1 or 2.

4. The compound according to any one of claims 1 to 3, wherein in formula (B), Acy, L1 and L2 are linked in order by an amide bond, and the C-terminus of L2 is linked to the ε-amino group of the Lys residue at position 26 of the GLP-1 analogue.

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

6. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

7. 7. The drug formulation of claim 6, wherein the pharmaceutically acceptable excipient is selected from one or more of a buffering agent, a preservative, a tonicity agent, a stabilizer, and a chelating agent, preferably the pharmaceutically acceptable excipient is a buffering agent, a preservative, and a tonicity agent.

8. the tonicity agent is selected from one or more of sodium chloride, propylene glycol, mannitol, sorbitol, glycerin, glucose and xylitol, preferably propylene glycol, mannitol or sodium chloride; and / or the preservative is selected from one or more of phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, and benzyl alcohol, preferably phenol or m-cresol; and / or the buffering agent 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; The drug formulation according to any one of claims 6 to 7.

9. The drug formulation according to any one of claims 6 to 8, wherein the pH of the formulation is about 6.0 to about 10.0, preferably about 6.5 to about 10.0, preferably about 6.5 to about 9.5, preferably about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.

1.

10. about 0.1 to 1.2 mM, preferably about 0.2 to 1 mM, preferably about 0.3 to 0.7 mM, more preferably about 0.48 to 0.6 mM of a compound according to any one of claims 1 to 5; an isotonicity agent at 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 selected from one or more of propylene glycol, glycerin, mannitol or sodium chloride; a preservative at 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, more preferably about 50-60 mM, preferably selected from one or more of phenol or m-cresol; a buffering agent selected from one or more of sodium acetate, citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate, at about 3-35 mM, preferably about 5-20 mM, more preferably about 5-15 mM, more preferably about 7-10 mM; and The pH is about 6.0 to about 10.0, preferably about 6.5 to about 9.5, preferably about 6.5 to about 8.5, more preferably about 7.0 to about 8.5, more preferably about 7.0 to about 8.1, and even more preferably about 7.3 to about 8.1; Drug formulations.

11. N-ε of about 0.3 to 0.7 mM, more preferably about 0.48 to 0.6 mM ２６ -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε ２６ -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, about 180-200 mM, more preferably about 183-195 mM propylene glycol; about 45-60 mM, more preferably about 50-60 mM, of phenol; a buffering agent that is about 5-15 mM, preferably about 7-10 mM disodium hydrogen phosphate; and The pH 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. Drug formulations.

12. N-ε of about 0.5 mM ２６ -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε ２６ -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, about 184 mM propylene glycol; about 58.5 mM phenol; about 10 mM disodium hydrogen phosphate; and The pH 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; Drug formulations.

13. N-ε of about 2.0 mg / mL ２６ -[2-(2-[2-(2-[2-(2-[4-(21-carboxyheneicosanoylamino)-4(S)-carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N-ε ２６ -[2-(2-[2-(4-[19-carboxynonadecanoylamino]-4(S)-carboxybutanoylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, about 14 mg / mL propylene glycol; about 5.5 mg / mL phenol; about 1.42 mg / mL disodium hydrogen phosphate; and The pH 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; Drug formulations.

14. 6. A method for the preparation of a pharmaceutical composition comprising a compound according to any one of claims 1 to 5 and an acylated insulin, wherein the acylated insulin is preferably B29K(N(ε)-docosandioyl-γGlu-OEG), desB30 human insulin, A14E, B16H, B25H, B29K(N(ε)-eicosandioyl-γGlu-2xOEG), desB30 human insulin or B29K(N(ε)-docosandioyl-γGlu-12xPEG), desB30 human insulin, More preferably, the acylated insulin is an acylated insulin whose parent insulin is a naturally occurring insulin or insulin analogue and which contains at least one lysine residue, the acyl moiety of which is linked to the amino group of a lysine residue or the N-terminal amino acid residue of the parent insulin, and which acyl moiety has formula (A), i.e., III-(II) ｍ -(I) ｎ - (A) however, m is 0 or an integer from 1 to 10, and n is an integer from 5 to 30 (preferably 5 to 20), I is a neutral, alkylene glycol-containing amino acid residue; II is an acidic amino acid residue, III is an aliphatic diacid containing 20 to 24 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; III, II and I are connected by an amide bond, and The order of appearance of II and I in formula (A) is independently interchangeable; Drug composition.

15. n is an integer from 5 to 15, preferably n is 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, preferably n is 5, 6, 7, 8, 9, 10, 11 or 12, preferably n is 5, 6, 7, 8, 9 or 10, preferably n is 5, 6, 7, 8 or 9, preferably n is 5, 6, 7 or 8; and / or m is an integer from 1 to 6, preferably m is 1, 2, 3 or 4, preferably m is 1 or 2, preferably m is 1, and / or III is an aliphatic diacid containing 20 to 23 carbon atoms, preferably III is an aliphatic diacid containing 20, 21 or 22 carbon atoms, provided that in form, a hydroxy group has already been removed from one of the carboxy groups of said aliphatic diacid; and / or The insulin parent contains one lysine residue.

15. The pharmaceutical composition of claim 14.

16. Iは、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ４ -O-(CH ２ ) ３ -NH-CO-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-CH ２ -O-CH ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ -NH-CO-CH ２ -O-CH ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-CO-(CH ２ ) ２ -CO-、-HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-CO-CH ２ -O-CH ２ -CO-、-HN-(CH ２ ) ３ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ３ —NH—CO—CH ２ -O-CH ２ -CO-, -HN-(CH ２ ) ３ -O-(CH ２ ) ３ -O-CH ２ -CO-, or -HN-(CH ２ ) ４ -O-(CH ２ ) ４ -O-CH ２ -CO-, and preferably I is -HN-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-, and / or II is an amino acid residue selected from γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp, and / or III is HOOC-(CH ２ ) １８ -CO-, HOOC-(CH ２ ) １９ -CO-, HOOC-(CH ２ ) ２０ -CO-, HOOC-(CH ２ ) ２１ -CO- or HOOC-(CH ２ ) ２２ -CO-.

16. The pharmaceutical composition of claim 14 or 15.

17. The pharmaceutical composition according to any one of claims 14 to 16, wherein formula (A) is linked by the C-terminus of I to the amino group of the lysine residue or the N-terminal amino acid residue of the parent insulin.

18. The pharmaceutical composition of any one of claims 14 to 17, wherein the acyl moiety is linked to the epsilon amino group of a lysine residue of the parent insulin.

19. The pharmaceutical composition according to any one of claims 14 to 18, wherein the lysine residue of the parent insulin is located at position B29.

20. 20. The pharmaceutical composition of any one of claims 14 to 19, wherein the parent insulin is selected from the following insulin or insulin analogs: desB30 human insulin, A14E, B16H, B25H, desB30 human insulin, A14E, B16E, B25H, desB30 human insulin, human insulin, A21G human insulin, A21G, desB30 human insulin, or B28D human insulin.

21. The acylated insulins include B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin, B29K(N(ε)-eicosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin, B29K(N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin, B29K(N(ε)-eicosandioyl-5xOEG-γGlu), desB30 Human insulin, B29K (N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin, B29K (N(ε)-eicosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin, B29K (N(ε)-eicosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin, B29K (N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-βAsp-6xOEG), desB30 human insulin insulin, B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αGlu-6xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin, B29 K (N(ε)-eicosandioyl-αAsp-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H,B29K (N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16H, B 25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin, A14E, B16H, B2 5H, B29K (N(ε)-eicosandioyl-αGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin, A14E, B16 H, B25H, B29K (N(ε)-eicosandioyl-αAsp-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin, A14E, B16E,B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin, A14 E, B16E, B25H, B29K (N(ε)-eicosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-5xOEG), desB30 human insulin, A 14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-5xOEG), desB30 human insulin Insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-6xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-γGlu-8xOEG),desB30 human insulin, B29K (N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin, B29K (N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin, B29K (N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin, B29K (N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin, B29K (N(ε)-eicosandioyl-βAsp-7xOEG), desB 30 human insulin, B29K (N(ε)-eicosandioyl-βAsp-8xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human Insulin, B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-αAsp-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε) -eicosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin, A14E, B16H, B25H,B29K (N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-βAsp-8 xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin, A14E, B16H, B2 5H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-αAsp-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG) G), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin, A14E, B16E, , B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin , A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-βAsp-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-αAsp-8xOEG), desB 30 human insulin, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin,B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin, B29K (N(ε)-docosandioyl-5xOEG-γGlu-γGlu), desB30 human insulin, B29K (N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl- desB30 human insulin, B29K (N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αAsp-6xOEG) , desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG) , desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu-γGlu),desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-6xOEG), desB30 human insulin Human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-6xOEG), desB3 0 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-6xOEG), des sB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-6xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-5xOEG-γGlu-γGlu),desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-6xOEG), des B30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-6xOEG) G), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-7xOEG-γGlu), desB3 0 human insulin, B29K (N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin, B29K (N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin, B29K (N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin, B29K (N(ε)-docosandioyl-βAsp-7xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-βAsp-8xOEG), desB30 human insulin,B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-αAsp-7xOEG ), desB30 human insulin, B29K (N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu- γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin, A14E, B16 H, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAsp-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-βAs, p-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-8xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-7xOEG-γGlu-γGlu), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-βAsp-8xOEG),desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αGlu-αGlu-8xOEG ), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-αAsp-8xOEG), desB30 human insulin, B29K (N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin, B29K (N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29 K (N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-6xOEG), desB30 human insulin, B29K (N(ε)-heneicosandioyl-γGlu-5xOEG). desB30 human insulin, B29K (N(ε)-heneicosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu-7xOEG),desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-8xOEG), desB30 human insulin, B29K (N(ε)-tricosandioyl-γGlu-5xOEG), desB30 human insulin, B29K (N(ε)-tricosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tricosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B 29K (N(ε)-trichosanedioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosanedioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosanedioyl-γGlu-6xOEG), desB30 human insulin, B29K (N(ε)-trichosandioyl-γGlu-7xOEG), desB30 human insulin, B29K (N(ε)-trichosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-trichosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-trichosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-trichosandioyl-γGlu-8xOEG), d esB30 human insulin, B29K (N(ε)-tetracosanedioyl-γGlu-5xOEG), desB30 human insulin, B29K (N(ε)-tetracosanedioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16E, B25H,B29K (N(ε)-tetracosanedioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-6xOEG), desB30 human insulin, B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG), desB30 human insulin, B29K (N(ε)-tetracosanedioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosanedioyl-γGlu-7xOEG) G), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tetracosandioyl-γGlu-7xOEG), desB30 human insulin, or A14E, B16E, B25H, B29K (N(ε)-tetracosandioyl-γGlu-8xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-12xOEG), desB30 human insulin Insulin, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25 H, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H,B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-heneicosandioyl-γGlu desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tricosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-tetracosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin Human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-10xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-eicosandioyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin, A14E, B16E, B25H, B29 K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-docosandioyl-γGlu-11xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-heneicosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tricosandioyl-γGlu, -12xOEG), desB30 human insulin, A14E, B16E, B25H, B29K (N(ε)-tetracosandioyl-γGlu-12xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-10xO EG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-11xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-9xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin, B29K ( N(ε)-docosandioyl-γGlu-11xOEG), desB30 human insulin, B29K (N(ε)-heneicosandioyl-γGlu-12xOEG), desB30 human insulin, B29K (N(ε)-tricosandioyl-γGlu-12xOEG), desB30 human insulin, B29K (N(ε)-tetracosandioyl-γGlu -12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-18xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-24xOEG), desB30 human insulin, Preferably, the acylated insulin is B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin, B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, B29K(N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin, B29 K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-10xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu- 9xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-11xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-12xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-11xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, A14E,B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-7xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε) -docosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-9xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-9xOEG) EG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-11xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-12xOEG), desB30 human insulin A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-18xOEG), desB30 human insulin, Preferably, the acylated insulin is B29K(N(ε)-eicosandioyl-γGlu-5xOEG), desB30 human insulin, B29K(N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin, B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin, B29K(N(ε)-docosandioyl-γGlu-5xOEG), desB30 human insulin, B29K(N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin, B29K (N(ε)-docosandioyl-γGlu-8xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG ), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG). the insulin is selected from the group consisting of insulin A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-10xOEG), desB30 human insulin, insulin A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin, Preferably, the acylated insulin is A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-6xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-12xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-6xOEG), desB30 human insulin. , A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-10xOEG), desB30 human insulin, A14E, B16H, B25H, B29K (N(ε)-eicosandioyl-γGlu-10xOEG), desB30 human insulin, or A14E, B16H, B25H, B29K (N(ε)-docosandioyl-γGlu-12xOEG), desB30 human insulin, 15. The pharmaceutical composition of claim 14.

22. A compound according to any one of claims 1 to 5, a pharmaceutical formulation according to any one of claims 6 to 13, or a pharmaceutical composition according to any one of claims 14 to 21, for use as a drug.

23. A compound according to any one of claims 1 to 5, a pharmaceutical formulation according to any one of claims 6 to 13, or a pharmaceutical composition according to any one of claims 14 to 21, for use in the treatment or prevention of hyperglycemia, diabetes, and / or obesity.

24. Use of a compound according to any one of claims 1 to 5, a pharmaceutical formulation according to any one of claims 6 to 13 or a pharmaceutical composition according to any one of claims 14 to 21 in the preparation of a medicament for treating or preventing hyperglycemia, diabetes, and / or obesity.

25. A method for treating or preventing hyperglycemia, diabetes, and / or obesity, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 5, a pharmaceutical formulation according to any one of claims 6 to 13, or a pharmaceutical composition according to any one of claims 14 to 21.

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