Fatty acylation BimBH3 analogue containing D-type amino acid as well as preparation method and application of fatty acylation BimBH3 analogue

Abstract

The invention discloses a BimBH3 analogue containing D-type amino acid and N-terminal fatty acid acylation, a preparation method of the BimBH3 analogue and application of the BimBH3 analogue as a PTPN1 inhibitor to development of hypoglycemic drugs. The structural general formula of the novel BimBH3 mimic peptide analogue is shown as a formula I: # imgabs0 #, wherein Raa2 is equal to D-His, D-Leu, D-Pro, D-Ser, D-Arg, D-Gln, D-Lys, D-Trp, D-Val, D-Tyr, D-Ala, Raa3 is equal to D-Gln, Raa7 is equal to D-Arg, and Raa8 is equal to D-Allo-Ile. The mimic peptide compound is derived from a core region of a Bim-BH3 structural domain, different D-type amino acid site-specific replacement modification and N-terminal conjugated hexadecanoic acid modification are adopted for amino acids at the second, third, seventh and eighth sites respectively, the BimBH3 mimic peptide modified by the D-type amino acid is more stable and has higher metabolic stability, and the target binding capacity and inhibitory activity of the BimBH3 mimic peptide can still be reserved or even improved. The BimBH3 mimic peptide compound shows an excellent PTPN1 inhibitor, the ability of resisting protease hydrolase and various digestive enzymes is also remarkably improved, and the BimBH3 mimic peptide compound has longer pharmacological action time and can play a potential long-acting hypoglycemic role through subcutaneous injection or oral administration. The compound is completely prepared by a polypeptide solid-phase synthesis method, and an obtained crude product is subjected to semi-preparative liquid chromatography purification and freeze-drying 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 BimBH3 analogs containing D-amino acids and acylated with fatty diacids at the N-terminus, a preparation method thereof, and the development of hypoglycemic drugs for use as PTPN1 inhibitors. Background Art

[0002] Diabetes is a metabolic disorder caused by insufficient insulin secretion or pancreatic islet dysfunction, a chronic disease characterized by hyperglycemia. It has become a serious social health problem and a significant socioeconomic burden. According to IDF data, my country has the largest number of adults aged 20-79 with diabetes. Diabetes is a group of lifelong metabolic diseases caused by various factors and characterized by chronic hyperglycemia. It is the third leading non-communicable cause of death after cancer and cardiovascular disease, and poses a serious threat to human health. Therefore, the treatment of diabetes is one of the major issues that urgently need to be addressed in the medical field in my country and even globally (Diabetes Research and Clinical Practice 183, 109-119 (2022)).

[0003] In recent years, an increasing number of novel targets or novel mechanisms of action have been used to develop drugs for the treatment of type 2 diabetes. These drugs can be categorized into the following categories: ① Increasing insulin secretion and delaying intestinal glucose absorption, such as the glucagon-like peptide-1 (GLP-1) receptor; ② Reducing hepatic glucose production, such as glycogen phosphorylase (GP); ③ Improving carbohydrate and lipid metabolism, such as adenosine 5'-monophosphate-activated protein kinase (AMPK); and ④ Enhancing insulin sensitivity in target tissues, such as PTPN1. Among them, PTPN1 is a non-transmembrane protein tyrosine phosphatase (PTP) family that plays a negative regulatory role in the insulin and leptin signaling pathways. With the gradual deepening of understanding of the mechanism of action of PTPN1, researchers have found that PTPN1 is an ideal drug target for the treatment of diabetes and obesity (Nature Reviews Endocrinology 20, 366–378 (2024)). PTPN1 belongs to the protein tyrosine phosphatase family and is an enzyme that specifically hydrolyzes aromatic phosphates, such as phosphate groups on phosphotyrosyl (pTyr) residues. By dephosphorylating tyrosine residues on the insulin receptor or its substrates, it negatively regulates insulin signaling transduction. Overexpression of PTPN1 in tissue cells reduces the activity of PTK, preventing the insulin receptor from binding to insulin, thereby causing insulin resistance and ultimately leading to type 2 diabetes. Together with protein tyrosine kinases (PTKs), it maintains the balance of tyrosine protein phosphorylation, participates in cell signal transduction, and regulates cell growth, differentiation, metabolism, gene transcription, and immune response. PTPN1 is a negative regulator of insulin and leptin signaling, which can dephosphorylate activated insulin receptors (IRs) and insulin receptor substrates (IRSs), thereby blocking the activation of the downstream PI3K-Akt pathway and inhibiting the transport of type 4 glucose transporters and glucose uptake in insulin-sensitive cells. It is generally considered a therapeutic target for metabolic diseases such as diabetes and obesity (Journal of Medicinal Chemistry 66, 3030-3044 (2023)). Although PTPN1 inhibitors have great potential in the development of hypoglycemic drugs, and many compounds have shown high inhibitory activity, most inhibitors have disadvantages such as poor cell membrane permeability, poor specificity and selectivity, and toxic side effects due to their excessive polarity, resulting in their cessation in preclinical research. PTPN1 inhibitors mainly include inorganic small molecule compounds, organic compounds, and PTPN1 inhibitors from natural products.To date, several small molecule compounds (ertiprotafib, trodusquemine, and JTT-551) have entered clinical trials, but all have been discontinued due to poor efficacy or side effects (PLoS One 15, e0240044(2020)). The development of PTPN1 inhibitors is hampered by poor cell membrane permeability and selectivity. Therefore, it is imperative to address the shortcomings of existing PTPN1 inhibitory molecules and develop novel, highly selective, low-side-effect, and long-lasting PTPN1 inhibitors to meet the urgent clinical needs in China.

[0004] Peptides are a class of compounds composed of amino acids linked by peptide bonds, usually composed of 10-100 amino acid molecules, which are linked in the same way as proteins. Their relative molecular mass is less than 10,000 and they are chemically active substances that are ubiquitous in organisms. In recent years, with the development and maturity of peptide synthesis technology, peptide drugs have become one of the hot spots in drug research and development. Due to their wide indications, high safety and significant efficacy, they have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, cardiovascular and cerebrovascular diseases, hepatitis, diabetes, and AIDS, and have broad development prospects (Cytokine 82, 52-57 (2016)). However, the disadvantages of peptide drugs are also obvious. Compared with chemical drugs, peptide drugs have unstable physical and chemical properties, are easily oxidized and hydrolyzed, easily aggregated, have a short half-life, a fast clearance rate, are not easy to penetrate the cell membrane, and most drugs cannot be taken orally (Current Pharmaceutical Design 16, 3185-3203 (2010)). Therefore, based on the advantages and disadvantages of peptide drugs, it is very necessary to rationally design peptide drugs. Among them, the introduction of non-natural amino acids through amino acid replacement can greatly improve the performance of peptide drugs and significantly prolong the duration of peptide drugs in the body. In the natural environment, amino acids, except glycine, appear in two enantiomeric forms, L and D. Modifying peptides by incorporating D-amino acids to evade recognition by human and microbial proteases is an effective strategy to prevent premature hydrolysis and degradation of proteins. Peptides composed of D-amino acids themselves show resistance to enzymatic proteolysis, which is in sharp contrast to their L-amino acid counterparts. In addition, studies have shown that the introduction of non-natural amino acids can increase the affinity of peptides and prolong their half-life (Pharmacology Research & Perspectives 10:e00916(2022)).

[0005] The fatty-acylated BimBH3 analogs containing D-amino acids described herein are derived from the core region of the Bim-BH3 domain. They are modified by site-specific substitutions of different D-amino acids at positions 2, 3, 7, and 8, and by site-specific substitutions of different D-unnatural amino acids at positions 2, 3, 7, and 8, respectively. The N-terminus is conjugated with hexadecanoic acid. The D-amino acid modification enhances the stability of the BimBH3 mimetic peptides, while retaining or even improving their target binding and inhibitory activity. The BimBH3 mimetic peptide compounds described herein exhibit excellent PTPN1 inhibitory properties, significantly enhancing their resistance to proteases, hydrolases, and various digestive enzymes. They exhibit a prolonged duration of action and can potentially exert long-lasting hypoglycemic effects via subcutaneous or oral administration. Summary of the Invention

[0006] The present invention provides a class of BimBH3 analogs containing D-amino acids and acylated with a fatty diacid at the N-terminus, as well as methods for their preparation and application in the development of hypoglycemic drugs as PTPN1 inhibitors. These BimBH3 mimetic peptide analogs exhibit excellent PTPN1 inhibitory activity and resistance to degradation by various proteases, such as proteinase K, DPP-4, pepsin, and chymotrypsin. They also possess excellent in vitro plasma stability and a significantly prolonged half-life. They can be used to develop long-acting drugs for the prevention or treatment of diseases targeting PTPN1, preferably type 2 diabetes.

[0007] To achieve the above-mentioned objectives, the present invention utilizes the following technical solutions: The present invention discloses a class of BimBH3 analogs containing D-amino acids and acylated at the N-terminus with fatty acids that can act as PTPN1 inhibitors and treat type 2 diabetes, as well as their preparation methods and applications. The general structural formula of the BimBH3 mimetic peptide analog is shown in Formula I:

[0008]

[0009] Formula I. General structural formula of fatty acylated BimBH3 analog compounds containing D-amino acids.

[0010] Among them Raa 2 =D-His, D-Leu, D-Pro, D-Ser, D-Arg, D-Gln, D-Lys, D-Trp, D-Val, D-Tyr, D-Ala, Raa 3 =D-Gln, Raa 7 =D-Arg, Raa 8 =D-Allo-Ile.

[0011] The fatty acylated BimBH3 analogue containing D-amino acids is modified by acylation of the N-terminus with hexadecanoic acid, Raa2 Use D-His, D-Leu, D-Pro, D-Ser, D-Arg, D-Gln, D-Lys, D-Trp, D-Val, D-Tyr, D-Ala substitution modification, Raa 3 Using D-Gln, Raa 7 Using D-Arg, Raa 8 D-Allo-Ile was used for substitution modification.

[0012] Furthermore, the preparation method of the fatty acylated BimBH3 analogue containing D-amino acids comprises the following steps:

[0013] (1) At room temperature, a certain amount of CTC resin with a degree of substitution of 1.308 was weighed and placed in a peptide solid phase synthesis reactor and swelled with dimethylformamide / dichloromethane;

[0014] (2) adding a mixture of tetrahydropyridine and dimethylformamide to remove the Fmoc protecting group;

[0015] (1) Place the CTC resin in a peptide solid phase synthesizer at room temperature and swell the resin with dichloromethane or dimethylformamide;

[0016] (2) adding a mixture of tetrahydropyridine and dimethylformamide to remove the Fmoc protecting group;

[0017] (3) Add 2-3 times the molar amount of N-Fmoc-protected amino acids and fatty acids to the CTC resin, and add 2.4-4.8 times the molar amount of HoBt, DIC / HBTU, HoBt, DIEA / Oxyma, and DIC to the CTC resin, and bubble react in a peptide solid phase synthesis reactor at 40°C for 30-120 minutes;

[0018] (4) repeating steps (2) and (3) until the synthesis of the entire fatty acylated BimBH3 analog sequence containing D-amino acids is completed;

[0019] (5) Add the lysis solution to the product of step (4), place it in a shaker at 40°C for 2 hours, add absorbent cotton to a syringe to filter, add anhydrous ice ether to precipitate the solid, wash the precipitated solid, and vacuum dry it to obtain a crude product of the fatty acylated BimBH3 analog containing D-amino acids;

[0020] (6) The crude product of the fatty acylated BimBH3 analogue containing D-amino acids is purified by reverse phase preparative liquid chromatography, and the mobile phase solution of the target compound peak is collected. After acetonitrile and methanol are removed by rotary evaporation, the solution is freeze-dried to obtain a powdery or flocculent solid, thereby obtaining a pure product of the fatty acylated BimBH3 analogue containing D-amino acids.

[0021] Furthermore, the lysis solution includes triisopropylsilane, 3,6-dioxa-1,8-octanedithiol, water and trifluoroacetic acid.

[0022] Furthermore, in step (5), N2 is blown after filtration to remove excess trifluoroacetic acid.

[0023] The present invention also provides a drug or pharmaceutical composition comprising the long-acting hypoglycemic fatty-acylated BimBH3 analogue containing D-amino acids targeting PTPN1 as described in any one of claims 1-5 as an active ingredient, characterized in that the drug or pharmaceutical composition comprises any one of the long-acting hypoglycemic fatty-acylated BimBH3 analogues containing D-amino acids targeting PTPN1 and one or more pharmaceutically acceptable carriers or excipients.

[0024] Furthermore, the fatty acylated BimBH3 analogue containing D-amino acids is used to prepare an inhibitor for inhibiting PTPN1 activity;

[0025] Furthermore, the fatty acylated BimBH3 analogs containing D-amino acids are used to prepare drugs for preventing or treating diseases targeting PTPN1;

[0026] Furthermore, the disease includes one of cancer, diabetes, metabolic syndrome, bone dysplasia syndrome, Alzheimer's disease and obesity, wherein the preferred indication is type 2 diabetes;

[0027] Furthermore, the cancer includes one of pancreatic cancer, prostate cancer, liver cancer, kidney cancer, thyroid cancer, skin cancer, lymphoma, breast cancer, bladder cancer, esophageal cancer, gastrointestinal cancer, tumors of the central nervous system, and tumors of the peripheral nervous system.

[0028] Furthermore, the drug or pharmaceutical composition containing the fatty acylated BimBH3 analog containing D-amino acids as an active ingredient is administered orally or by injection.

[0029] Compared to the prior art, the present invention provides the following advantages: A class of D-amino acid-containing BimBH3 mimetic peptides targeting PTPN1, capable of long-lasting hypoglycemic effects via subcutaneous injection or oral administration, are obtained through a peptide solid-phase synthesis method. These mimetic peptide compounds are derived from the core region of the Bim-BH3 domain, with site-specific substitution modifications at amino acids 2, 3, 7, and 8 using different D-non-natural amino acids, and the N-terminus is conjugated with hexadecanoic acid. Experimental studies have demonstrated that these D-amino acid-modified fatty-acylated BimBH3 analogs are more stable and metabolically stable after D-amino acid modification, while retaining or even improving their target binding and inhibitory activity. These D-amino acid-modified BimBH3 analogs can be used as PTPN1 inhibitors in the development of therapeutics for diseases such as cancer and diabetes, particularly for metabolic syndromes characterized by insulin resistance, such as type 2 diabetes and obesity. The BimBH3 mimetic peptide compounds of the present invention exhibit excellent PTPN1 inhibitory properties, significantly enhancing their resistance to proteases and various digestive enzymes, and exhibiting a longer duration of action. Compared to currently available long-acting glucose-lowering drugs, the fatty-acylated BimBH3 analogs containing D-amino acids described herein consist of 12 amino acids, have a simpler structure, excellent metabolic stability, and oral potential. Their synthesis cost is significantly lower than that of currently marketed once-weekly GLP-1 receptor agonists such as semaglutide and tilpotide. Therefore, the fatty-acylated BimBH3 analogs containing D-amino acids described herein have significant development value and application prospects in the development of long-acting glucose-lowering drugs targeting PTPN1 for subcutaneous or oral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1. These are the experimental results of the DPP-4 enzyme metabolic stability test of the fatty acylated BimBH3 analogs D-9, D-10, D-11, D-12, and D-14 containing D-amino acids described in the present invention;

[0031] Figure 2. BLI technology was used to evaluate the interaction kinetics and affinity between the D-type BimBH3 mimetic and PTPN1 protein;

[0032] Figure 3. Molecular docking results of the fatty-acylated BimBH3 analogs D-11 (top) and D-14 (bottom) containing D-amino acids described herein with PTPN1 in MOE. Overview of the simulated molecular interactions of compounds D-11 and D-14 with the target protein PTPN1 (left); ligand-protein 3D interaction diagrams of PTPN1 and selected binding sites of BimBH3 analogs D-11 and D-14 (center); and ligand-protein 2D interaction diagrams of PTPN1 and selected binding sites of BimBH3 analogs D-11 and D-14 (right).

[0033] Figure 4 Effects of subcutaneous injection of different doses of the fatty acylated BimBH3 analog D-14 containing D-amino acids of the present invention on GTT in KM mice;

[0034] Figure 5 Effects of oral administration of different doses of the fatty acylated BimBH3 analog D-14 containing D-amino acids of the present invention on OGTT in KM mice; DETAILED DESCRIPTION

[0035] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The methods in the following embodiments are all conventional methods unless otherwise specified. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0036] Example 1: Synthesis of Fatty Acylated BimBH3 Analogs Containing D-Amino Acids

[0037] Taking compound D-1 as an example, the specific preparation process is as follows:

[0038] (1) Resin activation: Weigh the corresponding amount of CTC resin and place it in a manual peptide solid phase synthesizer. Add 5 ml of DCM to swell the resin for 1 h at room temperature.

[0039] (2) Attachment of Phe(F): Wash with DMF three times, add 3 times the molar amount of Fmoc-Phe-OH, 10 ml of DCM, and 3.6 times the molar amount of DIEA to the peptide solid phase synthesizer, respectively, and react at room temperature for 1 hour. After the reaction, wash with DMF three times. Block the resin with a blocking solution prepared with a ratio of DCM:MeOH:DIEA = 17:2:1, add 10 ml each time and block for 15 minutes, repeat twice, and wash with DMF three times. Add a mixture of 20% tetrahydropyridine and 80% DMF to remove the Fmoc protecting group twice (8 minutes + 15 minutes), wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0040] (3) Glu(E) attachment: Wash with DMF three times, add 2 times the molar amount of Fmoc-Glu(OtBu)-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, remove the Fmoc protecting group twice (8 min + 15 min) by adding a mixture of 20% tetrahydropyridine and 80% DMF, wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0041] (4) Attachment of Asp(D): Wash with DMF three times, add 2 times the molar amount of Fmoc-Asp(OtBu)-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, add a mixture of 20% tetrahydropyridine and 80% DMF to remove the Fmoc protecting group twice (8 min + 15 min), wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0042] (5) Attachment of Gly(G): Wash with DMF three times, add 2 times the molar amount of Fmoc-Gly-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, remove the Fmoc protecting group twice (8 min + 15 min) by adding a mixture of 20% tetrahydropyridine and 80% DMF, wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0043] (6) Connecting Ile(I): Wash with DMF three times, add 2 times the molar amount of Fmoc-Ile-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, remove the Fmoc protecting group twice (8 min + 15 min) by adding a mixture of 20% tetrahydropyridine and 80% DMF, wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0044] (7) Arg(R) attachment: Wash with DMF three times, add 2 times the molar amount of Fmoc-Arg(Pbf)-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, remove the Fmoc protecting group twice (8 min + 15 min) by adding a mixture of 20% tetrahydropyridine and 80% DMF, wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0045] (8) Arg(R) attachment: Wash with DMF three times, add 2 times the molar amount of Fmoc-Arg(Pbf)-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, remove the Fmoc protecting group twice (8 min + 15 min) by adding a mixture of 20% tetrahydropyridine and 80% DMF, wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0046] (9) Attachment of Leu (L): Wash with DMF three times, add 2 times the molar amount of Fmoc-Leu-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, add a mixture of 20% tetrahydropyridine and 80% DMF to remove the Fmoc protecting group twice (8 min + 15 min), wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0047] (10) Glu(E) attachment: Wash with DMF three times, add 2 times the molar amount of Fmoc-Glu(OtBu)-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, remove the Fmoc protecting group twice (8 min + 15 min) by adding a mixture of 20% tetrahydropyridine and 80% DMF, wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0048] (11) Connecting Gln(Q): Wash with DMF three times, add 2 times the molar amount of Fmoc-Glu(OtBu)-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, add a mixture of 20% tetrahydropyridine and 80% DMF to remove the Fmoc protecting group twice (8 min + 15 min), wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0049] (12) Connecting D-His(H): Wash with DMF three times, add 2 times the molar amount of Fmoc-D-His-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, add a mixture of 20% tetrahydropyridine and 80% DMF to remove the Fmoc protecting group twice (8 min + 15 min), wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0050] (13) Connecting Ile(I): Wash with DMF three times, add 2 times the molar amount of Fmoc-Ala-OH, 2.4 times the molar amount of HoBt, 10 ml of DMF, and 2.4 times the molar amount of DIC, respectively, and react at 40°C for 30 min. Wash with DMF three times, remove the Fmoc protecting group twice (8 min + 15 min) by adding a mixture of 20% tetrahydropyridine and 80% DMF, wash with 10 ml of DMF three times, and detect with Kaiser's reagent.

[0051] (14) Palmitic acid ligation: Wash with DMF three times, add 3 times the molar amount of palmitic acid, 3.6 times the molar amount of HBTU, HoBt and DIEA respectively, dissolve in 10 ml of DMF, react with bubbling at 40 °C for 2 h, wash with DMF three times, and detect with Kaiser's reagent.

[0052] (15) Wash with 5 ml DMF 3 times, 5 ml DCM 3 times, and 5 ml MeOH 3 times, and drain.

[0053] (17) Cutting and cleaving the side chain protecting groups of the synthesized fatty acylated BimBH3 analogue containing D-amino acids: the product was dried with a vacuum pump and placed in a 10 mL centrifuge tube. A cutting solution prepared by 0.25 mL of triisopropylsilane, 0.25 mL of water, 0.25 mL of 3,6-dioxa-1,8-octanedithiol, and 9.25 mL of trifluoroacetic acid was added. The tube was shaken at 40°C for 2 h. Filtered with absorbent cotton in a syringe, the excess trifluoroacetic acid was blown off with N2, 30 mL of anhydrous ice ether was added, and the mixture was centrifuged at 3500 rpm for 3 min to obtain a white precipitate. The mixture was washed three times and dried under vacuum to obtain a crude product of the fatty acylated BimBH3 analogue containing D-amino acids.

[0054] (18) The crude product was purified by reverse phase preparative liquid chromatography (RP-HPLC), and the mobile phase solution of the target compound peak was collected. After rotary evaporation to remove acetonitrile and methanol, the product was freeze-dried to obtain a powder or flocculent solid, which was the pure fatty acylated BimBH3 analog containing D-amino acids. The structure was confirmed by mass spectrometry and high performance liquid chromatography.

[0055] The mass spectrometry data and HPLC purity analysis data of the 14 fatty acylated BimBH3 analog compounds containing D-amino acids described in the present invention are shown in Table 1.

[0056] Table 1. HPLC purity analysis data and mass spectrometry data of D-type BH3 mimetic peptide analogs

[0057]

[0058] Example 2: Protein tyrosine phosphatase 1B (PTPN1) inhibitory activity assay

[0059] In the present invention, MES buffer was used as the reaction system, PTPN1 was utilized, disodium p-nitrophenylphosphate (pNPP) was used as the specific substrate, the lead compound BimBH3-SM6 was selected as the positive control, and DMSO was used as the negative control. A screening model based on the enzyme reaction rate using a 96-well microplate as a carrier was established to search for PTPN1 inhibitors through enzymatic methods.

[0060] The specific implementation method is as follows: using a MES buffer system (20mM, pH 6.5), the reaction system totaling 200uL, the enzyme used in the system is human protein tyrosine phosphatase IB, the specific substrate is disodium p-nitrophenyl phosphate (pNPP), DMSO is used as a negative control, and sodium orthovanadate (2mM) is used as a positive control. The compound is dissolved in DMSO, and the gradient concentration is set to 10μM, 5uM, 1uM, 0.5μM, 0.1uM, 0.01μM, and 0.001μM, with three replicates per group. The microplate reader is preheated to 37°C in advance. The well plate with the sample loaded is placed in the microplate reader detector and the lid is closed. After creating a new detection plate, the detection wavelength is set to 405nm. Immediately select the detection well area and perform the absorbance test. The OD405 values ​​at different time points are read on the microplate reader at 1min, 5min, and 10min, and the rate of change of OD405 (△OD / min) is calculated. 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 enzyme activity is expressed as speed. The data of each group were analyzed using t-test. The formula for calculating the inhibition rate of the compound on PTPN1 is:

[0061] V=(OD 5min -OD 1min ) / 4*1000 or (OD 10min -OD 1min ) / 9*1000

[0062] Inhibition rate (%) = (v DMSO -v 样本 ) / v DMSO ×100%

[0063] Among them, OD 1min ,OD 5min ,OD 10min Respectively represent the absorbance values ​​measured at 405 nm for 1 min, 5 min, and 10 min of each group, v DMSO 、v 样本 The initial average reaction rates of the negative control group and the test compound are represented respectively. GraphPadPrism software was used to perform statistical inhibition data statistics and processing, and the inhibitory dose-effect curve was fitted and IC was calculated. 50 value.

[0064] Table 2. PTPN inhibitory activity of fatty acylated BimBH3 analogs containing D-amino acids

[0065]

[0066] nt: Compounds with inhibition rates lower than 50% in the initial screening were not subjected to IC 50 Determination of.

[0067] Example 3: Evaluation of the stability of fatty acylated BimBH3 analogs containing D-amino acids against enzyme metabolism.

[0068] Proteinase K

[0069] Weigh 2 mg of compound and dissolve it in TBS buffer to prepare a 0.4 mM stock solution. Measure 1 μL of Proteinase K and dilute it to 400 μL with TBS to obtain a 50 μg / mL stock solution of Proteinase K. Accurately measure 1600 μL of compound (0.4 mM) and 400 μL of Proteinase K stock solution (50 μg / mL) to create a 2000 μL reaction system. At various time points (0 h, 0.5 h, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, and 24 h), take 200 μL of sample and quench with 500 μL of quencher (0.2% TFA / CH3CN). The quenched sample is vortexed for 30 s and centrifuged at 14,000 rpm for 10 min. After centrifugal filtration, immediately analyze by RP-HPLC and integrate the remaining peak area of ​​the compound. GraphPad Prism 5 software was used to fit the residual peak area of ​​the compound at each time point into a curve, and the half-life (t 1 / 2 ) and calculated the increase or decrease times compared with SM-6 (relative stability times).

[0070] Chymotrypsin

[0071] Weigh 5 mg of compound and dissolve it in PBS to prepare a mother solution of compound at a concentration of 0.6 mM. Weigh 7.5 mg of chymotrypsin and dissolve it in 1 mL of PBS to prepare an enzyme solution of 1 μg / μL. Accurately measure 1500 μL of compound (0.6 mM) and 500 μL of enzyme solution (1 μg / μL) to mix to form a 2000 μL reaction system. At different time points (0h, 0.5h, 1h, 2h, 3h, 5h, 8h, 12h, and 24h), take 200 μL of sample and add 500 μL of quencher (0.2% TFA / CH3CN) for quenching. The quenched sample was vortexed for 30s, centrifuged at 14000 r / min for 10 min, and immediately analyzed by RP-HPLC after centrifugal filtration to integrate the remaining peak area of ​​the compound. GraphPad Prism 5 software was used to fit the remaining peak area of ​​the compound at each time point into a curve, and the half-life (t 1 / 2 ) and calculated the increase or decrease times compared with SM-6 (relative stability times).

[0072] Pepsin

[0073] Weigh 2 mg of compound and dissolve it in DMSO to prepare a 10 mM stock solution. Weigh 14.75 mg of pepsin and dissolve it in 35 mM HCl to 500 μL, yielding a 25 ug / μL stock solution. Accurately measure 50 μL of compound (40 mM), 136 μL of enzyme solution (25 μg / μL), and 1814 μL of 35 mM HCl to create a 2000 μL reaction system. At various time points (0 h, 0.5 h, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, and 24 h), 200 μL of sample was quenched with 500 μL of quencher (0.1 M Tris HCl (pH = 8.0)). The quenched sample was vortexed for 30 s, centrifuged at 14,000 rpm for 10 min, and immediately analyzed by RP-HPLC after centrifugation. The remaining peak area of ​​the compound was integrated. GraphPad Prism 5 software was used to fit the residual peak area of ​​the compound at each time point into a curve, and the half-life (t 1 / 2 ) and calculated the increase or decrease times compared with SM-6 (relative stability times).

[0074] DPP-4 enzyme

[0075] Weigh 1 mg of compound and dissolve it in 1 mL of phosphate buffer (pH = 7.0, 20 mM) to prepare a compound stock solution with a concentration of 1 mg / mL. Use a pipette to accurately measure 4.5 μL of DPP-4 enzyme (≥45 U / mg) and add it to the polypeptide sample solution and mix well. The reaction system was incubated in a 37°C water bath. At different time points (0h, 0.5h, 1h, 2h, 3h, 5h, 8h, 12h, and 24h), 100 μL of sample was taken and 200 μL of quencher (sodium bicarbonate (0.1M)) was added to terminate the reaction. The quenched sample was vortexed for 30s and centrifuged at 14000r / min for 10min. After centrifugation and filtration, it was immediately analyzed by RP-HPLC to integrate the remaining peak area of ​​the compound. GraphPadPrism5 software was used to fit the remaining peak area of ​​the compound at each time point to a curve, and the half-life (t 1 / 2 ) and calculated the increase or decrease times compared with SM-6 (relative stability times).

[0076] Table 3: Metabolic stability of selected BimBH3 analogs with good PPTN1 inhibition

[0077]

[0078] Example 4: Evaluation of Plasma Stability of Fatty Acylated BimBH3 Analog Compounds Containing D-Amino Acids.

[0079] The highly active fatty acylated BimBH3 analog containing D-amino acids was dissolved in 1 / 2PBS and 1 / 2H2O to a concentration of 0.4 mM and then incubated with 40% rat plasma. 300 uL of samples were taken at different time points (0h, 1h, 10h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h and 216h) and quenched by adding 300 uL of quencher (0.2% TFA / CH3CN). The quenched samples were vortexed for 30s, centrifuged at 15000 r / min and 4°C for 10 min, and analyzed by analytical HPLC after post-treatment. The percentage of compound remaining at each time point relative to the time origin (%) was determined by relative peak area. The remaining peak area of ​​the integrated compound was plotted against the reaction time, and the data were fitted to an exponential decay model in GraphPad Prism software to determine the half-life, which was used to evaluate plasma stability. The four selected D-form compounds all exhibited good plasma stability, with degradation t1 / 2 values ​​in rat plasma of 68.16h, 98.21h, 95.49h, and 100.62h, respectively. Compared with the analog BimBH3SM6, their plasma stability was improved by 5.27-, 8.25-, 8.02-, and 8.45-fold, respectively. This confirms our hypothesis that replacing natural L-form amino acids with unnatural D-form amino acids can yield analogs with higher target activity and plasma stability.

[0080] Table 4. In vitro plasma half-life data of four highly active PTPN1 inhibitor BimBH3 mimetic peptides

[0081]

[0082] Example 5: Determination of the affinity of fatty acylated BimBH3 analogs containing D-amino acids using biofilm interferometry (BLI)

[0083] Weigh 2 mg of compound and dissolve it in DMSO to prepare a 10 mM stock solution. Dilute 1 μL of the stock solution to 10 μM with MES buffer (20 mM, pH 6.5). For PTPN1, dilute to 10 μg / mL. Before the experiment begins, equilibrate the sample and buffer to room temperature. Pre-wet new biosensors for at least 10 minutes before use. Add 250 μL of buffer to the Max plate. Use tweezers to transfer the probe from the packaging tray to the buffered well. Add buffer, loading tube, and sample to a GatorBio 96-well black plate (Cat#06-0279). Avoid creating bubbles when adding buffer and sample to the Max plate or black microplate. Mark the buffer, loading, and sample wells on the 96-well plate diagram. In kinetic experiments, the molar concentration must be entered in the sample wells. Mark the probe positions on the Max plate diagram. Add regeneration solution and neutralization solution to the Max plate and mark the Regeneration and Neutralization well positions on the plate diagram. Set the Cycle according to the designed step positions and click the "Start" button to start the experiment.

[0084] Table 5. Kinetic parameters and affinity of four selected compounds to PTPN1 protein determined by BLI method

[0085]

[0086] Example 6: Prediction of mechanism of action using molecular docking

[0087] We used the Molecular Operating Environment (MOE) docking software to simulate the interaction between PTPN1 and compounds D-11 and D-14 with good PTPN1 inhibitory activity. The crystal structure of PTPN1 was downloaded from the Protein Data Bank (PDB) (https: / / www.rcsb.org / ), and the PDBID is 5K9V. After downloading the protein structure, it was imported into MOE, and the redundant chains were deleted and the protein structure was pre-processed. The protein was corrected for structural problems, protonated, unbound water molecules were deleted, and energy was optimized. In the set PTPN1 crystal structure, the active site is defined around the binding inhibitor. The corresponding structure of the D-type BimBH3 analog was drawn in ChemDraw 22.2.0 64-bit, saved in mol format, and opened and preprocessed in MOE. The PTPN1 complex-D-type BimBH3 was docked using MOE software under standard settings (T = 300K, pH 7.0). After docking, the docking results were displayed in the newly opened Database Viewer window. The list was sorted by the quality of the binding, primarily based on the docking score and the S column. A more negative value indicates a tighter binding of the ligand to the receptor. The molecular docking results of the selected D-11 and D-14 with PTPN1 can be seen from the docking result surface figure of D-type BimBH3 analogs with PTPN1. The preferred compounds D-11 and D-14 can both bind well to the surface or pocket of PTPN1. Among them, compound D-11 is predicted to interact with Glu26, Ala27, Ser28, Asp29, Phe30, Pro31, Asp53, His54, Ser55, Arg56, Ile57, Lys58, Leu71, Lys73, Met74, Glu75, Gln78, Arg79, Ser80, Pro206, His207, Gly209, Pro210, Leu251, Lys255, and Phe256, occupying the surface near the active site and binding to PTPN1. Compound D-14 is predicted to interact with Pro38, Lys39, Lys41, Asn42, Arg43, Asn44, Arg45, Tyr46, Arg47, Asp63, Asn64, Leu88, Pro89, Asn90, Thy91, Cys92, Gly93, His94, Leu119, Ile134, Phe135, Glu136, and Asp137, occupying the C-site surface of PTPN1 (via interactions with residues Pro38, Lys41, Asn42, Arg45, Tyr46, Arg47, Leu88, Pro89, and Asn90). These interaction patterns explain the binding affinity and specificity of the analogs and indicate that hydrogen bonds play an important role in stabilizing the ligand-receptor complex, which may be the key to the excellent PTPN1 inhibitory or regulatory activity of these analogs.

[0088] Example 7: Glucose tolerance test (GTT).

[0089] The preferred compound D-14 was administered subcutaneously or orally to evaluate its effect on an oral glucose tolerance test (GTT) in KM mice. For the oral glucose tolerance test, KM mice (male, 5-6 weeks old, 18-22 g) were fasted from 6:00 PM the day before the experiment until 9:00 AM the following morning. During the fasting period, the mice maintained normal drinking water. Twenty minutes before oral administration of starch, mice were administered different doses of compound D-14, either subcutaneously or orally. During the experiment, mice were gently lifted and gavage-administered with a 1 mL syringe (2 g / kg body weight) of starch solution according to standard gavage procedures. Blood glucose levels were measured by tail blood draw using an ACCU-CHEK Performa blood glucose meter (Roche DiabetesCare Gmbh, Sandhofer Strasse 11668305 Mannheim, Germany).

[0090] In the study of the preferred compound D-14, KM male mice were randomly divided into 4 groups. The mice were subcutaneously injected or orally with different doses of the compound. The blood glucose levels at different time points were measured and the area under the glucose curve was calculated based on the actual blood glucose values. When the compound D-14 was injected subcutaneously at doses of 0.05, 0.1 and 0.2 μM / kg, the AUC 0-120min The AUC values ​​were decreased by 7.1%, 15.6% and 26.2% respectively at oral doses of 10, 20 and 40 mg / kg. 0-120min These results indicate that compound D-14 exhibits a significant hypoglycemic effect at a dose of 0.2 μM / kg, and oral administration indicates that our synthesized D-amino acid-modified BimBH3 analogue has the potential to be an oral anti-type 2 diabetes drug.

[0091] 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 with 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 analogs containing D-amino acids and acylated N-terminal fatty acids, and a preparation method thereof and a method for developing hypoglycemic drugs as PTPN1 inhibitors. The general structural formula of the novel Bim BH3 mimetic peptide analog is shown in Formula I: Formula I. General structural formula of novel BimBH3 mimetic peptide compounds containing D-amino acids. where Raa 2 = D-His, D-Leu, D-Pro, D-Ser, D-Arg, D-Gln, D-Lys, D-Trp, D-Val, D-Tyr, D-Ala, Raa 3 = D-Gln, Raa 7 = D-Arg, Raa 8 = D-Allo-Ile.

2. The BimBH3 mimetic peptide analog targeting PTPN1 according to claim 1, characterized in that The N-terminus was modified by hexadecanoic acid acylation, Raa 2 Use D-His, D-Leu, D-Pro, D-Ser, D-Arg, D-Gln, D-Lys, D-Trp, D-Val, D-Tyr, D-Ala substitution modification, Raa 3 Using D-Gln, Raa 7 Using D-Arg, Raa 8 D-Allo-Ile is used for replacement modification. From the compounds, lead compounds that can retain or improve PTPN1 inhibitory activity and have excellent metabolic stability are screened, and can exert potential long-term hypoglycemic effects through subcutaneous injection or oral administration.

3. The BimBH3 mimetic peptide analog targeting PTPN1 according to claim 1, characterized in that The preparation method comprises the following steps: (1) At room temperature, a certain amount of CTC resin with a degree of substitution of 1.308 was weighed and placed in a peptide solid phase synthesis reactor, and swelled with dimethylformamide / dichloromethane; (2) adding a mixture of tetrahydropyridine and dimethylformamide to remove the Fmoc protecting group; (3) adding 2 to 3 times the molar amount of N-Fmoc-protected amino acids and fatty acids of the CTC resin, and adding 2.4 to 4.8 times the molar amount of HoBt, DIC / HBTU, HoBt, DIEA / Oxyma, and DIC of the CTC resin, and bubbling reaction in a peptide solid phase synthesis reactor at 40° C. for 30 min to 120 min; (4) repeating steps (2) and (3) until the synthesis of the entire PTPN1-targeting long-acting glucose-lowering fatty acylated BimBH3 analog sequence containing D-amino acids is completed; (5) The product of step (4) was dried with a vacuum pump and placed in a 10 mL centrifuge tube, a cutting solution was added, and the tube was placed in a shaker at 40° C. for 2 h, and absorbent cotton was added to the syringe for filtration, and anhydrous ice ether was added to precipitate the solid, and the precipitated solid was washed and vacuum dried to obtain a crude product of a BimBH3 mimetic peptide analog targeting PTPN1; (6) The crude product of the BimBH3 mimetic peptide analog targeting PTPN1 is purified by reverse phase preparative liquid chromatography, and the mobile phase solution of the target compound peak is collected, acetonitrile and methanol are removed by rotary evaporation, and then freeze-dried to obtain a powder or flocculent solid, thereby obtaining a pure compound.

4. The preparation method according to claim 3, characterized in that: The cutting fluid in the preparation step (5) includes triisopropylsilane, 3,6-dioxa-1,8-octanedithiol, water and trifluoroacetic acid.

5. The preparation method according to claim 3, characterized in that: In the step (5), after filtering, N2 is blown to remove excess trifluoroacetic acid.

6. A drug or pharmaceutical composition in which the BimBH3 mimetic peptide analog targeting PTPN1 with inhibitory function as claimed in any one of claims 1 to 5 is a pharmaceutically active ingredient, characterized in that: The medicine or pharmaceutical composition comprises any of the novel BimBH3 mimetic peptide analogs targeting PTPN1 and one or more pharmaceutically acceptable carriers, excipients or adjuvants.

7. Use of the BimBH3 mimetic peptide analog targeting PTPN1 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 targeting PTPN1 according to any one of claims 1 to 5 in the preparation of a drug for treating or preventing diseases related to PTPN1 protein as a target.

9. The use according to claim 8, characterized in that: The PTPN1 protein-related diseases include one of cancer, diabetes, metabolic disorders, obesity-induced bone hyperplasia syndrome and metabolic syndrome, among which a diabetes treatment drug with long-term subcutaneous injection (once a week) or oral (daily) administration is preferred.

10. The use according to claim 8, characterized in that: The administration method of the drug or pharmaceutical composition with the BimBH3 mimetic peptide analog targeting PTPN1 as the active ingredient is oral administration or subcutaneous injection.

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