BimBH3 analogue of double-site coupling fatty acid as well as preparation method and application of BimBH3 analogue

Abstract

The invention discloses a BimBH3 mimic peptide analogue of double-site coupling fatty acid, a preparation method of the BimBH3 mimic peptide analogue and application of the BimBH3 mimic peptide analogue serving as a novel PTPN1 inhibitor to development of long-acting hypoglycemic drugs. The structural general formula of the Bim BH3 mimic peptide analogue is as shown in formula I: # imgabs0 # mimic peptide compound is derived from a core region of a BimBH3 structural domain, the second amino acid is replaced and modified by Fmoc-Lys (Dde)-OH, the N end of a peptide chain is conjugated and modified by fatty monoacid or fatty diacid, the amino group of a side chain of lysine is conjugated and modified by fatty diacid, and the amino group of the side chain of lysine is conjugated and modified by fatty diacid. The compound has better metabolic stability, membrane permeability, pharmacokinetic characteristics and longer pharmacological action time, and shows an excellent long-acting hypoglycemic effect in the body of a type 2 diabetes mellitus model mouse. The compound is prepared by adopting a polypeptide solid-phase synthesis method, and a crude product is purified and freeze-dried to obtain a target compound.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a class of long-acting glucose-lowering dual-site coupled fatty acid BimBH3 mimetic peptides targeting PTPN1, and a preparation method and application thereof. Background Art

[0002] Diabetes is a global epidemic, and its incidence has been gradually increasing in recent years. Currently, there are about 500 million people with diabetes worldwide, and it is estimated that the number of patients will reach 643 million by 2030 and 783 million by 2045. According to the different clinical manifestations of diabetes, diabetes can be divided into three categories: type 1 diabetes, type 2 diabetes and gestational diabetes. About 90% of diabetic patients worldwide have type 2 diabetes, and the main characteristics of T2DM are insulin resistance and hyperglycemia. [Diabetes Res. Clin. Pract. 183, 109119 (2022)]

[0003] At present, the use of antidiabetic drugs is the preferred treatment option for improving hyperglycemia in diabetic patients. Common antidiabetic drugs in clinical practice can be divided into subcutaneous injection drugs (such as insulin and its analogs, GLP-1 drugs) and oral drugs (such as metformin, thiazolidinediones, etc.) according to the injection method. However, these drugs usually have some side effects and disadvantages, such as hypoglycemia, gastrointestinal adverse reactions, and obesity risks. The most popular GLP-1 long-acting glucose-lowering and weight-loss drugs also have problems such as complex structure, high cost, heavy medication burden, psychological problems such as depression and anxiety, and drug withdrawal rebound (weight loss). The development of long-acting glucose-lowering drugs based on new targets and unique mechanisms of action is still an urgent clinical need.

[0004] Non-receptor protein tyrosine phosphatase 1 (PTPN1, also known as PTP1B) is a member of the PTP family. It was first isolated and purified by Tonks et al. in human placental cells in 1988. It is mainly distributed in the endoplasmic reticulum of eukaryotic cells. The C-terminus of PTPN1 has 35 specific amino acid residues, which are used to anchor the endoplasmic reticulum and regulate enzyme activity through intracellular localization of subcellular structures; the N-terminus is the active domain that plays a catalytic role, and the extension direction is toward the cytoplasm. PTPN1 regulates and maintains cell homeostasis through dephosphorylation and participates in cell signal transduction. PTPN1 dephosphorylates phosphorylated insulin receptors, blocks the activation of signal pathways, and then inhibits the transport of glucose transporters and glucose uptake. Therefore, PTPN1 is a negative regulator of insulin action in the insulin receptor signaling pathway and plays an important role in insulin resistance. With the increasing research on PTPN1, PTPN1 has been shown to be closely related to diseases such as obesity, diabetes and cancer.

[0005] PTPN1 inhibitors achieve their inhibitory effects by inhibiting the enzymatic activity of PTPN1. The most studied PTPN1 inhibitors are difluoromethylene, phosphate, oxalylbenzoic acid, and phenoxyacetic acid. However, due to the highly conserved and positively charged catalytic pocket of PTPN1, PTPN1 inhibitors are limited. First, PTPN1 and PTPN2 have a high degree of homology, resulting in poor selectivity of small molecule inhibitors; second, because PTPN1 is an intracellular protein, some PTPN1 inhibitors with good activity have poor pharmacological activity due to poor membrane permeability.

[0006] The BH3 domain is a short α-helical fragment in the Bcl-2 family protein, which can mediate the interaction between the pro-apoptotic and anti-apoptotic proteins in the Bcl-2 family protein. The inventors' previous studies have found and identified that the Bcl-2 pro-apoptotic protein BimBH3 mimetic peptide is a new type of PTP1B inhibitor, which can improve the insulin resistance of HepG2 cells and significantly reduce the blood sugar of type 2 diabetes model mice. The pathological results show that it has a protective effect on pancreatic islet cells and has no obvious damage to liver and kidney tissues. In patients with type 2 diabetes, the main reason for poor blood sugar control is poor compliance with drug treatment, and long-acting anti-type 2 diabetes drugs are urgently needed clinically. Therefore, the development of novel structure, unique mechanism of action and long-acting hypoglycemic drugs based on BimBH3 mimetic peptides is still urgently needed clinically, and it is of great significance to promote basic and clinical research in the field of diabetes prevention and treatment in my country. [J Med Chem.2023,66(4):3030-3044].

[0007] The compound involved in the present invention is derived from the core region of the BimBH3 domain, and the second alanine is replaced with lysine, and a fatty acid molecule is coupled to the N-terminus to increase the albumin binding rate. Fatty acids of different lengths are also coupled to the lysine side chain to perform dual-site coupling of fatty acids, which can theoretically improve the metabolic stability and pharmacokinetic characteristics of the drug in vivo, thereby significantly prolonging the duration of drug efficacy and achieving long-term hypoglycemia once a week. In summary, this type of novel BimBH3 analog hypoglycemic polypeptide is a candidate drug molecule that targets PTPN1 and has the potential for long-term hypoglycemic effects once a week, and has the value of further developing long-acting polypeptide hypoglycemic drugs. Summary of the invention

[0008] The present invention describes a peptide analog compound derived from the core region of the Bim-BH3 domain, a synthesis method and an application thereof. The BimBH3 peptide analog with dual-site coupling of fatty acids has good PTPN1 and PTPN2 inhibitory activity and can resist degradation by a variety of proteases. Such peptide analogs have a longer pharmacological action time and achieve a long-term anti-type 2 diabetes effect.

[0009] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: The present invention discloses a novel dual-site coupled fatty acid-targeted PTPN1 long-acting hypoglycemic BimBH3 mimetic peptide and its preparation method and application. The structural formula of the Bim BH3 mimetic peptide analog is shown in Formula I:

[0010]

[0011] The BH3 mimetic peptide is derived from the core region of the BimBH3 domain, with the two-position alanine replaced by lysine, and the N-terminus and the lysine side chain are conjugated with small molecule fatty acids for dual-site coupling of fatty acids.

[0012] Further, the small molecule fatty acids at the X position of the conjugate are palmitic acid or octadecane dioic acid. The small molecule fatty acids at the Y position of the conjugate are malonic acid, glutaric acid, azelaic acid, sebacic acid, dodecanedioic acid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid, and docosanedioic acid.

[0013] Furthermore, the preparation method of the novel dual-site coupled fatty acid BimBH3 mimetic peptide comprises the following steps:

[0014] (1) Place the CTC resin in a manual peptide solid phase synthesizer at room temperature and swell the resin with DCM;

[0015] (2) Add 2-3 times the molar amount of the resin N-Fmoc protected amino acid or fatty acid and 2.4 times the equivalent of HOBt, DIC or Oxyma, DIC, and react by bubbling at 40°C for 30 min-60 min;

[0016] (3) adding piperidine / DMF mixture to remove Fmoc protecting group for 8+15 min;

[0017] (4) repeating steps (2) and (3) until the synthesis of the entire peptide analog backbone sequence is completed;

[0018] (5) Add 2% hydrazine hydrate / DMF solution to remove the protecting group of the lysine side chain Dde, add 2 equivalents of fatty acid and 3 equivalents of Oxyma and DIC, and bubble nitrogen at 40°C for 60 minutes; the complete peptide sequence is obtained.

[0019] (6) adding the lysate to the product of step (5), shaking on a shaker at 40° C. for 2 h, filtering, adding anhydrous icy ether to precipitate a solid, washing, and vacuum drying to obtain a crude product of a peptidomimetic analog;

[0020] (7) The crude peptide analog is purified by reverse phase preparative liquid chromatography and freeze-dried to obtain a pure product of the BimBH3 mimetic peptide analog with dual-site coupling of fatty acids.

[0021] Furthermore, in step (6), the cleavage solution is trifluoroacetic acid: triisopropylsilane: 3,6-dioxa-1,8-octanedithiol: pure water = 92.5:2.5:2.5:2.5.

[0022] Furthermore, in step (6), after the cracking and filtration, N2 is blown to remove trifluoroacetic acid.

[0023] The present invention provides a medicine or pharmaceutical composition containing all the novel dual-site coupled fatty acid-targeted PTPN1 long-acting glucose-lowering BimBH3 mimetic peptide active ingredients described in claims 1-5, characterized in that the medicine or pharmaceutical composition comprises any of the dual-site coupled fatty acid-targeted PTPN1 long-acting glucose-lowering BimBH3 mimetic peptides and one or more pharmaceutically acceptable carriers or excipients.

[0024] Furthermore, the dual-site coupled fatty acid BimBH3 mimetic peptide analog is used in the preparation of an inhibitor for inhibiting PTPN1 activity;

[0025] Furthermore, the BimBH3 mimetic peptide analogs with dual-site coupling of fatty acids are used in the preparation of drugs for preventing or treating diseases targeting PTPN1;

[0026] Furthermore, the diseases include type 2 diabetes, cancer, obesity and Alzheimer's disease, among which long-acting anti-type 2 diabetes therapeutic drugs are preferred. ;

[0027] Furthermore, the dual-site fatty acid-coupled BimBH3 mimetic peptide is administered orally or by injection;

[0028] Compared with the prior art, the advantages of the present invention are: the present invention obtains a new type of long-acting hypoglycemic BimBH3 analog peptides of dual-site coupled fatty acids targeting PTPN1 through a polypeptide solid phase synthesis method, the polypeptide analogs are derived from the core region of the BimBH3 domain, the second alanine is replaced by lysine, and the N-terminus is modified by coupling small molecule fatty acids with the lysine side chain. Compared with the long-acting hypoglycemic drugs currently on the market, the target is novel, the mechanism of action is unique, the structure is simpler, and the preparation cost is low. The present invention proves through experiments that the dual-site coupled fatty acid BimBH3 analog peptide has a significant inhibitory effect on PTPN1, can become an excellent PTPN1 inhibitor, and is used in the development of drugs for related diseases such as diabetes, cancer, obesity, etc. with PTPN1 as the target. It is worth noting that the present invention obtains a BimBH3 analog of dual-site coupled fatty acids with a longer pharmacological action time and a long-acting hypoglycemic effect once a week by inhibiting the PTPN1 target through activity screening. Therefore, the dual-site coupled fatty acid BimBH3 analogues of the present invention have great development value and commercial prospects in the field of long-acting hypoglycemic drugs targeting PTPN1. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a secondary structure characterization diagram of the far-ultraviolet circular dichroism (CD) spectrum of the analogue of the present invention;

[0030] Figure 2 This is a diagram showing the long-acting hypoglycemic effect of the analog D6 of the present invention in vivo;

[0031] Figure 3 This is a diagram of the in vivo pharmacokinetic effect of the analog D6-F of the present invention; DETAILED DESCRIPTION

[0032] The technical scheme of the present invention is further described in detail below in conjunction with specific embodiments. The methods in the following embodiments are all conventional methods unless otherwise specified.

[0033] Example 1

[0034] The specific preparation process of the peptide mimetic is as follows:

[0035] (1) Place the CTC resin in a manual peptide solid phase synthesizer at room temperature and swell the resin with DCM;

[0036] (2) Add 2-3 times the molar amount of the resin N-Fmoc protected amino acid or fatty acid and 2.4 times the equivalent of HOBt, DIC or Oxyma, DIC, and react by bubbling at 40°C for 30 min-60 min;

[0037] (3) adding piperidine / DMF mixture to remove Fmoc protecting group for 8+15 min;

[0038] (4) repeating steps (2) and (3) until the synthesis of the entire peptide analog backbone sequence is completed;

[0039] (5) Add 2% hydrazine hydrate / DMF solution to remove the protecting group of the lysine side chain Dde, add 2 equivalents of fatty acid and 3 equivalents of Oxyma and DIC, and bubble nitrogen at 40°C for 60 minutes; the complete peptide sequence is obtained.

[0040] (6) adding the lysate to the product of step (5), shaking on a shaker at 40° C. for 2 h, filtering, adding anhydrous icy ether to precipitate a solid, washing, and vacuum drying to obtain a crude product of a peptidomimetic analog;

[0041] (7) The crude peptide analog is purified by reverse phase preparative liquid chromatography and freeze-dried to obtain a pure product of the BimBH3 mimetic peptide analog with dual-site coupling of fatty acids.

[0042] The mass spectrometry data and HPLC purity analysis data of the 19 BimBH3 mimetic peptide compounds described in the present invention are shown in Table 1.

[0043] Table 1. Mass spectrometry data and HPLC purity analysis data of BimBH3 mimetic peptide analogs.

[0044]

[0045] Example 2: Protein tyrosine phospholipase 1B (PTPN1) / T cell protein tyrosine phosphatase (PTPN2) inhibitory activity assay

[0046] In the present invention, MES buffer is used as the reaction system, human protein tyrosine phosphatase 1B (PTPN1) is utilized, disodium p-nitrophenyl phosphate (pNPP) is used as the specific substrate, the lead compound SM-6 is selected as the positive control, and DMSO is used as the negative control. A screening model based on the enzyme reaction rate using a 96-well plate as a carrier is established, and PTPN1 inhibitors are sought through enzymatic methods.

[0047] The specific implementation method is: using MES buffer system (25mM, pH6.5), 86ul MES buffer, 4ul compound, 10μL pNPP (77mM), 100μL PTPN1 solution (50nM) were added to the 96-well plate in sequence, and the total reaction volume was 200μL. Each group was paralleled 3 times, with DMSO as the negative control, and the cells were shaken on a shaker at 25°C for 1min, and the OD values ​​of 1min, 5min, and 10min were read on the microplate reader. 405 Value, calculate OD 405The rate of change v (△OD / min). The reaction rate of each well in the initial stage is linearly correlated. The slope of the linear part of the kinetic curve determines the reaction rate of PTPN1, and the speed represents the enzyme activity. The obtained data are expressed as `x±SD, and each group of data is analyzed using t-test. The formula for calculating the inhibition rate of the compound on PTPN1 is:

[0048] Inhibition rate (%) = (v DMSO -v 样本 ) / v DMSO ×100%

[0049] Among them, v DMSO 、v 样本 Represent the initial average reaction rate of the negative control group and the test compound, respectively. GraphPad Prism software was used to perform statistical inhibition rate data statistics and processing, fit the inhibitory dose-effect curve and calculate IC 50 The inhibition results are shown in Table 2.

[0050] Table 2. Inhibition results of the tested peptide analogs on PTPN1 activity

[0051]

[0052] / : The inhibition rate of the peptide analog compound on PTPN1 was less than 50% at the initial screening concentration of 20 μmol / L, and the IC50 value was no longer determined.

[0053] Example 4: Evaluation of the inhibitory effect of albumin on the activity of the tested peptide analogs on PTPN1

[0054] The BimBH3 analogs D5, D6 and D16 screened by enzyme activity were re-measured for target inhibitory activity of the compounds after adding 2% HSA to the system using the determination method in Specific Implementation Case 3. The results are shown in Table 3. In the absence of albumin, the analogs have high inhibitory activity against PTPN1. After adding albumin, the activity of the analogs is significantly reduced, indicating that the analogs can effectively bind to human plasma albumin and exert long-term biological activity in vivo and in vitro.

[0055] Table 3. Effect of albumin on the inhibitory activity of the tested peptide analogs on PTPN1

[0056]

[0057] Example 5: Secondary structure of a dual-site modified BimBH3 mimetic peptide analog

[0058] The BimBH3 analogs screened by enzyme activity were determined by far-UV circular dichroism (CD) spectroscopy. The results are shown in the attached figure. Figure 1As shown, the selected dual-site fatty-acylated BimBH3 analogs did not have typical secondary structures (eg, α-helix, β-sheet) in buffer solution.

[0059] Example 6: Evaluation of DPP-IV Enzyme Stability of Dual-site Modified BimBH3 Peptide Mimics

[0060] The compounds screened by enzyme activity were subjected to DPP-IV enzyme stability assay, the screened compounds were configured to a certain concentration, incubated with the enzyme, samples at different time points were taken to quench the reaction with a quencher, and analyzed by HPLC after treatment. The remaining compound (%) at each time point relative to the time origin was determined by the relative peak area. The data were fitted with the exponential decay model in the GraphPad Prism software to determine the half-life to evaluate its stability. Then it was compared with the original compound. The enzyme stability data of the three double-site coupled fatty acid BimBH3 mimetic peptide compounds described in the present invention are shown in Table 4. As shown in Table 4, the stability of analogs D5 and D6 was significantly improved. The results show that fatty acylation and site-specific substitution of amino acids at position 2 significantly improve the anti-metabolic stability of the mimetic peptide to DPP-IV.

[0061] Table 4. Stability results of tested peptide analogs against DPP IV enzyme degradation

[0062]

[0063] Example 7: Evaluation of metabolic stability of dual-site modified BimBH3 mimetic peptide compounds.

[0064] The compounds screened out by enzyme activity were subjected to in vitro enzyme stability assay, the screened out compounds were configured to a certain concentration, incubated with the enzyme, samples at different time points were taken to quench the reaction with a quencher, and analyzed by HPLC after treatment. The remaining compound (%) at each time point relative to the time origin was determined by the relative peak area. The data were fitted with the exponential decay model in the GraphPadPrism software to determine the half-life, which was used to evaluate its stability. Then it was compared with the original compound. The enzyme stability data of the three double-site coupled fatty acid BimBH3 mimetic peptide compounds described in the present invention are shown in Table 5.

[0065] Table 5. Proteolytic stability of tested peptide analogs

[0066]

[0067] Example 8: Evaluation of the long-acting hypoglycemic activity of highly active dual-site modified BimBH3 mimetic peptide compounds in vivo

[0068] Evaluation of hypoglycemic effect in vivo: Male C57BL / Ks db / db mice, 8-9 weeks old, were selected and kept normally in an animal room with a constant temperature of 24±2.0℃ and a normal daylight cycle. After 2 weeks of adaptive feeding, the mice were randomly divided into parallel control group (normal saline), positive control group (semaglutide 0.1μmol / kg) and treatment group (BimBH3 mimetic peptide compound 0.2 / 0.1μmol / kg). Five mice were in a group, with a total of 4 groups, and the drug was administered once a week by subcutaneous injection (1mL / 10g mouse body weight), and the treatment cycle was 8 weeks. The blood glucose value was measured by taking blood from the tail of the mouse using ACCU-CHEK Performa blood glucose meter (Roche Diabetes Care GmbhSandhofer Strasse 11668305Mannheim, Germany), and the changes in fasting blood glucose (FBG) levels of type 2 diabetes model mice in each group were monitored.

[0069] The results of the in vivo long-acting hypoglycemic activity of the highly active dual-site modified PTPN1 inhibitor BimBH3 mimetic peptide compound described in the present invention are shown in the attached Figure 2 As shown. The fasting blood glucose level before the experiment was measured on the first day in each group, and the drug was administered once every seven days. From the experimental results, it can be seen that the fasting blood glucose of the model mice in the parallel control group continued to increase, which was consistent with the pathological characteristics of type 2 diabetes, while the fasting blood glucose level of the mice in the high-dose group was significantly lower than that in the parallel control group, and the effect was similar to that of the positive drug, and the blood glucose in the 0.1 μmol / kg dose group also decreased significantly, indicating that the dual-site coupled fatty acid BimBH3 mimetic peptide compound (D6) with excellent PTPN1 inhibitory activity in the present invention showed a long-term anti-type 2 diabetes therapeutic effect once a week, and has a very high development prospect and application value.

[0070] Example 9: In vivo pharmacokinetic evaluation of dual-site modified BimBH3 mimetic peptide compounds

[0071] The present invention studies the bioavailability and long-term effect of the Bim BH3 analogs with dual-site coupling of fatty acids in vivo. The ninth position Ala in the analog D6 dodecapeptide sequence, which has little effect on the activity, is replaced by Lys, and a 5-FAM fluorescent group (D6-F) is coupled to the Lys side chain (see Appendix). Figure 3 A). After subcutaneous injection, D6-F is almost completely absorbed after 2 hours and completely absorbed after 4 hours (Attached Figure 3 C). And it is mainly distributed in the liver, kidneys, and muscles (attached Figure 3D). The fluorescently labeled peptide analog D6-F was used for fluorescence quantification to evaluate the preliminary pharmacokinetic properties. The plasma concentration-time course was analyzed using non-compartmental fitting to obtain pharmacokinetic parameters. The analog D6-F existed in the body for a very long time, with a half-life of 226.2 hours ( Figure 3 B). The half-life of D6-F was increased by more than three times compared with the palmitic acid-modified BimBH3 peptide analog A2-19. The peak plasma drug concentration Cmax of D6-F was 444.2 ng / mL, and its AUC data showed the ultra-long-acting potential of this new type of PTPN1 / PTPN2 inhibitor.

[0072] In summary, the present invention provides a type of dual-site coupled fatty acid long-acting hypoglycemic BimBH3 mimetic peptide targeting PTPN1 and its preparation method and application. The dual-site coupled fatty acid BimBH3 mimetic peptide analog has good PTPN1 / PTPN2 enzyme inhibitory activity, and the mimetic peptide D6 exhibits excellent anti-protease hydrolysis stability. At the same time, it exhibits excellent once-a-week long-acting hypoglycemic effect in type 2 diabetic db / db mice, and can be used for the development of long-acting drugs for the prevention or treatment of type 2 diabetes, with high development prospects and excellent application value.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. The present invention discloses a type of BimBH3 mimetic peptide analogs with dual-site coupling of fatty acids, a preparation method thereof, and an application thereof as a novel PTPN1 inhibitor in the development of long-acting hypoglycemic drugs. The general structural formula of the Bim BH3 mimetic peptide analogs is shown in Formula I:

2. The preparation method and application of the BimBH3 mimetic peptide targeting PTPN1 with dual-site coupling of fatty acids according to claim 1, characterized in that: The N-terminus of the peptide chain is conjugated with hexadecanoic acid or octadecanoic acid, and the side chain of lysine is conjugated with malonic acid, glutaric acid, azelaic acid, sebacic acid, dodecanedioic acid, hexadecanoic acid, octadecanoic acid, eicosanedioic acid, and docosanedioic acid of different carbon chain lengths, wherein the preferred carbon chain lengths are: conjugate X uses long-chain hexadecanoic acid, and conjugate Y uses medium-chain dodecanedioic acid.

3. According to claim 1, the dual-site coupled fatty acid-targeted BimBH3 mimetic peptide for PTPN1, the preparation method comprising the following steps: (1) Place the CTC resin in a peptide solid phase synthesizer at room temperature and swell it with dichloromethane; (2) adding 2 times the molar amount of the resin N-Fmoc protected amino acid and 2.4 times the molar amount of the resin DIEA in the solvent of dichloromethane and bubbling reaction at room temperature for 60-90 minutes; (3) adding a piperidine / dimethylformamide mixture to remove the Fmoc protecting group; (4) Add 2 times the molar amount of the resin to the N-Fmoc protected amino acid or fatty acid and 2.4-4.8 times the molar amount of the resin to the HOBt, DIC or Oxyma, DIC, and react at 40° C. for 30 min-60 min; (5) repeating steps (3) and (4) to synthesize a peptide-mimicking backbone sequence; (6) adding a 2% hydrazine hydrate / dimethylformamide mixture to remove the Dde protection of the Lys side chain; (7) Add 2 times the molar amount of the resin fatty acid and 2.4 times the molar amount of the resin Oxyma / DIC, and react by bubbling at 40°C for 60-90 min to obtain the complete peptide sequence; (8) adding the lysate to the product of step (7), shaking at 40° C. for 2 h, filtering, adding anhydrous icy ether to precipitate the solid, washing, and vacuum drying to obtain a crude product of the peptidomimetic analog; The crude product of the polypeptide analog is purified by reverse phase preparative liquid chromatography and freeze-dried to obtain a flocculent or powdery solid, that is, a pure product of the BimBH3 mimetic peptide analog with dual-site coupling of fatty acids.

4. The preparation method according to claim 3, characterized in that: The cleavage solution in step (5) is trifluoroacetic acid: triisopropylsilane: 3,6-dioxa-1,8-octanedithiol: pure water in a ratio of 92.5:0.25:0.25:0.25; the excess trifluoroacetic acid in step (8) is removed by blowing with nitrogen.

5. All the novel dual-site coupled fatty acid-targeted long-acting hypoglycemic BimBH3 mimetic peptides described in claims 1-5 have been shown to have excellent metabolic stability, membrane permeability, pharmacokinetic characteristics and longer pharmacological action time, and have shown excellent long-acting hypoglycemic effect in type 2 diabetes model mice.

6. The drug or pharmaceutical composition containing all novel dual-site coupled fatty acid-targeted PTPN1 long-acting hypoglycemic BimBH3 mimetic peptide active ingredients described in claims 1-5, characterized in that: The medicine or pharmaceutical composition comprises any of the dual-site coupled fatty acid-targeted PTPN1 long-acting glucose-lowering BimBH3 mimetic peptides and one or more pharmaceutically acceptable carriers or excipients.

7. Use of the BimBH3 mimetic peptide analogue with dual-site coupling of fatty acids according to any one of claims 1 to 5 in the preparation of an inhibitor for inhibiting PTPN1 activity.

8. Use of the BimBH3 mimetic peptide analog with dual-site coupling of fatty acids according to any one of claims 1 to 5 in the preparation of a drug for preventing or treating a disease targeting PTPN1.

9. The use according to claim 8, characterized in that: The diseases include type 2 diabetes, cancer, obesity and Alzheimer's disease, among which long-acting anti-type 2 diabetes therapeutic drugs are preferred.

10. The use according to claim 8, characterized in that: The administration method of the dual-site coupled fatty acid BimBH3 mimetic peptide is oral administration or injection.

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