Novel acylated insulin analogues

Abstract

It provides novel acylated insulin analogs, specifically side chain compounds that can be used to prepare acylated insulin analogs, acylated insulin analogs and their pharmaceutical compositions, medical uses, administration methods and preparation methods. The acylated insulin analogs can be used for the treatment of diabetes. It has the effect of a once-weekly preparation or a long-acting insulin preparation, and can be used for treatment once a week or less frequently, which enhances the compliance of diabetic patients.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of China Patent Application No. 202110570030.6, filed with the State Intellectual Property Office of China on May 24, 2021, the entirety of which is incorporated herein by reference.

[0002] The present invention relates to the field of biopharmaceuticals. In particular, the present invention relates to novel acylated insulin analogues. More particularly, the present invention relates to side chain compounds that can be used to prepare acylated insulin analogues, to acylated insulin analogues, and to pharmaceutical compositions, medical uses, methods of administration and methods of preparation thereof. [Background technology]

[0003] Treatment of both type I and type II diabetes is increasingly dependent on so-called intensive insulin therapy, under which patients are treated with multiple daily insulin injections, including using one or two long-acting insulin injections daily to meet basal insulin needs, supplemented with large amounts of fast-acting insulin to meet meal-related insulin needs.

[0004] Many diabetic patients require insulin injections two to four times a day, weekly, monthly and annually. Because patient compliance is low, long-term subcutaneous injections cause some damage to the skin, and the discomfort of daily large-volume injections can be alleviated by using longer-acting insulin analogs, there is a need for insulin analogs that can be injected at least once a week.

[0005] Although Chinese Patent No. 105636979 discloses new derivatives of insulin analogues, their duration of action is still not ideal, and there remains an urgent need for a basal insulin preparation that can be administered once a week or even less frequently. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Chinese Patent No. 105636979 [Non-patent literature]

[0007] [Non-Patent Document 1] Glendorf T, Sorensen AR, Nishimura E, Pettersson I, and Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B chain α-Helix for Receptor Binding:Biochemistry.2008;47(16):4743-51 Summary of the Invention [Problem to be solved by the invention]

[0008] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. [Means for solving the problem]

[0009] In a first aspect of the present invention, a compound of formula (I): WXYZR (I) (In the formula, W is a fatty acid or diacid having 10 to 20 carbon atoms and the structure is -CO(CH2) n COOH, n is an integer between 10 and 20; X is a diamino compound containing a carboxylic acid group, and the carbon atom connecting the carboxylic acid group can be a chiral or achiral carbon, and is represented by the formula (a1), (a2) and (a3): [ka] wherein s is an integer between 2 and 20; in some embodiments, s is 2-10; in other embodiments, s is 2-8; and in still other embodiments, s is 4; one of the amino groups in X is connected to one of the acyl groups in W to form an amide bond; Y is -A(CH2) m B-, where m is an integer between 1 and 10; in some embodiments, m is an integer between 1 and 6; in some embodiments, m is 2; A and B are absent or are -CO-; Z is -(OEG) p and p is an integer between 1 and 3; in some embodiments, p is 2 and the OEG structure is [ka] or n can be an integer between 4 and 30, and R is a leaving group; in some embodiments, R is an activated ester group; The linking groups between W, X, Y and Z are amide peptide bonds or peptide bonds. Provided herein are novel side chain compounds having the structure shown in

[0010] Additionally, the side chain compound of the present invention has the following structural formula: [ka] (In the formula, n is an integer between 14 and 20, s is an integer between 2 and 4, m is an integer between 1 and 4, and p is 2; R is a group: [ka] (selected from may have:

[0011] In some embodiments of the invention, n is an integer between 14 and 20, s is an integer between 2 and 4, m is 2, and p is 2; R [ka] It is.

[0012] In some embodiments of the invention, the side chain compound has the following structural formula: [ka] (wherein n is an integer between 16 and 18, and R is [ka] is) has.

[0013] In some embodiments of the invention, the side chain compound of the invention is the following compound: [ka] [ka] (Wherein, R is [ka] is) is selected from one of the following:

[0014] In yet another embodiment of the invention, the side chain compound has the following structural formula: [ka] (Wherein, R is [ka] is) has.

[0015] In yet another embodiment of the invention, the side chain compound has the following structural formula: [ka] (Wherein, R is [ka] is) has.

[0016] In a second aspect of the present invention, a novel acylated insulin analogue is proposed, obtainable by acylation reaction between the side chain compound of the present invention and a human insulin analogue, the structure of which is represented by formula (II): WXYZM (II) (In the formula, W is a fatty acid or diacid having 10 to 20 carbon atoms and the structure is -CO(CH2) n COOH, n is an integer between 10 and 20; X is a diamino compound containing a carboxylic acid group, and the carbon atom connecting the carboxylic acid group can be a chiral or achiral carbon, and is represented by the formula (a1), (a2) and (a3): [ka] (wherein s is an integer between 2 and 20, in some embodiments, s is 2 to 10, and in other embodiments, s is 2 to 8), and one of the amino groups in X is connected to one of the acyl groups in W to form an amide bond; Y is -A(CH2) m B-, where m is an integer between 1 and 10, and in some embodiments m is an integer between 1 and 6, and A and B are absent or -CO-; Z is -(OEG) p and p is an integer between 1 and 3, and in some embodiments, p is 2, and the OEG structure is [ka] In other embodiments, p can be an integer between 4 and 30; the linking groups between W, X, Y and Z are amide (peptide) bonds; M is a human insulin analogue) As shown in.

[0017] In some embodiments of the invention, the acylated insulin analogue has the following structure: [ka] (In the formula, n is an integer between 14 and 20, s is an integer between 2 and 8, m is an integer between 1 and 6, and p is an integer between 1 and 3.) The compound has a side chain of

[0018] In some embodiments of the invention, the acylated insulin analogue has the following structure: [ka] (In the formula, n is an integer between 14 and 20, s is an integer between 2 and 8, m is an integer between 1 and 6, and p is an integer between 1 and 3.) The compound has a side chain of

[0019] The acylated insulin analogues of the present invention are obtained by an acylation reaction between a side chain compound of the present invention and a human insulin analogue, the human insulin analogue having an A chain and a B chain, the amino acid sequence of the A chain being shown in SEQ ID NO:1 and the amino acid sequence of the B chain being shown in SEQ ID NO:2 or SEQ ID NO:3, and the human insulin analogue being connected to the side chain compound by an amide bond through the ε nitrogen of the lysine residue at position B29.

[0020] A chain: GIVEQCCTSICSLEQLENYCN (SEQ ID NO: 1)

[0021] B chain: FVNQHLCGSHLVEALELVCGERGFHYTPK (SEQ ID NO: 2)

[0022] B chain: FVNQHLCGSHLVEALHLVCGERGFHYTPK (SEQ ID NO: 3)

[0023] In some embodiments of the invention, the acylated insulin analogues of the invention have the following structural formula: A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) nCO-NHC(COOH)(CH2) S CH2NH-CO(CH2) m CO-(OEG) p ), desB30 human insulin analog, or A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) n CO-NHC(COOH)(CH2) S CH2NH-CO(CH2) m CO-(OEG) p ), desB30 human insulin analogue (wherein n is an integer between 14 and 20, s is an integer between 2 and 8, m is an integer between 1 and 6, and p is 2); it should be noted that the C atom connecting the carboxyl group in -NHC(COOH)(CH2)SCH2NH- can be in D-, L- or racemic form.

[0024] In some embodiments of the invention, n is an integer between 14 and 18, s is an integer between 3 and 4, m is an integer between 2 and 4, and p is 2.

[0025] Further, the acylated insulin analogues of the present invention include the following compounds: A14E,B16E,25H,B29K(N(ε)-COOH(CH2) 18 CO-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-D-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 CO-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 CO-D-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 14 CO-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 14 CO-D-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 14 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Dab-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 CO-L-Dab-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 14 CO-L-Dab-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)3CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16CO-L-Lys-CO(CH2)3CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Dab-CO(CH2)3CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)4CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Dab-CO(CH2)4CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 CO-L-Dab-CO(CH2)4CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-D-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 16 CO-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 16 CO-D-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 16 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 14 CO-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 14 CO-D-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 14 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Dab-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 16 CO-L-Dab-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 14 CO-L-Dab-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)3CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 16 CO-L-Lys-CO(CH2)3CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2)18 CO-L-Dab-CO(CH2)3CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)4CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Dab-CO(CH2)4CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 16 CO-L-Dab-CO(CH2)4CO-(OEG)2), desB30 human insulin analog selected from any one of the following: "-Lys-" means a connection via an achiral lysine, "-L-Lys-" means a connection via an L-chiral lysine, and "-D-Lys-" means a connection via a D-chiral lysine; "Dab" means 2,4-diaminobutyric acid, "-L-Dab-" means attachment via an L-chiral Dab, and "-D-Dab-" means attachment via a D-chiral Dab.

[0026] In some embodiments of the invention the acylated insulin analogue is the following compound: A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analog; A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analog is selected from one of the following:

[0027] In yet another embodiment of the invention the acylated insulin analogue is the following compound: A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue, A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analog may be selected from any one of the following:

[0028] Here, A14E, B16E, B25H, B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analog has the following formula: [ka] It has the structure shown in A14E, B16H, B25H, B29K(N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analog has the following formula: [ka] It has the structure shown in:

[0029] A third aspect of the invention provides a pharmaceutical composition comprising a side chain compound of the invention and an acylated insulin analogue.

[0030] A fourth aspect of the invention proposes the use of the side chain compounds, acylated insulin analogues and pharmaceutical compositions of the invention in the manufacture of a medicament for treating or preventing diabetes in a subject; Diabetes refers to type I and type II diabetes.

[0031] A fourth aspect of the present invention provides a method for treating or preventing diabetes in a subject, comprising administering to the subject a therapeutically effective amount of a side chain compound, an acylated insulin analogue or a pharmaceutical composition of the present invention; Diabetes refers to type I and type II diabetes.

[0032] A fourth aspect of the present invention proposes the side chain compounds, acylated insulin analogues and pharmaceutical compositions of the present invention for use in treating or preventing diabetes in a subject; Diabetes refers to type I and type II diabetes.

[0033] A fifth aspect of the invention provides methods of administration of the side chain compounds, acylated insulin analogues and pharmaceutical compositions of this invention, wherein the compounds, acylated insulin analogues and pharmaceutical compositions are administered twice a week, once a week or less frequently.

[0034] A sixth aspect of the present invention proposes a process for the preparation of novel acylated insulin analogues of formula (II), comprising the step of carrying out an acylation reaction using a side chain compound of formula (I) and a human insulin analogue; the human insulin analogue has an A chain and a B chain, the amino acid sequence of the A chain being shown in SEQ ID NO:1 and the amino acid sequence of the B chain being shown in SEQ ID NO:2 or SEQ ID NO:3.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides a novel acylated human insulin analogue that can be used for the treatment of diabetes and has a longer duration of action for controlling glucose compared to the current once-daily preparation (insulin degludec), which can be used as a once-weekly preparation or a longer-acting insulin preparation that can be administered subcutaneously once a week or less frequently, providing a satisfactory therapeutic effect in diabetic patients requiring basal insulin therapy and improving patient compliance.

[0037] In the process of describing the present invention, relevant terms in this article are explained and illustrated, but these are only for the convenience of understanding the scheme, and should not be considered as limitations on the protection scheme of the present invention.

[0038] As used herein, "insulin analogue" refers to a polypeptide having a form that can be obtained by deletion and / or replacement of at least one amino acid residue present in naturally occurring insulin, such as the molecular structure of the human insulin structure, and / or by addition of at least one amino acid residue derived from naturally occurring insulin.

[0039] "DesB30 insulin" and "desB30 human insulin" refer to naturally occurring insulin or analogs thereof that lack the B30 amino acid residues.

[0040] The term "diabetes" includes type I diabetes, type II diabetes, gestational diabetes (during pregnancy) and other conditions that cause hyperglycemia. The term is used for metabolic disorders in which the pancreas produces insufficient amounts of insulin, or the body's cells are unable to respond appropriately to insulin, preventing the cells from absorbing glucose. As a result, glucose accumulates in the blood. Type I diabetes, also known as insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes, is caused by B-cell destruction, which often results in absolute insulin deficiency. Type II diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with significant insulin resistance and therefore relative insulin deficiency due to insulin resistance and / or significant insulin secretion abnormalities.

[0041] "A14E,B16E,B25H,B29K (N(ε)-eicosandioyl-L-Lys-succinic acid-2xOEG),desB30 human insulin" is a human insulin in which the amino acid Y at position A14 of human insulin is mutated to E, the amino acid Y at position B16 of human insulin is mutated to E, the amino acid F at position B25 of human insulin is mutated to H, and the amino acid K at position B29 of human insulin is mutated to the ε nitrogen (N ε It is modified on the cytosine triphosphate (referred to as CYP2Q10) by acylation with eicosanedioyl-L-Lys-succinic acid-2xOEG residues, which means that the amino acid T at position B30 of human insulin is deleted.

[0042] "OEG" is [2-(2-aminoethoxy)ethoxy]ethylcarbonyl; 2xOEG or (OEG)2 together refer to two OEG.

[0043] "Su" is succinimidyl-1-yl 2,5-dioxo-pyrrolidin-1-yl.

[0044] "Osu" refers to succinimidyl-1-yloxy=2,5-dioxo-pyrrolidin-1-yloxy. [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 shows the change in blood glucose in C57 mice after a single subcutaneous administration. [Diagram 2] FIG. 1 shows time and drug concentration data in IVPK in SD rats. [Diagram 3] FIG. 1 shows random blood glucose change curves of repeated administration to T1DM mice. [Figure 4] FIG. 1 shows random blood glucose change curves of repeated administration to T1DM mice. [Diagram 5] FIG. 1 shows time and drug concentration curves in SC.PK in SD rats. [Figure 6] FIG. 1 shows time and drug concentration curves in SCPK in C57BL6 mice. [Figure 7] FIG. 1 shows time and drug concentration curves in IVPK in beagles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0046] The solutions of the present invention are described below in conjunction with the embodiments. Examples of such embodiments are illustrated in the drawings, and the same or similar reference numbers refer to the same or similar components or components with the same or similar functions throughout. If no specific techniques or conditions are shown in the examples, the techniques or conditions described in the technical literature or product specifications will be used. The reagents or equipment used without manufacturer's indication are conventional products available on the market. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0047] Example 1 Preparation of insulin mutant analogs (A14E, B16E, B25H, B29K, desB30 human insulin analogs) The construction of vectors for insulin analogs, yeast expression, processing and purification can be carried out using standard techniques that are readily recognized by those skilled in the art. A non-limiting example of the preparation of insulin analogs has been described previously (Glendorf T, Sorensen AR, Nishimura E, Pettersson I, and Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B chain α-Helix for Receptor Binding: Biochemistry. 2008; 47(16): 4743-51). In short, using a yeast expression system, the A single chain and the B single chain of long-acting insulin were connected through an artificially designed C peptide, and a spacer peptide was added to increase the stability of the precursor protein and increase the expression of the target protein. Through enzymatic cleavage and subsequent purification, both the spacer peptide and the C peptide were cleaved in the downstream purification process to obtain long-acting insulin analogs. The complete conversion to the double-stranded DesB30 analog was verified by MALDI-TOF MS, and its purity was tested by RP-HPLC under acidic and neutral conditions. The engineered strain obtained by screening the transfected host bacteria can be fermented at high density with high expression levels and low fermentation costs. The designed genes facilitate the development of a simple and efficient purification process.

[0048] 1) Construction of recombinant expression vector The total synthesis of the target gene was entrusted to General Biosystems (Anhui) Co., Ltd., the target gene sequence and vector pPIC9K were digested with restriction enzymes BamHI and EcoRI (TAKARA), the digestion product was purified and recovered using a gel extraction kit according to the manufacturer's instructions. The vector was ligated using DNA ligation kit Ver2.1 (TAKARA) according to the manufacturer's instructions and transformed into competent cells DH5a. A single colony on the plate was randomly picked and the target gene was sequenced by Guangzhou Aike Biotechnology Co., Ltd. to verify its accuracy, and then the correct expression vector was extracted and verified using an Omega plasmid extraction kit. After linearization with the restriction enzyme SalI (Takara), the expression vector was purified and recovered using a gel extraction kit according to the manufacturer's instructions, and stored at -20°C for future use.

[0049] 2) Construction of recombinant engineering strains and protein fermentation expression The linearized recombinant expression plasmid was added to Pichia pastoris GS115 competent cells (Invitrogen) transformed by electroshock method, and electroshock was performed using a MicroPulser (Bio-Rad, 165-2100) device. After electroshock, 1mL of pre-chilled 1mol / L sorbitol was added, and the bacterial suspension was transferred to a sterile centrifuge tube, recovered and cultured in a shaker at 30℃ and 220rpm for 2 hours, then coated with MD medium plates and inverted cultured at 30℃ in an incubator. The transformants grown on the plates were screened for high-copy recombinants using Geneticin G418 (Merck).

[0050] The above screened recombinants were cultured in shaker flasks, fermented, single colonies were picked and inoculated into YPD medium for cultivation, shaken in a shaker at 30°C and 220 rpm for about 2 days, the seed liquid obtained by cultivation was inoculated into BMGY medium (buffered glycerol complex medium) at a ratio of 1:100, incubated in a shaker at 30°C and 220 rpm for about 24 hours with shaking, then anhydrous methanol was added at 1% of the volume of the fermentation medium to induce protein expression, anhydrous methanol was replenished every 12 hours, and then fermentation was terminated after 120 hours of induction. The fermentation broth was collected and centrifuged at 6000 rpm for 6 minutes, and the supernatant was collected. The supernatant was subjected to cation chromatography, enzyme digestion, polymer chromatography, ultrafiltration, and freeze-drying. The purity of the freeze-dried sample was detected by 90% by HPLC, and the molecular weight was detected by MALDI-TOF MS. The detected molecular weight of A14E, B16E, B25H, Des (B30) human insulin analogue was 5628.41 Da, the theoretical value was 5628.39 Da, and the detected value was consistent with the theoretical value; the detected molecular weight of A14E, B16H, B25H, Des (B30) human insulin analogue was 5637.06 Da, the theoretical value was 5636.31 Da, and the detected value was consistent with the theoretical value.

[0051] Example 2 Preparation of long-acting insulin 2.1 A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 Preparation of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analog (1)COOH(CH2) 18 Preparation process of CO-L-Lys-CO(CH2)2CO-(OEG)2-OSu side chain compound [ka]

[0052] a) ZCX-A00 (40 g, 58.39 mmol), ZCX-B00 (31.80 g, 233.56 mmol), Dowex50 WX2-100 acid cation resin (60 g) and 360 mL of n-octane were added to a three-necked round-bottom flask, the temperature was raised to 110° C., and the mixture was stirred and maintained at reflux for 72 hours. The heating power was turned off, and the mixture was continued to stir and return to room temperature. The filtrate was discarded by suction filtration to obtain a filtration residue, and then 360 mL of dichloromethane was added to the filtration residue and stirred at room temperature for 2 hours, and then the filtration residue was discarded by suction filtration, and the obtained filtrate was concentrated to dryness in vacuum to obtain a solid crude product. 60 mL of isopropanol was added to the solid crude product to recrystallize it to obtain 16.19 g of product ZCX-01.

[0053] ESI-MS m / z: 433.33 [M+H] + , which was consistent with the theoretical value.

[0054] b) ZCX-01 (10.0 g, 23.11 mmol) and 130 mL of dichloromethane were added to a 250 mL one-neck flask, then N-hydroxysuccinimide (2.93 g, 25.42 mmol) and dicyclohexylcarbodiimide (5.72 g, 27.72 mmol) were added, and the mixture was reacted at 30° C. for 24 hours. The mixture was then filtered to remove precipitate, distilled, and concentrated to dryness to obtain a solid crude product. 60 mL of isopropanol and 60 mL of n-heptane were added to the solid crude product to recrystallize it, obtaining 10.15 g of product ZCX-02.

[0055] ESI-MS m / z: 530.32 [M+H] + , which was consistent with the theoretical value.

[0056] c) ZCX-02 (10.6 g, 20 mmol), ZCX-C00 (lysine derivative, 8.2 g, 22 mmol) and 150 mL of dichloromethane were added to a 250 mL one-neck round-bottom flask, the mixture was stirred at room temperature, and then 5.5 mL of triethylamine was added. 2N hydrochloric acid solution was added to the mixture to adjust the pH to 1-2, the mixture was kept stirring for 30 minutes, then separated, the aqueous phase was discarded, and the organic phase was concentrated to dryness in vacuum and purified by column chromatography to obtain the product Nα-(long aliphatic chain diacid)-L-Lys-1-benzyl ester-6-Boc.

[0057] ESI-MS m / z: 752.52 [M+H] + , which was consistent with the theoretical value.

[0058] d) H NMR data of ZCX-03 showed that the structure obtained was the target product ZCX-03. [ka]

[0059] 1H-NMR (400 MHz, CDCl3) δ 7.37 (s, 10H), 6.08 (d, J = 7.2 Hz, 1H), 5.19 (dd, J = 26.8, 13.8 Hz, 4H), 4.66 (dd, J = 12.5, 7.4 Hz, 1H), 4.54 (s, 1H), 3.07 (d, J = 6.0 Hz, 2H), 2.37 (t, J = 7.5 Hz, 2H), 2.29 - 2.17 (m, 2H), 1.87 (d, J = 34.8 Hz, 1H), 1.73 (d, J = 14.2 Hz, 1H), 1.68 - 1.58 (m, 4H), 1.46 (s, 11H), 1.28 (d, J = 12.9 Hz, 30H).

[0060] ZCX-03 (11.5 g, 15 mmol), 55 mL of trifluoroacetic acid and 55 mL of dichloromethane were added to a one-neck round-bottom flask, and then the flask was placed in a 0°C low-temperature tank, and the mixture was stirred and reacted for 1 hour. After the reaction was essentially complete by TLC detection, the reaction system was concentrated to dryness in vacuum to obtain a viscous liquid, and then 200 mL of dichloromethane was added to dissolve, and the mixture was washed with saturated NaHCO3 solution, then separated, and the organic phase was washed twice with saturated brine, separated, and the organic phase was concentrated to dryness in vacuum, and recrystallized with absolute ethanol to obtain 8.35 g of product ZCX-C04.

[0061] ESI-MS m / z: 651.56 [M+H] + , which was consistent with the theoretical value.

[0062] e) ZCX-C04 (8.00 g, 12.31 mmol), 150 mL of dichloromethane and 3 mL of triethylamine were added to a one-neck round-bottom flask, the mixture was stirred at room temperature to dissolve, and then succinic anhydride (2.46 g, 24.62 mmol) was added. After the addition, the mixture was stirred and reacted at 30° C. for 24 hours. After the reaction was essentially complete by TLC detection, 10 mL of 2N HCl solution was added to adjust the pH to 1-2, then the mixture was stirred for 30 minutes, separated, the aqueous phase was discarded, and the organic phase was washed once with saturated brine, then separated, the organic phase was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated to dryness in vacuum to obtain a crude solid product. The crude solid product was recrystallized with absolute ethanol to obtain 8.9 g of product ZCX-C05.

[0063] ESI-MS m / z 751.03[M+H] + , which was consistent with the theoretical value.

[0064] H NMR data of ZCX-05 showed that the obtained structure was the target product ZCX-05.

[0065] 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 3.7 Hz, 10H), 6.39 - 6.26 (m, 2H), 5.19 (q, J = 12.2 Hz, 2H), 5.13 (s, 2H), 4.66 (td, J = 8.4, 4.6 Hz, 1H), 3.24 (d, J = 59.8 Hz, 2H), 2.74 - 2.65 (m, 2H), 2.51 (dd, J = 10.4, 5.6 Hz, 2H), 2.37 (t, J = 7.5 Hz, 2H), 2.30 - 2.23 (m, 2H), 1.85 (d, J = 34.4 Hz, 1H), 1.65 (d, J = 24.7 Hz, 5H), 1.52 (d, J = 36.5 Hz, 2H), 1.38 - 1.22 (m, 30H).

[0066] f) ZCX-05 (8.5 g, 11.32 mmol) and 130 mL of dichloromethane were added into a 250 mL one-neck flask, followed by the addition of N-hydroxysuccinimide (1.95 g, 16.98 mmol) and dicyclohexylcarbodiimide (3.50 g, 16.98 mmol). The mixture was reacted continuously at 30° C. for 24 hours, then filtered to remove precipitate, distilled and concentrated to dryness to obtain crude solid product ZCX-06, which was used directly in the next step without purification.

[0067] To the solid crude product ZCX-06 obtained in the previous step, [2-(2-{2-[2-(2-aminoethoxy)ethoxy]acetylamino}ethoxy)ethoxy]acetic acid (alternative name is H-2xOEG-OH) (3.73 g, 11.32 mmol) and 130 mL of dichloromethane were added, the mixture was stirred at room temperature for 10 minutes, and then 2.4 mL of triethylamine was added. After the addition, the mixture was stirred and reacted at 30° C. for 24 hours. After the reaction was essentially complete by TLC detection, 10 mL of 2N HCl solution was added to the mixture, stirred for 30 minutes, then separated, the aqueous phase was discarded, the organic phase was washed twice with saturated brine, separated, the aqueous phase was discarded, the organic phase was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated to dryness in vacuum to obtain a crude solid product. The crude solid product was purified by column chromatography to give 5.50 g of product ZCX-07.

[0068] ESI-MS m / z 1042.59[M+H] + , which was consistent with the theoretical value.

[0069] g) ZCX-07 (5.00 g, 4.80 mmol) and 130 mL of dichloromethane were added to a 250 mL one-neck flask, followed by the addition of N-hydroxysuccinimide (0.83 g, 7.2 mmol) and dicyclohexylcarbodiimide (1.49 g, 7.2 mmol). The mixture was reacted continuously at 30° C. for 24 hours, then filtered to remove precipitate, distilled, and concentrated to dryness to obtain a solid crude product. The solid crude product was recrystallized by adding 50 mL of isopropanol and 50 mL of n-heptane to obtain 4.30 g of product ZCX-08.

[0070] ESI-MS m / z 1139.10[M+H] + , which was consistent with the theoretical value.

[0071] The H NMR data of ZCX-08 indicated that the obtained structure was the target product ZCX-08.

[0072] 1H NMR (400 MHz, CDCl3) δ 7.36 (s, 10H), 7.27 - 7.22 (m, 1H), 6.63 (s, 1H), 6.29 (dd, J = 11.9, 6.7 Hz, 2H), 5.18 (t, J = 9.8 Hz, 2H), 5.12 (s, 2H), 4.61 (td, J = 7.9, 5.1 Hz, 1H), 4.51 (s, 2H), 4.02 (s, 2H), 3.83 - 3.76 (m, 2H), 3.70 - 3.66 (m, 4H), 3.61 (dd, J = 8.7, 4.0Hz, 4H), 3.56 - 3.48 (m, 4H), 3.47 - 3.40 (m, 2H), 3.23 - 3.12 (m, 2H), 2.87 (s, 4H), 2.52 (d, J = 5.2 Hz, 2H), 2.47 (d, J = 5.4 Hz, 2H), 2.36 (t, J = 7.5 Hz, 2H), 2.23 (t, J = 7.6 Hz, 2H), 1.80 (s, 1H), 1.64 (dd, J = 14.5, 7.3 Hz, 5H), 1.53 - 1.44 (m, 2H).

[0073] h) ZCX-08 (1.40 g, 1.22 mmol), 10% Pd / C (0.12 g), 0.1 mL of trifluoroacetic acid, 30 mL of THF and 10 mL of methanol were added to a one-neck round-bottom flask, the flask was flushed with hydrogen three times, sealed with a hydrogen balloon, and the mixture was placed at 30° C. and stirred for 6 hours for hydrogenation and debenzylation. After the reaction was essentially complete by TLC detection, the mixture was filtered to remove the 10% Pd / C, and 120 mL of n-heptane was added dropwise to the organic filtrate and continued to stir. During the addition, a solid precipitated, and after the addition was completed, the mixture was stirred at room temperature for 0.5 hours, filtered, and 0.83 g of COOH(CH2) was added. 18 The product ZCX-09, CO-L-Lys-CO(CH2)2CO-(OEG)2-Osu aliphatic side chain, was obtained.

[0074] ESI-MS m / z 959.45[M+H] + , which was consistent with the theoretical value.

[0075] H NMR data of ZCX-09 indicated that the obtained structure was the target product ZCX-09.

[0076] 1H NMR (400 MHz, DMSO) δ 4.63 (d, J = 25.1 Hz, 2H), 3.88 (s, 2H), 3.28 (dd, J = 11.5, 5.7 Hz, 2H), 3.19 (dd, J = 11.3, 5.6 Hz, 2H), 2.83 (s, 3H), 2.22 - 2.14 (m, 2H), 2.10 (t, J = 7.3 Hz, 2H), 1.60 - 1.42 (m, 4H), 1.26 (d, J = 24.8 Hz, 26H).

[0077] (2)A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 Preparation of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analog A14E, B16E, B25H, Des(B30) Human insulin (60 mg, 0.01 mmol) was dissolved in a solution of 5 mL of pure water and 2 mL of DMF, and the mixture was placed in a 10°C low-temperature reaction bath, and then 100 μl of triethylamine was added dropwise to adjust the pH to 11.50. COOH(CH2) 18 The CO-L-Lys-CO(CH2)2CO-(OEG)2-OSu side chain compound (14.37 mg, 0.015 mmol) was dissolved in 3 mL of DMF to form a side chain mixed solution, and under stirring, the side chain mixed solution was rapidly added to the above reaction system, and the pH of the reaction system was kept constant at 11.00-11.50 using 1N NaOH solution. After the addition, timing was started, and after 1.0 hour of reaction, the pH of the solution was adjusted to 7.0-7.5 with 1N HCl solution. The reaction was terminated to obtain a crude product solution of the acylation of the reactive protein, and the reaction process was controlled by RP-HPLC.

[0078] (3)A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18Purification of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analog The above protein acylation crude product solution was diluted with water to make the organic phase content about 15% (v / v), filtered through a 0.45 μm filter membrane, and then purified by RP-HPLC to obtain a purified solution.

[0079] (4)A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 Ultrafiltration and lyophilization of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analogue The above purified solution was replaced with water for injection using an ultrafiltration membrane packaging system, and then freeze-dried to obtain 23 mg of lyophilized product, the molecular structure of the resulting human insulin analogue was as follows: [ka]

[0080] (5)A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 Structural confirmation of CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 18 The measured mass spectrum of the desB30 human insulin analog was 6471.42 Da, which was consistent with the theoretical molecular weight of 6471.64 Da. It can be abbreviated as insulin-a3, A14E, B16E, B25H, B29K (N(ε)-COOH(CH2) 18 It was shown that the CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue was successfully prepared.

[0081] 2.2 A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16Preparation of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analog (1)COOH(CH2) 16 Preparation process of CO-L-Lys-CO(CH2)2CO-(OEG)2-OSu side chain compound [ka]

[0082] COOH(CH2) 16 The preparation of CO-L-Lys-CO(CH2)2CO-(OEG)2-Osu side chain (designated ZCY-09) was carried out by the procedure described in Example 2.1. 18 The preparation method of CO-L-Lys-CO(CH2)2CO-(OEG)2-OSu side chain compound is similar, and the structure and MS test of the prepared target product are shown below.

[0083] ESI-MS m / z 931.40[M+H] + , which was consistent with the theoretical value.

[0084] H NMR data of ZCY-09 indicated that the obtained structure was the target product ZCY-09. [ka]

[0085] 1H NMR (400 MHz, DMSO) δ 4.60 (s, 2H), 3.85 (d, J = 26.1 Hz, 2H), 2.83 (d, J = 4.1 Hz, 5H), 2.28 (p, J = 7.9 Hz, 4H), 2.18 (t, J = 7.3 Hz, 2H), 2.10 (t, J = 7.3 Hz, 2H).

[0086] (2)A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 Preparation of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analog A14E, B16E, B25H, Des(B30) Human insulin (60 mg, 0.01 mmol) was dissolved in a solution of 5 mL of pure water and 2 mL of DMF, and the mixture was placed in a 10°C low-temperature reaction bath, and then 100 μl of triethylamine was added dropwise to adjust the pH to 11.50. COOH(CH2) 16 CO-L-Lys-CO(CH2)2CO-(OEG)2-Osu side chain (13.95 mg, 0.015 mmol) was dissolved in 3 mL of DMF to form a side chain mixed solution. Under stirring, the side chain mixed solution was added quickly to the above reaction system, and the pH of the reaction system was kept constant at 11.00-11.50 using 1N NaOH solution. After the addition, timing was started, and after 1.0 h of reaction, the pH of the solution was adjusted to 7.0-7.5 with 1N HCl solution. The reaction was terminated to obtain a crude product solution of reactive protein acylation, and the reaction process was controlled by RP-HPLC.

[0087] (3)A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 Purification of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analog The above protein acylation crude product solution was diluted with water to an organic phase content of about 15% (v:v), filtered through a 0.45 μm filter membrane, and then purified by RP-HPLC to obtain a purified solution.

[0088] (4)A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 Ultrafiltration and lyophilization of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analogue The above purified solution was replaced with water for injection using an ultrafiltration membrane packaging system, and then freeze-dried to obtain 18 mg of lyophilized product, the molecular structure of the resulting human insulin analogue is as follows: [ka]

[0089] (5)A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 Structural confirmation of CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue A14E,B16E,B25H,B29K(N(ε)-COOH(CH2) 16 The measured mass spectrum of the desB30 human insulin analog was 6443.40 Da, which was consistent with the theoretical molecular weight of 6443.41 Da. It can be abbreviated as insulin-a2, A14E, B16E, B25H, B29K (N(ε)-COOH(CH2) 16 It was shown that the CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue was successfully prepared.

[0090] 2.3 A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 Preparation of CO-L-Lys-CO(CH2)2CO-(OEG)2),desB30 human insulin analog COOH(CH2) 18 The preparation of the CO-L-Lys-CO(CH2)2CO-(OEG)2-OSu side chain was carried out using the COOH(CH2) 18 The preparation method is the same as that of the CO-L-Lys-CO(CH2)2CO-(OEG)2-OSu side chain compound.

[0091] A14E, B16H, B25H, Des(B30) Human insulin (60 mg, 0.01 mmol) was dissolved in a solution of 5 mL of pure water and 2 mL of DMF, and the mixture was placed in a 10° C. low-temperature reaction bath, and then 100 μL of triethylamine was added dropwise to adjust the pH to 11.50. Nα-(eicosanedioic acid)-Nε-(OCCH2CH2CO-(2xOEG-OSu)-L-Lys side chain (14.37 mg, 0.015 mmol) was dissolved in 3 mL of DMF to form a side chain mixed solution. The side chain mixed solution was rapidly added to the above reaction system under stirring, and the pH of the reaction system was kept constant at 11.00-11.50 using 1N NaOH solution. After the addition, timing was started. After 1.0 h of reaction, the pH of the solution was adjusted to 7.0-7.5 with 1N HCl solution. The reaction was terminated to obtain a crude product solution of reactive protein acylation, and the reaction process was controlled by RP-HPLC.

[0092] The above protein acylation crude product solution was diluted with water to make the organic phase content about 15% (v:v), filtered through a 0.45 μm filter membrane, and then purified by RP-HPLC to obtain a purified solution.

[0093] The above purified solution was replaced with water for injection using an ultrafiltration membrane packaging system, and then freeze-dried to obtain 16 mg of lyophilized product, the molecular structure of the resulting human insulin analogue was as follows: [ka]

[0094] A14E,B16H,B25H,B29K(N(ε)-COOH(CH2) 18 The measured mass spectrum of the desB30 human insulin analog was 6480.10 Da, which was consistent with the theoretical molecular weight of 6480.10 Da. It can be abbreviated as insulin-a10, A14E, B16H, B25H, B29K (N(ε)-COOH(CH2) 18It was shown that the CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analogue was successfully prepared.

[0095] 2.4 A14E, B16E, B25H, B29K(N ε Preparation of -eicosanedioyl-gGlu-2xOEG), DesB30 human insulin analogue (1) Preparation of 19-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid tert-butyl ester: (Alternative names are t Bu-eicosanedioyl-gGlu)(O t Bu)-2xOEG-Osu) TSTU (1.50 g) and DIPEA (0.91 mL) were added to a solution containing 19-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid tert-butyl ester (3.0 g, purchased from Shanghai Topbiochem Technology Co., Ltd.) in acetonitrile (60 ml), and the mixture was stirred at room temperature overnight and then concentrated in vacuo. 0.1N HCl aqueous solution (100 mL) and ethyl acetate (200 mL) were added to the residue, then separated, the aqueous phase was extracted with ethyl acetate (50 mL), the organic phases were combined, washed once with saturated brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo to give 3.21 g of an oily liquid. ESI-MS m / z 972.30[M+H] + , which was consistent with the theoretical value.

[0096] (2) 19-((S)-1-carboxy-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid: (Alternative name is eicosanedioyl-gGlu-2xOEG-Osu) t Bu-eicosanedioyl-gGlu)(O t Bu)-2xOEG-Osu (3.0 g) was added to trifluoroacetate (66 mL) and the mixture was stirred at room temperature for 45 min. After the reaction was completed by TLC detection, the mixture was concentrated in vacuum to give an oily liquid, and then concentrated three times with toluene to give a solid. It was recrystallized using isopropyl alcohol and filtered to give 2.35 g of a white solid.

[0097] ESI-MS m / z 860.60[M+H] + , which was consistent with the theoretical value.

[0098] (3) (A14E, B16E, B25H, B29K (N ε Preparation of -eicosanedioyl-gGlu-2xOEG), DesB30 human insulin analogue A14E, B16E, B25H, Des(B30) Human insulin (60mg, 0.01mmol) was dissolved in a solution of 5ml of pure water and 2mL of DMF, the mixture was placed in a 10℃ low temperature reaction bath, and then 100μL of triethylamine was added dropwise to adjust the pH to 11.50. Eicosanedioyl-gGlu-2xOEG-Osu aliphatic side chain (15.00mg, 0.017mmol) was dissolved in 3mL of DMF to form a side chain mixed solution, and under stirring, the side chain mixed solution was added quickly to the above reaction system, and the pH of the reaction system was kept constant at 11.00-11.50 using 1N NaOH solution. After the addition, timing was started, and after 1.0 hour of reaction, the pH of the solution was adjusted to 7.0-7.5 with 1N HCl solution. The reaction was terminated to obtain a crude product solution of reactive protein acylation, and the reaction process was controlled by RP-HPLC.

[0099] (4) A14E, B16E, B25H, B29K(N ε Preparation of (Eicosanedioyl-gGlu-2xOEG)DesB30 human insulin analog The above protein acylation crude product solution was diluted with water to an organic phase content of about 15% (v:v), filtered through a 0.45 μm filter membrane, and then purified by RP-HPLC to obtain a purified solution.

[0100] (5) A14E, B16E, B25H, B29K(N ε -Eicosanoidioyl-gGlu-2xOEG), DesB30 human insulin analogue ultrafiltration and lyophilization The above purified solution was replaced with water for injection using an ultrafiltration membrane packaging system, and then freeze-dried to obtain 9.3 mg of lyophilized product, the molecular structure of the resulting human insulin analogue was as follows: [ka]

[0101] (6) A14E, B16E, B25H, B29K(N ε -Eicosanedioyl-gGlu-2xOEG), structure confirmation of DesB30 human insulin analogue A14E, B16E, B25H, B29K(N ε -eicosandioyl-gGlu-2xOEG), the measured mass spectrum of the DesB30 human insulin analog was 6372.28 Da, which was consistent with the theoretical molecular weight of 6372.33 Da. It can be abbreviated as insulin-a1, A14E, B16E, B25H, B29K (N ε -eicosandioyl-gGlu-2xOEG), the target product of DesB30 human insulin analogue was shown to be successfully prepared.

[0102] 2.5 A14E, B16H, B25H, B29K(N εPreparation of -eicosanedioyl-gGlu-2xOEG), DesB30 human insulin analogue (1) Preparation of 19-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid tert-butyl ester: (Alternative names are t Bu-eicosanedioyl-gGlu)(O t Bu)-2xOEG-Osu) TSTU (1.50 g) and DIPEA (0.91 mL) were added to a solution containing 19-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid tert-butyl ester (3.0 g, purchased from Shanghai Topbiochem Technology Co., Ltd.) in acetonitrile (60 mL), and the mixture was stirred at room temperature overnight and then concentrated in vacuo. 0.1N HCl aqueous solution (100 mL) and ethyl acetate (200 mL) were added to the residue, then separated, the aqueous phase was extracted with ethyl acetate (50 mL), the organic phases were combined, washed once with saturated brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo to give 3.21 g of an oily liquid. ESI-MS m / z 972.30[M+H] + , which was consistent with the theoretical value.

[0103] (2) 19-((S)-1-carboxy-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)nonadecanoic acid: (Alternative name is eicosanedioyl-gGlu-2xOEG-Osu) tBu-eicosanedioyl-gGlu)(O t Bu)-2xOEG-Osu (3.0 g) was added to trifluoroacetate (66 mL) and the mixture was stirred at room temperature for 45 min. After the reaction was completed by TLC detection, the mixture was concentrated in vacuum to give an oily liquid, and then concentrated three times with toluene to give a solid. It was recrystallized using isopropyl alcohol and filtered to give 2.35 g of a white solid. ESI-MS m / z 860.60[M+H] + , which was consistent with the theoretical value.

[0104] (3) (A14E, B16H, B25H, B29K (N ε Preparation of -eicosanedioyl-gGlu-2xOEG), DesB30 human insulin analogue A14E, B16H, B25H, Des(B30) Human insulin (60 mg, 0.01 mmol) was dissolved in a solution of 5 mL of pure water and 2 mL of DMF, the mixture was placed in a 10 °C low-temperature reaction bath, and then 100 μL of triethylamine was added dropwise to adjust the pH to 11.50. Eicosanedioyl-gGlu-2xOEG-OSu aliphatic side chain (15.00 mg, 0.017 mmol) was dissolved in 3 mL of DMF to form a side chain mixed solution, and under stirring, the side chain mixed solution was quickly added to the above reaction system, and the pH of the reaction system was kept constant at 11.00-11.50 using 1N NaOH solution. After the addition, timing was started, and after 1.0 h of reaction, the pH of the solution was adjusted to 7.0-7.5 with 1N HCl solution. The reaction was terminated to obtain a crude product solution of the acylation of the reactive protein, and the reaction process was controlled by RP-HPLC.

[0105] The above protein acylation crude product solution was diluted with water to an organic phase content of about 15% (v:v) and filtered through a 0.45 μm filter membrane.

[0106] The above purified solution was replaced with water for injection using an ultrafiltration membrane packaging system, and then freeze-dried to obtain 13.21 mg of lyophilized product, the molecular structure of the resulting human insulin analogue was as follows: [ka]

[0107] A14E, B16H, B25H, B29K(N ε -eicosandioyl-gGlu-2xOEG), the measured mass spectrum of the DesB30 human insulin analog was 6381.01 Da, which was consistent with the theoretical molecular weight of 6381.51 Da. ε -eicosandioyl-gGlu-2xOEG), the target product of DesB30 human insulin analogue was shown to be successfully prepared.

[0108] Example 3 In vitro bioactivity testing of insulin The acylated insulin analogs of the present invention can activate cells transfected with insulin receptor B to produce insulin receptor autophosphorylation, and can also reversibly bind to human serum albumin (HSA). The phosphorylation level of insulin receptor B was detected by Cisbio's Rhospho-IR beta (Tyr1150 / 1151) kit method to evaluate the biological activity of insulin. Cells were seeded in a 96-well plate overnight, and after removing the serum in the medium, 40 μl of serum-free medium was added to the culture for about 4 hours. Then, a dilution series of insulin derivatives was prepared using blank solution (0.6% casein, 0.06 mg / mL EDTA, 1xDPBS) and incubated with the cells in the 96-well plate for 5 minutes in a CO2 incubator (37°C, 5% CO2). The liquid in the 96-well plate was poured off, and a mixture of 100 μL of lysis buffer (2% Triton X-100, 150 mM NaCl, 50 mM HEPES, pH=7) and inhibitor (blocking reagent in the kit) was added to lyse the cells, and then the plate was shaken at 350 rpm for 30 minutes. The insulin receptor phosphorylation level in the supernatant after cell lysis was measured, and the relative activity (percent (%)) was evaluated by fitting a curve to the data using nonlinear regression in Graphpad Prism 5 software. A related assay was also used in which the blank solution also contained 1.5% HSA to simulate physiological conditions. The change in phosphorylation level of the insulin-activated insulin receptor of the present invention was detected as an indirect reflection of albumin binding activity.

[0109] [Table 1]

[0110] Remarks: (1) A14E, B16E, B25H, B29K(N ε -eicosanedioyl-gGlu-2xOEG), DesB30 human insulin analog, this compound is abbreviated as insulin-a1.

[0111] (2): A14E, B16H, B25H, B29K(N ε -Eicosanedioyl-gGlu-2xOEG), DesB30 human insulin analog, this compound is abbreviated as Icodec.

[0112] From the data in Table 1, it can be seen that the in vitro activity of the new aliphatic side chain acylation to prepare the new insulin drugs insulin-a3 and insulin-a10 is significantly reduced compared with recombinant human insulin or insulin degludec under the conditions of 0% HSA and 1.5% HSA. The main reason is that the side chain has a certain effect on the in vitro activity, since the side chain binds strongly to albumin and weakly to the receptor of the insulin precursor, which also reveals the different binding ability of the new insulin drugs and albumin. In this repeated administration experiment of C57BL6 mice modeled by STZ, it can be seen that the effective glucose control effect of the control insulin-a1 can be maintained for 3 days / time, and the effective glucose control effect of insulin-a3 can be maintained for 4-5 days / time. It can be seen that insulin-a3 has a longer glucose control maintenance time than the control insulin-a1, and the effect is better. Therefore, when the insulin precursor is the same, this reversible binding ability is better in the new insulin drugs.

[0113] Example 4 Hypoglycemic effects of test drugs on normal C57BL / 6 mice (1) Test product [Table 2]

[0114] Among them, degludec means insulin degludec, and icodec means A14E, B16H, B25H, B29K (N ε-eicosanoidioyl-gGlu-2xOEG), DesB30 human insulin analogue, insulin-a1 refers to the long-acting insulin A14E, B16E, B25H, B29K (N ε -eicosanedioyl-gGlu-2xOEG), DesB30 human insulin analog. Insulin-a3 is A14E, B16E, B25H, B29K (N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analog. Insulin-a4 is A14E, B16E, B25H, B29K (N(ε)-COOH(CH2) 16 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analog. Insulin-a10 is A14E, B16H, B25H, B29K (N(ε)-COOH(CH2) 18 CO-L-Lys-CO(CH2)2CO-(OEG)2), desB30 human insulin analog.

[0115] (2) Sample composition Different insulin analogues API used in the pharmacological experiments were formulated to the desired concentrations using PBS buffer.

[0116] (3) Laboratory animals [Table 3]

[0117] (4) Experimental Method SPF grade C57BL / 6 mice were housed in suitable vivarium with a barrier environment with a housing temperature of 20-26°C, humidity of 40-70%, and a 12 h / 12 ​​h day / night time, and the mice had free access to standard food and autoclaved water. After a 3-day isolation period and a 2-day acclimation period, random blood glucose was measured and the mice were weighed. The mice were divided into six groups according to random blood glucose and body weight. Animal grouping and dosing are shown in Table 4: [Table 4]

[0118] A single subcutaneous injection (SC) was used to administer the corresponding vehicle or drug. The control group was administered with the vehicle PBS without fasting throughout the entire process, and the mice were allowed to eat and drink ad libitum. Random blood glucose levels of C57 mice were measured before administration and at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 48, 54, 72 and 96 hours after administration.

[0119] All raw data were entered into an Excel file and expressed as mean ± SEM. Statistical analysis of the data was performed using Graphpad Prism 7.0 software, one-way or two-way ANOVA comparison method, and P<0.05 was used as the criterion for significant differences.

[0120] (5) Results Compared with the control group, 1 hour after administration, the blood glucose of the insulin degludec group, icodec group, insulin-a1 group, insulin-a3 group, insulin-a4 group and insulin-a10 group was significantly reduced; 2 hours after administration, the blood glucose of the mice in the insulin degludec group reached the lowest level and then rose slowly, while the blood glucose of the mice in the other five groups continued to decline slowly; 10 hours after administration, the blood glucose of the insulin degludec group gradually recovered, the blood glucose of the insulin-a4 group reached the lowest level and gradually recovered, and the blood glucose of the icodec group, insulin-a1 group, insulin-a3 group and insulin-a10 group still maintained a slow decline; 24 hours after administration, the blood glucose of the mice in the insulin degludec group returned to normal, the blood glucose of the insulin-a4 group gradually recovered, the blood glucose of the insulin-a1 group and insulin-a3 group reached the lowest level, and the blood glucose between the two groups was significantly reduced. There was no significant difference in blood glucose, and blood glucose gradually recovered during follow-up, but blood glucose in the Icodec group and insulin-a10 group continued to decline slowly; 48 hours after administration, blood glucose in the insulin-a1 group and insulin-a4 group returned to normal, blood glucose in the insulin-a3 group showed a tendency to rise, but blood glucose was still at a low level, blood glucose in the Icodec group reached the lowest level and gradually recovered, but blood glucose in the insulin-a10 group continued to decline slowly; 72 hours after administration, blood glucose in the insulin-a3 group remained low, blood glucose in the Icodec group gradually recovered, but blood glucose in the insulin-a10 group reached the lowest level and then gradually increased; 96 hours after administration, blood glucose in the other groups except the insulin-a10 group returned to normal levels, and blood glucose in the insulin-a10 group gradually increased, but had not yet reached the normal level. Specific data are shown in Table 5 and Figure 1. [Table 5]

[0121] The results show that in this single-dose experiment in normal C57BL / 6 mice, the effective blood glucose control time of insulin degludec is 24 hours, the effective blood glucose control time of insulin-a4 is 48 hours, the effective blood glucose control time of insulin-a1 is 72 hours, the effective blood glucose control time of icodec and insulin-a3 are both 96 hours, and the effective blood glucose control time of insulin-a10 is more than 96 hours. Compared with icodec, the effect of insulin-a3 on blood glucose control is slightly worse, but still has the same effective blood glucose control time as icodec, while the effect of insulin-a10 on blood glucose control is consistent with the trend of icodec and can be maintained for a longer time.

[0122] Example 5 Hypoglycemic effects of test drugs on STZ-induced type I diabetes mellitus (T1DM) in C57BL / 6 mice (1) Test product [Table 6]

[0123] (2) Sample composition Different insulin analogues API used in the pharmacological experiments were formulated to the desired concentrations using PBS buffer.

[0124] (3) Laboratory animals [Table 7]

[0125] (4) Experimental Method SPF grade C57BL / 6 mice were housed in a suitable breeding box in a barrier environment with a breeding temperature of 20-26 °C, humidity of 40-70%, and a day-night time of 12 h / 12 ​​h, and the mice had free access to standard food and autoclaved sterilized water. After a 3-day isolation period and a 2-day acclimation period, the mice were fasted for 12 h and intraperitoneally injected with 130 mg / kg streptozotocin solution (STZ, 13 mg / mL, in citrate buffer) or citrate buffer (control group). Random blood glucose and fasting blood glucose were detected 3 and 7 days after administration of streptozotocin, and mice with random blood glucose values ​​above 25 mmol / L and fasting blood glucose values ​​above 11.1 mmol / L were selected as T1DM model mice for follow-up experiments. On the day before administration, random blood glucose was monitored and the mice were weighed. The mice were randomly divided into four groups according to blood glucose and body weight.

[0126] Animal groupings and treatments were as follows: [Table 8]

[0127] Subcutaneous administration (SC) was used to administer the corresponding vehicle or drug once every 4-5 days for a total of four doses. Mice were allowed to eat and drink ad libitum throughout the experiment. Random blood glucose was assessed before the first dose and at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 48, 72, and 96 hours after dosing, as well as before the second, third, and fourth doses and at 1, 2, 4, 6, 8, 24, 48, 72, 96, and 120 hours after dosing.

[0128] All raw data were entered into an Excel file and expressed as mean ± SEM. Statistical analysis of the data was performed using Graphpad Prism 7.0 software, one-way or two-way ANOVA comparison method, and P<0.05 was used as the criterion for significant differences.

[0129] (5) Results Specific data are shown in Table 9 and FIG. [Table 9]

[0130] The results showed that compared with the model group, the blood glucose of insulin-a1 was significantly lowered 24 hours after each administration, reached the lowest level, then slowly rose, and reached the normal level 72 hours after administration; the blood glucose of insulin-a3 was significantly lowered 24 hours after the first and second administration, reached the lowest level, then slowly rose, and reached the normal level 96 hours after administration. With the increase in the number of administrations, the effective glucose control time of insulin-a3 was extended after the third and fourth administrations, reaching the normal level only 120 hours after administration, and after each administration, the lowering effect of insulin-a3 on blood glucose was better than insulin-a1.

[0131] In conclusion, the glucose control effect and effective glucose control time of insulin-a3 were significantly better than those of insulin-a1.

[0132] Example 6 Hypoglycemic effects of test drugs on STZ-induced type I diabetes mellitus (T1DM) in C57BL / 6 mice (1) Test product [Table 10]

[0133] (2) Sample composition Different insulin analogues API used in the pharmacological experiments were formulated to the desired concentrations using PBS buffer.

[0134] (3) Laboratory animals [Table 11]

[0135] (4) Experimental Method SPF grade C57BL / 6 mice were housed in appropriate breeding boxes in a barrier environment with a breeding temperature of 20-26 °C, humidity of 40-70%, and a day-night time of 12 h / 12 ​​h, and the mice had free access to standard food and autoclaved sterilized water. After a 3-day isolation period and a 2-day acclimation period, the mice were fasted for 12 h and intraperitoneally injected with 130 mg / kg of streptozotocin solution (STZ, 13 mg / mL, in citrate buffer). Random blood glucose and fasting blood glucose were detected 3 and 7 days after administration of streptozotocin, and mice with random blood glucose values ​​above 25 mmol / L and fasting blood glucose values ​​above 11.1 mmol / L were selected as T1DM model mice for follow-up experiments. On the day before administration, random blood glucose was monitored and the mice were weighed. The mice were divided into six groups according to random blood glucose and body weight.

[0136] Animal groupings and treatments are shown in Table 12: [Table 12]

[0137] Subcutaneous administration (SC) was used to administer the corresponding vehicle or drug once every 4-5 days for a total of three doses. Mice were allowed to eat and drink ad libitum throughout the experiment. Random blood glucose was assessed before the first dose and 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 48, 72, and 96 hours after the dose, as well as before the second and third doses and 0.5, 1, 2, 6, 24, 48, 72, 96, and 120 hours after the dose.

[0138] All raw data were entered into an Excel file and expressed as mean ± SEM. Statistical analysis of the data was performed using Graphpad Prism 7.0 software, one-way or two-way ANOVA comparison method, and P<0.05 was used as the criterion for significant differences.

[0139] (5) Results Specific data are shown in Table 13 and Figure 3. [Table 13]

[0140] Note: *P<0.05, **P<0.01, ***P<0.001 vs. model. Compared with the model group, the blood glucose of the three doses of Icodec-250, 500 and 1000 nmol / kg was significantly lowered 24 hours after each administration, reached the lowest level, and then rose slowly. Throughout the entire experimental period, the lowering effect of Icodec on blood glucose and the effective blood glucose control time were dose-dependent, i.e., the higher the dose, the stronger the lowering effect and the longer the blood glucose control time. At a dose of 1000 nmol / kg, the effective blood glucose control time can reach 96 hours.

[0141] Compared with the model group, the blood glucose of insulin-a3 significantly decreased 24 hours after each administration, reached the lowest level, and then slowly increased, and blood glucose returned to normal levels 96 hours after the first and second administrations. With increasing number of administrations, the effective glucose control time of insulin-a3 was extended after the third administration, reaching normal levels only 120 hours after administration. At the same time, the blood glucose of insulin-a10 significantly decreased 24 hours after each administration, reached the lowest level, and then slowly increased, and blood glucose returned to normal levels 96 hours after the first administrations. With increasing number of administrations, the effective glucose control time of insulin-a10 was extended after the second and third administrations, reaching normal levels only 120 hours after administration. Compared with the Icodec-1000nmol / kg group, the glucose control effect of insulin-a3 was slightly worse, but better than the Icodec-500nmol / kg group, and its effective glucose control time could be maintained for 96-120 hours; the glucose control effect of insulin-a10 was equivalent to that of Icodec-1000nmol / kg, and its effective glucose control time could be maintained for 120 hours.

[0142] In conclusion, insulin-a3 and insulin-a10 can still achieve similar or better hypoglycemic effects when doses are less than twice as high as Icodec.

[0143] Example 7 PK study of intravenous injection in rats (1) Test product [Table 14]

[0144] (2) Sample composition Different insulin analogues API used in the pharmacological experiments were formulated to the desired concentrations using PBS buffer.

[0145] (3) Laboratory animals [Table 15]

[0146] (4) Experimental Method Four male SD rats (2 rats / group) were administered a single intravenous (iv) dose of 10 nmol / kg insulin-a1 or insulin-a3, and blood was collected 0.083, 0.25, 0.5, 1, 2, 5, 7, and 24 hours after administration. Plasma was centrifuged, and the concentrations of insulin-a1 or insulin-a3 in the plasma were detected by LC-MS / MS.

[0147] (5) Experimental results The results in Figure 4 and Table 16 showed that the AUClast and Cmax of insulin-a3 were slightly higher than those of insulin-a1, and the higher Cmax indicated that the plasma binding may be higher. Furthermore, the half-lives of insulin-a1 and insulin-a3 were 15.3±4.8 hours and 11.2±1.9 hours, respectively. In conclusion, insulin-a3 and insulin-a1 have similar effects on PK in rats.

[0148] [Table 16]

[0149] Example 8 In vivo subcutaneous injection PK study in rats (1) Test product [Table 17]

[0150] (2) Sample composition Different insulin analogues API used in the pharmacological experiments were formulated to the desired concentrations using PBS buffer.

[0151] (3) Laboratory animals [Table 18]

[0152] (4) Experimental Method Nine SD rats (3 rats / group) were administered a single subcutaneous (SC) dose of 10 nmol / kg insulin-a1, insulin-a10 and icodec, and blood was collected at 1, 2, 5, 24, 31, 48, 72, 96 and 120 hours, plasma was centrifuged and the concentrations of insulin-a1, insulin-a3 and icodec in plasma were detected.

[0153] (5) Experimental results The results in Figure 5 and Table 19 show that the AUClast and Cmax of insulin-a1 and insulin-a10 are slightly higher than those of icodec, and the higher Cmax indicates that the plasma binding may be higher. Furthermore, the subcutaneous half-life of insulin-a1 and insulin-a10 was 21 hours and 17.2 hours, respectively. In conclusion, the effect of insulin a-10 on PK in mice is better than that of the control icodec.

[0154] [Table 19]

[0155] Example 9 PK study of subcutaneous injection in C57BL6 mice (1) Test product [Table 20]

[0156] (2) Sample composition Different insulin analogues API used in the pharmacological experiments were formulated to the desired concentrations using PBS buffer.

[0157] (3) Laboratory animals [Table 21]

[0158] (4) Experimental Method Six C57 mice (3 mice / group) were administered a single subcutaneous (SC) dose of 10 nmol / kg insulin-a1 or insulin-a3, and blood was collected 1, 2, 5, 24, 31, 55, and 72 hours after administration, and plasma was centrifuged to detect the concentrations of insulin-a1 or insulin-a3 in the plasma.

[0159] (5) Experimental results The results in FIG. 6 and Table 22 show that the AUC last and C max indicates slightly higher C max showed that plasma binding may be higher, and insulin receptor activity studies with 1.5% HSA also demonstrated that insulin-a3 has better albumin binding effect, reflecting a longer duration of efficacy. Furthermore, the subcutaneous half-lives of insulin-a1 and insulin-a3 were 14.3 and 18.6 hours, respectively. In conclusion, the PK effect of insulin-a-3 in mice is not inferior to that of the control insulin-a1.

[0160] [Table 22]

[0161] Example 10 PK experiment in beagle dogs (1) Test product [Table 23]

[0162] (2) Sample composition Different insulin analogues API used in the pharmacological experiments were formulated to the desired concentrations using PBS buffer.

[0163] (3) Laboratory animals [Table 24]

[0164] (4) Experimental Method Two beagle dogs (one in each group) were used in a double-cycle crossover design with a washout period of one week. In each cycle, a single dose of 10 nmol / kg of icodec or insulin-a10 was administered into the lateral small saphenous vein of the hind limb. Blood was collected at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 6 hours, 8 hours, 24 hours, 30 hours, 48 ​​hours, 72 hours, and 96 hours after administration, and the plasma was centrifuged to detect the concentrations of icodec or insulin-a10 in the plasma.

[0165] (5) Experimental results [Table 25]

[0166] The results in Table 25 and Figure 7 show that insulin-a10 has a higher C max are equivalent, and the AUC last Although the C value was slightly lower, the half-life of insulin-a10 was 46 ± 10.8 h, which was significantly longer than the half-life of icodec, 35.7 ± 6.7 h. In conclusion, the C value of insulin-a10 in beagles was significantly higher than that of icodec, 35.7 ± 6.7 h. max is the C of Icodek max and has a longer half-life.

[0167] Throughout this specification, reference to "embodiments," "some embodiments," "one embodiment," "another example," "example," "particular example," or "some examples" means that a particular feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of the disclosure. Thus, the appearance of phrases such as "in some embodiments," "in one embodiment," "in an embodiment," "in another example," "in an example," "in a particular example," or "in some examples" in various places throughout this specification do not necessarily refer to the same embodiment or example of the disclosure. Moreover, particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples. Furthermore, one skilled in the art may combine and combine different embodiments, examples, or features thereof, as long as they are not mutually inconsistent.

[0168] While illustrative embodiments have been shown and described, the above embodiments should not be construed as limiting the present disclosure, and it will be recognized by those skilled in the art that changes, substitutions, and modifications can be made to the embodiments without departing from the spirit, principles, and scope of the present disclosure.

Claims

1. Formula (I): W-X-Y-Z-R (I) (wherein, W is a fatty acid or diacid having 10 to 20 carbon atoms and the structure is -CO(CH 2 ) n COOH, n is an integer between 10 and 20; X is a diamino compound containing a carboxylic acid group, and the carbon atom connecting the carboxylic acid group can be a chiral carbon or an achiral carbon, and has the structures shown in formula (a1), (a2) and (a3) 【Chemical 1】 (wherein s is an integer between 2 and 20, preferably 2 to 10, more preferably 2 to 8), and one of the amino groups in X is connected to one of the acyl groups in W to form an amide bond; Y is -A(CH 2 ) m B- (wherein m is an integer between 1 and 10, preferably an integer between 1 and 6, A and B are absent, or are -CO-) Z is -(OEG) p where p is an integer between 1 and 3, preferably 2, and the OEG structure is [Chemical 2] and; R is a leaving group, preferably an activated ester group; the linking groups between W, X, Y and Z are amide peptide bonds or peptide bonds) A novel side chain compound having the structure shown.

2. Formula (I): W-X-Y-Z-R (I) (wherein, W is a fatty acid or fatty diacid having 10 to 20 carbon atoms, and its structure is -CO(CH 2 ) n COOH, and n is an integer between 10 and 20; X is a diamino compound containing a carboxylic acid group, and the carbon atom connecting the carboxylic acid group can be a chiral carbon or an achiral carbon, and has the structures shown in formula (a1), (a2) and (a3) [Chemical Formula 3] (wherein s is an integer between 2 and 20, preferably 2 to 10, more preferably 2 to 8), and one of the amino groups in X is connected to one of the acyl groups in W to form an amide bond; Y is -A(CH 2 ) m B- (wherein m is an integer between 1 and 10, preferably an integer between 1 and 6, A and B are absent or -CO-) Z is -(OEG) p where p is an integer between 4 and 30, and the OEG structure is 【Chemical Formula 4】 and; R is a leaving group, preferably an activated ester group; the linking groups between W, X, Y and Z are amide peptide bonds or peptide bonds) A novel side chain compound having the structure shown.

3. The following structural formula: 【Chemical Formula 5】 (wherein n is an integer between 14 and 20, s is an integer between 2 and 4, m is an integer between 1 and 4, p is 2, R is the following group: ​ selected from; preferably, n is an integer between 16 and 18, s is an integer between 2 and 4, m is 2, p is 2, preferably, R is 【Chemical Formula 7】 ) The compound according to claim 1, having.

4. The following compounds: 【Chemical Formula 8-1】 【Chemical Figure 8-2】 (wherein R is 【Chemical Formula 9】 ) selected from any one of; preferably, having the following structural formula: 【Chemical 10】 ; more preferably, having the following structural formula: 【Chemical 11】 The compound according to claim 1, having.

5. Obtained by an acylation reaction between the side chain compound according to claim 1 and a human insulin analog, formula (II): W-X-Y-Z-M (II) (wherein, W is a fatty acid or fatty diacid having 10 to 20 carbon atoms, and its structure is -CO(CH 2 ) n COOH, and n is an integer between 10 and 20; X is a diamino compound containing a carboxylic acid group, and the carbon atom connecting the carboxylic acid group can be a chiral carbon or an achiral carbon, and has the structures shown in formula (a1), (a2) and (a3) 【Chemical 12】 (wherein s is an integer between 2 and 20, preferably 2 to 10, more preferably 2 to 8), having a structure shown by the formula, and one of the amino groups in X is connected to one of the acyl groups in W to form an amide bond; Y is -A(CH 2 ) m B- (wherein m is an integer between 1 and 10, preferably an integer between 1 and 6, A and B are absent, or -CO-) Z is -(OEG) p where p is an integer between 1 and 3, preferably 2, and the OEG structure is 【Chemical 13】 and; the linking group between W, X, Y and Z is an amide bond or a peptide bond; M is a human insulin analog) A novel acylated insulin analog having a structure shown by the formula.

6. The side chain compound has the following structure: 【Chemical 14】 having; preferably, the side chain compound has the following structure: 【Chemical Formula 15】 having (wherein n is an integer between 14 and 20, s is an integer between 2 and 8, m is an integer between 1 and 6, and p is an integer between 1 and 3), The acylated insulin analog according to claim 5.

7. The human insulin analog M has an A chain and a B chain, the amino acid sequence of the A chain is shown in SEQ ID NO: 1, the amino acid sequence of the B chain is shown in SEQ ID NO: 2 or SEQ ID NO: 3, and the human insulin analog is connected to the side chain compound by an amide bond through the ε nitrogen of the lysine residue at position B29. The acylated insulin analog according to claim 5.

8. The following structural formula: A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) n CO-NHC(COOH)(CH 2 ) S CH 2 NH-CO(CH 2 ) m CO-(OEG) p ), desB30 human insulin analog, or A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) n CO-NHC(COOH)(CH 2 ) S CH 2 NH-CO(CH 2 ) m CO-(OEG) p ), desB30 human insulin analog (wherein n is an integer between 14 and 20, s is an integer between 2 and 8, m is an integer between 1 and 6, and p is 2) The acylated insulin analog according to claim 5, having.

9. The following structural formula: A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) n CO-NHC(COOH)(CH 2 ) S CH 2 NH-CO(CH 2 ) m CO-(OEG) p ), desB30 human insulin analog, or A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) n CO-NHC(COOH)(CH 2 ) S CH 2 NH-CO(CH 2 ) m CO-(OEG) p ), desB30 human insulin analog (wherein n is an integer between 14 and 18, s is an integer between 3 and 4, m is an integer between 2 and 4, and p is 2) The acylated insulin analog according to claim 5, having.

10. Selected from any one of the following compounds: A14E, B16E, 25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-D-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-D-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 14 CO-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 14 CO-D-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 14 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Dab-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-L-Dab-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 14 CO-L-Dab-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 3 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-L-Lys-CO(CH 2 ) 3 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Dab-CO(CH 2 ) 3 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 4 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Dab-CO(CH 2 ) 4 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-L-Dab-CO(CH 2 ) 4 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-D-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-D-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 14 CO-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 14 CO-D-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 14 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Dab-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-L-Dab-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 14 CO-L-Dab-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 3 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-L-Lys-CO(CH 2 ) 3 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Dab-CO(CH 2 ) 3 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 4 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Dab-CO(CH 2 ) 4 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 16 CO-L-Dab-CO(CH 2 ) 4 CO-(OEG) 2 ), desB30 human insulin analog ; preferably, selected from any one of the following compounds: A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16E, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog; A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog ; more preferably, selected from any one of the following compounds: A14E, B16E, B25H, B29K(N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog A14E, B16H, B25H, B29K (N(ε)-COOH(CH 2 ) 18 CO-L-Lys-CO(CH 2 ) 2 CO-(OEG) 2 ), desB30 human insulin analog The acylated insulin analog according to claim 5.

11. A pharmaceutical composition comprising the side chain compound according to claims 1 to 4 and the acylated insulin analog according to claims 5 to 10.

12. The side chain compound according to claims 1 to 4, the acylated insulin analog according to claims 5 to 10, and the pharmaceutical composition according to claim 11 for use in treating or preventing diabetes in a subject, wherein the diabetes refers to type I and type II diabetes, the side chain compound, the acylated insulin analog, and the pharmaceutical composition.

13. The compound, acylated insulin analog, and pharmaceutical composition for use according to claim 12, which are administered twice a week, once a week, or less frequently.

14. A method for preparing the novel acylated insulin analog of formula (II) according to claim 5, comprising the step of performing an acylation reaction using the side chain compound of formula (I) and the human insulin analog according to claim 1; The method, wherein the human insulin analog has an A chain and a B chain, the amino acid sequence of the A chain is shown in SEQ ID NO: 1, and the amino acid sequence of the B chain is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

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