Novel insulin derivative and use thereof

Abstract

Provided are a novel insulin derivative and a use thereof. Comparing the novel insulin derivative with human insulin, by carrying out amino acid mutation on sites B17, B25, and other sites such as the A14 site, the A19 site, the B3 site, and the B4 site, the novel insulin derivative has good performance of resisting in-vivo protease degradation; by means of an acylation reaction, a fatty acid side chain is connected at the B29 site, so that the fatty acid side chain can reversibly bind to albumin, further increasing the presence duration in the body, and thereby achieving a long-acting action mechanism. The novel insulin derivative shows obvious action characteristics of long-acting insulin, and can be used for developing a human insulin analogue administered once a week.

Description

Novel insulin derivatives and their uses Technical Field

[0001] The present invention relates to the field of new derivatives of human insulin analogs, and in particular to new insulin derivatives and uses thereof. Background Art

[0002] Insulin is a protein hormone secreted by pancreatic beta cells in response to endogenous or exogenous substances such as glucose, lactose, ribose, arginine, and glucagon. Insulin is the only hormone in the body that lowers blood sugar. Exogenous insulin is primarily used to treat diabetes and related conditions. Human insulin is composed of 51 amino acids, divided into two chains, A and B. Chain A contains 21 amino acids, and chain B contains 30 amino acids. The two chains are linked by two pairs of disulfide bonds, and the A chain itself also contains a pair of disulfide bonds. With the continuous development and widespread application of genetic engineering technology, insulin analogs for various uses have been prepared by adding, subtracting, or exchanging one or more amino acid residues.

[0003] Methods for expressing insulin precursors in Escherichia coli or Saccharomyces cerevisiae have been disclosed in many patents, such as U.S. Patent No. 5,962,267, WO95 / 16708, EP0055945, EP0163529, EP0347845, and EP0741188. The construction, expression, isolation, and purification of vectors for insulin analogs (such as DesB30 human insulin, or analogs thereof in which one or more amino acids in the A chain or B chain, or both the A chain and the B chain, are replaced with other amino acids) can be performed using techniques well known to those skilled in the art, such as the well-known technique disclosed in U.S. Patent No. 6500645, by expressing a DNA sequence encoding the target insulin analog in a suitable host cell to produce the insulin analog.

[0004] In the literature (Glendorf T, Sorensen AR, Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding; Biochemistry, 2008, 47:4743-4751), overlap extension PCR was used to introduce mutations into an insulin-encoding vector. The insulin analog was expressed in Escherichia coli as a proinsulin-like fusion protein with a small Arg-Arg C-peptide. The expressed fusion protein was cleaved using Lys-C, Kex2, and recombinant trypsin, and the single-chain precursor was enzymatically converted to the double-chain DesB30 human insulin analog.

[0005] Currently, a variety of insulin preparations with varying durations of action are available on the market. These preparations are primarily categorized by duration of action, including long-acting, intermediate-acting, and rapid-acting insulin preparations. Even the longest-acting insulin preparations currently available require daily injections, requiring many patients to undergo two to four injections daily. Over time, needle holes can develop at the injection site, leading to the risk of local skin hardening. Skin hardening can impair insulin absorption, causing blood sugar fluctuations and hypoglycemia. Frequent injections also create significant inconvenience for patients. A basal insulin preparation that can be injected once a week would significantly improve the quality of life for many patients.

[0006] WO1995007931A1 discloses insulin detemir, a long-acting insulin currently on the market, with a duration of action of up to 24 hours. Its molecular structure consists of a C14 fatty acid chain attached to the lysine residue at position B29 of human insulin depleted of B30. WO2005012347A1 discloses another long-acting insulin currently on the market, insulin degludec. Although also injected once daily, it has a flatter pharmacodynamic curve and a longer duration of action, allowing a second injection up to 42 hours after the first. Its molecular structure consists of a C16 fatty diacid attached to the lysine residue at position B29 of human insulin depleted of B30 via a glutamic acid linker. CN101573133B and WO2009 / 01042B disclose PEGylated insulins, which extend the duration of action of insulin by attaching PEG to the N-terminus of insulin. Technical issues

[0007] Among the currently marketed basal insulins and their analogs, degludec insulin has the longest half-life, approximately 25 hours, and a duration of action exceeding 42 hours. However, it can only be administered once daily. To date, no basal insulin product has been marketed with an injection cycle exceeding two days. Technical Solutions

[0008] In response to the shortcomings of the existing technology, the present invention provides a new type of insulin derivative. The present invention replaces the A chain or B chain of human insulin, or replaces one or more amino acids in the A chain and B chain at the same time, so that it has good resistance to protease degradation in the body; at the same time, it has good water solubility, and then connects a fatty acid side chain at the B29 position through an acylation reaction, so that it reversibly binds to albumin, further increasing its presence time in the body, thereby achieving a long-acting mechanism of action. The insulin derivative of the present invention exhibits obvious long-term insulin action characteristics and can be developed as a human insulin analog for once-weekly administration.

[0009] The technical solutions of the present invention are as follows:

[0010] An insulin derivative comprising at least one amino acid substitution in the A chain and / or B chain of human insulin; wherein the epsilon amino group of the Lys (K) residue at position 29 of the B chain of the insulin derivative is acylated to add a fatty acid side chain, and the Thr (T) residue at position 30 is deleted. The amino acid sequences of the A chain and B chain of human insulin are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0011] Furthermore, the substituent groups of the acylation reaction include an acyl group and a linker group; the acyl group includes a C18-C22 fatty acid; and the linker group includes γGlu and 2-5 OEG amino acid residues, wherein γGlu represents glutamic acid and OEG represents ethylene glycolamine.

[0012] Furthermore, Leu (L) at position 17 of the B chain of the insulin derivative is replaced with an acidic, basic or non-polar amino acid; the acidic amino acids include Asp (D) and Glu (E); the basic amino acids include His (H) and Arg (R); and the non-polar amino acids include Trp (W).

[0013] Furthermore, the insulin derivative further comprises Phe (F) at position 25 of the B chain being replaced by an acidic amino acid or a basic amino acid; the basic amino acid comprises His (H); and the acidic amino acid comprises Glu (E).

[0014] Furthermore, the insulin derivative further comprises one or more amino acid substitutions selected from the group consisting of:

[0015] Asn (N) at position 3 of chain B is replaced with Asp (D), His (H), or Glu (E);

[0016] Gln (Q) at position 4 of chain B was replaced with His (H);

[0017] Ala (A) at position 14 of chain B was replaced with Gly (G);

[0018] Tyr (Y) at position 14 of chain A is replaced with Asp (D) or Glu (E);

[0019] Gln (Q) at position 15 of chain A was replaced by Glu (E);

[0020] Tyr (Y) at position 19 of chain A is replaced with Asp (D) or His (H).

[0021] An insulin derivative, wherein Tyr (Y) at position 14 of the A chain of the insulin derivative is replaced by Asp (D) or Glu (E); Phe (F) at position 25 of the B chain is replaced by His (H) or Glu (E); and / or Tyr (Y) at position 16 of the B chain is replaced by His (H) or Glu (E); and / or Asn (N) at position 3 of the B chain is replaced by Glu (E) or His (H); and a fatty acid side chain is added to the epsilon amino group of the Lys residue at position 29 of the B chain of the insulin derivative through an acylation reaction, and Thr (T) at position 30 is deleted.

[0022] Furthermore, the insulin derivative is selected from any one of the following compounds:

[0023] Compound 1: B17E, B29K (N ε eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (indicates that the amino acid L at position 17 of the B chain in human insulin has been mutated to E, the amino acid K at position 29 of the B chain has been acylated to attach an eicosanedioyl-gGlu-2xOEG via the nitrogen of its ε-position residue, and the amino acid T at position 30 of the B chain has been deleted) (the amino acid sequences of the A chain and the B chain are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.).

[0024] Compound 2: B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (The amino acid sequences of the A and B chains are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.)

[0025] Compound 3: B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (The amino acid sequences of the A and B chains are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively.)

[0026] Compound 4: B17E, B29K (N ε Eicosanedioyl-γGlu-3xOEG), DesB30 human insulin;

[0027] Compound 5: B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-3xOEG), DesB30 human insulin;

[0028] Compound 6: B17E, B25H, A14E, B29K (N εEicosanedioyl-γGlu-3xOEG), DesB30 human insulin;

[0029] Compound 7: B17E, B29K (N ε Docosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0030] Compound 8: B17E, B25H, B29K (N ε Docosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0031] Compound 9: B17E, B25H, A14E, B29K (N ε Docosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0032] Compound 10: B17H, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (The amino acid sequences of the A and B chains are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively.)

[0033] Compound 11: B17D, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0034] Compound 12: B3H, B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (The amino acid sequences of the A and B chains are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively.)

[0035] Compound 13: B4H, B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0036] Compound 14: B17E, B25H, A14E, A19H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (The amino acid sequences of the A and B chains are shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively.)

[0037] Compound 15: B3E, B17E, B25H, A14E, B29K (N εEicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0038] Compound 16: B3D, B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0039] Compound 17: A14E, B14G, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0040] Compound 18: A14E, B17R, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0041] Compound 19: B14G, B17E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0042] Compound 20: A15E, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0043] Compound 21: A14E, B17W, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0044] Compound 22: A14E, B16H, B25E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0045] Compound 23: A14E, B16E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0046] Compound 24: A14E, B3E, B16E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0047] Compound 25: A14D, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin;

[0048] Compound 26: A14D, B3E, B16H, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0049] The amino acid sequences of the A chain and B chain of insulin are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0050] SEQ ID NO.1

[0051] SEQ ID NO.2

[0052] Compound 1: B17E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (SEQ ID NO. 3 and SEQ ID NO. 4)

[0053] SEQ ID NO.3

[0054] SEQ ID NO.4

[0055] Compound 2: B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (SEQ ID NO. 5 and SEQ ID NO. 6)

[0056] SEQ ID NO.5

[0057] SEQ ID NO.6

[0058] Compound 3: B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; (SEQ ID NO. 7 and SEQ ID NO. 8)

[0059] SEQ ID NO.7

[0060] SEQ ID NO.8

[0061] Compound 10, namely A14E, B17H, B25H, B29K (N εEicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0062] SEQ ID NO.9

[0063] SEQ ID NO.10

[0064] Compound 12, namely A14E, B3H, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0065] SEQ ID NO.11

[0066] SEQ ID NO.12

[0067] Compound 14, namely A14E, A19H, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0068] SEQ ID NO.13

[0069] SEQ ID NO.14

[0070] The sequences of compounds 4-9, 11, 13, 15-26 can all be obtained by the above-mentioned replacement of the sequence of human insulin. At the same time, the positions described in the present invention are counted from the N-terminus, for example, the "Asn at position 3 of the B chain is replaced by Asp, His or Glu" means that the amino acid Asn at position 3 is replaced by Asp, His or Glu starting from the N-terminus. The "B17E" means that the B chain is counted starting from the N segment, and the amino acid at position 17 is replaced by E. "DesB30" refers to a natural insulin B chain or its analogues lacking the B30 amino acid. The B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin is a derivative of human insulin, wherein the amino acid at position 14 in the A chain is substituted with E, and the amino acid at position 17 in the B chain is substituted with E, the amino acid at position 25 is replaced with H, and the amino acid at position 30 in the B chain is deleted.

[0071] The amino acids described herein are designated by three-letter or single-letter codes. Their meanings are those known in the art. Specifically, they are as follows: Alanine (Ala / A); Arginine (Arg / R); Aspartic acid (Asp / D); Cysteine ​​(Cys / C); Glutamine (Gln / Q); Glutamic acid (Glu / E); Histidine (His / H); Glycine (Gly / G); Asparagine (Asn / N); Tyrosine (Tyr / Y); Proline (Pro / P); Serine (Ser / S); Methionine (Met / M); Lysine (Lys / K); Valine (Val / V); Isoleucine (Ile / I); Phenylalanine (Phe / F); Leucine (Leu / L); Tryptophan (Trp / W); Threonine (Thr / T).

[0072] The present invention also protects the use of the above-mentioned insulin derivatives in the preparation of drugs for treating or preventing diabetes, and the action period thereof can reach 1 week. Beneficial effects

[0073] 1. Compared with human insulin, the novel insulin derivatives of the present invention are characterized by mutations of the amino acid Leu (L) at position B17 to amino acids such as Asp (D), His (H), Glu (E), Arg (R), and Trp (W); mutations of Phe (F) at position B25 to amino acids such as His (H) and Glu (E); and amino acid mutations at other positions such as A14, A19, B3, and B4. These mutations can significantly improve enzymatic cleavage by in vivo degradative enzymes, giving the compound excellent resistance to in vivo protease degradation, thereby increasing its duration of action. Adding a long-chain fatty acid at position B29 through an acylation reaction can increase the compound's binding ability and competitiveness with albumin in the body, reduce its binding ability to receptors on the surface of hepatocytes, thereby reducing receptor-mediated hepatocyte endocytosis and degradation, and increasing its duration of action in the peripheral circulatory system.

[0074] 2. The present invention is a series of basal insulin analog derivatives that can be injected once a week at most. They have a long pharmacokinetic profile. Patients using this product only need to inject once a week to achieve satisfactory therapeutic effects, greatly reducing the number of injections, improving the patient's quality of life, and reducing the risk of skin sclerosis and post-sclerosis complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG1 is a schematic structural diagram of compound 3 of the present invention.

[0076] FIG2 is a schematic diagram of the fatty acid side chain structure of compound 3 of the present invention linked to the epsilon amino group of the lysine residue at position 29 of the B chain.

[0077] Figure 3 shows the peptide maps of Compound 3 obtained in Example 3 of the present invention and Compound 3 before acylation after digestion and identification using V8 enzyme commonly used in insulin products, and further identification by peptide mapping mass spectrometry.

[0078] In the figure: (A) is the picture before acylation; (B) is the picture after acylation.

[0079] FIG4 is a digestion time curve of degludec insulin, compound 1, compound 2 and compound 3 by trypsin.

[0080] FIG5 is a digestion time curve of trypsin on degludec insulin, compound 3, compound 10, compound 12 and compound 14.

[0081] Figure 6 is the time curve of the hypoglycemic effect of insulin degludec, compound 1, compound 2, and compound 3 in rats. Modes for Carrying Out the Invention

[0082] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0083] Example 1

[0084] Compound 1, namely B17E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0085] The name is as follows: N-{ε-B29}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[GluB17],des-Thr B30-insulin (human) The structure is as follows:

[0086] The preparation method is as follows:

[0087] (1) Preparation of B17E, DesB30 human insulin

[0088] Using genetic engineering techniques well known in the art, the target protein, B17E, or DesB30 insulin precursor, is expressed in the genetically engineered E. coli BL21 strain using a DNA sequence encoding the desired protein. The expression vector employed can be a PET series vector. The cells are then transformed into competent E. coli and positive clones expressing the precursor protein are screened. The E. coli are then cultured in a fermenter at 37°C to express the precursor protein, and the precursor protein inclusion bodies are collected.

[0089] The collected precursor protein is renatured to obtain mature protein, which is enzymatically digested at room temperature using recombinant trypsin or lysine endopeptidase, and separated to obtain B17E and DesB30 human insulin with a purity exceeding 95%.

[0090] (2) Preparation of eicosanedioyl-γGlu-2xOEG-OSu

[0091] Eicosanedioyl-γGlu-2xOEG-OSu was synthesized by solid-phase synthesis using a procedure well known to those skilled in the art, comprising coupling a 9-Fmoc protected amino acid to a polystyrene 2-chlorotrityl chloride resin. The methods used can be found in Organic Synthesis on Solid Phase, FZ Dorwald, Wiley-VCH, 2000. ISBN 3-527-29950-5; Peptides: Chemistry and Biology, N. Sewald & H.-D. Jakubke, Wiley-VCH, 2002, ISBN 3-527-30405-3; and The Combinatorial Chemistry Catalog 1999, Novabiochem AG. After the 9-Fmoc amino acid is attached to the resin, the 9-Fmoc group is removed (deprotected) using, for example, a secondary amine, piperidine or diethylamine, followed by coupling of the next 9-Fmoc protected amino acid and subsequent deprotection. The synthetic sequence is terminated by coupling a mono-tert-butyl protected fatty diacid to yield mono-tert-butyl eicosanedioate. The compound is cleaved from the resin using 0.5% to 5% TFA / DCM (trifluoroacetic acid in dichloromethane). Finally, the C-terminal carboxyl group is activated, for example as an N-hydroxysuccinimide ester (OSu), which can be used directly or after purification as a coupling reagent or after deprotection for attachment to human insulin or insulin analogs to yield insulin analog derivatives.

[0092] LC-MS (electrospray): m / z = 859.02

[0093] (3)B17E, B29K(N ε Preparation of Eicosanedioyl-γGlu-2xOEG), DesB30 Human Insulin

[0094] Dissolve B17E, DesB30 human insulin (3 g, 0.53 mmol) in 100 ml of purified water, then add acetonitrile (100 ml) and adjust the pH to 11.5-12.0 with 1 M Na2CO3 solution. Dissolve eicosanedioyl-γGlu-2xOEG-OSu (0.75 g, 0.87 mmol) in 3.8 ml of N-methylpyrrolidone and slowly add it to the B17E, DesB30 human insulin solution while stirring. Use 1 M Na2CO3 solution to maintain the reaction pH at 11.5-12.0. Reaction for 30 minutes can obtain B17E, B29K (N ε Eicosanedioyl-γGlu-2xOEG)DesB30 human insulin.

[0095] (4)B17E, B29K(N ε Purification of Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin

[0096] The protein was purified on an AKTA purification instrument using sourceQ30 medium.

[0097] Column: 160ml (2.6*30cm)

[0098] Buffer A: 20 mM Tris 42% ethanol solution pH 7.0

[0099] Buffer B: 20 ​​mM Tris 500 mM ammonium acetate 42% ethanol pH 7.0

[0100] Gradient: 0-80% phase B, 15CV

[0101] Flow rate: 25ml / min

[0102] This chromatography procedure yields approximately 95% pure protein molecules. Freeze-drying allows for the production of pure protein for various assays. Mass spectrometry allows for molecular weight identification and confirmation of compound accuracy. This allows for subsequent in vitro and in vivo activity studies.

[0103] Example 2

[0104] Compound 2, namely B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0105] The designation is as follows: N-{ε-B29}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[GluB17, HisB25], des-Thr B30-insulin (human)

[0106] The structure is as follows:

[0107] The preparation method is as follows:

[0108] (1) Preparation of B17E, B25H, and DesB30 human insulin

[0109] Using genetic engineering techniques well known in the art, the target protein, namely B17E, B25H, or Des-B30 insulin precursors, is expressed in genetically engineered E. coli using a DNA sequence encoding the desired protein. The expression vector, such as a PET series vector, is then transformed into competent E. coli, and positive clones expressing the precursor protein are screened. The E. coli is then cultured in a fermenter at 37°C to express the precursor protein, and the precursor protein inclusion bodies are collected.

[0110] The collected precursor protein is renatured to obtain mature protein, which is enzymatically digested at room temperature using recombinant trypsin or lysine endopeptidase, and separated to obtain B17E, B25H, and DesB30 human insulin with a purity exceeding 95%.

[0111] (2) Preparation of eicosanedioyl-γGlu-2xOEG-OSu

[0112] Eicosanedioyl-γGlu-2xOEG-OSu can be synthesized by solid-phase synthesis using procedures well known to those skilled in the art, including coupling a 9-fluorenylmethoxycarbonyl-protected amino acid to a polystyrene 2-chlorotrityl chloride resin. The methods used can be found in Organic Synthesis on Solid Phase, FZ Dorwald, Wiley-VCH, 2000. ISBN 3-527-29950-5; Peptides: Chemistry and Biology, N. Sewald & H.-D. Jakubke, Wiley-VCH, 2002, ISBN 3-527-30405-3; and The Combinatorial Chemistry Catalog 1999, Novabiochem AG; and references therein. After the 9-Fmoc amino acid is attached to the resin, the 9-Fmoc group is removed (deprotected) using, for example, a secondary amine, piperidine or diethylamine, followed by coupling of the next 9-Fmoc protected amino acid and subsequent deprotection. The synthetic sequence is terminated by coupling a mono-tert-butyl protected fatty diacid to obtain mono-tert-butyl eicosanedioate. The compound is cleaved from the resin using 0.5% to 5% TFA / DCM (trifluoroacetic acid in dichloromethane). Finally, the C-terminal carboxyl group is activated, for example as an N-hydroxysuccinimide ester (OSu), which can be used directly or after purification as a coupling reagent, or after deprotection for attachment to insulin or other insulin analogs to obtain insulin analog derivatives.

[0113] LC-MS (electrospray): m / z = 859.02

[0114] (3)B17E, B25H, B29K(N ε Preparation of Eicosanedioyl-γGlu-2xOEG), DesB30 Human Insulin

[0115] B17E, B25H, DesB30 human insulin (3 g, 0.53 mmol) were dissolved in 100 ml of purified water, and then acetonitrile (100 ml) was added and the pH was adjusted to 11.5-12.0 with 1 M Na2CO3 solution. Eicosanedioyl-γGlu-2xOEG-OSu (0.75 g, 0.87 mmol) was dissolved in 3.8 ml of N-methylpyrrolidone and then slowly added to the B17E, B25H, DesB30 human insulin solution while stirring. 1 M Na2CO3 solution was used to maintain the reaction pH at 11.5-12.0. The reaction was continued for 30 minutes to obtain B17E, B25H, B29K (N εEicosanedioyl-γGlu-2xOEG)DesB30 human insulin (Compound 2).

[0116] (4)B17E, B25H, B29K(N ε Purification of Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin

[0117] The protein was purified on an AKTA purification instrument using sourceQ30 medium.

[0118] Column: 160ml (2.6*30cm)

[0119] Buffer A: 20 mM Tris 42% ethanol solution pH 7.0

[0120] Buffer B: 20 ​​mM Tris 500 mM ammonium acetate 42% ethanol pH 7.0

[0121] Gradient: 0-80% phase B, 15CV

[0122] Flow rate: 25ml / min

[0123] This chromatography procedure yields approximately 95% pure protein molecules. Freeze-drying allows for the production of pure protein for various assays. Mass spectrometry allows for molecular weight identification and confirmation of compound accuracy. This allows for subsequent in vitro and in vivo activity studies.

[0124] Example 3

[0125] Compound 3, namely A14E, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0126] The designation is as follows: N-{ε-B29}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[GluA14, GluB17, HisB25], des-Thr B30-insulin (human)

[0127] The structure is as follows:

[0128] The preparation method is as follows:

[0129] (1) Preparation of A14E, B17E, B25H, and DesB30 human insulin

[0130] Using genetic engineering techniques well known in the art, the target protein, namely A14E, B17E, B25H, or Des-B30 insulin precursor, is expressed in genetically engineered E. coli using a DNA sequence encoding the desired protein. The expression vector, such as a PET series vector, is then transformed into competent E. coli, and positive clones expressing the precursor protein are screened. The E. coli is then cultured in a fermenter at 37°C to express the precursor protein, and the precursor protein inclusion bodies are collected.

[0131] The collected precursor protein is renatured to obtain mature protein, the protein is enzymatically digested with recombinant trypsin or lysine endopeptidase at room temperature, and the purity of A14E, B17E, B25H, DesB30 human insulin with a purity of more than 95% is obtained through separation.

[0132] (2) Preparation of eicosanedioyl-γGlu-2xOEG-OSu

[0133] Eicosanedioyl-γGlu-2xOEG-OSu can be synthesized by solid-phase synthesis using procedures well known to those skilled in the art, including coupling a 9-fluorenylmethoxycarbonyl-protected amino acid to a polystyrene 2-chlorotrityl chloride resin. The methods used can be found in Organic Synthesis on Solid Phase, FZ Dorwald, Wiley-VCH, 2000. ISBN 3-527-29950-5; Peptides: Chemistry and Biology, N. Sewald & H.-D. Jakubke, Wiley-VCH, 2002, ISBN 3-527-30405-3; and The Combinatorial Chemistry Catalog 1999, Novabiochem AG; and references therein. After the 9-Fmoc amino acid is attached to the resin, the 9-Fmoc group is removed (deprotected) using, for example, a secondary amine, piperidine or diethylamine, followed by coupling of the next 9-Fmoc protected amino acid and subsequent deprotection. The synthetic sequence is terminated by coupling a mono-tert-butyl protected fatty diacid to obtain mono-tert-butyl eicosanedioate. The compound is cleaved from the resin using 0.5% to 5% TFA / DCM (trifluoroacetic acid in dichloromethane). Finally, the C-terminal carboxyl group is activated, for example as an N-hydroxysuccinimide ester (OSu), which can be used directly or after purification as a coupling reagent, or after deprotection for attachment to insulin or other insulin analogs to obtain insulin analog derivatives.

[0134] LC-MS (electrospray): m / z = 859.02

[0135] (3)A14E, B17E, B25H, B29K(N ε Preparation of Eicosanedioyl-γGlu-2xOEG), DesB30 Human Insulin

[0136] A14E, B17E, B25H, DesB30 human insulin (3 g, 0.53 mmol) was dissolved in 100 ml of purified water, and then acetonitrile (100 ml) was added and the pH was adjusted to 11.5-12.0 with 1 M Na2CO3 solution. Eicosanedioyl-γGlu-2xOEG-OSu (0.75 g, 0.87 mmol) was dissolved in 3.8 ml of N-methylpyrrolidone and then slowly added to the A14E, B17E, B25H, DesB30 human insulin solution while stirring. 1 M Na2CO3 solution was used to maintain the reaction pH at 11.5-12.0. After reacting for 30 minutes, A14E, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG)DesB30 human insulin (Compound 3).

[0137] (4)A14E, B17E, B25H, B29K(N ε Purification of Eicosanedioyl-γGlu-2xOEG), DesB30 Human Insulin The protein was purified on an AKTA purification instrument using sourceQ30 medium.

[0138] Column: 160ml (2.6*30cm)

[0139] Buffer A: 20 mM Tris 42% ethanol solution pH 7.0

[0140] Buffer B: 20 ​​mM Tris 500 mM ammonium acetate 42% ethanol pH 7.0

[0141] Gradient: 0-80% phase B, 15CV

[0142] Flow rate: 25ml / min

[0143] This chromatography procedure yields approximately 95% pure protein molecules. Freeze-drying allows for the production of pure protein for various assays. Mass spectrometry allows for molecular weight identification and confirmation of compound accuracy. This allows for subsequent in vitro and in vivo activity studies.

[0144] Example 4

[0145] Compound 10, namely A14E, B17H, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0146] The designation is as follows: N-{ε-B29}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[GluA14, HisB17, HisB25], des-Thr B30-insulin (human)

[0147] The structure is as follows:

[0148] The preparation method is as follows:

[0149] (1) Preparation of A14E, B17H, B25H, and DesB30 human insulin

[0150] Using genetic engineering techniques well known in the art, the target protein, namely A14E, B17H, B25H, or Des-B30 insulin precursor, is expressed in genetically engineered E. coli using a DNA sequence encoding the desired protein. The expression vector, such as a PET series vector, is then transformed into competent E. coli, and positive clones expressing the precursor protein are screened. The E. coli is then cultured in a fermenter at 37°C to express the precursor protein, and the precursor protein inclusion bodies are collected.

[0151] The collected precursor protein is renatured to obtain mature protein, the protein is enzymatically digested with recombinant trypsin or lysine endopeptidase at room temperature, and A14E, B17H, B25H, DesB30 human insulin with a purity exceeding 95% is obtained through separation.

[0152] (2) Preparation of eicosanedioyl-γGlu-2xOEG-OSu

[0153] Eicosanedioyl-γGlu-2xOEG-OSu can be synthesized by solid-phase synthesis using procedures well known to those skilled in the art, including coupling a 9-fluorenylmethoxycarbonyl-protected amino acid to a polystyrene 2-chlorotrityl chloride resin. The methods used can be found in Organic Synthesis on Solid Phase, FZ Dorwald, Wiley-VCH, 2000. ISBN 3-527-29950-5; Peptides: Chemistry and Biology, N. Sewald & H.-D. Jakubke, Wiley-VCH, 2002, ISBN 3-527-30405-3; and The Combinatorial Chemistry Catalog 1999, Novabiochem AG; and references therein. After the 9-Fmoc amino acid is attached to the resin, the 9-Fmoc group is removed (deprotected) using, for example, a secondary amine, piperidine or diethylamine, followed by coupling of the next 9-Fmoc protected amino acid and subsequent deprotection. The synthetic sequence is terminated by coupling a mono-tert-butyl protected fatty diacid to obtain mono-tert-butyl eicosanedioate. The compound is cleaved from the resin using 0.5% to 5% TFA / DCM (trifluoroacetic acid in dichloromethane). Finally, the C-terminal carboxyl group is activated, for example as an N-hydroxysuccinimide ester (OSu), which can be used directly or after purification as a coupling reagent, or after deprotection for attachment to insulin or other insulin analogs to obtain insulin analog derivatives.

[0154] LC-MS (electrospray): m / z = 859.02

[0155] (3)A14E, B17H, B25H, B29K(N ε Preparation of Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin A14E, B17H, B25H, DesB30 human insulin (3 g, 0.53 mmol) was dissolved in 100 ml of purified water, and then acetonitrile (100 ml) was added and the pH was adjusted to 11.5-12.0 with 1 M Na2CO3 solution. Eicosanedioyl-γGlu-2xOEG-OSu (0.75 g, 0.87 mmol) was dissolved in 3.8 ml of N-methylpyrrolidone and then slowly added to the A14E, B17E, B25H, DesB30 human insulin solution while stirring. The reaction pH was maintained at 11.5-12.0 with 1 M Na2CO3 solution. The reaction was continued for 30 minutes to obtain A14E, B17E, B25H, B29K (N εEicosanedioyl-γGlu-2xOEG)DesB30 human insulin (Compound 10).

[0156] (4)A14E, B17H, B25H, B29K(N ε Purification of Eicosanedioyl-γGlu-2xOEG)DesB30 Human Insulin The protein was purified on an AKTA purification instrument using sourceQ30 medium.

[0157] Column: 160ml (2.6*30cm)

[0158] Buffer A: 20 mM Tris 42% ethanol solution pH 7.0

[0159] Buffer B: 20 ​​mM Tris 500 mM ammonium acetate 42% ethanol pH 7.0

[0160] Gradient: 0-80% phase B, 15CV

[0161] Flow rate: 25ml / min

[0162] This chromatography procedure yields approximately 95% pure protein molecules. Freeze-drying allows for the production of pure protein for various assays. Mass spectrometry allows for molecular weight identification and confirmation of compound accuracy. This allows for subsequent in vitro and in vivo activity studies.

[0163] Example 5

[0164] Compound 12, namely A14E, B3H, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0165] The designation is as follows: N-{ε-B29}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[GluA14, HisB3, GluB17, HisB25], des-Thr B30-insulin (human)

[0166] The structure is as follows:

[0167] The preparation method is as follows:

[0168] (1) Preparation of A14E, B3H, B17E, B25H, and DesB30 human insulin

[0169] Using genetic engineering techniques well known in the art, the target protein, namely A14E, B3H, B17E, B25H, or Des-B30 insulin precursor, is expressed in genetically engineered E. coli using a DNA sequence encoding the desired protein. The expression vector, such as a PET series vector, is then transformed into competent E. coli, and positive clones expressing the precursor protein are screened. The E. coli is then cultured in a fermenter at 37°C to express the precursor protein, and the precursor protein inclusion bodies are collected.

[0170] The collected precursor protein is renatured to obtain mature protein, the protein is enzymatically digested with recombinant trypsin or lysine endopeptidase at room temperature, and the purity of A14E, B3H, B17E, B25H, DesB30 human insulin with a purity of more than 95% is obtained through separation.

[0171] (2) Preparation of eicosanedioyl-γGlu-2xOEG-OSu

[0172] Eicosanedioyl-γGlu-2xOEG-OSu can be synthesized by solid-phase synthesis using procedures well known to those skilled in the art, including coupling a 9-fluorenylmethoxycarbonyl-protected amino acid to a polystyrene 2-chlorotrityl chloride resin. The methods used can be found in Organic Synthesis on Solid Phase, FZ Dorwald, Wiley-VCH, 2000. ISBN 3-527-29950-5; Peptides: Chemistry and Biology, N. Sewald & H.-D. Jakubke, Wiley-VCH, 2002, ISBN 3-527-30405-3; and The Combinatorial Chemistry Catalog 1999, Novabiochem AG; and references therein. After the 9-Fmoc amino acid is attached to the resin, the 9-Fmoc group is removed (deprotected) using, for example, a secondary amine, piperidine or diethylamine, followed by coupling of the next 9-Fmoc protected amino acid and subsequent deprotection. The synthetic sequence is terminated by coupling a mono-tert-butyl protected fatty diacid to obtain mono-tert-butyl eicosanedioate. The compound is cleaved from the resin using 0.5% to 5% TFA / DCM (trifluoroacetic acid in dichloromethane). Finally, the C-terminal carboxyl group is activated, for example as an N-hydroxysuccinimide ester (OSu), which can be used directly or after purification as a coupling reagent, or after deprotection for attachment to insulin or other insulin analogs to obtain insulin analog derivatives.

[0173] LC-MS (electrospray): m / z = 859.02

[0174] (3)A14E, B3H, B17E, B25H, B29K(N ε Preparation of Eicosanedioyl-γGlu-2xOEG), DesB30 Human Insulin

[0175] A14E, B3H, B17E, B25H, DesB30 human insulin (3 g, 0.53 mmol) was dissolved in 100 ml of purified water, and then acetonitrile (100 ml) was added and the pH was adjusted to 11.5-12.0 with 1 M Na2CO3 solution. Eicosanedioyl-γGlu-2xOEG-OSu (0.75 g, 0.87 mmol) was dissolved in 3.8 ml of N-methylpyrrolidone and then slowly added to the A14E, B17E, B25H, DesB30 human insulin solution while stirring. 1 M Na2CO3 solution was used to maintain the reaction pH at 11.5-12.0. The reaction was continued for 30 minutes to obtain A14E, B3H, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin (Compound 12).

[0176] (4)A14E, B3H, B17E, B25H, B29K(N ε Purification of Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin

[0177] The protein was purified on an AKTA purification instrument using sourceQ30 medium.

[0178] Column: 160ml (2.6*30cm)

[0179] Buffer A: 20 mM Tris 42% ethanol solution pH 7.0

[0180] Buffer B: 20 ​​mM Tris 500 mM ammonium acetate 42% ethanol pH 7.0

[0181] Gradient: 0-80% phase B, 15CV

[0182] Flow rate: 25ml / min

[0183] This chromatography procedure yields approximately 95% pure protein molecules. Freeze-drying allows for the production of pure protein for various assays. Mass spectrometry allows for molecular weight identification and confirmation of compound accuracy. This allows for subsequent in vitro and in vivo activity studies.

[0184] Example 6

[0185] Compound 14, namely A14E, A19H, B17E, B25H, B29K (N εEicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

[0186] The designation is as follows: N-{ε-B29}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[GluA14, HisA19, GluB17, HisB25], des-Thr B30-insulin (human)

[0187] The structure is as follows:

[0188] The preparation method is as follows:

[0189] (1) Preparation of A14E, A19H, B17E, B25H, and DesB30 human insulin

[0190] Using genetic engineering techniques well known in the art, the target protein, namely A14E, A19H, B17E, B25H, or Des-B30 insulin precursor, is expressed in genetically engineered E. coli using a DNA sequence encoding the desired protein. The expression vector, such as a PET series vector, is then transformed into competent E. coli, and positive clones expressing the precursor protein are screened. The E. coli is then cultured in a fermenter at 37°C to express the precursor protein, and the precursor protein inclusion bodies are collected.

[0191] The collected precursor protein is renatured to obtain mature protein, the protein is enzymatically cleaved at room temperature using recombinant trypsin or lysine endopeptidase, and the purity of A14E, A19H, B17H, B25H, DesB30 human insulin with a purity of more than 95% is obtained through separation.

[0192] (2) Preparation of eicosanedioyl-γGlu-2xOEG-OSu

[0193] Eicosanedioyl-γGlu-2xOEG-OSu can be synthesized by solid-phase synthesis using procedures well known to those skilled in the art, including coupling a 9-fluorenylmethoxycarbonyl-protected amino acid to a polystyrene 2-chlorotrityl chloride resin. The methods used can be found in Organic Synthesis on Solid Phase, FZ Dorwald, Wiley-VCH, 2000. ISBN 3-527-29950-5; Peptides: Chemistry and Biology, N. Sewald & H.-D. Jakubke, Wiley-VCH, 2002, ISBN 3-527-30405-3; and The Combinatorial Chemistry Catalog 1999, Novabiochem AG; and references therein. After the 9-Fmoc amino acid is attached to the resin, the 9-Fmoc group is removed (deprotected) using, for example, a secondary amine, piperidine or diethylamine, followed by coupling of the next 9-Fmoc protected amino acid and subsequent deprotection. The synthetic sequence is terminated by coupling a mono-tert-butyl protected fatty diacid to obtain mono-tert-butyl eicosanedioate. The compound is cleaved from the resin using 0.5% to 5% TFA / DCM (trifluoroacetic acid in dichloromethane). Finally, the C-terminal carboxyl group is activated, for example as an N-hydroxysuccinimide ester (OSu), which can be used directly or after purification as a coupling reagent, or after deprotection for attachment to insulin or other insulin analogs to obtain insulin analog derivatives.

[0194] LC-MS (electrospray): m / z = 859.02

[0195] (3)A14E, A19H, B17E, B25H, B29K(N ε Preparation of Eicosanedioyl-γGlu-2xOEG), DesB30 Human Insulin

[0196] A14E, A19H, B17H, B25E, DesB30 human insulin (3 g, 0.53 mmol) was dissolved in 100 ml of purified water, and then acetonitrile (100 ml) was added and the pH was adjusted to 11.5-12.0 with 1 M Na2CO3 solution. Eicosanedioyl-γGlu-2xOEG-OSu (0.75 g, 0.87 mmol) was dissolved in 3.8 ml of N-methylpyrrolidone and then slowly added to the A14E, A19H, B17E, B25H, DesB30 human insulin solution while stirring. 1 M Na2CO3 solution was used to maintain the reaction pH at 11.5-12.0. The reaction was continued for 30 minutes to obtain A14E, A19H, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG)DesB30 human insulin (Compound 14).

[0197] (4)A14E, A19H, B17E, B25H, B29K(N ε Purification of Eicosanedioyl-γGlu-2xOEG)DesB30 Human Insulin

[0198] The protein was purified on an AKTA purification instrument using sourceQ30 medium.

[0199] Column: 160ml (2.6*30cm)

[0200] Buffer A: 20 mM Tris 42% ethanol solution pH 7.0

[0201] Buffer B: 20 ​​mM Tris 500 mM ammonium acetate 42% ethanol pH 7.0

[0202] Gradient: 0-80% phase B, 15CV

[0203] Flow rate: 25ml / min

[0204] This chromatography procedure yields approximately 95% pure protein molecules. Freeze-drying allows for the production of pure protein for various assays. Mass spectrometry allows for molecular weight identification and confirmation of compound accuracy. This allows for subsequent in vitro and in vivo activity studies.

[0205] Comparative Example 1

[0206] Degludec insulin (Novo Nordisk A / S batch number: KP54898-1) was purchased as comparative example 1.

[0207] Test Case

[0208] (1) Digestion identification:

[0209] Compound 3 obtained in Example 3 of the present invention was digested and identified using the V8 enzyme commonly used in insulin products: an appropriate amount of the compound was added to a 0.1% trifluoroacetic acid solution to prepare a solution containing 10 mg per ml. 20 μl of the solution was added to 20 μl of 0.2 mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7.3), 20 μl of 0.1% V8 enzyme solution, and 140 μl of water. The mixture was mixed and placed in a 37°C water bath for 2 hours. 3 μl of phosphoric acid was then added to prepare the test solution. Another appropriate amount of compound 3 before acylation was prepared in the same manner as a control solution. Following the method under the potency determination section, 0.2 mol / L sulfate buffer (pH 2.3)-acetonitrile (90:10) was used as mobile phase A, and acetonitrile-water (50:50) was used as mobile phase B. Gradient elution was performed according to Table 1.

[0210] Table 1 Gradient elution program

[0211] Take 25 μl of each of the reference solution and the test solution and inject them into the liquid chromatograph. Record the chromatograms. The digested protein peptides are further identified by peptide mapping mass spectrometry. The obtained peptide mapping data are shown in Figure 3.

[0212] (2) Comparative study of enzyme digestion:

[0213] Enzymatic analysis was performed on the different compounds of the present application and comparative example 1 using in vivo protein digestion enzymes such as trypsin and carboxypeptidase A (CPA). The analysis of the resistance of comparative example 1 and the different compounds of the present application to in vivo protease digestion is shown in Figures 4, 5 and Table 2.

[0214] Table 2 90% digestion time of trypsin for different compounds of the present application and comparative example 1

[0215] The results showed that after the B17 position mutated into Glu or other acidic, basic, or non-polar amino acids, the anti-enzyme activity increased. After adding other sites that mutated into other acidic, basic, or non-polar amino acids, the anti-enzyme degradation activity would also gradually increase on this basis.

[0216] (3) Mouse hypoglycemic test:

[0217] BALB / c mice were tested for hypoglycemic effect. The duration of hypoglycemic effect of different compounds of the present application and comparative example 1 was tested. Comparative example 1 was administered at 1 mg / kg, and the compound of the present application was administered at 3 mg / kg. The hypoglycemic effect period in mice is shown in Table 3.

[0218] Table 3 Effect time of different compounds of the present application and comparative example 1 on hypoglycemic effect in mice

[0219] The results showed that after the B17 position was mutated into Glu or other acidic, basic, or non-polar amino acids, the action period in mice was much better than that in Comparative Example 1; after further increasing the mutations of other sites into other acidic, basic, or non-polar amino acids, the action period of each compound in mice was further extended, and the action period in mice could reach more than 40 hours.

[0220] (4) Rat hypoglycemic test:

[0221] SD rats were tested for hypoglycemic effects. The duration of hypoglycemic effects of the different compounds of the present application and comparative example 1 was tested. Comparative example 1 was administered at 0.75 mg / kg, and the compound of the present application was administered at 3 mg / kg. Some of the results are shown in FIG6 , and the duration of hypoglycemic effects in rats is shown in Table 4 .

[0222] Table 4 Effect of different compounds of the present invention and comparative example 1 on hypoglycemic effect in rats

[0223] The results showed that after the B17 position was mutated into Glu or other acidic, basic, or non-polar amino acids, its in vivo action period in rats was far superior to that of degludec insulin in Comparative Example 1; after further increasing the mutations of other sites into other acidic, basic, or non-polar amino acids, the action period of each compound in rats was further extended, and the action period in rats could reach more than 60 hours.

[0224] The above test results show that when the B17 site is mutated to Glu, it is very helpful in resisting enzyme reactions in the body and prolonging the hypoglycemic effect period in animals. Therefore, this product intends to protect the B17 site mutation to Glu, His, and Asp mutations, further protecting compound 3 and its corresponding compound mutations, compound 12 and its corresponding covers. These compounds have the potential to be developed into once-weekly human insulin analogs.

[0225] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment. It is understood that other improvements and variations directly derived or imagined by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the scope of protection of the present invention.

Claims

1. An insulin derivative, characterized in that The insulin derivative replaces at least one amino acid in the A chain and / or B chain of human insulin; and the epsilon amino group of the Lys residue at the 29th position of the B chain of the insulin derivative is acylated to add a fatty acid side chain, and the Thr at the 30th position is deleted.

2. The insulin derivative according to claim 1, characterized in that The substituent groups of the acylation reaction include acyl and linker groups; the acyl includes C18-C22 fatty acids; the linker group includes γGlu and 2-5 OEG amino acid residues, wherein γGlu represents glutamic acid and OEG represents ethylene glycolamine.

3. The insulin derivative according to claim 1, characterized in that The Leu at position 17 of the B chain of the insulin derivative is replaced by an acidic, basic or non-polar amino acid; the acidic amino acids include Asp and Glu; the basic amino acids include His and Arg; and the non-polar amino acids include Trp.

4. The insulin derivative according to claim 3, characterized in that The insulin derivative further comprises that Phe at position 25 of the B chain is replaced by an acidic or basic amino acid; the basic amino acid comprises His; and the acidic amino acid comprises Glu.

5. The insulin derivative according to claim 4, characterized in that The insulin derivative further comprises one or more amino acid substitutions selected from the group consisting of: Asn at position 3 of chain B was replaced with Asp, His, or Glu; The Gln at position 4 of the B chain was replaced with His; Ala at position 14 of the B chain was replaced with Gly; Tyr at position 14 of chain A was replaced with Asp or Glu; Gln at position 15 of chain A was replaced by Glu; Tyr at position 19 of chain A is replaced with Asp or His.

6. The insulin derivative according to claim 1, characterized in that The Tyr at position 14 of the A chain of the insulin derivative is replaced by Asp or Glu; at the same time, the Phe at position 25 of the B chain is replaced by His or Glu, and / or the Tyr at position 16 of the B chain is replaced by His or Glu, and / or the Asn at position 3 of the B chain is replaced by Glu or His.

7. The insulin derivative according to any one of claims 1 to 6, characterized in that The insulin derivative is selected from any one of the following compounds: Compound 1: B17E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 2: B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 3: B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 4: B17E, B29K (N ε Eicosanedioyl-γGlu-3xOEG), DesB30 human insulin; Compound 5: B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-3xOEG), DesB30 human insulin; Compound 6: B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-3xOEG), DesB30 human insulin; Compound 7: B17E, B29K (N ε Docosanedioyl-γGlu-2xOEG), DesB30 Human insulin; Compound 8: B17E, B25H, B29K (N ε Docosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 9: B17E, B25H, A14E, B29K (N ε Docosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 10: B17H, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 11: B17D, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 12: B3H, B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 13: B4H, B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 14: B17E, B25H, A14E, A19H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 15: B3E, B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 16: B3D, B17E, B25H, A14E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 17: A14E, B14G, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 18: A14E, B17R, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 19: B14G, B17E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 20: A15E, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 21: A14E, B17W, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 22: A14E, B16H, B25E, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 23: A14E, B16E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 24: A14E, B3E, B16E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 25: A14D, B17E, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin; Compound 26: A14D, B3E, B16H, B25H, B29K (N ε Eicosanedioyl-γGlu-2xOEG), DesB30 human insulin.

8. Use of the insulin derivative according to any one of claims 1 to 7 in the preparation of a drug for treating or preventing diabetes.

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