Insulin derivatives and their use

Abstract

Equation (I): [Formula 1] Insulin derivatives of TIFF2026517987000033.tif51143 or their salts or solvates are provided. The insulin derivative of formula (I) has better efficacy or effect, a longer duration of action, and superior bioavailability and safety.

Description

[Technical Field]

[0001] This invention relates to the field of pharmaceuticals, and more specifically to novel insulin derivatives, their salts or solvates, pharmaceutical compositions, and their uses. [Background technology]

[0002] Insulin (INS) is a peptide hormone secreted by pancreatic β-cells in the pancreas. Its physiological effects include promoting cellular glucose uptake, enhancing glycogen synthesis, and inhibiting gluconeogenesis, thereby maintaining normal blood glucose levels and modulating carbohydrate, lipid, and protein metabolism. Structurally, insulin is a heterodimer consisting of two peptide chains, each containing 21 and 30 amino acids, linked by two interchain disulfide bonds. Chain A contains intrachain disulfide bonds. The specific amino acid sequence is as follows: Chain A: 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) B chain: 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) That is correct.

[0003] In diabetic patients, insulin cannot function properly due to insulin deficiency, insulin resistance, and loss of β-cell function. As a result, glucose in the blood cannot be utilized, leading to elevated blood glucose levels and hyperglycemia. Ultimately, glucose is excreted through urine, contributing to the development of various complications. Therefore, insulin therapy, which can regulate blood glucose levels to a normal range through insulin administration, is necessary for patients with abnormal insulin production (Type I) or insulin resistance (Type II).

[0004] Currently available insulin products are primarily categorized into five types: rapid-acting insulin, rapid-acting insulin, intermediate-acting insulin, long-acting insulin, and combination / pre-mixed insulin. However, these insulin products have a relatively short duration of action and require subcutaneous injection at least once a day, which causes patients various injection-related discomforts. Therefore, researchers are striving to develop insulin analogs that have superior efficacy, extended duration of action, and reduced injection frequency in order to enhance patient adherence to medication.

[0005] WO2018109162A1 discloses Icodec, an ultra-long-acting insulin analog administered once a week, which is currently undergoing clinical trials in multiple countries worldwide. Its structural formula is as follows:

[0006] [ka]

[0007] CN105061601A discloses an insulin conjugate of an immunoglobulin fragment that exhibits a longer duration of action compared to conventional, unmodified insulin.

[0008] CN101573133B and WO2009 / 010428 disclose PEG (PEGylated) extended insulins that exhibit a longer duration of action compared to conventional unmodified insulin.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0010] Therefore, there is still a widespread need in the current market for insulin analogs with better drug effects, extended duration of action, and reduced dosing frequency.

Means for Solving the Problems

[0011] In view of the state of the art, an object of the present invention is to provide a novel insulin derivative that exhibits better drug effects or efficiency, longer duration of action, and beneficial bioavailability and safety.

[0012] The present invention relates to the following formula (I):

[0013]

Chemical Formula

[0014] (wherein, Z is CH3 or COOH; n is 17, 18, 19, 20, or 21; X is either γ-Glu or is absent; Y is either -NH-(CH2)2-O-(CH2)2-O-CH2-CO-NH-(CH2)2-O-(CH2)2-O-CH2-CO- or not present; R is an insulin precursor in which the ε-amino group of the lysine side chain at position 29 of the B chain is connected to Y via an amide bond, and the insulin precursor provides an insulin derivative having A14E,B16H,B25H,desB30 human insulin (selected from A14E,B16H,B25H,desB27,desB30 human insulin).

[0015] In this invention, The A chains of human insulin A14E, B16H, B25H, and desB30 are as follows: 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); As stated above, The B chains of human insulin A14E, B16H, B25H, and desB30 are as follows: 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) That is correct.

[0016] In this invention, The A chains of human insulin are A14E, B16H, B25H, desB27, and desB30, as follows: 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); As stated above, The B chains of human insulin A14E, B16H, B25H, and desB30 are as follows: 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) That is correct.

[0017] In this invention, as one embodiment, the insulin derivative of formula (I) is as follows: Compound 1:A14E,B16H,B25H,B29K(N ε -[1-(19-carboxyl-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-carboxyl-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-carboxyl-nonadecyloxy)-β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB27,desB30 human insulin; Compound 4:A14E,B16H,B25H,B29K(N ε -[1-(19-carboxyl-nonadecyloxy)-β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB27,desB30 human insulin; Selected from.

[0018] In this invention, as one embodiment, the insulin derivative of formula (I) is as follows: Compound 2:A14E,B16H,B25H,B29K(N ε -[1-(19-carboxyl-nonadecyloxy)-β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB30 human insulin; Selected from.

[0019] In another embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of an insulin derivative of formula (I) or a salt or solvate thereof, and one or more pharmaceutically acceptable excipients, diluents, carriers or vehicles.

[0020] In one embodiment of the present invention, the pharmaceutical composition is an injectable or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, liquid, microneedle, suspension, or syrup.

[0021] In the present invention, as one embodiment, the pharmaceutical composition is in the form of microcapsules, microspheres, nanoparticles, or liposomes.

[0022] In one embodiment of the present invention, the pharmaceutical composition is intended for oral administration, inhalation administration, transdermal administration or parenteral administration, and parenteral administration is selected from intraperitoneal, intramuscular, intra-arterial, intravenous, subcutaneous or intradermal injection.

[0023] In one embodiment of the present invention, the pharmaceutical composition is administered at a frequency of at least once a day, once a week, or once a month.

[0024] The present invention provides the use of an insulin derivative of formula (I) or a salt or solvate thereof in the preparation of a pharmaceutical for the treatment, prevention or alleviation of diseases including diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity and related disorders.

[0025] The present invention provides a combination of an insulin derivative of formula (I) or a salt or solvate thereof with other drugs for treating the same or related disorders. Other drugs include, but are not limited to, metformin, sulfonylurea, SGLT-1 / 2 inhibitors, DPP-4 inhibitors, insulin and its analogues, GLP-1 and its analogues, GCG and its analogues, GIP and its analogues, FGF-21 and its analogues, or multi-target drugs from among the above drugs, or two or more of them.

[0026] Insulin derivatives of formula (I) according to the present invention exhibit better drug efficacy or efficiency, longer duration of action, and beneficial bioavailability and safety. [Brief explanation of the drawing]

[0027] [Figure 1] Figures 1a and 1b show the effects of subcutaneously administered solvent, Icodec, and compound 2 on lowering blood glucose levels in normal SD rats in Experimental Example 4. [Figure 2] Figures 2a and 2b show the effects of subcutaneously administered solvent, Icodec, compound 3, and compound 4 on lowering blood glucose levels in normal SD rats in Experimental Example 4. [Figure 3] Figures 3a and 3b show the blood glucose-lowering effects of intravenous administration of the solvent, Icodec, and compound 2 in normal beagle dogs in Experimental Example 5. [Modes for carrying out the invention]

[0028] The following examples and experimental illustrations are provided to further illustrate the present invention, but are not intended to limit the effective scope of the invention. [Examples]

[0029] Example 1: Preparation of Compound 2 The molecular formula and structural formula of compound 2 are as follows: A14E, B16H, B25H, B29K(N ε -[1-(19-carboxyl-nonadecyloxy)-β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB30 human insulin;

[0030] [ka]

[0031] Preparation of fatty acid derivatives (1) Preparation of methyl 1,2,3,4-tetraacetoxy-D-glucopyranuronate

[0032] [ka]

[0033] A solution of D-glucuronolactone (CAS32449-92-6) (100 g, 567 mmol, 1.00 equivalent) in MeOH (300 mL) was mixed with NaOH (567 mg, 14.1 mmol, 0.025 equivalent). The mixture was stirred at 20°C for 16 hours. The mixture was concentrated under vacuum, and pyridine (179 g, 2.27 mol, 183 mL, 4.00 equivalent) and Ac2O (289 g, 2.84 mol, 265 mL, 5.00 equivalent) were added at 5°C. The resulting mixture was stirred at 20°C for 3 hours. The reaction mixture was cooled to 0°C and filtered to obtain a filter cake. The filter cake was pulverized with ethanol (100 mL) at 20°C for 10 hours. Next, the mixture was cooled to 0°C, filtered, and the filtered cake was concentrated under reduced pressure to obtain methyl 1,2,3,4-tetraacetoxy-D-glucopyranuronate (43.9 g) as a white solid. 1H NMR:400 MHz, CDCl3. δ: 5.77 (d, J = 7.6 Hz, 1H), 5.32 - 5.23 (m, 2H), 5.17 - 5.13 (m, 1H), 4.18 (d, J = 9.2 Hz, 1H), 3.75 (s, 3H), 2.12 (s, 3H), 2.04 - 2.04 (m, 9H).

[0034] (2) Preparation of methyl 1-bromo-2,3,4-triacetoxy-α-D-glucopyranuronate

[0035] [ka]

[0036] At 0°C, methyl 1,2,3,4-tetraacetoxy-D-glucopyranuronate (43.9 g, 116 mmol, 1.00 equivalent) was added to a solution of hydrogen bromide in acetic acid (150 mL, 33% wt). The mixture was stirred at 20°C for 3 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was pulverized with ethanol (50 mL) at 20°C for 10 hours, cooled to 0°C, and filtered. The filtered cake was concentrated under reduced pressure to obtain methyl 1-bromo-2,3,4-triacetoxyl-α-D-glucopyranuronate (28.5 g) as a white solid. 1 H NMR: 400 MHz, CDCl3. δ: 6.64 (d, J = 4.0 Hz, 1H), 5.62 (t, J = 9.6 Hz, 1H), 5.27 - 5.22 (m, 1H), 4.86 (dd, J1 = 4.0 Hz, J2 = 10.0 Hz, 1H), 4.59 (d, J = 10.4 Hz, 1H), 3.77 (s, 3H), 2.10 (s, 3H), 2.06 (d, J = 2.5 Hz, 6H).

[0037] (3) Preparation of tert-butyl 20-hydroxyicosanoate

[0038] [ka]

[0039] Eicosanedioic acid mono-tert-butyl (180 g, 451 mmol, 1.00 equivalent) and N-methylmorphine (54.8 g, 541 mmol, 1.10 equivalents) were added to a tetrahydrofuran solution (1000 mL). The mixture was cooled to -10 °C under nitrogen, and isobutyl chloroformate (61.6 g, 451 mmol, 1.10 equivalents) was added. The mixture was then stirred at -10 °C for 1 hour and filtered. At 0 °C, sodium borohydride (25.6 g, 677 mmol, 1.50 equivalents) was added to the filtrate under nitrogen. After the reaction was complete, the mixture was filtered and the filtrate was concentrated under reduced pressure to obtain a residue. tert-Butyl 20-hydroxyicosanoic acid (29.0 g) was obtained as a white solid by purification by column chromatography (silica, petroleum ether:ethyl acetate = 100:1:1, Rf = 0.27). 1 H NMR: 400 MHz, 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).

[0040] (4) Preparation of methyl 1-((20-(tert-butoxyl)-20-oxoicosyl)oxy)-2,3,4-triacetoxy-β-D-glucopyranuronate

[0041]

Chemical formula

[0042] 29.0 g, 75.5 mmol, 2.00 equivalents of tert-butyl 20-hydroxyicosanoate, 43.7 g, 188 mmol, 5.00 equivalents of silver oxide, 17.2 g, 67.9 mmol, 1.80 equivalents of iodine, and 102 g, 755 mmol, 20.00 equivalents of calcium sulfate were added to 150 mL of chloroform solution. The mixture was stirred under nitrogen at 20°C for 15 minutes. Then, a solution of methyl 1-bromo-2,3,4-triacetoxy-α-D-glucopyranuronate (15.0 g, 37.7 mmol, 1.00 equivalent) in 50 mL of chloroform solution was added. The resulting mixture was stirred at 30°C for 18 hours. After the reaction, the filtrate was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (silicon dioxide, petroleum ether:ethyl acetate = 100:1 to 1:1, petroleum ether:ethyl acetate = 3:1) (Rf = 0.30) to obtain 1-((20-(tert-butoxy)-20-oxoicosyl)oxy)-2,3,4-triacetoxy-β-D-glucopyranuronate methyl (9.28 g) as a white oily substance. LC-MS: m / z = 723.0 [M + Na] + This matches the theoretical value. 1 H NMR: 400 MHz, CDCl3.δ: 5.30 - 5.22 (m, 2H), 5.03 - 4.98 (m, 1H), 4.54 (d, J = 7.6 Hz, 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 = 7.6 Hz, 2H), 2.03 (d, J = 7.2 Hz, 9H), 1.60 - 1.53 (m, 5H), 1.44 (s, 9H), 1.27 - 1.25 (m, 30H).

[0043] (5) Preparation of 1-((20-(tert-butoxy)-20-oxoicosyl)oxy)-β-D-glucopyranuronic acid

[0044] [ka]

[0045] ((20-(tert-butoxy)-20-oxoicosyl)oxy)-2,3,4-triacetoxyl-β-D-glucopyranuronic acid methyl (9.28 g, 13.2 mmol, 1.00 equivalent) and sodium methoxide (35.7 mg, 662 μmol, 0.05 equivalent) were added to methanol solution (50 mL). The mixture was stirred at 20°C for 5 hours. Then, lithium hydroxide monohydrate (555 mg, 13.2 mmol, 1.00 equivalent) was added. The mixture was stirred at 20°C for 17 hours. The reaction mixture was concentrated under reduced pressure to obtain 1-((20-(tert-butoxy)-20-oxoicosyl)oxy)-β-D-glucopyranuronic acid (11.3 g) as a pale yellow solid. LC-MS: m / z = 599.3 [MH] - This matches the theoretical value. 1 H NMR: 400 MHz, MeOD.δ: 4.28 (d, J = 8.0 Hz, 1H), 3.92 - 3.68 (m, 1H), 3.65 - 3.48 (m, 1H), 3.45 - 3.40 (m, 2H), 3.38 (t, J = 9.2 Hz, 1H), 3.21 (dd, J1 = 7.6 Hz, J2 = 9.1 Hz, 1H), 2.20 (t, J = 7.6 Hz, 2H), 1.64 - 1.54 (m, 4H), 1.44 (s, 9H), 1.39 - 1.36 (m, 2H), 1.29 (br s, 28H).

[0046] Preparation of intermediate 1 of compound 2 (1) Resin preparation: 2-CTC resin (150.0 mmol, 1.00 equivalent, substitution: 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 under nitrogen at 20°C for 2 hours. Then methanol (150.0 mL) was added and stirring was continued for a further 30 minutes. The resin was then mixed with N,N-dimethylformamide (1500 mL) * 5) The material was washed, then filtered, to obtain the resin.

[0047] (2) Deprotection: A resin-containing solution of N,N-dimethylformamide (1500 mL, containing 20% ​​piperidine) was stirred under nitrogen for 30 minutes. Then the resin was removed from the N,N-dimethylformamide (1500 mL) * 5) The material was washed and filtered to obtain the resin.

[0048] (3) Coupling: Fmoc-Ile-OH (450 mmol, 3.00 equivalents), DIEA (900 mmol, 6.00 equivalents), and HBTU (427.5 mmol, 2.85 equivalents) were added to a resin-containing solution (1000 mL) of N,N-dimethylformamide. The mixture was stirred under nitrogen at 20°C for 30 minutes. The resin was then coupled to N,N-dimethylformamide (1500 mL) * It was cleaned in step 5).

[0049] (4) Repeat steps (2) to (3) above to perform coupling for the following materials.

[0050] [Table 1]

[0051] (5) The resin is mixed with methanol (2400 mL) *3) The peptide resin was washed and dried under vacuum to obtain the peptide resin. Next, cleavage buffer (2400 mL) (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% mercaptopropionic acid) was added at 20°C to the flask containing the side-chain protected peptide resin, and the mixture was stirred for 2 hours. The peptide fragments were precipitated with cold isopropyl ether (10000 mL), filtered, and the filter cake was collected. The filter cake was then washed twice with isopropyl ether (50000 mL).

[0052] (6) The filter cake was dried under vacuum for 2 hours, and then dissolved in N,N-dimethylformamide (DMF) (30.0 L). The solution was maintained at a constant temperature of 20°C, and 0.1 M methanol-iodine solution was added dropwise until the yellow color disappeared. After stirring for 2 minutes, 0.1 M aqueous solution of sodium thiosulfate was added dropwise until the yellow color disappeared. The mixture was then freeze-dried to obtain intermediate 1 (180 g) as a white solid. LC-MS: m / z = 920.5 [M + H] + This matches the theoretical value.

[0053] Preparation of intermediate 2 of compound 2 (1) Resin preparation: 2-CTC resin (70.0 mmol, 1.00 equivalent, substitution: 1.00 mmol / g), Fmoc-Gln(Trt)-OH (70.0 mmol, 1.00 equivalent), and DIEA (280 mmol, 4.00 equivalent) were added separately to a dichloromethane solution. The mixture was stirred under nitrogen at 20°C for 2 hours. Then methanol (70.0 mL) was added and stirred for another 30 minutes. The resin was then mixed with N,N-dimethylformamide (700 mL) * 5) The material was washed and filtered to obtain the resin.

[0054] (2) Deprotection: A resin-containing solution of N,N-dimethylformamide (700 mL, containing 20% ​​piperidine) was stirred under nitrogen for 30 minutes. Then the resin was removed from the N,N-dimethylformamide (700 mL * 5) The material was washed and filtered to obtain the resin.

[0055] (3) Coupling: Fmoc-Glu(tBu)-OH (210 mmol, 3.00 equivalents), DIEA (420 mmol, 6.00 equivalents), and HBTU (199.5 mmol, 2.85 equivalents) were added to a resin-containing solution (400 mL) of N,N-dimethylformamide. The mixture was stirred under nitrogen at 20°C for 30 minutes. The resin was then coupled to N,N-dimethylformamide (700 mL) * It was cleaned in step 5).

[0056] (4) Repeat steps (2) to (3) above to perform coupling for the following materials.

[0057] [Table 2]

[0058] (5) The resin is mixed with methanol (1000 mL) * 3) The peptide resin was washed and vacuum-dried to obtain the peptide resin. Next, 1000 mL of cleavage buffer (20% hexafluoroisopropanol / 80% dichloromethane) was added to the flask containing the side-chain protected peptide resin at room temperature, with stirring performed twice for 30 minutes each time. Intermediate 2 (61.77 g) was obtained as a white solid by pressure concentration. LC-MS: m / z = 943.7 [M + H] + This matches the theoretical value.

[0059] Preparation of intermediate 3 of compound 2 (1) Resin preparation: 2-CTC resin (20.0 mmol, 1.00 equivalent, substitution: 1.00 mmol / g), Fmoc-Asn(Trt)-OH (20.0 mmol, 1.00 equivalent), and DIEA (80.0 mmol, 4.00 equivalent) were added separately to a dichloromethane solution (150 mL). The mixture was stirred under nitrogen at 20°C for 2 hours. Then methanol (20.0 mL) was added and stirred for another 30 minutes. The resin was then mixed with N,N-dimethylformamide (300 mL) * 5) The material was washed and filtered to obtain the resin.

[0060] (2) Deprotection: A resin-containing solution of N,N-dimethylformamide (300 mL, containing 20% ​​piperidine) was stirred under nitrogen for 30 minutes. Then the resin was removed from the N,N-dimethylformamide (300 mL * 5) The material was washed and filtered to obtain the resin.

[0061] (3) Coupling: Fmoc-Cys(Trt)-OH (60.00 mmol, 3.00 equivalents), DIEA (120 mmol, 12.00 equivalents), and HBTU (57.0 mmol, 2.85 equivalents) were added to a resin-containing solution (300 mL) of N,N-dimethylformamide. The mixture was stirred under nitrogen at 20°C for 30 minutes. The resin was then coupled to N,N-dimethylformamide (300 mL) * It was cleaned in step 5).

[0062] (4) Repeat steps (2) to (3) above to perform coupling for the following materials.

[0063] [Table 3]

[0064] (5) A resin-containing solution of N,N-dimethylformamide (300 mL, containing 20% ​​piperidine) was stirred under nitrogen for 30 minutes. Then the resin was mixed with N,N-dimethylformamide (300 mL * 5) The material was washed and filtered to obtain the resin.

[0065] (6) The resin is mixed with methanol (2400 mL * 3) The peptide resin was washed and vacuum-dried to obtain the peptide resin. Next, 400 mL of cleavage buffer solution (95% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water) was added at 20°C to the flask containing the side-chain protected peptide resin, and the mixture was stirred for 2 hours. The peptide fragments were precipitated using cold isopropyl ether (2000 mL), filtered, and the filter cake was collected. The filter cake was then washed twice with isopropyl ether (1000 mL).

[0066] (7) The crude peptide is dried in a vacuum for 2 hours, then dissolved in N,N-dimethylformamide and purified by high-performance liquid chromatography to obtain intermediate 3 (1.9 g) as a white solid. LC-MS: m / z = 1210.0 [M + 2H] 2+ This matches the theoretical value.

[0067] [Table 4]

[0068] Preparation of intermediate 4 of compound 2 (1) Resin preparation: 2-CTC resin (2.00 mmol, 1.00 equivalent, substitution: 1.00 mmol / g), Fmoc-Lys(Dde)-OH (2.00 mmol, 1.00 equivalent), and DIEA (8.00 mmol, 4.00 equivalent) were separately added to a solution of dichloromethane (20.0 mL). The mixture was stirred under nitrogen at 20°C for 2 hours. Then methanol (4.00 mL) was added and stirred for another 30 minutes. The resin was then mixed with N,N-dimethylformamide (20.0 mL) * 5) The material was washed and filtered to obtain the resin.

[0069] (2) Deprotection: A resin-containing solution of N,N-dimethylformamide (20.0 mL, containing 20% ​​piperidine) was stirred under nitrogen for 30 minutes. Then the resin was removed from the N,N-dimethylformamide (20.0 mL * 5) The material was washed and filtered to obtain the resin.

[0070] (3) Coupling: Fmoc-Pro-OH (6.00 mmol, 3.00 equivalents), DIEA (6.00 mmol, 12.00 equivalents), and HBTU (5.70 mmol, 2.85 equivalents) were added to a resin-containing solution (10.0 mL) of N,N-dimethylformamide. The mixture was stirred under nitrogen at 20°C for 30 minutes. The resin was then mixed with N,N-dimethylformamide (20.0 mL) * 5) Wash.

[0071] (4) Repeat steps (2) to (3) above to perform coupling on the following materials (1 to 27).

[0072] [Table 5] TIFF2026517987000015.tif71170

[0073] (5) Add 3% hydrazine hydrate / N,N-dimethylformamide (20.0 mL) and react for 30 minutes. * 5) was cleaned. Repeat steps (2) to (3) above to couple the materials (28 to 31).

[0074] (6) Wash with methanol and dry under vacuum to obtain peptide resin. Then add 150 mL of cutting buffer solution (92.5% trifluoroacetic acid / 2.5% triisopropylsilane / 2.5% water / 2.5% mercaptopropionic acid) and stir the mixture at 20°C for 2 hours. Precipitate the peptide fragments by using cold isopropyl ether (750 mL), filter, and collect the filter cake. Wash this twice with isopropyl ether (400 mL). Dry the crude peptide under vacuum for 2 hours.

[0075] (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 dimethyl sulfoxide solution (25.0 mL). The mixture was stirred at 20°C for 10 minutes.

[0076] (8) The crude peptide was purified by preparative high-performance liquid chromatography to obtain intermediate 4 (1.9 g) as a white solid. LC-MS: m / z = 1095.8 [M + 4H] 4+ This matches the theoretical value.

[0077] [Table 6]

[0078] Preparation of intermediate 5 for compound 2 Intermediate 3 (243.0 mg, 1.10 equivalents), intermediate 4 (400.0 mg, 1.00 equivalent), and DIEA (11.80 mg, 1.00 equivalent) were added to dimethyl sulfoxide solution (10 mL). The mixture was stirred at 20°C for 10 minutes.

[0079] After the reaction, the mixture was filtered to remove insoluble substances. The crude peptide was purified by high-performance liquid chromatography to obtain intermediate 5 (200.5 mg) as a white solid. LC-MS: m / z = 1636.9 [M + 4H] 4+ This matches the theoretical value.

[0080] [Table 7]

[0081] Preparation of Compound 2 Intermediate 5 (200.5 mg, 1.00 equivalent), 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 equivalent) were mixed. The mixture was stirred at 20°C for 10 minutes.

[0082] After the reaction, the mixture was filtered to remove insoluble substances. The crude peptide was purified using high-performance liquid chromatography to obtain the final product, compound 2 (13.7 mg), as a white solid. LC-MS: m / z = 1636.6 [M + 4H] 4+ This matches the theoretical value.

[0083] [Table 8]

[0084] Example 2: Preparation of Compound 1 The molecular formula and structural formula of compound 1 are as follows: A14E, B16H, B25H, B29K(N ε -[1-(19-carboxyl-nonadecyloxy)-β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB30 human insulin

[0085] [ka]

[0086] Compound 1 was prepared by removing the Fmoc-γGlu(OtBu)-OH listed in Table 5, using the method described in Example 1. All other materials and preparation methods were left unchanged. LC-MS: m / z = 1604.3 [M + 4H] 4+ This matches the theoretical value.

[0087] Example 3: Preparation of Compound 3 The molecular formula and structural formula of compound 3 are as follows: A14E, B16H, B25H, B29K(N ε -[1-(19-carboxyl-nonadecyloxy)-β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB27,desB30 human insulin

[0088] [ka]

[0089] Compound 3 was prepared according to the method described in Example 1, but Fmoc-γGlu(OtBu)-OH and Fmoc-Thr(tBu)-OH in Table 5 were removed. All other materials and preparation methods remained unchanged. LC-MS: m / z = 1579.3 [M + 4H] 4+ This matches the theoretical value.

[0090] Example 4: Preparation of Compound 4 The molecular formula and structural formula of compound 4 are as follows: A14E, B16H, B25H, B29K(N ε -[1-(19-carboxyl-nonadecyloxy)-β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB27,desB30 human insulin;

[0091] [ka]

[0092] Compound 4 was prepared according to the method described in Example 1 by removing the Fmoc-Thr(tBu)-OH listed in Table 5. All other materials and preparation methods were left unchanged. LC-MS: m / z = 1611.6 [M + 4H] 4+ This matches the theoretical value.

[0093] Compounds 1, 2, 3, and 4, given by the present invention, are shown in Table 9.

[0094] [Table 9]

[0095] Experimental Example 1: Study on the glucose uptake ability of adipocytes by the compound of the present invention. 1. Induction of adipogenesis in 1.3T3-L1 mouse fetal fibroblasts 1) Cell plating: Adipocytes were plated in 200 μL / well of maintenance medium 1 (DMEM, 1% penicillin-streptomycin, 10% fetal bovine serum, 1 μg / mL human insulin) in 4 × 10⁶ cells. 4 Cells were seeded in 96-well cell culture plates at a cell / well density. Culture was performed in a cell incubator at 10% CO2 and 37°C.

[0096] 2) After 2 days, switch to standard medium 2 (DMEM, 1% biantibody, 10% fetal bovine serum) and continue culturing.

[0097] 3) After 2 days, the cells exhibit the adipocyte phenotype and are ready for the glucose uptake assay in adipocytes.

[0098] 2. Procedure for adipocyte glucose uptake assay 1) KRPH buffer preparation: 5mM Na2HPO4, 20mM HEPES, 1mM MgSO4, 1mM CaCl2, 136mM NaCl, 4.7mM KCl, pH 7.40.

[0099] 2) On the day of the assay, the adipocyte culture medium was replaced with serum-free DMEM medium 3, and the cells were starved for 2 hours.

[0100] 3) Sample Dilution: All sample storage solutions were diluted with KRPH buffer solution. Icodec, compound 1, and compound 2 were subjected to 3-fold serial gradient dilutions over 10 points at a starting concentration of 25 μM.

[0101] 4) Starvated adipocytes were processed using a Well Vario liquid workstation. After rinsing with KRPH buffer, 50 μL of gradient-diluted samples of various concentrations were added to each well in a double-well system for each concentration point. Incubation was performed for 10 minutes.

[0102] 5) 0.25 μCi [ 3 50 μL / well of KRPH buffer containing [H]-deoxyglucose and 50 μM deoxyglucose was added, and then incubated in a CO2 incubator for 20 minutes.

[0103] 6) The cells were rinsed three times with 10 mM glucose-containing DPBS pre-cooled to 4°C, using 200 μL per well for each wash.

[0104] 7) The adipocytes were lysed with 100 μL of 10% sodium hydroxide solution per well, and then shaken on a shaker for 20 minutes.

[0105] 8) The cut samples were transferred to scintillation vials, 2 mL of scintillation solution was added, and scintillation counting was performed using Tri-Carb.

[0106] Finally, the data was analyzed using GraphPad Prism 7.

[0107] [Table 10]

[0108] Results Analysis: The table clearly shows that compound 1 and compound 2 are superior to Icodec in terms of enhancing the glucose uptake capacity of adipocytes.

[0109] Experimental Example 2: Human Serum Albumin (HSA) Binding Study 1. To study the in vitro binding of insulin derivatives to human serum albumin (HSA) via the activation of insulin receptor B (IRB) phosphorylation.

[0110] Cells were seeded at a density of 10,000 cells per well in a 384-well cell culture plate using a medium containing 5% serum. The cells were incubated overnight in a 37°C incubator containing 5% CO2. Icodec, compound 1, and compound 2 were lysed and diluted in F12 medium containing 0.1% HSA and 0.1% casein, respectively. The starting concentration for the test was 60 μM, and all samples were subjected to a 4-fold serial gradient dilution over 10 points. The cell plate was removed from the incubator, the medium was removed by centrifugation using a plate washer, the diluted samples were immediately added, and the mixture was reacted at room temperature for 5 minutes. The treated cell samples were discarded by using a plate washer, cell lysis buffer was immediately added, and the mixture was lysed by shaking thoroughly on a shaker. The cell lysates were diluted with 1× cell lysis buffer. 10 μL of the diluted cell lysates were transferred to an Optiplate 384-well plate. The Acceptor Beads mixture was added, the plate was sealed with aluminum foil, the mixture was thoroughly mixed, and left to stand for incubation at room temperature. Then, the Donor Beads mixture (protected from light) was added, the mixture was thoroughly mixed, and left to stand for incubation at room temperature. The plate was read using an Envision instrument in AlphaScreen mode. Finally, the data was analyzed using a GraphPad Prism 7.

[0111] [Table 11]

[0112] Results Analysis: As shown in the table above, compound 1 and compound 2 exhibit stronger and tighter binding to HSA compared to Icodec.

[0113] Note: In the detection method described in this invention, 0.1% human serum albumin (HSA) is typically added during the insulin derivative-activated IRB phosphorylation assay. Fatty acid-acylated insulin can bind tightly to HSA, affecting the results by artificially reducing the apparent efficacy of the compound. This problem can be avoided by using 0.1% casein instead. The improvement observed after casein administration can be considered an indicator of relatively tight binding of the compound to serum albumin.

[0114] 2. Determination of in vitro binding of insulin derivatives to HSA via SPR. Binding of Icodec, compound 2, compound 3, and compound 4 to human serum albumin on the Biacore 8K instrument.

[0115] 2.1 Experimental Procedure 2.1.1 Protein coupling Human serum albumin is immobilized onto the surface of the S-series CM5 sensor chip according to the instructions for use of the Amine Coupling Kit, Type 2 (Cytiva). The specific procedure is as follows: a) EDC and NHS were mixed in a 1:1 ratio. Human serum albumin (HSA) was diluted to 20 μg / mL using a pH 4.5 acetic acid solution. The EDC / NHS mixture solution, HSA protein solution, and 1 M ethanolamine-hydrochloric acid solution were added to the detection plate according to the program settings.

[0116] b) The detection plate was placed in the sample compartment of the Biacore 8K. The EDC / NHS mixture solution, HSA protein solution, and 1M ethanolamine-hydrochloride solution were sequentially injected into the channels on the surface of the CM5 chip by the Biacore 8K according to the program. Through this process, the chip was activated, ligand-coupled, and deactivated. Each channel consisted of two circulation pools, with circulation pool 1 acting as a reference channel that only undergoes surface activation and deactivation without injection of the HSA protein solution. The specific parameters were as follows:

[0117] [Table 12]

[0118] 2.1.2 Sample Testing a) Stock solutions of Icodec, compound 2, compound 3, and compound 4 were diluted with running PBST buffer solution (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, 0.05% Tween-20). All samples were subjected to a 2x serial gradient dilution starting at 200 μM at six points.

[0119] b) The prepared sample Icodec, compound 2, compound 3, compound 4, and running buffer were transferred to a detection plate according to the program. The detection plate was placed in the sample compartment of the Biacore 8K, and the running buffer served as a 0 concentration reference sample.

[0120] c) Inject the sample sequentially into each channel of the chip according to the program and execute the detection program. The specific parameters are as follows:

[0121] [Table 13]

[0122] 2.1.3 Data Analysis Before analyzing the raw data, it needs to be processed according to the following steps: a) Set the start time for the injection of all concentrations of compounds and buffer solutions to 0 seconds.

[0123] b) Set the baseline response value for all concentrations and all channels to 0RU.

[0124] c) For each channel, the corresponding reference channel response value is subtracted from the test channel response value to obtain response values ​​for samples of different concentrations.

[0125] d) The final sensing map is obtained by subtracting the response value of the zero-concentration sample from the response values ​​of the sample at different concentrations.

[0126] Finally, the data was analyzed using BiaCore Insight Evaluation Software with a 1:1 coupled model for analysis.

[0127] [Table 14]

[0128] Results Analysis: This table clearly shows that compounds 2, 3, and 4 exhibit superior binding affinity to HSA compared to Icodec.

[0129] Experimental Example 3: Half-life of insulin derivatives Six healthy male beagle dogs, 8-9 months old and weighing 7-10 kg, were selected from a reserve group. During the experiment, the animals were housed individually with free access to food and water.

[0130] After a one-week adaptation period, the experiment was initiated. Beagles were divided into two treatment groups based on body weight, with three dogs per group. The administered dose was 4 nmol / kg for both groups. Icodec and compound 2 were administered intravenously in a solvent containing 5 mM disodium hydrogen phosphate, 140 mM sodium chloride, and 70 ppm Tween 20, with a solvent pH of 8.0.

[0131] Icodec and compound 2 were dissolved separately in a solvent to a concentration of 40 nmol / mL. The dosage volume was 0.1 mL / kg. A single intravenous dose was administered. Blood samples were collected at 0.05 hours, 0.167 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, 32 hours, 48 ​​hours, 72 hours, 96 hours, 144 hours, 192 hours, and 216 hours after administration to determine their plasma concentrations of the administered compounds. Animals were fasted at the time of sampling; at other time points, they had free access to food and water. Finally, the data were analyzed using Phoenix WinNonlin 6.3.

[0132] [Table 15]

[0133] Results Analysis: This table clearly demonstrates that compound 2 exhibits a superior half-life compared to Icodec in intravenous administration studies in normal beagles.

[0134] Experimental Example 4: Pharmacodynamic studies in normal SD rats Normal male SD rats aged 8-9 weeks and weighing 280-320g were selected. The animals were housed in an animal containment facility with strictly controlled environmental conditions, maintaining a temperature between 20°C and 24°C and a humidity between 30% and 70%. During the experiment, the animals were housed individually with free access to food and water.

[0135] After a one-week adaptation period, the experiment was initiated. Rats were assigned to solvent group 1, solvent group 2, and treatment group, with 8 rats per group, based on pre-administration body weight and blood glucose levels. All administered doses were 650 nmol / kg. These were either subcutaneous injections of the solvent or subcutaneous injections of Icodec, compound 2, compound 3, and compound 4, respectively. The solvents described in solvent group 1 and solvent group 2 contained 30 mM phenol and 1.6% (w / v) glycerol, and the pH of the solvent was 8.0.

[0136] The above drugs were dissolved in a solvent to a concentration of 216.7 nmol / mL. The dosage volume was 3 mL / kg. For single subcutaneous administration, with the administration time set as 0 hours, blood glucose levels were measured at 0 hours before administration, and at 4, 8, 24, 28, 48, 72, 96, and 120 hours after administration (no dietary restrictions). Dose-response curves of blood glucose over time were plotted for each single dose of insulin derivatives.

[0137] To demonstrate the effect of the insulin derivative of the present invention on blood glucose levels, the area under the blood glucose-time curve (AUC) was calculated for each individual dose-response curve from zero to the monitoring endpoint. A smaller AUC value indicates a better blood glucose-lowering effect and a more effective drug.

[0138] [Table 16]

[0139] [Table 17]

[0140] As shown in Table 4.1, Figures 1a and 1b, and Table 4.2, Figures 2a and 2b, the blood glucose-lowering effects of insulin derivative compounds 2, 3, and 4 of the present invention are superior to those of Icodec.

[0141] Experimental Example 5: Pharmacodynamic Study of Normal Beagles Twelve healthy male beagle dogs, 8-9 months old and weighing 7-10 kg, were selected from a reserve group. During the experiment, the animals were housed individually with free access to food and water.

[0142] After a one-week adaptation period, the experiment was initiated. Beagles were assigned to either a solvent group or a treatment group, with four dogs in each group, based on their pre-administration body weight and blood glucose levels. The administered dose was 21 nmol / kg in all cases. This consisted of intravenous injection of either the solvent or Icodec and compound 2, respectively. The solvent contained 5 mM disodium hydrogen phosphate, 140 mM sodium chloride, and 70 ppm Tween 20, with a solvent pH of 8.0.

[0143] The above drugs were dissolved in a solvent to a concentration of 210 nmol / mL. The dosage volume was 0.1 mL / kg. A single intravenous dose was administered, with the administration time set as the 0 point. Blood glucose levels were measured at 0 hours before administration, and at 2, 24, 48, and 72 hours after administration. The animals were fasted at the time of sampling; at other time points, they had free access to food and water. Dose-response curves of blood glucose over time were plotted for each single dose of insulin derivatives.

[0144] To demonstrate the effect of the insulin derivative of the present invention on blood glucose levels, the area under the blood glucose-time curve (AUC) was calculated for each individual dose-response curve from zero to the monitoring endpoint. A smaller AUC value indicates a better blood glucose-lowering effect and a more effective drug.

[0145] [Table 18]

[0146] As shown in Table 5.1 and Figures 3a and 3b, the blood glucose-lowering effect of insulin derivative compound 2 of the present invention is superior to that of Icodec.

Claims

1. Equation (I): 【Chemistry 1】 (In the formula, Z is CH ３ or COOH; n is 17, 18, 19, 20, or 21; X is either γGlu or does not exist; Y is -NH-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-NH-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO- or does not exist; (R is an insulin precursor in which the ε-amino group of the lysine side chain at position 29 of the B chain is connected to Y via an amide bond.) Insulin derivatives or their salts or solvates.

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 insulin derivative of formula (I) is Compound 1: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxyl-nonadesyloxy)-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-carboxyl-nonadesyloxy)-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-carboxyl-nonadesyloxy)-β-D-glucuronyl]-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB27,desB30 human insulin; Compound 4: A14E, B16H, B25H, B29K (N ε -[1-(19-carboxyl-nonadecyloxy)-β-D-glucuronyl]-γGlu-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetyl),desB27,desB30 Human Insulin The derivative according to claim 1, characterized by being selected from among.

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

5. A pharmaceutical composition comprising an effective amount of an insulin derivative of formula (I) or a salt or solvate thereof, and a pharmaceutically acceptable adjuvant, diluent, carrier, or excipient.

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

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

8. The pharmaceutical composition according to claim 5, intended for oral administration, inhalation administration, transdermal administration or parenteral administration, wherein the parenteral administration is selected from intraperitoneal, intramuscular, intra-arterial, intra-venous, subcutaneous, or intradermal injection.

9. The pharmaceutical composition according to claim 6, characterized in that it 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, further comprising metformin, sulfonylurea, SGLT-1 / 2 inhibitor, DPP-4 inhibitor, insulin and its analogues, GLP-1 and its analogues, GCG and its analogues, GIP and its analogues, FGF-21 and its analogues, or multi-target drugs from among the above drugs, or two or more of these.

11. Use of an insulin derivative of formula (I) or a salt or solvate thereof according to any one of claims 1 to 4, or a pharmaceutical composition according to any one of claims 5 to 10, in the preparation of a pharmaceutical for the treatment, prevention or reduction of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia or obesity.

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