Phenylboronic acid-based insulin derivative as well as preparation method and application thereof

Abstract

The invention provides an insulin derivative based on phenylboronic acid and a preparation method and application thereof, and belongs to the technical field of insulin derivatives. The boric acid-based insulin derivative provided by the invention has a good glucose response release function: a phenylboronic acid group is combined with in-vivo protein to generate a phenylboronic acid ester bond, so that an insulin-protein complex is generated in situ in blood. When the concentration of glucose in blood is high, the phenylboronic acid ester bond is specifically cut off by glucose, so that insulin is released. The phenylboronic acid-based insulin derivative provided by the invention realizes stable and rapid glucose-responsive insulin release, can stably and durably maintain the blood glucose of a diabetic subject in a normal range, and hardly causes hypoglycemia.

Description

[0001] The present application claims priority to the international patent application with the application number PCT / CN2024 / 115879, the title of "A benzene boronic acid-based insulin derivative and its preparation method and application", filed with the international patent office on August 30, 2024, the entire content of which is incorporated by reference in the application. TECHNICAL FIELD

[0002] The present application belongs to the technical field of insulin derivatives, and relates to a benzene boronic acid-based insulin derivative and its preparation method and application. BACKGROUND

[0003] Diabetes is a chronic disease characterized by high blood sugar caused by insufficient insulin secretion or utilization. At present, the main treatment for diabetes is to control the blood sugar level within a reasonable range through scientific and reasonable methods to prevent acute metabolic disorders and delay the occurrence and development of complications. Controlling blood sugar level is the core of diabetes treatment. Currently, the treatment drugs for diabetes include two categories: oral drugs and injection drugs.

[0004] Insulin is one of the important hormones in the human body that regulates blood sugar. Human insulin is composed of two peptide chains, A chain and B chain. The A chain has 11 21 amino acids, and the B chain has 15 30 amino acids, a total of 16 51 amino acids. The A chain and the B chain are connected by A7 (Cys)-B7 (Cys) and A20 (Cys)-B19 (Cys) to form two disulfide bonds, respectively. In addition, A6 (Cys)-A11 (Cys) in the A chain also exists a disulfide bond.

[0005] Insulin replacement therapy is currently the main means of treatment for type I and type II late-stage diabetes, but exogenous ordinary insulin replacement is difficult to match the different insulin needs caused by changes in blood sugar in the human body due to various factors (including dietary intake, physical exercise, stress, etc.), which will lead to the fact that even with a precise dosage design, diabetes patients will still inevitably experience high blood sugar or low blood sugar symptoms.

[0006] Chinese patent application 201280011736.8 discloses a human insulin analogue and its acylated product, which successfully improves the hypoglycemic effect of the insulin analogue by modifying human insulin with PEG and other groups.

[0007] Chinese patent application 202080026404.1 discloses a glucose-sensitive insulin derivative, which is obtained by a side chain modification method containing a benzene boronic acid lactone or benzene boronic acid group.

[0008] Zeng, Wang, et al. published a research paper entitled "Week-long norm glycaemia in diabetic mice and minipigs via a subcutaneous dose of a glucose-responsive insulin complex" in Nature Biomedical Engineering. By wrapping the insulin-glucose complex with a layer of polymer material modified by phenylboronic acid groups, an intelligent insulin controlled-release material responsive to blood glucose level is formed. However, this method relies on the micro-nano structure complex formed by the polymer material, which has certain biological safety hazards.

[0009] In summary, the above-mentioned prior art cannot give a phenylboronic acid-based responsive insulin derivative to achieve glucose concentration-responsive in vivo insulin intelligent release. SUMMARY

[0010] Therefore, in view of the fact that the prior art cannot provide a phenylboronic acid-based insulin derivative to achieve glucose concentration-responsive in vivo insulin intelligent release, the purpose of the present application is to provide a phenylboronic acid-based insulin derivative, its preparation method and application. As shown in the formula, after the phenylboronic acid-based insulin derivative enters the blood, the phenylboronic acid group can form borate ester in situ and be combined with each other; at the same time, when the glucose concentration is too high under physiological conditions, the bonding of phenylboronic acid and protein is replaced and cut off by glucose, releasing insulin, thereby playing a role in reducing blood glucose. This effect can significantly prolong the time of stable blood glucose and almost no hypoglycemia occurs. Figure 1

[0011] To achieve the above-mentioned purpose of the application, on the one hand, the present application provides a phenylboronic acid-based insulin derivative, the sequence structure of which is as follows: Among them, R 1 , R 2 , R 3 are independently selected from H or the structure shown in formula (I), and at least one group of R 1 , R 2 , R 3 is the structure shown in formula (I);

[0013] R 4 is selected from halogen, hydrocarbon group, nitro group, amide group, ester group, hydroxyl group or nitrile group.

[0014] In the sequence structure, R​1 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 1 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 2 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the B chain of insulin 2 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the B chain of insulin 3 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the B chain of insulin 3 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the B chain of insulin

[0015] The term "alkyl" as used herein, unless otherwise indicated, includes both branched and straight chain saturated aliphatic hydrocarbon groups having the indicated number of carbon atoms, including all isomers. Commonly used abbreviations for alkyl groups, for example, methyl can be represented by "Me" or CH3, ethyl by "Et" or CH2CH3, propyl by "Pr" or CH2CH2CH3, butyl by "Bu" or CH2CH2CH2CH3, and the like. For example, "C 1-4 alkyl" (or "C1-C4 alkyl") means a straight chain or branched chain alkyl group having the indicated number of carbon atoms, including all isomers. C 1-4 alkyl includes n-, i-, s- and t-butyl, n- and i-propyl, ethyl and methyl. The term "C 1-20 alkyl" and the like have similar meanings. In addition, commonly used abbreviations for alkyl groups also include: i-propyl can be represented by "i-Pr", n-propyl by "n-Pr", n-butyl by "n-Bu", t-butyl by "t-Bu", and the like.

[0016] The term "alkoxy" means a straight chain and branched chain alkyl group of the indicated number of carbon atoms attached through an oxygen bridge.

[0017] The term "halogen" (or "halo") means fluorine, chlorine, bromine and iodine (or fluorinated (F), chlorinated (Cl), brominated (Br) and iodinated (I), respectively).

[0018] All ranges recited herein are inclusive of the endpoints. For example, "n is an integer between 0 and 2" means that n can be 0, 1 or 2, unless otherwise indicated.

[0019] HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 1 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 2 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 3 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 1 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 2 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 3 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 1 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 2 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 3 HN is R group substitution on the terminal amino hydrogen of the N-terminus of the A chain of insulin 1R 2 R 3 Any one, any two, or any three groups in it have the structure shown in formula (I).

[0020] Furthermore, the R 1 R 2 R 3 Each is independently selected from the structure shown in H or formula (I) and R 1 R 2 R 3 At least two groups in it have the structure shown in formula (I), specifically, it refers to R 1 R 2 R 3 Any two or three groups in it have the structure shown in formula (I).

[0021] Furthermore, the R 1 R 2 R 3 Each is independently selected from the structure shown in H or formula (I) and R 1 R 2 R 3 The two groups in it have the structure shown in formula (I).

[0022] Furthermore, the R 1 R 2 R 3 Each is independently selected from the structure shown in H or formula (I), and the R 1 and R 2 R 3 One of the groups has the structure shown in formula (I).

[0023] Preferably, the R 1 and R 3 For the structure shown in equation (I), the R 2 For H.

[0024] Preferably, the halogen is selected from fluorine, chlorine or bromine.

[0025] Preferably, the R 4 It is selected from halogen, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, nitro, amide, ester, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, or nitrile.

[0026] More preferably, the R 4 Selected from halogens, C1-C 20 Alkyl, C1-C 20 cycloalkyl, C1-C 20 alkenyl, C1-C 20 alkynyl group, C1-C 20 Cycloalkenyl, C1-C20 cycloalkynyl, nitro, amido, ester, C1-C 20 alkoxy or nitrile.

[0027] More preferably, the structure represented by formula (I) is selected from one of the following structures:

[0028]

[0030] Further, the structure represented by formula (I) is: In this case, the sequence structure of the insulin derivative is ; wherein R 1 , R 2 , R 3 are independently selected from H or , and at least one of R 1 , R 2 , R 3 is . Specifically, it means that R 1 , R 2 , R 3 are independently selected from H or and any one, any two or three of R 1 , R 2 , R 3 is .

[0031] Further, R 1 , R 2 , R 3 are independently selected from H or and at least two of R 1 , R 2 , R 3 is , specifically, it means that any two or three of R 1 , R 2 , R 3 is .

[0032] Still further, R 1 , R 2 , R 3 are independently selected from H or and two of R 1 , R 2 , R 3 is .

[0033] Still further, the R 1 , R 2 , R 3 are each independently selected from H or and the R 1 and R 2 , R 3 one group is .

[0034] Further preferably, and as an example of the present application, R 1 is 3-fluoro-1-phenylboronic acid-4-acyl, R 2 is H, R 3 is 3-fluoro-1-phenylboronic acid-4-acyl.

[0035] In another aspect, the present application provides a method for preparing the above insulin derivative, comprising the steps of: mixing a phenylboronic acid compound with N,N,N',N'-tetramethyl-O-(N-succinimidyl) tetrafluoroborate, N,N-diisopropylethylamine and a solvent, reacting, adding an insulin solution, post-treatment, to obtain the insulin derivative; The structure of the phenylboronic acid compound is

[0036] The R 4 is selected from halogen, hydrocarbyl, nitro, amido, ester, hydrocarbyloxy or nitrile.

[0037] Preferably, the halogen is selected from fluorine, chlorine or bromine.

[0038] Preferably, the R 4 is selected from halogen, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, nitro, amido, ester, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy or nitrile.

[0039] Preferably, the R 4 is selected from halogen, C1-C 20 alkyl, C1-C 20 cycloalkyl, C1-C 20 alkenyl, C1-C 20 alkynyl, C1-C 20 cycloalkenyl, C1-C 20 cycloalkynyl, nitro, amido, ester, C1-C 20 alkoxy or nitrile.

[0040] Preferably, the structure represented by formula (II) is selected from one of the following structures:

[0041]

[0042]

[0044] Preferably, and as an example of the present application, the phenyl boronic acid compound is: .

[0045] Preferably, the solvent is selected from at least one of dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, acetonitrile and acetone; the molar ratio of the phenyl boronic acid compound, the N,N,N',N'-tetramethyl-O-(N-succinimidyl) tetrafluoroborate and the N,N-diisopropylethylamine is 1:0.8-1.2:1.1-1.5; The temperature of the reaction is 20-30°C; the concentration of insulin in the insulin solution is 0.2-100 mg / mL; The solvent of the insulin solution is selected from phosphate buffer or dimethyl sulfoxide; the post-treatment is selected from using dialysis or precipitation.

[0046] More preferably, the post-treatment is precipitation, and the precipitation is specifically precipitation using a precipitant, and the precipitant is a mixture of precipitant A and precipitant B, the precipitant A is selected from methyl tert-butyl ether and / or diethyl ether; the precipitant B is selected from at least one of isopropyl acetate, acetone and tetrahydrofuran.

[0047] More preferably, the post-treatment is dialysis, and the dialysis is dialysis in deionized water; the molecular weight cut-off of the dialysis bag of the dialysis is 1000-3500, and the time of the dialysis is 12-24 h; after the dialysis, a white flocculent precipitate is formed, which is the insulin derivative.

[0048] In another aspect, the present application provides use of the above-mentioned insulin derivative or the insulin derivative prepared by the above-mentioned preparation method in the preparation of a drug for treating diabetes.

[0049] Preferably, the diabetes is type I diabetes and / or type II diabetes.

[0050] In another aspect, the present application provides a drug, and the effective component includes the above-mentioned insulin derivative or the insulin derivative prepared by the above-mentioned preparation method.

[0051] The pharmaceuticals of the present application suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the pharmaceuticals can be in the form of a sterile powder for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the ultimate injectable form must be sterile, fluid and stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi; thus, the pharmaceuticals must be stored, for example, to prevent the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0052] The pharmaceuticals of the present application can be in a form suitable for local use, for example, as an aerosol, cream, ointment, lotion, powder or the like. Furthermore, the compositions can be in a form suitable for use in transdermal delivery devices. The pharmaceuticals of the present application can be prepared for use in such formulations by conventional methods. For example, creams or ointments having the required consistency can be prepared by mixing the hydrophilic material with water and about 5% to about 10% by weight of the compound.

[0053] The pharmaceuticals of the present application can be in a form suitable for rectal administration wherein the carrier is a solid. The composition will preferably be in unit dose form as for example a suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be prepared by first forming a mixture containing the composition in a soft or molten carrier, and then cooling the mixture to solidify the carrier.

[0054] Preferably, the pharmaceutical is a subcutaneous injection pharmaceutical.

[0055] Preferably, the method of preparing the pharmaceutical is resuspending the above insulin derivative using a sterile phosphate buffer or normal saline.

[0056] Compared with the prior art, the present application has the following beneficial effects: The insulin derivative based on phenylboronic acid provided by the present application has good glucose-responsive release performance, can quickly release insulin at high blood glucose concentration, and continuously and slowly release insulin at normal blood glucose concentration. The present application utilizes the in-situ combination of phenylboronic acid with proteins in the body, introduces a phenylboronic ester bond that can be cut by glucose, and realizes stable and rapid glucose-responsive insulin release. Under physiological conditions, the competitive reversible combination of glucose with 1,3-diol and phenylboronic acid realizes sugar response, prolongs the treatment time, and can quickly adjust blood glucose in diabetic mice, has a stable and lasting effect for more than 200 h, and almost does not cause hypoglycemia. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 is a schematic diagram of the mechanism of action of the insulin derivative based on phenylboronic acid of the present application with proteins in the body.

[0058] Figure 2High performance liquid chromatogram of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0059] Figure 3 Q-TOF mass spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0060] Figure 4 Circular dichroism spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0061] Figure 5 Scanning electron micrographs of the phenylboronic acid-based insulin derivative synthesized in Example 1 at different magnifications, where the scale bar in (a) is 25 μm and the scale bar in (b) is 100 μm.

[0062] Figure 6 MALDI-TOF mass spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0063] Figure 7 Solid state nuclear magnetic resonance boron spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0064] Figure 8 Primary and secondary mass spectra of the phenylboronic acid-based insulin derivative synthesized in Example 2, where (a) is the primary mass spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 2, (b) is the secondary mass spectrum of the A chain of the phenylboronic acid-based insulin derivative synthesized in Example 2, and (c) is the secondary mass spectrum of the B chain of the phenylboronic acid-based insulin derivative synthesized in Example 2.

[0065] Figure 9 Mass spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 3. Figure 10 Blood glucose level line graph of the phenylboronic acid-based insulin derivative synthesized in Example 1 at different doses in the treatment of type I diabetic mice.

[0066] Figure 11 Blood glucose level line graph of the phenylboronic acid-based insulin derivative synthesized in Example 2 at a dose of 12 mg / kg in the treatment of type I diabetic mice.

[0067] Figure 12 Blood glucose level line graph of the phenylboronic acid-based insulin derivative synthesized in Example 3 at a dose of 12 mg / kg in the treatment of type I diabetic mice.

[0068] Figure 13 Blood glucose level line graph of the commercial long-acting insulin analogue (insulin glargine) at different doses in the treatment of type I diabetic mice by single needle injection and multiple needle continuous injection, where (a) represents single needle injection and (b) represents multiple needle injection.

[0069] Figure 14 Figure 1 is a graph of hypoglycemia and death in type I diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1 and a commercial long-acting insulin analogue (insulin glargine); wherein (a) indicates the proportion of mice that did not experience hypoglycemia at 4 mg / kg, 8 mg / kg, 12 mg / kg of the insulin derivative provided in Example 1; (b) indicates the survival rate of mice at 4 mg / kg, 8 mg / kg, 12 mg / kg of the insulin derivative provided in Example 1; (c) indicates the proportion of mice that did not experience hypoglycemia at a single-needle treatment at 40 U / kg, 400 U / kg of insulin glargine; (d) indicates the survival rate of mice at a single-needle treatment at 40 U / kg, 400 U / kg of insulin glargine; (e) indicates the proportion of mice that did not experience hypoglycemia at a multi-needle treatment of insulin glargine; (f) indicates the survival rate of mice at a multi-needle treatment of insulin glargine.

[0070] Figure 15 Figure 2 is a graph of the intraperitoneal glucose tolerance test of type I diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1, wherein (a) is the detection result at 1 day after treatment, and (b) is the detection result at 5 days after treatment.

[0071] Figure 16 Figure 3 is a graph of the in vivo glucose response insulin release verification of type I diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1, wherein (a) is the detection result at 1 day after treatment, and (b) is the detection result at 5 days after treatment; BGL indicates blood glucose level, and PIL indicates plasma insulin level.

[0072] Figure 17 Figure 4 is a graph of the pharmacokinetic curve of type I diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1; wherein (a) is intravenous injection, and (b) is subcutaneous injection.

[0073] Figure 18 Figure 5 is a graph of the biological safety evaluation of type I diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1; wherein (a) indicates the content of each component in serum, ALT is alanine aminotransferase, AST is aspartate aminotransferase, BUN is blood urea nitrogen, CR is creatinine, ALP is alkaline phosphatase, and ALB is blood albumin; (b) indicates blood cell content, RBC is red blood cell, PLT is platelet, WBC is white blood cell, NEUT is neutrophil, LYMPH is lymphocyte, MONO is monocyte, and EO is eosinophil.

[0074] Figure 19 is a graph of blood glucose levels of insulin derivative based on phenylboronic acid treated type I diabetic piglets synthesized in Example 1; wherein (a) is the blood glucose level of piglet No. I before administration, (b) is the blood glucose level of piglet No. I after administration, (c) is the blood glucose level of piglet No. II before administration, (d) is the blood glucose level of piglet No. II after administration, (e) is the blood glucose level of piglet No. III before administration, and (f) is the blood glucose level of piglet No. III after administration.

[0075] Figure 20 is a graph of in vivo glucose response insulin release verification of insulin derivative based on phenylboronic acid treated type I diabetic piglets synthesized in Example 1; wherein (a) is the blood glucose level and plasma insulin level of piglet No. I after 24 hours of treatment, (b) is the blood glucose level and plasma insulin level of piglet No. II after 24 hours of treatment, (c) is the blood glucose level and plasma insulin level of piglet No. III after 24 hours of treatment. Wherein, BGL indicates blood glucose level, and PIL indicates plasma insulin level. DETAILED DESCRIPTION

[0076] Terms and statements of the present invention: 1. As used herein, the articles "a", "an", and "the": unless otherwise specified, include plural referents.

[0077] 2. As used herein, numerical ranges: unless otherwise specifically indicated, all ranges or ratios disclosed herein are to be understood to encompass any and all subranges or subratios subsumed therein. For example, a stated range or ratio of 1 to 30 should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 30; that is, all subranges, integers, decimals, or fractions, between 1 and 30, including the values of 1 and 30.

[0078] 3. As used herein, the terms "comprising", "including", "containing", "having", "can", "contain", and variations thereof, are open-ended linking verbs of inclusion, meaning that the terms "comprising", "including", "containing", "having", "can", "contain", and variations thereof should be construed to mean that the item or items listed after such verb are not the only possible items or components that can be included in the methods, compositions, and articles of manufacture described herein.

[0079] The following non-limiting examples can provide a more complete understanding of the application to one of ordinary skill in the art, but are not intended to limit the scope of the application in any way. The following description is merely exemplary in nature and is not intended to limit the scope of the application as it is defined by the claims. Many changes and modifications can be suggested to the application herein, and it is intended that the application encompass such changes and modifications as fall within the scope of the claims.

[0080] The application is further described in the following detailed examples. The various chemical reagents used in the examples of the application were obtained from common commercial sources unless otherwise specified. The amounts recited in the following examples are amounts by mass unless otherwise specified. Room temperature is understood to be 25°C unless otherwise specified.

[0081] The reagents or instruments used in the following examples are shown in Table 1.

[0082] Table 1

[0083] Example 1 Preparation of insulin derivative based on phenylboronic acid.

[0084] Take 4-carboxyl-3-fluorophenylboronic acid 70 mg and 2-succinimidyl-1,1,3,3- tetramethyl uronium tetrafluoroborate 90 mg, place them in a 5 mL sample bottle, add 3.0 mL of anhydrous dimethyl sulfoxide and 78 μL of N,N-diisopropylethylamine, stir until clear, and react at room temperature for 3 hours to obtain a first solution. Take 2 g of recombinant human insulin and place it in a 60 mL sample bottle, add 20 mL of anhydrous dimethyl sulfoxide, stir until the solution is clear to obtain a recombinant human insulin solution. Add the first solution dropwise to the recombinant human insulin solution, and react for 1 hour. After the reaction is complete, add the solution dropwise to a precipitant of isopropyl acetate:methyl tert-butyl ether = 4:1, stir for 1 hour, filter off the precipitate, and dry to obtain a crude product.

[0085] Purify the crude product using high performance liquid chromatography (HPLC) to obtain an insulin derivative based on phenylboronic acid. The HPLC results are shown in Table 2. Figure 2

[0086] Use Q-TOF mass spectrometry, circular dichroism, scanning electron microscopy, MALDI-TOF mass spectrometry, and solid state nuclear magnetic resonance boron spectrum to characterize the structure of the obtained insulin derivative based on phenylboronic acid. The results are shown in Table 3, Table 4, Table 5, Table 6, and Table 7, respectively. Figure 3 Figure 4 Figure 5 Figure 6 Figure 7

[0087] From the results of the above experimental characterization, the structure of the insulin derivative based on phenylboronic acid is: ​​​​​​ , where R 1 and R 3 It is 3-fluoro-1-phenylboronic acid-4-acyl, R 2 For H.

[0088] The R 1 HN represents the terminal amino hydrogen atom at the N-terminus of the insulin A chain. 1 Group substitution; the R 2 HN represents the terminal amino hydrogen atom at the N-terminus of the insulin B chain. 2 Group substitution; the NHR 3 To perform R-methods on the ε-amino hydrogen of lysine (abbreviated as K) at position 29 of the insulin B chain 3 Group substitution.

[0089] Example 2 Preparation of insulin derivatives based on phenylboronic acid.

[0090] Take 90 mg of 4-carboxy-3-fluorophenylboronic acid and 90 mg of 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroboronic acid ester, place them in a 10 mL sample vial, add 5.0 mL of anhydrous dimethyl sulfoxide and 100 μL of N,N-diisopropylethylamine, stir until clear, and react at room temperature for 4 hours to obtain the first solution. Take 2.5 g of recombinant human insulin and place it in a 60 mL sample vial, add 25 mL of PBS solution, and add an appropriate amount of 1 M sodium hydroxide solution to adjust the pH to ≈9 to obtain the recombinant human insulin solution. Add the first solution dropwise to the recombinant human insulin solution, and react for 2 hours. During the reaction, use 1 M hydrochloric acid and 1 M sodium hydroxide solution to control the pH of the solution between 8.5 and 9. After the reaction is completed, dialyze with pure water for 12 h, and freeze-dry to obtain the crude product. Separate and purify the crude product by liquid chromatography to obtain an insulin derivative based on phenylboronic acid.

[0091] The detection results of the primary and secondary mass spectrometry of the insulin derivative based on phenylboronic acid are as follows: Figure 8 As shown. Among them, Figure 8 In (a), the main peak is the peak from which two water molecules have been removed. Figure 8 (b) shows the secondary mass spectrometry results of the A chain of this phenylboronic acid-based insulin derivative. Figure 8 (c) shows the secondary mass spectrometry result of the B chain of this phenylboronic acid-based insulin derivative. Based on the above experimental results, the structure of the phenylboronic acid-based insulin derivative prepared in Example 2 is presumed as follows: , where R 1 and R 2 It is 3-fluoro-1-phenylboronic acid-4-acyl, R 3 For H.

[0092] said R 1 HN, R 2 HN and NHR 3 have the same meanings as the corresponding groups in Example 1.

[0093] Example 3 Preparation of insulin derivative based on phenylboronic acid.

[0094] Take 4-carboxyl-3-fluorophenylboronic acid 120 mg and 2-succinimidyl-1,1,3,3- tetramethyl uronium tetrafluoroborate 156 mg, place them in a 10 mL sample bottle, add 5.0 mL of anhydrous dimethyl sulfoxide and 132 μL of N,N-diisopropylethylamine, stir until clear, and react at room temperature for 3 hours to obtain a first solution. Take 2 g of recombinant human insulin and place it in a 60 mL sample bottle, add 20 mL of anhydrous dimethyl sulfoxide, stir until the solution is clear to obtain a recombinant human insulin solution. Add the first solution dropwise to the recombinant human insulin solution, and react for 2 hours. After the reaction is complete, add the solution dropwise to a precipitant of isopropyl acetate:methyl tert-butyl ether = 4:1, stir for one hour, filter to obtain a precipitate, and dry to obtain a crude product of insulin derivative based on phenylboronic acid. Purify the crude product by liquid chromatography to obtain an insulin derivative based on phenylboronic acid. The detection results of the mass spectrum of the insulin derivative based on phenylboronic acid are shown in Figure 3, wherein the main peak is a peak of two water molecules. According to the above experimental results, it is presumed that the structure of the insulin derivative based on phenylboronic acid prepared in Example 3 is as follows: Figure 9 wherein R 1 , R 2 and R 3 are all 3-fluoro-1-phenylboronic acid-4-acyl.

[0095] said R 1 HN, R 2 HN and NHR 3 have the same meanings as the corresponding groups in Example 1.

[0096] Example 1 1. Rodent pharmacodynamic study Select C57BL / 6J mice (purchased from Hangzhou Medical College), after fasting overnight, intraperitoneally inject Streptozocin (STZ) solution under light-proof conditions, the dose is 120 mg / kg, measure the blood glucose of the mice after one week and two weeks, if the blood glucose > 300 mg / dL, it indicates that the modeling is successful. Otherwise, the mice can be intraperitoneally injected with STZ solution again after two weeks, the dose is 50 mg / kg, and the blood glucose of the mice is measured again after one week and two weeks.

[0097] ​2. In vivo hypoglycemic study of type I diabetic mice The type I diabetic mouse model was established according to the above method, and the type I diabetic mice with blood glucose of 400-500 mg / dL were selected for evaluation of therapeutic effect. After subcutaneous injection of the insulin derivative prepared in Example 1, the blood glucose level of the diabetic mice was in the normal range of less than 200 mg / dL for more than 214 hours, which was significantly longer than that of insulin glargine. The specific results are shown in Table 1. Figure 13 Compared with the commercially available long-acting insulin (insulin glargine), the insulin analogue prepared in Example 1 showed faster blood glucose lowering effect and more stable blood glucose control ability, and no diabetic mice showed hypoglycemia (less than 50 mg / dL) (see Table 1). Figure 10

[0098] At the same time, the dose-effect relationship showed that the insulin derivative prepared in Example 1 did not cause hypoglycemia in the range of 4-12 mg / kg, while 40% of the mice showed hypoglycemia when insulin glargine was 400 U / kg (converted to mass unit about 13.8 mg / kg), and all mice showed hypoglycemia when insulin glargine was used to control blood glucose for one week (i.e. 40 U / kg was injected once a day), and 40% of the mice died due to severe hypoglycemia (see Table 2). Figure 14

[0099] In addition, using the same method to model diabetic mice, subcutaneous injection of the insulin derivatives prepared in Example 2 and Example 3 (dose of 12 mg / kg body weight), the blood glucose levels of the diabetic mice were Figure 11 and Figure 12 , respectively.

[0100] In Example 2, the 3-fluoro-1-phenylboronic acid-4-acyl modification site of the insulin derivative was changed from A1, B29 (R 1 , R 3 ) in Example 1 to A1, B1 (R 1 , R 2 ). The experimental results showed that under the same dosage, the blood glucose control effect of the insulin derivative provided in Example 1 was better than that of Example 2. This indicates that the selection of the modification site has a certain influence on the blood glucose control effect of the insulin derivative.

[0101] In Example 3, the 3-fluoro-1-phenylboronic acid-4-acyl modification site of the insulin derivative was changed from A1, B29 (R 1 , R 3 ) in Example 1 to A1, B1, B29 (R 1 , R 2 , R 3 ​​The results showed that, at the same dosage, the insulin derivative provided in Example 1 was more effective in controlling blood glucose than that in Example 3. This indicates that modification at site B1 may weaken the binding of insulin to proteins compared to insulin analogs modified at sites A1 or B29 by affecting the conformation of insulin or by competing with phenylboronic acid modified at sites A1 or B29.

[0102] 3. Intraperitoneal glucose tolerance test in type I diabetic mice A type 1 diabetic mouse model was established using the method described above. Five type 1 diabetic mice (n=5) with blood glucose levels of 400-500 mg / dL were selected to evaluate the blood glucose control effect. On days 1 and 5 after subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), mice were intraperitoneally injected with 1.5 g / kg glucose. Blood glucose levels were measured and recorded every 15 minutes for 2 consecutive hours.

[0103] Meanwhile, a group of healthy C57BL / 6J mice were selected and administered 1.5 g / kg of glucose intraperitoneally. The blood glucose levels of the mice were measured and recorded every 15 minutes for 2 hours as a control.

[0104] The results showed that glucose stimulation on day 1 after drug administration maintained blood glucose levels in mice within the normal range (50–200 mg / dL), and the glucose control effect (evaluated by the area under the blood glucose-time curve) was superior to that in normal mice. However, glucose stimulation on day 5 after drug administration resulted in a significant peak in blood glucose levels at 15 minutes, which returned to normal (below 200 mg / dL) after 1 hour and remained stable for 2 hours (see [link to relevant documentation]). Figure 15 The blood sugar control effect was still better than that of normal mice.

[0105] 4. Verification of insulin release in response to glucose in type 1 diabetic mice A type 1 diabetic mouse model was established according to the above method. Type 1 diabetic mice (n=5) with blood glucose levels of 400-500 mg / dL were selected for in vivo glucose response insulin release evaluation. On days 1 and 5 after subcutaneous injection of the insulin analog prepared in Example 1 (12 mg / kg), mice were intraperitoneally injected with 3.0 g / kg glucose. Blood glucose levels were measured and recorded every 15 minutes for 2 consecutive hours. Blood samples (20 μL from the orbital cavity, centrifuged to obtain plasma) were collected at 0, 15, 30, 60, and 120 minutes.

[0106] The insulin content in plasma was detected using a recombinant human insulin ELISA kit.

[0107] The results show that after the administration of glucose, the blood glucose of the mice shows a significant peak (higher than 300 mg / kg), and the insulin concentration in the plasma also changes synchronously with the change of blood glucose, and both of them reach the peak at 30 minutes. On the first day after administration, the peak of insulin is increased by 4-5 times compared with the initial value; on the fifth day after administration, the peak of insulin is increased by 2-3 times. At the same time, when the blood glucose gradually decreases to the initial value, the insulin concentration in the plasma also decreases to the initial level, verifying the glucose-responsive insulin release (see Figure 16 ).

[0108] 5. Pharmacokinetics of the insulin derivative prepared in Example 1 for treating type I diabetic mice The type I diabetic mouse model is established according to the above method, and the type I diabetic mice (n = 5) with blood glucose of 400-500 mg / dL are selected for pharmacokinetic evaluation. After subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), blood samples are taken at predetermined time points (orbital blood sampling, 20 μL, and plasma is taken after centrifugation). The monitoring is continuously performed for 17 days (see Figure 17 ).

[0109] Meanwhile, a group of healthy C57BL / 6J mice are selected, and 1 nmol / kg of the insulin derivative prepared in Example 1 is administered intravenously, and blood samples are taken at predetermined time points (orbital blood sampling, 20 μL, and plasma is taken after centrifugation). The monitoring is continuously performed for 21 days (see Figure 17 ).

[0110] The plasma insulin concentration-time curve data is analyzed by non-compartment model using Winnonlin. The half-life of the insulin derivative prepared in Example 1 reaches 79.0 ± 17.7 hours in the type I diabetic mouse model after subcutaneous injection, and reaches 93.7.0 ± 9.5 hours in the healthy mouse after intravenous injection, which is 1000 times of the ordinary human insulin (half-life of about 5 minutes).

[0111] 6. Biological safety evaluation of the insulin derivative prepared in Example 1 for treating type I diabetic mice The type I diabetic mouse model is established according to the above method, and the type I diabetic mice (n = 5) with blood glucose of 400-500 mg / dL are selected for biological safety evaluation.

[0112] Before administration, blood samples are collected from the mice. After subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), blood samples are collected on the seventh day. Blood-related biochemical indicators and blood routine tests are performed, respectively.

[0113] Meanwhile, a group of healthy C57BL / 6J mice were selected, and blood samples were collected. Blood-related biochemical indicators and routine blood tests were performed as controls.

[0114] The results showed that after subcutaneous injection of the insulin derivative prepared in Example 1, all indicators of the mice were similar to those of healthy mice (see [link to relevant documentation]). Figure 18 ).

[0115] Example 2 Drug efficacy studies in non-rodent animals 1. Establishment of a type I diabetic pig model Six-month-old Bama pigs were selected, fasted overnight, and then injected intravenously with STZ solution at a dose of 150 mg / kg. Blood glucose levels were measured one and two weeks after the injection. If the blood glucose level was >300 mg / dL, the model was considered to have been successfully established.

[0116] 2. In vivo glucose-lowering study in type I diabetic piglets A type 1 diabetic pig model was established using the method described above, and the treatment effect was evaluated in type 1 diabetic pigs. After subcutaneous injection of the insulin derivative prepared in Example 1, the blood glucose levels of the diabetic pigs were measured using a continuous glucose monitoring system (CGMS).

[0117] The results showed that subcutaneous injection of the insulin derivative prepared in Example 1 achieved therapeutic effects for 8, 5, and 6 days in piglets I, II, and III (see [link]). Figure 19 ).

[0118] 3. Verification of insulin release in response to glucose in type I diabetic piglets A type 1 diabetic pig model was established using the method described above. Type 1 diabetic piglets were selected for in vivo glucose response insulin release evaluation. On day 1 after subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), piglets were intravenously injected with 10% glucose (at a rate of 1 L / h) until their blood glucose level exceeded 300 mg / dL, at which point glucose injection was stopped. Blood glucose levels were measured and blood samples were taken from the piglets every 15 minutes for 2 consecutive hours.

[0119] The insulin content in plasma was detected using a recombinant human insulin ELISA kit.

[0120] The results showed that after glucose stimulation, the piglets exhibited a significant peak in blood glucose (above 300 mg / kg), and the plasma insulin concentration also changed synchronously with the blood glucose level, peaking at the same time as blood glucose reached its peak. In both piglets, the peak insulin level was 2-3 times higher than the initial value. Simultaneously, as blood glucose gradually decreased to its initial value, the plasma insulin concentration also decreased back to the initial level, verifying the glucose-responsive insulin release (see [link to original text]). Figure 20 ).

[0121] Finally, it should be noted that the above is merely to illustrate the technical solutions of the present application, and is not a limitation on the scope of protection of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. An insulin derivative based on phenylboronic acid, characterized in that, The sequence structure of the insulin derivative is as follows: ； Wherein, the R 1 R 2 R 3 Each is independently selected from the structure shown in H or formula (I), and R 1 R 2 R 3 At least one group in it has the structure shown in formula (I); The R 4 It is selected from halogen, hydrocarbon, nitro, amide, ester, hydroxyl, or nitrile groups.

2. The insulin derivative according to claim 1, characterized in that, The halogen is selected from fluorine, chlorine or bromine.

3. The insulin analogue according to claim 1, characterized in that, The R 4 It is selected from halogen, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, nitro, amide, ester, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, or nitrile.

4. The insulin derivative according to claim 3, characterized in that, The R 4 Selected from halogens, C1-C 20 Alkyl, C1-C 20 cycloalkyl, C1-C 20 alkenyl, C1-C 20 alkynyl group, C1-C 20 Cycloalkenyl, C1-C 20 Cycloalkynyl, nitro, amide, ester, C1-C 20 Alkyl or nitrile groups.

5. The insulin derivative according to claim 4, characterized in that, The structure shown in formula (I) is selected from one of the following structures: 。 6. The insulin derivative according to claim 5, characterized in that, R 1 It is 3-fluoro-1-phenylboronic acid-4-acyl, R 2 For H, R 3 It is a 3-fluoro-1-phenylboronic acid-4-acyl group.

7. A method for preparing the insulin derivative according to any one of claims 1-6, characterized in that, Includes the following steps: A phenylboronic acid compound was mixed with N,N,N′,N′-tetramethyl-O-(N-succinimide)tetrafluoroborate, N,N-diisopropylethylamine and solvent, reacted, insulin solution was added, and post-treatment was performed to obtain an insulin derivative. The structure of the phenylboronic acid compound is as follows: ； The R 4 It is selected from halogen, hydrocarbon, nitro, amide, ester, hydroxyl, or nitrile groups.

8. The preparation method according to claim 7, characterized in that, The solvent is selected from at least one of dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, and acetone; The molar ratio of the phenylboronic acid compound, the N,N,N′,N′-tetramethyl-O-(N-succinimide)tetrafluoroborate, and the N,N-diisopropylethylamine is 1:0.8-1.2:1.1-1.5; The reaction temperature is 20-30℃; The concentration of insulin in the insulin solution is 0.2-100 mg / mL; The solvent for the insulin solution is selected from phosphate buffer or dimethyl sulfoxide; The post-treatment is selected from dialysis or precipitation.

9. The preparation method according to claim 8, characterized in that, The post-treatment is precipitation, specifically precipitation using a precipitating agent. The precipitating agent is a mixture of precipitating agent A and precipitating agent B. Precipitating agent A is selected from methyl tert-butyl ether and / or diethyl ether; precipitating agent B is selected from at least one of isopropyl acetate, acetone, and tetrahydrofuran.

10. The preparation method according to claim 8, characterized in that, The post-treatment is dialysis, which is performed in deionized water; the molecular weight cutoff of the dialysis bag is 1000-3500, and the dialysis time is 12-24 hours; after dialysis, a white flocculent precipitate is formed, which is the insulin derivative.

11. The use of the insulin derivative according to any one of claims 1-6 or the insulin derivative prepared by the preparation method according to any one of claims 7-10 in the preparation of a drug for treating diabetes.

12. The application according to claim 11, characterized in that, The diabetes refers to type 1 diabetes and / or type 2 diabetes.

13. A drug, characterized in that, The active ingredient includes the insulin derivative as described in any one of claims 1-6 or the insulin derivative prepared by the preparation method described in any one of claims 7-10.

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