Insulin derivative and use thereof

Abstract

Provided is an insulin derivative of formula (I) or a salt or solvate thereof. The insulin derivative of formula (I) has better efficacy or effects, longer action time, and excellent bioavailability and safety.

Description

An insulin derivative and its application Technical Field

[0001] The present invention relates to the field of medicine, and specifically provides a new insulin derivative or its salt or solvate, and a pharmaceutical composition and application thereof. Background Art

[0002] Insulin (INS) is a peptide hormone secreted by pancreatic beta cells. Its physiological function is to regulate carbohydrate, lipid, and protein metabolism by promoting cellular glucose uptake, stimulating glycogen synthesis, and inhibiting gluconeogenesis to maintain normal blood glucose levels. Structurally, insulin is a heterodimer of two peptide chains of 21 and 30 amino acids linked by two interchain disulfide bonds. Chain A also has an intrachain disulfide bond. The specific amino acid sequence is as follows:

[0003] A chain:

[0004] Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 1)

[0005] B chain:

[0006] Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr (SEQ ID NO: 2)

[0007] In patients with diabetes, insulin does not function properly due to a lack of insulin, insulin resistance, and loss of β-cell function. Consequently, glucose in the blood cannot be utilized, causing elevated blood sugar levels and leading to hyperglycemia, which is ultimately excreted in the urine and contributes to the development of various complications. Therefore, insulin therapy is necessary for patients with abnormal insulin production (Type I) or insulin resistance (Type II), as insulin administration is required to regulate blood sugar to normal levels.

[0008] Currently available insulin products are mainly divided into five types: rapid-acting insulin, short-acting insulin, intermediate-acting insulin, long-acting insulin, and combination / premixed insulin. However, these insulin products have a short duration of action and require at least one subcutaneous injection daily, which causes patients a lot of injection-related discomfort. Therefore, researchers have been working to develop insulin analogs with better efficacy, longer duration of action, and less frequent injections to improve patient compliance.

[0009] WO2018109162A1 discloses an ultra-long-acting insulin analog, Icodec, for once-weekly administration. Clinical trials are currently underway in multiple countries around the world. The specific structural formula is as follows:

[0010] CN 105061601 A discloses an insulin conjugate of an immunoglobulin fragment, which has a longer duration of action than conventional unmodified insulin.

[0011] CN101573133B and WO2009 / 010428 disclose PEGylated insulins with a longer duration of action compared to conventional unmodified insulins.

[0012] Therefore, there is still a need on the market for insulin analogs with better efficacy, longer duration of action, and lower dosing frequency.

[0013] Summary of the Invention

[0014] In view of the current state of the art, the present invention aims to provide a novel insulin derivative having better efficacy or function, longer duration of action, excellent bioavailability and safety.

[0015] The present invention provides an insulin derivative having formula (I):

[0016] in,

[0017] Z is CH3 or COOH;

[0018] n is 17, 18, 19, 20 or 21;

[0019] X is γGlu or absent;

[0020] Y is -NH-(CH2)2-O-(CH2)2-O-CH2-CO-NH-(CH2)2-O-(CH2)2-O-CH2-CO-, or is absent;

[0021] R is an insulin parent, the ε-amino group of the lysine side chain at position 29 of its B chain is connected to Y by an amide bond, and the insulin parent is selected from: A14E, B16H, B25H, desB30 human insulin; A14E, B16H, B25H, desB27, desB30 human insulin.

[0022] In the present invention,

[0023] The A chain of human insulin A14E, B16H, B25H, desB30 is:

[0024] Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO. 3);

[0025] The B chain of A14E, B16H, B25H, and desB30 human insulin is: Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys (SEQ ID NO. 4).

[0026] In the present invention,

[0027] The A chain of human insulin A14E, B16H, B25H, desB27, desB30 is:

[0028] Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO. 5);

[0029] The B chain of human insulin A14E, B16H, B25H, desB27, desB30 is:

[0030] Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Pro-Lys (SEQ ID NO. 6).

[0031] In the present invention, as one embodiment, the insulin derivative of formula (I) is selected from:

[0032] Compound 1: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB30 human insulin;

[0033] Compound 2: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB30 human insulin;

[0034] Compound 3: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB27,desB30 human insulin;

[0035] Compound 4: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB27, desB30 human insulin.

[0036] In the present invention, as one embodiment, the insulin derivative of formula (I) is selected from:

[0037] Compound 2: A14E, B16H, B25H, B29K (N ε -[1-(19-Carboxy-nonadecyloxy)β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB30 human insulin.

[0038] Another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the insulin derivative of formula (I) or a salt or solvate thereof, and a pharmaceutically acceptable adjuvant, diluent, carrier or excipient.

[0039] In the present invention, as one of the embodiments, the pharmaceutical composition is an injection or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, microneedle, suspension or syrup.

[0040] In the present invention, as one embodiment, the pharmaceutical composition is in the form of microcapsules, microspheres, nanoparticles or liposomes.

[0041] In the present invention, as one embodiment, the pharmaceutical composition is used for oral administration, inhalation administration, transdermal administration or parenteral administration, and the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection.

[0042] In the present invention, as one embodiment, the pharmaceutical composition is administered at a frequency of at least once a day, once a week, or once a month.

[0043] The present invention provides use of an insulin derivative of formula (I) or a salt or solvate thereof in the preparation of a medicament, wherein the medicament is used to treat, prevent or alleviate diseases such as diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity, and the like.

[0044] The present invention provides an insulin derivative of formula (I) or a salt or solvate thereof in combination with other drugs for treating the same or related diseases, wherein the other drugs include but are not limited to metformin, sulfonylureas, SGLT-1 / 2 inhibitors, DPP-4 inhibitors, insulins, GLP-1s, GCGs, GIPs, FGF-21s, or multi-target drugs among the above drugs, or two or more of them.

[0045] The insulin derivative of formula (I) of the present invention has better pharmacodynamics or efficacy, longer duration of action, excellent bioavailability and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG1a and FIG1b show the hypoglycemic effects of vehicle, Icodec and compound 2 injected subcutaneously into normal SD rats in Experimental Example 4. FIG.

[0047] FIG2a and FIG2b show the hypoglycemic effects of vehicle, Icodec, compound 3 and compound 4 injected subcutaneously into normal SD rats in Experimental Example 4. FIG.

[0048] FIG3a and FIG3b show the hypoglycemic effects of vehicle, Icodec and Compound 2 injected intravenously into normal beagle dogs in Experimental Example 5. FIG3a and FIG3b show the hypoglycemic effects of vehicle, Icodec and Compound 2 injected intravenously into normal beagle dogs DETAILED DESCRIPTION

[0049] The following examples and test examples are used to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0050] Example 1: Preparation of Compound 2

[0051] The molecular formula and structural formula of compound 2 are as follows:

[0052] A14E,B16H,B25H,B29K(N ε-[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB30 human insulin

[0053] Preparation of fatty acid derivatives

[0054] (1) Preparation of 1,2,3,4-tetraacetoxy-D-pyranoglucuronic acid methyl ester

[0055] D-glucuronolactone (CAS 32449-92-6) (100 g, 567 mmol, 1.00 equiv) and sodium hydroxide (567 mg, 14.1 mmol, 0.025 equiv) were added to a methanol solution (300 mL), and the mixture was stirred at 20°C for 16 hours. The mixture was then concentrated in vacuo, and pyridine (179 g, 2.27 mol, 183 mL, 4.00 equiv) and acetic anhydride (289 g, 2.84 mol, 265 mL, 5.00 equiv) were added at 5°C, and stirred at 20°C for another 3 hours. The reaction mixture was cooled to 0°C and filtered to obtain a filter cake. The filter cake was slurried with ethanol (100 mL) at 20°C for 10 hours, cooled to 0°C, filtered, and concentrated under reduced pressure to obtain methyl 1,2,3,4-tetraacetoxy-D-pyranoglucopyranosidate (43.9 g) as a white solid.

[0056] 1 H NMR: 400MHz, CDCl3.δ: 5.77 (d, J=7.6Hz, 1H), 5.32-5.23 (m, 2H), 5.17-5.13 (m,1H),4.18(d,J=9.2Hz,1H),3.75(s,3H),2.12(s,3H),2.04-2.04(m,9H).

[0057] (2) Preparation of 1-bromo-2,3,4-triacetoxy-α-D-pyranoglucuronic acid methyl ester

[0058] Methyl 1,2,3,4-tetraacetoxy-D-glucopyranosyl ester (43.9 g, 116 mmol, 1.00 equiv) was added to a solution of hydrogen bromide and acetic acid (150 mL, 33% wt) at 0°C and stirred at 20°C for 3 hours. The reaction mixture was concentrated under reduced pressure to yield a residue. The residue was slurried with ethanol (50 mL) at 20°C for 10 hours, cooled to 0°C, filtered, and the filter cake was concentrated under reduced pressure to yield methyl 1-bromo-2,3,4-triacetoxy-α-D-glucopyranosyl ester (28.5 g) as a white solid.

[0059] 1 H NMR: 400MHz, CDCl3.δ: 6.64(d,J=4.0Hz,1H),5.62(t,J=9.6Hz,1H),5.27-5.22(m,1H),4.86(dd,J 1=4.0Hz, J2=10.0Hz, 1H), 4.59 (d, J=10.4Hz, 1H), 3.77 (s, 3H), 2.10 (s, 3H), 2.06 (d, J= 2.5Hz, 6H).

[0060] (3) Preparation of tert-butyl 20-hydroxy-eicosanoate

[0061] To a tetrahydrofuran solution (1000 mL) were added mono-tert-butyl eicosanedioate (180 g, 451 mmol, 1.00 equiv) and N-methylmorphine (54.8 g, 541 mmol, 1.10 equiv). The mixture was cooled to -10°C under nitrogen, and isobutyl chloroformate (61.6 g, 451 mmol, 1.10 equiv) was added. The mixture was then stirred at -10°C for 1 hour and filtered. Sodium borohydride (25.6 g, 677 mmol, 1.50 equiv) was added to the filtrate at 0°C under nitrogen. After the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure to yield a residue. Purification by column chromatography (silica, petroleum ether:ethyl acetate = 100:1:1, Rf = 0.27) afforded tert-butyl 20-hydroxyeicosanate (29.0 g) as a white solid.

[0062] 1 H NMR: 400MHz, CDCl3.δ: 3.64 (t, J = 6.8 Hz, 2H), 2.20 (t, J = 7.6 Hz, 2H), 1.60-1.53 ​​(m, 4H), 1.44 (s, 9H), 1.41-1.25 (m, 31H).

[0063] (4) Preparation of 1-((20-(tert-Butoxy)-20-oxoeicosyl)oxy)-2,3,4-triacetoxy-β-D-pyranoglucuronic acid methyl ester

[0064] To a chloroform solution (150 mL) was added tert-butyl 20-hydroxyeicosanoate (29.0 g, 75.5 mmol, 2.00 equiv), silver oxide (43.7 g, 188 mmol, 5.00 equiv), iodine (17.2 g, 67.9 mmol, 1.80 equiv), and calcium sulfate (102 g, 755 mmol, 20.00 equiv). The mixture was stirred at 20°C under nitrogen for 15 minutes. A chloroform solution (50 mL) containing methyl 1-bromo-2,3,4-triacetoxy-α-D-pyranoglucopyranosidate (15.0 g, 37.7 mmol, 1.00 equiv) was then added, and the mixture was stirred at 30°C for 18 hours. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The product was purified by column chromatography (silica, petroleum ether:ethyl acetate = 100:1 to 1:1, petroleum ether:ethyl acetate = 3:1) (Rf = 0.30) to give methyl 1-((20-(tert-butoxy)-20-oxoeicosyl)oxy)-2,3,4-triacetoxy-β-D-pyranoglucopyranosyl ester (9.28 g) as a white oil.

[0065] LC-MS: m / z = 723.0 [M+Na] + , which is consistent with the theoretical value.

[0066] 1 H NMR: 400MHz, CDCl3.δ: 5.30-5.22 (m, 2H), 5.03-4.98 (m, 1H), 4.54 (d, J = 7.6Hz, 1H), 4.04-4.02 (m, 1H), 3.91-3.88 (m, 1H), 3.76 (s, 3H), 3.48-3.46(m,1H),2.20(t,J=7.6Hz,2H),2.03(d,J=7.2Hz,9H),1.60-1.53(m,5H),1.44(s,9H),1.27-1.25(m,30H).

[0067] (5) Preparation of 1-((20-(tert-Butyloxy)-20-oxoeicosyl)oxy)-β-D-pyranoglucuronic acid

[0068] To a methanol solution (50 mL) was added methyl 1-((20-(tert-butoxy)-20-oxoeicosyl)oxy)-2,3,4-triacetoxy-β-D-glucopyranosyl ester (9.28 g, 13.2 mmol, 1.00 equiv) and sodium methoxide (35.7 mg, 662 μmol, 0.05 equiv), and the mixture was stirred at 20°C for 5 hours. Lithium hydroxide monohydrate (555 mg, 13.2 mmol, 1.00 equiv) was then added, and the mixture was stirred at 20°C for 17 hours. The reaction mixture was concentrated under reduced pressure to afford 1-((20-(tert-butoxy)-20-oxoeicosyl)oxy)-β-D-glucopyranosyl ester (11.3 g) as a pale yellow solid.

[0069] LC-MS: m / z = 599.3 [MH] - , which is consistent with the theoretical value.

[0070] 1 H NMR: 400MHz, MeOD.δ: 4.28(d,J=8.0Hz,1H),3.92-3.68(m,1H),3.65-3.48(m,1H),3.45-3.40(m,2H),3.38(t,J=9.2Hz,1 H),3.21(dd,J1=7.6Hz,J2=9.1Hz,1H),2.20(t,J=7.6Hz,2H),1.64-1.54(m,4H),1.44(s,9H),1.39-1.36(m,2H),1.29(br s,28H).

[0071] Preparation of intermediate 1 of compound 2

[0072] (1) Resin preparation: 2-CTC resin (150.0 mmol, 1.00 equivalent, substitution degree 1.00 mmol / g), Fmoc-Cys(Trt)-OH (150.0 mmol, 1.00 equivalent), and DIEA (600 mmol, 4.00 equivalent) were added to a dichloromethane solution (1500 mL). The mixture was stirred at 20°C under nitrogen for 2 h, then methanol (150.0 mL) was added and stirred for another 30 min. The resin was washed with N,N-dimethylformamide (1500 mL*5) and then filtered to obtain the resin.

[0073] (2) Deprotection: A solution of N,N-dimethylformamide (1500 mL, containing 20% ​​piperidine) containing the resin was stirred under nitrogen for 30 min, the resin was washed with N,N-dimethylformamide (1500 mL*5), and the resin was filtered to obtain the resin.

[0074] (3) Coupling: Fmoc-Ile-OH (450 mmol, 3.00 equiv), DIEA (900 mmol, 6.00 equiv), and HBTU (427.5 mmol, 2.85 equiv) were added to a solution of resin in N,N-dimethylformamide (1000 mL) and stirred at 20°C under nitrogen for 30 min. The resin was washed with N,N-dimethylformamide (1500 mL x 5).

[0075] (4) Repeat the above steps (2)-(3) to couple the following materials:

[0076] Table 1 Preparation materials of compound 2 intermediate 1

[0077] (5) Wash the resin with methanol (2400 mL x 3) and vacuum dry to obtain the peptide resin. Then, add 2400 mL of lysis buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% mercaptopropionic acid) to the flask containing the side-chain protected peptide resin at 20°C and stir for 2 hours. Precipitate the peptide using cold isopropyl ether (10,000 mL). Filter and collect the filter cake, then wash twice with isopropyl ether (50,000 mL).

[0078] (6) The filter cake was vacuum dried for 2 hours, dissolved in N,N-dimethylformamide (30.0 L), and 0.1 M iodine-methanol solution was added dropwise at 20°C until the yellow color persisted. After stirring for 2 minutes, 0.1 M sodium thiosulfate aqueous solution was added dropwise until the yellow color disappeared. The mixture was lyophilized to obtain Intermediate 1 (180 g) as a white solid.

[0079] LC-MS: m / z = 920.5 [M+H] + , which is consistent with the theoretical value.

[0080] Preparation of compound 2 intermediate 2

[0081] (1) Resin preparation: 2-CTC resin (70.0 mmol, 1.00 equivalent, substitution degree 1.00 mmol / g), Fmoc-Gln(Trt)-OH (70.0 mmol, 1.00 equivalent), and DIEA (280 mmol, 4.00 equivalent) were added to a dichloromethane solution. The mixture was stirred at 20°C under nitrogen for 2 h, and then methanol (70.0 mL) was added and stirred for another 30 min. The resin was washed with N,N-dimethylformamide (700 mL*5) and then filtered to obtain the resin.

[0082] (2) Deprotection: A solution of resin in N,N-dimethylformamide (700 mL, containing 20% ​​piperidine) was stirred under nitrogen for 30 min, and the resin was washed with N,N-dimethylformamide (700 mL*5) and filtered to obtain the resin.

[0083] (3) Coupling: Fmoc-Glu(tBu)-OH (210 mmol, 3.00 equiv), DIEA (420 mmol, 6.00 equiv), and HBTU (199.5 mmol, 2.85 equiv) were added to a solution of the resin in N,N-dimethylformamide (400 mL) and stirred at 20°C under nitrogen for 30 min. The resin was washed with N,N-dimethylformamide (700 mL x 5).

[0084] (4) Repeat the above steps (2)-(3) to couple the following materials:

[0085] Table 2 Preparation materials of compound 2 intermediate 2

[0086] (5) The resin was washed with methanol (1000 mL x 3) and vacuum dried to obtain the peptide resin. 1000 mL of lysis buffer (20% hexafluoroisopropanol / 80% dichloromethane) was then added to the flask containing the side-chain protected peptide resin at room temperature and stirred for 30 minutes twice. Concentration under pressure afforded Intermediate 2 (61.77 g) as a white solid.

[0087] LC-MS: m / z = 943.7 [M+H] + , which is consistent with the theoretical value.

[0088] Preparation of intermediate 3 of compound 2

[0089] (1) Resin preparation: 2-CTC resin (20.0 mmol, 1.00 equivalent, substitution degree 1.00 mmol / g), Fmoc-Asn(Trt)-OH (20.0 mmol, 1.00 equivalent), and DIEA (80.0 mmol, 4.00 equivalent) were added to a dichloromethane (150 mL) solution. The mixture was stirred at 20°C under nitrogen for 2 h, then methanol (20.0 mL) was added and stirred for another 30 min. The resin was washed with N,N-dimethylformamide (300 mL x 5) and then filtered to obtain the resin.

[0090] (2) Deprotection: A solution of N,N-dimethylformamide (300 mL, containing 20% ​​piperidine) containing the resin was stirred under nitrogen for 30 min, the resin was washed with N,N-dimethylformamide (300 mL*5), and the resin was filtered to obtain the resin.

[0091] (3) Coupling: Fmoc-Cys(Trt)-OH (60.00 mmol, 3.00 equiv), DIEA (120 mmol, 12.00 equiv), and HBTU (57.0 mmol, 2.85 equiv) were added to a solution of the resin in N,N-dimethylformamide (300 mL) and stirred at 20°C under nitrogen for 30 min. The resin was washed with N,N-dimethylformamide (300 mL x 5).

[0092] (4) Repeat the above steps (2)-(3) to couple the following materials:

[0093] Table 3 Preparation materials of compound 2 intermediate 3

[0094] (5) A solution of resin in N,N-dimethylformamide (300 mL, containing 20% ​​piperidine) was stirred under nitrogen for 30 min, and the resin was washed with N,N-dimethylformamide (300 mL*5) and filtered to obtain the resin.

[0095] (6) Wash the resin with methanol (2400 mL x 3) and vacuum dry to obtain the peptide resin. Then, add 400 mL of lysis buffer (95% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water) to the flask containing the side-chain protected peptide resin at 20°C and stir for 2 hours. Precipitate the peptide using cold isopropyl ether (2000 mL). Filter and collect the filter cake, then wash twice with isopropyl ether (1000 mL).

[0096] (7) The crude peptide was dried under vacuum for 2 h, dissolved in N,N-dimethylformamide, and purified by HPLC to obtain intermediate 3 (1.9 g) as a white solid.

[0097] LC-MS: m / z = 1210.0 [M+2H] 2+ , which is consistent with the theoretical value.

[0098] Table 4 Purification conditions of compound 2 intermediate 3

[0099] Preparation of intermediate 4 of compound 2

[0100] (1) Resin preparation: 2-CTC resin (2.00 mmol, 1.00 equivalent, substitution degree 1.00 mmol / g), Fmoc-Lys(Dde)-OH (2.00 mmol, 1.00 equivalent), and DIEA (8.00 mmol, 4.00 equivalent) were added to a dichloromethane solution (20.0 mL). The mixture was stirred at 20°C under nitrogen for 2 h, then methanol (4.00 mL) was added and stirred for another 30 min. The resin was washed with N,N-dimethylformamide (20.0 mL*5) and then filtered to obtain the resin.

[0101] (2) Deprotection: A solution of N,N-dimethylformamide (20.0 mL, containing 20% ​​piperidine) containing the resin was stirred under nitrogen for 30 min, and the resin was washed with N,N-dimethylformamide (20.0 mL*5) and filtered to obtain the resin.

[0102] (3) Coupling: Fmoc-Pro-OH (6.00 mmol, 3.00 equiv), DIEA (6.00 mmol, 12.00 equiv), and HBTU (5.70 mmol, 2.85 equiv) were added to a solution of the resin in N,N-dimethylformamide (10.0 mL) and stirred at 20°C under nitrogen for 30 min. The resin was washed with N,N-dimethylformamide (20.0 mL x 5).

[0103] (4) Repeat the above steps (2)-(3) to couple the following materials (1-27):

[0104] Table 5 Preparation materials of compound 2 intermediate 4

[0105] (5) Add 3% hydrazine hydrate / N,N-dimethylformamide (20.0 mL) and react for 30 min. Wash with N,N-dimethylformamide (20.0 mL*5). Repeat the above steps (2)-(3) to couple the material (28-31).

[0106] (6) Wash with methanol and vacuum dry to obtain the peptide resin. Then add 150 mL of lysis buffer (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% mercaptopropionic acid) and stir at 20°C for 2 hours. Use cold isopropyl ether (750 mL) to precipitate the peptide. Filter and collect the filter cake. Wash twice with isopropyl ether (400 mL). Dry the crude peptide in vacuum for 2 hours.

[0107] (7) Crude peptide (3.0 g), 2,2'-dithiodipyridine (154.7 mg, 2.00 equivalents) and DIEA (181.5 mg, 4.00 equivalents) were added to a dimethyl sulfoxide solution (25.0 mL), and the mixture was stirred at 20°C for 10 minutes.

[0108] (8) The crude peptide was purified by preparative HPLC to obtain intermediate 4 (1.9 g) as a white solid.

[0109] LC-MS: m / z = 1095.8 [M+4H] 4+ , which is consistent with the theoretical value.

[0110] Table 6 Purification conditions of compound 2 intermediate 4

[0111] Preparation of intermediate 5 of compound 2

[0112] Intermediate 3 (243.0 mg, 1.10 eq), Intermediate 4 (400.0 mg, 1.00 eq) and DIEA (11.80 mg, 1.00 eq) were added to a dimethyl sulfoxide solution (10 mL), and the mixture was stirred at 20° C. for 10 minutes.

[0113] After the reaction was completed, the mixture was filtered to remove insoluble matter and the crude peptide was purified by HPLC to obtain Intermediate 5 (200.5 mg) as a white solid.

[0114] LC-MS: m / z = 1636.9 [M+4H] 4+ , which is consistent with the theoretical value.

[0115] Table 7 Purification conditions of compound 2 intermediate 5

[0116] Preparation of compound 2

[0117] Intermediate 5 (200.5 mg, 1.00 eq), water (7.00 mL), acetonitrile (3.00 mL), hydrochloric acid (0.50 mL), acetic acid (2.00 mL) and iodine (64.6 mg, 10.00 eq) were mixed and stirred at 20°C for 10 minutes.

[0118] After the reaction was completed, the mixture was filtered to remove insoluble matter. The crude peptide was purified by HPLC to obtain the final product, compound 2 (13.7 mg), as a white solid.

[0119] LC-MS: m / z = 1636.6 [M+4H] 4+ , which is consistent with the theoretical value.

[0120] Table 8 Purification conditions of compound 2

[0121] Example 2 Preparation of Compound 1

[0122] The molecular formula and structural formula of compound 1 are as follows:

[0123] A14E,B16H,B25H,B29K(N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB30 human insulin

[0124] Compound 1 was prepared by referring to the method of Example 1, except that Fmoc-γGlu(OtBu)–OH in Table 5 was removed, and the remaining materials and preparation methods remained unchanged.

[0125] LC-MS: m / z = 1604.3 [M+4H] 4+ , which is consistent with the theoretical value.

[0126] Example 3 Preparation of Compound 3

[0127] The molecular formula and structural formula of compound 3 are as follows:

[0128] A14E,B16H,B25H,B29K(N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB27, desB30 human insulin

[0129] Compound 3 was prepared by referring to the method of Example 1, except that Fmoc-γGlu(OtBu)–OH and Fmoc-Thr(tBu)-OH in Table 5 were removed, and the other materials and preparation methods remained unchanged.

[0130] LC-MS: m / z = 1579.3 [M+4H] 4+ , which is consistent with the theoretical value.

[0131] Example 4 Preparation of Compound 4

[0132] The molecular formula and structural formula of compound 4 are as follows:

[0133] A14E,B16H,B25H,B29K(N ε-[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB27, desB30 human insulin

[0134] Compound 4 was prepared by referring to the method of Example 1, except for removing Fmoc-Thr(tBu)-OH in Table 5, and the other materials and preparation methods remained unchanged.

[0135] LC-MS: m / z = 1611.6 [M+4H] 4+ , which is consistent with the theoretical value.

[0136] Compound 1, Compound 2, Compound 3 and Compound 4 prepared in the present invention are shown in Table 9.

[0137] Table 9 Insulin derivatives and their molecular weight and purity

[0138] Test Example 1: Study on the sugar uptake capacity of adipocytes by the compounds of the present invention

[0139] 1. Adipogenic Induction of 3T3-L1 Mouse Embryonic Fibroblasts

[0140] 1) Cell plating: Adipocytes were plated in 96-well cell culture plates at a volume of 200 μL / well using maintenance medium 1 (DMEM, 1% double antibody, 10% fetal bovine serum, 1 μg / mL human insulin). 4×10 4 Cells / well were cultured in a 10% CO2, 37°C cell culture incubator.

[0141] 2) After two days, the cells were cultured in normal culture medium 2 (DMEM, 1% double antibody, 10% fetal bovine serum).

[0142] 3) After two days, the cells have an adipocyte phenotype and can be used for adipocyte glucose uptake testing.

[0143] 2. Adipocyte Glucose Uptake Test Procedure

[0144] 1) Preparation of KRPH buffer: 5 mM Na2HPO4, 20 mM HEPES, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, pH 7.40.

[0145] 2) On the day of the experiment, the culture medium of the adipocytes was replaced with serum-free DMEM medium 3 and starved for 2 hours.

[0146] 3) Sample dilution: All sample stock solutions were diluted with KRPH buffer, and Icodec, compound 1, and compound 2 were diluted 3-fold in a series, starting at 25 μM, for 10 points.

[0147] 4) Starved adipocytes were operated using a Well Vario liquid handling station, rinsed once with KRPH buffer, and 50 μL of a gradient diluted sample of different concentrations was added to each well, with duplicate wells at each concentration point, and incubated for 10 minutes.

[0148] 5) Add 50 μL / well containing 0.25 μCi [ 3 H]-deoxyglucose and 50 μM deoxyglucose in KRPH buffer and incubate in a CO2 incubator for 20 min.

[0149] 6) Rinse the cells three times with 4°C pre-cooled DPBS containing 10 mM glucose, 200 μL / well each time.

[0150] 7) Lyse the adipocytes with 100 μL / well of 10% sodium hydroxide solution and shake on a shaker for 20 minutes.

[0151] 8) Transfer the lysate sample to a scintillation tube, add 2 mL of scintillation fluid, and perform scintillation counting using Tri-Carb.

[0152] Finally, the data were analyzed using GraphPad Prism7.

[0153] Table 1.1 Average EC of insulin derivatives 50 value

[0154] Note: All the above compounds were tested at least 3 times independently.

[0155] Result analysis: It can be clearly seen from the table that compound 1 and compound 2 are superior to Icodec in promoting the sugar uptake ability of adipocytes.

[0156] Experimental Example 2: Human Serum Albumin (HSA) Binding Study

[0157] 1. Study on the in vitro binding of insulin derivatives to HSA by activating phosphorylation of human insulin receptor B (IRB)

[0158] Cells were plated in a 384-well cell culture plate with 10,000 cells per well in a 5% CO2, 37°C incubator overnight. Icodec, compound 1, and compound 2 were dissolved and diluted in F12 medium containing 0.1% HSA and 0.1% Casein, respectively. The starting concentration was 60 μM, and all samples were serially diluted 4-fold to 10 points. The cell plate was removed from the incubator, the medium was removed using a plate washer, and the diluted samples were immediately added. The reaction was allowed to incubate at room temperature for 5 minutes. The treated cell samples were removed using a plate washer, and cell lysis buffer was immediately added. The cells were shaken thoroughly for lysis. The cell lysate was diluted with 1× cell lysis buffer. 10 μL of the diluted cell lysate was transferred to an Optiplate 384-well plate, and the Acceptor Beads mixture was added. The plate was sealed with foil, mixed thoroughly, and incubated at room temperature. The Donor Beads mixture (protected from light) was then added. The Donor Beads mixture was mixed thoroughly and incubated at room temperature. The plates were read using the Envision instrument AlphaScreen mode, and the data were analyzed using GraphPad Prism 7.

[0159] Table 2.1 Comparison of the effects of casein and HSA on the phosphorylation of IRBs activated by insulin derivatives

[0160] Note: All the above compounds were tested at least 3 times independently.

[0161] Result analysis: From the above table, it can be seen that compared with Icodec, compound 1 and compound 2 bind more tightly to HSA.

[0162] Note: The assay described in this article typically uses 0.1% human serum albumin (HSA) when performing IRB phosphorylation assays activated by insulin derivatives. Fatty acid-acylated insulins bind tightly to HSA, distorting the results and making the compound appear less effective. Using 0.1% casein can avoid this issue. The improvement seen with casein can be considered an indicator of the relative tightness of the compound's binding to serum albumin.

[0163] 2. Determination of Insulin Derivatives Binding to HSA in Vitro by SPR

[0164] The binding of Icodec, compound 2, compound 3, and compound 4 to human serum albumin was performed on a Biacore 8K instrument.

[0165] 2.1 Test steps

[0166] 2.1.1 Protein coupling

[0167] Human serum albumin was immobilized on the surface of the S series sensor chip CM5 according to the instructions of the Amine coupling kit, type 2 (Cytiva). The specific steps are as follows:

[0168] a) Mix EDC and NHS in a 1:1 ratio. Dilute human HSA to 20 μg / mL with pH 4.5 acetic acid solution. Add the EDC / NHS mixture, HSA protein solution, and 1 M ethanolamine-HCl solution to the assay plate according to the protocol.

[0169] b) Place the assay plate in the Biacore 8K sample chamber. The Biacore 8K sequentially injects the EDC / NHS mixture, HSA protein solution, and 1M ethanolamine-HCl solution into the channels on the CM5 chip surface according to the program to activate the chip, couple the ligand, and deactivate the chip. Each channel consists of two flow cells, with flow cell 1 serving as the reference channel for surface activation and deactivation only, without injecting the HSA protein solution. Specific parameters are as follows:

[0170] Table 2.2 Ligand coupling parameters

[0171] 2.1.2 Sample testing

[0172] a) Icodec, compound 2, compound 3, and compound 4 stock solutions were diluted using PBST running buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, 0.05% Tween-20). All samples started at 200 μM and were serially diluted 2-fold to 6 points.

[0173] b) The prepared samples Icodec, compound 2, compound 3, compound 4, and running buffer were transferred to the assay plate according to the program settings, and the assay plate was placed in the sample compartment of the Biacore 8K, wherein the running buffer was used as a reference sample with a concentration of 0.

[0174] c) Run the detection program and inject the sample into each channel of the chip in sequence according to the program. The specific parameters are as follows:

[0175] Table 2.3 Sample testing conditions

[0176] 2.1.3 Data Analysis Before analyzing the raw data, it is necessary to process it according to the following procedures:

[0177] a) The start time of injection of all concentrations of compound and buffer was set to 0 seconds.

[0178] b) The baseline response of all channels at all concentrations was defined as 0 RU.

[0179] c) Subtract the corresponding reference channel response value from the test channel response value of each channel to obtain the response value of samples of different concentrations.

[0180] d) The final sensorgram was obtained by subtracting the response value of the zero concentration sample from the response value of the samples with different concentrations.

[0181] Finally, Biacore Insight Evaluation Software was used to analyze the data, and the 1:1 binding model was selected for analysis.

[0182] Table 2.4 Binding kinetics of insulin derivatives to HSA

[0183] Result analysis: It can be clearly seen from the table that compound 2, compound 3, and compound 4 have better binding ability to HSA than Icodec.

[0184] Test Example 3: Half-life of insulin derivatives

[0185] Six healthy male beagle dogs aged 8-9 months and weighing 7-10 kg were selected from the animal stock. During the experiment, the animals were housed in individual cages and had free access to food and water.

[0186] The experiment began after one week of animal adaptation. Beagle dogs were divided into two dosing groups based on body weight, with three animals in each group. Both groups received a 4 nmol / kg dose. Icodec and compound 2 were injected intravenously in a vehicle containing 5 mM sodium hydrogen phosphate (NaHPO), 140 mM sodium chloride, and 70 ppM Tween 20 (pH 8.0).

[0187] Icodec and compound 2 were dissolved in the solvent to a concentration of 40 nmol / mL, respectively, at a dosing volume of 0.1 mL / kg. A single intravenous administration was performed, and blood concentrations of the administered compound were measured at 0.05, 0.167, 0.5, 1, 2, 4, 6, 8, 24, 32, 48, 72, 96, 144, 192, and 216 hours after administration. Animals were fasting during sampling and had free access to food and water at other times. Data were analyzed using Phoenix WinNonlin 6.3.

[0188] Table 3.1 Half-life of insulin derivatives injected intravenously in normal beagle dogs

[0189] Result analysis: It can be clearly seen from the table that in the intravenous administration test in normal beagle dogs, the half-life of compound 2 is better than that of Icodec.

[0190] Experimental Example 4: Pharmacodynamic Study in Normal SD Rats

[0191] Normal male Sprague-Dawley rats, 8-9 weeks old and weighing 280-320 g, were selected. The animals were housed in a strictly controlled animal enclosure with a temperature maintained at 20-24°C and a humidity level maintained at 30-70%. During the experiment, the animals were housed in individual cages and had free access to food and water.

[0192] After one week of animal adaptation, the experiment began. Rats were divided into vehicle group 1, vehicle group 2, and a treatment group based on body weight and pre-dose blood glucose levels. Each group consisted of eight rats, each receiving a 650 nmol / kg dose. Vehicle, or Icodec, compound 2, compound 3, or compound 4 were subcutaneously injected. The vehicle in vehicle group 1 and vehicle group 2 consisted of 30 mM phenol, 1.6% (w / v) glycerol, and a pH of 8.0.

[0193] The above-mentioned drug was dissolved in a solvent to a concentration of 216.7 nmol / ml in a dosing volume of 3 mL / kg. A single subcutaneous administration was performed, with the dosing time being defined as zero, and blood glucose was measured at 0 h before dosing and 4 h, 8 h, 24 h, 28 h, 48 h, 72 h, 96 h, and 120 h after dosing (without food restriction). A dose-response curve of blood glucose versus time was plotted for each single dose of the insulin derivative.

[0194] To illustrate the effect of the insulin derivatives of the present invention on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each individual dose-response curve. A smaller AUC value indicates a better blood glucose-lowering effect and better efficacy.

[0195] Table 4.1 Effects of single administration of Icodec and compound 2 on blood glucose in SD rats (Mean ± SEM, n = 8)

[0196] Note: *P<0.05, **P<0.01 vs vehicle group 1

[0197] Table 4.2 Effects of single administration of Icodec, compound 3, and compound 4 on blood glucose in SD rats (Mean ± SEM, n = 8)

[0198] Note: *P<0.05, **P<0.01 vs vehicle group 2

[0199] As shown in Table 4.1, Figures 1a and 1b, and Table 4.2, Figures 2a and 2b, the insulin derivatives Compound 2, Compound 3, and Compound 4 of the present invention have better hypoglycemic efficacy than Icodec.

[0200] Experimental Example 5: Pharmacodynamic Study in Normal Beagle Dogs

[0201] Twelve healthy male beagle dogs aged 8-9 months and weighing 7-10 kg were selected from the animal stock. During the experiment, the animals were housed in individual cages and had free access to food and water.

[0202] The experiment began after one week of animal adaptation. Beagle dogs were divided into a vehicle group and a treatment group based on body weight and pre-dose blood glucose levels. Each group had four animals, and the dose was 21 nmol / kg. The animals were injected intravenously with either vehicle or Icodec or Compound 2. The vehicle consisted of 5 mM sodium hydrogen phosphate (NaHPO), 140 mM sodium chloride, and 70 ppM Tween 20 (pH 8.0).

[0203] The above-mentioned drug was dissolved in a solvent to a concentration of 210 nmol / ml, and the dosing volume was 0.1 mL / kg. A single intravenous administration was performed, and blood glucose was measured at 0 h before dosing, and 2 h, 24 h, 48 h, and 72 h after dosing, with the dosing time as zero. The animals were fasting during sampling and had free access to food and water at other times. A dose-response curve of blood glucose versus time was plotted for each single dose of the insulin derivative.

[0204] To illustrate the effect of the insulin derivatives of the present invention on blood glucose, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated for each individual dose-response curve. A smaller AUC value indicates a better blood glucose-lowering effect and better efficacy.

[0205] Table 5.1 Effect of single administration on blood glucose in beagle dogs (Mean ± SEM, n = 4)

[0206] Note: *P<0.05, **P<0.01 vs vehicle group

[0207] As shown in Table 5.1, Figures 3a and 3b, relative to Icodec, the insulin derivative compound 2 of the present invention has a better hypoglycemic effect than Icodec.

Claims

1. An insulin derivative of formula (I) or a salt or solvate thereof: in, Z is CH3 or COOH; n is 17, 18, 19, 20 or 21; X is γGlu or absent; Y is -NH-(CH2)2-O-(CH2)2-O-CH2-CO-NH-(CH2)2-O-(CH2)2-O-CH2-CO-, or is absent; R is the insulin parent, and the ε-amino group of the lysine side chain at position 29 of its B chain is connected to Y by an amide bond.

2. The derivative according to claim 1, characterized in that The insulin precursor is selected from: A14E, B16H, B25H, desB30 human insulin; A14E, B16H, B25H, desB27, desB30 human insulin.

3. The derivative according to claim 1, characterized in that The insulin derivative of formula (I) is selected from: Compound 1: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB30 human insulin; Compound 2: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB30 human insulin; Compound 3: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy desB27, desB30 human insulin; or Compound 4: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB27,desB30 human insulin.

4. The derivative according to claim 1, characterized in that The insulin derivative of formula (I) is selected from: Compound 2: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxy-nonadecyloxy)β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl), desB30 human insulin.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the insulin derivative of formula (I) or a salt or solvate thereof, and a pharmaceutically acceptable auxiliary material, diluent, carrier or excipient.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is an injection or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, microneedle, suspension or syrup.

7. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in the form of microcapsules, microspheres, nanoparticles or liposomes.

8. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is for oral administration, inhalation administration, transdermal administration or parenteral administration; preferably, the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection.

9. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is administered at a frequency of at least once a day, once a week, or once a month.

10. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes: metformin, sulfonylureas, SGLT-1 / 2 inhibitors, DPP-4 inhibitors, insulin, GLP-1, GCG, GIP or FGF-21 drugs, or multi-target drugs among the above drugs, or two or more of them.

11. Use of the insulin derivative of formula (I) or its salt or solvate according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 5 to 10 in the preparation of a medicament for treating, preventing or alleviating diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, or obesity.