Long-acting insulin compounds

Abstract

The present invention relates to the field of pharmaceutical synthesis and discloses a long-acting insulin compound. The insulin compound according to the present invention is used to prepare a pharmaceutical composition for treating diseases. The pharmaceutical composition is used to treat various diseases such as type 1 diabetes, type 2 diabetes, and gestational diabetes.

Description

[Technical Field]

[0001] [Cross reference] This application claims priority from a Chinese patent application filed with the State Intellectual Property Office of the People's Republic of China on August 10, 2022, bearing application number 202210956232.9 and entitled "Long-acting insulin compound," the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of pharmaceutical synthesis, and in particular to long-acting insulin compounds. [Background technology]

[0003] Insulin is a protein hormone secreted by pancreatic beta cells in the pancreas under the stimulation of endogenous or exogenous substances, such as glucose, lactose, ribose, arginine, and glucagon. Insulin is the only hormone that lowers blood glucose levels in the body and also promotes the synthesis of glycogen, fat, and protein. Exogenous insulin is primarily used to treat diabetes.

[0004] Unlike other recombinant protein drugs, insulin, as a hormone that regulates blood glucose levels, has a very narrow therapeutic window. Higher blood glucose levels can lead to hypoglycemia, shock, or death, while lower levels can lead to hyperglycemia. Therefore, blood glucose levels must be strictly controlled. Therefore, insulin cannot be administered intravenously; it must be administered subcutaneously and slowly released into the blood. Furthermore, because three daily carbohydrate intakes are regulated by mealtime insulin and basal blood glucose levels are regulated by basal insulin, the physiological insulin curve is very complex. Therefore, the design of rapid-acting and long-acting insulins is significant.

[0005] Insulin preparations are typically formulated at 100 IU / ml, and at this concentration, insulin typically exists in the form of a dimer followed by a hexamer. After subcutaneous injection, insulin gradually releases dimers and monomers from the hexamer, which then enter the bloodstream and exert their effects. Insulin has a duration of action of 4-6 hours, which is longer than that of mealtime insulin, but it is prone to hypoglycemia 2 hours after a meal and shorter than that of basal insulin. Protamine binds to insulin to form a precipitate, which allows the insulin to dissolve and be released slowly after subcutaneous injection, extending its duration of action to more than 10 hours. It is typically administered twice daily. While protamine insulin has solved some of these problems, it still does not match the physiological insulin secretion curve, and its precipitation properties require it to be formulated as a suspension, making it difficult to accurately control the dosage.

[0006] Sanofi's insulin glargine is manufactured by adding two alkaline amino acids, arginine, to the ends of its B chain, changing its isoelectric point from 5.4 to 6.7. This allows a precipitate to form near its isoelectric point after subcutaneous injection, which then slowly dissolves and is released into the bloodstream, providing a sustained-release effect similar to that of protamine. However, because insulin glargine cannot be dissolved unless it is prepared as an acidic product due to its change in isoelectric point, the asparagine at A21, which is prone to deamidation, was substituted with glycine (hence the name insulin glargine). Insulin glargine has a half-life of 12 hours and a duration of action of 20-24 hours, and is administered by once-daily injection.

[0007] Novo Nordisk developed a next-generation long-acting insulin, degludec, which successfully received marketing approval in 2013. Insulin degludec removes the threonine at position B30 and attaches a 16-carbon fatty acid chain to the lysine at position B29 via a glutamic acid linker, which can bind to albumin in plasma and extend the insulin's half-life. Its mechanism of action is the same as that of insulin detemir. In addition, insulin degludec exhibits a double hexamer form in the formulation by optimizing the ratio of linker, fatty acid chain, and zinc ion. After subcutaneous injection, the diffusion of phenol causes a conformational change in insulin degludec, which rapidly assembles from the double hexamer to a linear multihexamer, typically several thousand molecules long. The multihexamer then slowly releases hexamers, dimers, and monomers, allowing them to enter the bloodstream and exert their effects. These two mechanisms of action give insulin degludec a half-life of 24 hours and a duration of action of up to 42 hours.

[0008] The long-acting insulins mentioned above can achieve the goal of once-daily administration, but cannot achieve a longer duration of action such as once-weekly administration. Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the above circumstances, the present invention provides a long-acting insulin compound. [Means for solving the problem]

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical means. According to the present invention, Compounds are provided that include the following (I) or (II) or (III) or (IV) and / or (V) and / or (VI): (I) an amino acid sequence represented by formula I; [ka] [In formula I, X1 is selected from the group consisting of S, CH2, NH and CO; X2 is selected from the group consisting of S, CH2, NH and CO; AA1 is selected from the group consisting of Asp, Glu, and Ada; AA2 is selected from the group consisting of any proteinogenic amino acid other than Cys, any non-proteinogenic amino acid containing no SH group, or is absent; AA3 is selected from the group consisting of His, Tyr and Phe; AA4 is selected from the group consisting of Asp, Glu, and Ada; AA5 is selected from the group consisting of His, Tyr and Phe; AA6 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA7 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA8 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA9 is Thr or absent; AA10 is selected from the group consisting of Lys, Dah, Orn, Dab and Dap, or is absent; R1 and R2 are HO2C(CH2)n1CO-(γ-Glu)n2-(PEGn3(CH2)n4CO)n5-, R1 and R2 do not exist simultaneously.] (II) A sequence in which one or more amino acids are substituted, deleted, added, and / or substituted in the amino acid sequence shown in (I); (III) a sequence having 90% or more homology to the amino acid sequence shown in (I) or (II); (IV) a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of a compound of formula I; (V) Prodrugs based on compounds of formula I; (VI) Optionally, a mixture containing (I), (II), (III), (IV) and / or (V).

[0011] In some specific embodiments of the present invention, In formula I, when X1 is S, X2 is S or CH2; when X1 is CH2, X2 is S or CH2; When X1 is NH, X2 is CO; When X1 is CO, X2 is NH; When X1 and X2 are both S, AA11 is NH2; When X1 and X2 are not S at the same time, or when neither is S, AA11 is NH2 or OH.

[0012] In some specific embodiments of the present invention, n1 is an integer from 10 to 25, n2 is an integer from 1 to 5, n3 is an integer from 1 to 30, n4 is an integer from 1 to 5, The n5 is an integer of 1 to 5.

[0013] In some specific embodiments of the present invention, the pharmaceutically acceptable salts include those of the compound and Zn 2+ There is a complex formed from

[0014] According to the present invention, Step 1: producing a peptide resin by solid-phase polypeptide synthesis; and step 2, obtaining the compound by acid decomposition and purification.

[0015] According to the present invention, there is further provided the use of the following compounds (I) and / or (II) in the manufacture of a medicament or pharmaceutical composition for preventing and / or treating a disease: (I) the compound; (II) A compound produced by the above production method.

[0016] According to the present invention, there is further provided the use of the following compounds (I) and / or (II) in the manufacture of a medicament or pharmaceutical composition for preventing and / or treating diabetes: (I) the compound; (II) a compound produced by the production method; The diabetes includes type 1 diabetes, type 2 diabetes and / or gestational diabetes.

[0017] The present invention further provides a pharmaceutical agent comprising the compound and / or a compound produced by the production method, and pharmaceutically acceptable additives and / or auxiliaries.

[0018] According to the present invention, (I) the compound; and / or (II) a compound produced by the production method; and / or (III) Pharmaceutical compositions containing the agent, as well as any other active ingredients, are further provided.

[0019] According to the present invention, (I) the compound; and / or (II) a compound produced by the production method; and / or (III) the agent; and / or (IV) There is further provided a method for preventing and / or treating diabetes, such as type 1 diabetes, type 2 diabetes, and / or gestational diabetes, comprising administering the pharmaceutical composition to a subject. . [Effects of the Invention]

[0020] The present invention provides beneficial effects, including but not limited to the following: By providing a long-acting insulin compound with a longer half-life, it is expected to be widely applicable to the treatment of type 1 diabetes, type 2 diabetes, and gestational diabetes. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention discloses a long-acting insulin compound, and those skilled in the art can realize the present invention by referring to the contents of this specification and appropriately adjusting the process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and use according to the present invention have been described through preferred embodiments, but it is clear that those skilled in the art can implement and apply the technology of the present invention by making amendments, suitable changes, and combinations to the method and use described herein without departing from the content, spirit, and scope of the present invention.

[0022] The present invention aims to provide long-acting insulin compounds with longer half-lives.

[0023] According to the present invention, there are provided long-acting insulin compounds and uses thereof.

[0024] To achieve the above-mentioned objects, the present invention first provides a compound represented by the following formula I, a pharmaceutically acceptable salt, solvate, chelate, or non-covalent complex of this compound, a prodrug based on this compound, or any mixture of said forms:

[0025] [ka] [In formula I, When X1 is S, X2 is S or CH2; when X1 is CH2, X2 is S or CH2; When X1 is NH, X2 is CO; When X1 is CO, X2 is NH; AA1 is selected from the group consisting of Asp, Glu, and Ada; AA2 is selected from the group consisting of any proteinogenic amino acid other than Cys, any non-proteinogenic amino acid containing no SH group, or is absent; AA3 is selected from the group consisting of His, Tyr and Phe; AA4 is selected from the group consisting of Asp, Glu, and Ada; AA5 is selected from the group consisting of His, Tyr and Phe; AA6 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA7 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA8 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA9 is Thr or absent; AA10 is selected from the group consisting of Lys, Dah, Orn, Dab and Dap, or is absent; When X1 and X2 are both S, AA11 is NH2; When X1 and X2 are not S at the same time, or when neither of them is S, AA11 is NH2 or OH; R1 and R2 are HO2C(CH2)n1CO-(γ-Glu)n2-(PEGn3(CH2)n4CO)n5-, where n1 is an integer between 10 and 25, n2 is an integer from 1 to 5, n3 is an integer from 1 to 30, n4 is an integer from 1 to 5, n5 is an integer from 1 to 5, R1 and R2 do not exist simultaneously.]

[0026] According to the present invention, the compound according to the present invention and Zn 2+ Further provided is a complex formed from

[0027] According to the invention, the compound according to the invention and Zn 2+ Further provided are pharmaceutical compositions comprising the complex formed therefrom, as well as the use of a pharmaceutical composition of a compound according to the invention in the manufacture of a pharmaceutical composition for treating a disease.

[0028] The pharmaceutical composition is used to treat various diseases such as type 1 diabetes, type 2 diabetes, and gestational diabetes.

[0029] Many of the features of the present invention are described in detail below or may be realized in the examples of the present invention. Unless otherwise specified, the amounts of various components and reaction conditions used in this specification can be interpreted as "approximately" or "approximately." Similarly, unless otherwise specified, all numerical parameters recited below and in the claims are approximate parameters, and different numerical values may be obtained under different experimental conditions due to standard errors.

[0030] In the present specification, in the event of any discrepancy or doubt between a compound's chemical structure and its chemical name, the compound defined by the chemical structure shall prevail. The compounds described herein may contain one or more chiral centers and / or double bonds, and such structures may also exist as stereoisomers, such as double bond isomers (e.g., geometric isomers), optical enantiomers, or diastereoisomers. Therefore, any chemical structure within the scope of this specification, whether partially or entirely comprised of such analogous structures, includes all possible enantiomers and diastereoisomers of that compound, including any pure stereoisomer (e.g., pure geometric isomer, pure enantiomer, or pure diastereoisomer) and any mixture of such isomers. These racemic and stereoisomeric mixtures may also be further resolved into their component enantiomers or stereoisomers by various separation or chiral molecule synthesis procedures commonly employed by those skilled in the art.

[0031] The compounds of structural formula I include, but are not limited to, optical isomers, racemates, and / or other mixtures of these compounds. In such cases, single enantiomers or diastereoisomers, e.g., optically active isomers, can be obtained by asymmetric synthesis or racemic separation. Resolution of racemates can be achieved by various methods, e.g., conventional recrystallization or chromatography using a resolving agent. The compounds of formula I also include cis and / or trans isomers of double bonds.

[0032] Compounds according to the present invention include, but are not limited to, compounds of Formula I and all their pharmaceutically acceptable forms. The various pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent conjugates, prodrugs based on said substances, and mixtures of any of said forms.

[0033] The present invention further provides a method for producing a peptide resin by solid-phase polypeptide synthesis, followed by acidolysis of the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product, wherein the step of producing a peptide resin by solid-phase polypeptide synthesis involves sequentially coupling corresponding protected amino acids or fragments in the following sequence onto a support resin by coupling solid-phase synthesis to produce the peptide resin:

[0034] In the production method, the amount of the Fmoc-protected amino acid used is 1.2 to 6 times, and preferably 2.5 to 3.5 times, the total number of moles of the resin added.

[0035] In the production method, the substitution value of the carrier resin is 0.2 to 1.0 mmol / g of resin, and preferably 0.3 to 0.5 mmol / g of resin.

[0036] In a preferred embodiment of the present invention, the solid-phase coupling synthesis method involves removing the Fmoc protecting group from the protected amino acid-resin obtained in the previous step, followed by a coupling reaction with the next protected amino acid. The deprotection time for removing the Fmoc protecting group is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

[0037] The coupling reaction requires the addition of a condensation reagent. The condensation reagent is one selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, and is preferably N,N-diisopropylcarbodiimide. The molar amount of the condensation reagent used is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total number of moles of amino groups in the amino resin.

[0038] The coupling reaction requires the addition of an activating reagent, which is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of the activating reagent used is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total number of moles of amino groups in the amino resin.

[0039] In a preferred embodiment of the present invention, the reagent for removing the Fmoc protecting group is a mixed solution of PIP / DMF (piperidine / N,N-dimethylformamide). The mixed solution contains 10 to 30% (V) piperidine. The amount of the reagent for removing the Fmoc protecting group used is 5 to 15 mL per gram of amino resin, and preferably 8 to 12 mL per gram of amino resin.

[0040] Preferably, the peptide resin is acidolyzed, and the resin and side chain protecting groups are simultaneously removed to obtain a crude product.

[0041] More preferably, the acid decomposition agent used in the acid decomposition of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), and water, with a volume ratio of 80-95% TFA, 1-10% EDT, and the remainder being water.

[0042] More preferably, the mixed solvent has a volumetric blending ratio of 89 to 91% TFA, 4 to 6% EDT, and the remainder being water. Most preferably, the mixed solvent has a volumetric blending ratio of 90% TFA, 5% EDT, and the remainder being water.

[0043] The amount of the acid decomposition agent used is 4 to 15 mL per gram of peptide resin, and preferably 7 to 10 mL per gram of peptide resin. The decomposition time with an acid decomposition agent is 1 to 6 hours, preferably 3 to 4 hours, at room temperature. The crude product is further purified by high performance liquid chromatography and lyophilized to give the pure product.

[0044] The Japanese names corresponding to the English abbreviations referred to in this invention are shown in Table 1 below. [Table 1]

[0045] All raw materials and reagents used in the preparation of the long-acting insulin compounds of the present invention are commercially available. The present invention will now be further described with reference to the following examples. [Example 1] Preparation of Compound 1

[0046] [ka] wherein Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH is as shown in SEQ ID NO: 1. Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Glu-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys(PEGCHCO-γGlu-eicosanedioic acid)-NH is as set forth in SEQ ID NO: 2. 1. Synthesis of Peptide Resin

[0047] Rink Amide BHHA resin was used as a carrier resin, and the peptide resin was prepared by coupling with the protected amino acids shown in Table 2 below through Fmoc deprotection and coupling reaction. The protected amino acids corresponding to those used in this example are shown in Table 2 below.

[0048] [Table 2-1] [Table 2-2] (1) Linking the first protected amino acid of the main chain

[0049] 0.03 mol of the first protected amino acid and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF, and 0.03 mol of DIC was added slowly to the DMF solution of the protected amino acid while stirring. The reaction was carried out at room temperature for 30 minutes with stirring to obtain an activated protected amino acid solution, which was then ready for further use.

[0050] 0.01 mol of Rink amide MBHA resin (substitution value approximately 0.3 mmol / g) was taken and deprotected with 20% PIP / DMF solution for 25 minutes, washed, and filtered to obtain the Fmoc-removed resin.

[0051] The activated first protected amino acid solution was added to the resin from which Fmoc had been removed, and a coupling reaction was carried out for 60 to 300 minutes, followed by filtration and washing to obtain a resin containing one protected amino acid. (2) Linking of the 2nd to 29th protected amino acids of the main chain

[0052] The corresponding second to 29th protected amino acids were linked in order using the same method as used to link the first protected amino acid in the main chain, to obtain a resin containing 29 amino acids in the main chain. (3) Linking the first protected amino acid side chain

[0053] The side chain protecting groups were removed using 50% HFIP / DMM solution, which was repeated 5 times for 35 minutes each time, followed by filtration and washing to obtain the Mtt-deprotected resin, which was ready for further use.

[0054] 0.3 mol of 2,2'-dithiobipyridine was dissolved in an appropriate amount of DMF, and the mixture was added to the resin from which the Mtt protection had been removed. The mixture was stirred and reacted for 3 hours, filtered, washed, and the SH-activated resin was obtained and prepared for further use.

[0055] 0.03 mol of the first protected amino acid in the side chain (Fmoc-Cys-Asn(Trt)-OtBu) was dissolved in an appropriate amount of DMF and added to the SH-activated resin. 2 ml of DIPEA was added, and the mixture was stirred for 3 hours to react. After that, the mixture was filtered and washed to complete the coupling of the first protected amino acid in the side chain. (4) Linking the second protected amino acid in the side chain

[0056] 0.03 mol of the second protected amino acid and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF, and 0.03 mol of DIC was added slowly to the DMF solution of the protected amino acid while stirring. The mixture was stirred at room temperature for 30 minutes to react, yielding an activated protected amino acid solution.

[0057] The resin was deprotected using a 20% PIP / DMF solution for 25 minutes, washed, and filtered. The solution of the second protected amino acid in the activated side chain was added to the resin from which the Fmoc had been removed, and the coupling reaction was carried out for 60 to 300 minutes. The resin was then filtered and washed to obtain a resin containing the second protected amino acid in the side chain. (5) Linking of the 3rd to 20th protected amino acids in the side chain

[0058] The third to twentieth protected amino acids corresponding to the side chains were linked in order using the same method as that used to link the first protected amino acid to the main chain, to obtain a peptide resin. 2. Preparation of Crude Product

[0059] The peptide resin was taken and added to a cleavage reagent with a volume ratio of TFA:water:EDT = 95:5:5 (10 mL of cleavage reagent / g of resin), stirred thoroughly, and allowed to react at room temperature for 3 hours. The reaction mixture was filtered through a sand core funnel, the filtrate was collected, and the resin was washed three times with a small amount of TFA. The combined filtrate was concentrated under reduced pressure and precipitated by adding anhydrous diethyl ether. The precipitate was washed three times with anhydrous diethyl ether and dried in vacuo to obtain an off-white powder.

[0060] The obtained off-white powder was dissolved in a 20% aqueous DMSO solution, adjusted to pH 7.5 with aqueous ammonia, and stirred for 10 hours to allow the reaction to proceed. Next, glacial acetic acid was added until the acetic acid concentration reached 20%, and a saturated iodine / ethanol solution was added dropwise with stirring until complete cyclization occurred. The mixture was then concentrated under reduced pressure at 35-40°C to obtain a concentrated solution of the crude product. 3. Preparation of pure product

[0061] The concentrated solution of the crude product was taken and filtered through a 0.45 μm mixed microporous membrane, purified and ready for further use.

[0062] Purification was performed using high-performance liquid chromatography (HPLC) with 10 μm reverse-phase C18 packing, a 0.1% TFA / water solution-0.1% TFA / acetonitrile mobile phase, and a 30 mm x 250 mm column flow rate of 20 mL / min. Gradient elution was performed with cyclic sample loading. The crude product solution was injected into the column, and the mobile phase elution was initiated. The main peak was collected, and the acetonitrile was evaporated to obtain the purified intermediate concentrate.

[0063] The purified intermediate concentrate was filtered through a 0.45 μm filter membrane and prepared for further use. Salt exchange was performed by high-performance liquid chromatography (HPLC). The mobile phase system was 1% acetic acid / water solution in acetonitrile, the purification chromatography packing was 10 μm reverse-phase C18, and the flow rate of the 30 mm x 250 mm chromatographic column was 20 mL / min (the flow rate can be adjusted accordingly depending on the chromatographic column specifications). Gradient elution and cyclic sample loading were used to inject the sample into the chromatographic column. The mobile phase elution was initiated, and the chromatogram was collected to observe the change in absorbance. The main peak of the salt exchange was collected and the liquid phase purity was determined. The main peak solution of the salt exchange was combined and concentrated under reduced pressure to obtain a pure aqueous acetic acid solution, which was then lyophilized to yield 3.9 g of pure product. The purity was 97.7%, the overall yield was 6.1%, and the molecular weight was 6358.2 (100% M+H). [Example 2] Preparation of Compound 2

[0064] [ka] Here, Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH is as shown in SEQ ID NO: 1. Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Glu-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys-Thr-Lys(PEGCHCO-γGlu-eicosanedioic acid)-NH is as shown in SEQ ID NO: 3.

[0065] The preparation method was the same as in Example 1, and the protected amino acids shown in Table 3 below were used. [Table 3-1] [Table 3-2]

[0066] 4.7 g of pure product was obtained with a purity of 98.5%, a total yield of 7.1%, and a molecular weight of 6587.5 (100% M+H). [Example 3] Preparation of Compound 3

[0067] [ka] wherein Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Asp-Asn-OH is as shown in SEQ ID NO: 4. Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Glu-Leu-Val-Dap-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys(PEGCHCO-γGlu-eicosanedioic acid)-NH is as set forth in SEQ ID NO: 5.

[0068] (1) The preparation method was the same as in Example 1, except for the linking of the first protected amino acid in the side chain.

[0069] (2) Linking the first protected amino acid in the side chain 0.03 mol of the first protected amino acid and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF, and 0.03 mol of DIC was added slowly to the DMF solution of the protected amino acid while stirring. The mixture was stirred at room temperature for 30 minutes to react, yielding an activated protected amino acid solution.

[0070] The side chain protecting groups were removed using a 50% HFIP / DCM solution, which was repeated five times for 35 minutes each time, followed by filtration and washing. The activated first protected amino acid solution was added to the resin from which the Mtt protection had been removed, and the coupling reaction was carried out for 60 to 300 minutes. The resin was then filtered and washed to obtain a resin containing the first protected amino acid in the side chain.

[0071] The protected amino acids shown in Table 4 below were used. [Table 4-1] [Table 4-2]

[0072] 3.5 g of pure product was obtained with a purity of 98.8%, a total yield of 5.5%, and a molecular weight of 6337.2 (100% M+H). [Example 4] Preparation of Compound 4

[0073] The structure of compound 4 is 、 [ka] wherein Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Asp-Asn-OH is as shown in SEQ ID NO: 4. Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Glu-Leu-Val-Dap-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys-Thr-Lys(PEGCHCO-γGlu-eicosanedioic acid)-NH is as set forth in SEQ ID NO: 6.

[0074] The preparation method was the same as in Example 3, and the protected amino acids shown in Table 5 below were used. [Table 5-1] [Table 5-2]

[0075] 3.7 g of pure product was obtained with a purity of 98.1% and a total yield of 5.6% with a molecular weight of 6566.4 (100% M+H). [Example 5] Preparation of Compound 5

[0076] The structure of compound 5 is 、 [ka] wherein Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-L-4-bromo-2-aminobutyramido-Asn-OH is shown in SEQ ID NO: 7. Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Glu-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys(PEGCHCO-γGlu-eicosanedioic acid)-NH is as set forth in SEQ ID NO: 8.

[0077] (1) The preparation method was the same as in Example 1, except for the linking of the first protected amino acid in the side chain. (2) Linking the first protected amino acid in the side chain The side chain protecting groups were removed using 50% HFIP / DCM solution, which was repeated 5 times for 35 minutes each time, followed by filtration and washing to obtain the Mtt-deprotected resin, which was ready for further use.

[0078] 0.03 mol of the first protected amino acid in the side chain was dissolved in an appropriate amount of DMF and added to the resin from which the Mtt protection had been removed. After thorough stirring, 0.03 mol of N-methylmorpholine and 0.03 mol of lithium chloride were added, and the mixture was stirred for 6 hours to react, filtered, and washed to obtain a resin containing the first protected amino acid in the side chain.

[0079] The protected amino acids shown in Table 6 below were used. [Table 6-1] [Table 6-2]

[0080] 4.3 g of pure product was obtained with a purity of 97.9%, a total yield of 6.8%, and a molecular weight of 6340.2 (100% M+H). [Example 6] Preparation of Compound 6

[0081] The structure of compound 6 is 、 [ka] wherein Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-L-4-bromo-2-aminobutyramido-Asn-OH is as shown in SEQ ID NO: 7. Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Glu-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys-Thr-Lys(PEGCHCO-γGlu-eicosanedioic acid)-NH is as set forth in SEQ ID NO: 9.

[0082] The preparation method was the same as in Example 5, and the protected amino acids shown in Table 7 below were used. [Table 7-1] [Table 7-2]

[0083] 3.5 g of pure product was obtained with a purity of 98.6%, a total yield of 6.4%, and a molecular weight of 6569.5 (100% M+H). [Example 7] Preparation of Compound 7

[0084] The structure of compound 7 is 、 [ka] wherein Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-OH is as shown in SEQ ID NO: 10. Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Glu-Leu-Val-(S,S) DAS[Dde-Asn(Trt)-OtBu]-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys(PEGCHCO-γGlu-eicosanedioic acid)-NH is as set forth in SEQ ID NO: 11.

[0085] (1) The preparation method was the same as in Example 1, except for the linking of the first protected amino acid in the side chain.

[0086] (2) Linking the first protected amino acid in the side chain 0.03 mol of the first protected amino acid and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF, and 0.03 mol of DIC was added slowly to the DMF solution of the protected amino acid while stirring. The mixture was stirred at room temperature for 30 minutes to react, yielding an activated protected amino acid solution.

[0087] The side chain protecting groups were removed using a 2% hydrazine hydrate / DMF solution, which was repeated three times for 10 minutes each time, followed by washing and filtration. The activated side chain first protected amino acid solution was added to the resin from which Dde had been removed, and the coupling reaction was carried out for 60 to 300 minutes. The resin was then filtered and washed to obtain a resin containing the first protected amino acid in the side chain.

[0088] The protected amino acids shown in Table 8 below were used. [Table 8-1] [Table 8-2]

[0089] 3.7 g of pure product was obtained with a purity of 98.2%, a total yield of 5.9%, and a molecular weight of 6322.2 (100% M+H). [Example 8] Preparation of Compound 8

[0090] The structure of compound 8 is 、 [ka] wherein Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-OH is as shown in SEQ ID NO: 10. Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Glu-Leu-Val-(S,S)DAS[Dde-Asn(Trt)-OtBu]-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys-Thr-Lys(PEGCHCO-γGlu-eicosanedioic acid)-NH is as set forth in SEQ ID NO: 12.

[0091] The preparation method was the same as in Example 7, and the protected amino acids shown in Table 9 below were used. [Table 9-1] [Table 9-2]

[0092] 4.0 g of pure product was obtained with a purity of 97.5%, a total yield of 6.1%, and a molecular weight of 6551.4 (100% M+H). [Example 9] Measurement of preliminary pharmacokinetic properties

[0093] Male SD rats were administered a dose of 1 mg / kg subcutaneously. Blood was collected from the orbital vein of the rats before administration (0 hours) and at 1, 2, 3, 4, 8, 24, 48, 96, and 144 hours after administration. Plasma samples were collected by centrifugation.

[0094] The blood drug concentrations of the corresponding compounds in the plasma samples of SD rats were measured by liquid chromatography-mass spectrometry. The half-lives of the compounds after subcutaneous (SC) administration to SD rats are shown in Table 10 below.

[0095] [Table 10]

[0096] The above has provided a detailed description of the long-acting insulin compound according to the present invention. Although the principles and embodiments of the present invention have been described using specific examples in this specification, the description of the examples is only used to help understand the method and essence of the present invention. Here, it should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications are also included in the scope of the claims of the present invention.

Claims

1. A compound characterized in that it comprises the following (I) or (II) or (III) or (IV) and / or (V) and / or (VI): (I) an amino acid sequence represented by the following formula I: 【Chemical 1】 [In formula I, X １ is S, CH ２ , NH and CO; X ２ is S, CH ２ , NH and CO; AA1 is selected from the group consisting of Asp, Glu and Ada; AA2 is selected from the group consisting of any proteinogenic amino acid other than Cys, any non-proteinogenic amino acid containing no SH group, or is absent; AA3 is selected from the group consisting of His, Tyr and Phe; AA4 is selected from the group consisting of Asp, Glu and Ada; AA5 is selected from the group consisting of His, Tyr and Phe; AA6 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA7 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA8 is selected from the group consisting of any proteinogenic amino acid other than Cys and any non-proteinogenic amino acid containing no SH group; AA9 is Thr or absent, AA10 is selected from the group consisting of Lys, Dah, Orn, Dab and Dap, or is absent; R1 and R2 are HO ２ C(CH ２ )n1CO-(γ-Glu)n2-(PEGn3(CH ２ )n4CO)n5-, R1 and R2 do not exist simultaneously. (II) a sequence in which one or more amino acids are substituted, deleted, added, and / or substituted in the amino acid sequence shown in (I); (III) a sequence having a homology of 90% or more to the amino acid sequence shown in (I) or (II); (IV) a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of a compound of formula I; (V) Prodrugs based on the compounds of formula I; (VI) A mixture optionally comprising (I), (II), (III), (IV) and / or (V) above.

2. In formula I, X １ If S, then X ２ is S or CH ２ and X １ is CH ２ If X ２ is S or CH ２ and X １ is NH, then X ２ is CO, X １ is CO, then X ２ is NH, X １ and X ２ When both are S, AA11 is NH ２ and X １ and X ２ If both are not S, or if neither is S, then AA11 is NH ２ or OH.

3. n1 is an integer from 10 to 25, n2 is an integer from 1 to 5, n3 is an integer from 1 to 30, n4 is an integer from 1 to 5, The compound according to claim 1 or 2, wherein n5 is an integer of 1 to 5.

4. The pharmaceutically acceptable salt is a compound of the compound and Zn ２＋ The compound according to any one of claims 1 to 3, which is a complex formed from

5. Step 1: producing a peptide resin by solid-phase polypeptide synthesis; and step 2 of obtaining the compound by acid decomposition and purification.

6. Use of the following compounds (I) and / or (II) in the manufacture of a medicament or pharmaceutical composition for preventing and / or treating a disease: (I) A compound according to any one of claims 1 to 4; (II) A compound produced by the production method described in claim 5.

7. Use of the following compounds (I) and / or (II) in the manufacture of a medicament or pharmaceutical composition for preventing and / or treating diabetes: (I) A compound according to any one of claims 1 to 4; (II) A compound produced by the production method according to claim 5; The diabetes is type 1 diabetes, type 2 diabetes and / or gestational diabetes.

8. A drug comprising the compound according to any one of claims 1 to 4 and / or the compound produced by the production method according to claim 5, and pharmaceutically acceptable additives and / or auxiliaries.

9. (I) a compound according to any one of claims 1 to 4; and / or (II) A compound produced by the production method according to claim 5; and / or (III 9. A pharmaceutical composition comprising the drug of claim 8 and any other active ingredient.

10. (I) a compound according to any one of claims 1 to 4; and / or (II) A compound produced by the production method according to claim 5; and / or (III) a drug according to claim 8; and / or (IV) A method for preventing and / or treating diabetes, comprising administering the pharmaceutical composition according to claim 9 to a subject, The method, wherein said diabetes is type 1 diabetes, type 2 diabetes and / or gestational diabetes.

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